Vista regulatory t cell mediator protein, vista binding agents and use thereof.

Institute

Mexican Property

Industrial

PATENT TITLE NO. 342017 ___SE___

SÍCRíWa I heard ECONOMY

I

<img file="MX342017B_D0001.tif" />

Headlines):

TRUSTEES OF DARTMOUTH COLLEGE

Address:

Rope Ferry Road, # 6210, Hanover, New Hampshire, 03755-1404, USA

Denomination:

VISTA REGULATORY T-CELL MEDIATOR PROTEIN, VISTA LINKING AGENTS AND THE USE OF THEM.

Classification:

Int.CI.8: A61K38 / 17; A61K39 / 395; A61P19 / 02; A61P37 / 00; C07K14 / 47;

C07K16 / 18; C07K19 / 00

Inventor (s):

RANDOLPH J. NOELLE; LILI WANG

REQUEST

Number:

MX / a / 2012/011089

<img file="MX342017B_D0002.tif" />

international presentation:

March 2011

PRIORITY

Country:

Date:

Number:

US

US US

US March 2010 October 201 Qi January 26, 2011 March 2011

12/732,371

61/390,434

61/436,379

61/449,882

Validity: Twenty years

Expiration Date: March 25, 2031

The reference patent is granted based on articles 1, 2, section V, 6, section III, and 59 of the Industrial Property Law. Pursuant to article 23 of the Industrial Property Law, this patent is valid for twenty years, non-extendable, counted from the filing date of the international application and will be subject to the payment of the fee to keep the deechos, f ¢,

Whoever signs this title is Industrial Property (Official Journal of 01/26/2004, 06/16/2005, 25 / Ü1 / 2006, subsection a), 4th and 12th sections I and III 01707 / 2002.15 / 07 / 2004, 07/28/2004 and the Mexican Property Institute and 5th paragraph a) of the Agreement that delegates powers to the Deputy General Directors, Coordinator, Divisional Directors, Heads of Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

based on the provisions per us ai. 6 ° fractions lll and 7 ° bis 2 of l * &lt;Law of the Federation (UOF) 06/27/1991, retaken on 08/02/1994, 10/26/1996 , 12/26/1997, 05/17/1999, 2009/05/06, 01/06/2010 '06/08/2010 08/28/2010 01/27,2312 and 04/09/2012); items 1 °, 3®fiaction V<img file="MX342017B_D0003.tif" /> Regulation of the Mexican Institute of Industrial Property (DOF 12/14/1999, amended on <img file="MX342017B_D0004.tif" />

<img file="MX342017B_D0005.tif" />

<img file="MX342017B_D0006.tif" />

Arenal No. 55G. 1st floor

Col. Pueblo Santa María Tepepan,

Xochirnilco. CP '6020, Mexico City

Such. (55) 53 34 07 00 www.impi qob.mx

Issue Date: September 9, 2016

THE DIVISIONAL DIRECTOR OF PATENTS

<img file="MX342017B_D0007.tif" />

NAHANNY CANAL REYES

<img file="MX342017B_D0008.tif" />

<img file="MX342017B_D0009.tif" />

<img file="MX342017B_D0010.tif" />

IMPI 'ιμτ: Mexican ππο DE LA P.VjriEDAD

VISTA REGULATORY T-CELL MEDIATOR PROTEIN. AGÉ

<img file="MX342017B_D0011.tif" />

VIEW LINK AND USE OF THEM

Introduction

This application claims priority to No. U.S. Serial 12 / 732,371 filed March 26, 2010, No. US Provisional 61 / 390,434 filed October 6, 2010; no. United States Provisional 61 / 436,379 filed on January 26, 2011 and No. US Provisional 61 / 449,882 filed March 7, 2011. All of these applications are incorporated by reference in their entirety.

This application is related to the fact that we discovered, characterized and functionally defined a new ligand, an inhibitor of the lg-superfamily, structurally different, hematopoietically restricted designated as Suppressor of Activation of T cells that contains the V region of Immunoglobulin (VISTA, for its acronym in English) or PD-L3. The extracellular domain has homology to the PD-L1 ligand of the B7 family, and VISTA, like PD-L1, has a profound impact on immunity. However, unlike PD-L1, VISTA expression is exclusively within the hematopoietic compartment. Expression is most prominent in antigen-presenting myeloid cells (APC), although expression is also of great interest in CD4 + T cells and a subset of Foxp3 + regulatory T cells (Treg). An expression of VISTA or a soluble VISTA-lg fusion protein in APCs, potently inhibits the in vitro proliferation of T cells, production of cytokines and induces the expression of Foxp3 in T cells. In contrast, an anti-VISTA monoclonal antibody newly developed interfered with VISTA-induced immune suppression of in vitro T-cell responses in VISTA + APCs. Furthermore, anti-VISTA enhanced in vivo the development of experimental allergic encephalomyelitis T-cell-mediated autoimmune disease (EAE), and facilitated the development of a tumor-specific, protective immune response with subsequent tumor remission. Initial studies of VISTA - / - mice reveal the first signs of spontaneous inflammatory disease, and i

ΙΜΡΙ (^ «will determine its final pathological fate. Unlike all lasSíQtra ^^^^^ it is associated with the PD-ligand (for example, B7-H3, H4, H6) ^ the hematopoietic restriction of VISTA together with its repressive activities ^ profound and unique structural features illustrate that VISTA is a new, functionally non-redundant, central negative regulator of immunity, whose expression is mainly restricted to myeloid.

Background of the invention

Induction of an immune response requires the expansion, differentiation, contraction of T cells, and the establishment of memory T cells. T cells must encounter antigen presenting cells (APCs) and communicate through interactions with the T cell receptor (TCR) / major histocompatibility complex (MHC) on APCs. Once the TCR / MHC interaction is established, other sets of receptor-ligand contacts are required between the T cells and the APC, i.e., co-stimulation via CD154 / CD40 and CD28 / B7.1-B7. .two. Synergy between these contacts is suggested to give rise, in vivo, to a productive immune response capable of purifying pathogens and tumors, and in some cases capable of inducing autoimmunity.

Another level of control was identified, ie, regulatory T cells (Tregs). This specific subset of T cells is generated in the thymus, released in the periphery, and is capable of constant and inducible control of in vitro and in vivo T cell responses (Sakaguchi (2000) Cell 101 (5): 455- 8; Shevach (2000) Annu. Rev. Immunol. 18: 423-49; Bluestone and Abbas (2003) Nat. Rev. Immunol. 3 (3): 253-7). Tregs are represented by a CD4 + CD25 + phenotype and also express high levels of cytotoxic T lymphocytes associated with antigen-4 (CTLA-4), OX-40, 4-1BB and the glucocorticoid-inducible TNF receptor (GITR) associated protein. (McHugh, et al. (2002) Immunity 16 (2): 311-23; Shimizu, et al. (2002) Nat. Immun. 3 (2): 13542). Elimination of Treg cells on day 5 of neonatal thymectomy or depletion of antibodies using anti-CD25, result in the induction of autoimmune pathology and exacerbation of T cell responses to foreign antigens and autoantigens, including anti-tumor responses

<img file="MX342017B_D0012.tif" />

others (1995) J. Immunol. 155 (3): 1151-64; Jones, et al. (2002) Cancer Immun. 2: 1). Furthermore, Tregs were also involved in the induction and conservation of transplant tolerance (Hara et al. (2001) J. Immunol. 166 (6): 3789-3796; Wood and Sakaguchi (2003) Nat. Rev. Immunol. 3: 199-210), since the depletion of Tregs with anti-CD25 monoclonal antibodies resulted in ablation of transplant tolerance and rapid graft rejection (Jarvinen, et al. (2003) Transplantation 76: 1375-9) . Among the receptors expressed by Tregs, GITR appears to be an important component since in vitro or in vivo ligation of GITR on the surface of Tregs with an agonist monoclonal antibody results in rapid termination of Treg activity (McHugh, et al. (2002) above; Shimizu, et al. (2002) above), also resulting in autoimmune pathology (Shimizu, et al. (2002) above) and ablation of transplant tolerance.

Co-stimulatory and co-inhibitory ligands and receptors not only provide a "2nd signal" for T cell activation, but also a balanced network of positive and negative signal to maximize immune responses against infection by limiting autoimmunity. The best characterized co-stimulatory ligands are B7.1 and B7.2, which are expressed by professional APCs, and whose receptors are CD28 and CTLA-4 (Greenwald, RJ, Freeman, GJ, and Sharpe, AH (2005). Annu Rev Immunol 23,515-548; Sharpe, AH, and Freeman, GJ (2002) Nat Rev Immunol 2, 116-126). CD28 is expressed by naive and activated T cells and is critical for optimal T cell activation. In contrast, CTLA-4 is induced after T cell activation and inhibits T cell activation by binding to B7.1 /B7.2, thus affecting CD28-mediated costimulation. CTLA-4 also transduces negative signaling through its cytoplasmic motif ITIM (Teft, W. A., Kirchhof, MG, and Madreñas, J. (2006). Annu Rev Immunol 24.65-97; Teft, WA, Kirchhof, MG, and Madreñas, J. (2006). Annu Rev Immunol 24,65-97. KO the B7.1 / B7.2 mice are affected by the adaptive immune response, Borriello, F., Sethna, Μ. P, Boyd, SD, Schweitzer, A. Ν., ΤίνοΙ, EA, Jacoby, D., Strom, TB, Simpson, EM, Freeman, GJ, and Sharpe, AH (1997)) Immunity 6, 303-313; Freeman, GJ, Borriello, F.,

<img file="MX342017B_D0013.tif" />

INSTITUTE μΞ / iC ΆΌ

Hodes, RJ, Reiser, H., Hathcock, KS, Laszlo, G., McKnigfit ^ D? ÍMnMiL &amp; ^ ·.

Lombard, DB, and others (1993). Science 262,907-909), while KO mice

CTLA-4 cannot adequately control inflammation and the development of systemic autoimmune diseases (Chambers, CA, Sullivan, TJ, and Allison, I.

P. (1997) Immunity 7, 885-895; Tivol, EA, Borriello, F., Schweitzer, AN, Lynch, WP, Bluestone, J, A., and Sharpe, AH (1995) Immunity 3, 541-547; Waterhouse, P., Penninger, JM, Timms, E., Wakeham, A., Shahinian, A., Lee, KP, Thompson, C B., Griesser, H., and Mak, TW (1995). Science 270,985-988.

The B7 family ligands were expanded to include the co-stimulators B7-H2 (ICOS ligand) and B7-H3, as well as the co-inhibitors B7-H1 (PD-L1), B7-DC (PD-L2) , B7-H4 (B7S1 or B7x) and B7-H6 Brandt, CS, Baratin, M „Yi, EC, Kennedy, J., Gao, Z., Fox, B., Haldeman, B., Osuander, CD, Kaifu , T., Chabannon, C., and others (2009) J Exp Med 206, 1495-1503; Greenwald, RJ,

Freeman, GJ, and Sharpe, AH (2005) Annu Rev Immunol 23, 515-548.

The inducible co-stimulatory molecule (ICOS) is expressed in activated T cells and binds to B7-H2 Yoshinaga, SK, Whoriskey, JS, Khare, SD, Sarmiento, U., Guo, J., Horan, T., Shih , G., Zhang, M., Coccia, MA, Kohno, T., and others (1999). Nature 402, 827-832. ICOS is important for T-cell activation, differentiation and function, as well as essential for T-helper cell-induced activation of B cells, Ig class exchange, and germinal center (GC) formation Dong, C., Juedes , AE, Temann, UA, Shresta, S., Allison, JP, Ruddle, NH, and Flavell, RA (2001) Nature 409, 97-101; Tafuri, A, Shahinian, A., Bladt, F., Yoshinaga, SK, Jordana, M., Wakeham, A., Boucher, LM, Bouchard, D., Chan, V. S., Duncan, G., et al. (2001) Nature 409, 105-109; Yoshinaga, SK, Whoriskey, JS, Khare, SD, Sarmiento, U., Guo, J., Horan, T., Shih, G., Zhang, M., Coccia, MA, Kohno, T., and others (1999 ) Nature 402,827-832. On the other hand, programmed death 1 (PD-1), negatively regulates T cell responses. KO PD-1 mice develop lupus-like autoimmune disease, or autoimmune dilated cardiomyopathy based on genetic background Nishirnura, H., Nose, M., Hiai, H., Minato, N., and Honjo, T. ( 1999) Immunity 11, 141-151. Nishirnura, H., Okazaki, T., Tanaka, Y., Nakatani, K.,

Hara, M., Matsumori, A., Sasayama, S., Mizoguchi, N., and Hónjo, 'DELAPUOIir.DAO

INQVSTkUL '· -----—'

T. (2001) Science 291, 319-322. Autoimmunity most likely results from the loss of signaling of both the PD-L1 ligand cerneH ^ D ^ fcS: · ReulenteifféñteTel CD80 was identified as a second receptor for PD-L1 that transduces inhibitory signals in T cells Butte, MJ, Keir, Μ. E., Phamduy, TB, Sharpe, AH, and Freeman, GJ (2007) Immunity 27,111-122. The receptor for B7H3 and B7-H4 is still unknown.

The best characterized co-stimulatory ligands are B7.1 and B7.2 and belong to the Ig superfamily which is made up of many critical immune regulators, such as the B7 family ligands and receptors. Members of the Ig superfamily are expressed on professional antigen presenting cells (APCs), and their receptors are CD28 and CTLA-4. CD28 is expressed by naive and activated T cells and is critical for optimal T cell activation. In contrast, CTLA-4 is induced after T cell activation and inhibits T cell activation by binding to B7.1 / B7.2, affecting CD28-mediated co-stimulation. Knockout (KO) mice B7.1 and B7.2 are affected by the adaptive immune response, while KO CTLA-4 mice cannot adequately control inflammation and development of systemic autoimmune diseases. Over time the B7 family ligands expanded to include co-stimulatory ligands such as B7-H2 (ICOS ligand) and B7-H3, and co-inhibitory ligands such as B7-H1 (PD-L1), B7DC (PD -L2), B7-H4 (B7S1 or B7x), and B7-H6. As a consequence, additional receptors of the CD28 family were identified. ICOS is expressed on activated T cells and binds to B7-H2. ICOS is a positive co-regulator, important for T cell activation, differentiation and function. On the other hand, programmed death 1 (PD-1) negatively regulates T cell responses. KO PD-1 mice developed autoimmune disease similar to lupus, or with dilated cardiomyopathy. Unlike VISTA (the immunosuppressive molecule that is the focus of the present invention), the two inhibitory ligands of the B7 family, PD-L1 and PD-L2, have different expression patterns. PD-L2 is expressed by induction in DCs and macrophages, while PD-L1 is widely expressed both in cells

ΙΜΡΙ £ 1.

hematopoietic as non-hematopoie cell types ^ éW ^ f ^ onsisj ^^^ the

<img file="MX342017B_D0014.tif" />

Immunosuppressive role of the PD-1 receptor, studies using PD-L1 - / - and PD-L2 - / - mice showed that both ligands have overlapping roles in inhibiting T cell proliferation and cytokine production. PD-L1 deficiency improves disease progression in both the non-obese diabetic (NOD) model of autoimmune diabetes and the murine model of multiple sclerosis (experimental autoimmune encephalomyelitis; EAE). PD-L1 - / - cells produce elevated levels of pro-inflammatory cytokines in both models of the disease. Furthermore, studies in NOD mice demonstrated that tissue expression of PD-L1 (ie, within the pancreas) contributes exceptionally to its ability to regionally control inflammation. PD-L1 is also highly expressed in placental syncytlotrophoblasts, which critically control maternal immune responses to the allogeneic fetus.

Taking into account the strong impact of this family of molecules on the regulation of immunity, substantial efforts in murine cancer models showed that selecting this family of molecules can induce protective antitumor immunity. Studies involving anti-CTLA-4 documented improved therapeutic benefit in murine models and clinical trials of melanoma. Mice vaccinated with B 16-GM-CSF (Gvax) promote rejection of B 16 melanomas when combined with blocked CTLA-4 antibodies.

Antibodies against PD-1, as well as PD-L1 also documented anti-tumor immunity and improved host survival in a wide variety of murine tumor models. Finally, although CTLA-4 and PD-1 belong to the same family of co-inhibitory molecules, evidence suggests that they use different non-repetitive mechanisms to inhibit T-cell activation, and when used in combination there is synergy in the ability to anti-CTLA-4 and anti-PD-1 / L1 to improve host survival in murine melanoma.

Based on the above, the elucidation of another new member of the B7 type family and its ligands and modulators would be useful considering the important iritnühidá ^

<img file="MX342017B_D0015.tif" />

INSTITUTE

DE LA PRGé '! C¿ .. · or role of these family members in the regulation of' ta, especially T-cell immunity.

Summary of the invention

The present invention relates to therapeutic methods that modulate the activity and / or that specifically bind or block the binding of a specific regulatory T cell protein to its counterreceptor. This protein, called PD-L3 or VISTA, is a new and structurally different inhibitory ligand of the Ig-superfamily, whose extracellular domain has homology with the PD-L1 ligand of the B7 family. This molecule is referred to interchangeably in the present description as PD-L3 or VISTA or as suppressor of immunoglobulin V-domain T-cell activation (VISTA). VISTA is expressed primarily within the hematopoietic compartment and is highly regulated in myeloid APCs and T cells. The therapeutic intervention of the VISTA inhibitory pathway represents a new approach to modulate T cell-mediated immunity in the treatment of a wide variety of cancers.

The present invention relates in particular to the use of antibodies specific to VISTA or PD-L3 to treat specific cancers including bladder cancer, ovarian cancer, and melanoma.

Furthermore, the present invention relates in particular to the use of PD-L3 or VISTA proteins, especially VISTA multimeric proteins and viral vectors (eg adenovirus) that are expressed to treat the same conditions where immunosuppression is therapeutically desired , such as allergy, autoimmunity, and inflammatory conditions.

As described below, the expression of VISTA appears to be exclusive to the hematopoietic compartment and this protein is highly expressed in mature myeloid cells (CD11b^bnght), with lower expression levels in CD4 T cells⁺, T^reg and CD8 T cells⁺. Soluble VISTA proteins, for example, the soluble VISTA-Ig fusion protein, or the expression of VISTA in APCs, suppress the proliferation of CD4 T cells in vitro⁺ and CD8⁺ and the production of cytokines. It was also observed that 3ηίί-νΐ5ΥΑ, 'ροτ;^Μ^ τηρΙ

<img file="MX342017B_D0016.tif" />

<img file="MX342017B_D0017.tif" />

anti-VISTA (13F3) blocked in vitro suppression of VISTA-induced T cell responses in VISTA APCs⁺. Furthermore, an antiVISTA mAb was found to exacerbate EAE and increase the frequency of encephalitogenic Th17s in vivo.

Furthermore, as described in detail below, an antiVISTA mAb was found to induce tumor remission in multiple (4) murine tumor models.

The expression of VISTA in myeloid-derived suppressor cells (MDSC) in these models is extremely high, suggesting that MDSC VISTA * suppresses tumor-specific immunity. As shown in the present description,

VISTA exerts immunosuppressive activities on T cells both in vitro and in vivo in mice and humans (in vitro only) and is an important mediator in the control of the development of autoimmunity and the immune response to cancer.

Specifically, the data shows that:

(1) VISTA is a new member of the Ig superfamily and contains an Ig-V domain with distant sequence similarity to PD-L1. We describe in the present invention that when produced as an Ig fusion protein or when overexpressed in artificial APCs, VISTA inhibits both the proliferation of CD4 T cells⁺ and CD8⁺ mouse and human as cytokine production.

(2) The expression of VISTA in myeloid APCs is inhibitory in vitro for T cell responses.

(3) The expression of VISTA in MDSC in the tumor microenvironment is extremely high. Phenotypic and functional analysis of many cell surface molecules previously suggested that they are involved in MDSC-mediated T cell suppression: CD115, CD124, CD80, PD-L1, and PD-L2 were expressed by MDSC but no differences were found in the levels of its expression or ratio of positive cells between MDSC and cells from tumor-free mice lacking immunosuppressive activity. Therefore, we can predict that VISTA will be the main negative regulator of B7 in MDSCs.

(4) Antibody-mediated blocking of VISTA induces protective immunity against autologous tumor.

<img file="MX342017B_D0018.tif" />

negative labor therapist,

ΙΜϊη

Based on this, VISTA appears to be a molecular molecule.

D £ THE dominant PROPERTY in MDSCs that interferes with the development of antitumor WWtun. Therefore, blocking anti-VISTA antibodies will allow the development of protective anti-tumor immunity in humans and other mammals.

Therefore, the invention relates to methods of using soluble VISTA proteins, for example, fusion proteins and VISTA multimeric proteins comprising multiple copies of the VISTA extracellular domain or a fragment thereof, and VISTA binding agents, for example. example small molecules and antibodies, or fragments thereof, that bind or modulate (agonize or antagonize) the activity of VISTA as immune modulators and for the treatment of different cancers, for example bladder, ovary and lymphoma, autoimmune disease, allergy, infection, and inflammatory conditions, eg, multiple sclerosis and arthritis.

As described in detail below, this protein is a new inhibitory ligand, whose extracellular Ig-V domain has homology with the two known ligands of the B7 family, Kill Ligands 1 and 2 (PD-L1 and PD-L2). and presents unique sequence characteristics and different expression patterns in vitro and in vivo in the subsets of APC and T cells, (which distinguishes PD-L3 or VISTA from other ligands of the B7 family). This protein: shown to have a functional impact on the proliferation and differentiation of CD4 T cells⁺ and CD8⁺(suppresses the proliferation of CD4 T cells⁺ and CD8⁺, as well as the production of cytokines). Based on its expression pattern and inhibitory impact on T cells, PD-L3 or VISTA apparently functions as a regulatory ligand that negatively regulates T cell responses during analogous interactions between T cells and myeloid-derived APCs.

While PD-L3 or VISTA appears to be a member of the B7 family of ligands, unlike other ligands of the B7 family, this molecule contains a single Ig-V domain without an Ig-C domain, and is phylogenetically closer to the receptor for Programmed Death 1 (PD-1) of the B7 family. Based on this, PDL3 or VISTA, and agonists or antagonists specific to it, can be used to regulate the activation and differentiation of T cells, and mod ^^. More.

INDUSTRIAL -Vfc regulatory network that controls immune responses. In particular, the la

<img file="MX342017B_D0019.tif" />

einae

PD-L3 or VISTA and ΡΟΤΤίΓΎΗΤΑ agonists or antagonists; - prcforontemfínt^Q Antibodies specific to PD-L3 or VISTA are useful in modulating immune responses in autoimmunity, inflammatory responses and diseases, allergy, cancer, infectious disease, and transplantation.

Therefore, the present invention in part relates to compositions for example for therapeutic, diagnostic or immunomodulatory use containing a soluble PD-L3 or VISTA isolated fusion protein or protein, for example a soluble VISTA-fusion protein. Ig or a VISTA multimeric protein, which comprises an amino acid sequence that is preferably at least 70-90% identical to the human or murine PD-L3 or VISTA polypeptide discussed in secs. with nos. Ident .: 2, 4 or 5 or an ortholog, or fragment thereof encoded by a gene that specifically hybridizes to sec. with nos. Identification number 1 or 3 that modulates VISTA and a pharmaceutically acceptable carrier in vivo. In some embodiments, the soluble or multimeric VISTA protein can be linked directly or indirectly to a heterologous (non-VISTA) protein or can be expressed by a viral vector or a cell containing, for example, a transfected immune cell such as a cell. T.

The present invention also provides expression vectors comprising an isolated nucleic acid encoding a VISTA protein that is at least 70-90% Identical to the human or murine VISTA amino acid sequence set forth in secs. with nos. Identification code: 2, 4 or 5 or a fragment or ortholog thereof, which is optionally fused to a sequence encoding another protein such as an Ig polypeptide, for example an Fe region or a reporter molecule, and host cells that contain such vectors.

The present invention also specifically relates to an isolated binding agent, preferably an antibody or antibody fragment that specifically binds to a PD-L3 or VISTA protein comprising the amino acid sequence set out in secs. with nos. Ident .: 2, 4 or 5 or a fragment or variant ortholog of it. In a preferred embodiment, the agent

IMPI binding modulates in vitro or in vivo (agonizes or antagonizes) the industrial modalities Most preferred, the binding agent is an agonist or antagonist antibody. ———

<img file="MX342017B_D0020.tif" />

The present invention further provides methods for modulating a cellular immune response by contacting an immune cell in vitro or in vivo with a VISTA protein, or specific binding agent to it, in the presence of a primary signal such that a cell response is modulated. immune. (The interaction of VISTA or a modulator of VISTA transmits a signal to immune cells, which regulates immune responses. The PD-L3 or VISTA protein is expressed at high levels in antigen-presenting myeloid cells, including myeloid dendritic cells (DC) and macrophages, and at lower densities in CD4 + and CD8 + T cells. After immune activation, expression PD-L3 or VISTA is up-regulated in myeloid APCs, but down-regulated in CD4 + T cells). Therefore, the PD-L3 or VISTA nucleic acids and polypeptides of the present invention, and agonists or antagonists thereof, are useful, for example, in modulating the immune response.

In another aspect, this invention provides isolated nucleic acid molecules that encode VISTA polypeptides, preferably that encode soluble fusion proteins and VISTA multimeric proteins, as well as suitable nucleic acid fragments as primers or hybridization probes for the detection of acids. nucleic codes encoding PD-L3 or VISTA. In one embodiment, a PD-L3 or VISTA nucleic acid molecule of the invention is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% identical or more to the nucleotide sequence (eg, to the full length of the nucleotide sequence) encoding PD-L3 or VISTA in secs. with nos. Identification number: 1 or 3 shown in the present description or a supplement thereto.

In another embodiment, a PDL3 or VISTA nucleic acid molecule includes a nucleotide sequence that encodes a polypeptide having an amino acid sequence that has a specific percent identity to the amino acid sequence of Sec. with nos. ID: 2, 4 or 5. In a preferred embodiment,

IMPI

MEXICAN INSTITUTE

Ut LA l'ROÍ lfCAÜ industrial

<img file="MX342017B_D0021.tif" />

a nucleic acid molecule of PD-L3 or VISTA includes a nucleotide sequence that encodes a polypeptide that has an amino acid sequence of at least about 71%, 75% 80%, 85%, 90%, 95% 96%, 97 %, 98%, 99% identical or more to the full length of the amino acid sequence of secs. with nos. of ident .: 2, 4 or 5 or to the extracellular domain of these.

In another preferred embodiment, an isolated nucleic acid molecule encodes the human or murine amino acid sequence or VISTA or a conserved region or functional domain therein. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence that encodes a polypeptide having the amino acid sequence of secs. with nos. Ident .: 2, 4 or 5. In yet another preferred embodiment, the nucleic acid molecule is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 , 950, 1000, 1050, 1100, 1150 or more nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 , 950, 1000, 1050, 1100, 1150 or more nucleotides in length and encodes a polypeptide that has PD-L3 or VISTA activity or modulates PD-L3 or VISTA function (as described herein).

Another embodiment of the invention features nucleic acid molecules, preferably PD-L3 □ VISTA nucleic acid molecules, that specifically detect PD-L3 or VISTA nucleic acid molecules relative to nucleic acid molecules that do not encode PD polypeptides. -L3 or VISTA. For example, in one embodiment, said nucleic acid molecule is at least about 880, 900, 950, 1000, 1050, 1100, 1150 or more nucleotides in length and hybridizes under stringent conditions with a nucleic acid molecule encoding the polypeptide. shown in sec. with nos. of ident .: 2, 4 or 5, or a complement of this one. In another embodiment, said nucleic acid molecule is at least 20, 30, 40, 50, 100, 150, 200, 250, 300, or more nucleotides in length and hybridizes under stringent conditions with a nucleic acid molecule encoding a fragment. of PD-L3 or VISTA, for example, comprising at least 20, 30, 40,

<img file="MX342017B_D0022.tif" />

INSTITUTE MgCJCANO 'ín'dustkial

50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 7Ó0, 750, 800, 850,

900, 950 or more nucleotides in length, includes the least nucleuliduy-IG (ie?

contiguous) of the nucleic acid sequence described in secs. with nos. Identification: 1 and 3 encoding the PD-L3 or VISTA polypeptides in secs. with nos. Ident .: 2, 4 or 5, or a complement of these, and hybridizes under stringent conditions with a nucleic acid molecule comprising the nucleotide sequence shown in secs. with nos. Ident .: 1 or 3 or a complement of it.

In still other preferred embodiments, the nucleic acid molecule encoding a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of sec. with nos. Ident .: 2 or 4 or 5, wherein the nucleic acid molecule is hybridized under stringent conditions to a complement of a nucleic acid molecule comprising secs. with nos. Ident .: 1 or 3, or a complement of these.

Another embodiment of the invention provides an isolated nucleic acid molecule that is antisense to a PD-L3 or VISTA nucleic acid molecule, for example, it is antisense to the coding strand of a PD-L3 or VISTA nucleic acid molecule. as shown in sec. with no. of ident. : 1 or 3.

Another aspect of the invention provides a vector comprising a PD-L3 or VISTA nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector.

In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method of producing a polypeptide, preferably a PD-L3 or VISTA polypeptide, by culturing in a suitable medium, a host cell, eg, a mammalian host cell such as a non-human mammalian cell, of the invention. containing a recombinant expression vector, such that the polypeptide is produced.

τΜΡΤ

Another aspect of this invention counts £ dH^LPD-L3 or VISTA (for example, proteins, polypeptidQSu-DéDiidQS. or fragments or parts thereof). In one embodiment, an isolated PD-L3 or VISTA polypeptide or PD-L3 or VISTA fusion protein includes at least one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

In a preferred embodiment, a PD-L3 or VISTA polypeptide includes at least one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain, and has a sequence amino acids of at least about 71%, 75%, 80%, 85%, 90%, 91%, 92%, 93%. 94%, 95% 96% 97%, 98%, 99% identical or more to the amino acid sequence of secs. with nos. Identification number: 2 or 4 or 5. In another preferred embodiment, a PD-L3 or VISTA polypeptide includes at least one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain, and has an activity PD-L3 or VISTA (as described in the present description).

In yet another preferred embodiment, a PD-L3 polypeptide includes at least one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain, and is encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of secs. with nos. Identification number: 1 or 3.

In another embodiment, the invention features fragments or parts of the polypeptide having the amino acid sequence of secs. with nos. ident .: 2 or 4 or 5, wherein the fragment comprises at least 15 amino acids (ie contiguous amino acids) of the amino acid sequence of secs. with nos. Ident .: 2 or 4. In another embodiment, a PD-L3 or VISTA polypeptide comprises or consists of the amino acid sequence of secs. with nos. Identification number: 2, 4 or 5.

In another embodiment, the invention features a PD-L3 or VISTA polypeptide that is encoded by a nucleic acid molecule consisting of a sequence of

0.0 nucleotides of at least about 70%, 75%, 80%, 8 ^ / ί,^{! ί}9 ^; 9Í% / 9W ./ X.// Λ

....... -vp>

93%, 94%, 95%, 96%, 97%, 98%, 99% identical or more to a sequence, the nucleotides of secs. with nos. Ident .: 1 or 3, or a complement thereof. This invention also features a PD-L3 or VISTA polypeptide that is encoded by a nucleic acid molecule that consists of a nucleotide sequence that hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule that comprises the sequence of nucleotides of sec. with nos. ID number: 1 or 3.

The polypeptides of the present invention or parts thereof, eg, the biologically active portions thereof, can be operably linked to a non-PD-L3 or VISTA polypeptide (eg, heterologous amino acid sequences) to form fusion polypeptides. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind to polypeptides of the invention, preferably human PD-L3 or VISTA polypeptides.

The invention also relates to methods for selecting anti-PD-L3 or VISTA antibodies that have functional properties from panels of monoclonal antibodies produced against this protein or a PDL-3 or VISTA-Ig fusion protein based on the desired functional properties, for example modulating the specific effects of PD-L3 or VISTA on immunity, such as the suppressive effect of the protein on TCR activation, the suppressive effect of the protein on anti-CD3 CD4 T cell proliferative responses, the suppression of antigen-specific proliferative responses of analogous CD4 T cells, the suppressive effects of PD-L3 or VISTA on the expression of specific cytokines such as IL-2 and interferon gamma, and others. In a particularly preferred embodiment the anti-PD-L3 or VISTA antibodies for use as therapeutic agents will be selected those in vitro, in the presence of soluble PD-L3 or VISTA proteins, for example PD-L3 or VISTA fusion protein -lg to enhance the suppressive effects of PDL3 or VISTA-lg on immune functions related to PD-L3 or VISTA. It is preferred as highly unexpected (shown below) that these antibodies in vivo behave

<img file="MX342017B_D0023.tif" />

INDI ¡TUTO MEXICANA DE LA rP.OHÍCAO INUUwT RíAL contrary to what might be expected from its in vitro effect on immunity, that is, these monoclonal antibodies anti-o VISTA'SOñ ¥ h7ñLrñüSnpfesores'.

Furthermore, PD-L3 or VISTA polypeptides (or biologically active parts thereof) or modulators of PD-L3 or VISTA molecules, i.e., antibodies, such as those selected using the above methods can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

In another embodiment, a PD-L3 or VISTA protein is used as an inhibitory signal to inhibit or decrease immune cell activation. In this embodiment, the inhibitory signal binds to an inhibitory receptor (eg, CTLA-4 or PD-1) on an immune cell thereby antagonizing the primary signal that binds to an activating receptor (eg, via of a TCR, CD3, BCR, or Fe polypeptide). Inhibition includes, for example, inhibition of second messenger generation; an inhibition of proliferation; an inhibition of effector function in the immune cell, e.g. reduced phagocytosis, reduced antibody production, reduced cellular cytotoxicity, failure of the immune cell to produce mediators, (such as cytokines (e.g. IL-2) and / or mediators of allergic responses); or the development of anergia.

In particular embodiments, the primary signal is a ligand (eg, CD3 or anti-CD3) that binds to TCR and initiates a primary stimulation signal. Such TCR ligands are readily available from commercial sources and specific examples include the anti-CD3 OKT3 antibody, prepared from hybridoma cells obtained from the American Type Culture Collection, and the anti-CD3 monoclonal antibody G19-4. In an alternative embodiment, a primary signal is delivered to a T cell through other mechanisms, including a protein kinase C activator, such as a phorbol ester (eg, phorbol myristate acetate), and a calcium ionophore. (eg, ionomycin, which raises cytoplasmic calcium concentrations), or the like. The use of such agents bypass the TCR / CD3 complex but deliver a stimulatory signal to T cells. Other agents that act as primary signals can include natural and synthetic ligands. A natural ligand can include MHC with or without a displayed peptide. Other ligands may include, but are not

<img file="MX342017B_D0024.tif" />

ican polypeptide, growth factor, cytokine, chemoqw) a-, -glycopepticlo ·, · soluble receptor, steroid, hormone, mitogen, such as PHA, or other superantigen, peptide-MHC tetramers (Altman, et al. (1996) Science 274 (5284): 94-6) and soluble MHC dimers (Dal Porto, et al. (1993) Proc Nati Acad Sci USA. 90: 6671-

5).

The immune cells activated in accordance with the method of the present invention can be subsequently expanded ex vivo and used in the treatment and prevention of a variety of diseases; for example, human T cells that were cloned and expanded in vitro to retain their regulatory activity (Groux, et al. (1997) Nature 389 (6652): 737-42). Before expansion, a source of T cells is obtained from an individual (eg, a mammal such as a human, dog, cat, mouse, rat, or transgenic species thereof). T cells can be obtained from a number of sources, including mononuclear cells from peripheral blood, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, spleen tissue, tumors, or T cell lines. T cells can be obtained from a unit of blood collected from an individual using any number of techniques known to the skilled technician, such as ficoll ™ separation.

In another aspect, the present invention provides a method for detecting the presence of a PD-L3 or VISTA nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a nucleic acid molecule, protein. or PD-L3 or VISTA polypeptide, such that the presence of a PD-L3 or VISTA nucleic acid molecule, protein or polypeptide, is detected in the biological sample. This expression of PD-L3 or VISTA can be used to detect certain disease sites such as inflammatory sites.

In another aspect, the present invention provides a method for detecting the presence of PD-L3 or VISTA activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity.

<img file="MX342017B_D0025.tif" />

τ? I

XV.L, k 1M

INS'i I ri.'TO MrXtCANO: 'í)

L '£ LA? ;; O:' PD-L3 or VISTA item, such that the presence of PD-L3 or VlSTA activity is detected in the biological sample.

In another aspect, the invention provides a method for modulating the activity of PD-L3 or VISTA, which comprises contacting a cell capable of expressing PD-L3 or VISTA with an agent that modulates the activity of PDL3 or VISTA, preferably an anti- PD-L3 or VISTA such that the activity of PD-L3 or VISTA is modulated in the cell. In one embodiment, the agent inhibits the activity of PD-L3 or VISTA. In another embodiment, the agent stimulates the activity of PD-L3 or VISTA. In a further embodiment, the agent interferes with or enhances the interaction between a PD-L3 or VISTA polypeptide and its natural binding partner (s). In one embodiment, the agent is an antibody that specifically binds to a PD-L3 or VISTA polypeptide. In another embodiment, the agent is a peptide, mimetic peptide, or other small molecule that binds to a PD-L3 or VISTA polypeptide.

In yet another embodiment, the agent modulates PD-L3 or VISTA expression by modulating the transcription of a translation of a PD-L3 or VISTA gene, translation of a PD-L3 or VISTA mRNA, or post-translational modification of a PD-L3 or VISTA polypeptide. In another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a PD-L3 or VISTA mRNA or a PD-L3 or VISTA gene.

In one embodiment, the methods of the present invention are used to treat an individual having a disorder or condition characterized by the expression or activity of the aberrant, insufficient, or unwanted nucleic acid or polypeptide of PD-L3 or VISTA by administering to the individual. an agent that is a PDL3 or VISTA modulator. In a preferred embodiment, the PD-L3 or VISTA modulator is a PD-L3 or VISTA polypeptide, preferably a soluble fusion protein or VISTA multimeric protein or anti VISTA antibody as described below. In another embodiment, the PD-L3 or VISTA modulator is a PD-L3 or VISTA nucleic acid molecule, for example an adenovirus vector. In another embodiment, the invention further provides treating the individual with an additional agent that modulates an immune response.

c ^ rnprerKfe

<img file="MX342017B_D0026.tif" />

In yet another embodiment, the invention provides an INDUSTRIAL yadubaltíLfe, an antigen and an agent that modulates (enhances or inhibits) the activity of PD-L3 or VISTA. In a preferred embodiment, the vaccine inhibits the interaction between PD-L3 or VISTA and its natural binding partner (s).

The present invention also provides diagnostic assays to identify the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a PD-L3 or VISTA polypeptide, (ii) regulation defective of the gene, and (iii) aberrant post-translational modification of a PD-L3 or VISTA polypeptide, wherein a wild-type form of the gene encodes a polypeptide with PD-L3 or VISTA activity.

In another aspect, the invention provides methods of identifying a compound that binds to or modulates the activity of a PD-L3 or VISTA polypeptide, providing a reporter composition comprising a PD-L3 or VISTA polypeptide that has PD-L3 activity or VISTA, contacting the indicator composition with a test compound, and determining the effect of the test compound on PD-L3 or VISTA activity in the reporter composition to identify a compound that modulates the activity of a PD-L3 or VISTA polypeptide.

In one aspect, the invention features a method for modulating the interaction of PD-L3 or VISTA with its natural binding partner (s) in an immune cell comprising contacting an antigen presenting cell expressing PD-L3 or VISTA with an agent. selected from the group consisting of: a form of PD-L3 or VISTA, or an agent that modulates the interaction of PD-L3 or VISTA and its natural binding partner (s) such that the interaction of PD-L3 or VISTA with the (s) ) natural binding partner (s) in an immune cell. In a preferred embodiment, an agent that modulates the interaction of PD-L3 or VISTA and its natural binding partner (s) is an antibody that specifically binds to PDL3 or VISTA. In one embodiment, the interaction of PD-L3 or VISTA with its natural binding partner (s) is up-regulated. In another embodiment, the interaction of PDL3 or VISTA with its natural binding partner (s) is downregulated. In one embodiment, the method further comprises contacting the immune cell or the

IMPI

INSTITUTO MEXICANO DE LA PROPERTY antigen-presenting cell with an additional agent that modulates

<img file="MX342017B_D0027.tif" />

immune response.

In one embodiment, the contact step is performed in vitro. In another embodiment, the contacting step is performed in vivo. In one embodiment, the immune cell is selected from the group consisting of: a T cell, a monocyte, a macrophage, a dendritic cell, a B cell, and a myeloid cell.

In another aspect, the invention corresponds to a method for inhibiting or increasing activation in an immune cell that comprises increasing or inhibiting the activity or expression of PD-L3 or VISTA in a cell such that the activation of the cell is inhibited or increased. immune.

In yet another aspect, the invention corresponds to a vaccine comprising an antigen and an agent that inhibits the interaction between PD-L3 or VISTA and its natural binding partner (s).

In yet another aspect, the invention corresponds to a vaccine comprising an antigen and an agent that promotes the interaction between PD-L3 or VISTA and its natural binding partner (s).

In another aspect, the invention corresponds to a method for treating an individual who has a condition that would benefit from the positive regulation of an immune response that comprises administering an agent that inhibits the interaction between PD-L3 or VISTA and its (s ) natural binding partner (s) in immune cells of the individual such that a condition being treated would benefit from upregulation of an immune response. In a preferred embodiment, the agent comprises a blocking antibody or small molecule that binds to PD-L3 or VISTA and inhibits the interaction between PD-L3 or VISTA and its natural binding partner (s). In another embodiment, the method further comprises administering a second agent that positively regulates an immune response to the individual. In another aspect, the invention corresponds to a method for treating an individual who has a condition that would benefit from the negative regulation of an immune response that comprises administering an agent that stimulates the interaction between PDL3 OR VISTA and its partner (s). ) of natural attachment in the cells of the individual such that

ΙΜΡΙ »5ι

INSTITUTE MFXiC. '. A'u

Say LA IT.CHEI ·. '. U a condition in question would benefit from the regulation of an immune response. --—-------————— For example, the condition treated with the PD-L3 or VISTA protein or binding agents is selected from the group consisting of: a tumor, a pathogenic infection, a condition or inflammatory immune response, preferably less pronounced inflammatory conditions, or an immunosuppressive disease. Specific examples include multiple sclerosis, thyroiditis, rheumatoid arthritis, type II and type I diabetes, and cancers, both advanced and early forms, including metastatic cancers, such as bladder cancer, ovarian cancer, melanoma, lung cancer, and other cancers. wherein VISTA suppresses an effective antitumor response. In some examples, cells or a viral vector expressing a nucleic acid encoding an anti-VISTA antibody or VISTA fusion protein can be administered to the individual.

In one embodiment, the agent comprises an antibody or a small molecule that stimulates the interaction between PD-L3 or VISTA and its natural binding partner (s). In another embodiment, the method further comprises administering a second agent that negatively regulates an immune response to the individual such as a PD-L1, PD-L2 or CTLA-4 fusion protein or antibody specific to it.

Illustrative treatable conditions using PD-L3 or VISTA proteins, PD-L3 or VISTA binding agents or antagonists or agonists in accordance with the invention include by way of example transplantation, an allergy, infectious disease, cancer, and inflammatory or autoimmune disorders, for example, an inflammatory immune disorder. Specific examples of the above include type 1 diabetes, multiple sclerosis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, rheumatic diseases, allergic disorders, asthma, allergic rhinitis, skin disorders, gastrointestinal disorders such as Crohn's disease and ulcerative colitis. , transplant rejection, post-streptococcal and autoimmune renal failure, septic shock, systemic inflammatory response syndrome (SIRS), adult respiratory distress syndrome (ARDS) and poisoning: auto-inflammatory as well as degenerative bone and joint diseases including osteoarthritis, arthritis by faith

<img file="MX342017B_D0028.tif" />

IMPIí

<img file="MX342017B_D0029.tif" />

Ut LA t HUI Ir.U.VD V. “Crystal and capsulitis and other arthropathies. In addition, the methods and solutions can be used to treat tendonitis, ligamentitis, and traumatic artery injury _______________

In another aspect, the invention corresponds to a cell-based assay for screening compounds that modulate PD-L3 or VISTA activity comprising contacting a cell expressing a PD-L3 or VISTA target molecule with a test compound and determining the ability of the test compound to modulate the activity of the PD-L3 or VISTA target molecule.

In yet another aspect, the invention corresponds to a cell-free assay for screening compounds that modulate the binding of PD-L3 or VISTA to a target molecule, which comprises contacting a PD-L3 or VISTA polypeptide or the biologically active part thereof with a test compound and determine the ability of the test compound to bind the PD-L3 or VISTA polypeptide or the biologically active part thereof.

In another embodiment, the invention corresponds to a method of identifying a compound, for example, an anti-PD-L3 or VISTA antibody that modulates the effect of PD-L3 or VISTA on T cell activation or cytokine production in a first and second antigen concentration comprising contacting a T cell expressing a PD-L3 or VISTA target molecule with a test compound at a first antigen concentration, determine the ability of the test compound to modulate T cell proliferation or cytokine production at the first antigen concentration, contacting a T cell expressing a target PD-L3 or VISTA molecule with the test compound at a second antigen concentration antigen, and determine the ability of the test compound to modulate T cell proliferation or cytokine production at the second antigen concentration, thereby identifying a compound that modulates T cell activation or cytokine production at a first and second antigen concentration.

In other specific modalities, panels of anti PD-L3 or VISTA antibodies and PD-L3 or VISTA proteins are screened to select those that inhibit or promote the effects of PD-L3 or VISTA on CD4 + and CD8 + T cell differentiation, proliferation and / or production of cytokines in vitro or in vivo.

IMPI

INrCTfTI I-iVx urv. «... -

<img file="MX342017B_D0030.tif" />

Mexican INSTITUTE of PROPERTY

In preferred embodiments, the individual SviSTA proteins, core acids, and PD-L3 or VISTA-specific ligands, · pi «fefontementa.jQjs. Antibodies that have desired effects on the functions of PD-L3 or VISTA are used to treat conditions such as cancer, autoimmune diseases, allergy, inflammatory disorders or infection and more specifically disorders of the immune system, such as severe combined immunodeficiency, multiple sclerosis, lupus systemic erythematosus, type I diabetes mellitus, lymphoproliferative syndrome, inflammatory bowel disease, allergies, asthma, graft versus host disease, and transplant rejection; immune responses to infectious pathogens such as bacteria and viruses; and cancers of the immune system, such as lymphomas and leukemias).

Detailed Description of Drawings

Figure 1. Sequence analysis. A. The complete amino acid sequence of murine PD-L3 or VISTA. B. Alignment of extracellular Ig domains amino acid sequences between murine PD-L3 or VISTA and selected ligands of the B7 family, including B7-H1 (PD-L1), B7-DC (PD-L2), B7-H3, and B7-H4. C. Alignment of the Ig domain of PD-L3 or VISTA with B7 family receptors, including PD-1, CTLA-4, CD28, BTLA, and ICOS. Ig-V domain, Ig-C domain, "___. The alignment was performed using the MUSCLE (Multiple Sequence Comparison by Logarithm Expectations) algorithm. D. Sequence identity (%) of Ig-V domains between PD-L3 or VISTA and other B7 family ligands and receptors is calculated using the ClustalW2 program. E. Sequence homology between human and murine PD-L3 or VISTA. Identical residues are shaded black. Highly preserved and semi-preserved residues are shaded dark and light gray, respectively.

Figure 2. Phylogenetic analysis of mouse PD-L3 or VISTA with other members of the immunoglobulin (Ig) superfamily. Complete sequence of mouse PDL3 OR VISTA and other members of the Ig superfamily, including CD28, CTLA-4, ICOS, BTLA, PD-1. B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2, B7-H3, B7-H4, B7-1, B7-2, BTNL2, BTN3A3. BTN2A2 and BTN1A1, were analyzed using the

IMPI

INSTITUTO MEXICANO DE LA fKOriEDAD I INDUftUtól ·,

<img file="MX342017B_D0031.tif" />

PhyML (Phylogenetic Maximum Probability) algorithm. Branching were shown at the junctions of the branches of the tree —---------- Figure 3. Expression patterns in tissue and hematopoietic cells of PDL3 or VISTA A. RT-PCR of PD-L3 or VISTA complete a from mouse tissues. Lanes: (1) muscle (2) heart (3) eye (4) thymus (5) spleen (6) small intestine (7) kidney (8) liver (9) brain (10) mammary gland (11) lung (12 ) ovary (13) bone marrow. B. RT-PCR of PD-L3 or whole VISTA from purified hematopoietic cell types. The lanes (1) peritoneal macrophages (2) CD11b + splenic monocytes (3) CD11c + splenic DC (4) CD4 + splenic T cells (5) CD8 + splenic T cells (6) splenic B cells. EC. Flow cytometric analysis of PD-L3 or VISTA expression in splenic CD4 + and CD8 + T cells from thymus and spleen (C), in CD11b + monocytes (D), and in DC CD11c + subsets from spleen and peritoneal cavity (E) . F. Splenic B cells, NK cells, and granulocytes are also analyzed. G. Differential expression of PD-L3 or VISTA in hematopoietic cells from different tissue sites, include mesenteric LN, peripheral LN, spleen, blood, and peritoneal cavity. Representative data from at least 3 independent experiments are shown.

Figure 4. PD-L3 or VISTA gene array data from the GNF gene array database (Novartis Research Foundation Genomics Institute) as well as the GEO database (gene expresslon ómnibus) from NCBI.

Figure 5. Specificity of PD-L3 or VISTA to hamster monoclonal antibodies. Mouse EL4 cell lines overexpressing either PD-L1 or PD-L3 or VISTA fused to RFP were stained using hybridoma culture supernatants and analyzed by flow cytometry. Two representative positive clones are shown.

Figure 6. Comparison of the expression of PD-L3 or VISTA with other ligands of the B7 family in spleen cells cultured in vitro. Expression of PD-L3 or VISTA and other B7 family ligands (i.e., PD-L1, PD-L2, B7-H3, and B7-H4) in hematopoietic cell types, including CD4 + T cells, CD11 monocytes

OR

IM Pl Mexican institute

<img file="MX342017B_D0032.tif" />

INSTITUTO Mexicano bhi, and DC CD11c + were compared. The cells were isolated in vitro cultured for 24 hours, with and without activation.

CD4 + T cells were activated with plaque-bound nCD3 (5pg / ml), CD 11 bhi monocytes and CD11c + DCs were activated with IFN alpha (20 ng / ml) and LPS (200 ng / ml).

Representative results from three independent experiments are shown.

Figure 7. Comparison of in vivo expression patterns of PD-L3 or VISTA and other ligands of the B7 family during immunization. DO11.10 TCR transgenic mice were flank immunized with chicken ovalbumin (OVA) emulsified in complete Freund's adjuvant (CFA). Draining and non-draining lymph node cells were collected 24 hours after immunization, and analyzed by flow cytometry for the expression of PD-L3 or VISTA, PD-L1, and PD-L2. Representative results are shown from at least four independent experiments. A. A population of CD11 b + cells expressing a high level of PD-L3 or VISTA was induced into the draining lymph node at 24 hours after immunization with CFA / OVA, but not only with CFA. These cells are of mixed phenotype of F4 / 80 + macrophages and CD11C + dendritic cells. B. The expression of PD-L3 or VISTA, PD-L1 and PD-L2 in CD11 bhi monocytes, DC CD 11c + and CD4 + T cells were analyzed 24 hours after immunization.

Figure 8 Loss of PD-L3 or VISTA expression on activated CD4 + T cells in response to immunization. DO11.10 mice were flank immunized with chicken ovalbumin (OVA) emulsified in complete Freund's adjuvant (CFA). Draining and non-draining lymph node cells were harvested 48 hours after immunization, and analyzed for PD-L3 or VISTA expression by flow cytometry. Representative results of 2 independent experiments are shown.

Figure 9 The immobilized PD-L3 or VISTA-Ig fusion protein inhibited the proliferation of CD4 + and CD8 + T cells. A. CFSE-labeled CD4 + and CD8 + T cells were stimulated by plate-bound OCD3 with or without PD-L3 or VISTA-Ig coabsorbed. The percentage of cells with low CFSE was quantified and is shown in B.

IΜ ΡI

INSTITUTE .M5XICANO

C CD4 + T cells from PD-1 KO mice were also suppressed O

VISTA-lg. D. PD-L3 or VISTAJg_ mediated suppression is persistent and may act late. CD4 + T cells were activated in the presence of PD-L3 or VISTA-lg or

IgControl, either 72 hours (i), or for 24 hours (ii, ii and iv). Cells pre-activated 24 hours were harvested and re-stimulated under specific conditions for another 48 hours. Cell proliferation was analyzed at the end of the 72 hour culture, (ii) Pre-activation with PD-L3 or VISTA-lg and re-stimulation with anti CD3; (Ii) Pre-activation with ant¡CD3 and re-stimulation with PD-L3 or VISTA-lg. (iv) Preactivation with PD-L3 or VISTA-lg and re-stimulation with PD-L3 or VISTA-lg. Duplicate wells were analyzed for all conditions. Representative results of at least four experiments are shown.

Figure 10. Similar inhibitory effect of PD-L1-lg and PD-L3 OR VISTA-lg fusion proteins on cell proliferation of CD4 + T cells. The bulk purified CD4 + T cells were labeled with CFSE and stimulated with plate-bound DCD3 along with the titer of PD-L1-lg or PD-L3 or VISTA-lg fusion proteins. CFSE dilution was analyzed at 72 hours and the percentage of cells with low CFSE was quantified. Duplicate wells were analyzed for all conditions. Representative results of 2 independent experiments are shown.

Figure 11. Suppressive impact of PD-L3 or VISTA-lg on the proliferation of naïve and memory CD4 + T cells. A. Naive CD4 + (CD25-CD44lowCD62Lhi) and memory (CD25-CD44hiCD62Llow) T cell subsets were sorted, CFSE-labeled, and stimulated with plate-bound anti-CD3 (2.5 pg / ml) along with PD-L3 or VISTA-lg or IgControl in the indicated relationships. Cell proliferation was analyzed at 72 hours by examining the CFSE cleavage profile. The percentage of proliferated cells, as determined by the percentage of cells with low CFSE, is calculated and shown in B. Duplicate wells were analyzed for all conditions. Representative results of two independent experiments are shown.

Figure 12 PD-L3 or VISTA-Ig fusion protein suppressed early cell proliferation and activation of TCR, but indirectly induces apoptosis.

<img file="MX342017B_D0033.tif" />

Bulk purified CD4 + T cells were stimulated with amphi-Ct) 3 unicRT to the plate together with PD-L3 or VISTA-lg or IgControl in the 4-2-f2 roll; 5 pg / ml and 6 pg / ml, respectively. ). Cells were analyzed by flow cytometry at 24 hours and 48 hours for the expression of CD69, CD62L, and CD44. Cells were also stained for annexin-V the marker of early apoptosis, and the marker of cell death 7-Aminoactinomycin D (7-AAD). Representative results of two independent experiments are shown.

Figure 13. PD-L3 or VISTA-lg inhibited cytokine production by CD4 + and CD8 + T cells. AB. Bulk purified CD4 + T cells were stimulated with plaque-bound anti-CD3, and PD-L3 or VISTA-lg or IgControl in the indicated ratios. Culture supernatants were collected after 24 hours and 48 hours. The levels of IL-2 and IFN □ were analyzed by ELISA. CD. CD4 + T cells were classified into naive (CD25-CD44lowCD62Lhi) and memory (CD25-CD44h¡CD62Llow) cell populations. Cells were stimulated with plate-bound 0CD3 and PD-L3 or VISTA-lg or IgControl in a ratio of 1: 2. The culture supernatants were collected at 48 hours and analyzed by ELISA for the level of IL-2 and IFN □. E. Bulk purified CD8 + T cells were stimulated with plate-bound DCD3 and PD-L3 or VISTA-Ig or IgControl in the indicated ratios. IFN □ in the culture supernatant was analyzed by ELISA. For all conditions, supernatants from six duplicate wells were pooled for ELISA analysis. Representative results of at least three experiments are shown.

Figure 14. Suppression mediated by PD-L3 or VISTA-lg can overcome a moderate level of co-stimulation provided by CD28, but was completely reversed by a high level of co-stimulation as well as partially rescued by exogenous IL-2. AB. CD4 + T cells were activated by plate bound DCD3 together with either PD-L3 or VISTA-Ig or IgControl in 1-1 ratio and 1-2 ratios. For cytokine rescue, soluble mlL-2, mlL-7, mlL-15 and mlL-23 (all at 40ng / ml) (A) were added to the cell culture. To examine the effects of co-stimulation DCD28 (1 pg / ml) was immobilized together with the □ CD3 and Ig proteins at the indicated ratios (B). Cell proliferation was analyzed at

ΙΜΡΙ £ 3 ^

MEXICAN INSTITUTE Jí¿

OF THE PROPERTY

INDUSTRIAL hours examining the CFSE division profiles. CD. To examine the suppressive activity of PD-L3 or VISTA in the presence of lower levels of a? For stimulation, titrated amounts of OCD28 were coated together with anti-CD3 (2.5 pg / ml) and PD-L3 or VISTA-lg fusion proteins or IgControl fusion proteins (10 pg / ml) to stimulate the proliferation of CD4 + T cells. Cell proliferation was analyzed at 72 hours. The percentages of proliferated cells with low CFSE were quantified and are shown in D.

Duplicate wells were analyzed for all conditions. Representative CFSE profiles from three independent experiments are shown.

Figure 15. PD-L3 or VISTA expressed in antigen presenting cells suppressed the proliferation of CD4 T cells. AC CHO cell line stably expressing MHCII l-Ad molecule and costimulating molecule B7-2 was used as the parent cell line. Cells were transduced with retroviruses expressing either PD-L3 or VISTA-RFP or RFP control molecules. The transduced cells were sorted to achieve the level of homogeneous expression. To test their ability as antigen presenting cells, CHO-PD-L3 or VISTA cells or CHO-RFP cells were treated with mitomycin C and mixed with OVA-specific transgenic CD4 + D011.10 T cells, in the presence of the titrated amount of peptide. OVA. DO11 cell proliferation was analyzed at 72 hours, either by CFSE cleavage profiles (AB), or by incorporation of tritium (C). D. The bone marrow derived dendritic cells were transduced with retrovirus RFP or B7B-H5-RFP during the 10 day culture period. Transduced CD11c + RFP + DCs and non-transduced CD11c + RFP- DCs were sorted and used to stimulate OVA-specific transgenic CD4 + OTII T cells in the presence of a titrated amount of OVA peptide. Cell proliferation was analyzed on day 3 by examining CFSE cleavage. For all experiments, duplicate wells were analyzed for all conditions, and representative results from three independent experiments are shown.

Figure 16. Surface expression level of PD-L3 or VISTA in DCs derived from retrovirally transduced bone marrow. DC derived from

<img file="MX342017B_D0034.tif" />

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342017B_D0035.tif" />

Bone marrow (BMDC) were cultured in the presence of GM-CSF (20ng / ml) and transduced with either retrovirus RFP or PD-L3 or VISTA-RFP as described in

Methods. On day 10, the level of expression on the surface of PD-L3 or VISTA was analyzed in the cultured BMDC, and compared with freshly isolated peripheral macrophages.

Figure 17 shows that the anti-PDL3 mAb shows efficacy in a passive transfer EAE model. In this adoptive transfer EAE model, donor SJL mice were immunized with CFA and the PLP peptide. On day 10, the total draining LN lymphocytes were isolated and cultured in vitro with PLP peptide, IL-23 (20 ng / ml) and anti-IFNg (10 pg / ml) for 4 days. The expanded CD4 T cells were then purified and adoptively transferred into naïve recipient mice. Disease progression was controlled and scored with: 0, no disease, 0.5 loss of tail tone, 1: floppy tail; 2: floppy tail + paresis on hind limb; 2.5: 1 hind limb paralysis; 3: paralysis in both hind limbs; 3.5: forelimb weakness; 4: hindlimb paralysis + unilateral forelimb paralysis. The mice were sacrificed when the disease score reached 4. *, the mice were sacrificed.

Figure 18 shows that anti-PD-L3 or VISTA antibodies show efficacy (reduce arthritis symptoms) in an animal model of collagen-induced arthritis.

Figure 19 shows that VISTA expressed on antigen presenting cells suppresses the proliferation of CD4 + T cells.

Figure 20 shows that an anti-VISTA antibody inhibited tumor growth in mice transplanted with MB49 tumor cells.

Figure 21 shows the antitumor effect of VISTA mAbs in four different mouse antitumor models.

Figure 22 shows the potentiating effect of VISTA mAbs on the efficacy of a CD40 / TLR agonist vaccine.

Figure 23 shows the expression of VISTA in CNS cells.

of

<img file="MX342017B_D0036.tif" />

IMPIí

INSTTOrmy_ANDx_{ICAÍ &lt;(}, '

Figure 24 shows the impact of VISTA on ftes &amp; nQÍ and T cells in an EAE model.

Detailed Description of the Invention 'Definitions

Before describing the invention in more detail, the following definitions are provided.

As used in the present description the term "immune cell" includes cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mastoid cells, basophils, and granulocytes.

As used herein, the term "T cell" includes CD4 + T cells and CD8 + T cells. The term T cell also includes both type 1 helper T cells and type 2 helper T cells.

The term "antigen-presenting cell" includes professional antigen-presenting cells (eg, B lymphocytes, monocytes, dendritic cells, and Langerhans cells) as well as other antigen-presenting cells (eg, keratinocytes, endothelial cells, astrocytes, fibroblasts. , and oligodendrocytes).

The term "antigen" in the present description refers to the antigen where modulation of its immune response may be therapeutically desired. In the case of a desired enhanced immune response to the particular antigens of interest, illustrative are such antigens which include, but are not limited to, infectious disease antigens for which a protective immune response can be elicited. For example, the HIV antigens under consideration are the proteins gag, env, pol, tat, rev, nef, reverse transcriptase, and other components of HIV. The E6 and E7 proteins of the human papillomavirus are also under consideration. In addition, the herpes virus EBNA 1 antigen

<img file="MX342017B_D0037.tif" />

simple is also under consideration. Other viral antigens to consider are hepatitis viral antigens such as hepatitis B virus S, M, and TUET proteins, the pre-S antigen of hepatitis B virus, and other hepatitis, for example, hepatitis A, B, and hepatitis B virus. C, viral components such as hepatitis C viral RNA; influenza viral antigens, such as hemagglutinin, neuraminidase, nucleoprotein, M2, and other influenza viral components; measles viral antigens, such as measles virus fusion protein and other components of measles virus; rubella viral antigens such as E1 and E2 proteins and other components of the rubella virus;

rotavirus antigens, such as VP7sc and other rotavirus components; cytomegalovirus antigens such as envelope glycoprotein B and other components of cytomegalovirus antigens; respiratory syncytial viral antigens such as RSV fusion protein, M2 protein and other components of respiratory syncytial viral antigen; herpes simplex viral antigens such as immediate early proteins, glycoprotein D, and other components of herpes simplex viral antigen, varicella zoster viral antigens such as gpl, gpll, and other components of varicella zoster viral antigen; Japanese encephalitis viral antigens such as proteins E, ΜΕ, ME-NS1, NS 1, NS 1-NS2A, 80% E, and other components of Japanese encephalitis viral antigen; rabies viral antigens such as rabies glycoprotein, and the rabies nucleoprotein and other components of the rabies viral antigen; West Nile virus prM and E proteins and Ebola envelope protein. See Fundamental Virology, Second Edition, editors. Knipe, DM and Howley PM (Lippincott Williams &amp; Wilkins, New York, 2001) for additional examples of viral antigens. Furthermore, bacterial antigens are also described. Bacterial antigens that can be used in the compositions and methods of the invention include, but are not limited to, bacterial pertussis antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase, and other components of the bacterial antigen. of pertussis; diphtheria bacterial antigens such as diphtheria toxin or toxoid and other components of diphtheria bacterial antigens; bacterial tetanus antigens, such as tetanus toxin or toxoid and other constituents of 31

INSTITUTO MEXICANO tetanus bacterial antigen; bacterial antigens of éátrejítdó.ócostate &amp; oomo M proteins and other components of streptococcal bacterial antigen; Staphylococcal bacterial antigens such as IsdA, IsdB, SdrD, and SdrE; gram-negative bacilli bacterial antigens such as popolysaccharides, flagellin, and other gram-negative bacterial antigen components; Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65) the main secreted protein of 30 kDa, antigen 85A, ESAT-6, and other components of mycobacterial antigen; Helicobacter pylori bacterial antigen components; pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides, and other components of pneumococcal bacterial antigen; Haemophilus influenzae bacterial antigens such as capsular polysaccharides and other components of the Haemophilus influenzae bacterial antigen; anthrax bacterial antigens such as anthrax protective antigen, anthrax lethal factor, and other components of the anthrax bacterial antigen; Yersinia pestis F1 and V proteins; rickettsial bacterial antigens such as rOmps and other components of the rickettsial bacterial antigen. Also included with the bacterial antigens described herein are any other bacteria, mycobacterial antigens, mycoplasma, rickettsia, or chlamydia. Examples of protozoa and other parasitic antigens include, but are not limited to, Plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte / gamete surface antigens, pf 1 55 / RESA of blood stage and other components of plasmodium antigen; toxoplasma antigens such as SAG-1, p30, and other components of the Toxoplasma antigen; Schistosoma antigens such as glutathione-S-transferase, paramyosin, and other components of Schistosoma antigen; antigens from Leishmania major and other leishmania such as gp63, lipophosphoglycan and its associated protein and other components of Leishmania antigen; and Trypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDa antigen, and other components of the Trypanosoma antigen. Examples of fungal antigens include, but are not limited to, Candida species antigens, 32

<img file="MX342017B_D0038.tif" />

Aspergillus species, Blastomyces species, Histoplasma species, Coccidiodomicosis species, Malassezia furfuryotrasespecies, Exophiala werneckii and other species, Piedraia hortai and other species, Trichosporum beigelii and other species, Microsporum species, Trichophyton species, Eporothrmophyton species, Sporothrmophyton species schenckii and other species, Fonsecaea pedrosoi and other species, Wangiella dermatitidis and other species, Pseudallescheria boydii and other species, Madurella grísea and other species, Rhizopus species, Absidia species, and Mucor species. Examples of prion disease antigens include PrP, beta-amyloid, and other prion-associated proteins.

In addition to the infectious and parasitic agents mentioned above, another area of ​​desirable enhanced immunogenicity to a non-infectious agent is in the area of ​​non-proliferative diseases, including but not limited to cancer, in which cells expressing cells are desirably eliminated from the body. cancer antigens. Tumor antigens that can be used in the compositions and methods of the invention include, but are not limited to, prostate specific antigen (PSA), breast, bladder, ovary, testis, melanoma, telomerase; multidrug resistance proteins, such as P-glycoprotein, MAGE-1, alpha fetoprotein, carcinoembryonic antigen, p53 mutant, papillomavirus antigens, gangliosides, or other melanoma components that contain carbohydrates or other tumor cells. It is contemplated by the invention that antigens from any type of tumor cell can be used in the compositions and methods described in the present disclosure. The antigen can be a cancer cell, or immunogenic materials isolated from a cancer cell such as membrane proteins. The universal antigens sumivin and telomerase and the MAGE family of testicular cancer antigens are included. Antigens that have been shown to be involved in autoimmunity and may be used in the methods of the present invention to induce tolerance include, but are not limited to, myelin basic protein, oligodendrocyte myelin glycoprotein, and multiple sclerosis proteolipid protein and collagen Cll protein from rheumatoid arthritis.

<img file="MX342017B_D0039.tif" />

MEXICAN INSTITUTE

OF INDUSTRIAL PROPERTY

<img file="MX342017B_D0040.tif" />

The antigen can be a part of an infectious agent such as HZV-1. EBV, HBV, influenza virus, SARS virus, poxvirus, malaria, or HSV, by way of non-limiting examples, for which vaccines that strongly mobilize T-cell-mediated immunity (via dendritic cells) are needed.

The term tumor denotes at least one cell or cell mass in the form of a new tissue formation, in particular in the form of an excess of spontaneous, autonomous and irreversible growth, which is more or less uninhibited, of endogenous tissue, the growth of which as rule is associated with the more or less pronounced loss of specific functions of cells and tissues. This cell mass or cell is not effectively inhibited, as regards its growth, by itself or by the regulatory mechanisms of the host organism, eg, melanoma or carcinoma. Tumor antigens not only include antigens present in or on the malignant cells themselves, but also include antigens present in the supportive tissue of the tumor stroma including endothelial cells and other blood vessel components.

As used herein, the term "immune response" includes T-cell-mediated and / or B-cell-mediated immune responses that are influenced by modulation of T-cell co-stimulation. Illustrative immune responses include responses of B cells (eg, antibody production) T cell responses (eg, cytokine production, and cellular cytotoxicity) and activation of cytokine responsive cells, eg macrophages. As used in the present description, the term "negative modulation" with reference to the immune response includes a decrease in one or more immune responses, while the term "positive modulation" with reference to the immune response includes an increase in some or more immune responses. more immune responses. It will be understood that positive modulation of one type of immune response can lead to corresponding negative modulation of another type of immune response. For example, positive modulation of the production of certain cytokines (eg IL-10) can lead to negative modulation of the cellular immune response.

IMPI

<img file="MX342017B_D0041.tif" />

<img file="MX342017B_D0042.tif" />

As used herein, the term "redéjW ¥ o-stimulator" includes receptors that transmit an immune signal, eg, CD28 or ICOS. As used herein, the term "inhibitory receptors" includes receptors that transmit a negative signal to an immune cell.

As used herein, the term co-stimulating, with reference to activated immune cells, includes the ability of a costimulatory molecule to provide a second signal without receptor-mediated activation (a "co-stimulatory signal") that induces proliferation or effector function. For example, a co-stimulatory signal can result in the secretion of cytokines, for example, in a T cell that received a signal mediated by the T cell receptor. Immune cells that received a signal mediated by the cell's receptor, for example through an activating receptor, are referred to herein as "activated immune cells."

An inhibitory signal that is transduced by an inhibitory receptor can occur even if a co-stimulatory receptor (such as CD28 or ICOS) is not present on the immune cell and thus is not simply a function of competition between inhibitory receptors and receptors. co-stimulatory receptors for the binding of co-stimulatory molecules (Fallarino et al. (1998) J. Exp. Med. 188: 205). Transmission of an inhibitory signal to an immune cell can result in lack of response, anergy, or programmed cell death in the immune cell. Preferably, the transmission of an inhibitory signal operates through a mechanism that does not involve apoptosis.

As used herein the term "apoptosis" includes programmed cell death that can be characterized using procedures that are known in the art. Apoptotic cell death can be characterized by cell shrinkage, membrane blistering, and chromatin condensation culminating in cell fragmentation. Cells that undergo apoptosis also display a characteristic pattern of internucleosomal DNA cleavage.

The term "autoimmunity" or

INSTITUTO MEXICANO Yz. '' '' ·· DE LA I ROriOAD “disease or affection yetriffiüne ^ eiTfá present description is a disease or disorder that arises from and directed towards the tissues of the individual or a co-segregation or manifestation of these or condition that subsequently results. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis , psoriatic arthritis, vertebral arthritis, and juvenile rheumatoid arthritis, osteoarthritis, progredient chronic arthritis, deforming arthritis, primary chronic polyarthritis, reactive arthritis, and ankylosing spondylitis), hyperproliferative inflammatory skin diseases, psoriasis such as plaque psoriasis, gouty psoriasis, pustular psoriasis and nail psoriasis, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, dermatitis contact allergy, dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis (MS), such as multiple sclerosis Hawthorn MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriesclerosis, disseminated sclerosis, and ataxic sclerosis, inflammatory bowel disease (IBD) (eg, Crohn's disease, autoimmune mediated gastrointestinal diseases, colitis such as ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis, and transmural colitis, and inflammatory autoimmune disease of the intestine, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis), respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea choroiditis, iritis, a hematologic autoimmune disorder, rheumatoid spondylitis, sudden hearing loss, diseases mediated by IgE such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen encephalitis and limbic encephalitis and / or τ Κ ΖΓ Ό'Τ

λ. .1 f_ζ;

INSTITUTE ΜΕΧ1 / - '··_:ζ V ', 3

OF ^THE| NDUST'Í L brainstem encephalitis, uveitis, such as uveitis^Danterior, acute anterior uveitis, granulomatous uveitis, nongranulanidtuuu uveitis, rji'ULinÍKJijnica uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic, primary, or acute immune mediated glomerulonephritis, such as mediated chronic, acute, or immune GN glomerulonephritis, Membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, proliferative membranous-or membranous GN (MPGN), including Type I and Type II, and rapidly progressive GN, allergic conditions, allergic reaction, eczema including allergic or atopic eczema, asthma, such as bronchiole asthma, bronchial asthma, and autoimmune asthma, conditions involving T-cell infiltration and chronic inflammatory responses, chronic inflammatory lung disease, autoimmune myocarditis, deficiency of leukocyte adhesion, systemic lupus erythematosus (SLE) or systemic lupus erythematosus, such as cutaneous SLE, subacute cutaneous lupus erythematosus, Neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus (including nephritis, pediatric cerebritis, non-renal, extra-renal, discoid, alopecia), juvenile-onset diabetes mellitus (Type I), including insulin-dependent pediatric diabetes mellitus (IDDM), adult-onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis, including vasculitis (including large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis)), medium vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), microscopic polyarteritis, CNS vasculitis, cutaneous necrotizing vasculitis, or hypersensitivity, systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as vasculitis or Churg-Strass syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (pernicious anemia), Addison's disease, pure red blood cell anemia or aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, 37

<img file="MX342017B_D0043.tif" />

leukopenia, diseases involving leukocyte diapedesis, inflammatory disorders of the CNS, multiple organ injury syndrome such as those secondary to septicemia, trauma, or hemorrhage, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease , anti-phospholipid antibody syndrome, allergic neuritis, Bechet or Behcet disease, Castleman syndrome, Goodpasture syndrome, Reynaud syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as bullous pemphigoid and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigoid mucous membrane pemphigoid, and pemphigoid erythematosus), poliendocrinopathies autoimmune disease or syndrome, complex immune nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (as developed for example by patients with myocardial infarction), including thrombotic thrombocytopenic purpura (TTP) and autoimmune or immunity-mediated thrombocytopenia, such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, autoimmune diseases of the testicles and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurological paraneoplastic syndromes, Lambert-Lambert syndrome, or Lambert-Etin syndrome. stiff man or stiff person syndrome encephalomyelitis such as allergic encephalomyelitis or allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), myasthenia gravis, such as myasthenia gravis associated thymoma, cerebellar degeneration, neuromyotonia, opsoclonus syndrome or myoclonus opsoclonus (OMM), and sensory neuropathy, motor neuropathy syndrome, motor neuropathy Sheehan's, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active autoimmune hepatitis or chronic active hepatitis, lymphoid interstitial pneumonitis, obliterative bronchiolitis 38

<img file="MX342017B_D0044.tif" />

IMPI ,, · INSTITUTO MEXICANO M LA LWÍltDAO

INDUSTRIAL (non-transplant) versus NSIP, Guillain-I syndrome

Berger (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumonocyrrosis, autoimmune enteropathy syndrome, celiac disease, coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, diopathic sprue, cryoglobulinemia , amiolotrophic lateral sclerosis (ALS, Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AGED), autoimmune hearing loss, opsoclonus myoclonus syndrome (OME), polychondritis, such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, including monoclonal B-cell lymphocytosis (for example , benign monoclonal gammopathy and monoclonal gammopathy of uncertain significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscle disorders, deafness, blindness, periodic paralysis, and CNS channelopathies, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, hepatic autoimmune disorder, fibromyalgia, multiple endocrine insufficiency, Schmidt syndrome , adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as demyelinating autoimmune diseases, Diabetic nephropathy, Dressler's syndrome, Greata alopecia, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, pigeon keeper's lung, allergic granulomatous vasculitis, benign lymphocytic angeitis, Alport syndrome, alveolitis such as allergic alveolltis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis, aspergillosls, Sampter syndrome, Caplan syndrome, dengue fever, endocardiocarditis, fibrosis , diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, elevated and persistent erythema, erythroblastosis fetalis, eosinophilic fasciitis, 39 iMPIr Shulman's syndrome, Felty's syndrome, filariasis,^INe¡ig | S ^ í₁S ^ fSs' Such as Chronic Industrial Cielitis, Heterochronic Cielitis, Iridocyclitis, or Fuch Cielitis, Henoch-Schonlein Purpura, Human Immunodeficiency Virus (HIV) infection, ECHO virus infection, Cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, gonadal autoimmune insufficiency, Sydenham's chorea, post-streptococcal nephritis, ubiterans thromboangiitis, thyrotoxicosis, tabes dorsalis, chorioiditis, polymyalgia of giant cells, endocrine ophthalmopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, keratoconjunctivitis, epidemic isperiodic nephropathy and familial nephrochemic lesion minimal changes, isperhemic nephrocytic lesion benign, retinal autoimmunity, joint inflammation, bronchitis, Chronic obstructive airway disease, silicosis, aphthous, aphthous stomatitis, arteriosclerotic disorders, aspermiogenesis, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, phacoanaphylactic endophthalmia, allergic enteritis, erythema nodosum, idiopathic fatigue syndrome chronic, rheumatic fever, Hamman-Rich disease, sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, ileitis regionalis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Quervain's thyroiditis, acquired splenic atrophy, anti-malignant thymoma, antisrtility antibodies due to malignant disease , diseases associated with Epstein-Barr virus and SCID, acquired immunodeficiency syndrome (AIDS), Parasitic diseases such as Leishmania, toxic shock syndrome, food poisoning, conditions involving T-cell infiltration, delayed hypersensitivity adhesion deficiency and diseases involving multiple organs, antigen-antibody complex mediated diseases, antiglomerular basement membrane disease , allergic neuritis, autoimmune poliendocrinopathies, ovaritis, primary myxedema, atrophic gastritis 40 leukocytes, immune responses associated with acute cytokine- and T-lymphocyte-mediated, leukocyte diapedesis, lesion syndrome

ΙΜΡΙΟ3

INSTITUTO MEXICANO autoimmune, sympathetic ophthalmia, rheumatic diseases,^Db ¥ lf © Fmedad^:;Mixed connective tissue, nephrotic syndrome, insulitis, poliendocrine insufficiency, impatia. peripheral, polyglandular autoimmune type I syndrome, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, acquired epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent sinusitis, sinus or purulent noitis acute or chronic, ethmoidal, frontal, maxillary or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, eosinophilia with pulmonary infiltration, Eosinophilia-myalgia syndrome, Loffler syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma or granulomas containing eosinophils, anaphylaxis, seronegative spondyloarthritis, poliendococuotic autoimmune disease, chronic sclerosing cholangitis, episodes of episodes, mumps scleritis Bruton, transient hypogammaglobulinaemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasias, autoimmune disorders associated with collagen diseases, rheumatism, neurological disease, ischemic reperfusion disorder, reduced blood pressure response, vascular dysfunction, angiectasis, tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia and disease accompanying vascularization, disorders from allergic hypersensitivity, glomerulonephritis, reperfusion injury, reperfusion injury of the myocardium or other tissues, dermatoses with acute inflammatory components, acute purulent meningitis or other inflammatory diseases of the central nervous system, ocular and orbital inflammatory disorders, transfusion-associated granulocyte syndromes, cytokine-induced toxicity, severe acute inflammation, intractable chronic inflammation, pyelitis, pneumonocyrrosis, retinopathy diabetic, diabetic great artery disorder, endoarterial hyperplasia, peptic ulcer, valvulitis and endometriosis.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastema, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, lung cancer (including

INSTITUTO MEXICAL JO -, * ELAP «OPIF_:D ^ V___ small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma, of ^ -puJmói ^, dg 'peritoneal cancer, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer) , pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, uterine or endometrial carcinoma, salivary gland carcinoma, kidney or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade follicular non-Hodgkin lymphoma ( NHL) ¡Small lymphocytic NHL (SL); Follicular NHL intermediate grade, diffuse NHL intermediate grade, immunoblastic NHL high grade, NHL lymphoblastic high grade, NHL of small non-cleft high grade cells; Bulky disease NHL; Mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL); hairy cell leukemia, chronic myeloblastic leukemia, multiple myeloma, and post-transplant lymphoproliferative disorder (PTLD).

The phrase "allergic disease" refers to a disease that involves allergic reactions. More specifically, an "allergic disease" is defined as a disease for which an allergen is identified, where there is a strong correlation between exposure to that allergen and the initiation of the pathological change, and where that pathological change was shown to have an immune mechanism. . In the present description, an immunological mechanism means that leukocytes show an immune response to allergen stimulation. Examples of allergens include mite antigens and pollen antigens. Representative allergic diseases include bronchial asthma, allergic rhinitis, atopic dermatitis, and allergies to pollen and insects. Allergic diathesis is a genetic factor that can be inherited by children of allergic parents. Familial allergic diseases are also called atopic diseases, and the genetically transmitted factor causing it is atopic diathesis. "Atopic dermatitis" is a general term for an atopic disease, especially those

<img file="MX342017B_D0045.tif" />

ΤΓ

MEXICAN INSTITUTE

OF THE? ROH.: J; - 9 INDUL1 ^ l / .L

<img file="MX342017B_D0046.tif" />

diseases accompanied by symptoms of dermatitis. Preferred examples include the allergic condition that is selected from the group consisting of eczema, allergic rhinitis, hay fever, urticaria, and food allergies. Allergic conditions include eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives), and food allergies and other atopic conditions.

Asthma ”- refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively, associated with atopic or allergic symptoms.

The phrase "inflammatory conditions or inflammatory disease" in the present disclosure includes chronic or acute inflammatory diseases, including a disease or condition selected from the group comprising: rheumatic diseases (including but not limited to rheumatoid arthritis, osteoarthritis, psoriatic arthritis) spondyloarthropathies (including but not limited to ankylosing spondylitis, reactive arthritis, Reiter's syndrome), crystal arthropathies (including but not limited to gout, pseudogout, calcium pyrophosphate deposition disease), Lyme disease, polymyalgia rheumatica; connective tissue diseases (including but not limited to systemic lupus erythematosus, systemic sclerosis, polymyositis, dermatomyositis, Sjogren's syndrome); vasculitis (including, but not limited to polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome); inflammatory conditions including consequences of sarcoidosis, trauma or ischemia; vascular diseases, including atherosclerotic vascular disease, atherosclerosis, and occlusive vascular disease (including but not limited to atherosclerosis, ischemic heart disease, myocardial infarction, stroke, peripheral vascular disease), and vascular stent restenosis; eye diseases, including uveitis, corneal disease, iritis, iridocyclitis, and cataracts;

The term treatable cancer in the present invention includes, but is not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include bladder, ovarian, melanoma, squamous cell, lung cancer

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

<img file="MX342017B_D0047.tif" />

(including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, and various types of head and neck cancer, as well as B-cell lymphoma (including follicular / low-grade non-Hodgkin's lymphoma grade (NHL); small lymphocytic NHL (SL), follicular / intermediate grade NHL, intermediate grade diffuse NHL, and high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade non-cleft small cell NHL; Bulky disease NHL; Mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL); hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatosis, edema (such as that associated with brain tumors), and Meigs syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer. pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. In an illustrative embodiment (see working examples), the cancer is an early advanced (including metastasis) of the bladder, ovary, or melanoma. In another embodiment, the cancer is colorectal cancer. Cancer conditions susceptible to the treatment of the invention include metastatic cancers where expression of VISTA by myeloid derived suppressor cells suppresses anti-tumor responses and anti-invasive immune responses. The method of the present invention is particularly suitable for the treatment of vascularized tumors.

<img file="MX342017B_D0048.tif" />

The invention is also suitable for treating cancers in combination with chemotherapy or radiotherapy or other biological products and for enhancing their activity, that is, in individuals where expression of VISTA by myeloid derived suppressor cells suppresses antitumor responses and efficacy of chemotherapy or radiotherapy or biological efficacy. Any chemotherapeutic agent exhibiting anticancer activity can be used in accordance with the present invention. Preferably, the chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodopillotoxins, antibiotics, lasparaginase, topoisomerase inhibitors, interferons , platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressants, adrenocorticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analog. Most preferably, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel, and doxetaxel. Two or more chemotherapeutic agents can be used in a cocktail that is administered in combination with the administration of the anti-VEGF antibody. A preferred chemotherapy combination is based on fluorouracil, comprising 5-FU and one or more other chemotherapeutic agent (s). Suitable dosage regimens of combination chemotherapies are known in the art and are described in, for example, Saltz et al. (1999) Proc ASCO 18: 233a and Douillard et al. (2000) Lancet 355: 1041-7. The biological product can be other immune enhancers such as antibodies to PD-L1, PD-L2, CTLA-4 and PD-L1, PD-L2, CTLA-4 fusion proteins as well as cytokines, growth factor antagonists and agonists, hormones and anti-cytokine antibodies.

Depending on the shape of the PD-L3 or VISTA molecule that binds to a receptor, a signal can be transmitted (for example, by a multivalent form of a PD-L3 or VISTA molecule that results in receptor cross-linking or by a soluble form of PDL3 or VISTA that binds to Fe receptors on the

ΙΜΡΙΟΚΒ

Mexican INSTITUTE Va ». '-Jf

OF RACVJITY ~ antigen-presenting cells) or inhibit (for example, by'trha soluble, monovalent form of a molecule of PD-L3 O VISTA or ma form c ^ i.

VISTA which is altered using methods known in the art such that Fe receptors do not bind on antigen presenting cells), for example, competing with activating forms of PDL3 or VISTA molecules for receptor binding. However, there are cases where a soluble molecule can be stimulatory. The effects of various modulating agents can be easily demonstrated using routine screening tests, as described in the present disclosure.

As used herein, the term "activating receptor" includes immune cell receptors that bind to antigen, complex antigen (eg, in the context of MHC molecules), or antibodies. Such activating receptors include T cell receptors (TCR), B cell receptors (BCR), cytokine receptors, LPS receptors, complement receptors, and Fe receptors.

For example, T cell receptors are present on T cells and associate with CD3 molecules. T cell receptors are stimulated by antigen in the context of MHC molecules (as well as by polyclonal reagents that activate T cells). Activation of T cells through TCR results in numerous changes, for example, protein phosphorylation, lipid membrane changes, ion fluxes, cyclic nucleotide alterations, RNA transcription changes, changes in protein synthesis , and changes in cell volume.

The term B cell receptor (BCR) as used herein includes the transmembrane Ig complex (mlg) and other transmembrane polypeptides (eg, Ig alpha and Ig beta) found in B cells. The signal transduction function of mlg is triggered by the crosslinking of receptor molecules with oligomeric or multimeric antigens. B cells can also be activated by anti-immunoglobulin antibodies. After activation of BCR, numerous changes occur in B cells, including tyrosine phosphorylation.

<img file="MX342017B_D0049.tif" />

tNS ~^TUTO MEXICANO 02 i A FÍUl'MO INOÜí'IVJAL

The term Fe receptor (FcRs) includes the cell surface receptors for the Fe part of immunoglobulin molecules (tg) r ± os ^ Fe receptors found in many cells that participate in the immune response. Among the human FcRs that have been identified so far are those that recognize IgG (termed gamma FcR), IgE (epsilon FcR1), IgA (alpha FcR), and polymerized IgM / A (mu alpha FcR). FcRs are found in the following cell types: epsilon FcRI (mast cells), epsilon FcRIl (many leukocytes), alpha FcR (neutrophils), and mu alpha FcR (glandular epithelium, hepatocytes) (Hogg, N. (1988) Immunol Today 9: 185-86). The widely studied gamma FcRs are essential in cellular immune defenses, and are responsible for stimulating the release of inflammatory mediators and hydrolytic enzymes involved in the pathogenesis of autoimmune diseases (Unkeless, J. C (1988) Annu. Rev. Immunol. 6:25 1-87). Gamma FcRs provide a critical link between effector cells and Ig-secreting lymphocytes, as macrophage / monocyte, polymorphonuclear leukocytes, and natural killer cell (NK) gamma FcRs confer a specific IgG-mediated recognition element. Human leukocytes have at least three different receptors for IgG: gamma FcRI h (found on monocytes / macrophages), gamma FcRIl h (on monocytes, neutrophils, eosinophils, platelets, possibly B cells, and the K562 cell line), and gamma Fclll (in NK cells, neutrophils, eosinophils, and macrophages).

With respect to T cells, the transmission of a co-stimulatory signal to a T cell involves a signaling pathway that is not inhibited by cyclosporin A. Furthermore, a co-stimulatory signal can induce the secretion of cytokines (for example, IL-2 and / or IL-10) in a T cell and / or may prevent the induction of non-response to antigen, the induction of anergy, or the induction of cell death in the T cell.

As used herein, the term "inhibitory signal" refers to a signal transmitted through an inhibitory receptor molecule on an immune cell. Such a signal antagonizes a signal through an activating receptor (for example, through a TCR, CD3, BCR, or Fe molecule) and can

INSTITUTO MEXICANO C..-1

OF PROPERTY 'Z

INDUSTRIAL * - &lt;· - 'result, for example, in the inhibition of: the generation of the second messenger; proliferation, or effector function in the immune cell, - for example, reduced phagocytosis, antibody production, or cellular cytotoxicity, or the failure of the immune cell to produce mediators (such as cytokines (e.g., IL-2) and / or mediators of allergic responses); or the development of anergia.

As used herein, the term "non-responsive" includes the refractivity of immune cells to stimulation, eg, stimulation through an activating receptor or cytokine.

Lack of response can occur, for example, due to exposure to immunosuppressants or high doses of antigen.

As used herein, the term anergy or tolerance includes refractivity to stimulation mediated by the activating receptor. Such refractivity is generally antigen-specific and persists after exposure to the tolerance antigen has ceased. For example, anergy in T cells (as opposed to non-response) is characterized by a lack of cytokine production, eg, IL-2. T cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a co-stimulatory signal). Under these conditions, re-exposure of cells to the same antigen (even if re-exposure occurs in the presence of a co-stimulatory molecule) results in failure to produce cytokines and, thus, failure to proliferate. Energetic T cells, however, can accumulate responses to unconnected antigens and can proliferate if cultured with cytokines (eg, IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using a reporter cell line. Alternatively, a construct reporter gene can be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the IL2 5 'gene enhancer or by a multimer of the API sequence that can be found within the enhancer ( Kang et al. (1992) Science 257: 1134).

IMPI

INSTITUTO MEXICANO LE LA PROHuDAO

INDUSTRIAL

Modulation of a co-stimulatory signal results in modulation of the effector function of an immune cell. Thus, the term PD-L3 or VISTA activation includes the ability of a PD-L3 or VISTA polypeptide to bind to its natural binding partner (s), the ability to modulate the co-stimulatory or inhibitory signals of immune cells, and the ability to modulate the immune response.

Modulation of an inhibitory signal in an immune cell results in modulation of cytokine proliferation and / or secretion by an immune cell.

As used herein, a naturally occurring nucleic acid molecule refers to an RNA or DNA molecule that has a nucleotide sequence that occurs in nature (eg, encodes a natural protein).

As used herein, an antisense nucleic acid molecule comprises a nucleotide sequence that is complementary to a sense nucleic acid that encodes a protein, eg, complementary to the coding strand of a complementary, double-stranded cDNA molecule. to an mRNA sequence or complementary to the coding strand of a gene. As a consequence, an antisense nucleic acid molecule can hydrogen bond to a sense nucleic acid molecule.

As used herein, the term "coding region" refers to regions of a nucleotide sequence that comprises codons that are translated into amino acid residues, while the term "non-coding region" refers to regions of a nucleotide sequence that do not. they are translated into amino acids (eg, 5 'and 3' untranslated regions).

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule that was attached to it. One type of vector is a plasmid, which refers to a circular loop of double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced

<img file="MX342017B_D0050.tif" />

1IVK i. ki

INSTITUTE M / X // Λ '

DE LA EROPicO; '»INDUS i RueL ίΧ' / ·^&lt;5&lt;\ (eg, bacterial vectors having a replication of bacterial origin and episomal mammalian vectors). Other vectors - (peF ^! JemptO;^_Mammalian episomal vectors ^ elο) integrate into the genome of a host cell after introduction into the host cell, and thereby replicate along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as recombinant expression vectors or simply expression vectors. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, plasmid and vector can be used interchangeably since plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of such expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses), that serve equivalent functions.

As used herein, the term "host cell" is intended to refer to a cell into which a nucleic acid molecule of the invention was introduced, such as a recombinant expression vector of the invention. The terms host cell and recombinant host cell are used interchangeably in the present description. It should be understood that such terms refer not only to the cell of the particular individual but to the progeny or potential progeny of said cell. Because of certain modifications that may occur in successive generations either due to mutation or environmental influences, such progeny may, in fact, not be identical to the parent cell, but it is still included within the scope of the term used in the present description.

As used herein, a "transgenic animal" refers to a non-human animal, preferably a mammal, more preferably a mouse, in which one or more of the cells of the animal include a transgene. The term transgene refers to an exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that

<img file="MX342017B_D0051.tif" />

<img file="MX342017B_D0052.tif" />

remains in the genome of the mature animal, for example by directing the expression of an encoded gene product in one or more cell or tissue types of the transgenic animal.

As used herein, a "recombinant homologous animal" refers to a type of non-human transgenic animal, preferably a mammal, more preferably a mouse, in which an endogenous gene was altered by homologous recombination between the endogenous gene and a exogenous DNA molecule introduced into a cell of the animal, eg, an embryonic cell of the animal, prior to development of the animal.

As used herein, an "isolated protein" refers to a protein that is substantially free of other proteins, cellular material, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.

An isolated or purified protein or biologically active part thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the PD-L3 or VISTA protein is derived, or substantially free of chemical precursors or other products. chemicals when chemically synthesized. The term "substantially free of cellular material" includes PD-L3 or VISTA protein preparations in which the protein is separated from the cellular components of the cells from which it is isolated or produced recombinantly. In one embodiment, the term "substantially free of cellular material" includes PD-L3 or VISTA protein preparations that have less than about 30% (by dry weight) non-PD-L3 or VISTA protein (also referred to herein as a protein contaminant), more preferably less than about 20% non-PD-L3 or VISTA protein, even more preferably less than about 10% non-PD-L3 or VISTA protein, and most preferably less than about 5% non-PD-L3 or VISTA protein. When the PD-L3 or VISTA protein or biologically active part thereof is produced recombinantly, it is also preferred substantially free of culture medium, that is, the culture medium 51

<img file="MX342017B_D0053.tif" />

<img file="MX342017B_D0054.tif" />

DELA r «yi represents less than approximately 20%, with higher preference less than approximately 10%. and most preferably less &amp; jde-api = exiffleeternente ^%^m~ of the volume of the protein preparation.

The term "substantially free of chemical precursors or other chemicals" includes preparations of the PD-L3 or VISTA protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the term "substantially free of chemical precursors or other chemicals" includes preparations of the PD-L3 or VISTA protein that have less than about 30% (by dry weight) chemical precursors or non-PD-L3 or VISTA chemicals. , more preferably less than about 20% chemical precursors or non-PD-L3 or VISTA chemicals, even more preferably less than about 10% non-PD-L3 or VISTA chemical precursors or chemicals, and most preferably less than about 5% non-PD-L3 or VISTA chemical precursors or chemicals.

The term "antibody," as used herein, includes an antigen-binding part of an antibody (or simply part of an antibody), as well as whole antibody molecules. The term "antigen-binding part", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, PD-L3 or VISTA). It was shown that the antigen-binding function of an antibody can be performed by fragments of a whole antibody. Examples of binding fragments that fall within the term "antigen-binding part of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F fragment (abj2, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 341: 544-546). consisting of a VH domain; and (vi) an isolated complementarity determining region (CDR).

<img file="MX342017B_D0055.tif" />

MEXICAN INSTITUTE

OF INDUSTRIAL PEGPITTY

<img file="MX342017B_D0056.tif" />

Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be prepared as a single protein chain in which the VL and VH regions are linked. pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426, and Huston et al. (1988) Proc Nati. Acad. Sci. USA 85 : 58795883, and Osbourn et al. 1998 Nat. Biotechnol. 16: 778). Such single chain antibodies are also intended to be included within the term "antigen-binding part of an antibody." Any of the specific scFv VH and VL sequences can be linked to human immunoglobulin constant region cDNA or genomic sequences, to generate expression vectors that encode complete IgG molecules or other isotypes. VH and VL can also be used in the generation of Fabs, Fv, or other immunoglobular fragments either using protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed in a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to be paired with the complementary domains of another chain and creating two antigen-binding sites (see, for example, Holliger, P. et al. (1993) Proc Nati Acad Sci USA 90: 6444-6448; Poljak, RJ et al. (1994) Structure 2: 1121-1123).

Furthermore, an antigen-binding antibody or part thereof may be part of larger immunoadhesion molecules, formed by covalent or non-covalent association of the antibody or part of the antibody with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to generate a tetrameric scFv molecule (Kipriyanov, SM et al. (1995) Hum. Antibodies Hybridomas 6: 93101) and the use of a cysteine ​​residue, a marker peptide and a polyhistidine C-terminal tag to make bivalent and biotinylated scFv molecules

<img file="MX342017B_D0057.tif" />

(Kipriyanov, SM et al. (1994) Mol Immunol. 31: 1047-105.8). The parts of the antibody, such as Fab and F (ab¹) 2, can be prepared ~ a'partrf ^,of whole antibodies using conventional techniques, such as digestion of whole antibodies with papain or pepsin, respectively. Furthermore, the antibodies, parts of the antibody and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described in the present invention.

The antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic; or modified forms of these, eg, humanized, chimeric, etc. Preferably; the antibodies of the invention bind specifically or substantially specifically to PD-L3 or VISTA molecules. The terms monoclonal antibodies and monoclonal antibody composition, as used herein, refer to a population of antibody molecules that contain a single species of an antigen-binding site capable of immunoreacting with a particular epitope of an antigen. , whereas the term "polyclonal antibodies" and "polyclonal antibody composition" refer to a population of antibody molecules that contain multiple species of antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically exhibits a unique binding affinity for a particular antigen with which it immunoreacts.

The term "humanized antibody", as used herein, is intended to include antibodies generated by a non-human cell that have variable and constant regions that were altered to more closely resemble antibodies that would be generated by a human cell. For example, by altering the amino acid sequence of the non-human antibody to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (eg, randomly introduced mutations or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in CDRs. The term humanized antibody, as used herein

XV.EHJ

INSTITUTO MEXICANO ν ', 7Λ

DESCRIPTION OF THE FEOPSEEiAD A .V description, also includes antibodies in which CDR sequences derived from the germ line of another mammalian species, ΙθΐΈοηηό a mouse, rsé * laserf6eh the human framework sequences.

An isolated antibody, as used herein, is intended to refer to an antibody that is substantially free of other antibodies that have different antigenic specificities (for example, an isolated antibody that specifically binds to PD-L3 or VISTA is substantially free of antibodies that specifically bind to antigens other than PD-L3 or VISTA). Furthermore, an isolated antibody can be substantially free of other cellular materials and / or chemicals.

An oligomerization domain in the present disclosure refers to a domain that when attached to a VISTA extracellular domain or fragment thereof, facilitates oligomerization. Such oligomerization domains comprise alpha-helix self-association, eg, leucine zipper, which can be further stabilized by additional disulfide bridges. The domains are designed to be compatible with vector folding across a membrane, an ongoing thought to facilitate in vivo folding of the polypeptide into a functional binding protein. Examples of these are known in the art and include by way of example coils of GCN4, and COMP.

The coiled alpha-helical coil is probably the most widespread oligomerization subunit motif in proteins. As a consequence, the coiled spiral serves a variety of different functions. In several families of transcriptional activators, for example, in DNA short leucine zippers play an important role in the positioning of DNA-binding regions (Ellenberger et al., 1992, Cell 71: 1223-1237). The coiled coil is also used to form intermediate filament protein oligomers. Coiled coil proteins also appear to play an important role in both vesicle and membrane viral fusion (Skehel and Wiley, 1998, Cell 95: 871-874). In both cases, the hydrophobic sequences, embedded in the fusing membranes, are found at the same end of the rod-shaped complex composed of a bundle of many alpha-helices.

ΙΜΡΙ _, ιιι INSTITUTO MEXICANO Wjrf-. '' ', ZÍ

This molecular arrangement is believed to cause apposition at closure that the complexes assemble by membrane fusion. Spiral winding is frequently used to control oligomerization. It is found in many types of proteins, including transcription factors such as, but not limited to, GCN4, viral fusion peptides, SNARE complexes, and certain tRNA synthetases, among others. Very long coiled spirals are found in proteins such as tropomyosin, intermediate filaments, and components of the polar body of the spindle. The coiled spiral involves a series of alpha-helices that supercoil around each other in a highly organized manner associating in a parallel or an antiparallel orientation. Although dimers and trimers are the most common. The helices can be of the same or different proteins. Spiral winding is made up of component helices that come together to hide their hydrophobic seams. Since the hydrophobic seams are twisted around each helix, then the helixes are also twisted to wrap around each other, hiding the hydrophobic seams and forming a supercoil. This is the characteristic interdigitation of the side chains between neighboring helices, known as knob-in-hole packing, which defines the structure as a spiral wound. The helices do not have to run in the same direction for this type of interaction to occur, although parallel conformation is more common. The antiparallel conformation is very rare in trimers and unknown in pentamers, but more common in intramolecular dimers, where the two helices are frequently connected by a short loop. In the extracellular space, the heterotrimeric spiral coiling of the laminin protein plays an important role in the formation of basement membranes. Other examples are thrombospondins and oligomeric cartilage matrix protein (COMP) in which three (thrombospondins 1 and 2) or five chains (thrombospondins 3, 4 and COMP) are connected. The molecules have a bouquet-like appearance, and the reason for their oligomeric structure is probably the multivalent interaction of the C-terminal domains with cell receptors. The yeast transcriptional activator GCN4 is 1 of more than 30 identified eukaryotic proteins that contain the DNA-binding motif of the basic region of the leucine zipper (bZIP) (Ellenberger et al., 1992,

<img file="MX342017B_D0058.tif" />

Cell 71: 1223-1237). The bZIP dimer is a pair of alpha heli

DE LA PRÜÍÍLDAD 'a parallel spiral wound over its 34 carboxy-terminal residues and gradually diverge towards its amino terminal to pass through the rip-tnaycrr of the DNA-binding site. The interface in the dimerization of the spiral winding is oriented almost perpendicular to the axis of the DNAs, giving the complex the appearance of the letter T. The bZIP contains a 4-3 heptad repeat of hydrophobic and nonpolar residues that are packed together in an alpha-helical coil (Ellenberger et al., 1992, Cell 71: 1223-1237). The stability of the dimer results from the side-by-side packing of leucines and nonpolar residues at the a and d positions of the heptad repeat, as well as a limited number of intra- and interhelical salt bridges, which are shown in a crystalline structure of the peptide GCN4 of the leucine zipper (Ellenberger et al., 1992, Cell 71: 1223-1237). Another example is CMP (matrilin-1) isolated from cartilage of the bovine trachea as a homotrimer of subunits of mass 52,000 (Paulsson and Heinegard, 1981, Biochem J. 197: 367-375), where each subunit consists of a vWFA1 module, a single EGF domain, a vWFA2 modulus, and a spiral wound domain spanning five heptads (Kiss et al., 1989, J. Biol. Chem. 264: 8126-8134; Hauser and Paulsson, 1994, J. Biol. Chem. 269 : 25747-25753). Electron microscopy of purified CMP showed a bouquet-like trimer structure in which each subunit forms an ellipsoid emerging from a common point corresponding to the spiral winding (Hauser and Paulsson, 1994, J. Biol. Chem. 269: 25747-25753): The spiral wound domain in matrilin-1 has been extensively studied. The trimeric structure is preserved after complete reduction of interchain disulfide bonds under non-denaturing conditions (Hauser and Paulsson, 1994, J. Biol. Chem. 269: 25747-25753). Still another example is cartilage oligomeric matrix protein (COMP). A non-collagenous glycoprotein, COMP, was first identified in cartilage (Hedbom et al., 1992, J. Biol. Chem. 267: 6132-6136). The protein is a 524 kDa five subunit homo-pentamer consisting of an N-terminal region of heptad repeat (cc) followed by four domains (EF) similar to epidermal growth factor (EGF), seven domains (T3) that bind calcium and a globular C-terminal domain (TC). According to this domain organization, COMP belongs to the family of 57

<img file="MX342017B_D0059.tif" />

I Μ Ρ I &lt;?;

thrombospondins. Heptad repeats (abcdefg) .sub.n with res'ídúb ^ rpréfereritef

INDUSTRIAL ___ hydrophobic at the a and d positions of the helically wound spiral domains (Cohen and Parry, 1994, Science 263: 488-489). ReciehtémeñféréTWTnírTiu · Recombinant five-stranded spiral COMP (COMPcc) was crystallized and its structure resolved in 0.2 nm resolution (Malashkevich et al., 1996. Science 274: 761-765).

The term "family" when referring to the polypeptide and nucleic acid molecules of the invention is intended to mean two or more polypeptide or nucleic acid molecules that have a common structural domain or motif and that have sufficient amino acid or nucleotide sequence homology as defined. in the present description. Said family members can be of natural or non-natural origin and can be either of the same or different species. For example, a family may contain a first polypeptide of human origin, as well as other, distinct polypeptides of human origin, or alternatively, they can contain homologues of non-human origin, eg, monkey polypeptides. Members of a family may also have common functional characteristics.

For example, the PD-L3 or VISTA family of polypeptides of the present invention preferably comprises at least one signal peptide domain. As used herein, a signal sequence or signal peptide includes a peptide that contains about 15 or more amino acids that occur at the N-terminus of secretory and membrane-bound polypeptides and that contain a large number of residues of hydrophobic amino acids. For example, a signal sequence contains at least about 10-30 amino acid residues, preferably about 15-25 amino acid residues, more preferably about 18-20 amino acid residues, and even more preferably about 19 amino acid residues, and has at least about 35-65%, preferably about 38-50%, and more preferably about 40-45% hydrophobic amino acid residues (e.g. valine, leucine, isoleucine or phenylalanine). Said signal sequence, also known in the art as a ιμρι ^> »

INSTITUTO MEXICANO \ ~ \ ¿J | signal peptide, serves to target a polypeptide that contains dicfia'sequence to a lipid bilayer, and cleaves into secreted membrane-bound polypeptides. As described below a signal sequence was identified in the amino acid sequence of native human PD-L3 or VISTA and was also identified in the amino acid sequence of mouse native PD-L3 or VISTA.

Another embodiment of the invention, a PD-L3 or VISTA polypeptide of the present invention is identified based on the presence of a transmembrane domain. As used herein the term "transmembrane domain" includes an amino acid sequence approximately 15 amino acid residues in length that spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha helix structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids in a hydrophobic transmembrane domain are, for example, leucines, isoleucines, tyrosines, or tryptophanes. Transmembrane domains are described in, for example, Zagotta, WN et al. (1996) Annu. Rev. Neurosci. 19: 235-263, the contents of which are incorporated by reference into the present disclosure. The PDL3 transmembrane domain region is identified in the present disclosure (see, for example, Figure 1).

In another embodiment, a PD-L3 or VISTA molecule of the present invention is identified based on the absence of an Ig C domain and the presence of an IgV domain in the corresponding polypeptide or nucleic acid molecule. As used herein, the IgV and IgC domains are recognized in the art as member domains of the Ig superfamily. These domains correspond to structural units that have different folding patterns called Ig folds. Ig folds are composed of a sandwich of two beta sheets, each consisting of 510 amino acid antiparallel beta chains with a conserved disulfide bond between the two sheets in most, but not all, of the domains. The IgC domains of Ig molecules,

<img file="MX342017B_D0060.tif" />

<img file="MX342017B_D0061.tif" />

<img file="MX342017B_D0062.tif" />

INSTITUTO MEXICANO DE LA VEO HE DAD

TCR, and MHC share the same types of sequence patterns, and are called the C1 set in the Ig superfarnilia. Other IgC-caGn-within-other- sets. IgV domains also share sequence patterns and are called set V domains. IgV domains are longer than C domains and form an additional pair of beta chains. The amino acid residues of the native human and murine PD-L3 or VISTA polypeptide, which constitute the IgV domain can be seen in Figure 1. The presence of an IgV domain is likely required for the binding of PD-L3 or VISTA to its natural binding partner (s).

In another embodiment, a PD-L3 or VISTA molecule of the present invention is identified based on the presence of an extracellular domain in the corresponding polypeptide or nucleic acid molecule. As used herein, the term "extracellular domain" represents the N-terminal amino acids that extend like a tail from the surface of a cell. An extracellular domain of the present invention includes an IgV domain and can include a signal peptide domain. (See Figure 1).

In yet another embodiment, a PD-L3 or VISTA molecule of the present invention is identified based on the presence of a cytoplasmic domain in the corresponding polypeptide or nucleic acid molecule. As used herein, the term "cytoplasmic domain" represents the C-terminal amino acids, which extend like a tail in the cytoplasm of a cell intended to comprise cytoplasmic domains.

In a preferred embodiment, the PD-L3 or VISTA molecules of the invention include at least one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

The isolated polypeptides of the present invention, preferably PD-L3 or VISTA polypeptides, have an amino acid sequence sufficiently identical to the amino acid sequence of secs. with nos. of ¡dent. 2 or 4, or 5 or are encoded by a nucleotide sequence sufficiently identical to sec. with nos. of ident. 1 or 3 or a fragment or complement of it. As used in the present description, the term "sufficiently identical" refers to

IMPI

INSTITUTO MEXICANO DE LA PROPlEi> ΑΓ a first sequence of amino acids or nucleotides that contains a sufficient or minimal number of amino acid residues or -nucleotides identical or

<img file="MX342017B_D0063.tif" />

equivalents (eg, an amino acid residue having a similar side chain) to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and / or a common functional activity . For example, amino acid or nucleotide sequences that share common structural domains have at least 30%, 40%, or 50% homology, preferably 60% homology, most preferably 70% -80%, and even more preferably 90- 95% homology across the amino acid sequences of the domains and contain at least one or preferably two structural domains or motifs, which are defined herein as sufficiently identical.

Furthermore, amino acid or nucleotide sequences that share at least 30%, 40%, or 50% preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein. description as sufficiently identical.

As used interchangeably in the present description, PD-L3 or VISTA activity, PD-L3 or VISTA biological activity or PD-L3 or VISTA functional activity, refers to an activity exerted by a PD-L3 or VISTA protein, polypeptide or nucleic acid molecule in a PD-L3 or VISTA responsive cell or tissue, or in a PD-L3 or VISTA polypeptide binding partner, as determined in vivo, or in vitro, according to standard techniques. These activities include modulating CD4 + and CD8 + T cell proliferation and cytokine production. In another embodiment, a PD-L3 or VISTA activity is a direct activity, such as an association with a PD-L3 or VISTA binding partner. As used herein, a target molecule or binding partner is a molecule with which a PD-L3 or VISTA polypeptide binds or interacts in nature, i.e., expressed in a T cell, such that the function mediated by PD-L3 or VISTA. Alternatively, a PD-L3 or VISTA activity is an indirect activity, such as a cell signaling activity.

<img file="MX342017B_D0064.tif" />

<img file="MX342017B_D0065.tif" />

^EU 'A / ROFIIDAJJ' * *

inuuSTRlxi 'mediated by PD-L3 or VISTA polypeptide. Biological activities ae PD-L3 or VISTA are described in the present description. For example, PD-L3 or VISTA pOttpeptides and PD-L3 or VISTA agonists or antagonists of the present invention may have one or more of the following activities: (1) suppress or promote proliferation of CD4 + and CD8 + T cells, (2) suppress or promote cytokine production (3) function as a regulatory ligand that negatively regulates T cell responses during analogous interactions between T cells and Myeloid derived APCs (4) negatively regulate CD4 + T cell responses by early suppression of TCR activation and arrest of cell division, but with minimal direct impact on apoptosis, (5) suppressing or promoting antigen-specific T cell activation during analogous interactions between APCs and T cells and / or (6) suppressing or promoting T cell mediated immune responses; (7) modulate the activation of immune cells, eg, T lymphocytes, and (8) modulate the immune response, eg, inflammatory immune response of an organism, eg, a mouse or a human organism.

As a consequence, another embodiment of the invention features isolated PD-L3 or VISTA proteins and polypeptides that modulate one or more PD-L3 or VISTA activities. These polypeptides will include PD-L3 or VISTA polypeptides that have one or more of the following domains: a signal peptide domain, an IgV domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain, and, preferably, a PD activity. -L3 or VISTA.

Preferred PD-L3 or VISTA polypeptides may further have at least one extracellular domain, and one or more than a signal peptide domain, an IgV domain, a transmembrane domain, and a cytoplasmic domain, and are preferably encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising in the present disclosure a complement of the nucleotide sequence of secs. with nos. of ident. : 1 or 3. The nucleotide and amino acid sequence of the isolated and exemplified cDNA sequence of human and murine PD-L3 or VISTA and the amino acid sequence

INSTITUTE MFX'CANO predicted from human PD-L3 or VISTA polypeptide was included / a $ uWtla

<img file="MX342017B_D0066.tif" />

sequences of the present description.

<img file="MX342017B_D0067.tif" />

Human VISTA or PD-L3 or VISTA was identified as a positively regulated molecule in a T cell transcriptional profile screening. Our characterization of an identical 930 bp gene product recovered from a CD4 T cell cDNA library⁺ murines confirmed the size and sequence. In silico sequence and structural analysis predict a 309 amino acid type I transmembrane protein after maturation. Its extracellular domain contains a single 136 amino acid extracellular Ig-V domain, which binds to a 23 amino acid stem region, a 21 residue transmembrane segment, and a 97 amino acid cytoplasmic domain. The VISTA cytoplasmic tail does not contain any signaling domains. A BLAST sequence search with the VISTA Ig-V domain identified PD-L1 of the B7 family as the evolutionarily closest of the related protein with a significant E-value cutoff score. A sequence alignment based on the structure of VISTA with the B7 family members PD-L1, PD-L2, B7-H3, and B7-H4 highlights several amino acids that are conserved consistently in all Ig-V domains of the proteins.

Various aspects of the invention are described in more detail in the following subsections:

I. Isolated PD-L3 or VISTA nucleic acid molecules

One aspect of the invention corresponds to isolated nucleic acid molecules encoding PD-L3 or VISTA polypeptides or biologically active parts thereof, as well as nucleic acid fragments sufficient to use as hybridization probes to identify nucleic acid molecules encoding PD. -L3 or VISTA (eg, PD-L3 or VISTA mRNA) and fragments for use as PCR primers for amplification or mutation of PD-L3 or VISTA nucleic acid molecules. As used in the present description, the term nucleic acid molecule is intended to include DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and DNA or RNA analogs generated using nucleotide analogs. The nucleic acid molecule can be single-stranded

<img file="MX342017B_D0068.tif" />

preferably it is double stranded DNA.

The term "isolated nucleic acid molecule" includes nucleic acid molecules that are separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules that separate from the chromosome with which genomic DNA naturally associates. Preferably, an isolated nucleic acid molecule is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid molecule) in the genomic DNA of the organism from which it is derived. derives nucleic acid. For example, in various embodiments the isolated PD-L3 or VISTA nucleic acid molecule may contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of naturally-flanking nucleotide sequences. nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived. Furthermore, an isolated nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium, when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when synthesized. chemically.

A nucleic acid molecule of the present invention, for example, a nucleic acid molecule having the nucleotide sequence of Sec. with nos. Ident.:1, 3, or a portion thereof can be isolated using standard molecular biology techniques and the sequence information provided in the present disclosure. Using all or part of the nucleic acid sequence of secs. with nos. Ident.:1, or 3 as a hybridization probe, PD-L3 or VISTA nucleic acid molecules can be isolated using standard hybridization and cloning techniques (eg, as described in Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. Second, ed., Coid Spring Harbor Laboratory, Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY, 1989).

INSTITUTO MEXICANO V, *, '*' ¿Zt DE LA Ι'ΚΟΙΊΕΟΛΟ \ ¿

INDUSTRIAL ^ * »- 22! -In addition, a nucleic acid molecule includes all or part of sec.

with nos. Identification number: 1, 3, or an ortholog or variant can be isolated pui¹ the reaction¹ polymerase chain (PCR) using synthetic oligonucleotide primers designed based on the sequence of secs. with nos. Identification number: 1, 2, 3, 4 or 5.

A nucleic acid molecule of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecule thus amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to PD-L3 nucleotide sequences can be prepared by standard synthetic techniques, for example using an automated DNA synthesizer.

In a preferred embodiment, an isolated PD-L3 or VISTA-encoding nucleic acid molecule of the invention comprises the nucleotide sequence shown in secs. with nos. of ident.:1, or 3, or a fragment of it. In another embodiment, the nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement to the nucleotide sequence shown in secs. with nos. Ident.:1, or 3, or a part of any of these nucleotide sequences. A nucleic acid molecule that is complementary to the nucleotide sequence shown in sec. with nos. Ident.:1, or 3, is one that is sufficiently complementary to the nucleotide sequence shown in secs. with nos. Ident .: 1, or 3 such that it can hybridize to the nucleotide sequence shown in sec. with nos. ident.:1, or 3, respectively, thereby forming a stable duplex.

In yet another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or more to the complete nucleotide sequence shown in secs. with nos. Ident .: 1 or 3, or a part of any of these nucleotide sequences.

IMPI

Furthermore, the nucleic acid molecule of the invdñúíóft ^ üéttS

<img file="MX342017B_D0069.tif" />

only a part of the nucleic acid sequence of secs. with nos. of ident.:1, or 3, for example, a fragment that can be used as a probe or primer or a fragment that encodes a part of a PD-L3 or VISTA polypeptide, for example, a biologically active part of a PD-L3 or VISTA polypeptide. The nucleotide sequences determined from the cloning of the human PD-L2 gene are allowed for the generation of probes and primers designed for use in the identification and / or cloning of other members of the PD-L2 family, as well as homologues of PD-L3 or VISTA from other species. The probe / primer typically comprises the substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of secs. with nos. Ident .: 1, or 3; of an antisense sequence of secs. with nos. of ident.:1, 3, or a naturally occurring or mutant allelic variant of secs. with nos. of ident.:1, or 3.

In one embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence that is greater than about 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400 -450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900950 or more nucleotides in length and hybrid under stringent conditions hybridization with a nucleic acid molecule of secs. with nos. of ident.:1, or 3, or the complement of these. In a further embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence that is greater than about 880-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150 or more nucleotides. long and hybrid under stringent conditions of hybridization with a nucleic acid molecule of secs. with nos. of ident. : 1 or 3, or the complement of these. In yet another embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence that is greater than 50-100, 100-150, 150-200, 200-250, 250-300 or more nucleotides of

<img file="MX342017B_D0070.tif" />

ΙΜΡΙ: 5¾

INSTITUTE MS.KIC / 'NO length and hybridization under stringent hybridization conditions for a nucleic acid molecule comprising the coding region of soGt-god no. do- &lt;dont:! - 1-é 3, or a complement of these. In yet another embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence that is greater than about 50-100,100-150,150-200, 200-250, 250-300, 300-350, 350-400, 400- 450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750

800, 850-900, 900-950, or more nucleotides in length, includes at least about 15 nucleotides (ie, 15 contiguous) of the sequence comprising the coding region of secs. with nos. of ident.:1 or 3, or a complement thereof, and hybridized under stringent conditions with a nucleic acid molecule comprising the nucleotide sequence shown in secs. with nos. Ident .: 1, or 3 or a complement of these.

Probes based on PD-L3 or VISTA nucleotide sequences can be used to screen transcripts or genomic sequences encoding the same or homologous polypeptides. In preferred embodiments, the probe further comprises a label group attached to it, for example, the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used as part of a diagnostic test kit to identify cells or tissues that incorrectly express a PD-L3 or VISTA polypeptide, such as by measuring a level of a nucleic acid encoding PD-L3 or VISTA in a sample of cells from an individual by, for example, detecting PD-L3 or VISTA mRNA levels or determining whether a PD-L3 or VISTA genomic gene mutated or deleted.

A nucleic acid fragment encoding a biologically active part of a PD-L3 or VISTA polypeptide can be prepared by isolating a part of the nucleotide sequence of secs. with nos. of ident.:1, or 3, which encodes a polypeptide that has a biological activity of PD-L3 or VISTA (e.g., the ability to bind to its natural binding partner (s) and / or modulate the activity of cells immune), expressing the encoded part of the PD-L3 or VISTA polypeptide (eg, by recombinant expression in vitro) and evaluating the activity of the encoded part of the PD-L3 or VISTA polypeptide.

The invention further includes nucleic acid molecules that differ from the nucleotide sequence shown in secs. with nos. of ident.:1, or 3 due to the degeneracy of the genetic code and thus encode the same PD-L3 or VISTA polypeptides as those encoded by the nucleotide sequence shown in secs. with nos. of ident.:1, or 3. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence that encodes a polypeptide having an amino acid sequence shown in secs. with nos. Identification number: 2, 4 or 5.

In addition to the nucleotide sequences of PD-L3 or VISTA shown in sec. with nos. From ident: 1, and 3, it will be appreciated by those of skill in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of PDL3 or VISTA polypeptides may exist within a population (e.g. , the human population). Such genetic polymorphism in the PD-L3 or VISTA genes can exist between individuals within a population due to natural allelic variation. As used herein, the terms gene and recombinant gene refer to nucleic acid molecules that include an open reading frame that encodes a PD-L3 or VISTA polypeptide, preferably a mammalian PD-L3 or VISTA polypeptide, and it can further include non-coding regulatory sequences and introns.

Human or mouse PD-L3 or VISTA allelic variants include both functional and non-functional PD-L3 or VISTA polypeptides. Functional allelic variants are naturally occurring amino acid sequence variants of the human or mouse PD-L3 or VISTA polypeptide that retain the ability to bind to the natural PD-L3 or VISTA binding partner (s) and / or modulate. the proliferation of CD4 + and CD8 + T cells and the production of cytokines and the activation of lymphocytes. Functional allelic variants will typically contain only the conservative substitution of one or more amino acids from secs. with nos. Ident .: 2, 4 or 5, or substitution, deletion or insertion of non-critical residues in non-critical regions of the polypeptide.

i JL V1 riv

INSTITUTO MEXICANO V DE LA PROPERTY V. __1NDU._Tit,_TO,

Nonfunctional allelic variants are variant^NDcie^{!TO THE}naturally occurring amino acid sequence of PD-L3 polypeptide that do not have the ability to bind either the natural PD-L3 or VISTA binding partners, and / or modulate any of the PD-L3 or VISTA activities described herein invention. Non-functional allelic variants will typically contain a non-conservative substitution, deletion or insertion or premature truncation of the amino acid sequence of secs. with nos. ident.:2, 4 or 5, or a substitution, insertion or deletion at critical residues or critical regions of the polypeptide for example, in an IgV domain.

The present invention further provides non-human, non-mouse orthologs of human or mouse PD-L3 or VISTA polypeptide. Human or mouse PD-L3 or VISTA polypeptide orthologs are polypeptides that are isolated from non-human and non-mouse organisms and possess the same binding activity and / or activity that modulates lymphocyte activation, and the ability to to modulate the proliferation of CD4 + and CD8 + T cells and the production of cytokines such as the human and murine PD-L3 or VISTA polypeptides described in the present invention. Human or mouse PD-L3 polypeptide orthologs can easily be identified as comprising an amino acid sequence that is substantially identical to sec. with nos. of ident.:2, 4 or 5.

Furthermore, nucleic acid molecules that encode other members of the PD-L3 or VISTA family and thus have a nucleotide sequence that differs from the PD-L3 or VISTA sequences of Seq. with nos. Indent.:1, or 3 are intended to be within the scope of the invention. For example, another PDL3 or VISTA cDNA can be identified based on the nucleotide sequence of mouse or human PDL3 or VISTA. Furthermore, nucleic acid molecules that encode PD-L3 or VISTA polypeptides of different species, and thus have a nucleotide sequence that differs from the PD-L3 or VISTA sequences of secs. with nos. Ident.:1, or 3 are intended to be within the scope of the invention. For example, a monkey PD-L3 or VISTA cDNA can be identified based on the nucleotide sequence of mouse or human PD-L3 or VISTA.

<img file="MX342017B_D0071.tif" />

-IcÜAD vary

<img file="MX342017B_D0072.tif" />

Dt LA

The nucleic acid molecules corresponding to las^LNatural and homologous PD-L3 or VISTA cDNAs of the invention can be isolated based on their homology with the PD-L2 nucleic acids described in the present invention using the cDNAs described in the present invention, or a part of these, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. The nucleic acid molecules corresponding to the natural and homologous allelic variants of the PD-L3 or VISTA cDNAs of the invention can be further isolated by mapping to the same chromosome or locus as the PD-L3 or VISTA gene.

As a consequence in another embodiment an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybrid under stringent conditions to the nucleic acid molecule comprising the coding region of the nucleotide sequence of the sec. with nos. Ident.:1 or 3. In another embodiment, the nucleic acid is at least 700, 750, 800, 850, 880-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150 or more nucleotides in length.

As used herein, the term "hybrid under stringent conditions" is intended to describe the conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain mutually hybridized. Preferably the conditions are such that sequences of at least about 70%, more preferably at least about 80% even more preferably at least about 85% or 90% identical to each other remain mutually hybridized. Such stringent conditions are known to those of skill in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al., Editors., John Wiley 8i Sons, Inc (1995), sections 2, 4 and

6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Coid Spring Harbor Press, Coid Spring Harbor, NY (1989), Chapters 7, 9, and 11. A preferred, non-limiting example of stringent hybridization conditions includes 4-fold or 6-fold hybridization in sodium chloride / sodium citrate (SSC), approximately 65-70 degrees C (or 4-fold hybridization in SSC plus 50% formamide at approximately 42-50 degrees C) followed by one or more washes in 1 X SSGU at approximately 05-7 degrees C. A further preferred, non-limiting example of stringent hybridization conditions includes hybridization 6 times in SSC at 45 degrees C, followed by one or more washes 0.2 times in SSC, 0.1% SDS at 65 degrees C. A preferred, non-limiting example of highly stringent hybridization conditions includes 1-time hybridization in SSC at approximately 65-70 degrees C (or 1-time hybridization in SSC plus 50% formamide at approximately 42-50 degrees C) followed by one or more washes 0.3 times in SSC, approximately 65-70 degrees C. A preferred, non-limiting example of low stringency hybridization conditions includes 4-fold or 6-fold hybridization in SSC at approximately 5060 degrees C (or alternatively 6-fold hybridization in SSC plus 50% formamide at approximately 40-45 degrees C) followed by one or more washes 2 times in SSC, at approximately 50-60 degrees C. Intermediate ranges to the values ​​cited above, for example, 65-70 degrees C to 42-50 degrees C are also intended to be included by the present invention. SSPE (1 time in

SSPE is 0.15 M NaCl, 10 mM NaH₂PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1 time in SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for anticipated hybrids that are less than 50 base pairs in length should be 5-10 degrees C lower than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (degrees C) -2 (# of bases

A + T) +4 (# of bases G + C). For hybrids between 18 and 49 base pairs in length, Tm (degrees C) = 81.5 +16.6 (log 10 [Na +]) + 0.41 (% G + C) - (600 / N), where N is the number of bases in the hybrid, and [Na +1] is the concentration of sodium ions in the hybridization buffer ([Na +] for 1 time in SSC = 0.165M). It will also be recognized by the skilled practitioner that additional reagents can be added to hybridization and / or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, e.g., nitrocellulose or nylon membranes, including but are not limited to 71

<img file="MX342017B_D0073.tif" />

blocking agents (eg BSA or herring or salmon sperm carrier DNA), detergents (eg SDS), chelating agents (eg EDTA), Ficoll, PVP and the like. When using nylon membranes, in particular, a further preferred non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5 M NaHfePO ^ 7% SDS at approximately 65 degrees C, followed by one or more washes in 0.02 M NaH2PO4.1% SDS at 65 degrees C, see, for example, Church and Gilbert (1984) Proc. Nati. Acad. Sci. USA. 81: 1991-1995 (or alternatively 0.2 times SSC, 1% SDS).

Preferably, an isolated nucleic acid molecule of the invention hybridizes under stringent conditions to the sequence of secs. with nos. ident.:1, or 3 or corresponds to a nucleic acid molecule of natural origin. As used herein, a "naturally occurring nucleic acid molecule" refers to an RNA or DNA molecule that has a nucleotide sequence that occurs in nature (ie, encodes a natural polypeptide).

In addition to the naturally occurring allelic variants of the PDL3 or VISTA sequences that may exist in the population, the skilled technician will further appreciate that changes can be introduced by mutation in the nucleotide sequences of secs. with nos. of ident.:1 or 3, thereby leading to changes in the amino acid sequence of the encoded PD-L3 or VISTA polypeptides, without altering the functional capacity of the PD-L3 or VISTA polypeptides. For example, nucleotide substitutions that lead to amino acid substitutions at non-essential amino acid residues can be generated in the sequence of secs. with nos. Ident.:1, or 3. A nonessential amino acid residue is a residue that can be altered from the wild-type PD-L3 or VISTA sequence (eg, the sequence of Seqs. Ident .: 2, 4 or 5) without altering the biological activity, whereas an essential amino acid residue is required for biological activity. For example, the amino acid residues that are conserved among the PD-L3 or VISTA polypeptides of the present invention, eg, those present in an extracellular domain, are predicted not to be particularly susceptible to alteration. In addition,

IMPI

INSTITUTO MEXICANO Dl LA property of additional amino acid residues that are conserved among the 'poípeptide ^ of

<img file="MX342017B_D0074.tif" />

PD-L3 or VISTA of the present invention and other members of the PD L3 or VISTA family are likely not susceptible to alteration.

As a consequence, another aspect of the invention corresponds to nucleic acid molecules that encode PD-L3 or VISTA polypeptides that contain changes in amino acid residues that are not essential for activity. Said PD-L3 or VISTA polypeptides that differ in amino acid sequence from sec. with ID numbers: 2, 4, or 5 still retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence of at least about 71%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or more to sec. with nos. Identification number: 2, 4 or 5.

An isolated nucleic acid molecule encoding a PD-L3 or VISTA polypeptide identical to the polypeptide of secs. with nos. Ident.:2, 4, or 5 can be created by introducing one or more nucleotide substitutions, additions, or deletions in the nucleotide sequence of sec. with ID Nos .: 1 or 3 such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded polypeptide. Mutations can be introduced in secs. with nos. of ident. : 1 or 3 by standard techniques, such as site-directed mutagenesis and POR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted nonessential amino acid residues. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg asparagine, glutamine, serine, threonine, tyrosine , cysteine), nonpolar side chains (e.g. glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),

<img file="MX342017B_D0075.tif" />

Branched beta side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a PD-L3 or VISTA polypeptide is preferably substituted with another amino acid residue from the same side chain family. Alternatively, in another embodiment, random mutations can be introduced along all or part of a PD-L3 or VISTA coding sequence such as by saturation mutagenesis, and the resulting mutants can be screened for PD biological activity. -L3 or VISTA to identify mutants that retain activity. Following the mutagenesis of sec. with nos. Ident.:1, or 3, the encoded polypeptide can be recombinantly expressed and the activity of the polypeptide can be determined.

In a preferred embodiment, a PD-L3 or VISTA mutant polypeptide can be tested for the ability to bind to and / or modulate the activity of a natural PD-L3 or VISTA binding partner, to modulate intra- or intercellular signaling, modulate the activation of T lymphocytes, and / or modulate the immune response of an organism.

Still another aspect of the invention corresponds to isolated nucleic acid molecules encoding PD-L3 or VISTAPD-L3 or VISTA or VISTA fusion proteins. Said nucleic acid molecules, comprising at least a first nucleotide sequence encoding a PD-L3 or VISTAPD-L3 or VISTA or VISTA protein, polypeptide or peptide, operably linked to a second nucleotide sequence encoding a polypeptide or peptide protein not PD-L3 or VISTA, they can be prepared by standard recombinant DNA techniques.

In addition to nucleic acid molecules encoding the DP-L3 or VISTA polypeptides described above, another aspect of the invention corresponds to isolated nucleic acid molecules, which are antisense to it. An antisense nucleic acid comprises a nucleotide sequence that is complementary to a sense nucleic acid that encodes a polypeptide, for example, complementary to the coding strand of a double-stranded cDNA molecule or

<img file="MX342017B_D0076.tif" />

irliT! i U i O &lt;»'LA i • -.ai'? -j complementary to an mRNA sequence. CbtW &amp; dtts

<img file="MX342017B_D0077.tif" />

Antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to the complete PD-L3 or VISTA coding strand, or only to a part of it. In one embodiment, an antisense nucleic acid molecule is antisense to a coding region of the coding strand of a nucleotide sequence that encodes a PDL3 or

SIGHT. The term "coding region" refers to the region of the nucleotide sequence that comprises codons that are translated into amino acid residues.

In another embodiment, the nucleic acid molecule is antisense to a noncoding region of the coding strand of a nucleotide sequence that it encodes

PD-L. The term "non-coding region" refers to the 5 'and 3' sequences flanking the coding region that are not translated into amino acids (also referred to as 5 'and 3' untranslated regions). Given the coding strand sequences encoding human or mouse PD-L3 or VISTAPD-L3 or VISTA or VISTA of humans or mice described in the present description, the antisense nucleic acids of the invention can be designed according to the base pairing rules of Watson and Crick. The antisense nucleic acid molecule may be complementary to the entire coding region of the PD-L3 or VISTA mRNA, but more preferably it is an oligonucleotide that is antisense to only a part of the coding or non-coding region of the PD-L3 mRNA. or VIEW. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the PD-L3 or VISTA or VISTA mRNA.

An antisense oligonucleotide can be for example about 5, 10, 15,

20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acid molecule of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid molecule (for example, an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the acids. antisense and sense nucleics, for example, phosphorothioate derivatives and acridine substituted nucleotides can be used. The

<img file="MX342017B_D0078.tif" />

Examples of modified nucleotides that can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracilor * 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethyl-urine, 5-carboxymethylaminomethyl-urine, 5-carboxymethyl-urine carboxymethylaminomethyluracil, dihydrouracil, beta-Dgalactosylkeosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiuracil, beta-D-mannosylkeosine, 5'-methoxycarboxyrennethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine acid, 5-oxyacetic (v), wybutoxosine, pseudouracil, cheosin, 2-thiocytocin. 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino- 3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector in which a nucleic acid was subcloned in an antisense orientation (i.e., the RNA transcribed from the inserted nucleic acid will have an antisense orientation to a target nucleic acid. of interest described further in the next subsection).

The antisense nucleic acid molecules of the invention are typically administered to an individual or generated in situ such that they hybridize with or bind to cellular mRNA and / or genomic DNA encoding a PD-L3 or VISTAPD-L3 or VISTA or VISTA polypeptide to thereby inhibiting the expression of the polypeptide for example, by inhibiting transcription and / or translation. Hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the greater than double groove. propeller. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection into a tissue site. Alternatively, the antisense nucleic acid molecules can be modified in selected target cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, for example

INSTITUTE .V.Ea'C / NO and

FROM THE ΡΚυ?! Ί · Γ. ··.) ·> 5

<img file="MX342017B_D0079.tif" />

Binding antisense nucleic acid to peptides or antibodies that cell surface or antigens. Antisense nucleic acid molecules can also be delivered to cells using vectors described in the present invention. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs are preferred in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol promoter.

In yet another embodiment, the antisense nucleic acid molecule of the invention is an alpha anomeric nucleic acid molecule. An anomeric alpha nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15: 6625-6641 ). The antisense nucleic acid molecule may also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15: 6 13 1-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987) FEBS Lett. 2 15: 327-330).

In yet another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as mRNA, to which they have a complementary region. Thus, ribozymes (eg, hammerhead ribozymes (described in Haseloff and Gerlach (1988) Nature 334: 585-591)) can be used to catalytically cleave PD-L3 or VISTA mRNA transcripts to thereby inhibit translation of the PD-L3 or VISTA or VISTA mRNA. A ribozyme having specificity for a nucleic acid encoding PDL3 or VISTA can be designed based on the nucleotide sequence of a PD-L3 or VISTA cDNA described in the present disclosure (i.e., secs. with nos. Identification number: 1 or 3). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence that is cleaved in an mRNA encoding PD-L3 or VISTAPD-L3 or VIEW or VIEW. See, for example,

Cech et al., US Patent No. 4,987,071 and Cécti et al., US Patent No. 5,116,742. Alternatively, aLARNm do PD L3 or- VISTAIt can be used to select a catalytic RNA having specific ribonuclease activity from a group of RNA molecules. See, for example, Bartel, D. and Szostak, JW (1993) Science 261: 1411-1418.

Alternatively, expression of the PD-L3 or VISTA gene can be inhibited by targeting the complementary nucleotide sequences to the PD-L3 or VISTA regulatory region (eg, the PD-L3 or VISTA promoter and / or enhancers; to form triple helical structures that prevent transcription of the PDL3 gene in target cells See generally Helene, C (1991) Anticancer Drug Des 6 (6): 569-84, Helene, C. et al. (1992) Ann. NY Acad. Sci. 660: 27-36, and Maher, LJ (1992) Bioessays 14 (12): 807-15.

In yet another embodiment, the PD-L3 or VISTA nucleic acid molecules of the present invention can be modified at the base portion, sugar portion, or phosphate backbone, to improve, for example, stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup, B. and Nielsen, PE (1996) Bioorg. Med. Chem. 4 (1): 5-23) . As used herein, the terms peptide nucleic acids or PNAs refer to nucleic acids mimetics, for example DNA mimetics, in which the phosphate deoxyribose backbone is replaced by a pseudopeptide backbone and only the four nucleobases are conserved. natural. The neutral backbone of PNAs was shown to account for specific hybridization with DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup and Nielsen (1996) above and PerryO'Keefe et al. (1996) Proc Nati. Acad. Sci. USA. 93: 14670-675.

PNAs from PD-L3 or VISTA nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antigens or antisense agents for sequence-specific modulation of gene expression by, for example, inducing the

<img file="MX342017B_D0080.tif" />

arresting transcription or translation or inhibiting replication. PNAs from PD-L3 or VISTA nucleic acid molecules can also be used in the analysis of single base-paired mutations in a gene (eg, by PNA-directed PCR blocking); as "artificial restriction enzymes" when used in combination with other enzymes (eg, S1 nucleases (Hyrup and Nielsen (1996) above)); or as probes or primers for DNA sequencing or hybridization (Hyrup and Nielsen (1996) above; Perry-O'Keefe et al. (1996) above).

In another embodiment, PD-L3 or VISTA PNAs can be modified (for example, to improve their stability or cellular uptake), by attaching lipophilic or other helper groups to PNAs, by forming PNA-DNA chimeras, or by using of liposomes or other drug delivery techniques known in the art. For example, PNA-DNA chimeras can be generated from PDL-3 or VISTA nucleic acid molecules that can combine the advantageous properties of PNA and DNA. Such chimeras allow the recognition of DNA enzymes (eg, RNase H and DNA polymerases), to interact with the DNA part while the PNA part could provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of links between nucleobases, and orientation (Hyrup and Nielsen (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup and Nielsen (1996) above and Finn PJ et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA strand can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, for example, 5- (4-methoxytrityl) amino-S'-deoxy-thymidine phosphoramidite, can be used as a bridge between PNA and the 5 'end of DNA (Mag, M. et al. (1989) Nucleic Acids Res. 17: 5973-88). The PNA monomers are then gradually coupled to produce a chimeric molecule with a 5 'PNA segment and a 3' DNA segment (Finn PJ et al. (1996) above). Alternatively, the molecules

IΜ ΡI

MEXICAN INIT1TUTO θ ^THE|··/;

chimeric can be synthesized with a 5 'segment of DNA and a 3' segment of

PNA (Peterser, KH et al. (1975) Bioorganic Med. Cherrn ± ett5H449M4424) r

In other embodiments, the oligonucleotide may include other attached groups such as peptides (for example, to target host cell receptors in vivo), or agents that facilitate transport across the cell membrane (see, for example, Letsinger et al. (1989) Proc. Nati. Acad. Sci. USA 86: 65536556; Lemaitre et al. (1987) Proc Nati. Acad. Sci. USA 84: 648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier ( see, for example, PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization-trigger cleavage agents (See, for example, Krol et al. (1988) Biotechniques 6: 958-976) or intercalating agents (See, for example, Zon (1988) Pharrn. Res 5: 539-549). For this purpose, the oligonucleotide can be conjugated to another molecule (eg, a peptide, hybridization-triggering cross-linking agent, transport agent, or hybridization-triggering cleavage agent).

Alternatively, the expression characteristics of an endogenous PD-L3 or VISTA gene within a cell line or microorganism can be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element it is operably linked to the endogenous PD-L3 or VISTA gene. For example, an endogenous PD-L3 VISTA gene that is normally transcriptionally silent, that is, a PD-L3 or VISTA gene that is not normally expressed, or is expressed only at very low levels in a cell line or microorganism, is it can activate by inserting a regulatory element that is capable of promoting the expression of a gene product normally expressed in that cell line or microorganism. Alternatively, a transcriptionally silent endogenous PD-L3 or VISTA gene can be activated by insertion of a promiscuous regulatory element that works across cell types.

A heterologous regulatory element can be inserted into a stable cell line or cloned microorganism, such that it is operably linked to an endogenous PDL3 or VISTA gene, using techniques, such as homologous recombination.

IMPI f '' 7

INSTITUTO MEXICANO 7,; '-: ..-— - J i DE LA PROPERTY Directed, which are well known by persons with technical experience, are described, for example, in Chappel. United States patent tlniring nfim_r 5,272,071;

PCT Publication No. WO 91/06667, published May 16, 1991.

II. Isolated PD-L3 or VISTA polypeptides and anti-PD-L3 or VISTA antibodies

One aspect of the invention corresponds to isolated PD-L3 or VISTA polypeptides, and biologically active parts thereof, as well as polypeptide fragments suitable for use as immunogens to generate anti-PD-L3 or VISTA antibodies. In one embodiment, native PD-L3 or VISTA polypeptides can be isolated from cell or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, PD-L3 or VISTA polypeptides are produced by recombinant DNA techniques. As an alternative to recombinant expression, a PD-L3 or VISTA protein or polypeptide can be chemically synthesized using standard peptide synthesis techniques.

An isolated or purified polypeptide or biologically active part thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the PD-L3 or VISTA polypeptide is derived, or substantially free of chemical or other precursors. chemicals when chemically synthesized. The term "substantially free of cellular material" includes PD-L3 or VISTA polypeptide preparations in which the polypeptide is separated from the cellular components of the cells from which the cells are isolated or produced recombinantly. In one embodiment, the term "substantially free of cellular material" includes PD-L3 or VISTA polypeptide preparations that have less than about 30% (by dry weight) non-PD-L3 or VISTA protein (also referred to herein as a contaminating protein), more preferably less than about 20% non-PD-L3 or VISTA protein, even more preferably less than about 10% non-PD-L3 or VISTA protein, and most preferably less than about 5% non-PD-L3 or VISTA protein. When the PD-L3 or VISTA polypeptide or biologically active part thereof is produced recombinantly, it is preferably also substantially free of Mexican medium 'ROPiEDaD!

<img file="MX342017B_D0081.tif" />

IVj

INSTITUTE

Culture OE LA, that is, the culture medium represents less than 20%, more preferably less than about ~ 1Q ~% ^ y_CQn. most preferably less than about 5% by volume of the protein preparation.

The term "substantially free of chemical precursors or other chemicals" includes PD-L3 or VISTA polypeptide preparations in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide. In one embodiment, the term "substantially free of chemical precursors or other chemicals" includes PD-L3 or VISTA polypeptide preparations that have less than about 30% (by dry weight) chemical precursors or non-PD-L3 or VISTA chemicals. , more preferably less than about 20% chemical precursors or non-PD-L3 or VISTA chemicals, even more preferably less than about 10% non-PD-L3 or VISTA chemical precursors or chemicals, and most preferably less than about 5% non-PD-L3 or VISTA chemical precursors or chemicals.

As used herein, a biologically active part of a PD-L3 or VISTA polypeptide includes a fragment of a PDL3 OR VISTA polypeptide that participates in an interaction between a PD-L3 or VISTA molecule and a non-PD molecule. -L3 or VISTA, eg a natural PD-L3 or VISTA ligand. Biologically active parts of a PD-L3 or VISTA polypeptide include peptides that comprise amino acid sequences sufficiently identical to, or derived from, the amino acid sequence of the PD-L3 or VISTA polypeptide, for example, the amino acid sequence that shown in sec with nos. Identifier: 2, 4, or 5 that include fewer amino acids than full length PD-L3 or VISTA polypeptides, and exhibit at least one PD-L3 or VISTA polypeptide activity. Typically, biologically active portions comprise a domain or motif with at least one PD-L3 or VISTA polypeptide activity, e.g., modulating (suppressing) the proliferative responses of CD4 T cells to anti-CD3, suppressing the proliferative response of cells ig; ae.-

<img file="MX342017B_D0082.tif" />

Zion

IMPIf

INSTITUTE Mf.X'CAM))

OF THE PROPERTY analogous T CD4 in an antigen-specific way, specific cytokine effects, and others. A biologically active part of a PD-L3 or VISTA polypeptide can be a polypeptide that is, for example, 25, 50,

75, 100, 125, 150, 175, 200, 225 or more amino acids in length. Biologically active parts of a PD-L3 or VISTA polypeptide can be used as targets to develop agents that modulate an activity mediated by PD-L3 or

VISTA, for example, the activation of immune cells.

In one embodiment, a biologically active part of a PDL3 or VISTA polypeptide comprises at least a part of an extracellular domain. It should be understood that a preferred biologically active part of a PDL3 or VISTA polypeptide of the present invention may contain at least a part of an extracellular domain (eg, comprising an IgV), and one or more of the following domains: a domain signal peptide, a transmembrane domain, and a cytoplasmic domain. In addition, other biologically active parts, in which other regions of the polypeptide are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native PD-L3 or VISTA polypeptide.

In a preferred embodiment, the PD-L3 or VISTA polypeptide has an amino acid sequence that is shown in secs. with nos. Identification Number: 2, 4, or 5. In other embodiments, the PD-L3 or VISTA polypeptide is substantially identical to sec. with nos. ident: 2, 4, or 5, and preserves the functional activity of the sec. polypeptide. with nos. Ident .: 2, 4 or 5, as described above which still differs in amino acid sequence due to natural allelic variation or mutagenesis.

The polypeptide and nucleic acid sequences of the present invention can further be used as a look-up sequence to search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10. Nucleotide BLAST searches can be performed with the NBLAST Program, score = 100, word length = 12 for

IJMÍ F 1 obtain nucleotide sequences homologous to moleculesxde> acid nüCÍejcoqe ° DE LA i^:í <C'r.

PD-L3 or VISTA of the invention. BLAST searches for proteins can be performed with the XBLAST program, score = 100, wordlength ^ to obtain amino acid sequences homologous to PDL3 or VISTA polypeptide molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. See the Internet website of the National Center for Biotechnology Information.

The invention also provides PD-L3 or VISTA chimeric or fusion proteins. As used herein, a PD-L3 or VISTA chimeric protein or fusion protein comprises a PD-L3 or VISTA polypeptide operably linked to a non-PD-L3 or VISTA polypeptide. A PD-L3 or VISTA polypeptide refers to a polypeptide that has an amino acid sequence corresponding to a PD-L3 or VISTA molecule, while a non-PDL-3 or VISTA polypeptide refers to a polypeptide that has a sequence of amino acids corresponding to a polypeptide that is not substantially homologous to that of the PDL-3 or VISTA polypeptide, for example, a polypeptide that is different from the PD-L3 or VISTA polypeptide and is derived from the same or a different organism. Within a PD-L3 or VISTA fusion protein, the PD-L3 or VISTA polypeptide may correspond to all or a part of a PD-L3 or VISTA polypeptide. In a preferred embodiment, a PD-L3 or VISTA fusion protein comprises at least one biologically active part of a PD-L3 or VISTA polypeptide. In another preferred embodiment, a PD-L3 or VISTA fusion protein comprises at least two domains of a PD-L3 or VISTA polypeptide. Within the fusion protein, the term "operably linked" indicates that the PD-L3 or VISTA polypeptide and the non-PDL3 or VISTA polypeptide are fused to each other in frame. Non-PD-L3 or VISTA polypeptide can be fused to the N-terminal or C-terminal end of the PD-L3 polypeptide or

VISTA and corresponds to a portion that alters the solubWi ^ o ^ injda ^ of uOion, stability or valence of the PD-L3 or VISTA polypeptide.

For example, in one embodiment, the fusion protein is a GST-PD-L3 or VISTA fusion protein in which the PD-L3 or VISTA sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant PD-L3 or VISTA. In another embodiment, the fusion protein is a PD-L3 or VISTA polypeptide that contains a heterologous signal sequence at its N-terminus. In certain host cells (eg, mammalian host cells), the expression and / or secretion of PD-L3 or VISTA can be increased by the use of a heterologous signal sequence. In a preferred embodiment, the fusion protein is an Ig-PD-L3 or VISTA fusion protein in which the PD-L3 or VISTA sequences are fused to a part of an Ig molecule. The Ig part of the fusion protein may include an immunoglobulin constant region, for example, a C domain of gamma 1 or a C domain of human gamma 4 (for example, the hinge, CH2, and CH3 regions of human gamma 1 IgC or gamma 4 human IgC (see, eg, Capon et al., US Patent Nos. 5,116,964; 5,580,756; 5,844,095, and the like, which are incorporated herein by reference). A resulting fusion protein may have altered the solubility, binding affinity, stability, and / or valency of PD-L3 or VISTA (ie, the number of binding sites per molecule) and may increase the efficiency of protein purification.

In particular, preferred Ig fusion proteins with PD-L3 or VISTA include a portion of the extracellular domain of PD-L3 or VISTA coupled to an immunoglobulin constant region (eg, the Fe region). The immunoglobulin constant region may contain genetic modifications that reduce or eliminate the effector activity inherent in the immunoglobulin structure. For example, DNA encoding an extracellular portion of a PD-L3 or VISTA polypeptide can be linked to DNA encoding the hinge, CH2, and CH3 regions of modified human IgG gamma 1 and / or IgG gamma 4 by site directed mutagenesis. , for example, as taught in WO 97/28267. The PD-L3 or VISTA fusion proteins of the invention can be incorporated into

<img file="MX342017B_D0083.tif" />

pharmaceutical compositions and administered in vivo to an individual. PD-L3 or VISTA fusion proteins can be used to affect the biócfispóllfBilldyil of a PD-L3 or VISTA binding partner. The use of PD-L3 or VISTA fusion proteins may be therapeutically useful for the treatment of conditions or disorders that would benefit from modulation of the immune response. Additionally, the PD-L3 or VISTA fusion proteins of the invention can be used as immunogens to produce anti-PD-L3 or VISTA antibodies in an individual, to purify PD-L3 or VISTA binding proteins, and in assays for screens to identify molecules that inhibit the interaction of PD-L3 or VISTA with its natural binding partner.

Preferably, a PD-L3 or VISTA fusion or chimeric protein of the invention is produced by standard recombinant DNA techniques.

The present invention also pertains to PD-L3 or VISTA polypeptide variants that function either as PD-L3 or VISTA agonists (mimetics) or as PD-L3 or VISTA antagonists. PD-L3 or VISTA polypeptide variants can be generated by mutagenesis, eg, discrete site mutation or truncation of a PD-L3 or VISTA polypeptide. An agonist of PD-L3 or VISTA polypeptides can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a PD-L3 or VISTA polypeptide. An antagonist of a PD-L3 or VISTA polypeptide may inhibit one or more of the activities of the naturally occurring form of PD-L3 or VISTA polypeptide, for example, by competitively modulating a PD-L3 or VISTA-mediated activity of a PD-L3 or VISTA polypeptide. Thus, specific biological effects can be obtained by treatment with a limited function variant. In one embodiment, treating an individual with a variant that has a subset of the biological activities of the naturally occurring form of the polypeptide has fewer side effects in an individual relative to treatment with the naturally occurring form of the PD-polypeptide. L3 or VISTA.

In one embodiment, variants of a PD-L3 or VISTA polypeptide that function either as agonists (mimetics) of PD-L3 or VISTA or as

IMPI

INSTITUTO MEXICANO antagonists of PD-L3 or VISTA can be identified tam¡ ^ hdoÍífé ti

<img file="MX342017B_D0084.tif" />

combinatorial mutants, eg, truncation mutants of a PD-L3 or VISTA polypeptide for the agonist or antagonist activity of the PD-L3 or VISTA polypeptide. In one embodiment, a highly diverse library of PD-L3 or VISTA variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a highly diverse gene library. A highly diverse library of PD-L3 or VISTA variants can be produced, for example, by enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential PDL-3 sequences or

VISTA is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (eg, by phage display) that contain the set of PD-L3 or VISTA sequences in them. There are a variety of methods that can be used to produce libraries of potential PD-L3 or VISTA variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed on an automated DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. The use of a degenerate set of genes allows the provision, in a mixture, of all the sequences encoding the desired set of potential PD-L3 or VISTA sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (See, for example, Narang, SA (1983) Tetrahedron 39: 3; Itakura and others (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 11: 477).

In addition, libraries of fragments of a sequence encoding the PD-L3 or VISTA polypeptide can be used to generate a highly diverse population of PD-L3 or VISTA fragments for the subsequent screening and selection of variants of a PD-L3 polypeptide. L3 or VISTA. In one embodiment, a library of coding sequence fragments can be generated by treating a double-stranded PCR fragment of a PD-L3 or VISTA coding sequence with a nuclease under conditions where it is produced by cutting only about once per molecule, denaturing double-stranded DNA, renaturing

1JV1 rif

INSTI TUTO MEXiCZiNO \ DE LA PROPERTY

Ut LA t'KL / AlLi.At / DNA to form double-stranded DNA that can be^ESTUARY1ncratr — sense / antisense pairs of different cut products, removing parts-of ..simple ..

strand from the reformed duplexes by treatment with S1 nuclease, and ligating the resulting library of fragments into an expression vector. By this method, an expression library can be derived that encodes N-terminal, C-terminal and internal fragments of various sizes of the PD-L3 polypeptide or

SIGHT.

Various procedures are known in the art for screening gene products from combinatorial libraries produced by point mutations or truncation, and for screening cDNA \ libraries for gene products that have a selected property. Such techniques are adaptable for rapid screening of gene libraries generated by combinatorial mutagenesis of PD-L3 or VISTA polypeptides. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming the appropriate cells with the resulting vector library, and expressing the combinatorial genes. under conditions in which the detection of a desired activity facilitates the isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that increases the frequency of functional mutants in libraries, can be used in combination with screening assays to identify PD-L3 or VISTA variants (Arkin and Youvan (1992) Proc Nati. Acad. Sci. USA 89: 7811-7815; Delagrave et al. (1993) Protein Eng. 6 (3): 327-331).

In addition to PD-L3 or VISTA polypeptides consisting only of naturally occurring amino acids, PD-L3 or VISTA mimetic peptides are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are called mimetic or peptidomimetic peptides (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985) TINS ​​p.392; and Evans et al. (1987) J. Med. Chem. 30: 1229, which are incorporated herein by reference) and are developed by rCair · • fwi.lH.í ♦ iv- «

IMFIi

INSTITUTE MJX.'CANU usually with the help of molecular modeling compufáKzáWÉ¡ALos - mimetic peptides that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect.

Generally, mimetic peptides are structurally similar to a paradigm polypeptide (i.e., a polypeptide having biological activity or pharmacological activity), such as human or mouse PD-L3 or VISTA, but have one or more optionally substituted peptide linkages. by a link selected from the group consisting of: -CH2NH -, - CH2S -, - CH2-CH2 -, - CH.dbd.CH- (cis and trans), - COCH2 -, - CH (OH) CH2--, and --CH2SO-- , by methods known in the art and further described in the following references: Spatola,

AF in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins Weinstein, B., editor, Marcel Dekker, New York, p. 267 (1983); Spatola. AF, Vega Data (March 1983), Vol. 1, Number 3, Peptide Backbone Modifications; Morley, JS (1980) Trends. Pharm. Sci. Pp. 463-468; Hudson, D. et al. (1979) Int. J. Pept. Prot. Res. 14: 177-185 (-CH2NH-, CH2CH2-); Spatola, AF et al. (1986) Life. Sci. 38: 1243-1249 (-CH2-S); Hann, Μ. M. (1982) J. Chem. SoC

Perkin.Trans. I 307-314 (--CH-CH-, cis and trans); Almquist, RG and others (1980) J. Med. Chem. 23: 1392-1398 (--COCH2-); Jennings-White, C et al. (1982) Tetrahedron Lett. 23: 2533 (--COCH2--); Szelke. M. et al., European Patent Application No. EP 45665 (1982) CA: 97: 39405 (-CH (OH) CH2-); Holladay, MW et al. (1983) Tetrahedron. Lett. 24: 4401-4404 (-C (OH) CH2-); and Hruby, VJ (1982) Life Sci. 31: 189-199 (--CH2-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide bond is -CH2NH-. Such peptide mimetics can have significant advantages over polypeptide modalities, including for example: more economical production, higher chemical stability, better pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (eg, broad spectrum of biological activities), reduced antigenicity, and others. The labeling of the mimetic peptides usually involves the covalent attachment of one or more markers, directly or through a spacer (for example, an amide group) to position (s) that do not interfere with the mimetic peptide being predicted by activity data-quantitative structure and 89 "3-"

<img file="MX342017B_D0085.tif" />

molecular modeling. Said positions in general are positions that do not form direct contact with the macromolecule (s) an ^ qües ^ 'One ~ ehpepttdemimetic to produce the therapeutic effect. Derivatization (eg, labeling) of the peptide mimetic should not substantially interfere with the desired biological or pharmacological activity of the peptide mimetic.

Systematic substitution of one or more amino acids from an amino acid sequence of PD-L3 or VISTA with a D-amino acid of the same type (eg, D-lysine instead of L-lysine) can be used to generate more stable peptides. In addition, constrained peptides that comprise an amino acid sequence of PD-L3 or VISTA or a substantially identical sequence variation can be generated by methods known in the art (Rizo and Gierasch (1992) Annu. Rev. Biochem. 61: 387, incorporated herein by reference); for example, by adding internal cysteine ​​residues capable of forming intramolecular disulfide bridges that cyclize the peptide. The PD-L3 or VISTA polypeptide amino acid sequences identified in the present disclosure will enable those of skill in the art to produce polypeptides corresponding to the PD-L3 or VISTA peptide sequences and sequence variants thereof. Such polypeptides can be produced in prokaryotic or eukaryotic host cells by expressing polynucleotides encoding a PD-L3 or VISTA peptide sequence, often as part of a larger polypeptide. Alternatively, such peptides can be synthesized by chemical methods. Methods for the expression of heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art. Certain amino-terminal and / or carboxy-terminal modifications and / or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: improved stability, increased potency and / or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others. The peptides can be used therapeutically to treat disease, for example, by altering co-stimulation in a patient.

IM FI ¢ 57-5¾

INSTITUTO MSXICU-IO JA de LA Γλο, -ι sdau> .réreí

An isolated PD-L3 or VISTA polypeptide, or a part or fraⁱgmentd 'ofⁱThis can be used as an immunogen to generate antimprpnR q and iq bind PD-L3 or

VISTA using standard techniques for the preparation of polyclonal and monoclonal antibodies. A complete polypeptide of PD-L3 or VISTA can be used or, alternatively, the invention provides antigenic peptide fragments of PDL3 or VISTA for use as immunogens. In one embodiment, a PDL3 or VISTA antigenic peptide comprises at least 8 amino acid residues from the amino acid sequence shown in secs. with nos. Ident .: 2,4 or 5 and includes a PD-L3 or VISTA epitope such that an antibody raised against the peptide forms a specific immune complex with the PD-L3 or VISTA polypeptide.

Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Preferred epitopes that are included by the antigenic peptide are regions of PD-L3 or VISTA that are found in the extracellular domain of the polypeptide, eg, hydrophilic regions, as well as regions with high antigenicity.

A PD-L3 or VISTA immunogen is typically used to prepare antibodies by immunizing a suitable individual (eg, rabbit, goat, mouse, or other mammal) with the immunogen. An appropriate immunogenic preparation may contain, for example, the recombinantly expressed PD-L3 or VISTA polypeptide or a chemically synthesized PD-L3 or VISTA polypeptide. The preparation may further include an adjuvant, such as Freund's Complete or Incomplete Adjuvant, or similar immunostimulatory agent. Immunization of a suitable individual with an immunogenic preparation of PD-L3 or VISTA induces a polyclonal anti-PD-L3 or VISTA antibody response.

As a consequence, another aspect of the invention corresponds to anti-PD-L3 or VISTA antibodies. The term "antibody" as used herein, refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, that is, molecules that contain an antigen-binding site that specifically binds

INSTITUTO MEXICANO VT '. ·'. · - &lt;^xi

DE LA PAOMEDAO · * '' i

INDUSTRIAL * '&lt;., · - *' (immunoreacts with) an antigen, such as a PD-L3 or VISTA. Examples of immunologically active parts of the immunoglobutin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind to PD-L3 or VISTA molecules. The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a population of antibody molecules that contain only one species of antigen-binding site capable of immunoreacting with a particular epitope of PD-L3. or VIEW. A monoclonal antibody composition thus typically exhibits a unique binding affinity for a particular PD-L3 or VISTA polypeptide with which it immunoreacts.

Polyclonal anti-PD-L3 or VISTA antibodies can be prepared as described above by immunizing a suitable individual with a PD-L3 or VISTA immunogen, eg, a PD-L3 or VISTA-Ig fusion protein. The anti-PD-L3 or VISTA antibody titer in the immunized individual can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using immobilized PD-L3 or VISTA. If desired, antibody molecules directed against PD-L3 or VISTA can be isolated from the mammal (eg, from blood) and further purified by well-known techniques, such as protein A chromatography to obtain the fraction IgG. At an appropriate time after immunization, for example, when anti-PD-L3 or VISTA antibody titers are higher, antibody-producing cells can be obtained from the individual and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256: 495-497 (see also Brown et al. (1981) J. Immunol. 127: 539-46; Brown et al. (1980) J. Biol. Chem. 255: 4980-83; Yeh and others (1976) Proc Nati. Acad. Sci. USA 76: 2927-31, and Yeh et al. (1982) Int. J. Cancer 29: 269-75), the latest human B-cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4: 72), the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies and

INSTITUTE MsV

OH PJ.C.-,>

_1N............... 'i

Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trióWál techniques The technology for producing monoclonal antibody hybridomas is well-known (see, generally Kenneth RH in Monoclonal Antibodies: A New Dimension In Biological Analyzes, Plenum Publishing Corp., New York, NY (1980); Lemer,

EA (1981) Yale J. Biol. Med. 54: 387-402; Gefter, ML et al. (1977) Somatic Cell Genet. 3: 231-36). In summary, an immortal cell line (typically a myeloma) is fused with lymphocytes (typically splenocytes) from a mammal immunized with a PD-L3 or VISTA immunogen as described above, and the culture supernatants of the resulting hybridoma cells are They screen to identify a hybridoma that produces a monoclonal antibody that binds to PD-L3 or VISTA. Any of the many well-known protocols used to fuse lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-PD-L3 or VISTA monoclonal antibody (see, for example, Galfre, G. et al. (1977) Nature 266 : 55052; Gefter and others (1977) above; Lemer (1981) above; and Kenneth (1980) above). Furthermore, the person skilled in the art will appreciate that there are many variations of such methods that could also be useful. Typically, the immortal cell line (eg, a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be prepared by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin, and thymidine (HAT medium). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, for example, the myeloma lines P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Ag14. These myeloma lines are available from the ATCC. Typically, HAT-sensitive mouse myeloma cells are fused with mouse splenocytes using polyethylene glycol (PEG). The hybridoma cells resulting from the fusion are then selected using HAT medium, which kills the unproductive and unfused fused myeloma cells (the unfused splenocytes die after several days because they do not transform). Hybridoma cells that produce 93

T Μ Ρ ί monoclonal antibody of the invention are detected by sieving lé ^^ t ^ gtfeinté ^ de · j 'of the ΓΚΟΡ hybridoma culture for antibodies that bind to PD-L3^l^^JVtSTA, for example, using a standard ELISA assay. ____________—

Specific methods for producing antibodies that bind to PD-L3 or VISTA can be carried out using methods known in the art and as described in the Examples. As an alternative to preparing hybridomas that secrete monoclonal antibodies, an anti-PD-L3 monoclonal antibody can be identified and isolated by screening a combinatorial library of recombinant immunoglobulins (eg, a library of phage display antibodies) with PD-L3 or VISTA. to thereby isolate the members of the immunoglobulin library that bind to PD-L3 or VISTA. Kits for generating and screening phage display libraries are commercially available.

As noted, these antibodies are screened to identify those that bind to specific PD-L3 or VISTA epitopes, for example in the IgV domain or other specific domains and / or to select antibodies that have high affinity and avidity for the PD protein. -L3 or VISTA. Additionally, these antibodies are screened to identify those that modulate the specific functions and effects of PD-L3 or VISTA on immunity and immune cells in vitro and in vivo. For example, in case of a particular anti-PD-L3 or VISTA antibody, tests can be carried out to determine the modulatory effect on the immune functions negatively regulated by PD-L3 or VISTA including the production of cytokines by the cells. CD4 + or CD8 + T, CD28 co-stimulation, CD4 + T cell proliferation, and the proliferation of naive and memory CD4 + T cells, and others. In a preferred embodiment, assays are performed to identify the therapeutic potential of anti-PD-L3 or VISTA antibodies in vitro while the presence of PD-L3 or VISTA-lg enhances suppression by PD-L3 or VISTA- Ig, these PD-L3 or VISTA antibodies act in vivo in the opposite way, that is, they are immunosuppressive. The invention includes anti-VISTA antibodies and their uses, which specifically bind to the extracellular domain of 136 amino acids, for example antibodies to 1-50, 50-100, 100-136 amino acids, that bind

MEXICAN ^Α₍£? Ρ^Ρί5θΛΠ. · '*. '' • Vi * AÍ '^ OUSTNMt K> «Mylw - | jp specifically to IgV, antibodies that specifically bind to the stem region, antibodies that specifically bind to the region of Tr ^ nTsmembcana ^ and, antibodies that specifically bind the region cytoplasmic. These specific regions are identified in the application.

Furthermore, recombinant anti-PD-L3 or VISTA antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human parts, which can be prepared using recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using the methods described in Robinson et al., International Application No. PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al., PCT International Publication No. WO 86/01533; Cabilly et al., US Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc Nati. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526; Sun et al. (1987) Proc Nati. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; Shaw et al. (1988) J. Nati. Cancer Inst. 80: 1553-1559; Morrison, SL (1985) Science 229: 1202-1207; Oi et al. (1986) Biotechniques 4: 214; Winter, US Patent No. 5,225,539; Jones et al. (1986) Nature 321: 552-525; Verhoeyen et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141: 40534060.

An anti-PD-L3 or VISTA antibody (eg, monoclonal antibody) can be used to isolate PD-L3 or VISTA by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-PD-L3 or VISTA antibody can facilitate the purification of natural PD-L3 or VISTA from cells and recombinantly produced PDL3 or VISTA expressed in host cells. Additionally, an anti-PD-L3 or VISTA antibody can be used to detect PD-L3 or VISTA polypeptide (for example, in a cell lysate or supernatant

IMPI 'ST'TUTO MEXICANO DE La IKOEIEDAO INDUSTRIAL

<img file="MX342017B_D0086.tif" />

cell) to assess the abundance and expression pattern of the PD-L3 or VISTA polypeptide. Anti-PD-L3 or VISTA antibodies can be used to diagnose polypeptide levels in tissue as part of a clinical trial procedure, to determine, for example, the efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically binding) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone fluorescein, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; An example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 1125, 1131, S35, or H3.

III. Recombinant expression vectors and host cells

Another aspect of the invention corresponds to vectors, preferably expression vectors, that contain a nucleic acid molecule that encodes a PD-L3 or VISTA polypeptide (or a part of it). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it was linked. One type of vector is a plasmid, which refers to a circular loop of double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, where the additional DNA segments in the viral genome can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) integrate into the genome of a host cell after introduction into the host cell, and

HtJ Í'll · Μ ι.ι thus replicate together with the host genome

INSTITUTO MEXICANO vectors are capable of directing gene expression

<img file="MX342017B_D0087.tif" />

operationally. Such vectors are referred to in the present invention as expression vectors. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, plasmid and vector can be used interchangeably since plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses), that serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which are operably linked to the nucleic acid sequence being expressed. Within a recombinant expression vector, operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence (s) in a manner that allows expression of the nucleotide sequence (e.g., in a transcription / translation system in vitro or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) Methods Enzymol. 185: 3-7. Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression of the protein

INSTITUTO MEXICA.'IO desired, and the like. The expression vectors of the invention ihiroáüS ^ in host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described in the present invention (for example, PD-L3 or VISTA polypeptides, mutant forms of PD-L3 or VISTA polypeptides, fusion proteins and the like).

The recombinant expression vectors of the invention can be designed for the expression of PD-L3 VISTA polypeptides in prokaryotic or eukaryotic cells. For example, PD-L3 VISTA polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel (1990) above. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using regulatory sequences from the T7 promoter and T7 polymerase. Purified fusion proteins can be used in PD-L3 or VISTA activity assays (for example, direct assays or competitive assays are described in detail below), or to generate antibodies specific to PD-L3 or VISTA polypeptides, for example. In another embodiment, the PD-L3 or VISTA expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecI (Baldari et al. (1987) EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933943), pJRY88 (Schultz and others (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, California), and picZ (Invitrogen Corporation, San Diego, California). Alternatively, PD-L3 or VISTA polypeptides can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides in cultured insect cells (eg, Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-

39). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When using '- · (! C. I ··

........ ... '' $ In mammalian cells, the control functions of the expression vector are often provided by regulatory elements9-w^:ateS; - For-example_T-the.

Commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook, J. et al., Molecular Cloning: A Laboratory Handbook. 2nd edition, Coid Spring Harbor Laboratory, Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY 1989.

In another embodiment, the recombinant mammalian expression vector is capable of directing the expression of the nucleic acid preferentially in a particular cell type (eg, tissue-specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Caiame and Eaton (1988) Adv. Immunol 43: 235-275). In particular, the T cell receptor promoters (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Baneji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (eg, the neurofilament promoter; Byrne and Ruddle (1989) Proc Nati Acad Sci USA. 86: 54735477), pancreatic-specific promoters (Edlund et al. (1985), Science 230: 912-916), and mammary gland-specific promoters (eg, the whey promoter; U.S. Patent No. 4,873,316 and publication of European application No. 264,166). Developing regulated promoters also include, for example, the murine hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev .. 3: 537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a way that allows for

IMPI

INSTITUTE XK &amp; t: o.

expression (by transcription of the DNA molecule) of a² molecule d &amp; ARt4 &lt;which is antisense to PD-L3 or VISTA mRNA. Regulatory sequences operably linked to a cloned nucleic acid molecule in the antisense orientation can be selected to direct the continuous expression of the antisense RNA molecule in a variety of cell types, eg, viral promoters and / or enhancers, or can be they can select regulatory sequences to direct constitutive, tissue-specific, or cell-type-specific expression of the antisense RNA. The antisense expression vector can be in the form of a plasmid, phagemid, or recombinant attenuated virus in which the antisense nucleic acids are produced under the control of a high-efficiency regulatory region, the activity of which can be determined by the type of cell in the one where the vector is introduced. For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et al., Antlsense RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, volume 1 (1) 1986.

Another aspect of the invention corresponds to host cells in which a PD-L3 or VISTA nucleic acid molecule of the invention is introduced, for example, a PD-L3 or VISTA nucleic acid molecule within a vector of recombinant expression or a PDL3 or VISTA nucleic acid molecule that contains sequences that allow it to recombine homologously at a specific site in the host cell genome. The terms host cell and recombinant host cell are used interchangeably in the present description. Such terms are understood to refer not only to the particular target cell but to the progeny or potential progeny of said cell. Because certain modifications can occur in successive generations either by mutation or environmental influences, such progeny may, in fact, not be identical to the parent cell, but are still included within the scope of the term as used herein. . A host cell can be any prokaryotic or eukaryotic cell. Vector DNA can be introduced into prokaryotic or eukaryotic cells through conventional transformation or transfection techniques. As used in the present description,

100

ΙΜΡΙΟ>

INSTITUTO MÍX.ICA ·;? '. &lt;_ /:.:

OF THE PRC,? Lr ¿.. D '-.y ......

The terms transformation and transfection are intended to refer to a variety of techniques recognized in the art for introducing lipoid-JiucleiGG-fernaeum (eg, DNA) into a host cell, including co-precipitation with calcium phosphate or calcium chloride, transfection mediated by DEAE-dextran, lipofection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd edition, Coid Spring Harbor Laboratory, Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY, 1989), and other laboratory manuals. To identify and select for these integrants, a gene encoding a selectable marker (eg, antibiotic resistance) is generally introduced into host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin, and methotrexate. A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (ie, express) a PD-L3 or VISTA polypeptide. Accordingly, the invention further provides methods for producing a PD-L3 or VISTA polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a PD-L3 or VISTA polypeptide was introduced) in a suitable medium such that a PD-L3 or VISTA polypeptide is produced . In another embodiment, the method further comprises isolating a PD-L3 or VISTA polypeptide from the host cell or medium.

The host cells of the invention can be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which PD-L3 or VISTA coding sequences were introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous PD-L3 or VISTA sequences were introduced into their genome or homologous recombinant animals in which endogenous PD-L3 or VISTA sequences were altered. Said animals are useful to study the function and / or activity of a PD-L3 or VISTA and to identify and / or evaluate modulators of

101

INSTITUTO MEXICANO y..¿ - ·. _F._:.J

OF THE PROPERTY ·· ·>. ' • PD-L3 or VISTA activity. As used herein, a transgenic animal is a non-human animal, preferably a. mammalian nnn most preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is an exogenous DNA that is integrated into the genome of a cell from a transgenic animal that develops and remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types. or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous PD-L3 or VISTA gene was disrupted by homologous recombination between the endogenous gene. and an exogenous DNA molecule introduced into a cell of the animal, eg, an embryonic cell of the animal, prior to development of the animal. A transgenic animal of the invention can be created by introducing a nucleic acid encoding PD-L3 or VISTA into the male pronucleus of a fertilized oocyte, for example, by microinjection, retroviral infection, and allowing the oocyte to develop into a pseudopregnant female foster animal. . The PD-L3 or VISTA cDNA sequence of sec. with nos. Identification number: 1 or 4 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of a human PD-L3 or VISTA gene, such as a monkey or rat PD-L3 or VISTA gene, can be used as a transgene. Alternatively, a gene homologous to PD-L3 or VISTA, such as another member of the PD-L3 OR VISTA family, can be isolated based on hybridization to the PD-L3 OR VISTA cDNA sequences of sec. with nos. Ident .: 1 or 3 (further described above in subsection I) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence (s) can be operably linked to a PD-L3 or VISTA transgene to direct the expression of a PD-L3 or VISTA polypeptide in particular cells. Methods for generating transgenic animals by manipulating embryos and microinjection, particularly animals 102

<img file="MX342017B_D0088.tif" />

institute 'such as mice, have become conventional'⁷ describe, for example, in US Patent Nos. 4,736,866 and 4,870,009, both by Leder et al., US Patent No. 4,873,191 to Wagner et al. And in Hogan, B., Manipulating the Mouse Embryo, (Coid Spring Harbor Laboratory Press, Coid Spring Harbor, New York, 1986). Similar methods are used for the production of other transgenic animals. A founder transgenic animal can be identified based on the presence of a PD-L3 or VISTA transgene in its genome and / or expression of PD-L3 mRNA or

SEEN in tissues or cells of animals. A founder transgenic animal can then be used to breed additional animals that carry the transgene. In addition, transgenic animals that carry a transgene encoding a PD-L3 or VISTA polypeptide can be further crossed with other transgenic animals that carry other transgenes.

To create a homologous recombinant animal, a vector is prepared containing at least a portion of a PD-L3 or VISTA gene in which a deletion, addition, or substitution was introduced to thereby alter, eg, functionally disrupt, the PD-L3 or VISTA gene. The PD-L3 or VISTA gene can be a human or murine gene (for example, the cDNA of seq. IDs: 1 or 3)

In another embodiment, non-human transgenic animals can be produced containing selected systems that allow for regulated expression of the transgene. An example of such a system is the bacteriophage P1 recombinase cre / loxP system. For a description of the cre / loxP recombinase system, see, eg, Lakso et al. (1992) Proc Nati. Acad. Sci. USA. 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system from S. cerevisiae (O'Gorman et al. (1991) Science 251: 13511355. If a cre / loxP recombinase system is used to regulate the expression of the transgene, animals are required that contain transgenes encoding both the Ore recombinase and a selected polypeptide. Such animals can be provided by the construction of double transgenic animals, for example, coupling of two transgenic animals, one containing a

103

ΙΜΡΐΓ> 5λ '

MEXICAN INSTITUTE · '·· J

DELA PROPERTY - '··. ·. ··> The transgene that encodes a selected polypeptide and the otitP' ^ üe contains a transgene that encodes a recombinase. —— ——

Clones of the non-human transgenic animals described in the present invention can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385: 810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In summary, a cell, eg, a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter the G0 phase. The quiescent cell can then be fused, for example, through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to the morula or blastocyst stage and is then transferred to the pseudopregnant female foster animal. The offspring born from this female breeding animal will be a clone of the animal from which the cell is isolated, for example the somatic cell

IV. Pharmaceutical Compositions

PD-L3 or VISTA molecules, for example, PD-L3 or VISTA nucleic acid molecules, PD-L3 or VISTA polypeptide fragments, and anti-PD-L3 or VISTA antibodies (also referred to herein as active compounds or modulating agents) of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid, polypeptide or antibody molecule and a carrier, eg, a pharmaceutically acceptable carrier. As used herein the term "pharmaceutically acceptable carrier" includes any solvent, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarding agents, and the like, compatible with pharmaceutical administration. The use of such media and agents in pharmaceutically active substances is well known in the art. Except that any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated to the extent possible. Supplemental active compounds can also be incorporated into compositions.

104

<img file="MX342017B_D0089.tif" />

IMPIí

INSTITUTO MFX.ICAKO '-. DELA FROPIUMD. V.

As noted said compositions may comprise a desired antigen, for example, a tumor antigen or other immunomodulatory compounds, such as Toll-like receptor agonists, type 1 interferon, such as alpha and beta interferons, and CD40 agonists such as agonist antibodies to CD40 and antibody fragments, preferably human anti-CD40 agonist antibodies and antibody fragments or other immune enhancers or suppressors such as PD-L1, PD-L2, CTLA4 fusion proteins and antibodies specific to them.

In some preferred embodiments, the PDL3 OR VISTA-based composition or therapy may further include an antigen or other immune agonist. When the antigen is present in the composition or therapy, it can be administered in an amount that, in combination with the other components of the combination, is effective to generate an immune response against the antigen. For example, the antigen can be administered in an amount from about 100 .mu.g / kg to about 100 mg / kg. In some embodiments, the antigen can be administered in an amount from about 10 .mu.g / kg to about 10 mg / kg. In some embodiments, the antigen can be administered in an amount from about 1 mg / kg to about 5 mg / kg. The amount of particular antigen that constitutes an effective amount to elicit an immune response, however, depends to some extent on certain factors such as, for example, the particular antigen that is administered; the particular agonist being administered and the amount of these; the particular agonist being administered and the amount of it; the state of the immune system; the method and order of administration of the agonist and antigen; the species to which the formulation is administered; and the desired therapeutic result. As a consequence, it is not generally practical to determine the amount that constitutes an effective amount of the antigen. Those skilled in the art, however, can easily determine the appropriate amount with due consideration of such factors.

The antigen can be any material capable of raising a Th1 immune response, which can include one or more of, for example, a cell response.

105

<img file="MX342017B_D0090.tif" />

limited to peptides; polypeptides; lipids; glycolipids; polysaccharides; carbohydrates; polynucleotides; prions; live or inactivated bacteria; viruses or fungi; and bacterial, viral, fungal, protozoal, tumor-derived, or organism-derived antigens, toxins, or toxoids.

Furthermore, certain currently experimental antigens, especially materials such as recombinant proteins, glycoproteins, and peptides that do not cause a strong immune response, can be used in connection with adjuvant combinations of the invention. Illustrative experimental subunit antigens include those related to viral disease, such as adenovirus, AIDS, chickenpox, cytomegalovirus, dengue, feline leukemia, fowl plague, hepatitis A, hepatitis B, HSV-1, HSV-2, swine cholera, influenza A, influenza B, Japanese encephalitis, measles, parainfluenza, rabies, respiratory syncytial virus, rotavirus, wart, and yellow fever.

In one embodiment, the antigen can be a cancer antigen or a tumor antigen. The terms cancer antigen and tumor antigen are used interchangeably and refer to an antigen that is differentially expressed by cancer cells. Therefore, cancer antigens can be exploited to differentially target an immune response against cancer cells. Cancer antigens can thus potentially stimulate tumor-specific immune responses. Certain cancer antigens are encoded, but not necessarily expressed, by normal cells. Some of these antigens can normally be characterized as silent (that is, they are not expressed) in normal cells, those that are expressed only at certain stages of differentiation, and those that are temporarily expressed (for example, embryonic and fetal antigens). Other cancer antigens can be encoded by mutant cellular genes such as, for example, oncogenes (eg, activated ras oncogene), suppressor genes (eg, mutant p53), or fusion proteins resulting from interior deletions or chromosomal translocations.

106

OF The PkCH¹DAD {. '>>

INDUSTRl AL

Still other cancer antigens can be encoded by those transmitted by RNA and DNA tumor viruses.

Examples of tumor antigens include MAGE / MÁRT-TMeian-A, gp 100, dipeptidyl peptidase IV (DPPUV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC) -C017-1A / GA733, antigen carcinoembryonic (CEA) and its antigenic epitopes CAP-1 and CAP-2, etv6, am11, prostate-specific antigen (PSA) and its antigenic epitopes PSA1, PSA-2 and PSA-3, prostate-specific membrane antigen (PSMA) , T cell / CD3 receptor zeta chain, MAGE family of tumor antigens (eg, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE -A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGEC2, MAGE-C3, MAGE-C4, MAGE- C5), family of tumor antigens-GAGE (e.g. GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, epsilonchaderin, alpha-catenin, beta-catenin, gamma.-catenin, p120ctn, gp10.sup.Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Conexin 37, idiotype -lg, p15, gp75, gangliosides GM2 and GD2, viral products such as human papillomavirus proteins, Smad family tumor antigens, lmp-1, PIA, EBV-encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase , SSX-1, SSX-2 (HOM-MEL-40), SSX-3, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.

Cancers or tumors and specific tumor antigens associated with such tumors (but not exclusively) include acute lymphoblastic leukemia (etv6, am11, cyclophilin b), B-cell lymphoma (idiotype-lg), glioma (E-cadherin, alphacatenin, beta -catenin, gamma-catenin, p120ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family, HER2 / neu, c-erbB-2), cervical carcinoma (p53, p21ras), carcinoma colon (p21ras, HER2 / neu, c-erbB-2, MUC family), colorectal cancer (associated colorectal antigen (CRC) -CO171A / GA733, APC), choriocarcinoma (CEA), epithelial cell cancer (cyclophilin b),

107

IMPI0T ^

INSTITUTO MEXICANO gastric cancer (HER2 / neu, c-erbB-2, glycoprotein ga733), cahcw> K'épab3 € elute ^> (alpha-fetoprotein), Hodgkins lymphoma (lmp-1, EBNA-1). lung cancer (CEA, MAGE-3, NY-ESO-1), lymphoid cell-derived leukemia (cyclophilin b), melanoma (protein p5, gp75, oncofetal antigen, gangliosides GM2 and GD2, MelanA / MART-1, cdc27, MAGE-3, p21ras, gp100.sup.Pmel117), myeloma (MUC family, p21ras), non-small cell lung carcinoma (HER2 / neu, c-erbB-2), nasopharyngeal cancer (lmp-1, EBNA -1), ovarian cancer (MUC family, HER2 / neu, c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2 / neu. c-erbB-2, glycoprotein ga733), kidney cancer (HER2 / neu , c-erbB-2), squamous cell cancers of the cervix and esophagus (viral products such as human papillomavirus proteins), testicular cancer (NY-ESO-1), and T-cell leukemia (HTLV- epitopes). 1).

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for intradermal, or subcutaneous parenteral application may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and tonicity adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be contained in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when soluble in water) or dispersions and sterile powders for the extemporaneous preparation of solutions or dispersions.

108 l'f ffe'ii · (Sterile injectables. For intravenous administration, the ^ y ^ ljjpultts ^ d: the i'Z · fe include physiological saline, bacteriostatic water, Cremopho ^ EL'TM. (BAsFT Parsippany, NJ) or phosphate regulated saline (Pft &) - J ^ tnrin <s ir> <s level, the composition must be sterile and must be fluid as long as there is easy availability of syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures of these. The proper fluidity can be preserved, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol sorbitol, and sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (eg, modulating agents such as a PD-L3 or VISTA nucleic acid molecule, a fragment of a PD-L3 or VISTA polypeptide, an anti-PD-L3 antibody or VISTA, or a combination of an anti-PD-L3 or VISTA antibody and an anti-PD-L1 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are

109

IΜΜ1 vacuum drying and lyophilization producing a powder of any desired additional Ingredient from a previously filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a flowable vehicle for use as a mouthwash, wherein the compound in the flowable vehicle is applied orally and shaken and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjunct materials can be included as part of the composition. The lozenges, pills, capsules, troches, and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate or esterotes; a slip agent such as colloidal silicon dioxide; an educating agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

For administration by inhalation, the compounds are administered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, appropriate penetrating agents are used in the formulation to permeate the barrier. Such penetrating agents are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, derivatives of fusidic acid. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration,

110

I

IΜ ΡI

INSTITUTE MFXICAXX

OF THE PROPJELVi &lt;.

Active compounds are formulated into ointments, ointments, gels, or coffees and are generally known in the art. . ---------- The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal administration. In one embodiment, the active compounds are prepared with vehicles that will protect the compound against rapid clearance from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations will be apparent to those of skill in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting cells infected with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those of skill in the art, for example, as described in US Pat. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit dosages for treating the individual; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of composing said active compound for the treatment of individuals. .

111

A tut-ev -j¿a

<img file="MX342017B_D0091.tif" />

INSTITUTO MEXICANO DELA / KOPiEDAD INDUSTRIAL

<img file="MX342017B_D0092.tif" />

The toxicity and therapeutic efficacy of these compounds can be determined by standard pharmaceutical procedures in οΰΐΐΙνο ^^ ίΠΙτθβ ^ οexperimental animals. Data obtained from cell culture assays and animal studies can be used in formulation at a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration in the range that includes the IC50 (ie, the concentration of the test compound that achieves the maximum mean inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) is in the range of about 0.001 to 30 mg / kg of body weight, preferably about 0.01 to 25 mg / kg. body weight, more preferably about 0.1 to 20 mg / kg of body weight, and even more preferably about 1 to 10 mg / kg, 2 to 9 mg / kg, 3 to 8 mg / kg, 4 to 7 mg / kg, or 5 to 6 mg / kg of body weight. The experienced technician will appreciate that certain factors may influence the dosage required to effectively treat an individual, including but not limited to the severity of the disease or disorder, prior treatments, the individual's general health and / or age, and other diseases. present. In addition, treating an individual with a therapeutically effective amount of a protein, polypeptide, or antibody may include a single treatment or, preferably, it may include a series of treatments.

112

In a preferred example, an individual is treated or polypeptide in the range of about 0.1 to 20 mg / kg of body weight, - j ... jujn.-jrrr once a week for about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage can be apparent and apparent from the results of diagnostic tests as described in the present invention.

The present invention includes agents that modulate the expression or activity of PD-L3 or VISTA. An agent can, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds). that have a molecular weight approximately less than 10,000 grams per mole, organic or inorganic compounds that have a molecular weight approximately less than 5,000 grams per mole, organic or inorganic compounds that have a molecular weight approximately less than 1,000 grams per mole, organic or inorganic compounds that have a molecular weight approximately less than 500 grams per mole , and salts, esters, and other pharmaceutically acceptable forms of said compounds. It will be understood that the appropriate doses of small molecular agents depend on a number of factors within the scope of those of skill in medicine, veterinary medicine, or research. The small molecule dose (s) will vary, for example, depending on the identity, size and condition of the individual or sample to be treated, also depending on the route by which the composition is administered, if applicable, and the effect. that the physician wishes to have the small molecule on the nucleic acid or polypeptide of the invention.

113

<img file="MX342017B_D0093.tif" />

<img file="MX342017B_D0094.tif" />

I .NO

Illustrative dosages include milligram quantities of the small molecule per kilogram of individual or sample weight (oorejemoki. Approximately 1 microgram per kilogram to approximately 500 milligrams per kilogram, approximately 100 micrograms per kilogram to approximately 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). It is further understood that appropriate doses of a small molecule depend on the potency of the small molecule with respect to the expression or activity that is modulated. Such appropriate doses can be determined using the assays described in the present invention. When one or more of these small molecules must be administered to an animal (eg, a human) to modulate the expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian or researcher may, for example, prescribe a relatively low dose initially, then increasing the dose until an appropriate response is obtained. Furthermore, it is understood that the specific dose level for any particular animal individual will depend on a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the individual, the time of administration, route of administration, rate of excretion, any combination of drugs, and the degree of expression or activity that is modulated.

Furthermore, an antibody (or fragment thereof) can be conjugated to a therapeutic region such as a cytotoxin, a therapeutic agent, or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramycldin D, ethidium bromide, emetine, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxydione anthrazine, mitoxantrone, mitomycin, 1-dehydrochloride actinomycin, dehydrochloride procaine, tetracaine, lidocaine, propranolol, and puromycin and homologues thereof. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechlorethamine, thioepa

114

ΙΜΡΙ ^ Γ ^

INSTITUTO MEXICAN't fe .- j chlorambucil, melphalan, carmustine (BSNU) and lomustine (C'ÓÑ ^ T ^ cloteéfariiida, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (eg, vincristine and vinblastine).

The conjugates of the invention can be used to modify a given biological response, the region of the drug that is not constructed to limit the classical chemical therapeutic agents. For example, the drug region can be a protein or polypeptide that possesses a desired biological activity. Such polypeptides can include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, interferon alpha, interferon beta, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator; or biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-I), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte colony stimulating factor and macrophages f 'GM-CSF), granulocyte colony stimulating factor (G-CSF) or other growth factors. Techniques for conjugating such a therapeutic region with antibodies are well known.

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be administered to an individual by, for example, intravenous injection, local administration (see US Patent No. 5,328,470), or by stereostatic injection (see, for example, Chen et al. (1994) Proc Nati Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector may include the gene therapy vector in an acceptable diluent, or it may comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, when the complete gene delivery vector can be produced intact from recombinant cells, eg, retroviral vectors, the pharmaceutical preparation can include one or more cells.

115

INSTITUTE that produce the pharmaceutical géní &amp; ajNt ^ g administration system can be included in a container, container or dispenser along with instructions for administration.

V. Uses and Methods of the Invention

The PD-L3 or VISTA molecules, for example, the nucleic acid molecules, polypeptides, polypeptide homologs, and PD-L3 or VISTA antibodies and antibody fragments described in the present invention can be used in one or more of the following methods: a) screening tests; b) predictive medicine (eg, diagnostic assays, prognostic assays, clinical monitoring trials), and c) treatment methods (eg, therapeutic and prophylactic, eg, positively and negatively modulating the immune response). As described in the present invention, a PD-L3 or VISTA polypeptide of the invention has one or more of the following activities: 1) it binds to and / or modulates the activity of its natural binding partner (s), 2) it modulates intra- or intercellular signaling, 3) modulates T lymphocyte activation, 4) modulates the immune response of an organism, eg, a mammalian organism, such as a mouse or human. Isolated nucleic acid molecules of the invention can be used, for example, to express PD-L3 or VISTA polypeptide (for example, via recombinant expression vector in a host cell in gene therapy applications), to detect PD-L3 or VISTA mRNA (eg, in a biological sample) or a genetic alteration in a PD-L3 or VISTA gene, and to modulate PD-L3 or VISTA activity, as described below. PD-L3 or VISTA polypeptides can be used to treat conditions or disorders characterized by insufficient or excessive production of a PD-L3 or VISTA polypeptide or production of inhibitors of PD-L3 or VISTA. In addition, PD-L3 or VISTA polypeptides can be used to screen for the naturally occurring binding partner (s) of PD-L3 or VISTA, to screen drugs or compounds that modulate the activity of PD-L3 or VISTA. , as well as to treat conditions or disorders characterized by insufficient or excessive production of the PD-L3 or VISTA polypeptide or production of forms of PD-L3 or VISTA polypeptides that decreased, aberrantly or undesirably the activity in

116

<img file="MX342017B_D0095.tif" />

INSTITUTO M2XICA? Rj DE LA PROFIEDAD INDUSTRIAL

<img file="MX342017B_D0096.tif" />

IN DUSTRÍAL '· &lt;/. * comparison to wild-type PD-L3 or VISTA polypeptide (for example, immune system disorders such as severe combined immunodeficiency, multiple sclerosis, systemic lupus erythematosus, type I diabetes mellitus, lymphoproliferative syndrome, inflammatory bowel disease, allergies, asthma, graft versus host disease, and transplant rejection, immune responses to infectious pathogens such as bacteria and viruses; and cancers of the immune system such as lymphomas and leukemias). Furthermore, the anti-PD-L3 or VISTA antibodies of the invention can be used to detect and isolate PD-L3 or VISTA polypeptides, regulate the bioavailability of PD-L3 or VISTA polypeptides, and modulate the activity of PDL3 or VISTA, for example, by modulating the interaction between PD-L3 or VISTA and its natural binding partner (s).

A. Screening tests:

The invention provides a method (also referred to herein as a screening assay) for identifying modulators, i.e. candidates or test compounds or agents (eg, peptides, peptidomimetics, small molecules, or other drugs) that bind to PD-L3 or VISTA polypeptides, have a stimulating or inhibitory effect on, for example, PD-L3 or VISTA expression or PD-L3 or VISTA activity, or they have a stimulating or inhibitory effect on the interaction between PD-L3 or VISTA and its natural binding partner (s).

In one embodiment, the invention provides assays to screen the candidate or test compounds that bind to the PDL3 or VISTA protein or polypeptide or the biologically active portion thereof, eg, modulate the ability of the PD-L3 or VISTA polypeptide. to interact with your natural bonding partner (s). In another embodiment, the invention provides assays for screening candidates or test compounds that bind to or modulate the activity of a PD-L3 or VISTA protein or polypeptide or biologically active portion thereof. In a preferred embodiment, the invention provides assays to screen candidates or test compounds that have a stimulatory or inhibitory effect on immune functions downregulated by PD-L3 or VISTA as identified in the present disclosure or based on their effect on the interaction

117

MEXICAN INSTITUTE ^- - '') between PD-L3 or VISTA and its naturaf ^^ asurictone-BSO binding partner (s) associated with PD-L3 or VISTA include by way of example the inhibition of cytokine production (e.g., IL -2, interferon gamma by T cells, suppression of moderate CD28 costimulation, inhibition of CD4 + and CD8 + T cell proliferation, suppression of naive and memory CD4 + T cell proliferation, and suppression of TCR activation without induction of apoptosis. The test compounds of the present invention can be obtained using any of a number of approaches in combinatorial library methods known in the art, including: biological libraries; parallel spatially addressable solid phase or solution libraries; synthetic library methods requiring deconvolution; the single drop one compound library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches apply to peptides, non-peptide oligomers, or small molecule compound libraries (Lam, KS (1997) Anticancer Drug Des. 12: 145).

In one embodiment, an assay is a cell-based assay in which a cell expressing a PD-L3 or VISTA polypeptide or biologically active part thereof is contacted with a test compound, and the ability of the compound is determined. test to modulate PD-L3 or VISTA activity. Determination of the ability of the test compound to modulate PD-L3 or VISTA activity can be achieved by monitoring, for example, the ability of PD-L3 or VISTA to bind to its natural binding partner (s), and modulate the activity of immune cells. The immune cell can be, for example, a T cell, a B cell, or a myeloid cell. Determination of the ability of the test compound to modulate the binding of PD-L3 or VISTA to its counter-receptor (to be determined) can be carried out, for example, by coupling PD-L3 or VISTA with a radioisotope or enzymatic marker to monitor the ability of a test compound to modulate the binding of PD-L3 or VISTA to T cells expressing the PD-L3 or VISTA counter-receptor. Determination of the ability of the test compound to bind PD-L3 or VISTA can be

118

11V1 r jl

MFXiOANO INSTITUTE Vv 1

OF THE í'RCPiF.L'Ai'J ^ .7 '. r '. and 'to carry out, for example, coupling the compound with a radioisotope or enzymatic marker such that the binding of the compound to PD-L3 or VISTA can be determined by detecting the PD-L3 or VISTA compound labeled in a complex.

It is also within the scope of this invention to determine the ability of a compound to interact with PD-L3 or VISTA without the labeling of either of the interacting agents. For example, a microphysiometer can be used to detect the interaction of a compound with PD-L3 or VISTA without labeling either the compound or PD-L3 or VISTA (McConnell, HM et al. (1992) Science 257: 19061912). As used herein, a microphysiometer (eg, Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and PD-L3 or VISTA.

In another embodiment, an assay is a cell-based assay comprising contacting the T cell expressing a PD-L3 or VISTA binding partner with a test compound and determining the ability of the test compound to modulate (eg, stimulate or inhibit) the activity of the PD-L3 or VISTA binding partner. Determination of the ability of the test compound to modulate the activity of a PD-L3 or VISTA binding partner can be carried out, for example, by determining the ability of the PDL3 or VISTA polypeptide to bind or interact with the binding of PD-L3 or VISTA.

Determination of the ability of the PD-L3 or VISTA polypeptide, or a biologically active fragment thereof, to bind or interact with a PD-L3 or VISTA binding partner can be carried out by one of the methods described above. to determine direct union. In a preferred embodiment, the determination of the ability of the PD-L3 O VISTA polypeptide to bind or interact with a PD-L3 O VISTA binding partner can be carried out by determining the activity of the binding partner. For example, the activity of the binding partner can be determined by detecting the induction of a second cellular messenger (eg, tyrosine kinase or phosphatase activity), detecting the catalytic enzyme activity of an appropriate substrate, detecting

119

<img file="MX342017B_D0097.tif" />

INSTITUTO MEXICANO DE LA ΡΚΟΤ'ΙΕΓΆΙΤ induction of a reporter gene (comprising a target-sensitive regulatory element operably linked to a nucleic acid ^ u ^ xodjfica-4JP - R + ar <> aelor '· detectable, for example, luciferase ), or by detecting a target-regulated cellular response. For example, determination of the ability of the PD-L3 or VISTA polypeptide to bind or interact with a natural binding partner to PD-L3 or VISTA, can be carried out by measuring the ability of a compound to modulate co-stimulation or inhibiting immune cells in a proliferation assay, or interfering with the ability of a PD-L3 or VISTA polypeptide to bind to antibodies that recognize a part of the PD-L3 or VISTA polypeptide. In one embodiment, compounds that modulate T cell activation can be identified by determining the ability of a compound to modulate T cell proliferation or cytokine production. In a preferred embodiment, compounds that modulate T cell activation can be identified by determining the ability of a compound to modulate T cell proliferation or cytokine production at more than one antigen concentration.

In yet another embodiment, an assay of the present invention is a cell-free assay in which a PD-L3 or VISTA polypeptide or biologically active portion thereof is contacted with a test compound and the ability of the compound is determined. test for binding to PD-L3 or VISTA polypeptide or biologically active part thereof. Preferred biologically active moieties of PD-L3 or VISTA polypeptides used in the assays of the present invention include fragments that participate in interactions with other non-PD-L3 or VISTA molecules, for example, at least a portion of an extracellular domain that binds to a PD-L3 or VISTA binding partner. Binding of the test compound to the PD-L3 or VISTA polypeptide can be determined either directly or indirectly as described above.

In another embodiment, the assay is a cell-free assay in which a PD-L3 or VISTA polypeptide or biologically active part thereof is contacted with a test compound and the ability of the test compound to modulate ( eg, stimulate or inhibit) the activity of the PD-L3 or VISTA polypeptide or biologically active part thereof. Determining the

120

<img file="MX342017B_D0098.tif" />

<img file="MX342017B_D0099.tif" />

<img file="MX342017B_D0100.tif" />

MEXICAN INSTITUTE

f) And the FROÍIEDAiT INDUSTRIAL ability of the test compound to modulate the activity of a PD-L3 or VISTA polypeptide can be carried out, for example, by determining the ability of the PD-L3 or VISTA polypeptide to bind to a binding partner of PD-L3 or VISTA by one of the methods described above to determine direct binding. The cell-free assays of the present invention are amenable to the use of both soluble and / or membrane-bound forms of polypeptides (e.g., PD-L3 or VISTA polypeptides or biologically active portions thereof, or binding partners to which PD-L3 or VISTA joins). In the case of cell-free assays in which a membrane-bound form is used a polypeptide (e.g., a cell surface PD-L3 or VISTA), it may be desirable to use a solubilizing agent such that the form-bound to polypeptide membrane is kept in solution. Examples of such solubilizing agents include nonionic detergents such as noctylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoylN-methylglucamide, decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X114, Tesit, Isotridecipoli (ethylene glycol ether) n, 3 - [(3-cholamidopropyl) dimethylammonioj1-propane sulfonate (CHAPS), 3 - [(3-cholamidopropyl) dimethylammono] -2-hydroxy-1propane sulfonate ( CHAPSO), or N-dodecyl.dbd.NN-dimethyl-3-ammonium-1-propane sulfonate.

In more than one embodiment of the foregoing assay methods of the present invention, it may be desirable to immobilize either PD-L3 or VISTA or their binding partner to facilitate the separation of complex from non-complex forms of one or both polypeptides, as well as to accommodate automation of the assay. The binding of a test compound to a PD-L3 or VISTA polypeptide, or the interaction of a PD-L3 or VISTA polypeptide with its binding partner in the presence and absence of a candidate compound, can be carried out in any suitable container to hold reagents. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the polypeptides to bind to a matrix. For example, glutathione-S-transferase / PDL3 or VISTA fusion proteins or glutathione-S-transferase / binding partner fusion proteins are

121

<img file="MX342017B_D0101.tif" />

can adsorb on glutathlon sepharose beads (Sigma Cherfíical,

IMPI

MEXICAN INSTITUTE OF PROPERTY

Missouri) or glutathione derivatized microtiter plates, which are then combined with the test compound or test compound and either the non-adsorbed binding partner polypeptide or PD-L3 polypeptide or VISTA, and the mixture was incubated in conditions leading to complex formation (eg, under physiological conditions for salt and pH). Following incubation, the beads or wells of the microtiter plate are washed to remove any unbound components, the matrix is immobilized in the case of the beads, and complex formation is determined either directly or indirectly. , for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of PD-L3 or VISTA binding or activity determined using standard techniques. Other techniques for immobilizing polypeptides on arrays can also be used in the screening assays of the invention. In an alternative embodiment, determining the ability of the test compound to modulate the activity of a PD-L3 or VISTA polypeptide can be carried out by determining the ability of the test compound to modulate the activity of a downstream operating molecule. of PD-L3 or VISTA, for example, by interacting with the cytoplasmic domain of a PD-L3 or VISTA binding partner. For example, the levels of second messengers, the activity of the interacting molecule on an appropriate target, or the binding of the interactor to an appropriate target can be determined as described above.

In another embodiment, PD-L3 or VISTA expression modulators are identified with a method wherein a cell is contacted with a candidate compound and expression of the PDL3 or VISTA mRNA or polypeptide in the cell is determined. The level of expression of PD-L3 or VISTA mRNA or polypeptide in the presence of the candidate compound is compared to the level of expression of PD-L3 or VISTA mRNA or polypeptide in the absence of the candidate compound. The candidate compound can then be identified as a modulator of PD-L3 or VISTA expression based on this comparison if the change is statistically significant.

122

WICKED

In yet another aspect of the invention, polypepndMró ^ B ^ gL ^^ i ^^ can be used as bait proteins in a double-hybrid assay or triple-hybrid assay (see, for example, US Patent ^ ntdü ^ ñurnT ^ Ba.ál ^;

Zervos et al. (1993) Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054; Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696, and Brent W094 / 10300), to identify other polypeptides that bind to or interact with PD-L3 or VISTA (PD-L3 or VISTA binding proteins, binding partners to PD-L3 or VISTA, or PD-L3 or VISTA-bp) and engage in the activity of PD-L3 or VISTA. Such PD-L3 or VISTA binding proteins are also likely to be involved in signal propagation by PD-L3 or VISTA polypeptides or PDL3 or VISTA targets such as elements downstream of a pathway. PDL3 or VISTA mediated signaling. Alternatively, such PD-L3 or VISTA binding polypeptides can be inhibitors of PD-L3 or VISTA. The two-hybrid system relies on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. In summary, the assay uses two different DNA constructs. In one construct, the gene encoding a PD-L3 or VISTA polypeptide is fused to a gene encoding the DNA-binding domain of a known transcription factor (eg, GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, encoding an unidentified polypeptide (prey or sample) is fused to a gene encoding the activation domain of the known transcription factor. If the bait and prey polypeptides are able to interact, in vivo, to form a PD-L3 or VISTA-dependent complex, the DNA-binding and activation domains of the transcription factor are attracted in close proximity. This proximity allows the transcription of a reporter gene (eg, LacZ) that is operably linked to a transcription factor-responsive transcriptional regulatory site. Reporter gene expression can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene encoding the polypeptide that interacts with the PD-L3 or VISTA polypeptide.

123

MEXICAN INSTITUTE

OF THE PROPERTY

In another aspect, the invention corresponds to one or more of the assays described in the present invention. Rnr..ejfimplo,. A modulating agent can be identified using a cell-based or cell-free assay, and the ability of the agent to modulate the activity of a PD-L3 or VISTA polypeptide can be confirmed in vivo, for example, in an animal such as an animal model for cell transformation and / or tumorigenesis.

This invention further corresponds to new agents identified by the screening tests described above. As a consequence, it is within the scope of this invention to further use an identified agent in an appropriate animal model as described in the present invention. For example, an agent identified as described in the present invention (e.g., a PD-L3 or VISTA modulating agent, a PD-L3 or VISTA antisense nucleic acid molecule, a PD-L3 or VISTA specific antibody , or a PD-L3 or VISTA binding partner) can be used in an animal model to determine the efficacy, toxicity or side effects of treatment with said agent. Alternatively, an agent identified as described in the present invention can be used in an animal model to determine the mechanism of action of said agent. Furthermore, this invention corresponds to the uses of new agents identified by the screening assays described above for the treatments described in the present invention.

B. Detection Assays

The parts or fragments of the cDNA sequences identified in the present disclosure (and the corresponding complete gene sequences) can be used in various ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome, and thus locate gene regions associated with genetic disease; (ii) identify an individual from a tiny biological sample (tissue typing), and (iii) assist in the forensic identification of a biological sample. These applications are described in the following subsections.

1. Chromium omic mapping

124

IMPI

MEXICAN INSTITUTE OF PROPERTY

Once the sequence (or part of the sequence is isolated) this sequence can be used to map the location of the chromosome gene. This process is called chromosome mapping. As a consequence, the parts or fragments of The PD-L3 or VISTA nucleotide sequences described in the present invention can be used to map the location of the PD-L3 or VISTA genes on a chromosome. Mapping PD-L3 or VISTA sequences on chromosomes is an important first step in correlating these sequences with genes associated with disease. Briefly, PD-L3 or VISTA genes can be mapped onto chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the nucleotide sequences of PD-L3 or VISTA. Computer analysis of PD-L3 or VISTA sequences can be used to predict primers that do not span more than one exon in genomic DNA, thus complicating the amplification process. These primers can then be used for the PCR detection of somatic cell hybrids containing individual human chromosomes. Only those hybrids that contain the human gene corresponding to the PD-L3 OR VISTA sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals (eg, human and mouse cells). As human and mouse cell hybrids grow and divide, they gradually lose human chromosomes in a random order, but retain mouse chromosomes. Using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the only human chromosome containing the gene encoding the necessary enzyme will be preserved. Using various means, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a complete set of mouse chromosomes, allowing easy mapping of individual genes onto specific human chromosomes (D'Eustachio, P. et al. (1983) Science 220: 919-924). Somatic cell hybrids containing only human chromosome fragments can also be produced using human chromosomes with translocations and deletions.

<img file="MX342017B_D0102.tif" />

125 ___. ,,,. ... MEXICAN INSTITUTE

The PCR mapping of somatic cell hybrids ^ es. · M pro

<img file="MX342017B_D0103.tif" />

<img file="MX342017B_D0104.tif" />

Quick to assign a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler.

Using the nucleotide sequences of PD-L3 or VISTA to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies that can equally be used to map a PD-L3 or VISTA sequence onto its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc Nati. Acad.

Sci. USA. 87: 6223-27), pre-screening with the tagged chromosomes separated by flow and preselection by hybridization to specific chromosomes from libraries of

CDNA.

Fluorescent in situ hybridization (FISH) of a DNA sequence to a metaphase chromosome stretch can further be used to provide precise chromosomal location in a single step. Chromosome extensions can be prepared using cells whose division was blocked at metaphase by a chemical such as colcemide that disrupts the mitotic spindle. Chromosomes can be briefly trypsinized and then Giemsa stained. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases are more likely to bind to a single chromosomal location with sufficient signal intensity for easy detection. Preferably 1,000 bases, and more preferably 2,000 bases will be sufficient to achieve good results in a reasonable time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of basic techniques (Pergamon Press, New York 1988). Chromosome mapping reagents can be used individually to label a single chromosome or a single site on that chromosome, or panels of reagents can be used to label multiple sites and / or multiple chromosomes. Reagents corresponding to noncoding regions of genes are actually preferred for mapping purposes. The sequences

126 rntn? mtrxaetts »

IM PI ii xvi - · .'- ü.

encoders are more likely to be kept within^N3'ⁿs¡ ^ r ^^ atñd ^^ Íres, thus increasing the possibility of cross-hybridization during ir * -.Tr ​​»SS: nT -a Λ-Λ5Κrr mapping. ^— chromosomal.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with the genetic map data. Ultimately, complete gene sequencing from multiple individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

two. Tissue Typing.

The PD-L3 or VISTA sequences of the present invention can also be used to identify individuals from minute biological samples. In addition, the sequences of the present invention can be used to provide an alternative technique that determines the actual DNA sequence base by base of selected parts of an individual's genome. Thus, the nucleotide sequences of PD-L3 or VISTA described in the present invention can be used to prepare two PCR primers from the 5 'and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently the sequence.

Panels of corresponding DNA sequences from individuals, prepared in this way, can provide individual unique identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The PD-L3 or VISTA nucleotide sequences of the invention represent only parts of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the non-coding regions. Allelic variation between individual humans is estimated to occur with a frequency of approximately once per 500 bases. Each of the sequences described in the present invention can be used, to some extent, as a standard against which the DNA of an individual can be compared for identification purposes. Because the

127

IMPI

INSTITUTO MEXíC / .X'O DE LA) Xü ^ lLAO greater numbers of polymorphisms occur in the nócodifying regions; fewer sequences are needed to differentiate individuals. The seouonoioo we encoding sec. with nos. Identification numbers: 1 or 4 can conveniently provide identification of the positive individual with a panel of perhaps 10 to 1,000 primers yielding each of the 100 base non-coding amplified sequences. If the predicted coding sequences are used such as those of sec with nos. Identification Number: 3 or 6, a more appropriate number of primers for positive individual identification would be 500-2000.

If a panel of PD-L3 or VISTA nucleotide sequence reagents described in the present invention is used to generate a unique identification database for an individual, those same reagents can subsequently be used to identify that individual's tissue. Using the unique identification database, positive identification of the living or dead individual can be prepared from extremely small tissue samples.

3. Use of PD-L3 or VISTA sequences in Forensic Biology

DNA-based identification techniques can also be used in forensic biology. The sequences of the present invention can be used to provide polynucleotide reagents, eg, POR primers, targeting specific loci in the human genome, which can improve the reliability of DNA-based forensic identifications, eg, by providing another marker. identification (i.e., another DNA sequence that is unique to a particular individual). As mentioned above, the actual base sequence information can be used for identification as an accurate alternative to the patterns formed by the restriction enzyme generated fragments. Sequences targeting non-coding regions of sec. with nos. Identification numbers: 1 or 3 are particularly appropriate for this use as higher numbers of polymorphisms occur in the non-coding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the nucleotide sequences of PD-L3 or VISTA or parts thereof, eg, fragments derived from the non-coding regions of sec. with nos. ID: 1 or 3 that have a length of at least

128

ΙΜΡΙ

INSTITUTO MEXICANO \ 'Λ bases, preferably at least 30 bases. The sequences ^ indcleótMos give

PD-L3 or VISTA described in the present invention can be further used to provide polynucleotide reagents, e.g., labeled or markable probes that can be used in, e.g., an in situ hybridization technique, to identify a specific tissue, e.g. , lymphocytes. This can be very helpful in cases where a forensic pathologist presents with tissue of unknown origin. Panels of such PD-L3 or VISTA probes can be used to identify tissue by species and / or by organ type. Similarly, these reagents, for example PD-L3 or VISTA primers or probes can be used to screen the tissue culture for contamination (i.e., screen for the presence of a mixture of different cell types in a culture). .

C Predictive medicine

The present invention also corresponds to the field of predictive medicine, in which diagnostic tests, prognostic tests and monitoring of clinical trials are used for prognostic (predictive) purposes to thereby prophylactically treat an individual. As a consequence, one aspect of the present invention relates to diagnostic assays to determine the expression of the polypeptide and / or the nucleic acid of PD-L3 or VISTA as well as the activity of PD-L3 or VISTA, in the context of a sample. biological (e.g. blood, serum, cells, or tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted expression or activity of PD-L3 or VISTA. The invention also provides prognostic (or predictive) assays to determine whether an individual is at risk of developing a disorder associated with the expression or activity of nucleic acid, PD-L3 polypeptide, or VISTA. For example, mutations in a PD-L3 or VISTA gene can be tested in a biological sample. Such assays can be used for prognostic or predictive purposes to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with the expression or activity of the nucleic acid, PDL3 polypeptide, or VISTA.

129

<img file="MX342017B_D0105.tif" />

srm r-ι j .. í t »'_í! ¡y., ·.»>.? ·'>

Another aspect of the invention corresponds to the influence of agents (for example, drugs, compounds) on the expression or activity of PD-L3 or VISTA in clinical trials. These and other agents are described in more detail in the following sections.

1. Diagnostic tests

An illustrative method of detecting the presence or absence of the PD-L3 or VISTA polypeptide or nucleic acid in a biological sample involves obtaining a biological sample from a test individual and contacting the biological sample with a compound or agent capable of detecting the acid. nucleic or polypeptide of PD-L3 or VISTA (for example, MRNA or genomic DNA) encoding the PDL3 or VISTA polypeptide such that the presence of PD-L3 or VISTA nucleic acid or polypeptide is detected in the biological sample. A preferred agent for detecting PD-L3 or VISTA genomic mRNA or DNA is a labeled nucleic acid probe capable of hybridizing to PD-L3 or VISTA genomic mRNA or DNA. The nucleic acid probe can be, for example, the PD-L3 or VISTA nucleic acid pool set out in sec. with nos. Ident .: 1, or 3, or a portion thereof such as an oligonucleotide of at least 15, 30, 50, 100, 250, or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions PD-genomic DNA or mRNA L3 or VISTA. Other suitable probes for use in the diagnostic assays of the invention are described in the present invention. A preferred agent for detecting PD-L3 or VISTA polypeptide is an antibody capable of binding to PD-L3 or VISTA polypeptide, preferably an antibody with a detectable marker. The antibodies can be polyclonal, or more preferably monoclonal. An intact antibody, or a fragment thereof (eg, Fab or F (ab ') 2) can be used. The term "labeled," with respect to the probe or antibody, is intended to include direct labeling of the probe or antibody by coupling (i.e., physically binding) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescence-labeled secondary antibody and a DNA probe end-labeled with

130 biotin so that it can be detected with fluorescence-labeled spheptay. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from an individual, as well as tissues, rétofay, and ldCTBS present within an individual. That is, the detection method of the invention can be used to detect PD-L3 or VISTA mRNA, polypeptide, or genomic DNA, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for the detection of PD-L2 mRNA include Northem-type hybridizations and in situ hybridizations. In vitro techniques for the detection of PD-L3 or VISTA polypeptide include enzyme-linked immunosorbent assays (ELISA), immunoblotting, immunoprecipitations, and immunofluorescence. In vitro techniques for the detection of PD-L3 or VISTA genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for the detection of PD-L3 or VISTA polypeptide include the introduction of a labeled anti-PD-L3 or VISTA antibody into an individual. For example, the antibody can be labeled with a radioactive marker whose presence and location in an individual can be detected by standard imaging techniques. In one embodiment, the biological sample contains polypeptide molecules from the test individual. Alternatively, the biological sample may contain mRNA molecules from the test individual or genomic DNA molecules from the test individual. A preferred biological sample is a serum sample isolated from an individual by conventional means. In another embodiment, the methods further involve obtaining a control biological sample from a control individual, contacting the control sample with a compound or agent capable of detecting PD-L3 or VISTA polypeptide, mRNA, or genomic DNA, such that the presence of polypeptide , MRNA, or genomic DNA of PD-L3 or VISTA is detected in the biological sample, and comparing the presence of polypeptide, mRNA, or genomic DNA of PDL3 or VISTA in the control sample with the presence of polypeptide, MRNA or genomic DNA of PD-L3 or VISTA in the test sample.

The invention also includes kits for detecting the presence of PD-L3 or VISTA in a biological sample. For example, the kit may comprise a labeled compound or agent capable of detecting PD-L3 polypeptide or mRNA or

131 jMy P1

VISTA in a biological sample; means of determining ^ 1ferdáa »^ 0 ^ d '^ | Pf ^^ or VISTA in the sample, and means of comparing the amount of PD-' L3 or VISTA in the sample with a standard. The compound or agent may be released in a suitable container. The kit may further comprise instructions for the use of the kit that detects PD-L3 or VISTA polypeptide or nucleic acid.

two. Prognostic tests

The diagnostic methods described in the present invention can further be used to identify individuals who have or are at risk of developing a disease or disorder associated with aberrant or unwanted expression or activity of PD-L3 or VISTA. As used herein, the term aberrant includes PD-L3 or VISTA expression or activity that deviates from wild-type PD-L3 or VISTA expression or activity. Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity that does not follow the developmental pattern of wild-type expression or sub-cellular expression pattern. For example, aberrant expression or activity of PD-L3 or VISTA is intended to include cases where a mutation in the PD-L3 or VISTA gene causes the PD-L3 or VISTA gene to be under-expressed or over-expressed and situations where such mutations result in a non-functional PD-L3 or VISTA polypeptide or a non-functional polypeptide in wild-type form, for example, a polypeptide that does not interact with a PD-L3 or VISTA binding partner , or one that interacts with a non-PD-L3 or VISTA binding partner. As used herein, the term "unwanted" includes an unwanted phenomenon that is involved in a biological response such as activation of the immune cell. For example, the term "unwanted" includes an expression or activity of PD-L3 or VISTA that is undesirable in an individual.

The assays described in the present invention, such as the preceding diagnostic assays or the following assays, can be used to identify an individual who has or is at risk of developing a disorder associated with poor regulation in polypeptide activity or expression. nucleic acid PD-L3 or VISTA such as an autoimmune disorder, an immunodeficiency disorder, an autoimmune system disorder, allergic or inflammatory disorder or cancer. Thus, the present invention provides a method for identifying an aberrant or unwanted PD-L3 or VISTA expression or activity disorder in which a test sample is obtained from an individual and the PD-L3 polypeptide or nucleic acid L3 or VISTA (for example, MRNA or genomic DNA) is detected where the presence of PD-L3 or VISTA polypeptide or nucleic acid is diagnostic for an individual who has or is at risk of developing a disease or disorder associated with aberrant or unwanted expression or activity of PD-L3 or VISTA. As used in the present description, a "test sample" refers to a biological sample obtained from an individual of interest. For example, a test sample can be a biological fluid (eg, cerebrospinal fluid or serum), cell sample, or tissue.

In addition, the prognostic assays described in the present invention can be used to determine whether an individual can be administered an agent (eg, an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug. -candidate) to treat a disease or disorder associated with aberrant or unwanted PD-L3 or VISTA expression or activity. For example, such methods can be used to determine whether an individual can be effectively treated with an agent for an autoimmune disorder, immunodeficiency disorder, cancer of the immune system, or allergic or inflammatory disorder. Thus, the present invention provides methods for determining whether an individual can be effectively treated with an agent for a disorder associated with aberrant or undesired expression or activity of PDL-3 or VISTA in which a test sample is obtained and detects the expression or activity of the PD-L3 or VISTA polypeptide or nucleic acid (e.g. wherein the abundance of expression or activity of the PD-L3 or VISTA polypeptide or nucleic acid is diagnosed for an individual who may be administered the agent to treat a disorder associated with aberrant or unwanted expression or activity of PD-L3 or SIGHT). The methods of the invention can also be used to detect genetic alterations in a PD-L3 or VISTA gene, determining from that

133

<img file="MX342017B_D0106.tif" />

INSTiWC mode if an individual with the altered gene is at risk characterized by defective regulation in the activity of D-OlÍDentirln or ~ the expression of PD-L3 or VISTA nucleic acid, such as an autoimmune disorder, an immunodeficiency disorder , a cancer of the immune system, an allergic disorder, or an inflammatory disorder. The methods described in the present invention can be carried out, for example, by using prepackaged diagnostic kits comprising at least one antibody or nucleic acid probe reagent described in the present invention, which can be conveniently used , for example, in clinical practice to diagnose patients who have symptoms or a family history of a disease or condition that involves a PD-L3 or VISTA gene. Furthermore, any type of cell or tissue in which PD-L3 or VISTA is expressed can be used in the prognostic assays described in the present invention.

3. Monitoring of effects during clinical trials

Monitoring the influence of agents (for example, drugs) on the expression or activity of a PD-L3 or VISTA polypeptide (for example, modulation of cell proliferation and / or migration) can be applied not only in screening basic drugs, but also in clinical trials. For example, the efficacy of an agent determined by a screening assay as described in the present invention to increase gene expression, PD-L3 or VISTA polypeptide levels, or PD-L3 or VISTA upregulated activity, can be monitor in clinical trials individuals with decreased gene expression, PD-L3 or VISTA polypeptide levels, or negatively regulated PD-L3 or VISTA activity. Alternatively, the efficacy of an agent determined by a screening assay to decrease gene expression, PD-L3 or VISTA polypeptide levels, or PD-L3 or VISTA downregulated activity, can be monitored in clinical trials of individuals. exhibiting increased gene expression, PDL-3 or VISTA polypeptide levels, or PD-L3 or VISTA activity. As noted PD-L3 or VISTA is expressed on many types of hematopoietic cells, including APCs (macrophages and myeloid dendritic cells), and CD4 + T cells, and more specifically it is expressed

134

<img file="MX342017B_D0107.tif" />

iM? jU on the DC CD11c⁺, CD4 T cells⁺ (including both the 3 'and nTregs Foxp3⁺), CD8 T cells⁺, and Grl + granulocytes, and is expressed at low levels in B cells and NK cells. In such clinical trials, the expression or activity of a PD-L3 or VISTA gene, and preferably other genes that were involved in, for example, a PD-L3 or VISTA-associated disorder can be used as a reading or marker of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including those for PDL-3 or VISTA, that are modulated in cells can be identified by treatment with an agent (eg, compound, drug, or small molecule) that modulates PD-L3 or VISTA activity (eg, identified in a screening assay as described in the present invention). Thus, to study the effect of agents on disorders associated with PD-L3 or VISTA, for example in a clinical trial, cells can be isolated and RNA prepared and analyzed for PD-L3 or VISTA expression levels. and other genes involved in PD-L3 or VISTA associated disorder respectively. Gene expression levels (eg, a gene expression pattern) can be quantified by Northern membrane analysis or RT-PCR, as described in the present invention, or alternatively by measuring the amount of polypeptide produced, by one of the methods as described in the present invention, or by measuring the activity levels of PD-L3 or VISTA or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of cells to the agent. Consequently, this state of response can be determined prior to, and at various points during, the individual's treatment with the agent. In a preferred embodiment, the present invention provides a method of monitoring the efficacy of treating an individual with an agent (eg, an agonist, antagonist, peptide mimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate. identified by the screening assays described in the present invention) including the steps of (i) obtaining a pre-sample from an individual prior to administration of the agent; (ii) detect the level of expression of a PD-L3 or VISTA polypeptide, mRNA or genomic DNA in the sample prior to administration; (iii) obtain one or more

135 samples from the individual after administration; (ivj'-d ^ títíri after administration the level of expression or activity of polypeptide, mRNA. genomic xxJkDN of PD-L3 or VISTA in samples; (v) compare the level of expression or activity of the PD-L3 or VISTA polypeptide, mRNA, or genomic DNA of the sample prior to administration with that after administration of the PD-L3 or VISTA polypeptide, mRNA, or genomic DNA in the sample or samples, and (vi) alter the administration of the agent to the individual accordingly. For example, augmented administration of the agent may be desired to increase the expression or activity of PD-L3 or VISTA to higher levels than detected, ie, to increase the efficacy of the agent. Alternatively, decreased administration of the agent may be desired to decrease the expression or activity of PDL3 or VISTA to levels lower than detected, ie, to decrease the efficacy of the agent. According to one embodiment, the expression or activity of PD-L3 or VISTA can be used as an indicator of the efficacy of an agent, even in the absence of an observable phenotypic response.

D. Treatment Methods

The present invention provides both prophylactic and therapeutic methods for treating an individual at risk of (or susceptible to) a disorder characterized by insufficient or excessive production of PD-L3 or VISTA protein or production of forms of PD-L3 or VISTA proteins that they have aberrant or decreased activity compared to the wild-type protein of PD-L3 or VISTA. Furthermore, the anti-PD-L3 or VISTA antibodies of the invention can be used to detect and isolate PD-L3 or VISTA proteins, regulate the bioavailability of PD-L3 or VISTA proteins, and modulate the activity of PD-L3 or VISTA. for example, modulating the interaction of PD-L3 or VISTA with its counter receptor.

1. Prophylactic methods

In one aspect, the invention provides a method of preventing, in an individual, a disease or condition associated with aberrant or unwanted expression or activity of PD-L3 or VISTA, by supplying the individual with a PD-L3 or VISTA polypeptide or an agent. that modulates the expression of PD-L3 or VISTA or at least one activity of PD-L3 or VISTA. Individuals at risk of

136

IMPI

Mexican INSTITUTE

<img file="MX342017B_D0108.tif" />

ΙΛΓ THE

INDUSTRIAL a disease or disorder that is caused or contributed by aberrant or unwanted expression or activity of PD-L3 or VISTA can be identified under, for example, any or a combination of diagnostic or prognostic assays as described in the present invention . Administration of a prophylactic agent may occur before the manifestation of the characteristic symptoms of PD-L3 or VISTA aberration, such that a disease or disorder is prevented or, alternatively, its progression is delayed. Depending on the type of PD-L3 or VISTA aberration, for example, a PD-L3 or VISTA polypeptide, PD-L3 or VISTA agonist, or PD-L3 or VISTA antagonist agent (for example, an anti-PD antibody -L3 or VISTA) can be used to treat the individual. The appropriate agent can be determined based on the screening tests described in the present invention.

two. Therapeutic methods

An important aspect of the invention corresponds to the methods for modulating the expression or activity or interaction of PDL-3 or VISTA with its natural binding partners. In relation to PD-L3 or VISTA therapy, it was shown to inhibit CD28 co-stimulation, inhibit TCR activation of immune cells, inhibit proliferation of activated immune cells (CD4 + and CD8 + T cells), inhibit production of cytokines by T cells (IL-2, gamma interferon) and transmit an inhibitory signal to immune cells. As a consequence, the expression and / or activity of PD-L3 or VISTA, as well as the interaction between PD-L3 or VISTA and its binding partner (s) in T cells can be modulated to modulate the immune response. Because PD-L3 or VISTA binds to inhibitory receptors (on T cells), up-regulation of PD-L3 or VISTA activity should result in down-regulation of the immune response, while down-regulation of the PD-L3 or VISTA activity should result in up-regulation of immune responses. In a preferred embodiment, PD-L3 or VISTA binds to inhibitory receptors. As noted above, contrary to expectations the specific antibodies against PD-L3 or VISTA produced by the applicant in that in vitro (in the presence of PD-L3 or VISTA-lg) they improve the suppression activities of PD fusion proteins. -L3 or VISTA137

ΙΜΡΙ®3 lg (that is, these antibodies enhance PDL-3 or VISTA suppression \ rélác1onádá | ~ ^^ T3á activities such as the effects of PD-L3 or VISTA on cytokine production, T-cell proliferation, Difference or activation and other functions as noted above), behave in the opposite way to what would be expected in vivo, that is, these antibodies were found to be immunosuppressive in vivo.

The modulatory methods of the invention involve contacting a cell with a PD-L3 or VISTA polypeptide or agent that modulates one or more of the activity activities of the PD-L3 or VISTA polypeptide associated with the cell, for example, an agent that modulates the expression or activity of PD-L3 or VISTA and / or modulates the interaction of PD-L3 or VISTA and its natural binding partner (s). An agent that modulates the activity of the PD-L3 or VISTA polypeptide may be an agent as described in the present invention, such as a nucleic acid or polypeptide, a naturally-occurring binding partner of a PD-L3 polypeptide, or VISTA, a PD-L3 or VISTA antibody, a PD-L3 OR VISTA agonist or antagonist, a peptidomimetic of a PD-L3 or VISTA agonist or antagonist, a PD-L3 or VISTA peptidomimetic or other small molecule. The soluble forms of PD-L3 or VISTA can also be used to interfere with the binding of PD-L3 or VISTA to any of its natural binding partners or ligands.

An agent that modulates the expression of PD-L3 or VISTA is, for example, an antisense nucleic acid molecule, triple oligonucleotide, ribozymes, or recombinant vector for the expression of a PD-L3 or VISTA polypeptide. For example, an oligonucleotide complementary to the area around the translation initiation site of the PD-L3 or VISTA polypeptide can be synthesized. One or more antisense oligonucleotides can be added to the cell medium, typically at 200 pg / ml, or administered to a patient to prevent synthesis of a PD-L3 or VISTA polypeptide. The antisense oligonucleotide is absorbed by cells and hybridized with a PD-L3 or VISTA mRNA to prevent translation. Alternatively, an oligonucleotide that binds to the double-stranded DNA can be used to form a triple construct that prevents unwinding and transcription of the DNA. As a result of either, the synthesis of the PD-L3 or VISTA polypeptide is blocked.

138

<img file="MX342017B_D0109.tif" />

IMPI

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

When the expression of PD-L3 or VISTA is preferentially modulated, said modulation occurs in a different way than the annulment of the yei i ^,üe ”PD-L3 or VISTA.

Agents that modulate expression, by virtue of the fact that they control the amount of PD-L3 or VISTA in a cell, also modulate the total amount of PD-L3 or VISTA activity in a cell. In one embodiment, the PD-L3 or VISTA modulating agent stimulates one or more PD-L3 or VISTA activities. Examples of such stimulatory agents include active PD-L3 or VISTA polypeptide and a nucleic acid molecule encoding PD-L3 or VISTA that was introduced into the cell. In another embodiment, the agent inhibits one or more PD-L3 or VISTA activities. Examples of such inhibitory agents include PD-L3 or VISTA antisense nucleic acid molecules, anti-PD-L3 or VISTA antibodies, PD-L3 or VISTA inhibitors, and compounds identified in screening tests of individuals. In a further preferred embodiment, an inhibitory agent is a combination of an anti-PD-L3 or VISTA antibody and an anti-PDL1 or anti-PD-L2 antibody. These modulatory methods can be performed in vitro (eg, by contacting the cell with the agent) or, alternatively, by contacting an agent with cells in vivo (eg, by administering the agent to an individual). As such, the present invention provides methods of treating an individual afflicted with a condition or disorder that benefits from positive or negative modulation of a PD-L3 or VISTA polypeptide, for example, a disorder characterized by non-expression or activity. desired, insufficient or aberrant of a PD-L3 or VISTA polypeptide or nucleic acid molecule. In one embodiment, the method involves administering an agent (eg, an agent identified by a screening assay described in the present invention), or combination of agents that modulate (eg, positively or negatively regulate) the expression or activity of PD-L3 or VISTA. In another embodiment, the method involves administering a PDL3 or VISTA polypeptide or nucleic acid molecule as therapy to compensate for the reduced, aberrant, or unwanted expression or activity of PD-L3 or VISTA.

139

Diseases of the individual that can be

<img file="MX342017B_D0110.tif" />

PD-L3 binding or VISTA are identified above and include various inflammatory, autoimmune, cancerous, allergic and infectious disorders. A particularly preferred indication is multiple sclerosis.

Stimulation of PD-L3 or VISTA activity is desirable in situations where PD-L3 or VISTA is abnormally downregulated and / or where increased PD-L3 or VISTA activity is likely to have an effect. beneficial. Similarly, inhibition of PD-L3 or VISTA activity is desirable in situations where PD-L3 or VISTA is abnormally upregulated and / or where decreased PD-L3 or VISTA activity is likely to have a beneficial effect. Illustrative agents for use in the negative modulation of PD-L3 or VISTA (i.e., PD-L3 or VISTA antagonists) include, for example, antisense nucleic acid molecules, antibodies that recognize and block PD-L3 or VISTA, combinations of antibodies that recognize and block PD-L3 or VISTA and antibodies that recognize and block against PD-L3 or VISTA receptors, and compounds that block the interaction of PD-L3 or VISTA with its naturally occurring binding partner (s) in an immune cell (e.g., soluble monovalent molecules, of PD-L3 or VISTA, soluble forms of molecules of PD-L3 or VISTA that do not bind to Fe receptors on antigen-presenting cells; soluble forms of PD-L3 or VISTA binding partners; and compounds identified in individual screening assays). Illustrative agents for use in the positive modulation of PD-L3 or VISTA (i.e., PD-L3 or VISTA agonists) include, for example, nucleic acid molecules encoding PD-L3 or VISTA polypeptides, multivalent forms of PDL3 or VISTA, compounds that increase the expression of PDL3 or VISTA, compounds that improve the interaction of PD-L3 or VISTA with its naturally occurring binding partners and cells that express PD-L3 or VISTA.

3. Negative regulation of immune responses

There are numerous embodiments of the invention for up-regulation of the inhibitory function of a PD-L3 or VISTA polypeptide to thereby down-regulate immune responses. Negative regulation can be

140

<img file="MX342017B_D0111.tif" />

<img file="MX342017B_D0112.tif" />

in the form of inhibiting or blocking an immune response and in involving that the induction of an immune response is prevented. The functions of activated immune cells can be inhibited by negatively regulating immune cell responses or by inducing specific anergy in immune cells, or both. For example, in embodiments where PD-L3 or VISTA binds to an inhibitory receptor, forms of PD-L3 or VISTA that bind to the inhibitory receptor, eg, multivalent PD-L3 or VISTA on a cell surface, can be used to negatively modulate the immune response. In one embodiment of the invention, an activating antibody used to stimulate PD-L3 or VISTA activity is a bispecific antibody. For example, such an antibody may comprise a PD-L3 or VISTA binding site and another binding site that targets a cell surface receptor on an immune cell, for example a T cell, a B cell, or a myeloid cell. . In one embodiment, said antibody, in addition to comprising a PD-L3 or VISTA binding site, may further comprise a binding site that binds to a B cell antigen receptor, a T cell antigen receptor, or an Fe receptor, to target the molecule to a specific cell population. The selection of this second antigen for the bispecific antibody provides flexibility in the selection of the cell population that is targeted for inhibition. Agents that promote PD-L3 or VISTA activity or enhance the interaction of PD-L3 or VISTA with its natural binding partners (eg, PD-L3 or VISTA activating antibodies or PD-L3 activating small molecules L3 or VISTA) can be identified by their ability to inhibit immune cell proliferation and / or effector function, or to induce anergy when added to an in vitro assay. For example, cells can be cultured in the presence of an agent that stimulates signal transduction through an activating receptor. A number of art-recognized readings of cell activation can be employed to measure, for example, cell proliferation or effector function (eg, antibody production, cytokine production, phagocytosis) in the presence of the activating agent. The ability of a test agent to block this activation can be easily determined by measuring the agent's ability to effect a decrease in proliferation or effector function being measured. In a

141

JLK j. κ<img file="MX342017B_D0113.tif" /> s; · '' A - Λ'Λ {; - £ ·. DV ^ ** ^ *** ^^ modality, at low antigen concentrations, interactions of Tak immune cells with PD-L3 or VISTA strongly inhibit B7-C.D9ft-eu * signals. - modality, at high antigen concentrations, interactions of immune cells with PD-L3 or VISTA can reduce cytokine production, but not inhibit T cell proliferation. Consequently, the ability of a test compound to block activation can be determined by measuring cytokine production and / or proliferation at different antigen concentrations.

In one embodiment of the invention, tolerance against specific antigens is induced by co-administration of an antigen with a PD-L3 agonist or VISTA. For example, tolerance can be induced to specific polypeptides. In one embodiment, immune responses to foreign allergens or polypeptides to which an immune response is undesirable can be inhibited. For example, patients receiving Factor VIII frequently generate antibodies against this clotting factor. Co-administration of an agent that stimulates PD-L3 or VISTA activity or interaction with its natural binding partner (s), with recombinant factor VIII (or physically binding PD-L3 or VISTA to Factor VIII, for example, by cross-linking) can result in negative modulation of the immune response.

In one embodiment, a PD-L3 or VISTA agonist and another agent that can block the activity of co-stimulatory receptors on an immune cell can be used to negatively modulate immune responses. Illustrative molecules include: agonist forms of other PD ligands, soluble forms of CTLA-4, anti-B7-1 antibodies, anti-B7-2 antibodies, or combinations of these. Alternatively, two separate peptides (eg, a PD-L3 or VISTA polypeptide with polypeptide forms that block B7-2 and / or B7-1), or a combination of antibodies (eg, activating antibodies against a PDL3 or VISTA polypeptide with monoclonal antibodies blocking anti-B7-2 and / or anti-B7-1) can be combined as a single composition or administered separately (simultaneously or sequentially) to down-regulate responses immune mediated by immune cells in an individual. Also, an amount

142

<img file="MX342017B_D0114.tif" />

<img file="MX342017B_D0115.tif" />

therapeutically active one or more peptides having a PD-L3 or VISTA polypeptide activity, in addition to one or more polypeptides having B7-1 and / or B7-1 activity, can be used in conjunction with other negative modulating reagents to influence immune responses. Examples of other negative modulating reagents include antibodies that block a co-stimulatory signal (eg, against CD28 or ICOS), antibodies that activate an inhibitory signal through CTLA4, and antibodies against other immune cell markers (eg, against CD40, CD40 ligand, or cytokines), fusion proteins (eg, CTLA4-Fc or PD-1-Fc), and immunosuppressive drugs (eg, rapamycin, cyclosporin A, or FK506). PD-L3 or VISTA polypeptides may also be useful in the construction of therapeutic agents that block immune cell function by killing cells. For example, parts of a PD-L3 or VISTA polypeptide can be linked to a toxin to prepare a cytotoxic agent capable of triggering the destruction of the cells to which it binds.

To prepare cytotoxic agents, the polypeptides of the invention can be linked, or operably linked, to toxins using procedures that are known in the art. A wide variety of toxins are known that can be conjugated to the polypeptides or antibodies of the invention. Examples include: toxins derived from numerous useful plants, fungi, or even bacteria, which, by way of example, include: various A-chain toxins in particular ricin A-chain; ribosome inactivation proteins such as saporin or gelonin; alpha-sarcin; aspergilline; restrictocin, and ribonucleases such as placental, angiogenic ribonuclease, diphtheria toxin, or pseudomonas exotoxin. A preferred region of toxin for use in connection with the invention, the deglycosylated A chain, is the A chain of the toxin that was treated to modify or remove carbohydrate residues. (US Patent No. 5,776,427).

Infusion of one or a combination of such cytotoxic agents (for example, PD-L3 or VISTA-ricin (alone or in combination with PD-L1-ricin), into a patient can result in the death of immune cells, in particular to

143

IMPIgS * thOTryr / ... &lt;WCANO a consequence of the fact that activated immune cells express higher amounts of PD-L3 or VISTA binding partners. For example, because PD-1 is induced on the surface of activated lymphocytes, a PD-L3 polypeptide or

VISTA can be used to direct the depletion of these specific cells by Fc-R dependent mechanisms or by ablation by conjugating a cytotoxic drug (eg, ricin, saporin, or calicheamicin) to the PD-L3 polypeptide or

SIGHT. In another embodiment, the toxin can be conjugated to an anti PD-L3 or VISTA antibody to target the death of the antigen presenting cell expressing PD-L3 or VISTA. In an additional mode, PD-L3 or

VISTA-antibody-toxin can be a bispecific antibody. Such bispecific antibodies are useful for targeting a specific cell population, for example, using a marker that is only found on a certain cell type, for example B lymphocytes, monocytes, dendritic cells, or Langerhans cells. The immune responses that negatively regulate by activating the Activity of PD-L3 or VISTA or the interaction of the immune cell with PDL3 or VISTA (and thus stimulating the negative signaling function of PD-L3 or VISTA) is useful in modulation negative immune response, for example in tissue, skin and organ transplantation situations, graft versus host disease (GVHD), or allergies, or in autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis. For example, blocking immune cell function results in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant begins through its recognition as foreign by immune cells, followed by an immune reaction that destroys the transplant. Administration of a molecule that promotes the activity of PD-L3 or VISTA, or the interaction of PD-L3 or VISTA with its natural binding partner (s), can inhibit the generation of a costimulatory signal in cells. immune (such as a soluble, multimeric form of a PD-L3 or VISTA polypeptide) alone or in conjunction with another negative modulating agent before or at the time of transplantation. Furthermore, the promotion of PDL3 or VISTA activity may also be sufficient to anergize immune cells, thereby inducing tolerance in an individual.

144

<img file="MX342017B_D0116.tif" />

To achieve sufficient immunosuppression or tolerance in an individual, it may also be desired to block the co-stimulatory function of other molecules. For example, one may wish to block the function of B7-1 and B7-2 by administering a soluble form of a combination of peptides having an activity for each of these antigens or by blocking antibodies against these antigens (separately or together in a single composition. ) before or at the time of transplantation. Alternatively, it may be desirable to promote the inhibitory activity of PD-L3 or VISTA and inhibit a co-stimulatory activity of B7-1 and / or B7-2. Other negative modulating agents that can be used in connection with the negative modulating methods of the invention include, for example, agents that transmit an inhibitory signal through CTLA4, soluble forms of CTLA4, antibodies that activate an inhibitory signal through CTLA4, blocking antibodies against other immune cell markers, or soluble forms of other receptor ligand pairs (for example, agents that alter the interaction between CD40 and CD40 ligand (eg, ligand anti-CD40 antibodies)), antibodies against cytokines, or immunosuppressive drugs. For example, the activating activity of PD-L3 or VISTA or the interaction of PD-L3 or VISTA with its natural binding partner (s) is useful in the treatment of autoimmune disease. Many autoimmune disorders are the result of inappropriate activation of immune cells that are reactive against the tissue itself and that promote the production of cytokines and autoantibodies involved in the pathology of diseases. Preventing the activation of autoreactive immune cells can reduce or eliminate the symptoms of the disease. The administration of agents that promote the activity of PD-L3 or VISTA or the interaction of PD-.L3 or VISTA with its natural binding partner (s), can induce antigen-specific tolerance of autoreactive immune cells that could lead long-term to disease relief. Furthermore, the co-administration of agents that block the co-stimulation of immune cells by altering the receptor-ligand interactions of B7 molecules with co-stimulatory receptors may be useful in inhibiting the activation of immune cells to prevent the production of autoantibodies or cytokines. that can be involved in the disease process. The efficacy of reagents in preventing or alleviating disorders

145 Autoimmune can be determined using a number of Hren ^ * anim models

INNs characterized by human autoimmune diseases, experimental murine autoimmune encephalitis, systemic lupus erythematosus in MRL / lpr / lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and experimental murine myasthenia gravis (see Paul editor, Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Inhibition of immune cell activation is therapeutically useful in treating allergies and allergic reactions, for example, by inhibiting IgE production. An agent that promotes PD-L3 or VISTA activity or interaction of PD-L3 or VISTA with its natural binding partner (s) may be administered to an allergic individual to inhibit immune cell-mediated allergic responses in the individual. Stimulation of PD-L3 or VISTA activity or interaction with its natural binding partner (s) can be accompanied by allergen exposure in conjunction with appropriate MHC molecules. Allergic reactions can be systemic or local in nature, depending on the route of entry of the allergen and the pattern of deposition of IgE in mastoid or basophil cells. Thus, immune cell-mediated allergic responses can be inhibited locally or systemically by administration of an agent that promotes PD-L3 or VISTA activity or PD-L3 or VISTA-immune cell interactions.

Inhibition of immune cell activation through stimulation of PD-L3 or VISTA activity or interaction of PD-L3 or VISTA with its natural binding partner (s), may also be therapeutically important in pathogenic infections of immune cells (for example, by viruses or bacteria). For example, in acquired immunodeficiency syndrome (AIDS), viral replication is stimulated by the activation of immune cells. Stimulation of PD-L3 or VISTA activity can result in inhibition of viral replication and thereby ameliorate the course of AIDS.

Negative regulation of an immune response through stimulation of PD-L3 or VISTA activity or interaction of PD-L3 or VISTA with its (s) 146

<img file="MX342017B_D0117.tif" />

natural binding partner (s) may also be useful in treating an autoimmune attack of autologous tissues. Thus, conditions 'that are caused or' exacerbated by autoimmune attack (eg, heart disease, myocardial infarction, or atherosclerosis) can be corrected or ameliorated by increasing the activity of PD-L3 or VISTA or binding of PD-L3 or SEE your natural bonding partner. It has been addressed within the scope of the invention, therefore, to modulate conditions exacerbated by autoimmune attack, such as autoimmune disorders (as well as conditions such as heart disease, myocardial infarction and arteriosclerosis), stimulating the activity of PD- L3 or VISTA or interaction of PDL3 or VISTA with its counter receiver.

Four. Positive regulation of immune responses

Inhibition of PD-L3 or VISTA activity or interaction of PD-L3 or VISTA with its natural binding partner (s), is also useful in therapy as a means of positive regulation of the immune response. Positive regulation of the immune response can be in the form of enhancing an existing immune response or producing an initial immune response. For example, enhancing an immune response through inhibition of PD-L3 or VISTA activity is useful in cases of infections with microbes, eg, bacteria, viruses or parasites, or in cases of immunosuppression. For example, in one embodiment, an agent that inhibits the activity of PD-L3 or VISTA, for example, an antibody that does not activate (i.e., a blocking antibody) against PD-L3 or VISTA, or a soluble form of PD-L3, or VISTA, is therapeutically useful in situations where up-regulation of antibody and cell-mediated responses, resulting in more rapid or complete clearance of a virus, bacterium, or parasite, may be beneficial. These conditions include viral skin diseases such as Herpes or shingles, in which case the agent can be administered topically to the skin. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis can be alleviated by the systemic administration of such agents. In certain cases, it may also be desired to administer other agents that positively regulate immune responses, for example,

147

I .Μ ΡI

INSTITUTO MEXICANO D £ Ι.Λ rtOi IEOAD Cv. »'Forms of B7 family members that transduce point ^^' itaveS ^ ee² co-stimulatory receptors, to also increase the immune response

Alternatively, immune responses can be enhanced in an infected patient by killing the patient's immune cells, contacting the immune cells in vitro with an agent that inhibits the activity of PD-L3 or VISTA or interaction of PD-L3 or VISTA with their (s ) natural binding partner (s), and reintroducing stimulated immune cells into the patient in vitro. In another embodiment, a method of enhancing immune responses involves isolating infected cells from a patient, eg, virus-infected cells, transfecting them with a nucleic acid molecule that encodes a form of PD-L3 or VISTA that cannot bind to their natural binding partner (s), such that the cells express all or part of the PD-L3 or VISTA molecule on their surface, and reintroduce the transfected cells into the patient. Transfected cells may be able to prevent an inhibitory signal, and thereby cells activate immune cells in vivo.

An agent that inhibits the activity of PD-L3 or VISTA or interaction of PD-L3 or VISTA with its natural binding partner (s), can be used prophylactically in vaccines against various polypeptides, for example, polypeptides derived from pathogens. . Immunity against a pathogen, eg, a virus, can be induced by vaccination with a viral polypeptide together with an agent that inhibits the activity of PD-L3 or VISTA, in an appropriate adjuvant. Alternatively, a vector comprising genes encoding both a pathogenic antigen and a form of PD-L3 or VISTA that blocks the interaction of PD-L3 or VISTA with immune cells can be used for vaccination. Nucleic acid vaccines can be administered by a variety of means, for example, by injection (eg, Intramuscular, intradermal, or the biolistic injection of DNA-coated gold particles into the epidermis with a gene gun using an accelerator. of particles or compressed gas to inject the particles into the skin (Haynes et al. (1996) J. Biotechnol. 44:37)). Alternatively, nucleic acid vaccines can be administered non-invasively. For example, pure or lipid-formulated DNA can be delivered to the system

148

<img file="MX342017B_D0118.tif" />

or direct to another site, eg Peyer's patches by oral administration of DNA (Schubbert (1997) Proc Nati. Acad-. Sui. UOA 94.001) r Attenuated microorganisms can be used for administration to mucosal surfaces Sizemore et al. (1995) Science 270: 29).

In another embodiment, the antigen in the vaccine is a self-antigen. Said vaccine is useful in modulating the tolerance of an organism. Immunization with a self-antigen and an agent that blocks the activity of PD-L3 or VISTA or interaction of PD-L3 or VISTA with its natural binding partner can break tolerance (i.e., interfere with the tolerance of a self-antigen ). Such a vaccine can also include adjuvants such as alum or cytokines (eg, GM-CSF, IL-12. B7-1, or B7-2). In one embodiment, an agent that inhibits PD-L3 or VISTA activity or interaction of PD-L3 or VISTA with its natural binding partner (s), can be administered with MHC class I polypeptides, for example , a cell transfected to co-express a PD-L3 or VISTA polypeptide or blocking antibody and the MHC class I polypeptide chain alpha, and microglobulin beta 2 that result in the activation of T cells and provide immunity from the infection. For example, viral pathogens for which vaccines are useful include: hepatitis B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria, and schistosomiasis.

In another application, inhibition of PD-L3 or VISTA activity or interaction of PD-L3 or VISTA with its natural binding partner (s) may be useful in treating tumor immunity. Tumor cells (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, or carcinoma) can be transfected with a nucleic acid molecule that inhibits the activity of PD-L3 or VISTA. These molecules can be, for example, nucleic acid molecules that are antisense to PD-L3 or VISTA, or they can encode anti-PD-L3 or VISTA antibodies that are not activated. These molecules can also be the variable region of an anti-PD-L3 or VISTA antibody. If desired, tumor cells can be transfected with other polypeptides that activate co-stimulation (eg, B7-1 or B7-2). Transfected tumor cells are returned to the patient, resulting in inhibition (eg, local inhibition) of PD-L3 activity or

149

I

INSTITUTE

OF THE PROI'IEOAD

INDUSTRIAL

<img file="MX342017B_D0119.tif" />

SIGHT. Alternatively, gene therapy techniques can be used to target a tumor cell for in vivo transfection.

Stimulation of an immune response to tumor cells can also be achieved by inhibiting the activity of PD-L3 or VISTA or interaction of PD-L3 or VISTA with its natural binding partner (s) by treating a patient with an agent that inhibits the activity of PD-L3 or VISTA or interaction of PD-L3 or VISTA with its natural binding partner (s). Preferred examples of such agents include, for example, antisense nucleic acid molecules, antibodies that recognize and block PD-L3 or VISTA, and compounds that block the interaction of PD-L3 or VISTA with its partner (s). naturally occurring binding partners in an immune cell (eg, soluble monovalent PD-L3 or VISTA molecules; soluble forms of PD-L3 or VISTA molecules that do not bind to Fe receptors on antigen-presenting cells; soluble forms of PD-L3 or VISTA binding partner (s) and compounds identified in individual screening assays). In addition, tumor cells that lack MHC class I or MHC class II molecules, or fail to express sufficient amounts of MHC class I or MHC class II molecules can be transfected with the nucleic acid that encodes all or part of ( for example, a part of the cytoplasmic-truncated domain) of an MHC class I alpha chain polypeptide and microglobulin beta2 polypeptide or an MHC class II alpha chain polypeptide and an MHC class II beta chain polypeptide to thereby express MHC polypeptides class I or MHC class II on the cell surface. Expression of appropriate MHC class I or II in conjunction with a PD-L3 or VISTA inhibiting polypeptide or antisense nucleic acid induces a T cell-mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct that blocks expression of an MHC class II-associated polypeptide, such as the invariant chain, can also be co-transfected with a DNA encoding a polypeptide that inhibits PD-L3 or VISTA or nucleic acid or antisense to promote the presentation of tumor associated antigens and induce tumor specific immunity. The expression of B7-1 by murine B7-negative tumor cells was shown to induce specific T-cell-mediated immunity accompanied by

150 cr LA ^V!.: AL) tumor rejection and prolonged protection to tumor challenge in ratóhés {Chéni't. et al. (1992) Cell 71: 1093-1102; Townsend, SE and AllisQjxJ ^ P.41993i) .- Science— 259: 368-370; Baskar, S. et al. (1993) Proc Nati. Acad. Sci. 90: 5687-5690). Thus, the induction of an immune response mediated by immune cells in a human individual may be sufficient to overcome the tumor-specific tolerance in the individual. In another embodiment, the immune response can be stimulated by inhibition of PD-L3 or VISTA activity or interaction of PD-L3 or VISTA with its natural binding partner (s), such that pre-tolerance is exceeded. -existing. For example, immune responses against antigens for which an individual cannot increase a significant immune response, for example tumor-specific antigens, can be induced by administering an agent that inhibits PD-L3 or VISTA activity or PD- capacity. L3 or VISTA to bind with its natural binding partner, can be used as adjuvants to increase responses to foreign antigens in the active immunization process.

In one embodiment, immune cells are obtained from an individual and cultured ex vivo in the presence of an agent that inhibits the activity of PD-L3 or VISTA or interaction of PD-L3 or VISTA with its partner (s) of natural binding, to expand the immune cell population. In a further embodiment, the immune cells are then administered to an individual. Immune cells can be stimulated to proliferate in vitro, for example, by providing immune cells with a primary activation signal and a co-stimulatory signal, as is known in the art. Various forms of PD-L3 or VISTA polypeptide or agents that inhibit PD-L3 or VISTA activity can also be used to co-stimulate proliferation of immune cells. In one embodiment, the immune cells are cultured ex vivo according to the methods described in PCT application no. WO 94/29436. The co-stimulatory molecule can be soluble, bound to a cell membrane, or bound to a solid surface, such as a bead.

In a further embodiment, performing any of the methods described in the present invention, it is within the scope of the invention to positively regulate an immune response by administering one or more additional agents. For example, the use of other known agents to stimulate the immune response,

151

<img file="MX342017B_D0120.tif" />

Such as cytokines, adjuvants, or stimulatory forms of costimulatory molecules or their ligands can be used in conjunction with an agent that inhibits the activity of PD-L3 or VISTA or interaction of PD-L3 or VISTA with its partner (s) of natural union.

E. Identification of cytokines modulated by modulation of PD-L3 or VISTA activity or interactions of PD-L3 or VISTA with its counter-receptor in T cells.

The PD-L3 or VISTA molecules described in the present invention can be used to identify cytokines that are produced by or whose production is enhanced or inhibited in immune cells in response to modulation of PD-L3 or VISTA activity or interaction. PD-L3 or VISTA with your natural bonding partner (s). Immune cells can be stimulated in vitro below the potymus with a primary activation signal, for example, T cells can be stimulated with phorbol ester, anti-CD3 antibody or preferably antigen, in association with an MHC molecule class II and given a co-stimulatory signal, for example, by a stimulatory form of the B7 family antigen, for example by a cell transfected with nucleic acid encoding a B7 polypeptide and expressing the peptide on its surface, or by a soluble stimulatory form of the peptide. The cells can then be contacted with cells expressing PD-L3 or VISTA (eg, antibodies against PD-L3 or VISTA). Known cytokines released into the media can be identified by ELISA or by the ability of an antibody that blocks the cytokine to inhibit the proliferation of immune cells or the proliferation of other cell types that are induced by the cytokine. For example, an IL-4 ELISA kit available from Genzyme (Cambridge, Massachusetts), as is an IL-7 blocking antibody. Blocking antibodies against IL-9 and IL-12 are available from the Institute of Genetics (Cambridge, Massachusetts). The effect to stimulate or block PD-L3 or VISTA activity or the interaction of PD-L3 or VISTA and its binding partner (s) on the cytokine profile can then be determined. As noted above and shown in the examples, PD-L3 or VISTA apparently suppresses the expression of interferon IL-2 and gamma by immune cells.

152

I Μ ΡI institute * ⁱ . · Η

DE LA? I (OPk '-. V &lt;í V \ ~ _t »AÍF

An in vitro immune cell co-stimulation assay described above can also be used in a method to identify new cytokines that can be modulated by modulation of PD-L3 or VISTA activity. For example, where stimulation of the CD28 / CTLA4 pathway appears to enhance IL-2 secretion, stimulation of the ICOS pathway appears to increase IL-10 secretion (Hutloff et al. (1999) Nature 397: 263). If a particular activity after co-stimulation induced, for example, immune cell proliferation, it cannot be inhibited by adding blocking antibodies to known cytokines, the activity may result from the action of an unknown cytokine. Following co-stimulation, this cytokine can be purified from the media by conventional methods and its activity measured by its ability to induce proliferation of immune cells.

To identify cytokines that may play a role in tolerance induction, an in vitro T cell co-stimulation assay can be used as described above. In this case, the T cells can be given the primary activation signal and contact a selected cytokine, but cannot give the co-stimulatory signal. After the immune cells are washed and rested, they can be challenged again with both a primary activation signal and a co-stimulatory signal. If the immune cells did not respond (eg, proliferate or produce cytokines) they became tolerant and the cytokine did not prevent the induction of tolerance. However, if the immune cells respond, the induction of tolerance was prevented by the cytokine. Those cytokines that are capable of inhibiting tolerance induction can be blocked in vivo in conjunction with reagents that block B lymphocyte antigens as a more efficient means of inducing tolerance in transplant recipients or individuals with autoimmune diseases. For example, a cytokine that blocks an individual's antibody could be administered in conjunction with an agent that promotes PD-L3 or VISTA activity or interaction of PD-L3 or VISTA with a binding partner.

Thus, to summarize we have now identified a new member of the Programmed Death Ligand (PDL) family that is expressed by Treg cells. This

153 new protein was designated PD-L3 or VISTA. The receivers

ΙΜΡΙβ ^ Χ

MEXICAN INSTITUTE AND -^J &lt;'j

OF THE ITOI-I. ' J / JJ \ of this PD-L plant are type I transmembrane proteins that contain a single IgV domain, while the ligands are type I transmembrane proteins that express both an IgV and an IgC extracellular domain. Similar to other members of the PDL family, PD-L3 or VISTA co-stimulates in vitro proliferation of aCD3 from T cells. Furthermore, the expression of PD-L3 or VISTA is increased in activated aCD3 Treg and reduced in the presence of aGITR.

A second protein, similar to TNF, was also identified as up-regulated after aCD3 / aGITR stimulation. This protein was designated Treg-sTNF. These proteins may be involved in contact and paracrine dependent suppression of immunity and are therefore useful for modulating (eg, inhibiting or stimulating) an immune response and in the treatment of diseases and conditions involving Treg signaling. For example, the PD-L3 or VISTA protein can be used as a co-stimulatory signal to stimulate or enhance immune cell activation. PDL-3 or VISTA proteins and PD-L3 or VISTA binding agents and PD-L3 or VISTA agonists and antagonists are especially useful in the treatment of immune conditions where regulation of T-cell immunity is desired, for example. example, modulation of T cell activation, differentiation and proliferation, and in particular modulation of CD4 + and CD8 + T cell proliferation, production of cytokines, and T cell responses during analogous interactions between T cells and myeloid derived APCs.

This invention is further illustrated by the following examples, which are not to be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures and Sequence Listing, are incorporated into the present disclosure by reference.

Examples

The following Materials and Methods were used in the examples that follow:

Materials and methods

154

<img file="MX342017B_D0121.tif" />

INSTITUTO MEXICANA DE LA PROPitPAD INDUSTRIAL

<img file="MX342017B_D0122.tif" />

Xpr sion profiles

To facilitate comparisons with established Treg cell expression profiles, standard growth and activation conditions were employed (McHugh, et al. (2002) above). In summary, new isolated Treg cells (-96% positive) were inoculated at 106 / ml in complete RPMI medium supplemented with 10% fetal bovine serum and 100 units of IL-2 in a 24-well plate precoated with anti-CD3 with or without anti-GITR (DTA-I) (Shimizu, et al. (2002) above). The cells were cultured at 37 ° C for 0 and 12 hours, the RNA was purified and subsequently analyzed using an array of A430 oligonucleotides from the Affymetrix® mouse genome.

Comparing data from the resting or activated CD4 + CD25 + T cell pools, gene expression patterns were found to be similar to those established in the art (Gavin, et al. (2002) above; McHugh, et al. (2002) above ). To identify genes regulated by GIRT signaling, gene expression profiles were compared between different cell populations with or without anti-GITR treatment. A list of known as well as unknown genes were compiled including previously uncharacterized PD-L3 or VISTA and Treg-sTNF.

Mice

C57BL / 6 mice, and CD4 OTII transgenic mice were purchased from the Jackson Laboratory. FoxP3-GFP knock-in reporter mice were previously described by Fontenot, JD, Rasmussen, JP, Williams, LM, Dooley, JL, Farr, AG, and Rudensky, AY (2005). Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22, 329-341 and were generously provided by Alexander Rudensky, University of Washington School of Medicine, Seattle, Washington. KO PD-1 mice were generously provided by Dr. Tasuku Honjo (Kyoto University, Japan) Nishimura, H., Nose, M., Hiai, H., Minato, N., and Honjo, T. (1999). Development of lupus-like autoimmune disease by disruption of the PD-1 gene encoding and ITIM motif-carrying immunoreceptor. Immunity 11, 141-151; Nishimura, H., Okazaki, T. Tanaka, Y,. Nakatani, K., Hara, M., Matsumori, A., Sasayama, S., Mizoguchi, A.,

155

ΜΡΙ

INSTITUTO MEXICANO DE LA FKÜHl I 'AD INDUSTRIAL

Hiai, H., Minato, N., and Honjo, T. (2001). Autoimmune dilated cardiomvoDathv in PD-1 receptor-deficient mice. Science 291, 319-322. All animals were kept in a pathogen-free facility at Dartmouth School of Medicine.

Acs, cell lines, and reagent:

The antibodies aCD3 (2C11), aCD28 (PV-I), aCD4 (GK1.5). aCD8 (53-6.7), aCD11b (M1 / 70), aF4 / 80 (BM8), aCD11c (N418), αΝΚ1.1 (PK136), aGrl (RB6-8C5), aPD-L1 (MIN5), aPD-L2 (TY25), aB7-H3 (M3.2D7), aB7-H4 (188) were purchased from Bioscience. LPS (Sigma), recombinant murine IFNG (Peprotech), human IL-2 (Peprotech), soluble PD-L1-lg fusion protein (R&amp;D Systems) were used at the indicated concentrations. Freund's complete adjuvant (CFA) and chicken ovalbumin (OVA) were purchased from Sigma. The CHO cell line expressing the MHC II l-Ad molecule and co-stimulatory molecule B7-2 was kindly provided by Dr. Arlene Sharpe (Harvard Medical School).

Molecular cloning of PD-L3 or VISTA, production of retroviru and retroviral transduction of cells

PD-L3 or whole VISTA was cloned from purified murine CD4 + T cells. Total RNA was isolated from CD4 + T cells using the Qiagen RNAminit kit. The cDNA was generated using the Bio-Rad iScript ™ cDNA Synthesis Kit. PD-L3 or full-length VISTA was amplified and cloned into the EcoRI-Xhol sites of a retroviral vector pMSCV-IRESGFP Zhang. X., and Ren, R. (1998) .Bcr-Abl efficiently induces a myeloproliferative disease and production of excess interleukin3 and granulocyte-macrophage colony stimulating factor in mice: a novel model for chronic myelogenous leukemia. Blood 92,3829-3840, in which the IRES-GFP fragment was replaced by RFP, thus resulting in a PD-L3 or VISTA fusion protein fused to the N-terminus of RFP. Free helper retroviruses were generated in HEK293T cells by transient transfection of the retroviral vector PD-L3 or VISTA-RFP together with an ecotrophic packaging vector pCL-Eco (IMGENEX corp.). Retroviral transduction of murine T cell line EL4 cells, or bone marrow derived DCs were carried out by centrifuging the infection at 2000 rpm for 45 min at RT in the presence of 8 pg / ml of polybrene (Sigma).

<img file="MX342017B_D0123.tif" />

156

Production of protein d fu ion PD-L3 or VISTA-Ig

<img file="MX342017B_D0124.tif" />

INSTITUTE M'-XiCAMO

OF THE INDUSTRIAL RCHEDAD

<img file="MX342017B_D0125.tif" />

The extracellular domain of PD-L3 or VISTA (amino acids 32-190) was amplified and cloned into the Spel-BamHI sites of the parental vector CDM7B Hollenbaugh, D. Douthwright, J., McDonald, V., and Aruffo, A. (1995 ). J Immunol Methods 188, 1-7. This vector contains the mutant form of the constant and hinge regions of human IgG1, which greatly reduced binding to Fe receptors. The resulting vector CDM7B-PD-L3 or VISTA was co-transfected with a DHFR expression vector pSVdhfr (Mclvor, RS, and Simonsen, CC (1990)). Nucleic Acids Res 18,7025-7032 in the CHO (dhfr-) cell line (ATCC # CRL-9096). Stable CHO cell clones expressing PD-L3 or VISTA-lg were selected in nucleotide-free MEM-alpha medium (Invitrogen). Further amplification with 0.5-1 μΜ methotrexate (Sigma M9929) yielded clones expressing high levels of soluble PDL3 or VISTA-lg fusion protein. The fusion protein was further purified from the culture supernatant using standard protein G column affinity chromatography.

Generation of PD-L3 OR VISTA monoclonal antibodies

Armenian hamsters were immunized 4 times weekly with EL4 cells overexpressing PD-L3 or VISTA-RFP, then boosted with CFA-emulsified PD-L3 or VISTA-lg fusion protein. Four weeks after the boost, the hamsters were boosted again with the soluble PDL3 or VISTA-Ig fusion protein. Four days after the last boost, the hamster spleen cells were harvested and fused with the myeloma cell line SP2 / 0-Agl4 (ATCC # CRL-1581) using standard hybridoma fusion techniques Shulman, M., Wilde, CD , and Kohler, G. (1978). A better cell line for making hybridomas secreting specific antibodies. Nature 276, 269-270. Hybridoma clones secreting PD-L3 OR VISTA specific antibodies were selected after limiting dilution and screened by both ELISA and flow cytometry methods.

RNA and RT-PCR

Total RNA from various mouse tissue samples or purified hematopoietic cell types were collected using the TrizolTM method.

157

<img file="MX342017B_D0126.tif" />

(Invitrogen) following the instructions of the companies. The cDNAs were prepared

Using the ¡ScriptTM cDNA Synthesis Kit (Bio-I

CDNAs (10ng) from tissue were used by RT-PCR reactions to amplify the entire PDL3 or VISTA. The PCR products were observed after running through a 1% agarose gel.

Flow cytometry

Flow cytometric analysis was performed on the FACSCAN using the CelIQuest program (BD Bioscience). Data analysis was performed using the FlowJo program (Treestar).

Cell preparation

CD4 + T cells were isolated from naive mice using the total CD4 + T cell isolation kit (Miltenyi). When indicated, the enriched CD4 + T cells were flow-separated into naive (CD441ow CD25-CD62Lhi) and memory (CD44hi CD25-CD62Llow) populations. For in vitro proliferation assays, CD4 + T cells were labeled with 5 µΜ of CFSE (Molecular Probes) for 10 minutes at 37C, and washed twice before being stimulated.

In vitro plaque-bound T-cell activation assay

Purified CD4 + T cells (100,000 cells per well) were cultured in 96-well flat-bottom plates in the presence of anti-CD3 (clone 2C11) and either PD-L3 or VISTA-lg or control-lg in the ratios indicated concentration. For example, for a wide range titration, 96-well plates were coated with 2.5 µg / ml of aCD3 mixed together with 1.25 µg / ml (2: 1 ratio),

2.5 pg / ml (1: 1 ratio), 5 pg / ml (1: 2 ratio), or 10 yg / ml (1: 4 ratio) of PD-L3 protein or VISTA-lg or control-lg in PBS at 4 ° C overnight. The wells were washed 3 times with PBS before adding the CD4 + T cells. The replicate cultures were in complete RPMI1640 medium supplemented with 10% FBS, 10 mM HEPES, 50 and M β-ΜΕ, penicillin / streptomycin / Lglutamine. When indicated, either 100 U / ml human IL-2 (PeproTech) or titrated amount of CCD28 (clone PV-1, cell Βίο X) were coated along with CCD3

158

<img file="MX342017B_D0127.tif" />

to rescue the inhibitory effects of PD-L3 or VISTA-lg. Cultures were analyzed on day 3 by CFSE profiles or as indicated according to kinetics.

Culture of bone marrow-derived DC, retroviral transduction, and stimulation of transgenic CD4 + T cells

Bone marrow-derived DCs were generated as described by Lutz, MB, Kukutsch, N., Ogilvie, AL, Rossner, S., Koch, F., Romani, N., and Schuler, G. (1999). An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 223, 77-92; They are, YI, Egawa, S., Tatsumi, T., Redlinger, RE, Jr., Kalinski, P., and Kanto, T. (2002). A novel bulk culture method for generating mature dendritic cells from mouse bone marrow cells. J. Immunol. Methods 262, 145-157, with some modifications. Briefly, on day 0, bone marrow cells were isolated from the tibia and femur by washing them with a 27G needle. After lysis of the red blood cells, 1-2 x 106 bone marrow cells were resuspended in 1 ml of RPMI 1640 complete medium containing 20 ng / ml of GM-CSF (Peprotech Inc.), in cell culture plates. of 6x wells (Nunc, Inc.). 2 ml of supernatant containing either retrovirus RFP or PD-L3 or VISTA-RFP was added to the bone marrow cells. Polybrene (Sigma) was also added at a final concentration of 8 pg / ml. The infection was carried out by centrifuging the plate at 2000 rpm for 45 min at RT. Fresh medium was added before the cells were then cultured for another 2 hours. The similar infection procedure was repeated on day + 1, day + 3, day + 5, and day + 7. Slightly adherent cells (90% are CD11c +) were harvested on day 10+ and CD11c + RFP + cells were double positive and used to stimulate OT-II transgenic CD4 + T cells. For OT-II T cell proliferation assays, 100,000 CFSE-labeled CD4 + OT-II T cells were cultured in round bottom 96-well plates with 30,000 BMDC rated RFP + or PD-L3 or VISTA-RFP +, in the presence of the amount Titrated synthetic peptide OVA323-339 (Anaspec). The proliferation of OT-II T cells was analyzed within 72 hours by examining the CFSE profiles.

PD-L3 or VISTA expression studies in response to immunization

159

INSTITUTO MEXIC. '. H'C F - / |

To immunize DO 11.10 transgenic mice, 300 ^ WOUSA \ SigffifiHe emulsified with CFA (200 μΙ), and injected subcutaneously into the flanks of the mice. The desiccated and non-desiccated inguinal lymph nodes were harvested at the indicated time intervals. Cell suspensions alone were prepared and analyzed by flow cytometry for the expression of PD-L3 or VISTA and other surface markers.

PD-L3 or VISTA inhibitory activity

The inhibitory activity of PD-L1 was revealed using antigen-presenting cells that in vitro overexpress PD-L1 with the CD4 + and CD8 + T-cell antigen receptor of transgenic T cells and antigenic stimulation (Carter, et al. (2002 ) Eur. J. Immunol. 32: 634-43). In the same way, the lentivector described in the present invention, which expresses the complete PD-L3 or VISTA, is transduced in cell lines that express the major histocompatibility complex (MHC) class II and MHC class I. The response of TEa- Tg or 2C transgenic T cells to antigen presented by empty transduced vector or PD-L3 or VISTA-transduced antigen presenting cells is determined according to established methods.

Protein Expression. Expression patterns in lymphoid, monocyte, and dendritic cell subsets, as well as non-hematopoietic tissues, were determined by RT-PCR and immunoblot analysis using standard protocols in conjunction with a rabbit antibody to aPD-L3 or VISTA described in present invention.

Production of monoclonal antibodies. PD-L3 or VISTA is overexpressed in murine B cell line A20, and the recombinant cell line was used to immunize Armenian hamsters. After 5x cell immunization, hamsters were boosted with purified PD-L3 or VISTA-Ig fusion protein emulsified with CFA. Four weeks later, a final boost was provided with PD-L3 or VISTA-lg. Subsequently, fusions of hamster splenocytes with SP2 / 0 cells were performed on day 4. Sixteen different clones were identified that recognize by ELISA the fusion protein PD-L3 or VISTA-lg, as well as PD-L3 or VISTA stained but no PD-L1 overexpressed in the cell line

160

<img file="MX342017B_D0128.tif" />

Murine t ΕΜ. Eleven of the clones were subcloned with

<img file="MX342017B_D0129.tif" />

evaluation of its ability to stain endogenous PD-L3 or VISTA in cells and tissues, and to block the functions of PD-L3 or VISTA.

Proliferation assays:

In vitro CD4 T cell proliferation assays were designed to screen for the activity of the Mab of PD-L3 or VISTA. In this assay, T cells were stimulated with immobilized anti-CD3 in microplate wells, which cross-links T cell receptors. Using a PD-L3 or VISTA-Ig fusion protein, which is composed of the extracellular domain of PD- L3 or VISTA fused with the Fe part of human IgG, the activity of the mAb of PD-L3 or VISTA was detected in two different configurations. First, when mAb was co-immobilized with aCD3, it potently inhibited T cell proliferation, only in the presence of added soluble PD-L3 or VISTA-Ig fusion protein. This activity was dependent on the ability of PD-L3 or VISTA-lg to bind to the mAb immobilized in the well. Using this form of assay, clones that had high, intermediate, or low suppressive activity were identified. Second, when mAb was added as a soluble reagent for the assay, it exerted potent suppressive activity on T cell proliferation, synergizing with the immobilized PD-L3 or VISTA-Ig fusion protein. In this form of assay, clones were identified that had different suppressive activities.

EXAMPLE 1: Cloning and sequence analysis of PD-L3 or VISTA.

PD-L3 or VISTA and Treg-sTNF were identified by the global transcriptional profile of Treg at rest, Treg activated with aCD3, and Treg activated with aCD3 / aGITR. aGITR was selected during this analysis as an inducer of GITR on Treg that was shown to extinguish its contact-dependent suppressive activity (Shimizu, et al. (2002) above). PD-L3 or VISTA and Treg-sTNF were identified on the AFFIMETRIX® DNA arrays based on their unique expression patterns (Table 1). PD-L3 or VISTA showed increased expression in Treg activated by aCD3 and reduced expression in the presence of aGITR; and TregsTNF showed increased expression dependent on aCD3 / aGITR.

161

ΙΜΡϊΒΒ

Purified Τ CD4 + CD25 + cells were stimulated ιϋήηΒ ^ ουΙτΰστΙυΐΈΜτύθ overnight with none, aCD3 or aCD3 / aGITR, and the RNA was ajs] ¿jaaca-el-anátt «ts by real-time PCR. The expression listed is related to actin.

TABLE 1

<td rowspan="2">MRNA</td><td colspan="3">Relative Expression</td>

<td>None</td><td>□ CD3</td><td>aCD3 / aGITR</td>

<td>PD-L3 or VISTA</td><td> 6</td><td> 10</td><td> 7</td>

<td>T^reg-sTNF</td><td> 0.2</td><td> 0.3</td><td> 1.5</td>

Affimetrix analysis of resting-function activated CD25 + CD4 + nTregs revealed expression of a gene product (cDNA RIKEN 4632428N05, or 4632428N05Rik) with unknown function but sequence homology to the Ig superfamily.

More specifically, a 930 bp gene product was cloned from the CD4 + T cell cDNA library, which correlates to size and predicted sequence. In silico sequence and structural analysis predicts a 309 amino acid transmembrane protein after maturation, with a 159 amino acid extracellular domain, a 22 amino acid transmembrane domain, and a 95 amino acid cytoplasmic tail (Figure 1A). Amino acid sequence alignment reveals an immunoglobulin- (lg) V-like extracellular domain homologous to B7 family ligands such as PD-L1, PD-L2, B7-H3, and B7-H4, as well as B7-H4 receptors. the B7 family (ie PD -1, CTLA-4, CD28, BTLA, ICOS) (Figure 1B-C). Although the sequence identity of the Ig-V domains between the B7 family ligands and receptors is generally not very high (&lt;40%), the Ig-V domain of 4632428N05Rik has the highest homology to the ligands of the B7 PD-L1 and PD-L2 family. The sequence alignment also reveals several highly conserved cisternae (Figure 1B) that are important for the formation of intra-chain disulfide bonds, which is 162 characteristic of the B7 Sica and other family of ligands, (2Óü3 ^^^ r ^ unii ^^^ 49861. ___________

The extracellular domain of 4632428N05Rik contains only the Ig-V domain but lacks the Ig-C domain (Figure 1B-C). This unique trait is characteristic of B7 family receptors, and distinguishes 4632428N05Rik from all other B7 family ligands, which contain both Ig-V and Ig-C domains Freeman, GJ (2008). Proc. Nati Acad Sci USA 105, 10275-10276; Lazar-Molnar and others, (2008). Proc. Nati Acad Sci USA 105.10483-10488; Lin et al., (2008), Proc Nati Acad Sci USA 105, 3011-3016; Schwartz et al., (2001), Nature, 410,604-608; Stamper et al., (2001), Nature, 410, 608-61. Consequently, phylogenetic analysis using the PhyML (Maximum Phylogenetic Probability) algorithm placed 4632428N05Rik in a closer evolutionary distance with B7 family receptors, particularly PD-1, than with B7 family ligands ( Figure 2) Guindon, S., and Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 52.696704. However, the cytoplasmic tail of PD-L3 or VISTA does not contain any of the signaling domains (e.g. ITIM, ITAM or ITSM), which are the signature domains of the Sharpe, AH, and Freeman B7 family receptors, GJ (2002). The B7-CD28 superfamily. Nat Rev Immunol. 2, 116-126. It is therefore hypothesized that despite its close evolutionary relationship with the inhibitory PD-1 receptor, 4632428N05Rik represents a new member of the B7 ligand family. Based on these structural and phylogenetic characteristics, this molecule was named as the PD-1 expressing ligand (PD-L3 or VISTA). PD-L3 or VISTA is highly conserved also between mouse and human orthologs, sharing 77% sequence identity (Figure 1D).

The nucleic acid sequence encoding mouse PD-L3 or VISTA is set forth herein as sec. with no. ID: 1 and the mouse PD-L3 or VISTA protein sequence is set forth as sec. with no. Identification number: 2.

The human homologue of PD-L3 or VISTA is located on chromosome 10 (72.9 Mb) and consists of 6 exons thereby generating a transcript of 4689 bases in length that codes for a residual protein 311. The sequence

163 coding for human homologous mRNA is provided in éFGé'ÓBari

ΙΜΡΙΓ

MEXICAN INSTITUTE γ. OF THE PROHE FAD

<img file="MX342017B_D0130.tif" />

Accession number NM_022153 and the sequence of the protein is given oemo

NP_071436. The nucleic acid sequence encoding human PD-L3 or VISTA is set forth herein as sec. with no. Identification No. 3 and the sequence of the human PD-L3 OR VISTA protein is set forth as sec. with no. Identification Number: 4. Mouse and human genes share 74% and are 68% identical on the protein level. Homologs were also identified in Rattus norvegicus on chromosome 20 (27.7 Mb; GenBank accession number

BC098723), as well as Fugu rubripes and Danio rerio. In particular embodiments, the PD-L3 or VISTA proteins of the present share share the common amino acid sequence set forth in sec. with no. Identification number: 5.

EXAMPLE 2: PD-L3 OR VISTA expression studies by RT-PCR analysis and flow cytometry.

As shown in the experiments in Figure 3, RT-PCR analysis was used to determine the expression pattern of PD-L3 or VISTA mRNA in mouse tissues (Figure3A). PD-L3 or VISTA is expressed primarily in hematopoietic tissues (spleen, thymus, bone marrow), or tissues with extensive leukocyte infiltration (ie, lung). Weak expression was also detected in non-hematopoietic tissues (ie, heart, kidney, brain, and ovary). Analysis of various types of hematopoietic cells reveals the expression of PD-L3 or VISTA in peripheral macrophages, splenic monocytes CD11b +, DC CD11c +, CD4 + T cells and CD8 + T cells, but lower expression level in B cells (Figure 3B). This expression pattern is broadly consistent also with the GNF (Novartis Research Foundation Genomics Institute) gene matrix database Su et al., (2002), Acad Proc Nati Sci USA 99,4465-4470, as well as NCBI's GEO (Gene Expression Omnibus) database (Figure 4AD).

To study protein expression, the hamster monoclonal antibodies 8D8 and 6E7 specific to PD-L3 or VISTA were produced. Specificity is demonstrated by positive staining in EL4 T cells

164 murines that overexpress PD-L3 or VISTA, but of EL4 that overexpress PD-L1 (Figure 5).

<img file="MX342017B_D0131.tif" />

Both polyclonal and monoclonal antibodies were raised against PD-L3 or VISTA. Using a rabbit anti-PD-L3 or VISTA antibody, the PDL3 or VISTA protein was localized to lymphoid organs and found prominently in brain tissue. Of the monoclonal antibodies identified, the specificity of an aPD-L3 or VISTA clone 8D8 was further evaluated. In this analysis, clone 8D8 was tested for binding against a panel of PD-L-like Ig-fusion protein molecules including CTLA-4, PD-1, PD-L1, PD-L2, B7-1 and B7-2, PD-L3 or VISTA and hlg. The results of this analysis indicate that 8D8 aPDL-3 was very specific for PD-L3 or VISTA.

Specifically, using the anti-PD-L3 mAb or VISTA clone 8D8, the expression of PD-L3 OR VISTA was analyzed in hematopoietic cells by flow cytometry. Foxp3GFP mice lacking reporter were used to distinguish CD4 + nTreg (34). In peripheral lymphoid organs (spleen and lymph nodes), significant expression is observed in all subsets of CD4 + T cells (see total CD4 + T cells, or naïve Foxp3- and nTreg Foxp3 + cells, and CD4 + memory T cells ), while CD8 + T cells express the markedly lower amount of PD-L3 or surface VISTA (Figure 3C). In the thymus, PD-L3 or VISTA expression is negative on CD4 + CD8 + double positive thymocytes, low on CD4 positive single cells, and detectable on CD8 positive single cells. Next, a strong correlation of high PD-L3 or VISTA expression with marker CD11b can be observed for both splenic and peripheral cells, which include both F4180 macrophages and CD11c + myeloid DCs (Figure 3D-E). On the other hand, B cells and NK cells are mostly negative for the expression of PD-L3 or VISTA. A small percentage of Gr-1 + granulocytes also express PD-L3 VISTA (Figure 3F).

A differential expression pattern is shown in the same cell line from different lymphoid organs (Figure 3G). For CD4 + T cells and intermediate CD11 b monocytes, the expression level follows the pattern of mesenteric lymph node> peripheral LN and spleen> of the peritoneal cavity and blood.

165 iMPi heard

This pattern is less pronounced for cells suggesting that PD-L3 OR VISTA expression in certain cell types can be regulated by cell maturity and / or tissue microenvironment.

In addition to freshly isolated cells, PD-L3 O VISTA expression was analyzed in splenic CD4 + T cells, CD11bhi monocytes and CD11c + CDs in in vitro culture with and without activation (Figure 6). Spleen cells were cultured either with medium, or with anti-CD3 (for T cell activation), or with IFNQ and LPS (for monocyte and DC activation) for 24 hours before being analyzed for expression of PD-L3 or VISTA or other B7 family ligands (eg PD-L1, PD-L2, B7-H3 and B7-H4). This comparison revealed different expression patterns between these molecules. PD-L3 or VISTA expression is rapidly lost in all cell types after in vitro culture, regardless of activation status. In contrast, PD-L1 expression is positively regulated in CD4 + T cells after stimulation, or in CD11bh¡ and DC CD11c + monocytes after culture in medium alone, and the physiognomy of stimulation is also improved. The expressions of PD-L2, B7-H3 and B7H4 are not prominent under the culture conditions used. The loss of expression of PD-L3 or VISTA in vitro is unique compared to other ligands of the B7 family, but could reflect suboptimal culture conditions that fail to mimic the tissue microenvironment.

To direct how the expression of PD-L3 or VISTA can be regulated in vivo, TCR CD4 DO11.10 transgenic mice were immunized with chicken ovalbumin analog antigen (OVA) emulsified with complete Freund's adjuvant (CFA). At 24 hours after immunization, draining lymph node cells were analyzed for expression of PD-L3 or VISTA (Figure 7A). Immunization with antigen (CFA / OVA) without adjuvant only dramatically increased the CD11b + PD-L3 or VISTA + myeloid cell population, which contained a mixed population of F4 / 80 + macrophages and CD11c + DC. Further comparison with PD-L1 and PD-L2 reveals that although PD-L1 has the highest level of constitutive expression, PD-L3 or VISTA is the most highly up-regulated during said inflammatory immune response (Figure 7B). Collectively, these

166

<img file="MX342017B_D0132.tif" />

IMPI

INSTITUTO MEXICANO DE LA F'ROPIEOAO data strongly suggest that the expression of myeloid PD-L3 or VlST / Ten is tightly regulated by the immune system, which may contribute to its role in the control of the immune response and regulation of the immunity of T cells

In contrast to its increased expression in APCs, PD-L3 OR VISTA expression is decreased in CD4 + DO11.10 T cells in a late time interval after immunization (i.e. 48 hours but not 24 hours) ( Figure 8). This result suggests that the in vivo expression of PD-L3 or VISTA in CD4 T cells can be regulated by their activation state and cytokine microenvironment during an active immune response.

EXAMPLE 3: Functional impact of PD-L3 or VISTA signaling on CD4 + and CD8 + T cell responses

A PD-L3 or VISTA-lg fusion protein was produced to examine the regulatory roles of PD-L3 or VISTA in CD4 + T cell responses. The PD-L3 or VISTA-Ig fusion protein contains the extracellular domain of PD-L3 or VISTA fused to the Fe region of human IgG1. When immobilized on the microplate, PD-L3 or VISTA-lg but not the Ig control suppressed the proliferation of bulk purified CD4 + and CD8 + T cells in response to stimulation of anti-CD3 bound to the plate, as determined by cell division arrested (Figure 9AB). The PD-L3 or VISTA-lg fusion protein did not affect the absorption of the anti-CD3 antibody to the plastic wells, as determined by ELISA (data not shown), thus excluding the possibility of non-specific inhibitory effects. CD4 + KO PD-1 T cells were also suppressed (Figure 9C), indicating that PD-1 is not the receptor for PD-L3 or VISTA. The inhibitory effect of PD-L1-lg and PD-L3 OR VISTA-lg was also directly compared (Figure 10). When amounts of titrated Ig fusion proteins were absorbed to the microplates along with □ CD3 to stimulate CD4 +, PD-L3 or VISTA-lg T cells it showed similar inhibitory efficacy to PD-L1-lg fusion protein.

Because bulk purified CD4 + T cells contain several subsets, the effect of PD-L3 or VISTA-lg was evaluated on subsets of naive (CD25-CD44lowCD62Lhi) and memory (CD25167 sorted CD4 + T cells).

<img file="MX342017B_D0133.tif" />

CD44hiCD62Llow) (Figure 11). It is shown that PD-L3 or VISTA can^YouÉúprirTfir% proliferation of both subsets, although with many hands affirm <=> n the memory cells.

To further understand the PD-L3 or VISTA-mediated suppression mechanism, the expression of markers of early TCR activation and apoptosis were measured followed by T cell activation in the presence or absence of PD-L3 or VISTA-lg. Consistent with the negative effect on cell proliferation, there is a global suppression on the expression of the early activation markers CD69, CD44, CD62L (Supplementary Figure 12A). On the other hand, the PD-L3 or VISTA-lg fusion protein did not induce apoptosis. On the contrary, less apoptosis was observed (as determined by the percentage of 7AAD-annexin V + cells) in the presence of PD-L3 or VISTA or VISTA-lg than in the control-lg, both in the early stage (24 hours) and late (48 hours) of TCR activation (Figure 12B). For example, in the 24 hour time interval, in the total uncontrolled population, ~ 27% of cells were apoptotic in the presence of PD-L3 or VISTA or VISTA-lg while ~ 39% of control cells were apoptotic . When examining cells within the living cell R1 channel, it is evident that PD-L3 or VISTA or VISTA-lg strongly inhibited activation-induced cell death (ACID), as approximately 72.6% control cells became apoptotic. while only 43.5% of the cells were apoptotic when treated with PD-L3 or VISTA or VISTA-lg. Similar results were observed for the 48 hour time interval. Thus, it appears that PD-L3 or VISTA or VISTA negatively regulates CD4 + T cell responses by suppressing early TCR activation and arresting cell division, but with minimal direct effect on apoptosis. This suppression mechanism is similar to that of B7-H4 Sica, GL, Choi, IH, Zhu, G., Tamada, K., Wang, SD, Támura, H., Chapovai, AI, Flies, DB, Bajorath, J. , and Chen, L. (2003). B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity 18, 849-861.

A 2-step assay was developed to determine whether PD-L3 or VISTA or VISTA-lg can suppress pre-activated CD4 T cells, and how persistent their suppressive effect is. It is shown that the suppressive effect of PD-L3 or VISTA or

168

<img file="MX342017B_D0134.tif" />

MEXICAN INSTITUTE

OF THE PKOI'IC; The VISTA-lg fusion protein persists after its withdrawal at 24 hours after activation (Figure 9D). Furthermore, both naive and pre-activated CD4i T cells can be suppressed by PD-L3 or VISTA or VISTA-lg (Figure 9Di, 9Di¡¡ and 9Div).

Next, the effect of PD-L3 or VISTA or VISTA-lg on the cytokine production of CD4 + T cells was analyzed. PD-L3 or VISTA or VISTA-lg suppressed the production of Th1 cytokines IL-2 and IFNalpha from culture of purified CD4 + T cells in bulk (Figure 13A-B). The effect of PD-L3 or VISTA or VISTA was further tested in the separate naive (CD25-CD44lowCD62Lhi) and memory (CD25-CD44hiCD62Llow) CD4 + T cell populations. Memory CD4 + T cells are shown to be the main source for cytokine production within the CD4 + T cell compartment, and PD-L3 or VISTA or VISTA can suppress this production (Figure 13C-D). The similar inhibitory effect of PDL3 or VISTA or VISTA on IFNalpha production from CD8 + T cells is also shown (Figure 13E). This inhibitory effect of PD-L3 or VISTA or VISTA on cytokine production by CD4 + and CD8 + T cells is consistent with the hypothesis that PD-L3 or VISTA or VISTA is an inhibitory ligand that negatively regulates immune responses.

Next, studies were designed to determine the factors that are capable of overcoming the inhibitory effect of PD-L3 or VISTA or VISTA. Since PD-L3 or VISTA or VISTA suppressed IL-2 production, and IL-2 is essential for T cell survival and proliferation, we hypothesized that IL-2 may evade the inhibitory activity of PD-L3 or VISTA or VISTA. As shown in Figure 14A exogenous IL-2, but not IL-15, IL-7, or IL-23, partially reversed the suppressive effect of PD-L3 or VISTA or VISTA-lg on cell proliferation. Incomplete rescue by high levels of IL-2 indicates that PD-L3 or VISTA or VISTA signaling targets broader T-cell activation pathways than just IL-2 production. On the other hand, the potent costimulation signal provided by the agonist anti-CD28 antibody completely reversed the suppression mediated by PD-L3 or VISTA or VISTA-lg (Figure 14B), while intermediate levels of co-stimulation are still suppressed by the

169 PD-L3 or VISTA or VISTA signaling (Figure 14C).

<img file="MX342017B_D0135.tif" />

PD-L3 or VISTA or VISTA mediated immune suppression may be more effective in less inflammatory conditions, but will inevitably be squashed by strong positive co-stimulatory signals. In this sense, PD-L3 or VISTA or VISTA shares this characteristic with other suppressor ligands of the B7 family such as PD-L1 and B7-H4 Sica and others, (2003), Immunity, 18,849-861. Carter and others, (2002),

Eur J Immunol 32, 634-643.

In addition to the PD-L3 or VISTA or VISTA-lg fusion protein, it is necessary to confirm that PD-L3 or VISTA or VISTA expressed in APCs can suppress the activation of antigen-specific T cells during analogous interactions between APCs and T cells For this purpose, PD-L3 or VISTA or VISTA-RFP or RFP control protein was over-expressed by retroviral transduction in an artificial antigen presenting cell line (CHO-APC) that stably expresses MHCII and B7-2 molecules Latchman et al. , (2001). Nat. Immunol 2, 261-268. One of the problems in the expression of PD-L3 or VISTA or VISTA in CHO is that the majority of PD-L3 or VISTA or VISTA fails to localize on the cell surface, perhaps due to the foreign environment that lacks support for surface localization. PD-L3 or VISTA or VISTA (data not shown). Although there are no clear motifs present in the cytoplasmic tail of PD-L3 or VISTA or VISTA to suggest the mode of regulation, we speculate that the tail may play a role for its intracellular localization. Consequently, a tailless mutant of PD-L3 or VISTA or VISTA was designed and found to successfully localize the CHO cell surface (data not shown).

To stimulate the T cell response, CHO-PD-L3 or VISTA or VISTA or CHO-RFP cells were incubated together with CD4 + DO 11.10 T cells in the presence of the antigenic peptide OVA. As shown in Figure 15 AC, CHO-PD-L3 or VISTA or VISTA induced less DO11.10 cell proliferation than CHORFP cells. This suppressive effect is more pronounced at lower concentrations of peptide, consistent with the notion that a stronger stimulatory signal can overcome the suppressive effect of PD-L3 or VISTA or VISTA.

170

Furthermore, the inhibitory effect of PD-L3 or VISTÁo VISTA complete on natural APCs was confirmed. In vitro cultured bone Id niédirtlT dendritic-elerivated cells (BMDC) do not express high levels of PD-L3 or VISTA or VISTA (Figure 16). PD-L3 or VISTA or VISTA-RFP or RFP was expressed in BMDC by retroviral transduction during the 10-day culture period. The transduced cells were ranked for homogeneity based on RFP expression. The level of expression of PD-L3 or VISTA or VISTA in the transduced DC was estimated by staining with anti-PD-L3 or VISTA or VISTA mAb, and it was found to be similar to the level of freshly isolated peritoneal macrophages, from that mode within the physiological range of expression (Figure 16). The sorted BMDCs were then used to stimulate OVA-specific transgenic CD4 + T cells (OTII) in the presence of the OVA peptide (Figure 15D). They show that the expression of PD-L3 or VISTA or VISTA on BMDC suppressed the proliferative responses of analogous CD4 + T cells. This result is consistent with previous data using PD-L3 or VISTA or VISTA-lg fusion protein and CHO-APC cells, suggesting that PD-L3 or VISTA or VISTA may suppress T cell-mediated immune responses.

EXAMPLE 4: Evaluation of anti-PD-L3 or VISTA or VISTA antibodies in the Multiple Sclerosis (EAE) animal model

Because TPD-L3 OR VISTA or VISTA mAbs appear to suppress T cell responses in vivo, nPD-L3 OR VISTA or VISTA was tested to evaluate whether it can inhibit a T cell mediated autoimmune disease. Using the model of the Experimental Allergic Encephalomyelitis (EAE), the functional effect of CPDL-L3 mAbs on inflammatory diseases was determined. EAE is a widely used murine model of human autoimmune disease of multiple sclerosis. EAE can be induced either by immunization with myelin antigens in adjuvant or by adoptive transfer of specific T cells to myelin, resulting in inflammatory infiltrates of various effector T cells and B cells, and macrophages, and demyelination of the system. central nervous.

171

WICKED

MEXICAN INSTITUTE

The AcM aPDL-L3 was tested in the passive model of EAÉ^lp^TO^^{? s}YOU ^ eOTFW-^:'^> induction of anaphylaxis due to the injection of large amounts of mAb as foreign antigen. In this adoptive transfer EAE model, SJL donor mice were immunized with CFA and PLP peptide. On day 10, total lymphocytes were isolated from drainage LN, and cultured in vitro with PLP peptide, IL-23 (20 ng / ml) and anti-IFNg (10 pg / ml) for 4 days. The expanded CD4 T cells were then purified, and adoptively transferred into naïve recipient mice. This analysis indicated that aPDL-L3 mAb delayed disease onset as well as reduced disease severity, thereby significantly shifting the disease progression curve (Figure 17). Furthermore, it reduced severity in a large percentage of the mice and greatly increased survival from about 22% to more than 75%. This demonstrated activity of the aPDL-L3 mAb in EAE is consistent with in vitro data, and the use of this reagent as a new immunoregulatory reagent in various inflammatory diseases is shown.

EXAMPLE 5: EXPRESSION OF VIEW IN THE CNS

The expression of VISTA in the CNS was carried out as well. These tests revealed that in mice with the disease, VISTA expression is markedly reduced (76% -> 33%) in CD11b + cells (Figure 23), consequently with the hypothesis that loss of VISTA can be tolerated. to improve inflammation. This is interesting, and of likely functionally important when we contrast inflammatory myeloid cells in the present invention, with MDSC in tumors expressing extremely high levels of VISTA. EAE mice were reported to have high numbers of myeloid derived suppressor cells (MDSC CDL11 b + Ly-6Chigh) in the spleen that are potently suppressive for T-cell activation and may moderate disease 32. Our data strongly support the hypothesis that VISTA may play a role in myeloid-mediated suppression of EAE.

EXAMPLE 6: The effect of VISTA on the fate and function of T cells in EAE.

We also conducted experiments testing the effect of VISTA on T cell fate and function in EAE. We wish to assess whether VISTA alters the

172

<img file="MX342017B_D0136.tif" />

<img file="MX342017B_D0137.tif" />

Ρ growth of pathogenic T cells, encephalitogenesis ^ expar T cells, T cell polarity, longevity and the conversion of Teff -> Treg. We studied the effect of VISTA blockade on T cell fate in EAE. Consistent with the higher disease score, CNS analysis at the end of the disease course confirmed significantly more infiltration of IL17A-producing CD4 + T cells (from 0.66—> 11%) in the 13F3 (VISTA) treated group ( Figure 24).

EXAMPLE 7: PD-L3 OR VISTA or VISTA transgenic and knock-out mice

Using lentiviral embryo infection, four transgenic mice were produced that omnipresently express PD-L3 or VISTA or VISTA. These mice express full PD-L3 or VISTA or VISTA under the control of the human elongation factor 1 promoter. These mice were generated using the lentiviral vector pWPT. Similar to other members of the PD-L1 family (Appay, et al. (2002) J. Immunol. 168: 5954-8), it is contemplated that PD-L3 or VISTA or VISTA will function as a negative regulator in vivo while functioning to costimulate the proliferation of aCD3 T cells in vitro. In this regard, these mice are expected to spontaneously develop autoimmunity and in vivo immune responses in PD-L3 or VISTA or VISTA transgenic mice (i.e. humoral immune responses, priming T cells, etc.) are assessed to assess the growth of systemic autoimmune disease.

For knockout mice, PD-L3 or VISTA or VISTA is inactivated by homologous recombination. A BAC clone containing the complete sequence of PDL3 or VISTA or VISTA was purchased from INVITROGEN ™ (Carlsbad, CA). A vector targeting PD-L3 or VISTA or VISTA was generated by inserting a 1.6 kb fragment located on the 5 'side of the second exon of the PD-L3 or VISTA or VISTA gene upstream the neomycin gene and the 5 kb fragment. located on the 3 'side of the third exon of the PD-L3 or VISTA or VISTA gene downstream of the neomycin gene. B6-derived embryonic stem cells (ES) are electroporated with the vector targeting PD-L3 or VISTA or VISTA and recombinant clones are selected. The selected clones are then injected into the C57BL / 6 blasts and the resulting chimeric male pups are crossed with mice.

173

ΙΜΡΙ FLP suppressors to remove the neomycin cassette. The '^ r ^^^^ d ^^ telo. ^ Targeted in offspring is determined by PCR from genomic DNA. The second and third exons contain the domain of PD-L3 or VISTA or VIsia, therefore the resulting mice have only the inactivated form of the PD-L3 or VISTA or VISTA molecule.

The overall immune capacity of PD-L3 OR VISTA or VISTA deficient mice is determined as with other PD-L - / - mice, including evaluation of T cell responses to antigen, humoral immune responses, overt autoimmunity (e.g., Systemic Lupus Erythematosus, inflammatory bowel disease), and an increased susceptibility to induced autoimmune disease (experimental autoimmune encephalomyelitis) (Chen (2004) above).

EXAMPLE 8: Specific antibodies to PD-L3 OR VISTA or VISTA tested in the collagen-induced arthritis animal model.

As shown in the experiments in Figure 18, male DBA / 1J mice were immunized at the base of their tails with 100 µl of emulsion containing 100 µg of type II chick collagen (C-ll) in CFA (Mycobacterium tuberculosis 3.5 mg / ml) and boosted via IP with 100 pg of aqueous Cll on day 21 after immunization. Mice in each treatment group (n = 6) were either untreated (NT- black circles), injected with 300 pg hamster IgG (Ig Ham black squares), or injected with 300 pg monoclonal antibody 7c9 (red triangle) or 13F3 (green triangle), as indicated. The injections were administered every 2 days. Arthritic swelling was scored on a scale of 0-4 for each leg of each mouse on the days indicated.

The arthritis score shown is the total score for all the feet of the mice in each treatment group divided by the number of mice in the group.

EXAMPLE 9: Blocking of VISTA by a VISTA-specific monoclonal antibody improves the in vitro responses of T cells.

174

<img file="MX342017B_D0138.tif" />

<img file="MX342017B_D0139.tif" />

<img file="MX342017B_D0140.tif" />

lsstí tute

The specific VISTA mAb (13F3) was identified as neutralizing that mediated by VISTA (Figure 19). Myeloid CD11 b? .. sa APCs purified from naive mice to stimulate CD4 T cells⁺ OT-II transgenic plants in the presence or absence of 13F3. Consequently with its neutralizing effect, 13F3 enhanced the proliferation of T cells stimulated by CD11 b myeloid cells.^hl, which were shown to express high levels of VISTA.

EXAMPLE 10: Anti-VISTA improves antitumor immunity.

Due to the ability of anti-VISTA to enhance T cell activation, we evaluated whether anti-VISTA can enhance the protective immune response to an immunogenic tumor. One model in which we have a great deal of experience is carcinoma of the bladder, MB49. MB49 expresses the male antigen, and thus is modestly immunogenic in female mice, although it will grow and kill female mice if there is no immune intervention. To test the efficacy of VISTA therapy, female mice were administered subcutaneously (sq) with MB49 tumor cells and treated with VISTA. Days after that, the tumor size was measured until the mice had to be sacrificed. As can be easily seen in Figure 20 anti-VISTA therapy greatly impairs tumor growth. We believe it is due to the ability of anti-VISTA to enhance cell-mediated immune responses (MIC).

EXAMPLE 11: Effect of VISTA on tumor regression in 4 murine tumor model.

Experiments on the immunogenic bladder carcinoma tumor MB49 showed that the use of mAb 13F3 neutralizes VISTA and protects the host from tumor growth. The data indicate that VISTA has considerable performance as a negative immunoregulator in a tumor microenvironment due to its extremely high expression of MDSCs. Studies examining the effect of mouse anti-VISTA on the growth of immunogenic (MB49) and highly non-immunogenic (B16) tumor models will further confirm the efficacy of a VISTA therapy, clarify on the mechanism of

175

INSTITUTO MEXICANO action, and will provide the basis for selecting the dose justification for each tumor model detailed below.

MB49 in female mice: We have already shown efficacy in this murine model. The MDSCs in this model also express high levels of VISTA (not shown). In this model, due to the presence of the HY antigen, the MB49 tumor is modestly immunogenic. Since

<td>Tumor Name</td><td>Kind of tumor</td><td>Guest</td><td>Groups</td><td>essays</td>

<td>MB49</td><td>Carcinoma of bladder</td><td>Female B6</td><td rowspan="4">rVIEW ig control</td><td rowspan="4">Tumor development Survival immune / autoimmune assays</td>

<td>MB49</td><td>Carcinoma of bladder</td><td>Male B6</td>

<td>B16.F10</td><td>Melanoma</td><td>Female or male B6</td>

<td>ID8</td><td>Ovarian cancer</td><td>Female B6</td>

We know that VISTA therapy is effective, we will use this model as a positive control to determine the dosage (1-100 ug / mouse; and time (day of tumor inoculation, or 4, 7, 10 days after tumor; therapeutic intervention ) of anti-VISTA therapy.

MB49 in male mice: Using effective dose and time in female mice, the efficacy of anti-VISTA therapy is determined in male mice (in which the tumor is less immunogenic).

B16 melanoma: The anti-CTLA-4 mAb was very effective in this model, and represents a non-immunogenic tumor where the mouse model has been valuable in predicting success in humans. Dosage and timing regimens will be similar to those shown to be effective in model MB49.

ID8 ovarian carcinoma: It is in this model that VISTA expression was shown to be extremely high in MDSCs. Tumor-bearing mice are

176 treat with aVISTA at the time of inoculation and after inoculation.

<img file="MX342017B_D0141.tif" />

Methods. WT B6 mice are used to determine the optimal dose and tlétrrpb of anti-VISTA treatment for remission of all indicated murine tumor models. The models used are listed in the table above.

The dose and time test readings are the tumor growth kinetics. For MB49 and B16 studies, all tumor studies are performed via the intradermal inoculation (id) route and therefore the tumor size can be easily measured. Tumor measurements are collected every 2-3 days with a caliper. In each of these models, the effect of anti-VISTA or control antibody will be tested for its ability to delay tumor growth or facilitate tumor regression. The growth of ID8 will continue using a whole body imaging transduced ID8 luciferase using an IVIS workstation. In addition, the survival of the host will also be determined.

Data on tumor growth are expressed as mean tumor volume ± SEM and differences between groups will be analyzed by two-tailed ANOVA. Probability values ​​(p) less than 0.05 are considered statistically significant. Survival data are analyzed using the Kaplan-Meier method with the Wilcoxon scale test and the logarithmic scale test used to verify the significance of the difference in survival between the groups. In the B16 models, the frequencies of mice developing vitiligo are determined.

Using these methods, reduced tumor growth and / or tumor regression is obtained in mice treated with anti-VISTA mAb compared to mice treated with Ab control in various of the non-immunogenic tumor models. Anti-VISTA treatment had already been shown to retard tumor growth in an immunogenic tumor model. As each of these tumor models have their own specific growth kinetics and, anticipated dependence on VISTA to confer tumor growth and

177

J hΛ ΡI .77 —...............

suppressing immunity, the mouse mAb will be administered yarséá at the tumor mori1emff> a and inoculum or at times after this. Additionally, at least 3 different concentrations of a VISTA mAb are tested to determine the optimal dose for therapeutic benefit.

As shown in Figure 21A-E, treatment with VISTA mAb reduced tumor growth in all these 4 tumor models where mice were inoculated either by the sq route with tumor cells of A. MB49, B.MCA105 , or C.EG7, or ip route with D. ID8-luciferase tumor cells, and treated with mAb VISTA 13F3 on alternate days (300pg), beginning on day +1. The subcutaneous growth of the tumor was monitored. For the ID8-luciferase tumor, mice were imaged on day 30 using IVIS Xenogen. E. The expression of VISTA on myeloid leukocytes in tumor bearing mice was also determined. The LN drainage and tumor tissues (ascites) were analyzed for the expression of VISTA. These findings show that VISTA expressed in MDSC is a major suppressor molecule that interferes with the development of protective anti-tumor immunity, and VISTA alleviates this suppressive activity allowing immune intervention and reduced tumor growth. These results also support the conclusion that VISTA in myeloid cells in autoimmune disease plays a critical role in regulating the extent of inflammation.

EXAMPLE 12: Synthesis of oligomeric VISTA and VISTA fusion proteins useful for the treatment of autoimmunity.

Soluble VISTA-lg in vitro is not suppressive and its binding to cells cannot be readily detected. In contrast, this plastic-bound molecule is profoundly suppressive. Furthermore, studies using VISTA-lg in vivo did not show overt activity (data not shown). Regarding these studies, the VISTA-lg that was created has mutations in the CH2-CH3 domain that exclude FcR binding, and therefore is not cytophilic in vivo. Recent studies showed that tetrameric PD-L1 binds 100X more (Kd 6x 10-8 M) than monomeric PD-L126 to PD-1, and that its binding to cells was easily detectable. Tetrameric PD-L1 was not tested in vivo, but was shown to block functional suppression in vitro.

178

<img file="MX342017B_D0142.tif" />

<img file="MX342017B_D0143.tif" />

INSTITUTO MEXICANO DE LA RCOVIEDAD INDUSTRIAL by the native PD-L1. Using similar methods, the oligomers that will direct the VISTA pathway and induce a potent niinuiiusupréSói'á ιιΊ víticF and in vivo activity were prepared.

Said oligomers are constructed using the VISTA monomeric extracellular domain or a fragment thereof, for example, at least 50, 75, 100, 125, 150, 175 or 200 amino acids long whose extracellular domain or a part thereof is used as building blocks for the oligomer. In these methods, the inventors take advantage of well-established MHC tetramer technologies. In these methods, the VISTA ectodomain construct or fragment is linked to the N-terminus of a variety of oligomerization domains (identified above) to generate a series of Vista complexes with valencies ranging from divalent to heptavalent.

Thus, a series of non-covalent oligomers is created as a function of the high affinity of the spirally wound domains that direct the stable formation of the dimeric, trimeric, tetrameric, pentameric, and heptameric assembly. These oligomeric constructs are expressed in a host cell, eg, E. coli. When the expression is carried out in E. coli the expressed oligomers are then refolded and purified from inclusion bodies using standard laboratory protocols. This approach has routinely produced high quality material for biological and structural analysis, including peptide-MHC complexes and trimeric GITRL66. Isolated oligomeric proteins are then evaluated by SDS-PAGE, analytical gel filtration, analytical ultracentrifugation, and mass spectrometry. These quality control measures guarantee the availability of homogeneous materials, well characterized by in vitro and in vivo studies. Parallel organization of these constructs results in molecules in which the valence equals the oligomeric state, as each individual VISTA complex is positioned to productively interact with the cell surface attached to the VISTA receptor. The above constructs possess extreme stability and homogeneity of the oligomeric state. (spirally wound non-covalent oligomerization domains typically exhibit

179 melting temperatures exceeding 100 ° C, except heptamer which has a melting temperature of 95 ° C.

Furthermore, the dimeric VISTA-Ig is tetramerized in either cytophilic or nocytophilic. In-frame VISTA Fe fusion constructs with IgG1 Fe (both wild-type IgGI and IgG1 that do not bind to existing FcR) are modified with an N-terminal BirA site for enzymatic biotinylation and cloned into the pRES2 vector. -EGFP. Enzymatic biotinylation will allow for single, specific residue modification and orientation after multimerization with avidin. This approach was used for the generation of numerous Ig fusion proteins, including B7-1, PD-L1, PD-L2, and TIM-3. The expressed proteins are then enzymatically biotinylated in vitro, purified by HPLC size exclusion, and tetramerized using PE-avidin. The resulting tetramers that are cytophilic or not are evaluated in vivo.

These engineered multimeric VISTA proteins are useful in the treatment of autoimmunity and other conditions where intervention in the VISTA pathway is therapeutically justified in immunosuppression.

EXAMPLE 13: VISTA adenoviral vectors to induce immunosuppression.

Gene transfer using recombinant adeno-associated virus (AAV) has had great technological development in gene therapy. Specifically, AAV-mediated gene delivery of the PD-L1 gene, or CTLA4-lg and CD40-lg achieved therapeutic efficacy in autoimmune disease models of lupus and heart transplantation. These methods will be used to administer either complete VISTA, or VISTA oligomeric ectodomains, and their therapeutic effects are evaluated in the EAE model. The recombinant adenovirus vector expressing either full-length murine VISTA, or VISTA oligomeric ectodomain, is created using the Adeno-X ™ expression system (Clontech) according to the manufacturer's instructions. Briefly, VISTA is cloned into an expression vector based on pAdDEST, E1 and E3 deleted, under the control of the human cytomegalovirus (CMV) promoter. VISTA and control of lacZ-expressing adenoviruses are then purified from cell lysates. For systemic overexpression of VISTA, adenovirus was administered to mice by intravenous injection into the

180

IMPI

TAIL VEIN INSTITUTE (1 x 109 plaque-forming units [PfuJT'ya'Bea á

<img file="MX342017B_D0144.tif" />

shortly after disease induction through var.unariAn after disease onset. Control mice will receive 100 µl of

PBS. The development and alterations of the disease are monitored both in mice

SJL as in C57BL / 6 mice, which present the different disease progression pattern, and which represent two different forms of clinical manifestation of human MS patients.

EXAMPLE 14: Functional studies with engineered proteins and adenoviral vectors.

Mice are also administered (5-100 ug protein / mouse 3 x week) with engineered VISTA and / or adenoviral vectors. Following administration, T cell expansion, differentiation, as well as EAE development are determined.

EXAMPLE 15: Structural studies in VISTA and molecular determination of determinants of the function of VISTA

Affinity, specificity, oligomeric state, and the formation and localization of organized signaling complexes are critical contributors to immune function. All these characteristics impact immune signaling and regulation, as the organization of the receptor-ligand ectodomains directly control the recruitment, organization and function of cytoplasmic signaling and non-covalently associated structural molecules. The high resolution of the VISTA crystal structure is determined using techniques including bacterial, insect and mammalian expression systems, as well as high throughput crystallization and structure determination approaches. To validate the crystallographically observed disulfide bond pattern, we will exploit high resolution mass spectrometry using approaches that have been successfully supported by published studies of TIM-3 and human DcR359. Based on these structural results, a series of mutants with altered oligomeric properties are designed, as well as mutants in the vicinity of any of the perturbed regions of the VISTA IgV domain. These mutant proteins will provide additional and direct mechanistic understanding in the 181

<img file="MX342017B_D0145.tif" />

1MPI

MEXICAN INSTITUTE

OF THE FKOHtDAO

INDUSTRIAL VISTA function and should be useful in therapeutics where immunosuppression is desired, such as the autoimmune, allergic and inflammatory diseases identified in the present disclosure. These mutants, especially oligomers are tested in vitro in systems and evaluated in animal models of autoimmune and inflammatory diseases to evaluate the immunosuppressive effect on disease progression, disease remission or in protecting the animal from developing the autoimmune condition or inflammatory.

These oligomeric VISTA proteins will activate the VISTA pathway and function as a target of immune intervention in autoimmunity. This intervention will suppress immunity and exert a therapeutic benefit in autoimmune disease and other conditions where autoimmune suppression is desired. This is achieved by administering the oligomerized VISTA proteins in different autoimmune and inflammatory models such as animal models of EAE and collagen-induced arthritis. In addition, as discussed above, adenoviral vectors that overexpress full VISTA or oligomers of VISTA are constructed and tested in vivo. These studies will confirm the immunosuppressive effects of the VISTA oligomers.

EXAMPLE 16: Experiments using the conditional transgenic mouse strain that over-expresses VISTA (VISTA transgenic mouse strain: R26StopFLVISTA (VISTA).

A targeted construct containing the complete VISTA cDNA preceded by a termination cassette flanked by loxP, was targeted within the ubiquitous expressed ROSA26 locus. Multiple offspring correctly targeted to R26StopFL / -Vista were born, and raised in the suppressor strain CMV-Cre. Preliminary data on the VISTA x CMV-cre confirms GFP and enhanced VISTA expression. Studies on the immune status of these mice (T cell responses to antigen, antibody titers, etc.) will confirm a suppressive phenotype. The VISTA strain will cross-link CD4-cre, CD11c-cre, and Lys-Cre to determine whether the location of the VISTA expression lineage influences suppression. The phenotype and function of the T cells are also determined if the overexpression of VISTA results in the generation of aTreg. In these studies of

182

IMPI

INSTt^ri * TO MEXICAN

Tregs from the OVA-cre immune strain x VISTA is transferred $ dot> t¡

<img file="MX342017B_D0146.tif" />

WT hosts, to see if antigen immunization in the presence of over-expressed VISTA induces antigen-specific Tregs. This should verify that VISTA impacts Treg differentiation.

Furthermore, the studies are carried out in the EAE model by means of which the impact of the VISTA proteins on different lineages (by cross-linking with CD4-, CD11c-, Lys-cre) with respect to the development of the disease is evaluated. Since the disease can be suppressed by the restricted overexpression lineage of VISTA mutants or in the CMV x VISTA mutant, temporal control of the development of the disease is also using Cre-ERT2x VISTA. Through the administration of tamoxifen we can induce the overexpression of VISTA before, or at the onset of the disease or at the peak of the disease to determine if VISTA may have an effect on the induction and / or effector phases of immunity. Using chimeric BM mice, the temporally-restricted overexpression of VISTA can be restricted to the hematopoietic compartment. During an assessment of controlling the time window VISTA is overexpressed, VISTA is genetically activated, then with the administration of the anti-VISTA mAb it is serologically turned off. These studies will determine where and when VISTA has to act to control the development and progression of autoimmune disease.

EXAMPLE 17: Effect of anti-VISTA antibodies on the CD40 / TLR agonist vaccine

As shown in Figure 22, experiments were performed that tested the effect of anti-VISTA antibodies on the efficacy of the vaccine. These results show that anti-VISTA improves the therapeutic efficacy of a CD40 / TLR vaccine. C57BL / 6 mice were challenged with 1x10 5 square metastatic melanoma B16.F10 cells per sq route Four days later, the mice were vaccinated with 100 pg of tumor-associated AV antigen, 100 pg of aCD40 FGK45 (CD40 agonistic antibody), and 100 pg S-27609 (TLR7 agonist) with or without anti-VISTA (20 ug x 3 / week). Tumor growth was monitored by caliper measurements.

183

Having described the invention,

IMPI

INSTITUTO MEXICANO provide

<img file="MX342017B_D0147.tif" />

claims. These claims are intended to cover all generic and specific features described in the present invention, and all statements of the scope which, in terms of language, can be said to fall between them.

184