Optimization of human antibodies that bind lymphocyte activation gene-3 (lag-3), and uses thereof

Background to the invention

Therapeutic antibodies are one of the fastest growing segments of the pharmaceutical industry. To maintain strength (ie activity) and minimize immunogenicity, antibodies and other protein drugs must be protected against physical and chemical degradation during production and storage. In fact, one of the main difficulties in developing antibody-containing therapeutics is a potential immunogenic reaction when administered to a patient, which can lead to rapid clearance or even cause life-threatening side effects including anaphylactic shock. The immunogenicity of the antibody is influenced by various factors, such as physiochemical properties (e.g., purity, stability or solubility), clinical factors (e.g., dose, route of administration,

Immunogenicity of antibodies and / or loss of antibody activity are often the result of deamination. Deamination is a process of chemical degradation occurring spontaneously in proteins (eg antibodies). The deamination removes the amino acid functional group, such as asparagine or glutamine, which damages the amide-containing side chains. What, in turn, causes structural and biological changes in the whole protein, creating heterogeneous forms of the antibody. Deamination is one of the most common post-translational modifications occurring in therapeutic antibodies produced by recombination.

For example, the heterogeneity of the heavy chain of the monoclonal antibody h1B4 (a humanized anti-CD18 antibody) as a result of deamination during cell culture, as reported by Tsai et al. (Pharm Res 10 (11): 1580 (1993)). In addition, the reduction / loss of biological activity as a result of deamination is a recognized problem. For example, Kroon et al. characterized a number of deamination sites in the therapeutic antibody OKT3, and reported that trials from the OKT3 production series (14 months to 3 years) had an activity below 75% (Pharm Res 9 (11): 1386 (1992), page 1389, second column) . In addition, OKT3 trials showing large amounts of oxidized peptides in their maps had clearly reduced activity in the study of antigen binding strength (page 1390, first column). The authors summarized that specific sites of chemical modification occurring during storage of OKT3 were identified during peptide mapping and correlated with observed changes in chemical analyzes and biological studies of the antibody (page 1392, second column). A loss of biological activity was also reported for variously differently deaminated therapeutic proteins, which included recombinant human DNase (deoxyribonuclease) (Cacia et al. (1993) J. Chromatogr. 634: 229-239) and recombinant soluble CD4 (Teshima et al. (1991) Biochemistry 30: 3916-3922).

In general, deamination is an important and unpredictable problem in the pharmaceutical industry. In particular, efforts to monitor variability caused by deamination in antibody-containing therapeutics, as well as FDA concerns about such variability, increased costs and delayed clinical trials. In addition, modifications to solve the problem, including shifting conditions (e.g., temperature, pH, and cell type) associated with the formation of recombinants and / or alteration of amino acids sensitive to deamination (e.g., site-directed mutagenesis) can negatively affect stability and activity, particularly when the changes affect the antibody-determining regions (CDRs) of the antibody. Accordingly, there is a need for more stable versions of therapeutic antibodies.

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summary

The present invention provides isolated monoclonal antibodies (e.g., human monoclonal antibodies), and antigen-binding portions thereof that bind human LAG-3 as defined in the claims. The monoclonal antibodies of the present invention have optimized physical stability compared to previously described anti-LAG-3 antibodies. In particular, the invention relates to a modified form of the 25F7 antibody (US 2011/0150892 A1) which has significantly improved thermal and chemical stability compared to the unmodified antibody. Specifically, by altering the critical binding region of the heavy chain of the CDR2 domain of the 25F7 antibody, the modified antibody has been shown to have significantly higher thermal and chemical stability, reduced deamination, higher thermal reversibility and lower aggregation. At the same time, it was surprisingly observed that the modified antibody retained the same high affinity binding to human LAG-3, including the ability to inhibit LAG-3 binding to class II histocompatibility complex (MHC) molecules and stimulate antigen-specific T cell responses. a significant increase in the stability and preservation of the binding / biological activity of the modified antibody was surprising, especially in light of the criticality of the CDRs for antibody function.

Antibodies of the present invention can be used in many applications, including detection of LAG-3 protein and stimulation of antigen-specific T cell response in cancer patients or viral infection.

The heavy chain CDR1, CDR2 and CDR3 regions of an antibody, or antigen-binding portion thereof, of the invention comprise the amino acid sequences of SEQ ID NOs: 15, 16, and 17, respectively. The light chain CDR1, CDR2 and CDR3 regions of an antibody, or antigen binding portion thereof, of the invention comprise the amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively. In one embodiment, an isolated monoclonal antibody (e.g., a human antibody), or antigen binding portion thereof, of the invention has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In another embodiment, the antibody further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

In a preferred embodiment, the antibody exhibits increased physical properties (i.e., thermal and chemical stability) compared to the 25F7 antibody, while still retaining the same binding affinity for human LAG-3 as 25F7. For example, the antibody exhibits reduced sequence variation in the region of the heavy CDR2 chain due to deamination, compared to the 25F7 antibody, e.g. about 2.5% or less amino acid sequence modifications after 12 weeks at 4 ° C (i.e. under stability testing conditions at " real time "as described herein) and / or about 12.0% or less amino acid sequence modifications after 12 weeks at 40 ° C (i.e. under accelerated stress conditions as described herein) while still maintaining binding affinity for human LAG- 3 with a KD of at least 1 x 10^-7 M or below (more preferably, K D 1 x 10^-8 M or below, KD 5 x 10^-9 M or below, KD 1 x 10^-9 M or below). In another embodiment, the antibody exhibits a thermal reversibility of at least 40% in PBS at pH 8.0.

In another embodiment, the antibody has a higher melting point (indicative of higher overall in vivo stability) compared to the unmodified antibody (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3: 361-71). In one embodiment, the antibody exhibits TM1 (initial deployment temperature) higher than 60 ° C, e.g. higher than 65 ° C, or greater than 70 ° C. The melting point of the antibody can be measured using differential techniques such as differential scanning calorimetry (Chen et al. (2003) Pharm Res 20: 1952-60; Ghirlando et al. (1999) Immunol Lett 68: 47-52) or circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40: 343-9).

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In another embodiment, the antibody is characterized by resistance to rapid degradation. Degradation (degradation) of the antibody can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander AJ and Hughes DE (1995) Anal Chem 67: 3626-32).

In another embodiment, the antibody has a minimum aggregation effect of e.g. 25% or less, 20% or less, 15% or less, or 10% or less, 5% or less, or 4% or less. Aggregation can lead to the triggering of an unwanted immune response and / or altered or unfavorable pharmacokinetic properties. The aggregation can be measured by several known techniques, e.g. size exclusion (SEC) in high performance liquid chromatography (HPLC) and light scattering.

In another embodiment, the antibody further comprises one of the following:

(a) binds to the monkey LAG-3;

(b) not associated with the mouse LAG-3;

(c) inhibits the binding of LAG-3 to class II molecules of the major histocompatibility complex (MHC); and (d) stimulates immune responses, particularly an antigen-specific T-cell response. Preferably, the antibody exhibits at least two of properties (a), (b), (c) and (d). More preferably, the antibody exhibits at least three of properties (a), (b), (c) and (d). Most preferably, the antibody exhibits all four of properties (a), (b), (c) and (d).

In another embodiment, the antibody stimulates an antigen-specific T cell response, such as production of interleukin-2 (IL-2) in an antigen-specific T cell response. In another embodiment, the antibody stimulates an immune response such as an anti-tumor reaction (e.g., inhibiting tumor growth in an in vivo tumor transplant model) or autoimmune response (e.g., development of diabetes in NOD mice).

In another embodiment, the antibody binds to a LAG-3 epitope comprising the amino acid sequence PGHPLAPG (SEQ ID NO: 21). In another embodiment, the antibody binds to a LAG-3 epitope comprising the amino acid sequence HPAAPSSW (SEQ ID NO: 22) or PAAPSSWG (SEQ ID NO: 23).

In another embodiment, the antibody stains the pituitary tissue in immunohistochemistry, or does not stain the pituitary tissue in immunohistochemistry.

Antibodies of the invention may be full-length antibodies, e.g., IgG1, IgG2 or IgG4 isotypes, optionally serine-to proline mutation in the heavy-chain constant region of the hinge region (at the position corresponding to position 241, as described in Angal et al. (1993) Mol. Immunol. 30: 105-108), so that the heterogeneity of the sulphide bridges inside the heavy chain is reduced or removed. In one aspect, the isotype of the constant region is IgG4 with a mutation of amino acid residues 228, e.g. S228P. Optionally, the antibodies can be antibody fragments, such as Fab, Fab 'or Fab'2 fragments or single chain antibodies.

In another aspect of the invention, the antibody (or antigen-binding portion thereof) is part of an immunoconjugate that comprises a therapeutic agent, e.g. a cytotoxin or a radioactive isotope, conjugated to an antibody. In another aspect, the antibody is part of a bispecific molecule comprising a second functional molecule (e.g., a second antibody) having a different binding specificity than said antibody or antigen binding portion thereof.

Also provided are compositions comprising antibodies, or antigen binding portions thereof, immunoconjugates, or bispecific molecules of the invention, optionally in a pharmaceutically acceptable carrier formulation.

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Also provided are nucleic acid molecules encoding antibodies or antigen binding portions thereof (e.g., variable and / or CDR regions) according to the invention, as well as expression vectors comprising such nucleic acids and host cells that contain such expression vectors. The present invention also relates to methods of preparing anti-LAG-3 antibodies using host cells comprising expression vectors, including the possible steps of: (i) expressing the antibody in a host cell; and (ii) isolating the antibody from the host cell.

In another aspect, the present invention provides the LAG-3 antibodies of the invention for use in methods of stimulating an immune response. In one embodiment, the method comprises stimulating an antigen-specific T cell reaction through the contacted T cells with the antibody of the invention, such that it is stimulated by an antigen-specific T cell reaction. In a preferred embodiment, the production of interleukin-2 is stimulated by antigen-specific T cells. In another embodiment, the patient is a cancer patient and the immune anti-cancer reaction is stimulated. In another embodiment, the patient is a patient infected with the virus and the immune anti-viral reaction is stimulated.

Still in another embodiment, the invention provides an antibody, or antigen-binding portion thereof, for use in a method of inhibiting tumor cell growth in a patient comprising administering to the patient an antibody, or antigen-binding portion thereof, to inhibit tumor growth in the patient. And still in another embodiment, the invention provides an antibody, or antigen-binding portion thereof, for use in a method of treating a viral infection in a patient comprising administering to the patient an antibody, or antigen-binding portion thereof, to cure the patient's viral infection. In another embodiment, the methods include administering a composition, a bispecific molecule or an immunoconjugate of the invention.

In yet another embodiment, the invention provides an antibody, or antigen-binding portion thereof, for use in a method of stimulating an immune response in a patient comprising administering to the patient an antibody, or antigen-binding portion thereof, and at least one additional immunostimulatory antibody, such as an anti-human antibody. PD-1, anti-PD-1 antibody and / or anti-CTLA-4 antibody, so as to stimulate the immune response of a patient, for example to inhibit tumor growth or stimulate an antiviral response. In one embodiment, the additional immunostimulatory antibody is an anti-PD-1 antibody. In another embodiment, the additional immunostimulatory agent is an anti-PD1 antibody. In yet another embodiment, an additional immunostimulatory agent is an anti-CTLA-4 antibody. In yet another embodiment, the antibody, or antigen-binding portion thereof, of the invention is administered with a cytokine (e.g., IL-2 and / or IL-21), or a costimulatory antibody (e.g., anti-CD 137 and / or anti-CD antibody). -GITR). Antibodies can be, e.g., human antibodies, chimeric antibodies or humanized antibodies.

The present invention provides anti-LAG-3 antibodies and compositions of the invention for use in the aforementioned methods or for the preparation of a medicament for use in the aforementioned methods (e.g., for treatment).

Other properties and advantages of the present invention will prove to be indisputable after reading the detailed description and examples which should not be regarded as exhaustive.

A brief description of the drawings

Figure 1A shows the nucleotide sequence (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) of the human antibody heavy chain variable region

Monoclonal 25F7. The CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 6) and CDR3 (SEQ ID NO: 7) and germline V, D and J areas were designated. CDR regions were determined using the Kabat system (Kabat et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, Department of Health and Social Services, USA, NIH Publication No. 913242).

Figure 1B shows the nucleotide sequence (SEQ ID NO: 3) and the amino acid sequence (SEQ ID NO: 4) of the heavy chain variable region of the human monoclonal antibody 25F7. The CDR1 (SEQ ID NO: 8), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO: 10) and germ line V, D and J sites were designated. The heavy and light full-length sequence the in-acid 25F7 antibodies are shown in SEQ ID NOs: 32 and 34, respectively.

Figure 2A shows the amino acid sequence (SEQ ID NO: 12) of the heavy chain variable region of the human monoclonal antibody LAG3.5. CDR1 regions (SEQ ID NO: 15), CDR2 (SEQ ID NO: 16) and CDR3 (SEQ ID NO: 16) were determined. The heavy and light chain of the full-length amino acid sequence of the LAG3.5 antibody is shown in SEQ ID NO: 35 and 37 respectively.

Figure 2B shows the nucleotide sequence (SEQ ID NO: 13) and the amino acid sequence (SEQ ID NO: 14) of the kappa light chain variable region of the human monoclonal antibody LAG3.5. The CDR1 regions (SEQ ID NO: 18), CDR2 (SEQ ID NO: 19) and CDR3 (SEQ ID NO: 20) were determined.

Figure 3 shows the amino acid sequence of the heavy chain variable region CDR2 variants LAG-3 LAG3.5 (SEQ ID NO: 16), LAG3.6 (SEQ ID NO: 24), LAG3.7 (SEQ ID NO: 25), and LAG3 .8 (SEQ ID NO: 26), compared to the amino acid sequence of the CDR2 heavy chain variable region of 25F7 antibody (LAG3.1) (SEQ ID NO: 6) and the corresponding human germline sequence (SEQ ID NO: 27). The CDR2 heavy chain variable region of the LAG3.5 antibody differs from the heavy chain variable region CDR2 of the 25F7 antibody with arginine (R) at position 54 (versus asparagine (N)) and serine (S) at position 56 (versus asparagine (N)). The remaining CDRs of LAG3.5 and 25F7 antibodies are identical.

Figures 4A and 4B show graphs of binding activity (EC50 and affinity, respectively) of LAG3.1 (25F7), LAG3.2, LAG3.5, LAG3.6, LAG3.7, and LAG3.8 antibodies with activated human CD4 + T cells.

Figures 5A, B, C, D, and E show thermal melting curves (i.e., thermal stability) of LAG3.1 (25F7), LAG3.5, LAG3.6, LAG3.7, and LAG3.8, respectively.

Figures 6A, B, C, D, and E show the thermal reversibility curves (i.e., thermal stability) of the LAG3.1 (25F7), LAG3.5, LAG3.6, LAG3.7, and LAG3.8 antibodies, respectively.

Figure 7 is a graph of the binding activity of LAG3.1 (25F7) and LAG3.5 antibodies with activated human CD4 + T cells and antigen binding (Biacore).

Figure 8 shows the results of peptide mapping using mass spectrometry (chemical modifications / molecular stability) of LAG3.1 (25F7) and LAG3.5 mappers for deamination and isomerization after incubation for 5 days under accelerated stress as described in this disclosure.

Figure 9 is a graph comparing the hydrophilicity profiles of the LAG3.1 (25F7) and LAG3.5 antibodies.

In Figures 10A, B, C, and D are graphs comparing the affinity and physical stability (i.e., thermal and chemical stability) of LAG3.1 and LAG3.5 antibodies at 4C and 40C, both in stability studies under accelerated stress conditions. and "real-time" conditions as described in this disclosure.

Figures 11A and B are graphs comparing the percentage of amino acid sequence modifications of LAG3.1 and LAG3.5 antibodies at 4C ° and 40C °.

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Detailed description of the invention

In order to better understand the text, some terms were first defined. Additional definitions are included in detailed descriptions.

The terms "25F7", "25F7 antibody", "LAG3.1 antibody" and "LAG3.1" are given for the anti-human LAG-3 antibody described in US2011 / 0150892 A1. The nucleotide sequence (SEQ ID NO: 1) encoding the 25F7 heavy chain variable region (LAG3.1) and the corresponding amino acid sequence (SEQ ID NO: 2) is shown in Figure 1A (with CDR sequences designated as SEQ ID NO: 4, 5, and 7, respectively). The nucleotide sequence (SEQ ID NO: 3) encoding the 25F7 light chain variable region (LAG3.1) and the corresponding amino acid sequence (SEQ ID No. 4) is shown in Figure 1B (with CDR sequences designated as SEQ ID NOs: 8, 9, and 10, respectively).

The term "LAG-3" refers to the gene-3 of lymphocyte activation (Lymphocyte Activation Gene-3). The term "LAG-3" includes variants, isoforms, homologs, orthologs and paralogs. For example, antibodies specific for the human LAG-3 protein may in some cases cross-react with the non-human LAG-3 protein. In other embodiments, antibodies specific for human LAG-3 may be completely specific for human LAG-3 and may not show cross-reactivity, characteristic of other species or other forms of cross-reactivity, or may cross-react with LAG-3 from certain other species. but not react with all other species (eg cross-react with the monkey LAG-3 but not react with the mouse LAG-3). The term "human LAG-3" refers to the human LAG-3 sequence, such as the total amino acid sequence of a human LAG-3 with accession number to Genbank NP_002277 (SEQ ID NO: 29). The term "mouse LAG-3" refers to the mouse LAG-3 sequence, such as the total amino acid sequence of the mouse LAG-3 with the accession number of the Genbank NP_032505. LAG-3 is also known in the art, e.g. as CD223. Sequence of human LAG- 3 may differ from the human LAG-3 with the accession number of Genbank No. NP_002277 by the presence of, e.g., conserved mutations or mutations in unconserved regions, and LAG-3 generally performs the same biological function as the human LAG-3 with the Genbank accession number For example, the biological function of human LAG-3 is the occurrence of an epitope in the extracellular domain of LAG-3, which binds specifically to the antibody,

The term & quot; LAG-3 monkey & quot; is intended to include LAG-3 proteins expressing in the old and new world monkeys, including, but not limited to, LAG-3 cynomologous LAG-3 and Rhesus monocyte LAG-3. 3 is the amino acid sequence of Rhesus LAG-3, which is also deposited at Genbank under accession number XM_001108923.An other representative amino acid sequence of the monkey LAG-3 is an alternative clone rhesus pa23-5 as described in US 2011/0150892 A1. Such alternative rhesus sequence shows the difference of a single amino acid at position 419, compared to the sequence deposited at Genbank.

The particular sequence of human LAG-3 will generally be at least 90% identical to the human LAG-3 amino acid sequence found in Genbank accession number NP_002277 and will contain amino acid residues that identify amino acid sequences as human in comparison to the amino acid sequences of LAG-3. in other species (e.g. mice). In particular cases, the human LAG-3 may be at least 95% or even at least 96%, 97%, 98% or 99% identical to the amino acid sequence of LAG-3 located in Genbank with accession number NP_002277. In particular embodiments, the human LAG-3 sequence will show no more than 10 amino acid differences compared to the LAG-3 sequence found in Genbank with accession number NP_002277. In embodiments, human LAG-3 may display no more than 5 or even no more than 4, 3, 2,

- EP 2867 258 amino acids compared to the LAG-3 sequence located in Genbank with accession number NP_002277. The percent identity can be determined based on the description in the present disclosure.

The term "immune response" refers to the action of, e.g., lymphocytes, antigen presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by said cells or liver (including antibodies, cytokines and additional), resulting in selective destruction, destruction or elimination of invasive pathogens from the human body, cells or tissues infected with pathogens, cancer cells or, in cases of autoimmune or pathological inflammation, normal cells or tissues.

"Antigen-specific T-cell reaction" refers to a T-cell response that is the result of T-cell stimulation by an antigen for which this T-cell is specific. Non-limiting examples of T cell responses to specific antigen stimulation include proliferation and cytokine production (e.g. IL-2).

called complementarity determining regions (CDRs), separated by more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to the tissue or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complementary system. arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to the tissue or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complementary system. arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to the tissue or host factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complementary system.

The term "antigen-binding portion" (or simply "part of an antibody") as used in the present disclosure defines one or more antibody fragments that retain the ability to specifically bind to an antigen (e.g., LAG-3 protein). It has been demonstrated that the function of an antibody that allows binding to an antigen can be performed by full length antibody fragments. Examples of binding fragments are defined by the term "antigen-binding portion" of an antibody and include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) the F (ab ') fragment²a divalent fragment comprising two Fab fragments joined by a disulphide bridge in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and CH1 domains; (v) an Fv fragment consisting of the VL and VH domains of a single antibody arm, (vi) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of the VH domain; (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, a variable heavy chain region comprising one variable domain and two constant domains. Furthermore, although the two domains of the Fv, VL and VH fragment are encoded by separate genes, they can be joined using recombination methods with a synthetic linker, so that they can be made from one protein chain, wherein the VL and VH regions pair to form monovalent molecules (known as one Fv chain (scFv), see e.g. Bird et al (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies can also be considered as an "antigen-binding fragment of an antibody". Antibody fragments are obtained by conventional techniques known to those skilled in the art, and fragments are screened for suitability, as are intact antibodies.

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As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic properties (e.g., an isolated antibody that specifically binds the LAG-3 protein is substantially free of antibodies specifically binding to antigens other than LAG-3 proteins). However, an isolated antibody that binds specifically to the human LAG3 protein may show cross-reactivity with other antigens, such as other species of LAG-3 proteins. Furthermore, the isolated antibody may essentially be free of other cellular material and / or chemical compounds.

The terms "monoclonal antibody" or "monoclonal antibody composition" as used in the disclosure refer to preparations of antibody molecules with a single molecular composition. The monoclonal antibody composition exhibits single binding specificity and affinity with a particular epitope.

The term "human antibody" as used in the present disclosure is intended to mean variable antibody regions in which both the framework and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, then the constant region also comes from human germline immunoglobulin sequences. The human antibodies of the invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or directed mutagenesis in vitro or somatic mutation in vivo). However, the term "human antibody" as used in the present disclosure does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice,

The term "human monoclonal antibody" refers to antibodies having a single binding specificity, the regions and variables in which both the framework and the CDR regions are isolated from the human germline immunoglobulin sequence. In one embodiment, human monoclonal antibodies are produced by a hybridoma comprising B cells obtained from a transgenic non-human animal species, e.g. a transgenic mouse with a genome comprising a human heavy chain transgene and a light chain transgene associated in an immortalized cell.

by dividing the human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable regions in which framework and CDR regions are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or when using a transgenic animal for human Ig somatic mutation in vivo), whereby the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that after isolation from the VH and VL sequences, and compared to the human germline sequences, they may not be naturally occurring in the human germline repertoire in vivo. Such recombinant human antibodies have variable regions in which framework and CDR regions are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or when using a transgenic animal for human Ig somatic mutation in vivo), whereby the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that after isolation from the VH and VL sequences, and compared to the human germline sequences, they may not be naturally occurring in the human germline repertoire in vivo. Such recombinant human antibodies have variable regions in which framework and CDR regions are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or when using a transgenic animal for human Ig somatic mutation in vivo), whereby the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that after isolation from the VH and VL sequences, and compared to the human germline sequences, they may not be naturally occurring in the human germline repertoire in vivo.

The term "isotype" as used herein refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region genes.

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The terms & quot; antigen recognition antibody & quot; and & quot; antigen specific antibody & quot; are interchangeable with the term & quot; antibody specific for antigen. & Quot;

The term "human antibody derivatives" refers to any modified form of a human antibody, e.g. an antibody conjugate or other agent, or antibody.

The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted into human framework sequences. Additional framework modifications can be made within human framework sequences.

The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from other species, and the constant region sequences are derived from other species, such as antibodies in which the variable region sequences are derived from the murine antibody and the constant region sequences are derived from a human antibody .

The term antibody used in the present disclosure which "binds specifically to human LAG-3" refers to an antibody that binds to a human LAG-3 protein (and possibly to a LAG-3 protein of one or more non-human species), but not is largely bound to non-LAG-3 proteins. Preferably, the antibody should bind to the human LAG-3 protein with "high affinity", i.e. at K D 1 x 10^-7 M or less, more preferably 1 x 10^-8 M or less, more preferably 5 x 10^-9 M or less, more preferably 1 x 10^-9 M or below.

The term "does not bind significantly" to a protein or cell, as used in the present disclosure, means that it does not bind or that it does not bind high affinity to the protein or cells, i.e. binds to the protein or cells at a KD value 1 x 10^-6 M or above, more preferably 1 x 10^-5 M or above, more preferably 1 x 10^-4 M or above, more preferably 1 x 10^-3 M or above, and more preferably of the order of 1 x 10^-2 M or above.

The term "Kassoc" or "Ka" as used in the present disclosure relates to an association index of a particular interaction between a given antibody and an antigen, while the term "Kdis" or "Kd," refers to the rate of dissociation constant of a given antibody with an antigen. The term "K D", as used in the present disclosure, refers to the dissociation constant obtained from the ratio Kd to Ka (e.g., Kd / Ka) and is expressed in molar concentration (M). K D values ​​for antibodies can be determined using methods known in the art. A preferred method of determining the KD value of an antibody is the use of surface plasmon resonance, preferably using a biosensor system, such as the Biacore system.^®.

The term "high affinity" of an IgG antibody refers to an antibody with a K D of 1x10^-7 M or less, more preferably 5 x 10^-8 M or below, even more preferably 1x10^-8 M or below, even more preferably 5 x 10^-9 M or below, and even more preferably 1 x 10^-9 M or below for the target antigen. However, "high affinity binding" may differ in the case of isotypes of other antibodies. For example, "high affinity binding" for the IgM isotype refers to an antibody with a K D value of 10^-6 M or below, more preferably 10^-7 M or below, even more preferably 10^-8 M or below.

The term "deamination" is a process of chemical degradation occurring spontaneously in proteins (e.g., antibodies). As a result of deamination, an amino acid functional group such as asparagine or glutamine is removed, which damages the amide-containing side chains. Particularly, the asparagine side chain attacks the adjacent peptide group to form a symmetrical intermediate of succinimide imide (succinimide). Symmetry of intermediate results in two hydrolysis products, aspartate or iso-aspartate. A similar reaction can also occur in the side chains, and partially converts to iso-aspartate. In the case of glutamine, the rate of deamidation is generally ten times lower than asparagine, however, the mechanism is essentially the same, requiring only water molecules to pass.

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The term "subject" or "subject" refers to a human or non-human animal. The term "non-human animal" includes vertebrates, e.g. mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians and reptiles, although mammals such as non-human primates, sheep are preferred. , dogs, cats, cows and horses.

Various aspects of the present invention are described in detail in the following sections in the following subsections.

Anti-LAG-3 antibodies have increased stability and favorable functional properties

Antibodies of the present invention bind specifically to human LAG-3 and have optimized stability compared to the previously described anti-LAG-3 antibodies, especially when compared to the 25F7 antibody (LAG3.1). Optimization includes reduced deamidation (e.g., increased chemical stability) and increased thermal refolding (e.g., increased physical stability) while still maintaining high affinity binding to human LAG-3.

Methods for identifying deamidation sites are known in the art (see, e.g., ion-exchange chromatography, reversed phase and hydrophobic interaction, and peptide mapping of proteolytic digest products (LC-MS)). Suitable tests to measure physically stability e.g. analysis of melting points and / or refolding of antibody structure after denaturation (e.g., percent reversibility, as described, e.g., Example 3, Section 3).

Binding to human LAG-3 can be assessed using one or more techniques well known in the art. For example, the antibody may be tested in a flow cytometry test in which the antibody reacts with a human LAG-3 cell line, such as CHO cells, that are transfected to have LAG-3 expression on their cell surfaces (e.g., human LAG-3 , or LAG-3 monkey (e.g., Rhesus macaque or Japanese macaque or mouse LAG-3). Other suitable cells for use in a flow cytometry study include CD4-stimulated CD4-activated T cells.⁺, with native LAG-3 expression. In addition, or optionally, binding of the antibody, which includes binding kinetics (e.g., K D), can be tested in BIAcore studies. Still other suitable binding studies include the ELISA, for example, using recombinant LAG-3 protein.

Antibodies of the invention preferably bind to the human LAG-3 protein KD 1 x 10^-7 M or lower, more preferably 1 x 10^-8 M or lower, 5 x 10^-9 M or lower, or 1 x 10^-9 M or lower.

Typically, the antibody binds to LAG-3 in lymphoid tissues, such as tonsil, spleen or thymus, which can be detected in immunochemistry. In one embodiment, the antibody stains the pituitary tissue (e.g., they are retained in the pituitary gland) as measured by immunohistochemistry. In another embodiment, the antibody does not stain the pituitary tissue (e.g., they are not retained in the pituitary) as measured by immunohistochemistry.

Additional functional properties include cross-reactivity with LAG-3 from other species. For example, the antibody may bind to simian LAG-3 (e.g. cynomolgus monkey, rhesus) but does not bind substantially to LAG-3 from mouse LAG-3. Preferably, the antibody of the invention binds human LAG-3 with high affinity.

Other functional properties include the ability of the antibody to stimulate an immune response, such as an antigen specific T cell reaction. This can be examined, for example, by assessing the ability of the antibody to stimulate the production of interleukin-2 (IL-2) by reacting antigen-specific T cells. In certain embodiments, the antibody binds to human LAG-3 and stimulates a T cell reaction specific for the antigen. In other embodiments, the antibody binds to human LAG-3 but does not stimulate the reaction of antigen specific T cells. Other means of assessing the ability of an antibody to stimulate an immune response include studies of its tumor / tumor growth inhibitory capacity, such as in a tumor inoculated tumor model in vivo (see e.g.

Example 6) or the ability to stimulate an immune response, such as the ability to facilitate the development of autoimmune diseases in an autoimmune model, e.g., the ability to facilitate the development of diabetes in the NOD mouse model.

Preferred antibodies of the invention are human monoclonal antibodies. Additional or alternatively, the antibodies may be, e.g., chimeric antibodies or humanized monoclonal antibodies.

Monoclonal antibody LAG3.5

A preferred antibody of the invention is a human monoclonal antibody

LAG3.5, characterized structurally and chemically, as described below and in the following examples. The VH amino acid sequence for LAG3.5 is shown in SEQ ID NO: 12 (Figure 2A). The VL amino acid sequence for LAG3.5 is shown in SEQ ID NO: 14 (Figure 2B).

The VH and VL sequences (or CDR sequences) or other anti-LAG-3 binding antibodies of human LAG-3 can be "mixed and aligned" with the VH and VL (or CDR sequences) of the LAG3.5 antibody. Preferably, the VH and VL chains (or CDR sequences of such chains) are mixed and matched, the VH sequence from the specific VH / VL pairing should be replaced by the structurally similar VH sequence. Likewise, preferably the VL sequence from a given VH / VL coupling is replaced by a structurally similar VL sequence.

Accordingly, in one embodiment, the antibodies of the invention, or antigen binding portions thereof, include:

(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

(i.e., VH from LAG3.5); and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14 (i.e., VL of LAG3.5) or VL of another anti-LAG3 antibody (i.e., that differs from LAG3.5);

where the antibody binds specifically to human LAG-3.

Antibodies of the invention, or antigen binding portions thereof, include:

(a) heavy chain regions of CDR1, CDR2 and CDR3 comprising the amino acid sequences LAG3.5, SEQ ID NO: 15, 16 and 17, respectively; and (b) the light chain CDR1, CDR2 and CDR3 regions comprising the amino acid sequences LAG3.5, SEQ ID NOs: 18, 19 and 20, respectively;

where the antibody binds specifically to human LAG-3.

Further, an antibody or antigen binding a portion thereof is described herein which comprises the CDR2 heavy chain variable region of the LAG3.5 antibody consisting of the CDRs of other human LAG-3 binding antibodies, e.g. CDR1 and / or CDR3 from the heavy chain variable region, and or CDR1, CDR2, and / or CDR3 of the light chain variable region of a different anti-LAG-3 antibody.

In addition, it is well known in the art that the CDR3 domain alone, independently of the (y) CDR1 and / or CDR2 domains, can determine the specificity / binding of the antibody of the related antigen and that a plurality of antibodies with the same binding specificity can be predicted based on the common CDR3 sequence. . See, e.g., Klimka et al., British J. of Cancer B3 (2): 252-260 (2000); Beiboer et al., J. Mol. BioL 296: 833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Barbas et al., J. Am. Chem. Soc. 116: 2161-2162 (1994); Barbas et al., Proc. Natl. Acad. Sci. USA 92: 2529-2533 (1995); Ditzel et al., J. Immunol. 157: 739-749 (1996); Berezov et al., BIAjournal 8: Scientific Review 8 (2001); Igarashi et al., J. Biochem (Tokyo) 117: 452-7 (1995); Bourgeois et al., J. Virol 72: 807-10 (1998); Levi et al., Proc. Natl. Acad. Sci. USA 90: 4374-8 (1993); Polymenis and Stoller, J. Immunol. 152: 5218-5329 (1994) and Xu and

- 12 - EP 2867258 Davis, Immunity 13: 37-45 (2000). See also, US Pat. Nos. 6,951,646; 6,914,128; 6,090,382; 6,818,216; 6,156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185.

Further, antibodies described herein, including CDR2 of the LAG3.5 heavy chain variable region and at least CDR3 of the heavy and / or light chain variable region of LAG3.5 (SEQ ID NOs: 17 and / or 20), or CDR3 of the variable region of the chain a heavy and / or a light other LAG-3 antibody, where the antibody may specifically bind to human LAG-3. Such antibodies preferably (a) compete for binding to; (b) retain functional characteristics; (c) bind to the same epitope; and / or (d) have similar binding affinity as LAG3.5. Further, the antibodies may comprise the CDR2 of the heavy chain variable region of LAG3.5 (SEQ ID NO: 17 and / or 20), or CDR2 of the light chain variable region of another LAG-3 antibody, where the antibody may specifically bind to human LAG-3. Additionally,

Conservative modifications

In addition, antibodies that comprise heavy and / or light chain variable region sequences, or CDR1, CDR2 and CDR3 sequences differing from the sequences are described herein.

LAG3.5 with one or more conservative modifications. Preferably, however, residues 54 and 56 in the VH CDR2 remain as arginine and serine, respectively (i.e. they are not mutated). Those of skill in the art understand that specific conservative sequence modifications can be made without removing the antigen binding. See, e.g., Brummell et al. (1993) Biochem 32: 1180-8; de Wildt et al. (1997) Prot. Eng. 10: 835-41; Komissarov et al. (1997) J. Biol. Chem. 272: 26864-26870; Hall et al. (1992) J. Immunol. 149: 1605-1612; Kelley and O'Connell (1993) Biochem. 32: 6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10: 341-6 and Beers et al. (2000) Clin. Can. Res. 6: 2835-43. Accordingly, the antibody described herein comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences and / or a light chain variable region comprising CDR1 sequences,

(a) the heavy chain variable region CDR1 comprises SEQ ID NO: 15, and / or its conservative modifications in addition to positions 54 and 56; and / or (b) the heavy chain variable region CDR3 comprises SEQ ID NO: 17, and its conservative modifications; and / or (c) the light chain variable region CDR1 and / or CDR2 and / or CDR3 sequences comprise SEQ ID NO: 18, and / or, SEQ ID NO: 19, and / or, SEQ ID NO: 20, and / or its conservative modifications; and (d) the antibody specifically binds to human LAG-3.

In addition, or optionally, the antibody may have one or more of the following functional properties described above, such as high affinity binding to human LAG-3, binding to simian LAG-3, binding to mouse LAG-3, inhibitory ability to bind LAG-molecules 3 with class II molecules of the major histocompatibility complex (MHC) and / or the ability to stimulate antigen specific T cell responses.

The antibody described herein can be, for example, human, humanized or chimeric antibody.

As used in the present disclosure, the term "conservative sequence modifications" is used to define amino acids that do not significantly affect or alter the binding characteristics of an antibody containing an amino acid sequence. Such conservative modifications rely on substitution, addition and removal of amino acids. Modifications can be introduced into the antibody of the invention using standard techniques

EP 2867 258 known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families contain amino acids with basic side chains (e.g., lysine, arginine, histidine), acid side chains (e.g. aspartic acid, glutamic acid) with uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), branched side chains (e.g., threonine, valine, isoleukin) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the described CDR regions of the antibody of the invention may be replaced with another amino acid residue from the same side chain family, and the modified antibody may be tested for functional maintenance (e.g., the functions described above) by using the functional tests described earlier.

Constructed and modified antibodies

Antibodies can be prepared using an antibody having at least one V H and / or V L sequence of LAG3.5 as a starting material to construct a modified antibody. The antibody may be engineered by modifying at least one residue in one or both variable regions (e.g., V H and / or V L), e.g. in at least one CDR region, and / or in at least one framework region. Additionally or alternatively, the antibody may be constructed by modifying residues in the constant region (constant regions), e.g. to alter one or more functions of the antibody.

In some embodiments, a CDR graft can be made to construct variable antibody regions. Interactions between antibodies and target antigens occur, mostly via amino acid residues, located on the six heavy chain and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences in the CDRs are more diverse between individual antibodies than sequences outside of the CDRs. Because CDR sequences are responsible for most of the interaction between the antibody and the antigen, it is possible to express recombinant antibodies imitating the specific properties of naturally occurring antibodies by constructing expression vectors containing CDR sequences from specific,

The present invention relates to isolated monoclonal antibodies or antigen binding portions thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising the sequences of SEQ ID NO: 15, 16, 17, respectively a light chain variable region comprising CDR1, CDR2 sequences. , and CDR3 comprising the sequences of SEQ ID NO: 18, 19, 20, respectively (i.e., CDRs of LAG3.5). When such antibodies comprise the VH and VL CDR sequences of monoclonal antibodies LAG3.5, they may contain differing framework sequences of these antibodies.

Such framework sequences can be obtained from public DNA databases or published sources containing information on the germline antibody gene sequence. For example, human germline DNA sequences for human genes in the light and heavy chain variable regions can be found in the human germline database "VBase" (available online on the website<a href="http://www.mrc-cpe.cam.ac.uk/vbase">www.mrc-cpe.cam.ac.uk/vbase</a>), as well as in Kabat et al. (1991), cited above; Tomlinson et al. (1992) "The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with

- 14 - EP 2867258 Different Hypervariable Loops "J. Mol. Biol. 227: 776-798; and Cox et al. (1994)" A Directory of Human Germ-line VH Segments Reveals a Strong Bias in their Usage "Eur. J Immunol. 24: 827836. As another example, human germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain sequences discovered in the mouse Huo7 HuMAb germline are available in the accompanying Genbank under accession numbers 1-69 (NG_0010109, NT_024637 and BC070333), 3-33 (NG_0010109 and NT_024637) and 3-7 (NG_0010109 and NT_024637). Thus in another example, the following heavy chain sequences discovered in the germline of mouse HCo12 HuMAb are available in the accompanying Genbank at access numbers 1-69 (NG_0010109,NT_024637 and BC070333), 5-51 (NG_0010109 and NT_024637), 4-34 (NG_0010109 and NT_024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).

Protein antibody sequences are compared to a database of compiled protein sequences by the sequence similarity search method, called Gapped BLAST (Altschul et al. (1997) supra), which is known to those skilled in the art.

Preferred framework sequences for use in the antibodies of the present invention are structurally similar to the framework sequences used by selected antibodies of the present invention, e.g., similar to VH 4-34 framework sequences and / or VK L6 framework sequences used by the preferred monoclonal antibodies of the present invention. The VH CDR1, CDR2, and CDR3 and VK CDR1, CDR2 and CDR3 sequences may be grafted onto framework regions having an identical sequence with the germline germline gene from which the framework sequence is derived, or CDR sequences may be grafted onto framework regions containing one or more mutations compared to germline sequences. For example, it was found

Another type of variable region modification is the mutation of amino acid residues in the VH and / or VL regions of CDR1, CDR2 and / or CDR3 to improve one or more binding properties (e.g., affinities) of the antibody of interest. Site-directed mutagenesis or PCR mutagenesis can be performed to introduce mutations, and the binding effect of the antibody or other functional characteristics of interest can be assessed by in vitro or in vivo assays as described in the text and Examples. Preference is given to modifications (discussed earlier). Mutations may be a substitution, addition or deletion of amino acids, but substitutions are preferred. In addition, usually no more than one, two, three or four residues change in the variable region of the CDR.

Accordingly, anti-LAG-3 monoclonal antibodies or antigen-binding portions thereof comprising a heavy chain variable region comprising: (a) a V H CDR1 region comprising SEQ ID NO: 15 or an amino acid sequence from one, two, three, four or a five-fold substitution, deletion or addition of amino acids in comparison with SEQ ID NO: 15; (b) a VH CDR2 region comprising SEQ ID NO: 16 or an amino acid sequence with one, two, three, four or five fold substitution, deletion or addition of amino acids compared to SEQ ID NO: 16; (preferably, positions 54 and 56 are the same as in SEQ ID NO: 16); (c) a VH CDR3 region comprising SEQ ID NO: 17 or an amino acid sequence with one, two, three, four or five substitutions, deletions or additions of amino acids in comparison with SEQ ID NO: 17; (d) a VL CDR1 region comprising SEQ ID NO: Or an amino acid sequence with one, two, three, four or five fold substitutions, deletions or additions of amino acids compared to SEQ ID NO: 18; (e) a VL CDR2 region comprising SEQ ID NO: 19 or an amino acid sequence with one, two, three, four or five fold substitution, deletion or addition of amino acids compared to SEQ ID NO: 19; (f) a VL CDR3 region comprising SEQ ID NO: 20 or an amino acid sequence with one, two, three, four or five fold substitution, deletion or addition of amino acids compared to SEQ ID NO: 20. deletion or addition of amino acids in comparison with SEQ ID NO: 19; (f) a VL CDR3 region comprising SEQ ID NO: 20 or an amino acid sequence with one, two, three, four or five fold substitution, deletion or addition of amino acids compared to SEQ ID NO: 20. deletion or addition of amino acids in comparison with SEQ ID NO: 19; (f) a VL CDR3 region comprising SEQ ID NO: 20 or an amino acid sequence with one, two, three, four or five fold substitution, deletion or addition of amino acids compared to SEQ ID NO: 20.

- EP 2867258

The engineered antibodies described in the work include antibodies in which framework residues have been modified in VH and / or VK, e.g. to improve antibody properties. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one of the methods consists in a "back-mutation" of one or more framework residues corresponding to a germline sequence. Specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework antibodies with the germline sequences from which the antibody is derived.

Another way of modifying the framework is to mutate one or more residues in the framework region to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. Such a method is also called "deimmunization" and has been described with further details in US Patent Publication No. 20030153043.

In addition or alternatively to modifications performed in the framework regions, the antibodies of the invention may be engineered to contain modifications in the Fc region, typically to effect alteration of one or more of the antibody's functional properties, such as biological serum half-life, complement fixation, receptor binding. Fc, and / or cytotoxicity of the antigen-dependent cell. Furthermore, the antibody of the invention may be chemically modified (e.g., one or more chemical molecules may be attached to the antibody) or modified to alter glycosylation, and again to alter one or more of the antibody's chemical properties. Each of the given embodiments is described in detail below.

In a preferred embodiment, the antibody is an IgG4 antibody isotype comprising serine mutation to proline at the position corresponding to position 228 (S228P; EU index) in the heavy chain hinge region constant region. The mutation reported has been found to remove the heterogeneity of disulfide bridges within the heavy chain at the hinge region (Angal et al. Above, position 241 based on the Kabat numbering system).

In one embodiment, the CH1 hinge region is modified such that the number of cysteine ​​residues in the hinge region changes, e.g., increases or decreases. The method is described in greater detail in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the CH1 hinge region is altered, e.g. to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the antibody Fc hinge region is mutated to shorten the biological half-life of the antibody. Specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface domain of the hinge Fc fragment, resulting in a weakening of protein A (SpA) binding relative to the native Fc binding domain of the SpA binding. The method is described in more detail in US Patent No. 6,165,745.

In another embodiment, the antibody is modified to extend its biological half-life. Various methods are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in US Patent No. 6,277,375. Alternatively, to extend the biological half-life, the antibody in the CH1 or CL region may be altered to include a salvage receptor binding epitope derived from two loops of the CH2 domain of the immunoglobulin Fc region (IgG) as described in US Patent No. 5,869,046 and 6,121,022.

In yet other embodiments, the Fc region is changed by replacing at least one amino acid residue to alter effector function (s) of the antibody.

For example, one or more amino acids selected from amino acid residues 234,

235, 236, 237, 297, 318, 320 and 322 can be replaced by another amino acid residue so that the antibody has altered affinity to the effector ligand, but retained its ability

- binding of the parent antibody to the antigen. The effector ligand that affects affinity may be, for example, the Fc receptor of the complement C1 component. The method is described in more detail in US Patent Nos. 5,624,821 and 5,648,260.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 may be replaced with another amino acid residue so that the antibody has an altered C1q binding and / or reduced or removed complement dependent cytotoxicity (CDC). The method is described in more detail in U.S. Patent No. 6,194,551.

In another example, one or more amino acid residues at amino acid positions 231 and 239 are changed to effect alteration of the complement binding capacity of the antibody. The method is described in more detail in PCT publication WO 94/29351.

In yet another example, the Fc region has been modified to increase the ability of the antibody to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or to increase the affinity of the antibody to the Fcy receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252,

254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330,

331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. The method describes more particularly in PCT publication WO 00/42072. In addition, binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al. (2001) J. Biol. Chem. 276: 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 are shown to improve binding to FcyRIII. In addition, the following binding mutants are shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A

In yet another embodiment, the glycosylation of the antibody is modified. One can, e.g., produce an aglycosylated antibody (e.g., an antibody lacking glycosylation). Glycosylation can be altered, e.g. to increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved, e.g., by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made resulting in the elimination of one or more variable regions of the framework glycosylation sites to eliminate glycosylation from there. Such aglycosylation may increase the affinity of the antibody for the antigen. See, e.g., U.S. Patents 5,714,350 and 6,350,861.

Additionally or alternatively, an antibody with an altered type of glycosylation may be constructed, such as a hypofucosylated antibody with reduced amounts of fucosyl residues or an antibody with increased GlcNac bisection structures. Such altered glycosylation patterns have been demonstrated to increase the ability of the antibodies to ADCC. Such modifications can be obtained, e.g., by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation mechanisms have been described by those skilled in the art; may be used as host cells in which the expression of the recombinant antibodies described in the present disclosure can be provided to produce an antibody with altered glycosylation. For example, the Ms704 cell lines, Ms705 and Ms709 do not have the FUT8 fucosyltransferase gene (alph (1,6) fucosyltransferase), therefore, antibodies expressed in the Ms704, Ms705 and Ms709 cell lines do not have fucose on their carbohydrates. The cell lines Ms704, Ms705 and Ms709 FUT8^{- / -} was constructed by targeted destruction of the FUT8 gene in CHO / DG44 cells with two substitution vectors (see US Patent Publication No. 20040110704 and YamaneOhnuki et al. (2004) Biotechnol Bioeng 87: 614-22). As another example, in EP 1,176,195 a cell line with a functionally damaged FUT8 gene encoding a fucosyl transferase is shown so that antibodies expressing in such a cell line exhibit

Hypofucosylation by reducing or eliminating the enzyme with alpha α-1,6 linkage. EP 1,176,195 also describes cell lines with low enzyme activity for adding fucose to N-acetylglucosamine that binds to the Fc region of the antibody or is devoid of enzymatic activity, e.g. YB2 / 0 rat myeloma cell line (ATCC CRL 1662). PCT Publication WO 03/035835 describes a variant of the CHO cell line, Lec13 cells with a reduced ability to attach fucose to carbohydrates linked to Asn (297), also causing hypofucosylation of antibodies expressed in the host cell (see also Shields et al. (2002) J. Biol. Chem. 277: 26733-26740). Antibodies with a modified glycosylation profile can also be produced in hen eggs, as described in PCT publication WO 06/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells such as Lemna. Methods for the production of antibodies in plant systems are disclosed in the US patent application corresponding to Alston &amp; Bird LLP application identification number 040989/314911, attached to the file August 11, 2006. PCT Publication WO 99/54342 describes a cell line engineered to express glycosyl transferases that modify glycoproteins (e.g., β (1,4) -acetylglucosaminyltransferase III (GnTIII)) such that expressing antibodies in engineered cell lines exhibit increased bisecting of GlcNac structures that result in increased ADCC activity of antibodies (see also Umana et al (1999) Nat. Biotech. 17: 176180). Alternatively, the fucose residues in the antibody can be removed with the fucosidase enzyme; for example.

Other antibody modifications contemplated in the present disclosure are pegylations. The antibody may be pegylated to, e.g., extend the biological half-life of the antibody (e.g., serum). The PEGylation of an antibody, or a fragment thereof, typically involves reaction with a polyethylene glycol (PEG) such as a reactive ester or aldehyde derivative.

PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. Preferably, the pegylation should be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is used to describe any form of PEG used to derivatize other proteins, such as (C 1 -C 10) an alkoxy or an aryloxy-polyethylene glycol or a polyethylene glycolmaleimide. In some embodiments, the antibody to be pegylated is an aglycosylated antibody. Protein pegylation methods are known to those skilled in the art and can be used with the antibodies described in this work. See, e.g., EP 0 154 316 and EP 0 401 384.

Physical properties of antibodies

Antibodies of the invention can be characterized based on various chemical properties to detect and / or differentiate their different classes.

For example, antibodies can contain one or more glycosylation sites, both in the light chain variable region and in the heavy chain variable region. Glycosylation sites may result in increased immunogenicity of the antibody or a change in the pK of the antibody as a result of altered antigenic binding (Marshall et al. (1972) Annu Rev

Biochem 41: 673-702; Gala and Morrison (2004) J Immunol 172: 5489-94; Wallick et al. (1988) JExp Med 168: 1099-109; Spiro (2002) Glycobiology 12: 43R-56R; Parekh et al. (1985) Nature 316: 452-7; Mimura et al. (2000) Mol Immunol 37: 697-706). It is known that glycosylation occurs in motifs containing the NXS / T sequence. In some cases, it is better to have an anti-LAG-3 antibody that does not contain variable region glycosylation. This can be achieved by selecting antibodies that do not contain a glycosylation motif in the variable region or by mutating residues in the glycosylation region.

In a preferred embodiment, the antibodies do not contain asparagine isomeric sites. Asparagine deamination can occur in the NG or DG sequence and causes

- formation of an isoaspartic acid residue that introduces the looping of the polypeptide chain and reduces its stability (isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pI) that is generally in the pH range between 6 and 9.5. The pI point for the IgG1 antibody is usually in the pH range of 7-9.5, while the pI for the IgG4 antibody is usually in the pH range 68. It is believed that the pI antibodies outside the normal range may have some development and instability in vivo. . Therefore, it is preferred to have an anti-LAG-3 antibody with a pI value in the neutral range. This can be achieved by selecting antibodies with pI in the normal range or by mutating charged surface residues.

Nucleic acid molecules encoding antibodies of the invention

In another aspect, the present invention provides nucleic acid molecules encoding the heavy and light chain variable regions, or CDR regions, of the antibodies of the invention. Nucleic acids may exist in whole cells, in a cell lysate or in partially purified or substantially pure form. A nucleic acid is "isolated" or "provided in a substantially pure form" after purification from other cell components or other contaminants, e.g. other cellular nucleic acids or proteins using standard techniques, e.g. alkali treatment / SDS, CsCl binding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art.

See, Ausubel, et al., Ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and

Wiley Interscience, New York. Nucleic acids of the invention may be, e.g. DNA or RNA, may also contain or not intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressing in hybridomas (e.g., hybridomas prepared from transgenic mice containing human immunoglobulin genes as described below), cDNAs encoding antibody light and heavy chains generated by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library, (e.g., using phage display techniques), the nucleic acid encoding such antibodies can be recovered from the gene library.

Preferred nucleic acid molecules of the invention include molecules that encode the VH and VL sequences of monoclonal antibodies LAG3.5 (SEQ ID NOs: 12 and 14, respectively). After obtaining the DNA fragments encoding the VH and VL segments, the resulting DNA fragments can be further manipulated using standard recombinant DNA techniques, e.g. conversion of variable region genes into full-length gene chains, Fab fragments or the scFv gene. In said manipulations, a DNA fragment encoding VL- or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively coupled", as used in this context, means that these two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in the frame.

The isolated VH region encoding DNA can be transformed into a full-length heavy chain gene by coupling the DNA encoding the VH with another DNA molecule encoding the heavy chain constant regions (CH1, CH2 and CH3). Sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al (1991), supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The variable region of the heavy chain may be, e.g., a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but most preferably a constant region of IgG1 or IgG4. In the case of the fragment gene

The heavy chain of the Fab heavy chain, the DNA encoding the VH, may be operably coupled to another DNA molecule encoding only the constant region of the CH1 heavy chain.

The isolated VL-encoding DNA can be transformed into a full-length light chain gene (as well as a Fab light chain gene) by operatively coupling DNA encoding the VL with another DNA molecule encoding the light chain constant region, CL. Sequences of human heavy chain constant region genes are known in the art (see e.g. Kabat et al., Supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region may be a kappa or lambda constant region.

To create the scFv gene, DNA fragments encoding VH and VL are operably linked to another fragment encoding an elastic linker, e.g. encoding the amino acid sequence (Gly4-Ser) 3, so that the VH and VL sequences can be expressed as a continuous single protein chain with regions. VL and VH connected by a flexible linker. (see, e.g., Bird et al (1988) Science 242: 423-426, Huston et al. (1988) Proc. Natl Acad. Sci USA 85: 5879-5883; McCafferty et al., (1990) Nature 348 : 552-554).

Production of monoclonal antibodies

The described monoclonal antibodies (mAbs) can be produced by the well-known hybridization technique of the somatic cell (hybridoma) Kohler and Milstein (1975) Nature 256: 495. Other embodiments regarding monoclonal antibodies include viral or oncological transformation of B lymphocytes and phage display technique. Chimeric or humanized antibodies are well known to those skilled in the art. See, e.g., U.S. Patent No. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

Human monoclonal antibodies directed against human LAG-3 can be produced using transgenic or transchromosomic mice having parts of the human immune system instead of part of the mouse system. The transgenic or transchomosome mice described include mice referred to in the text as HuMAb Mouse® and KM Mouse®, respectively, and collectively referred to as "human Ig mice"

The HuMAb Mouse® mouse (Medarex®, Inc.) has a minimum human immunoglobulin gene coding for non-engineered heavy chain (μ and γ) and light κ sequences together with targeted mutations inactivating endogenous μ and κ loci (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859). Accordingly, mice exhibit reduced expression of mouse IgM or κ and in response to immunization, introduced heavy and light chain transgenes are subject to class exchange and somatic mutation to produce human high affinity monoclonal IgGK antibodies (Lonberg et al. (1994), supra; in Lonberg (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding and Lonberg (1995) Ann. NY Acad Sci. 764: 536-546). The preparation and use of HuMAb Mouse® mice and the genomic modifications of these mice are described in the entries presented to Taylor et al. (1992) Nucleic Acids Research 20: 6287-6295; Chen et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152: 29122920; Taylor et al. (1994) International Immunology 6: 579-591; and Fishwild et al. (1996) Nature Biotechnology 14: 845-851. See further, U.S. Patent No. 5,545,806; 5,569,825; USA 90: 3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152: 29122920; Taylor et al. (1994) International Immunology 6: 579-591; and Fishwild et al. (1996) Nature Biotechnology 14: 845-851. See further, U.S. Patent No. 5,545,806; 5,569,825; USA 90: 3720-3724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152: 29122920; Taylor et al. (1994) International Immunology 6: 579-591; and Fishwild et al. (1996) Nature Biotechnology 14: 845-851. See further, U.S. Patent No. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and 5.545.807; PCT Publication No. WO 92/03918; WO 93/12227; WO 94/25585; WO 97/13852; WO 98/24884; WO 99/45962 and WO 01/14424.

Further, human antibodies can be transferred, with the mouse having human immunoglobulin sequences, to transgenes and transchromosomes, so that the mouse has a human heavy chain transgene and a human light chain transchromosome. The mouse is determined

EP 2867258 in the present invention as the mouse "KM mouse®", and a detailed description thereof can be found in PCT WO 02/43478. The modified form of the described mouse, which further comprises the homozygous disruption of the endogenous FcyRIIB receptor gene is also described in PCT WO 02/43478 with reference in the present invention as "KM mouse / FCGR2D®." In addition, mice with HCo7 heavy chain transgenes may be used, or HCo12 or both.

Additional transgenic animals that can be used to produce human antibodies include Xenomouse mice (Abgenix, Inc., U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963), "TC mice" (Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97: 722-727) and cows with heavy and light chain transchromosomes (Kuroiwa et al. (2002) Nature Biotechnology 20: 889-894, PCT publication WO 02/092812).

Further, human monoclonal antibodies can also be prepared by phage display methods for screening libraries of human immunoglobulin genes. See, e.g., U.S. Patent No. 5,223,409; 5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6.593,081.

The described human monoclonal antibodies can also be prepared using SCID mice with remodeled human immune cells to generate a human antibody response to immunization. See, e.g., U.S. Patent Nos. 5,476,996 and 5,698,767.

Further, human anti-LAG-3 antibodies can be prepared using phage display, where the phages include nucleic acids coding for antibodies produced by transgenic animals previously immunized with LAG-3. Preferably, the transgenic animal is a mouse

HuMab, KM, or Kirin. See, e.g., U.S. Patent No. 6,794,132.

Immunization of mice with human IG

Mice with human Ig can be immunized with a purified or enriched LAG-3 antigen preparation, a recombinant LAG-3 protein or cells expressing the LAG-3 protein. See, e.g., Lonberg et al. (1994), supra; Fishwild et al. (1996), supra; PCT publications WO 98/24884 or WO 01/14424. Preferably, 6-16 week old mice are immunized with 5-50 μg LAG-3 protein. Optionally, a part of LAG-3 conjugated to the non-LAG-3 polypeptide is used,

Transgenic mice can be immunized intraperitoneal (IP) or intravenously (IV) with LAG-3 antigen with Freund's adjuvant, followed by subsequent immunizations with IP or IV antigen in incomplete Freund's adjuvant. Adjuvants other than Freunda or whole cells without adjuvant. Plasma may be screened by ELISA analysis and cells from mice with appropriate titers of anti-LAG-3 immunoglobulin may be used for fusions.

Generation of hybridomas that produce human monoclonal antibodies

To generate hybridomas that produce human monoclonal antibodies, splenocytes and / or lymph node cells of the immunized mouse can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened to produce antibodies specific for the antigen. The preparation of hybridomas is well known in the art. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

Manufacture of transfectomas producing monoclonal antibodies of the invention

Antibodies of the invention can also be produced in host cell transfectomas, by using, e.g., recombinant DNA techniques and methods for transfection of genes known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encodes partial and full-length light and heavy chains

EP 2867258 obtained by standard molecular biology techniques can be placed in one or more expression vectors to allow genes operably linked to transcriptional and translational sequences. In this context, the term "surgical coupling" is used to determine the ligation of an antibody gene with a vector so that the transcription and translation control sequences in the vector perform their function of regulating the transcription and translation of the antibody gene.

The term "regulatory sequence" includes promoters, enhancers and other elements of expression control (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, e.g., in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Preferred regulatory sequences for the expression of a mammalian host cell contain viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers were isolated from cytomegalovirus (CMV), Simian 40 (SV40), adenovirus (e.g. major late promoter) adenovirus (AdMLP) and polyoma. Optionally, non-viral regulatory sequences, such as the ubiquitin promoter or the β-globin promoter, may be used. regulatory elements consisting of sequences from a variety of sources, such as the SRα promoter system containing the SV40 early promoter sequences, and the long terminal repeat (LRTR) of type 1 human leukemia virus T cells (Takebe et al. (1988) Mol. Cell. Biol 8: 466-472). The expression vector and expression control sequences are selected to be compatible in the expression of the host cell used.

The light chain gene and the antibody heavy chain gene can be introduced into the same or separate expression vectors. In preferred embodiments, the variable regions are used to create the full-length genes of any antibody isotype when placed in expression vectors already encoding the heavy and light chain constraints of the desired isotype, such that the VH segment is operably linked to the segment. (a) CH in the vector, and the VK segment is operably linked to the CL segment in the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates the secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector, in a way that for the signal peptide to connect to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (e.g., a non-immunoglobulin protein signal peptide).

In addition to antibody chain genes and regulatory sequences, recombinant expression vectors may have additional sequences that regulate vector replication in host cells (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. No. 4,399,216, 4,634,665 and 5,179,017). For example, a typical selectable marker gene provides drug resistance, such as G418, hygromycin or methotrexate, to a host cell with an inserted vector. Preferred selectable marker genes include dihydrofolate reductase (DHFR) gene (for use in dhfr-host host cells with methotrexate selection / amplification) and neo gene (for G418 selection).

To express the light and heavy chains, the expression vector (s) encoding the light and heavy chains is transfected into the host cell using standard techniques. Various forms of the term "transfection" cover a wide variety of commonly used techniques for introducing exogenous DNA into a prokaryotic or eukaryotic host cell, such as, e.g., electroporation, calcium phosphate precipitation, DEAEdextran transfection, and the like. Although it is theoretically possible to express the antibodies described in both eukaryotic and prokaryotic host cells, the expression of antibodies in eukaryotic cells, and more preferably in mammalian host cells, is most advantageous because such eukaryotic cells, in particular mammalian cells,

- EP 2867258

Preferred host cells for expressing recombinant antibodies are Chinese hamster ovary cells (CHO cells) (e.g., CHO dhfr cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. US 77: 4216-4220, used with a DHFR selection marker, e.g. as described in RJ Kaufman and PA Sharp (1982) J. Mol. Biol. 159: 601621), NSO myeloma cells, COS cells and SP2 cells. Particularly, when using NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When the recombinant expression vectors encoding the antibody genes are introduced into mammalian host cells, the antibodies are produced by multiplying host cells in sufficient time, that the expression of the antibody in the host cell or more preferably the secretion of antibodies into the culture medium where the host cells are propagated. Antibodies can be recovered from the culture medium by standard purification methods.

immunoconjugates

Antibodies of the invention may be conjugated to a therapeutic agent to form immunoconjugates, such as antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, smaller DNA groove-binding agents, DNA intercalators, DNA-crosslinking agents, histone deacetylase inhibitors, inhibitors of nuclear export, proteasome inhibitors, topoisomerase inhibitors (I or II), heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In ADC, antibodies and therapeutic agents are preferably coupled via a cleavable linker, such as a peptidyl, disulphide or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala -AlaAsn, Cit-Cit, Val-Lys, Lys, Cit, Cheese or Glu. The ADC can be prepared as described in US Patent No. 7,087,600; 6,989,452; and 7,129,261; PCT publications WO 02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; and WO 08/103693; US Patent Publication No. 20060024317; 20060004081; and 20060247295.

Bispecific molecules

In another aspect, the present disclosure relates to bispecific molecules comprising one or more antibodies of the invention conjugated to at least one other antibody or antigen binding portion thereof to form a bispecific molecule that binds to at least two binding sites or target molecules. Thus, as used herein, a "bispecific molecule" includes molecules characterized by three or more specificities. In a preferred embodiment, the bispecific molecule includes a first LAG-3 specific binding and a second linkage specific for a releasing molecule that includes cytotoxic effector cells that kill the target cell expressing LAG-3. Examples of suitable releasing molecules are CD64, CD89, CD16 and CD3. See e.g.

In one embodiment, the bispecific molecule has, in addition to anti-specific binding and anti-LAG-3 specificity, a third specificity. The third specificity may be with an anti-boosting factor (EF) e.g. a molecule binding to a surface protein involved in cytotoxic activity and thereby increasing the immune response against the target cell. For example, part of the anti-boosting factor (anti-EF) can bind to cytotoxic T cells (e.g., by D2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) or other immune cells, cause enhanced immune response against the cell target.

Bispecific molecules can exist in many different formats and sizes. From one end of the size spectrum, the bispecific molecule retains the traditional format

The antibodies, except that instead of two arms with identical specificity, have two binding arms, each of which has a different specificity. At the other end are bispecific molecules having two single-chain antibody fragments (scFv) linked by a peptide chain, the so-called Bs construct (scFv) 2 Intermediate size molecules have two different F (ab) fragments coupled with a peptidyl linker. Bispecific molecules of these and other formats can be prepared as a result of genetic engineering, somatic hybridization or chemical methods. See, e.g., Kufer et al, cited above; Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998); and van Spriel et al., Immunology Today, 21 (8), 391-397 (2000), and references therein.

Pharmaceutical compositions

In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more antibodies of the present invention prepared together with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present invention may also be administered in combination therapy with, for example, another immunostimulatory agent, an anti-cancer agent, an antiviral agent or a vaccine such that the anti-LAG-3 antibody enhances the immune response to the vaccine.

The pharmaceutical composition may contain many excipients. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, binders, dispersions or suspending agents, solubilizers, dyes, flavors, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents , and their combinations. The selection and use of suitable excipients is discussed in Gennaro, ed., Remington: The Science and Practice of Pharmacy, ed. 20 (Lippincott Williams &amp; Wilkins 2003).

Preferably, the pharmaceutical composition should be suitable for intravenous, intramuscular, subcutaneous, parenteral, paraspinal or skin application (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated with material to protect against acids or other natural conditions that may lead to its deactivation. The term "parenteral administration" as used in the text means other methods of administration of drugs than enteral or topical, usually in the form of an injection and includes without limitation intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, retro-orbital, intracardiac, intradermal, intraperitoneal, endotracheal, subcutaneous, subcuticular , intraspinal, subcapsular, subarachnoid, intraspinal, epidural and homeostatic injections or infusions.

The pharmaceutical compositions of the invention may include pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" means a salt that retains the required biological activity of the parent compound and has no undesired toxicological effect. Examples of such salts are salts with added acid and salts with addition of bases. Salts with the addition of acids, among others salts derived from non-toxic inorganic acids, such as hydrochloric acid, nitric phosphoric acid, sulfuric acid, hydrobromic acid, hydrogen iodide, phosphonic acid and the like, as well as acids derived from non-toxic organic acids such as mono-aliphatic dicarboxylic acids, aliphatic phenyl-substituted carboxylic acids, aliphatic hydroxy acids. carboxylic acids, aromatic acids, sulfonic acids, aliphatic and aromatic, and the like. Salts with the addition of bases

among others salts derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, a

EP 2867 258 also includes nontoxic organic amines such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

The pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They can also be prepared in a microemulsion, liposome or other ordered structure suitable to achieve a high drug concentration.

The amount of active ingredient that can be combined with the carrier material to achieve a unit dosage form varies depending upon the host treated and the particular mode of administration and will generally be the amount of the composition causing the therapeutic effect. Generally, from one hundred percent, the amount will range from about 0.01% to about ninety-nine percent of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

The dosage regimen is adapted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses over a certain time interval, or a dose proportional reduced or increased dose, as indicated depending on the requirements of the given therapeutic situation. It is especially preferred to formulate the parenteral composition in a unit dosage form that provides easy administration and unification of the dose. The form of the drug in the present disclosure means physically discrete units adapted for uniform dosing to patients; each unit contains a fixed amount of active compound calculated to provide the required therapeutic effect in association with the required pharmaceutical carrier. Alternatively, the antibody may be administered in the form of a sustained release composition. In this case, less frequent feeding is required.

When the antibody is administered, the dosage ranges from about 0.0001 - 100 mg / kg, and more usually in the range of 0.01 - 5 mg / kg, of the host's body weight. For example, doses may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg or 10 mg / kg body weight within 1-10 mg / kg body weight. An exemplary treatment regimen consists of administering the drug once a week, once every two weeks, once every three weeks, four weeks, once a month, once every three months or once every three to six months. Preferred dosage regimes for anti-LAG-3 antibodies of the invention include 1 mg / kg body weight or 3 mg / kg body weight administered intravenously, combined with antibody administration, according to the following dosage regimens: (i) six doses every four weeks, then every three months; (ii) every three months; (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight every three weeks. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, and in some methods, about 25-300 μg / ml.

A "therapeutically effective dose" of the anti-LAG-3 antibody of the invention preferably results in alleviation of disease symptoms, an increase in the frequency and duration of asymptomatic periods or prevents weakness or disability as a result of the disease. For example, in the treatment of tumor patients, a "therapeutically effective dose" preferably inhibits tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80 % for non-treated patients. A therapeutically effective amount of the therapeutic compound can reduce the size of the tumor, or otherwise alleviate the symptoms in a patient who is usually a human, but may be another mammal.

The pharmaceutical composition may be a controlled release formulation, which includes implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene / vinyl acetate (EVA), polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. See e.g.

Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc.,

New York, 1978.

- EP 2867258

The therapeutic compositions may be administered with medical devices such as (1) needless hypodermic devices (e.g., US 5,399,163; 5,383,851; 5,312,335;

5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) microinfusion pumps (US 4,487,603); (3) percutaneous devices (US 4,486,194); (4) infusion apparatus (US 4,447,233 and 4,447,224); and (5) osmotic devices (US 4,439,196 and 4,475,196).

In some embodiments, human monoclonal antibodies can be prepared to ensure adequate distribution in vivo. For example, to ensure the passage of the therapeutic compounds of the invention through the blood-brain barrier, they can be prepared in liposomes that can additionally contain targeting molecules to increase selective transport to specific cells or organs. See e.g. US

4,522,811; 5,374,548; 5,416,016; and 5,399,331; VV Ranade (1989) J. Clin. Pharmacol. 29: 685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038; Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180; Briscoe et al. (1995) Am. J. Physiol. 1233: 134; Schreier et al. (1994) J Biol. Chem. 269: 9090; Keinanen and Laukkanen (1994) FEBS Lett. 346: 123; and Killion and Fidler (1994) Immunomethods 4: 273.

Application of the invention

Antibodies (compositions, bispecific and immunoconjugates) of the present invention have many in vitro and in vivo applications, including, for example, LAG-3 detection or enhancement of the immune response by LAG-3 block. In preferred embodiments, the antibodies are human antibodies. Such antibodies can be administered to cells in vitro or ex vivo culture or human, e.g. in vivo, to increase immunogenicity in a variety of situations. Accordingly, the antibody, or antigen-binding portion thereof, of the invention may be used in a method of modifying an immune response in a patient comprising administering to the patient an antibody, or antigen-binding portion thereof, such that the patient's immune response is modified. Preferably, the reaction is enhanced, stimulated or stronger.

Preferred patients are people who need to enhance the immune response. The methods are particularly suitable for the treatment of patients - people with disorders that can be treated by increasing the immune response (e.g., a T cell mediated immune response). In addition, the methods are particularly useful in the treatment of cancer in vivo. To obtain an antigen specific enhancement, the anti-LAG-3 antibodies can be administered together with the antibody of interest, or the antigen may already be present in the patient being treated (e.g., a cancer patient or a viral infection). When anti-LAG-3 antibodies are administered in combination with another agent, they can be used in any order or simultaneously.

In addition, methods for detecting the presence of a human LAG-3 antigen in an assay or measuring a human LAG-3 antigen are described herein, including contact with a test and a control sample, with a human monoclonal antibody or antigen binding portion thereof specifically binding to human LAG-3 in conditions that allow the formation of an antibody complex or a portion thereof with human LAG-3. Subsequently, complex formation is detected when the difference between the formation of the complex as compared to the control indicates the presence of human LAG-3 antigen in the sample. In addition, the anti-LAG-3 antibodies of the invention can also be used to purify human LAG-3 by purification in immunoaffinity chromatography.

The demonstrated ability of the anti-LAG-3 antibodies of the invention to inhibit LAG-3 binding to class II major histocompatibility complex (MHC) molecules and to stimulate antigen-specific T cell responses, the antibodies of the invention can also be used in in vitro and in vivo methods to stimulate, enhance or increase the reaction of antigen-specific T cells. In one embodiment, the method comprises stimulating an antigen-specific T-cell reaction through contacted T-cells with

According to the invention, an antibody according to the invention is stimulated to stimulate the antigen specific T-cell reaction. Any suitable antigen-specific T-cell response indicator can be used to measure antigen-specific T cell responses. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of the antibody and / or increased cytokine production in the presence of the antibody. In a preferred embodiment, the production of interleukin-2 is stimulated by antigen-specific T cells.

The invention provides an antibody of the invention for use in a method of stimulating an immune response (e.g., a specific T cell response to an antigen) in a patient comprising administering to the patient an immune stimulating antibody in a subject (e.g., a T cell specific reaction to an antigen) In a preferred embodiment, the patient is a cancer patient and an anti-cancer immune reaction is stimulated. In another preferred embodiment, the patient is a patient infected with the virus and the antiviral response is stimulated.

Still in another embodiment, the invention provides an antibody of the invention for use in a method of inhibiting the growth of tumor cells in a patient comprising administering an antibody to the patient so as to inhibit tumor growth in the patient. And in yet another embodiment, the invention provides an antibody of the invention for use in a method of treating a viral infection in a patient comprising administering to the patient an antibody of the invention, such as to cure a viral infection in the treated subject.

These and other methods of the invention are discussed in detail below.

Tumor

Blockade of LAG-3 by antibodies can increase the immune response to cancer cells in a patient. In one aspect, the present invention provides an anti-LAG-3 antibody of the present invention for use in treating a patient in vivo to inhibit tumor growth. The anti-LAG-3 antibody can be used alone to inhibit the growth of cancerous tumors. Alternatively, the anti-LAG-3 antibody can be used in combination with other immunogenic agents, standard cancer treatment or other antibodies as described below.

Accordingly, in one embodiment, the invention provides an anti-LAG-3 antibody, or antigen-binding portion thereof, for use in a method of inhibiting tumor cell growth in a patient comprising administering to the patient a therapeutically effective amount of an anti-LAG-3 antibody or antigen-binding portion thereof. . Preferably, the human anti-LAG-3 antibody (such as any human anti-human LAG-3 antibodies is described herein). Additional or alternatively, the antibodies may be, e.g., chimeric or humanized anti-LAG-3 antibodies.

Preferably, tumors the growth of which can be inhibited using the described antibodies of the invention include tumors that are usually responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include saithe (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., non-prostate adenocarcinoma), breast cancer, colorectal cancer and lung cancer (e.g. small cell lung cancer). In addition, the present invention includes tumor-resistant (malignant) diseases that are refractory or recurrent, the growth of which can be inhibited using the described antibodies of the invention.

Examples of other cancers that can be treated using the antibodies of the invention include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant skin or eye melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer (anal region), cancer stomach, testicular cancer, cancer of the fallopian tubes, endometrial cancer, cervical cancer, cancer

cancers of environmental origin including asbestos-borne cancer, and combinations of these tumors. The present invention is also useful in the treatment of metastatic tumors, particularly metastatic tumors expressing PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144).

Optionally, anti-LAG-3 antibodies can be combined with an immunological agent, such as tumor cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune-stimulating cytokines (He et al. ( 2004) J. Immunol. 173: 4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antibody peptides, such as gp100 peptides, MAGE, Trp-2, MART1 and / or tyrosinase antigens, or tumor cells transfected to express the GM-CSF cytokine (as discussed below).

As has been shown in humans, some cancers are immunogenic such as melanomas. By raising the threshold of T-cell activation as a result of LAG-3 blockade, the cancer reaction of the host can be activated.

The LAG-3 block is probably more effective when combined with the vaccination protocol. Many strategies for experimental vaccination against cancer have been developed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 6062; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738, see also Restifo, N. and Sznol, M., Cancer Vaccines, R. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer: Principles and Practice of Oncology, fifth edition). In one strategy, the vaccine was prepared using autologous or allogeneic tumor cells. Such cell vaccines have been shown to be most effective when tumor cells are transduced, to express GM-CSF. As has been shown, GM-CSF is a potent activator of antigenic presentation during cancer vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci USA 90: 3539-43).

Study of gene expression and large-scale gene expression patterns in various cancers leads to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, such tumor-specific antigens are differentiating antigens expressed in tumors and in a cell from which the tumor has multiplied, for example gp100 melanocyte antigens, MAGE and Trp2 antigens. More importantly, many of these antigens can be demonstrated as targets of tumor-specific T cells found in the host. The LAG-3 block can be used in combination with a collection of recombinant proteins and / or peptides expressed in the tumor to generate an immune response to these proteins. These proteins are usually reviewed by the immune system as their own antigens and therefore tolerated. Tumor antigens may include white telomerase, which is necessary for the synthesis of chromosomes telomers and which is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (Kim et al (1994) Science 266: 2011-2013). (These somatic tissues can be protected against immune attack by various means). The antigen may also be "neo-antigens" expressed in tumor cells because of somatic mutations (These somatic tissues can be protected against immune attack by various means). The antigen may also be "neo-antigens" expressed in tumor cells because of somatic mutations (These somatic tissues can be protected against immune attack by various means). The antigen may also be "neo-antigens" expressed in tumor cells because of somatic mutations

2867258 altering the protein sequence or forming proteins conjugated between two unbound sequences (i.e., bcr-abl on the Philadelphia chromosome) or the idiotype of B cell tumors.

Other cancer vaccines may include viral proteins that are involved in human cancers, such as human papillomavirus (HPV) viruses, hepatitis viruses (HBV and HCV) and Kaposi's sarcoma virus (KHSV). Another form of tumor specific antigen can be used in conjunction with the LAG-3 blockade are purified heat shock proteins (HSP) isolated from tumor tissue. Heat shock proteins contain fragments of tumor cell proteins and these HSP cells are highly effective in delivering antigen presenting cells for tumor resistance (Suot &amp; Srivastava (1995) Science 269: 1585-1588; Tamura et al. (1997) Science 278 117-120).

Dendritic cells (DCs) are potent antigen presenting cells that can be used to obtain the first antigen-specific reaction. DCs can be made ex vivo and provided with various peptide proteins and antigens as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be transduced by genetic methods to also display these tumor antigens. DC can also be directly coupled to tumor cells for immunization (Kugler et al. (2000) Nature Medicine 6: 332-336). DC immunization as a vaccination method can be effectively combined with LAG-3 blockade to activate stronger anti-cancer reactions.

The LAG-3 blockade can also be combined with standard cancer treatment. The LAG-3 blockade can also be effectively combined with chemotherapy regimens. In these cases, it may be likely to reduce the dose of the chemotherapy agent administered (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is the LAG-3 antibody in combination with decarbaZine in the treatment of melanoma. Other example of such a combination is the anti-LAG-3 antibody in combination with interleukin-2 (IL-2) in the treatment of melanoma. The rationale for the scientific use of combined LAG-3 blockade and chemotherapy is that cell death, as a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway. Other combination therapies, which may cause synergy with LAG-3 blockade through cell death, irradiation, surgery, and hormone deprivation. Each of these protocols forms a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with LAG-3 blockade. Inhibition of angiogenesis leads to the death of tumor cells that can nourish the tumor antigen on the antigen presentation pathways of the host.

LAG-3 blocking antibodies can also be used in combination with bispecific antibodies targeting effectors expressing the Fcca or Fcc receptor into tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target microphages to tumor sites. Targeting can more effectively activate tumor-specific reactions. The T cell arm of these reactions should be increased by the use of the LAG-3 block. Optionally, the antigen may be delivered directly to dendritic cells (DCs) using bispecific antibodies that bind to the tumor antigen and a specific dendritic cell surface marker.

Cancers avoid host immune surveillance using a wide variety of mechanisms. Many of these mechanisms can be overcome by inactivating tumor-expressed proteins that are immunosuppressive. This includes, but is not limited to, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard &amp; O'Garra (1992) Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies to each of these molecules can be used in combination with anti-LAG-3 to counteract the action of immunosuppressive drugs and to support host tumor immune responses.

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Other antibodies that activate host immune responses can be used in combination with anti-LAG-3. This includes surface molecules of dendritic cells that activate DC activity and antigen presentation. Anti-CD40 antibodies can effectively replace T cell helper activity (Ridge et al. (1998) Nature 393: 474-478) and can be used in combination with LAG-3 antibodies (Ito et al. (2000) Immunobiology 201 (5) 52740). Activating antibodies against co-stimulatory molecules of T cells, such as CTLA-4 (e.g., US Patent No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169),

4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff et al. (1999) Nature 397: 262-266) may also provide increased levels of T cell activation.

Bone marrow transplantation is currently used in the treatment of various hematopoietic cancers. While graft versus host disease (GVHD) is a consequence of such treatment, therapeutic benefits can be obtained from graft vs cancer reactions. The LAG-3 blockade can be used to increase the efficacy of T cell-specific tumor cells.

There are also many protocols of experimental treatment including ex vivo activation and expansion of antigen-specific T cells and the transfer of these cells to recipients to stimulate tumor-specific T cells against antigen (Greenberg &amp; Riddell (1999) Science 285: 546-51). The methods provided can also be used to activate T cell responses to infectious agents such as CMV. Activation ex vivo in the presence of anti-LAG-3 antibodies may increase the frequency and activity of adoptively transferred T-cells.

Infectious diseases

In addition, the methods described herein have been used to treat patients exposed to particular toxins or pathogens. Accordingly, a method is described herein for treating an infectious disease in a patient, which comprises administering to the patient an anti-LAG-3 antibody, or antigen-binding portion thereof, such that the patient is being treated for an infectious disease. Preferably, the human antibody is an anti-human LAG-3 antibody (such as any human anti-LAG-3 antibodies described herein). Additionally or alternatively, the antibody may e.g. be a chimeric or humanized antibody.

Similarly to its use for tumors as described above, the LAG-3 intermediate blocking antibody can be used alone or as an adjuvant, in combination with vaccines, to stimulate an immune response to toxin and auto-antigen pathogens. Examples of pathogens where such a therapeutic approach may be particularly useful include pathogens that currently do not have an effective vaccine, or pathogens in which conventional vaccines are less than completely effective. These include, but are not limited to, HIV, viral hepatitis (A, B and C), influenza, herpes, lamblia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. The LAG-3 blockade is particularly useful against known infections caused by factors such as HIV that present altered antigens in the course of infections.

Some examples of pathogenic viruses that cause infections treated with anti-human LAG-3 antibodies, or antigen-binding parts thereof, of the invention include HIV, hepatitis viruses (A, B or C), herpes viruses (e.g., VZV, HSV-1, HAV- 6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza viruses, flaviviruses, echoviruses, rhinoviruses, Coxsackie viruses, coronaviruses, respiratory syncytial virus, parotidal gland virus, rotavirus, measles virus, rubella virus, parvoviruses , vaccinia virus, HTLV virus, dengue virus, papillomavirus, infectious mollusc virus, spinal cord antigens, rabies virus, JC virus and arbitis encephalitis.

- EP 2867258

Some examples of pathogenic bacteria causing infections treated by the methods described herein include chlamydia, rickettsiae, mycobacteria, staphylococci, streptococci, pneumococci, meningococcus and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, bacillus botulinum toxin, anthrax, plague, leptospirosis and borreliosis (Lyme disease).

Some examples of pathogenic fungi causing infections treated by the methods described herein include: Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucorales species (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treated with the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii , Nippostrongylus brasiliensis.

In all of the above methods, LAG-3 blockade can be combined with other types of immunotherapy, such as cytokine treatment (e.g., interferonamis, GM-CSF, G-CSF, IL2), or bispecific antibody therapy, which provides increased presentation of tumor antigens (see e.g. Holliger (1993) Proc. Natl Acad. Sci USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123).

Autoimmune reactions

Anti-LAG-3 antibodies can trigger and increase autoimmune reactions. Of course, the induction of an anti-cancer reaction using peptide tumor cell vaccines reveals that many anti-cancer reactions involve the body's response. (van Elsas et al.

(2001) J. Exp. Med. 194: 481-489; Overwijk, et al. (1999) Proc. Natl. Acad. Sci. USA 96: 29822987; Hurwitz, (2000) above; Rosenberg &amp; White (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4). Therefore, one can contemplate the use of anti-LAG-3 blockade in combination with various auto-proteins to develop protocols to successfully generate immune reactions against these self-proteins in the treatment of the disease. For example, there is abnormal accumulation of Aβ-peptide in amyloid deposits in the brain in Alzheimer's disease; anti-amyloid antibody reactions can purify existing amyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other personal proteins such as IgE for the treatment of allergy and asthma as well as TNFα for the treatment of rheumatoid arthritis may also be used as targets. Ultimately, anti-LAG-3 antibodies can induce antibody responses to various hormones. Neutralizing antibody responses to sex hormones can be used in contraception. Neutralizing the antibody response to hormones and other soluble factors necessary for the growth of certain tumors can also be considered as possible vaccination targets.

Methods analogous to those described above for the use of anti-LAG-3 antibodies can be used to induce autoimmune autoimmune reactions in the treatment of patients with abnormal accumulation of other auto-antigens, such as amyloid deposits, including Aβ in Alzheimer's disease, cytokines such as TNFα , and IgE.

vaccines

Anti-LAG-3 antibodies can be used to stimulate antigen-specific immune reactions by co-administering the anti-LAG-3 antibody with the antigen of interest (e.g., a vaccine). Accordingly, a method is described herein for enhancing an immune response to an antigen in a patient that comprises administering to the patient: (i) an antigen; and (ii) an anti-LAG-3 antibody, or antigen binding portion thereof, such that

- The immune response to the antigen of the patient is enhanced. Preferably the human antibody is an anti-human LAG-3 antibody (such as any human anti-LAG-3 antibodies described herein). Additionally or alternatively, the antibody may e.g. be a chimeric or humanized antibody. The antigen may be, for example, a tumor tumor antigen, a viral antigen, a bacterial antigen or an antigen from a pathogen. Unlimited examples of such antigens include those discussed in the above chapters, such as tumor antigens (or tumor vaccines) discussed above, or antigens from viruses, bacteria or other pathogens described above.

The described suitable routes for administering antibody compositions (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) of the invention in vivo and in vitro are well known in medicine and can be selected by skilled workers. For example; compositions containing antibodies can be administered by injection (e.g., intravenous or subcutaneous). The appropriate dosage of the particles depends on the age and weight of the patient and the concentration and / or composition of the particular antibody-containing composition.

As previously described, the anti-LAG-3 human antibodies of the invention can be administered together with one or more drugs, e.g. a cytotoxic agent, a radiotoxic or an immunosuppressant. The antibody may be combined with the drug (as an immune complex) or administered separately in addition to the drug. In the latter case (administered separately), the antibody should be used before, after or in parallel with the drug or along with other known forms of treatment, e.g. anti-cancer therapy, e.g. irradiation. Such therapeutic agents include, but are not limited to, anti-cancer agents, e.g. doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine and hydroxyurea cyclophosphamide, which are administered separately are effective only at the toxic or sub-toxic level for the patient. Cisplatin is administered intravenously at doses of 100 mg / kg once every four weeks, and adriamycin administered intravenously at doses of 60-75 mg / ml every 21 days. Combined use of human anti-LAG-3 antibodies or antigen-binding portions thereof, in accordance with the present invention, in combination with drugs used in chemotherapy, provides two anti-cancer substances that operate using various mechanisms of cytotoxic action on human tumor cells. Such combined drug delivery can solve problems arising from the development of drug resistance or changes in the antigenicity of tumor cells that cause them to stop responding to antibodies. and adriamycin administered intravenously at doses of 60-75 mg / ml every 21 days. Combined use of human anti-LAG-3 antibodies or antigen-binding portions thereof, in accordance with the present invention, in combination with drugs used in chemotherapy, provides two anti-cancer substances that operate using various mechanisms of cytotoxic action on human tumor cells. Such combined drug delivery can solve problems arising from the development of drug resistance or changes in the antigenicity of tumor cells that cause them to stop responding to antibodies. and adriamycin administered intravenously at doses of 60-75 mg / ml every 21 days. Combined use of human anti-LAG-3 antibodies or antigen-binding portions thereof, in accordance with the present invention, in combination with drugs used in chemotherapy, provides two anti-cancer substances that operate using various mechanisms of cytotoxic action on human tumor cells. Such combined drug delivery can solve problems arising from the development of drug resistance or changes in the antigenicity of tumor cells that cause them to stop responding to antibodies. acting using various mechanisms of cytotoxic action on human tumor cells. Such combined drug delivery can solve problems arising from the development of drug resistance or changes in the antigenicity of tumor cells that cause them to stop responding to antibodies. acting using various mechanisms of cytotoxic action on human tumor cells. Such combined drug delivery can solve problems arising from the development of drug resistance or changes in the antigenicity of tumor cells that cause them to stop responding to antibodies.

Also included within the scope of the present invention are kits comprising antibody compositions of the invention (e.g., human antibodies, multispecific or bispecific molecules, or immunoconjugates), and instructions for using them. The kit may further comprise one or more additional reagents, such as one or more of the described additional human antibodies of the invention (e.g., a human antibody with complementary activity, binding to the LAG-3 antigen epitope, differing from the first human antibody). Sets usually contain a label indicating the use of the contents of the kit. The term label includes any subtitles or saved material supplied with the kit, or in any other way accompanying the kit.

Combination therapy

In another aspect, the invention provides an anti-LAG-3 antibody (or antigen-binding portion thereof) of the invention for use in a combination therapy method in which the anti-LAG-3 antibody (or antigen-binding portion thereof) is co-administered with one or more other the number of additional antibodies that are effective in stimulating the immune responses thus further enhancing, stimulating or increasing the immune responses of the patient. In one embodiment, the invention provides an anti-LAG-3 antibody of the invention for use in a method of stimulating an immune response in a patient comprising

- administration of an anti-LAG-3 antibody and at least one additional immunostimulatory antibody, such as an anti-PD-1 antibody, anti-PD-1 antibody and / or anti-CTLA-4 antibody, to stimulate an immune response in the patient for example to inhibit tumor growth or stimulate antiviral response. In another embodiment, the patient is administered an anti-LAG-3 antibody and an anti-PD-1 antibody. In yet another embodiment, the patient is administered an anti-LAG3 antibody and an anti-PD-L1 antibody. In yet another embodiment, the patient is administered an anti-LAG-3 antibody and an anti-CTLA-4 antibody. In one embodiment, the anti-LAG-3 antibody is a human antibody, such as an antibody of the present disclosure. Alternatively, the anti-LAG-3 antibody may be, for example, a chimeric or humanized antibody (e.g. prepared from a mouse anti-LAG-3 mAb). In another embodiment, at least one additional immunostimulatory antibody (e.g., anti-PD-1 antibody, anti-PD-L1 and / or anti-CTLA-4) is a human antibody. Alternatively, at least one additional immunostimulatory antibody may be, for example, a chimeric or humanized antibody (e.g., prepared from a mouse anti-PD-1 antibody, anti-PD-L1 and / or anti-CTLA-4).

In addition, a method of treating a hyperproliferative disease is described herein (e.g.

cancer), comprising administering the LAG-3 antibody and the CTLA-4 antibody to the patient. The LAG-3 antibody may be administered in a subtherapeutic dose, the anti-CTLA4 antibody is administered in a subtherapeutic dose, or both are administered in a subtherapeutic dose.

Furthermore, a method for altering an adverse event associated with the treatment of a hyperproliferative disease with an immunostimulatory drug is described herein, comprising administering a LAG-3 antibody and a sub-therapeutic dose of a CTLA-4 antibody to a patient. The patient can be a human. The anti-CTLA4 antibody may be a human sequence of a 10D1 monoclonal antibody (described in PCT publication WO 01/14424) and the anti-LAG-3 antibody may be a human monoclonal antibody sequence such as the LAG3.5 antibodies described herein. Other anti-CTLA-4 antibodies, encompassed by the methods of the present invention, include, for example, those disclosed in: WO 98/42752; WO 00/37504; U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc. Natl. Acad. Sci. USA 95 (17): 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22 (14S): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res. 58: 5301-5304. In certain embodiments, the anti-CTLA-4 antibody binds to human CTLA-4 with a KD of 5 x 10^-8 M or below, binds to human CTLA-4 at a KD of 1x10^-8 M or below, binds to a human CTLA-4 KD of the order of 5 x 10^-9 M below, or is associated with human CTLA-4 at a KD of the order of 1 x 10^-8 M and 1 x 10^-10 M or below.

In addition, a method of treating a hyperproliferative disease is described herein (e.g.

tumors), comprising administering the LAG-3 antibody and the PD-1 antibody to the patient.

The anti-LAG-3 antibody may be administered in a subtherapeutic dose, the anti-PD-1 antibody is administered in a subtherapeutic dose, or both are administered in a subtherapeutic dose.

In addition, the method described herein modifies adverse events associated with the treatment of a hyperproliferative disease with an immunostimulatory drug, comprising administering anti-LAG-3 antibody and a subtherapeutic dose of an anti-PD-1 antibody to a patient. The patient can be a human. The anti-PD-1 antibody may be a human monoclonal antibody sequence and the anti-LAG-3 antibody may be a human monoclonal antibody sequence, such as the LAG3.5 sequences described herein. The human anti-PD-1 antibody sequence sequences include 17D8, 2D3, 4H1, 5C4 and 4A11 , described in PCT publication WO 06/121168. Other anti-PD-1 antibodies include, e.g., lambrolizumab (WO2008 / 156712), and AMP514 (WO2010 / 027423, WO2010 / 027827, WO2010 / 027828, WO2010 / 098788). The anti-PD1 antibody may bind to human PD-1 at KD 5 x 10^-8 M or below, with human PD-1 at KD 1 x 10^-8 M or below, with human PD-1 at KD 5 x 10^-9 M or below, or may bind to human PD-1 at K_D between 1 x 10^-8 M or i 1 x 10^-10 M or below.

- EP 2867258

Furthermore, a method of treating a hyperproliferative disease (e.g., a cancer) is described herein, comprising administering a LAG-3 antibody and a PD-L1 antibody to a patient. The LAG-3 antibody may be administered in a subtherapeutic dose, the anti-PD-L1 antibody may be administered in a subtherapeutic dose, or both are administered in a subtherapeutic dose. In addition, the method described herein modifies adverse events associated with the treatment of a hyperproliferative disease with an immunostimulatory drug, comprising administering anti-LAG-3 antibody and a subtherapeutic dose of an anti-PD-L1 antibody to a patient. The patient can be a human. The anti-PD-L1 antibody may be a human monoclonal antibody sequence and the anti-LAG-3 antibody may be a human sequence of a monoclonal antibody, such as LAG3 described herein. Examples of human anti-PD-L1 antibody sequences include 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4, described in PCT publication WO 07/005874. Other anti-PD-L1 antibodies include, e.g., MPDL3280A (RG7446) (WO2010 / 077634), MEDI4736 (WO2011 / 066389), and MDX1105 (WO20071005874). The anti-PD-L1 antibody described may bind to human PD-L1 at a KD of 5 x 10^-8 M or below, with human PD-L1 at KD 1 x 10^-8 M or below, with human PD-L1 at KD 5 x 10^-9 M or below, or bind to human PD-L1 at KD between 1 x 10^-8 M and 1 x 10^-10 M or below.

Blockade of LAG-3 and one or more secondary target antigens, such as CTLA-4 and / or PD-1 and / or PD-L1 by antibodies, can increase the immune response to cancer cells in a patient. Tumors, the growth of which can be inhibited using the described antibodies of the disclosure, include tumors that are usually responsive to immunotherapy. Representative examples of tumors for treatment with the combination therapy of the present disclosure include cancers specifically mentioned in monotherapy discussion with anti-LAG-3 antibodies.

In certain embodiments, a combination of therapeutic antibodies herein can be administered simultaneously as one composition in a pharmaceutically acceptable carrier, or simultaneously as separate compositions with each antibody in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies can be administered sequentially. For example, an anti-CTLA-4 antibody and an anti-LAG-3 antibody may be administered sequentially, such as an anti-CTLA-4 antibody administered first and an anti-LAG-3 antibody as a second or an anti-LAG-3 antibody administered as first and anti-CTLA-4 antibody as second. Additionally or optionally, the anti-PD-1 antibody and the anti-LAG-3 antibody can be administered sequentially, such as the first anti-PD-1 antibody and the anti-LAG-3 antibody second or the first anti-LAG-3 antibody and the anti-PD-1 antibody second. Additionally or optionally, the anti-PD-L1 antibody and the anti-LAG-3 antibody can be administered sequentially, such as the anti-PD-L1 antibody administered first and the anti-LAG-3 antibody as second or the anti-LAG-3 antibody administered sequentially. first and the anti-PD-L1 antibody as the second.

Furthermore, if more than one dose of combination therapy is administered sequentially, the order of sequential administration may be reversed or maintained in the same order at each time point of administration, sequential administration may have been combined with concurrent administration or any combination thereof. For example, the first administration of a combination of anti-CTLA-4 antibody and anti-LAG-3 antibody may be simultaneous, the second may be sequential with the first anti-CTLA-4 and anti-LAG-3 second, and the third may be sequential with the first anti -LAG-3 and anti-CTLA-4 second, etc. Additionally or optionally, the first administration of the anti-PD-1 antibody and anti-LAG-3 antibody combination may be simultaneous, the second may be sequential with the first anti-PD-1 and anti-LAG-3 second, and the third can be sequential with the first anti-LAG-3 and anti-PD-1 second,

A typical dosing regimen may comprise first sequential administration with a first anti-LAG-3 and a second anti-CTLA-4, (and / or anti-PD-1 and / or anti-PD-L1), and sequential administration may be simultaneous.

Optionally, the combination of an anti-LAG-3 antibody and one or more additional antibodies (e.g., anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies) can be further combined with an immunological agent, such as such as tumor cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune-stimulating cytokines (He et al. (2004) J. Immunol. 173: 4919-28). Non-limiting examples of tumor vaccines that may be used include melanoma antibody peptides, such as gp100 peptides, MAGE, Trp-2, MART1 and / or tyrosinase antigens, or tumor cells transfected to express the GM-CSF cytokine (as discussed below) .

Combined LAG-3 and CTLA-4 blockade and / or PD-1 and / or PD-L1 blockade can also be further combined with standard cancer treatment. For example, the combined LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade can also be effectively combined with chemotherapeutic regimens. In such cases, it is possible to reduce the dose of another chemotherapeutic agent administered in combination with the present disclosure (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is the combination of anti-LAG-3 antibody and anti-CTLA-4 antibodies and / or anti-PD-1 antibodies and / or anti-PD-L1 antibodies further in combination with decarbaZine in the treatment of melanoma. Another example is the combination of anti-LAG-3 antibody and anti-CTLA-4 antibodies and / or anti-PD-1 antibodies and / or anti-PD-L1 antibodies further in combination with interleukin-2 (IL-2) in the treatment of melanoma . The scientific justification underlying the use of LAG-3 blockade and blockade of CTLA-4 and / or PD-1 and / or PD-L1 in chemotherapy, which is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway. Other combination therapies that can synergize with the combined LAG-3 blockade and CTLA4 and / or PD-1 and / or PD-L1 through cell death are irradiation, surgery, and hormone deprivation. Each of these protocols forms a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with a combination of LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade. Braking aNogogenesis leads to the death of tumor cells that can be the source of tumor antigen administered to the host antigen presentation pathway.

The combination of LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blocking antibodies can also be used in combination with bispecific antibodies targeting effectors expressing the Fcα or Fcγ receptor into tumor cells (see, e.g., U.S. Patent No. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. The T cell of these reactions should be increased by the use of LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade.

In another example, the combination of anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies may be used in combination with anti-neoplastic antibodies such as Rituxan® (rituximab), Herceptin. ® (trastuzumab), Bexxar® (tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab), Lymphocide® (eprtuzumab), Avastin® (bevacizumab), and Tarceva® (erlotinib), and the like. By way of example, without resorting to theory, it is concluded that treatment with an anti-cancer antibody or a toxin-conjugated antitumor antibody can lead to the death of a tumor cell (e.g., tumor cells) which should increase the CTLA-4 dependent immune response, PD-1 , PD-L1 or LAG-3. In an exemplary embodiment, the treatment of a hyperproliferative disease (e.g.

Anti-PD-1 and / or anti-PD-L1, simultaneously or sequentially or any combination thereof, which may enhance the anti-tumor immune response of the host against the tumor.

Cancers avoid host immune surveillance using a wide variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins that are expressed in tumors and that are immunosuppressive. These include, but are not limited to, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard &amp; O'Garra (1992) Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). In another example, the antibodies of each of these molecules can be further used in combination with a combination of anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies to counteract the action of immunosuppressive drugs. and to foster host anti-cancer immune responses.

Other antibodies that can be used to activate the host immune reaction can be further used with a combination of anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies. This includes surface molecules of dendritic cells that activate DC activity and antigen presentation. Anti-CD40 antibodies (Ridge et al., Supra) can be used with a combination of anti-LAG-3 and anti-CTLA-4 and / or anti-PD1 and / or anti-PD-L1 antibodies. Other activation of antibodies against co-stimulatory molecules of T cells Weinberg et al., Supra, Melero et al., Supra, Hutloff et al., Supra) may also provide increased levels of T cell activation.

As discussed above, bone marrow transplantation is currently used in the treatment of various hematopoietic cancers. The combined blockade of LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 can be used to increase the efficiency of T-cells specific for donor cancer.

Many experimental protocols include ex vivo activation and expansion of antigen-specific T cells and the transfer of these cells to recipients to stimulate T-cell specific antigen against cancer (Greenberg &amp; Riddell, supra). The methods provided can also be used to activate T cell responses to infectious agents such as CMV. It can be assumed that ex vivo activation in the presence of anti-LAG-3 and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies may increase the frequency and activity of adoptively transferred T-cells.

In addition, the method described herein modifies adverse events associated with the treatment of a hyperproliferative disease with an immunostimulatory drug, including the administration of anti-LAG-3 antibody and a subtherapeutic dose of anti-CTLA-4 and / or anti-PD-1 and / or anti-PD antibody. L1 to the patient. For example, such methods include methods for reducing cases of intestinal inflammation or diarrhea induced by an immunostimulatory therapeutic antibody by administering a non-resorbable steroid to a patient. Since any patient who receives an immunostimulatory therapeutic antibody is at risk of developing intestinal inflammation or diarrhea induced by such an antibody, the entire patient population is suitable for treatment according to the methods of the present invention. Although steroids have been administered for the treatment of IBD and the prevention of exacerbation of IBD, they have not been used to prevent (disease, or occurrence) IBD in patients who have not been diagnosed with IBD. Significant side effects associated with steroids, even non-absorbable steroids, discouraged prophylactic use.

In further embodiments, a combination of LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade (i.e., anti-LAG-3 immunostimulatory therapeutic antibodies and anti-CTLA-4 and / or anti-PD-1 antibodies) and / or anti-PD-L1) can be further combined using any non-resorbable steroid. As used herein, "non-resorbable steroid" is a glucocorticoid having extensive first-pass metabolism such that subsequent metabolism in the liver, steroid bioavailability is low, i.e. below 20%. In one embodiment of the invention, the non-absorbable steroid is budesonide.

- EP 2867258

Budesonide is a topical glucocorticosteroid that is metabolized extensively, mainly in the liver, after oral administration. ENTOCORT EC® (Astra-Zeneca) is a pH-dependent and time-dependent oral budesonide formulation designed to optimize drug delivery to the ileum and the colon. ENTOCORT EC® is approved in the US for the treatment of mild to moderate forms of Crohn's disease including ileum and / or ascending colon The usual ENTOCORT EC® oral dose for the treatment of Crohn's disease is 6 to 9 mg / day. ENTOCORT EC® is released in the intestines before absorption and retention through the intestinal mucosa. After passing through the target intestinal mucosa, ENTOCORT EC® is metabolized extensively through the cytochrome P450 system in the liver to metabolites with low glucocorticoid activity. Because, bioavailability is low (around 10%). The low bioavailability of budesonide gives a better therapeutic index compared to glucocorticoids with less extensive first-pass metabolism. Budesonide causes fewer side effects, including less suppression of the hypothalamus, than systemically acting corticosteroids. However, chronic administration of ENTOCORT EC® may cause systemic glucocorticoid effects such as excessive secretion of adrenocortical cortices and suppression of the adrenal cortex. See PDR ed. 58 2004; 608,610. than systemically acting corticosteroids. However, chronic administration of ENTOCORT EC® may cause systemic glucocorticoid effects such as excessive secretion of adrenocortical cortices and suppression of the adrenal cortex. See PDR ed. 58 2004; 608,610. than systemically acting corticosteroids. However, chronic administration of ENTOCORT EC® may cause systemic glucocorticoid effects such as excessive secretion of adrenocortical cortices and suppression of the adrenal cortex. See PDR ed. 58 2004; 608,610.

In further further embodiments, the combination of LAG-3 and CTLA-4 and / or PD-1 and / or PD-L1 blockade (i.e., anti-LAG-3 immunostimulatory therapeutic antibodies and anti-CTLA-4 and / or anti-block antibodies) PD-1 and / or anti-PD-L1) in combination with a non-resorbable steroid can be further combined with the use of salicylate. Salicylates include 5-ASA compounds such as, for example, sulfasalazine (AZULFIDINE®, Pharmacia &amp; UpJohn); olsalazine (DIPENTUM®, Pharmacia &amp; UpJohn); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.) and mesalamine (ASACOL®, Procter &amp; Gamble Pharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc., ROWASA®, Solvay).

According to the methods of the present invention, salicylate administered in combination with anti-LAG-3 antibodies and anti-CTLA-4 and / or anti-PD-1 and / or anti-PD-L1 antibodies and a non-absorbable steroid may comprise overlapping or sequential administration of salicylate and a non-absorbable steroid to reduce the number of intestinal inflammations induced by immunostimulatory antibodies. Thus, for example, methods for reducing the number of intestinal inflammations induced by immunostimulatory antibodies of the present invention include the administration of a salicylate and a non-resorbable steroid simultaneously or sequentially (e.g., salicylate administered 6 hours after a non-resorbable steroid) or any combination thereof. In addition, according to the present invention, salicylate and non-absorbed steroid may be administered by the same route (e.g.

The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. Particular reference is made to PCT publication WO 09/045957, WO 09/073533, WO 09/073546, and WO 09/054863.

Examples

Example 1: Design of LAG3.1 variants (antibody 25F7)

Variants of 25F7, a previously described anti-LAG-3 antibody, referred to herein as LAG3.1, were constructed by first analyzing the amino acid sequence of the antibody for potential decomposition sites. Expression of mutagenesis of the targeted VH LAG3.1 region was performed using the QuikChange II XL® Site-Directed targeted mutagenesis kit.

Mutagenesis Kit (Agilent Technologies). The changed VH regions were then subcloned into

U.S. EPO (EMD Millipore) containing the human IgG4-S228P constant region. Various heavy chain vectors, each of which was cotransfected with a vector expressing the LAG3.1 kappa chain into CHO-S cells, and then stable pools (harvested) were selected for expression.

Five potential motivations for deamination were identified in the variable region of the heavy CDR2. These sites are in positions 52, 54, 56, 58, and 60 of the variable region of the heavy chain LAG3.1 (SEQ ID NO: 2) (see Figure 1A). Particularly, deamination of the "NG" sequence in VH CDR2 (SEQ ID NO: 6) was observed under all conditions, as well as further sequence isomerization. The deamination of the starting material was about 10%. Furthermore, it was found that the sequence "NG" does not correspond to the germline sequence (see figure 3). However, the compliance of the germline sequence was at the glycosylation site, and therefore, was not included in the antibody variants.

Four variants (here in relation to LAG3.5, LAG3.6, LAG3.7 and LAG3.8) were designed, two of which related to potential deamination motifs (positions 54 and 56), as can be seen in Figure 3. The options were matrix of conditions, as summarized in Table 1 below, and analyzed the following parameters: (a) chemical and thermal stability (physical stability); (b) size exclusion chromatography (aggregation); (c) isoelectric focusing (IEF) (load unbalance); (d) Biacore activity (binding and functional activity) and (e) peptide mapping in mass spectrometry (chemical modifications / molecular stability).

Table 1

<td>Buffer</td><td>Acetate (100nM NaCl, 3% w / v mannitol, 0.03% Tween-20)</td><td>Citrate (100nM NaCl, 3% w / v mannitol, 0.03% Tween-20)</td>

<td>PH</td><td>5.5, 6.0, 6.5, 7.0</td><td>5.5, 6.0, 6.5, 7.0</td>

<td>Temperature</td><td>4 ° C and 37 ° C</td><td>4 ° C and 37 ° C</td>

<td>Time</td><td>0, 4, 8, 12 weeks</td><td>0, 4, 8, 12 weeks</td>

Example 2: Characteristics of LAG-3 variants

1. Activation binding of human CD4 + T cells

To study the binding capacity of antibody variants to native human LAG-3 on the surface of activated human T cells, normal peripheral blood mononuclear cells were stimulated on 15 cm tissue culture plates at a density of 2x10 6 cells / ml with a combination of anti-CD3 antibodies (eBioscience, no. cat. 16-0037-85) and anti-CD28 (BD Bioscience, cat. no. 555725) present in a solution of 5 μg / ml and 3 μg / ml, respectively. After three days of stimulation the cells were expanded, washed 1x with PFAE 1x buffer (1x PBS + 2% FBS, 0.02% sodium azide, 2mM Na EDTA), and resuspended in 1x PFAE buffer for color.

Regarding the binding reaction, the LAG3.1 variants were diluted in series with cold PFAE 1x buffer, and then 50 μl of the diluted antibody solution was mixed with 50 μl of anti-human CD4 labeled Fitc (BD Bioscience, Cat. No. 555346) dissolved in 1:16 in 1x buffer PFAE. Regarding the binding reaction, 100 μl of this diluted antibody mixture was added to 2x10⁵ cells and the resulting mixture was incubated at 4 ° C for 30 minutes. The cells were then washed twice with 1x PFAE buffer. Diluted 1: 200 PE-labeled Goat anti-human Fcy-specific antibody (Jackson ImmunoResearch, Cat. No. 109-116-170) was added and the resulting mixture was incubated for 30 minutes at 4 ° C and then washed twice with cold 1x PFAE buffer. After the final wash, 150 μl of cold was added

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1x PFAE for each antibody binding solution and analysis was performed in flow cytometry on a FACSCanto flow cytometer (BD Bioscience).

The results of the flow cytometry analysis are summarized in Figure 4A, which is a graph indicating the EC50 for antibody binding to activated human CD4 + T cells. Figure 4B is a graph indicating the binding of antibodies to the soluble human LAG-3 / Fc antigen in BIACORE. It has been shown that the affinity of LAG3.5 and LAG3.8 binding is slightly lower, compared to LAG3.1, while their dissociation constants are slightly higher compared to LAG3.1.

2. Physical stability

Thermal stability and thermal denaturation of the variants were tested using the Microcal VP-DSC. Specially, each of the variants was diluted in PBS (Mediatech cat. No. 21-040-CV lot number 21040139). The final sample concentration was 250 μg / ml after dilution in PBS. The sample was scanned to 74 ° C, cooled to 25 ° C, and again heated to 74 ° C. The PBS buffer was used as a blank control. The data fits the model with states of more than two (non-2-state) and a tailored curve derived by the Origin software.

As summarized in Table 2 and shown in Figure 5, LAG3.5 has a higher melting point of TM2 than LAG3.1, indicating higher overall stability.

Table 2

<td>Has b</td><td>Tm1 (° C)</td><td>Tm2 (° C)</td>

<td></td><td>Corresponds to CH2 and / or Fab domains</td><td>Corresponds to the CH3 and / or Fab domains</td>

<td>LAG3.1</td><td>70.7</td><td>75.7</td>

<td>LAG3.5</td><td>70.5</td><td>76.3</td>

<td>LAG3.6</td><td>67.8</td><td>70.8</td>

<td>LAG3.7</td><td>69.4</td><td>73.5</td>

<td>LAG3.8</td><td>70.3</td><td>75.4</td>

Antibody re-folding after denaturation is the reverse measure of long-term aggregation potential. Accordingly, the LAG-3 variants were also examined and compared in terms of thermal reversibility. Specifically, the antibodies were heated to 74 ° C and cooled to room temperature before heating again to 74 ° C. The ratio of the area under the curve of the second to the first thermogram is an evaluation of thermal reversibility, which is a direct measurement of conformational reversibility

As summarized in Table 3 and shown in Figure 6, LAG3.5 has a markedly higher thermal reversibility than other variants. It should be noted that the percent reversibility for LAG3.5 (47%) was greater than twice the reversibility for LAG3.1 (20%). Thermal reversibility is strongly correlated with the strength of long-term aggregation. Lower reversibility corresponds to potential higher aggregation. Based on this observation, LAG3.1 should potentially have significantly higher aggregation over time as compared to LAG3.5. Similarly, all other variants should potentially show significantly higher aggregation over time compared to LAG3.5.

Table 3

<td>Has b</td><td>Thermal reversibility (%)</td>

<td>LAG3.1</td><td>20</td>

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<td>Has b</td><td>Thermal reversibility (%)</td>

<td>LAG3.5</td><td>47</td>

<td>LAG3.6</td><td>0</td>

<td>LAG3.7</td><td>11</td>

<td>LAG3.8</td><td>26</td>

3. Aggregation

Variants were also tested for stability as a measure of aggregation using standard size exclusion chromatography (SEC-HPLC) according to the following protocol: antibody testing was dissolved to 1.0 mg / ml with phosphate buffer PBS in 10 μl was used in HPLC (Waters, model 2795) . Separation was performed in a gel filtration column (TOSOH Bioscience, TSKgel G3000 SWxl, 7.8mm x 300mm, Product No. 08541) using 0.1M sodium phosphate, 0.15M sodium chloride, 0.1M sodium sulfate, pH 7.2 as mobile phase. The analyte was detected by monitoring UV absorption at 280 nm, and the percentage composition of the peak area of ​​the antibody was determined using Empower software. As seen in Table 4, substantially LAG3.5 showed clearly reduced aggregation compared to LAG3.1.

Table 4

<td>Attempt</td><td>IgG Monomer (% area peak)</td><td>Aggregate IgG (% area peak)</td>

<td>LAG3.1</td><td>90</td><td>10</td>

<td>LAG3.5</td><td>96</td><td>4</td>

<td>LAG3.6</td><td>96</td><td>4</td>

<td>LAG3.7</td><td>95</td><td>5</td>

<td>LAG3.8</td><td>95</td><td>5</td>

Example 3: Selection of the variant

Based on the investigations described here, a variant of the LAG3.5 antibody for further analysis was chosen for further investigation, due to its significantly improved physical and chemical stability compared to the unmodified form (LAG3.1), especially its high conformational coiling capacity (thermal reversibility) ). The analysis included a two-step approach (a) accelerated stress followed by (b) a 12-week real-time stability assessment. Particularly, LAG3.5 was incubated at 1.0 mg / ml at pH 8.0, 50 mM ammonium bicarbonate for 5 days at 40 ° C. The degree of modification after 5 days was analyzed, as well as the effect on activity and stability. The LAG3.5 variant was then tested for stability in PBS in real time in PBS for 12 weeks and then analyzed. The results of these tests are described below.

1. Antigen binding

As seen in Figure 7 (and Table 5), no changes in antigen binding were observed after 5 days. As can also be seen in Figures 10A and B, LAG3.5 showed no change in antigen binding or physical stability after 12 weeks. In particular, LAG3.5 maintains a higher affinity than LAG3.8 throughout the 12 week period at both 4 ° C and 40 ° C.

EP 2867258

- 40 Table 5

<td>Clone ID</td><td>Antigen</td><td>Kd x 10^-9 (M)</td><td>kon x 10⁴ (1 / Ms)</td><td>Koff x 10^-4 (1 / s)</td>

<td rowspan="2">Lag3.1</td><td>PBS</td><td>0.21</td><td>166</td><td>3.44</td>

<td>pH 8</td><td>0.20</td><td>184</td><td>3.61</td>

<td rowspan="2">Lag3.5</td><td>PBS</td><td>0.25</td><td>130</td><td>3.22</td>

<td>pH 8</td><td>0.20</td><td>148</td><td>2.98</td>

<td rowspan="2">Lag3.8</td><td>PBS</td><td>0.25</td><td>147</td><td>3.68</td>

<td>pH 8</td><td>0.25</td><td>162</td><td>4.02</td>

2. Chemical modifications / molecular stability

Peptide mapping in mass spectrometry was used to analyze the chemical / molecular stability of LAG3.5 compared to LAG3.1. Specifically, the purified antibody was reduced, alkylated, dialyzed and digested with trypsin (Promega Cat # V5111) and GlucC (Roche Cat. # 11047817001). Digestion was analyzed in nano-LC mass spectrometry

MSMS (Thermo Fisher LTQ Orbitrap).

As seen in Figure 8, LAG3.1 showed increased heterogeneity in VH compared to LAG3.5 when subjected to accelerated stability at higher pH, resulting in deamination of aspartic residues (step 1). The mass change resulting from the isomerization could not be detected under the current experimental conditions. The change in percent is expressed as the proportion of all changes combined to the parent peak.

Furthermore, as seen in Figure 11, LAG3.1 showed increased heterogeneity in VH compared to LAG3.5 when subjected to accelerated stability over 12 weeks, both at 4 ° C and 40 ° C (step 2.

3. Physical stability

Thermal reversibility was measured in PBS and pH 8.0. In both conditions, LAG3.5 again showed approximately twice as high levels of refolding as compared to LAG3.1. Particularly, as shown in Tables 6-8, LAG3.5 showed 43% refolding compared to 18% for LAG3.1 in PBS. LAG3.5 also showed 48% refolding compared to 29% for LAG3.1 at pH 8.0.

Table 6 - DSC: melting

<td>Has b</td><td>Conditions</td><td>Tm1</td><td>tm2</td>

<td>Lag3.1</td><td>PBS</td><td>70.7</td><td>75.7</td>

<td>Lag3.1</td><td>pH 8</td><td>70.4</td><td>75.6</td>

<td>Lag3.5</td><td>PBS</td><td>70.8</td><td>76.4</td>

<td>Lag3.5</td><td>pH 8</td><td>70.5</td><td>76.3</td>

Table 7-Fluorol-2: development

<td>Mab / mutants</td><td>Point middle (M)</td><td>Aggregation (M)</td>

<td>Lag3.1 PBS</td><td>1.99</td><td>-</td>

<td>Lag3.1 pH8</td><td>2.08</td><td>-</td>

- EP 2867258

<td>Mab / mutants</td><td>Point middle (M)</td><td>Aggregation (M)</td>

<td>Lag3.5 PBS</td><td>1.86</td><td>-</td>

<td>Lag3.5 pH8</td><td>2.00</td><td>-</td>

Table 8: DSC: rewind

<td>Has b</td><td>% reversibility of PBS</td><td>% reversibility of pH8</td>

<td>Lag3.1</td><td>18</td><td>29</td>

<td>Lag3.5</td><td>43</td><td>48</td>

4. Incompatibility of cargo

For assessing charge incompatibility (heterogeneity), the variants were analyzed using isoelectric focusing (IEF) with standard markers pI 5.5 and pI 10.0 as compared to LAG3.1. Briefly, the antibody solutions were applied on a 1 mm thick IEF pI 37 gel (Invitrogen, Cat. # EC6648BOX) together with pI 3-10 markers (SERVA, Cat. # 39212). Electrophoresis was performed using IEF 3-7 cathode buffer (Invitrogen, cat. # LC5370) and IEF anode buffer (Invitrogen, Cat. # LC5300) and using electrical current in a constant order of 100 V for 1 h, constant 200 V for 1 h. and constant 500 V for 30 min. IEF gels were stained with Coomassie blue to detect protein strips and decolorized with a solution of acetic acid and methanol. IEF gels were analyzed using ImageQuant TL software. Based on this analysis (no data provided),

5. HIC-HPLC

To assess solubility, the variants were analyzed using standard hydrophobic interaction chromatography (HIC-HPLC) according to the following protocol: 50 μl of 2M ammonium sulfate was added to a 50 μl aliquot of the test antibody at 1 mg / ml. 80 μl of the test sample was then used in HPLC (Waters, model 2795) connected in line with an HIC column (TOSOH Bioscience, Ether-5PW TSK-gel, 7.5mm x 75mm, Product No. 07573). The sample was eluted at 1.0 ml / min with a gradient of 100% A buffer (2M ammonium sulfate, 0.1M sodium phosphate, pH 7.0) to 100% buffer B (0.1M sodium phosphate, pH 7.0) for 50 minutes. Antibodies were detected by monitoring UV absorption at 280 nm, and the data was analyzed using Empower software. As shown in Figure 9, the hydrophilicity of LAG3.5 exhibited solubility at high concentrations of ammonium sulfate.

Example 4: Reversible inhibition of T cell dependent immune response

LAG3.5 activity was determined on the basis of a functional study using a murine hybridoma specific for T cell antigen (3A9). Hybridoma 3A9 exhibits a T cell receptor specific for chicken lysozyme peptide (HEL48-62) and secretes IL-2 when cultured with antigen presenting cells (LK35.2) of pulsating peptide, matching MHC. Because huLAG-3-Fc is capable of binding to a MHC class II positive class B cell line, the expression of huLAG-3 on line 3A9 should exert inhibitory effect by binding to class II on the mouse display line.

Cells 3A9. Inhibition was reversed by LAG-3 blockade using LAG3.5. Therefore, inhibition of dependent LAG-3 for LAG3.5 has been demonstrated.

Example 5: Activation of t cells by LAG3.5

The functional activity of LAG3.5 on primary T cells was evaluated using human PBMC cultures stimulated with SEB superantigen. Total PBMCs were isolated from the blood of eighteen human donors and stimulated for 72 hours in one of the study formats: (i) a constant amount of antibody (20 μg / ml) and serial dilution of SEB, or (ii) a fixed amount of SEB (85 μg / ml) and serial dilutions of the antibody. The secreted IL-2, as a measure of T cell activity, was monitored by ELISA analysis. Anti-PD-1 antibody and Ipilimumab were used as positive controls, and LAG3.5 activity in combination with anti-PD-1 or anti-CTLA-4 was also evaluated for a subset of donors.

Increased IL-2 secretion was observed in the range of SEB concentrations in fifteen out of eighteen donors treated with only LAG3.5 compared to treatment with control antibody isotype. In most cases, the stimulation was less than that observed for anti-PD-1 or Ipilimumab treatment. Regarding LAG3.5, the results of the two test formats (described above) were consistent with each other. In addition, in 5 out of 6 donors tested, the combination of LAG3.5 with anti-PD-1 or Ipilimumab resulted in higher levels of stimulation than was observed for the isotype of the control antibody combined with anti-PD-1 or Ipilimumab. The obtained data explained that LAG3.5 can act in the study of a normal human T-cell and can further activate the reactions dependent on the inhibition of anti-PD-1 or CTLA-4 activity.

CONCLUSION IN THE SEQUENCE

<td>SEQ ID NO:</td><td>DESCRIPTION</td><td>SEQUENCE</td>

<td>1</td><td>V_H on 25F7 (LAG3.1)</td><td></td>

> 1408_LAG-3_403_25F7.1_VH1_NT

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACC

CTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGATTACTACTGGAA

CTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAA

TCATAATGGAAACACCAACTCCAACCCGTCCCTCAAGAGTCGAGTCACCCTA

TCACTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGGTCTGTGACCG

CCGCGGACACGGCTGTGTATTACTGTGCGTTTGGATATAGTGACTACGAGTA

CAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

<td>2</td><td>V_H aa 25F7</td><td></td>

> 1408_LAG-3_403_25F7.1_VH1_AA

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINH

NGNTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNW

FDPWGQGTLVTVSS

<td>3</td><td>V_K at 25F7</td><td></td>

> 1408_LAG-3_403_25F7.1_VK1_NT

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAA

GAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTG

GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCC

AACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACA

GACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATT

ACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTTGGCCAGGGGACCAACCT

GGAGATCAAA

- EP 2867258

<td>4</td><td>V_K aa 25F7</td><td></td>

> 1408_LAG-3_403_25F7.1_VK1_AA

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRAT

GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK

<td>5</td><td>V_H CDR1 aa 25F7</td><td>DYYWN</td>

<td>6</td><td>V_H CDR2 aa 25F7</td><td>EINHNGNTNSNPSLKS</td>

<td>7</td><td>V_H CDR3 aa 25F7</td><td>GYSDYEYNWFDP</td>

<td>8</td><td>V_K CDR1 aa 25F7</td><td>RASQSISSYLA</td>

<td>9</td><td>V_K CDR2 aa 25F7</td><td>DASNRAT</td>

<td>10</td><td>V_K CDR3 aa 25F7</td><td>QQRSNWPLT</td>

<td>11</td><td>V_H on LAG3.5</td><td></td>

V_H the LAG3.5 caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtc cttcagtgattactactggaactggatccgccagcccccagggaaggggctggagtggattggggaaatcaatcatcgtggaa gcaccaactccaacccgtccctcaagagtcgagtcaccctatcactagacacgtccaagaaccagttctccctgaagctgaggt ctgtgaccgccgcggacacggctgtgtattactgtgcgtttggatatagtgactacgagtacaactggttcgacccctggggcc agggaaccctggtcaccgtctcctca

<td>12</td><td>V_H aa LAG3.5</td><td></td>

V_H aa LAG3.5

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGE

INHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYS

DYEYNWFDPWGQGTLVTVSS

<td>13</td><td>V_K on LAG3.5</td><td></td>

V_K the LAG3.5 gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagt attagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccact ggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgca gtttattactgtcagcagcgtagcaactggcctctcacttttggccaggggaccaacctggagatcaaa

<td>14</td><td>V_K aa LAG3.5</td><td></td>

V_K aa LAG3.5

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYD

ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQ

GTNFEIK

<td>15</td><td>V_H CDR1 aa LAG3.5</td><td>DYYWN</td>

<td>16</td><td>V_H CDR2 aa LAG3.5</td><td>EINHRGSTNSNPSLKS</td>

<td>17</td><td>V_H CDR3 aa LAG3.5</td><td>GYSDYEYNWFDP</td>

<td>18</td><td>V_K CDR1 aa LAG3.5</td><td>RASQSISSYLA</td>

<td>19</td><td>V_K CDR2 aa LAG3.5</td><td>DASNRAT</td>

<td>20</td><td>V_K CDR3 aa LAG3.5</td><td>QQRSNWPLT</td>

<td>21</td><td>LAG-3 epitope</td><td>PGHPLAPG</td>

<td>22</td><td>LAG-3 epitope</td><td>HPAAPSSW</td>

<td>23</td><td>LAG-3 epitope</td><td>PAAPSSWG</td>

<td>24</td><td>V_H CDR2 aa LAG3.6</td><td>EIIHSGSTNSNPSLKS</td>

- EP 2867258

<td>25</td><td>V_H CDR2 aa LAG3.7</td><td>EINHGGGTNSNPSLKS</td>

<td>26</td><td>V_H CDR2 aa LAG3.8</td><td>EINHIGNTNSNPSLKS</td>

<td>27</td><td>VH CDR2 aa human line embryonic (HUMAN GERMLINE)</td><td>GEINHSGSTNY</td>

<td>28</td><td></td><td></td>

<td>29</td><td>Human LAG-3 aa</td><td></td>

sequence of human LAG-3 aa

MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDL

SLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGP

GGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALS

CRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPV

RESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPL

TVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLE

DVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSG

QERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTE

LSSPGAQRSGRAPGALPAGHLLLFLTLGVLSLLLLVTGAFGFHLWRRQWRPRRF

SALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL *

<td>thirty</td><td>V_H CDR2 aa LAG3.2</td><td>VIWYDGSNKYYADSVKG</td>

<td>31</td><td>V_H LAG3.1 on</td><td></td>

LAG3.1HC

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACC

CTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGATTACTACTGGAA

CTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAA

TCATAATGGAAACACCAACTCCAACCCGTCCCTCAAGAGTCGAGTCACCCTA

TCACTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGGTCTGTGACCG

CCGCGGACACGGCTGTGTATTACTGTGCGTTTGGATATAGTGACTACGAGTA

CAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCT

AGCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCT

CCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC

GGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC

CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG

TGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACA

AGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCC

CATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCT

GTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTC

ACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAAC

TGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG

GAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC

AGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCC

TCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG

AGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACC

AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGT

GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC

CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGAC

AAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG

GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAT

GA

<td>32</td><td>V_H LAG3.1 aa</td><td></td>

TRANSLATION \ OF \ LAG3.1HC

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINH

NGNTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNW

- EP 2867258

FDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS

WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV

DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE

DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC

KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD

IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMH

EALHNHYTQKSLSLSLGK *

<td>33</td><td>V_L LAG3.1 on</td><td></td>

LAG3.1LC

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAA

GAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTG

GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCC

AACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACA

GACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATT

ACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTTGGCCAGGGGACCAACCT

GGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCT

GATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT

TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT

CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCT

ACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACA

AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA

GAGCTTCAACAGGGGAGAGTGTTAG

<td>34</td><td>V_L LAG3.1 aa</td><td></td>

TRANSLATION \ OF \ LAG3.1LC

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRAT

GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVA

APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE

QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC *

<td>35</td><td>V_H LAG3.5 aa</td><td></td>

heavy chain sequence LAG3.5 - complete

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGE

INHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYS

DYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT

YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT

LMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY

RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT

LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK *

<td>36</td><td>V_H LAG3.5 on</td><td></td>

sequence of the heavy chain LAG3.5 - complete caggtgcagctacagcagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtc cttcagtgattactactggaactggatccgccagcccccagggaaggggctggagtggattggggaaatcaatcatcgtggaa gcaccaactccaacccgtccctcaagagtcgagtcaccctatcactagacacgtccaagaaccagttctccctgaagctgaggt ctgtgaccgccgcggacacggctgtgtattactgtgcgtttggatatagtgactacgagtacaactggttcgacccctggggcc agggaaccctggtcaccgtctcctcagctagcaccaagggcccatccgtcttccccctggcgccctgctccaggagcacctcc

- 46 - EP2867258 gagagcacagccgccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg accagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccag cagcttgggcacgaagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagttgagtccaa atatggtcccccatgcccaccatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaagga cactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaact ggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtca gcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctc catcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggaga tgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgg gcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagag cctctccctgtctctgggtaaatga

<td>37</td><td>V_L LAG3.5 aa</td><td></td>

sequence of the kappa LAG3.5 chain - complete

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYD

ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQ

GTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

DNAFQSGNSQESVTEQDSKDSTYSFSSTFTFSKADYEKHKVYACEVTHQG

FSSPYTKSFNRGEC *

<td>38</td><td>V_L LAG3.5 on</td><td></td>

sequence of the kappa chain LAG3.5 - complete gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagt attagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccact ggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgca gtttattactgtcagcagcgtagcaactggcctctcacttttggccaggggaccaacctggagatcaaacgtacggtggctgca ccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatccca gagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagc aaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaa gtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag

SEQUENCE LIST <110>

<120> OPTIMIZATION OF ANTIBODIES THAT BIND LYMPHOCYTE ACTIVATION GENE-3 10 (LAG-3), AND USES THEREOF <130> 11911-WO-PCT <140>

<141> 2013-05-30 <150> 61 / 667,058 <151> 2012-07-02 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 360 <212> DNA <213> Artificial Sequence < 220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polynucleotide"

<220>

<221> CDS <222> (1) .. (360)

- 47 EP 2867258 <400> 1

<td>cag</td><td>gtg</td><td>cag</td><td>eta</td><td>cag</td><td>cag</td><td>TGG</td><td>GGC</td><td>GCA</td><td>GGA</td><td>cOT</td><td>TTG</td><td>aag</td><td>Cct</td><td>teg</td><td>gag</td><td>48</td>

<td>Gin</td><td>val</td><td>Gin</td><td>Leu</td><td>Gin</td><td>Gin</td><td>Trp</td><td>Gly</td><td>ala</td><td>Gly</td><td>Leu</td><td>Leu</td><td>lys</td><td>Pro</td><td>Cheese</td><td>Glu</td><td></td>

<td>1</td><td></td><td></td><td></td><td>5</td><td></td><td></td><td></td><td></td><td>10</td><td></td><td></td><td></td><td></td><td>15</td><td></td><td></td>

<td>acc</td><td>cOT</td><td>tcc</td><td>ctc</td><td>acc</td><td>TGC</td><td>get</td><td>gtc</td><td>tat</td><td>GGT</td><td>ggg</td><td>tcc</td><td>ttc</td><td>agt</td><td>gat</td><td>trays</td><td>96</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>ala</td><td>val</td><td>Tyr</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>phe</td><td>Cheese</td><td>Asp</td><td>Tyr</td><td></td>

144 tgg aac tgg atc cgc cg ccc ccggg aagggg cgg gag tgg att Tyr Trp Asn Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp How much

40 45

<td>ggg</td><td>gaa</td><td>atc</td><td>aat</td><td>cat</td><td>aat</td><td>GGA</td><td>aac</td><td>acc</td><td>aac</td><td>tcc</td><td>aac</td><td>ccg</td><td>tcc</td><td>ctc</td><td>aag</td><td>192</td>

<td>Gly</td><td>Glu</td><td>How much</td><td>own</td><td>His</td><td>own</td><td>Gly</td><td>own</td><td>Thr</td><td>own</td><td>Cheese</td><td>own</td><td>Pro</td><td>Cheese</td><td>Leu</td><td>lys</td><td></td>

<td></td><td>50</td><td></td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td><td></td><td></td>

<td>agt</td><td>ega</td><td>gtc</td><td>acc</td><td>eta</td><td>tCA</td><td>eta</td><td>gac</td><td>ACG</td><td>tcc</td><td>aag</td><td>aac</td><td>cag</td><td>ttc</td><td>tcc</td><td>cOT</td><td>240</td>

<td>Cheese</td><td>Arg</td><td>val</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Asp</td><td>Thr</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe</td><td>Cheese</td><td>Leu</td><td></td>

<td>65</td><td></td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td><td></td>

<td>aag</td><td>cOT</td><td>agg</td><td>tet</td><td>gtg</td><td>acc</td><td>gee</td><td>GCG</td><td>gac</td><td>ACG</td><td>get</td><td>gtg</td><td>tat</td><td>trays</td><td>tgt</td><td>GCG</td><td>288</td>

<td>lys</td><td>Leu</td><td>Arg</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>ala</td><td></td>

336 ttt gga tat agt gac tac gag tac aac tgg

Phe Gly Tyr Cheese Asp Tyr Glu Tyr Asn Trp

100 105 gga acc ctg gtc acc gtc tcc tca

Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 2 <211> 120 <212> PRT <213> Artificial Sequence <220>

ttc gac ccc Phe Asp Pro tgg hg cag Trp Gly Gin 110

360 <221> source <223> / note = "Description of Artificial Sequence: Synthetic polypeptide"

<400> 2

- EP 2867258

<td rowspan="2">Gin 1</td><td rowspan="2">val</td><td rowspan="2">Gin</td><td colspan="7">Leu Gin Gin Trp Gly Ala Gly</td><td rowspan="2">Leu</td><td colspan="2" rowspan="2">Leu Lys</td><td rowspan="2">Pro</td><td colspan="2" rowspan="2">Glu 15 cheese</td>

<td colspan="2">5</td><td colspan="5">10</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>ala</td><td>val</td><td>Tyr</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>phe</td><td>Cheese</td><td>Asp</td><td>Tyr</td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>Tyr</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Trp</td><td>How much</td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td>

<td>Gly</td><td>Glu</td><td>How much</td><td>own</td><td>His</td><td>own</td><td>Gly</td><td>own</td><td>Thr</td><td>own</td><td>Cheese</td><td>own</td><td>Pro</td><td>Cheese</td><td>Leu</td><td>lys</td>

<td></td><td>50</td><td></td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Arg</td><td>val</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Asp</td><td>Thr</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe</td><td>Cheese</td><td>Leu</td>

<td>65</td><td></td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

<td>lys</td><td>Leu</td><td>Arg</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>ala</td>

<td></td><td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td></td><td>95</td><td></td>

<td>phe</td><td>Gly</td><td>Tyr</td><td>Cheese</td><td>Asp</td><td>Tyr</td><td>Glu</td><td>Tyr</td><td>own</td><td>Trp</td><td>phe</td><td>Asp</td><td>Pro</td><td>Trp</td><td>Gly</td><td>Gin</td>

<td></td><td></td><td></td><td>100</td><td></td><td></td><td></td><td></td><td>105</td><td></td><td></td><td></td><td></td><td>110</td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>Leu</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>115</td><td></td><td></td><td></td><td></td><td>120</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 3 <211> 321 <212> DNA <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polynucleotide" <220>

<221> CDS <222> (1) .. (321)

- EP 2867258

<td colspan="16">&lt;400&gt; 3</td>

<td rowspan="2">gaa Glu 1</td><td rowspan="2">att How much</td><td rowspan="2">gtg val</td><td rowspan="2">TTG Leu</td><td rowspan="2">aca Thr 5</td><td rowspan="2">cag Gin</td><td rowspan="2">tct Cheese</td><td rowspan="2">cca gcc Pro Ala</td><td rowspan="2">acc Thr 10</td><td rowspan="2">cOT Leu</td><td rowspan="2">tct Cheese</td><td colspan="2">ttg tct</td><td colspan="2">cca ggg</td><td rowspan="2">48</td>

<td>Leu</td><td>Cheese</td><td>Pro 15</td><td>Gly</td>

<td>gaa</td><td>aga</td><td>gcc</td><td>acc</td><td>ctc</td><td>tcc</td><td>TGC</td><td>agg gcc</td><td>agt</td><td>cag</td><td>agt</td><td>att</td><td>agc</td><td>agc</td><td>trays</td><td>96</td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg Ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Tyr</td><td></td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td><td></td>

<td>tta</td><td>gcc</td><td>TGG</td><td>trays</td><td>caa</td><td>cag</td><td>aaa</td><td>cct ggc</td><td>cag</td><td>GCT</td><td>ccc</td><td>agg</td><td>ctc</td><td>ctc</td><td>atc</td><td>144</td>

<td>Leu</td><td>ala</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro Gly</td><td>Gin</td><td>ala</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Leu</td><td>How much</td><td></td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td><td></td>

<td>tat</td><td>gat</td><td>GCA</td><td>tcc</td><td>aac</td><td>agg</td><td>gcc</td><td>act ggc</td><td>atc</td><td>ca</td><td>gcc</td><td>agg</td><td>ttc</td><td>agt</td><td>GGC</td><td>192</td>

<td>Tyr</td><td>Asp</td><td>ala</td><td>Cheese</td><td>own</td><td>Arg</td><td>ala</td><td>Thr Gly</td><td>How much</td><td>Pro</td><td>ala</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td></td>

<td></td><td>50</td><td></td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td><td></td><td></td>

<td>agt</td><td>ggg</td><td>tct</td><td>ggg</td><td>aca</td><td>gac</td><td>ttc</td><td>act ctc</td><td>acc</td><td>atc</td><td>agc</td><td>agc</td><td>eta</td><td>gag</td><td>Cct</td><td>240</td>

<td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Pro</td><td></td>

<td>65</td><td></td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td><td></td>

<td>gaa</td><td>gat</td><td>ttt</td><td>GCA</td><td>gtt</td><td>tat</td><td>trays</td><td>tgt cag</td><td>cag</td><td>cgt</td><td>agc</td><td>aac</td><td>TGG</td><td>Cct</td><td>ctc</td><td>288</td>

<td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys Gin</td><td>Gin</td><td>Arg</td><td>Cheese</td><td>own</td><td>Trp</td><td>Pro</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td></td><td>95</td><td></td><td></td>

<td>act</td><td>ttt</td><td>GGC</td><td>cag</td><td>ggg</td><td>acc</td><td>aac</td><td>cg gag</td><td>atc</td><td>aaa</td><td></td><td></td><td></td><td></td><td></td><td>321</td>

<td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>own</td><td>Leu Glu</td><td>How much</td><td>lys</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td>100</td><td></td><td></td><td></td><td>105</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polypeptide" <400> 4

Glu Ile Val Leu Thr Gin Cheese Pro Ala Thr Leu Cheese Leu Cheese Pro Gly 15 10 15

Glu Arg Ala Thr Leu Cheese Cys Arg Ala Cheese Gin Cheese Ile Cheese Ser Tyr 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile 35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60

Cheese Gly Cheese Gly Thr Asp Phe Thr Leu Thr Ile Cheese Cheese Leu Glu Pro 65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Leu 85 90 95

Thr Phe Gly Gin Gly Thr Asn Leu Glu Ile Lys 100 105 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220>

- 50 EP 2867258 <221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 5

Asp Tyr Tyr Trp Asn

5 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 6

Glu Ile Asn His Asn Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys Ser 15 10 15 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 7

Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro

10 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 8

Arg Ala Ser Gin Ser Ile Ser Ser Tyr Leu Ala 15 10 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 9

Asp Ala Ser Asn Arg Ala Thr <210> 10 <211> 9 <212> PRT

- 51 EP 2867258 <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 10

Gin Gin Arg Cheese Asn Trp Pro Leu Thr

5 <210> 11 <211> 360 <212> DNA <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polynucleotide" <400> 11

<td>caggtgcagc</td><td>tacagcagtg</td><td>gggcgcagga</td><td>ctgttgaagc</td><td>cttcggagac</td><td>cctgtccctc</td><td>60</td>

<td>acctgcgctg</td><td>tctatggtgg</td><td>gtccttcagt</td><td>gattactact</td><td>ggaactggat</td><td>ccgccagccc</td><td>120</td>

<td>ccagggaagg</td><td>ggctggagtg</td><td>gattggggaa</td><td>atcaatcatc</td><td>gtggaagcac</td><td>caactccaac</td><td>180</td>

<td>ccgtccctca</td><td>agagtcgagt</td><td>caccctatca</td><td>ctagacacgt</td><td>ccaagaacca</td><td>gttctccctg</td><td>240</td>

<td>aagctgaggt</td><td>ctgtgaccgc</td><td>cgcggacacg</td><td>gctgtgtatt</td><td>actgtgcgtt</td><td>tggatatagt</td><td>300</td>

<td>gactacgagt</td><td>acaactggtt</td><td>cgacccctgg</td><td>ggccagggaa</td><td>ccctggtcac</td><td>cgtctcctca</td><td>360</td>

<210> 12 <211> 120 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polypeptide" <400> 12

52 EP2867258

Gin Val Gin Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Cheese Phe Cheese Asp Tyr 20 25 30

Tyr Trp Asn Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45

Gly Glu Ile Asn His Arg Gly Cheese Thr Asn Cheese Asn Pro Cheese Leu Lys 50 55 60

Cheese Arg Val Thr Leu Cheese Leu Asp Thr Cheese Lys Asn Gin Phe Cheese Leu 65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gin 100 105 110

Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 13 <211> 321 <212> DNA <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polynucleotide" <220>

<221> CDS <222> (1) .. (321) <400> 13

<td rowspan="2">gaa Glu 1</td><td rowspan="2">att How much</td><td rowspan="2">gtg val</td><td rowspan="2">TTG Leu</td><td rowspan="2">aca Thr 5</td><td rowspan="2">cag Gin</td><td rowspan="2">tct Cheese</td><td colspan="2" rowspan="2">cca gcc Pro Ala</td><td rowspan="2">acc Thr 10</td><td rowspan="2">cOT Leu</td><td rowspan="2">tct Cheese</td><td colspan="2">ttg tct</td><td colspan="2">cca ggg</td><td rowspan="2">48</td>

<td>Leu</td><td>Cheese</td><td>Pro 15</td><td>Gly</td>

<td>gaa</td><td>aga</td><td>gcc</td><td>acc</td><td>ctc</td><td>tcc</td><td>TGC</td><td>agg</td><td>gcc</td><td>agt</td><td>cag</td><td>agt</td><td>att</td><td>agc</td><td>agc</td><td>trays</td><td>96</td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Tyr</td><td></td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td><td></td>

<td>tta</td><td>gcc</td><td>TGG</td><td>trays</td><td>caa</td><td>cag</td><td>aaa</td><td>Cct</td><td>GGC</td><td>cag</td><td>GCT</td><td>ccc</td><td>agg</td><td>ctc</td><td>ctc</td><td>atc</td><td>144</td>

<td>Leu</td><td>ala</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro</td><td>Gly</td><td>Gin</td><td>ala</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Leu</td><td>How much</td><td></td>

- EP 2867258

<td></td><td></td><td>35</td><td></td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td><td></td>

<td>tat</td><td>gat</td><td>GCA</td><td>tcc</td><td>aac</td><td>agg</td><td>gcc</td><td>act</td><td>GGC</td><td>atc</td><td>ca</td><td>gcc</td><td>agg</td><td>ttc</td><td>agt</td><td>GGC</td><td>192</td>

<td>Tyr</td><td>Asp</td><td>ala</td><td>Cheese</td><td>own</td><td>Arg</td><td>ala</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro</td><td>ala</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td><td></td>

<td></td><td>50</td><td></td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td><td></td><td></td>

<td>agt</td><td>ggg</td><td>tct</td><td>ggg</td><td>aca</td><td>gac</td><td>ttc</td><td>act</td><td>ctc</td><td>acc</td><td>atc</td><td>agc</td><td>agc</td><td>eta</td><td>gag</td><td>Cct</td><td>240</td>

<td>Cheese</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Pro</td><td></td>

<td>65</td><td></td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td><td></td>

<td>gaa</td><td>gat</td><td>ttt</td><td>GCA</td><td>gtt</td><td>tat</td><td>trays</td><td>tgt</td><td>cag</td><td>cag</td><td>cgt</td><td>agc</td><td>aac</td><td>TGG</td><td>Cct</td><td>ctc</td><td>288</td>

<td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>Arg</td><td>Cheese</td><td>own</td><td>Trp</td><td>Pro</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td></td><td>95</td><td></td><td></td>

<td>act</td><td>ttt</td><td>GGC</td><td>cag</td><td>ggg</td><td>acc</td><td>aac</td><td>cOT</td><td>gag</td><td>atc</td><td>aaa</td><td></td><td></td><td></td><td></td><td></td><td>321</td>

<td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>own</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td>100</td><td></td><td></td><td></td><td></td><td>105</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 14 <211> 107 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polypeptide"

<td colspan="3">&lt;400&gt; 14</td><td colspan="2" rowspan="2">Leu Thr 5</td><td rowspan="2">Gin</td><td rowspan="2">Cheese</td><td colspan="2" rowspan="2">Pro Ala</td><td colspan="2" rowspan="2">Thr Leu 10</td><td colspan="2" rowspan="2">Leu cheese</td><td rowspan="2">Cheese</td><td rowspan="2">Pro 15</td><td rowspan="2">Gly</td>

<td>Glu 1</td><td colspan="2">Ile Val</td>

<td>Glu</td><td>Arg</td><td>ala</td><td>Thr 20</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>Arg</td><td>ala 25</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>How much</td><td>Cheese thirty</td><td>Cheese</td><td>Tyr</td>

<td>Leu</td><td>ala</td><td>Trp 35</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lys</td><td>Pro 40</td><td>Gly</td><td>Gin</td><td>ala</td><td>Pro</td><td>Arg 45</td><td>Leu</td><td>Leu</td><td>How much</td>

<td>Tyr</td><td>Asp 50</td><td>ala</td><td>Cheese</td><td>own</td><td>Arg</td><td>ala 55</td><td>Thr</td><td>Gly</td><td>How much</td><td>Pro</td><td>ala 60</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td>

<td>Cheese 65</td><td>Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp 70</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much 75</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Glu</td><td>Pro 80</td>

<td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>val 85</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin 90</td><td>Arg</td><td>Cheese</td><td>own</td><td>Trp</td><td>Pro 95</td><td>Leu</td>

<td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>own</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td></td><td></td><td></td><td></td><td></td>

100 105 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 15

Asp Tyr Tyr Trp Asn

5 &amp; ​​lt; 210 &amp; gt; 16

- EP 2867258 <211> 16 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 16

Glu Ile Asn His Arg Gly Cheese Thr Asn Ser Asn Pro Ser Leu Lys Ser 15 10 15 <210> 17 <211> 12 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 17

Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro

10 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 18

Arg Ala Ser Gin Ser Ile Ser Ser Tyr Leu Ala

10 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 19

Asp Ala Ser Asn Arg Ala Thr

5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 20

EP 2867258

Gin Gin Arg Cheese Asn

Trp

Pro

- 55 Leu Thr <210> 21 <211> 8 <212> PRT <213> Homo sapiens <400> 21

Pro Gly His Pro Leu Ala Pro Gly

5 <210> 22 <211> 8 <212> PRT <213> Homo sapiens <400> 22

His Pro Ala Ala Pro Ser Ser Trp

5 <210> 23 <211> 8 <212> PRT <213> Homo sapiens <400> 23

Pro Ala Ala Pro Cheese Cheese Trp Gly

5 <210> 24 <211> 16 <212> PRT <213> Artif icial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 24

Glu Ile Ile His Cheese Gly Cheese Thr Asn Cheese Asn Pro Cheese Leu Lys Cheese 15 10 15 <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 25

Glu Ile Asn His Gly Gly Thr Asn Ser Asn Pro Ser Leu Lys Ser 15 10 15 <210> 26 <211> 16 <212> PRT <213> Artificial Sequence <220>

<221> source

- 56 EP 2867258 <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 26

Glu Ile Asn His Ile Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys Ser 15 10 15 <210> 27 <211> 11 <212> PRT <213> Homo sapiens <400> 27

Gly Glu Ile Asn His Cheese Gly Ser Thr Asn Tyr

10 <210> 28 <211> 15 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 28

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Cheese 15 10 15 <210> 29 <211> 525 <212> PRT <213> Homo sapiens <400> 29

EP 2867258

<td rowspan="2">Underworld 1</td><td colspan="10">- 57 -</td>

<td>Trp</td><td>Glu</td><td>Ala Gin Phe 5</td><td>Leu Gly Leu</td><td>Leu 10</td><td>phe</td><td>Leu Gin</td><td>Pro</td><td>Leu 15</td><td>Trp</td>

<td>val</td><td>ala</td><td>Pro</td><td>Val Lys Pro 20</td><td>Leu Gin Pro 25</td><td>Gly</td><td>ala</td><td>Glu Val</td><td>Pro thirty</td><td>val</td><td>val</td>

<td>Trp</td><td>ala</td><td>Gin 35</td><td>Glu Gly Ala</td><td>Pro Ala Gin 40</td><td>Leu</td><td>Pro</td><td>Cys Ser 45</td><td>Pro</td><td>Thr</td><td>How much</td>

<td>Pro</td><td>Leu 50</td><td>Gin</td><td>Asp Leu Cheese</td><td>Leu Leu Arg 55</td><td>Arg</td><td>ala</td><td>Gly Val 60</td><td>Thr</td><td>Trp</td><td>Gin</td>

<td>His 65</td><td>Gin</td><td>Pro</td><td>Asp Ser Gly 70</td><td>Pro Pro Ala</td><td>ala</td><td>ala 75</td><td>Pro Gly</td><td>His</td><td>Pro</td><td>Leu 80</td>

<td>ala</td><td>Pro</td><td>Gly</td><td>Pro His Pro 85</td><td>Ala Ala Pro</td><td>Cheese 90</td><td>Cheese</td><td>Trp Gly</td><td>Pro</td><td>Arg 95</td><td>Pro</td>

<td>Arg</td><td>Arg</td><td>Tyr</td><td>Thr Val Leu 100</td><td>Cheese Val Gly 105</td><td>Pro</td><td>Gly</td><td>Gly Leu</td><td>Arg 110</td><td>Cheese</td><td>Gly</td>

<td>Arg</td><td>Leu</td><td>Pro 115</td><td>Leu Gin Pro</td><td>Arg Val Gin 120</td><td>Leu</td><td>Asp</td><td>Glu Arg 125</td><td>Gly</td><td>Arg</td><td>Gin</td>

<td>Arg</td><td>Gly 130</td><td>Asp</td><td>Phe Ser Leu</td><td>Trp Leu Arg 135</td><td>Pro</td><td>ala</td><td>Arg Arg 140</td><td>ala</td><td>Asp</td><td>ala</td>

<td>Gly 145</td><td>Glu</td><td>Tyr</td><td>Arg Ala Ala 150</td><td>Val His Leu</td><td>Arg</td><td>Asp 155</td><td>Arg Ala</td><td>Leu</td><td>Cheese</td><td>Cys 160</td>

<td>Arg</td><td>Leu</td><td>Arg</td><td>Leu Arg Leu 165</td><td>Gly Ala</td><td>Cheese 170</td><td>Underworld</td><td>Thr Ala</td><td>Cheese</td><td>Pro 175</td><td>Pro</td>

<td>Gly</td><td>Cheese</td><td>Leu</td><td>Arg Ala Ser 180</td><td>Asp Trp Val 185</td><td>How much</td><td>Leu</td><td>Asn Cys</td><td>Cheese 190</td><td>phe</td><td>Cheese</td>

<td>Arg</td><td>Pro</td><td>Asp 195</td><td>Arg Pro Ala</td><td>Cheese Val His 200</td><td>Trp</td><td>phe</td><td>Arg Asn 205</td><td>Arg</td><td>Gly</td><td>Gin</td>

<td>Gly</td><td>Arg 210</td><td>val</td><td>Pro Val Arg</td><td>Glu Cheese Pro 215</td><td>His</td><td>His</td><td>His Leu 220</td><td>ala</td><td>Glu</td><td>Cheese</td>

<td>phe 225</td><td>Leu</td><td>phe</td><td>Leu Pro Gin 230</td><td>Val Ser Pro</td><td>Underworld</td><td>Asp 235</td><td>Gly cheese</td><td>Pro</td><td>Trp</td><td>Gly 240</td>

<td>Cys</td><td>How much</td><td>Leu</td><td>Thr Tyr Arg</td><td>Asp Gly Phe</td><td>own</td><td>val</td><td>Ser Ile</td><td>Underworld</td><td>Tyr</td><td>own</td>

EP 2867258

245

- 58 250 255

Leu Thr Val Leu Gly Leu 260

Glu Pro Pro Thr Pro 265

Leu Thr Val Tyr Ala 270

Gly Ala Gly Ser Arg Val 275

Gly Leu Pro Cys Arg 280

Leu Pro Ala Gly Val 285

Gly Thr Arg Ser Phe Leu 290

Thr Ala Lys Trp Thr 295

Pro Pro Gly Gly Gly 300

Pro Asp Leu Val Thr 305 310

Gly Asp Asn Gly Asp 315

Phe Thr Leu Arg Leu 320

Glu Asp Val Ser Gin Ala 325

Gin Ala Gly Thr Tyr 330

Thr Cys His Ile His 335

Leu Gin Glu Gin Gin Leu 340

Asn Ala Thr Val Thr 345

Leu Ala Ile Ile Thr 350

Val Thr Pro Lys Ser Phe 355

Gly Cheese Pro Gly Cheese 360

Leu Gly Lys Leu Leu 365

Cys Glu Val Thr Pro Val 370

Gly Gin Glu Arg 375

Phe Val Trp Cheese Ser 380

Leu Asp Thr Pro Ser Gin 385 390

Arg Cheese Phe Cheese Gly 395

Pro Trp Leu Glu Ala 400

Gin Glu Ala Gin Leu Leu 405

Gin Pro Trp Gin 410

Cys Gin Leu Tyr Gin 415

Gly Glu Arg Leu Leu Gly 420

Ala Ala Val Tyr Phe 425

Thr Glu Leu Cheese Cheese 430

Pro Gly Ala Gin Arg Cheese 435

Gly Arg Ala Pro Gly 440

Ala Leu Pro Ala Gly 445

His Leu Leu Leu Phe Leu 450

Thr Leu Gly Val Leu 455

Leu Leu Leu Leu 460

Val Thr Gly Ala Phe Gly 465 470

Phe His Leu Trp Arg 475

Arg Gin Trp Arg Pro 480

Arg Arg Phe Ser Ala Leu 485

Lys Ile Glu Glu Leu 500

Glu Gin Gly Ile His 490

Glu Gin Glu Pro Glu 505

Pro Pro Gin Ala Gin 495

Pro Glu Pro Glu Pro 510

Glu Pro Glu Pro Glu Pro 515 <210> 30 <211> 17 <212> PRT <213> Artificial Sequence

Glu Pro Glu Pro Glu 520

Gin Leu 525

- EP 2867258 <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<400> 30

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 15 10 15

Gly <210> 31 <211> 1344 <212> DNA <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polynucleotide"

<400> 31 caggtgcagc acctgcgctg ccagggaagg ccgtccctca aagctgaggt gactacgagt gctagcacca agcacagccg tggaactcag ggactctact tacacctgca aaatatggtc ttcctgttcc tgcgtggtgg ggcgtggagg cgtgtggtca tgcaaggtct gggcagcccc aaccaggtca tgggagagca gacggctcct aatgtcttct ctctccctgt <210> 32 <211>447 tacagcagtg tctatggtgg ggctggagtg agagtcgagt ctgtgaccgc acaactggtt agggcccatc ccctgggctg gcgccctgac ccctcagcag acgtagatca ccccatgccc ccccaaaacc tggacgtgag tgcataatgc gcgtcctcac ccaacaaagg gagagccaca gcctgacctg atgggcagcc tcttcctcta catgctccgt ctctgggtaa gggcgcagga gtccttcagt gattggggaa caccctatca cgcggacacg cgacccctgg cgtcttcccc cctggtcaag cagcggcgtg cgtggtgacc caagcccagc accatgccca caaggacact ccaggaagac caagacaaag cgtcctgcac cctcccgtcc ggtgtacacc cctggtcaaa ggagaacaac cagcaggcta gatgcatgag atga ctgttgaagc gattactact atcaatcata ctagacacgt gctgtgtatt ggccagggaa ctggcgccct gactacttcc cacaccttcc gtgccctcca aacaccaagg gcacctgagt ctcatgatct cccgaggtcc ccgcgggagg caggactggc tccatcgaga ctgcccccat ggcttctacc tacaagacca accgtggaca gctctgcaca cttcggagac ggaactggat atggaaacac ccaagaacca actgtgcgtt ccctggtcac gctccaggag ccgaaccggt cggctgtcct gcagcttgggtggacaagag tcctgggggg cccggacccc agttcaactg agcagttcaa tgaacggcaa aaaccatctc cccaggagga ccagcgacat cgcctcccgt agagcaggtg accactacac cctgtccctc ccgccagccc caactccaac gttctccctg tggatatagt cgtctcctca cacctccgag gacggtgtcg acagtcctca cacgaagacc agttgagtcc accatcagtc tgaggtcacg gtacgtggat cagcacgtac ggagtacaag caaagccaaa gatgaccaag cgccgtggag gctggactcc gcaggagggg acagaagagc

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1344

- EP 2867258 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polypeptide"

<td colspan="3">&lt;400&gt; 32</td><td colspan="10" rowspan="2">Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys</td><td rowspan="3">Pro</td><td rowspan="3">Cheese 15</td><td rowspan="3">Glu</td>

<td colspan="2" rowspan="2">Gin Val 1</td><td rowspan="2">Gin</td>

<td colspan="4">5</td><td colspan="6">10</td>

<td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>ala</td><td>val</td><td>Tyr</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>phe</td><td>Cheese</td><td>Asp</td><td>Tyr</td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>Tyr</td><td>Trp</td><td>own</td><td>Trp</td><td>How much</td><td>Arg</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td><td>Trp</td><td>How much</td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td>

<td>Gly</td><td>Glu</td><td>How much</td><td>own</td><td>His</td><td>own</td><td>Gly</td><td>own</td><td>Thr</td><td>own</td><td>Cheese</td><td>own</td><td>Pro</td><td>Cheese</td><td>Leu</td><td>lys</td>

<td></td><td>50</td><td></td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Arg</td><td>val</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Asp</td><td>Thr</td><td>Cheese</td><td>lys</td><td>own</td><td>Gin</td><td>phe</td><td>Cheese</td><td>Leu</td>

<td>65</td><td></td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

<td>lys</td><td>Leu</td><td>Arg</td><td>Cheese</td><td>val</td><td>Thr</td><td>ala</td><td>ala</td><td>Asp</td><td>Thr</td><td>ala</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>ala</td>

<td></td><td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td></td><td>95</td><td></td>

<td>phe</td><td>Gly</td><td>Tyr</td><td>Cheese</td><td>Asp</td><td>Tyr</td><td>Glu</td><td>Tyr</td><td>own</td><td>Trp</td><td>phe</td><td>Asp</td><td>Pro</td><td>Trp</td><td>Gly</td><td>Gin</td>

<td></td><td></td><td></td><td>100</td><td></td><td></td><td></td><td></td><td>105</td><td></td><td></td><td></td><td></td><td>110</td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>Leu</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td>ala</td><td>Cheese</td><td>Thr</td><td>lys</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>val</td>

115 120 125

EP 2867258

<td rowspan="2">phe</td><td rowspan="2">Pro Leu 130</td><td colspan="10">- 61 -</td><td rowspan="2">ala</td>

<td>Ala Pro</td><td>Cys</td><td>Cheese 135</td><td>Arg</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Glu 140</td><td>Cheese</td><td>Thr Ala</td>

<td>Leu 145</td><td>Gly Cys</td><td>Leu Val</td><td>lys 150</td><td>Asp</td><td>Tyr</td><td>phe</td><td>Pro</td><td>Glu 155</td><td>Pro</td><td>val</td><td>Thr Val</td><td>Cheese 160</td>

<td>Trp</td><td>Asn Cheese</td><td>Gly Ala 165</td><td>Leu</td><td>Thr</td><td>Cheese</td><td>Gly</td><td>val 170</td><td>His</td><td>Thr</td><td>phe</td><td>Pro Ala 175</td><td>val</td>

<td>Leu</td><td>Gin Cheese</td><td>Gly 180 cheese</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Leu 185</td><td>Cheese</td><td>Cheese</td><td>val</td><td>val</td><td>Thr Val 190</td><td>Pro</td>

<td>Cheese</td><td>Cheese Ser 195</td><td>Leu Gly</td><td>Thr</td><td>lys</td><td>Thr 200</td><td>Tyr</td><td>Thr</td><td>Cys</td><td>own</td><td>val 205</td><td>Asp His</td><td>lys</td>

<td>Pro</td><td>Asn 210 cheese</td><td>Thr Lys</td><td>val</td><td>Asp 215</td><td>lys</td><td>Arg</td><td>val</td><td>Glu</td><td>Cheese 220</td><td>lys</td><td>Tyr Gly</td><td>Pro</td>

<td>Pro 225</td><td>Cys Pro</td><td>Pro Cys</td><td>Pro 230</td><td>ala</td><td>Pro</td><td>Glu</td><td>phe</td><td>Leu 235</td><td>Gly</td><td>Gly</td><td>Pro Ser</td><td>val 240</td>

<td>phe</td><td>Leu Phe</td><td>Pro Pro 245</td><td>lys</td><td>Pro</td><td>lys</td><td>Asp</td><td>Thr 250</td><td>Leu</td><td>Underworld</td><td>How much</td><td>Arg 255</td><td>Thr</td>

<td>Pro</td><td>Glu Val</td><td>Thr Cys 260</td><td>val</td><td>val</td><td>val</td><td>Asp 265</td><td>val</td><td>Cheese</td><td>Gin</td><td>Glu</td><td>Asp Pro 270</td><td>Glu</td>

<td>val</td><td>Gin Phe 275</td><td>Asn Trp</td><td>Tyr</td><td>val</td><td>Asp 280</td><td>Gly</td><td>val</td><td>Glu</td><td>val</td><td>His 285</td><td>Asn Ala</td><td>lys</td>

<td>Thr</td><td>Lys Pro 290</td><td>Arg Glu</td><td>Glu</td><td>Gin 295</td><td>phe</td><td>own</td><td>Cheese</td><td>Thr</td><td>Tyr 300</td><td>Arg</td><td>Val Val</td><td>Cheese</td>

<td>val 305</td><td>Leu Thr</td><td>Val Leu</td><td>His 310</td><td>Gin</td><td>Asp</td><td>Trp</td><td>Leu</td><td>own 315</td><td>Gly</td><td>lys</td><td>Glu Tyr</td><td>lys 320</td>

<td>Cys</td><td>Lys Val</td><td>Asn 325 cheese</td><td>lys</td><td>Gly</td><td>Leu</td><td>Pro</td><td>Cheese 330</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Lys Thr 335</td><td>How much</td>

<td>Cheese</td><td>Lys Ala</td><td>Lys Gly 340</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Glu 345</td><td>Pro</td><td>Gin</td><td>val</td><td>Tyr</td><td>Thr Leu 350</td><td>Pro</td>

<td>Pro</td><td>Gin 355</td><td>Glu Glu</td><td>Underworld</td><td>Thr</td><td>lys 360</td><td>own</td><td>Gin</td><td>val</td><td>Cheese</td><td>Leu 365</td><td>Thr Cys</td><td>Leu</td>

<td>val</td><td>Lys Gly</td><td>Phe Tyr</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>How much</td><td>ala</td><td>val</td><td>Glu</td><td>Trp</td><td>Glu Cheese</td><td>own</td>

- 62 - EP 2867258

<td></td><td>370</td><td></td><td></td><td></td><td></td><td>375</td><td></td><td></td><td></td><td></td><td>380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Gin</td><td>Pro</td><td>Glu</td><td>own</td><td>own</td><td>Tyr</td><td>lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>val</td><td>Leu</td><td>Asp</td><td>Cheese</td>

<td>385</td><td></td><td></td><td></td><td></td><td>390</td><td></td><td></td><td></td><td></td><td>395</td><td></td><td></td><td></td><td></td><td>400</td>

<td>Asp</td><td>Gly</td><td>Cheese</td><td>phe</td><td>phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Arg</td><td>Leu</td><td>Thr</td><td>val</td><td>Asp</td><td>lys</td><td>Cheese</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td>405</td><td></td><td></td><td></td><td></td><td>410</td><td></td><td></td><td></td><td></td><td>415</td><td></td>

<td>Trp</td><td>Gin</td><td>Glu</td><td>Gly</td><td>own</td><td>val</td><td>phe</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>val</td><td>Underworld</td><td>His</td><td>Glu</td><td>ala</td><td>Leu</td>

<td></td><td></td><td></td><td>420</td><td></td><td></td><td></td><td></td><td>425</td><td></td><td></td><td></td><td></td><td>430</td><td></td><td></td>

<td>His</td><td>own</td><td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Gly</td><td>lys</td><td></td>

<td></td><td></td><td>435</td><td></td><td></td><td></td><td></td><td>440</td><td></td><td></td><td></td><td></td><td>445</td><td></td><td></td><td></td>

<td colspan="2">&lt;210&gt; 33</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<211> 645 <212> DNA <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polynucleotide" <400> 33

<td>gaaattgtgt</td><td>tgacacagtc</td><td>tccagccacc</td><td>ctgtctttgt</td><td>ctccagggga</td><td>aagagccacc</td><td>60</td>

<td>ctctcctgca</td><td>gggccagtca</td><td>gagtattagc</td><td>agctacttag</td><td>cctggtacca</td><td>acagaaacct</td><td>120</td>

<td>ggccaggctc</td><td>ccaggctcct</td><td>catctatgat</td><td>gcatccaaca</td><td>gggccactgg</td><td>catcccagcc</td><td>180</td>

<td>aggttcagtg</td><td>gcagtgggtc</td><td>tgggacagac</td><td>ttcactctca</td><td>ccatcagcag</td><td>cctagagcct</td><td>240</td>

<td>gaagattttg</td><td>cagtttatta</td><td>ctgtcagcag</td><td>cgtagcaact</td><td>ggcctctcac</td><td>ttttggccag</td><td>300</td>

<td>gggaccaacc</td><td>tggagatcaa</td><td>acgtacggtg</td><td>gctgcaccat</td><td>ctgtcttcat</td><td>cttcccgcca</td><td>360</td>

<td>tctgatgagc</td><td>agttgaaatc</td><td>tggaactgcc</td><td>tctgttgtgt</td><td>gcctgctgaa</td><td>taacttctat</td><td>420</td>

<td>cccagagagg</td><td>ccaaagtaca</td><td>gtggaaggtg</td><td>gataacgccc</td><td>tccaatcggg</td><td>taactcccag</td><td>480</td>

<td>gagagtgtca</td><td>cagagcagga</td><td>cagcaaggac</td><td>agcacctaca</td><td>gcctcagcag</td><td>caccctgacg</td><td>540</td>

<td>ctgagcaaag</td><td>cagactacga</td><td>gaaacacaaa</td><td>gtctacgcct</td><td>gcgaagtcac</td><td>ccatcagggc</td><td>600</td>

<td>ctgagctcgc</td><td>ccgtcacaaa</td><td>gagcttcaac</td><td>aggggagagt</td><td>gttag</td><td></td><td>645</td>

<210> 34 <211> 214 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polypeptide" <400> 34

EP 2867258

- 63 Glu Ile Val Leu Thr Gin Cheese Pro Ala Thr Leu Cheese Leu Cheese Pro Gly 15 10 15

Glu Arg Ala Thr Leu Cheese Cys Arg Ala Cheese Gin Cheese Ile Cheese Ser Tyr 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile 35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60

Cheese Gly Cheese Gly Thr Asp Phe Thr Leu Thr Ile Cheese Cheese Leu Glu Pro

70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Leu

90 95

Thr Phe Gly Gin Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110

Pro Ser Val Phe Ile Phe Pro Pro. Ser. Glu Gin Leu Lys Cheese Gly 115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Cheese Gly Asn Cheese Gin

145 150 155 160

Glu Cheese Val Thr Glu Gin Asp Cheese Lys Asp Ser Thr Tyr Cheese Leu Cheese

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

Ala Cys Glu Val Thr His Gin Gly Leu Cheese Ser Pro Val Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys 210 <210> 35 <211> 447 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polypeptide" <400> 35

EP 2867258

- 64 Gin Val Gin Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Cheese Phe Cheese Asp Tyr 20 25 30

Tyr Trp Asn Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45

Gly Glu Ile Asn His Arg Gly Cheese Thr Asn Cheese Asn Pro Cheese Leu Lys 50 55 60

Cheese Arg Val Thr Leu Cheese Leu Asp Thr Cheese Lys Asn Gin Phe Cheese Leu 65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gin 100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ser Ser Thr Lys Gly Pro Ser Val 115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175

Leu Gin Cheese Cheese Gly Leu Tyr Cheese Leu Cheese Cheese Val Val Thr Val Pro 180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220

Pro Cys Pro Pro Cys Pro Al Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240

EP 2867258

- 65 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255

Pro Glu Val Thr Cys Val Val Val Asp. Val Ser Gin Glu Asp Pro Glu 260 265 270

Val Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285

Thr Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300

Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Cheese Ser Ile Glu Lys Thr Ile

325 330 335

Lys Al Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 340 345 350

Pro Ser Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu 355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp. Ile Ala Val Glu Trp. Glu Ser Asn 370 375 380

Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430

His Asn His Tyr Thr Gin Lys Leu Ser Leu Leu Ser Leu Gly Lys 435 440 445 <210> 36 <211> 1344 <212> DNA <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polynucleotide" <400> 36

- 66 - EP 2867258

<td>caggtgcagc</td><td>tacagcagtg</td><td>gggcgcagga</td><td>ctgttgaagc</td><td>cttcggagac</td><td>cctgtccctc</td><td>60</td>

<td>acctgcgctg</td><td>tctatggtgg</td><td>gtccttcagt</td><td>gattactact</td><td>ggaactggat</td><td>ccgccagccc</td><td>120</td>

<td>ccagggaagg</td><td>ggctggagtg</td><td>gattggggaa</td><td>atcaatcatc</td><td>gtggaagcac</td><td>caactccaac</td><td>180</td>

<td>ccgtccctca</td><td>agagtcgagt</td><td>caccctatca</td><td>ctagacacgt</td><td>ccaagaacca</td><td>gttctccctg</td><td>240</td>

<td>aagctgaggt</td><td>ctgtgaccgc</td><td>cgcggacacg</td><td>gctgtgtatt</td><td>actgtgcgtt</td><td>tggatatagt</td><td>300</td>

<td>gactacgagt</td><td>acaactggtt</td><td>cgacccctgg</td><td>ggccagggaa</td><td>ccctggtcac</td><td>cgtctcctca</td><td>360</td>

<td>gctagcacca</td><td>agggcccatc</td><td>cgtcttcccc</td><td>ctggcgccct</td><td>gctccaggag</td><td>cacctccgag</td><td>420</td>

<td>agcacagccg</td><td>ccctgggctg</td><td>cctggtcaag</td><td>gactacttcc</td><td>ccgaaccggt</td><td>gacggtgtcg</td><td>480</td>

<td>tggaactcag</td><td>gcgccctgac</td><td>cagcggcgtg</td><td>cacaccttcc</td><td>cggctgtcct</td><td>acagtcctca</td><td>540</td>

<td>ggactctact</td><td>ccctcagcag</td><td>cgtggtgacc</td><td>gtgccctcca</td><td>gcagcttggg</td><td>cacgaagacc</td><td>600</td>

<td>tacacctgca</td><td>acgtagatca</td><td>caagcccagc</td><td>aacaccaagg</td><td>tggacaagag</td><td>agttgagtcc</td><td>660</td>

<td>aaatatggtc</td><td>ccccatgccc</td><td>accatgccca</td><td>gcacctgagt</td><td>tcctgggggg</td><td>accatcagtc</td><td>720</td>

<td>ttcctgttcc</td><td>ccccaaaacc</td><td>caaggacact</td><td>ctcatgatct</td><td>cccggacccc</td><td>tgaggtcacg</td><td>780</td>

<td>tgcgtggtgg</td><td>tggacgtgag</td><td>ccaggaagac</td><td>cccgaggtcc</td><td>agttcaactg</td><td>gtacgtggat</td><td>840</td>

<td>ggcgtggagg</td><td>tgcataatgc</td><td>caagacaaag</td><td>ccgcgggagg</td><td>agcagttcaa</td><td>cagcacgtac</td><td>900</td>

<td>cgtgtggtca</td><td>gcgtcctcac</td><td>cgtcctgcac</td><td>caggactggc</td><td>tgaacggcaa</td><td>ggagtacaag</td><td>960</td>

<td>tgcaaggtct</td><td>ccaacaaagg</td><td>cctcccgtcc</td><td>tccatcgaga</td><td>aaaccatctc</td><td>caaagccaaa</td><td>1020</td>

<td>gggcagcccc</td><td>gagagccaca</td><td>ggtgtacacc</td><td>ctgcccccat</td><td>cccaggagga</td><td>gatgaccaag</td><td>1080</td>

<td>aaccaggtca</td><td>gcctgacctg</td><td>cctggtcaaa</td><td>ggcttctacc</td><td>ccagcgacat</td><td>cgccgtggag</td><td>1140</td>

<td>tgggagagca</td><td>atgggcagcc</td><td>ggagaacaac</td><td>tacaagacca</td><td>cgcctcccgt</td><td>gctggactcc</td><td>1200</td>

<td>gacggctcct</td><td>tcttcctcta</td><td>cagcaggcta</td><td>accgtggaca</td><td>agagcaggtg</td><td>gcaggagggg</td><td>1260</td>

<td>aatgtcttct</td><td>catgctccgt</td><td>gatgcatgag</td><td>gctctgcaca</td><td>accactacac</td><td>acagaagagc</td><td>1320</td>

<td>ctctccctgt</td><td>ctctgggtaa</td><td>atga</td><td></td><td></td><td></td><td>1344</td>

<210> 37 <211> 214 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polypeptide" <400> 37

Glu Ile Val Leu Thr Gin Cheese Pro Ala Thr Leu Cheese Leu Cheese Pro Gly 15 10 15

EP 2867258

- 67 Glu Arg Ala Thr Leu Ser Cys Arg Ala Cheese Gin Ser Ile Ser Ser Tyr 20 25 30

Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile 35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60

Cheese Gly Cheese Gly Thr Asp Phe Thr Leu Thr Ile Cheese Cheese Leu Glu Pro

70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Leu

90 95

Thr Phe Gly Gin Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110

Pro Ser Val Phe Ile Phe Pro Pro. Ser. Glu Gin Leu Lys Cheese Gly 115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Cheese Gly Asn Cheese Gin

145 150 155 160

Glu Cheese Val Thr Glu Gin Asp Cheese Lys Asp Ser Thr Tyr Cheese Leu Cheese

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190

Ala Cys Glu Val Thr His Gin Gly Leu Cheese Ser Pro Val Thr Lys Ser 195 200 205

Phe Asn Arg Gly Glu Cys 210 <210> 38 <211> 645 <212> DNA <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic polynucleotide" <400> 38

<td></td><td>- 68 -</td><td>EP 2867258</td>

<td>gaaattgtgt</td><td>tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc</td><td>60</td>

<td>ctctcctgca</td><td>gggccagtca gagtattagc agctacttag cctggtacca acagaaacct</td><td>120</td>

<td>ggccaggctc</td><td>ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc</td><td>180</td>

<td>aggttcagtg</td><td>gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct</td><td>240</td>

<td>gaagattttg</td><td>cagtttatta ctgtcagcag cgtagcaact ggcctctcac ttttggccag</td><td>300</td>

<td>gggaccaacc</td><td>tggagatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca</td><td>360</td>

<td>tctgatgagc</td><td>agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat</td><td>420</td>

<td>cccagagagg</td><td>ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag</td><td>480</td>

<td>gagagtgtca</td><td>cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg</td><td>540</td>

<td>ctgagcaaag</td><td>cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc</td><td>600</td>

<td>ctgagctcgc</td><td>ccgtcacaaa gagcttcaac aggggagagt gttag</td><td>645</td>

<210> 39 <211> 5 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 39

Pro Val Gly Val Val

5 <210> 40 <211> 13 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 40

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro 15 10 <210> 41 <211> 11 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 41

Gly Glu Ile Asn His Asn Gly Asn Thr Asn Ser

10 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence

- 69 EP 2867258 <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 42

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser 15 10 <210> 43 <211> 11 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 43

Gly Glu Ile Ile His Ser Gly Ser Thr Asn Ser 15 10 <210> 44 <211> 11 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 44

Gly Glu Ile Asn His Gly Gly Gly Thr Asn Ser 15 10 <210> 45 <211> 11 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400>

Gly Glu Ile Asn His Ile Gly Asn Thr Asn Ser

10 <210> 46 <211> 14 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <220>

<221> MOD_RES <222> (4) .. (4) <223> Isomerized residue

- EP 2867 258 <400> 46

How much Asn His Asp Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys 15 10 <210> 47 <211> 14 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 47

How much Asn His Asp Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys 15 10 <210> 48 <211> 14 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 48

How much Asn His Asn Gly Asn Thr Asp Ser Asn Pro Ser Leu Lys 15 10 <210> 49 <211> 14 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 49

Ile Asp As Asg Gly Asn Thr Asn Ser Asn Pro Ser Leu Lys 15 10 <210> 50 <211> 14 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <220>

<221> MOD_RES <222> (8) .. (8) <223> Isomerized residue <400> 50

How much Asn His Arg Gly Ser Thr Asp Ser Asn Pro Ser Leu Lys 15 10 <210> 51

- 71 EP 2867258 <211> 14 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide" <400> 51

How much Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys 15 10 <210> 52 <211> 14 <212> PRT <213> Artificial Sequence <220>

<221> source <223> / note = "Description of Artificial Sequence: Synthetic peptide"

<220>

<221> MOD_RES <222> (2) .. (2) <223> Isomerized residue <400> 52

Ile Asp Arg Gly Ser Thr Asp Ser Asn Pro Ser 15 10

<img file="PL2867258T3_D0001.tif" />

LEGAL PATENT LAW "BELLEPAT"

Izabela Szych niska-Ha wranek ul Słowackieao 44, 37-700 Prz * 'nvśl tel. (016) 742-37-77 fax: (016) 376--72-87 mobile (0608) 503-081 e-maH <a href="mailto:t-eliepat@op.pl">t-eliepat@op.pl</a> NIP: 795-207-16-72 REGON: 1803505 (6

Proxy:

<img file="PL2867258T3_D0002.tif" />

EP 2867258