MX355418B

Antibodies that bind csf1r.

Abstract

The present invention relates to antibodies that bind to CSF1 R. Heavy chains and antibody light chains are also provided which are capable of forming antibodies that bind to CSF1R. Polynucleotides encoding antibodies to CSF1R are provided. Polynucleotides encoding heavy chains and light chains of antibody are also provided. Treatment methods that use antibodies to CSF1R are provided. The methods include, but are not limited to, methods for treating rheumatoid arthritis, bone loss, and multiple sclerosis.

MX355418B, drawing sheet 1 — Sheet 1 of 232

Term

4.6 yearsleft in the term

Expires 4 May 2031.

23 claims: 4 independent, 19 dependent

1
Habiéndose descrito la invención como antecede, se reclama como propiedad lo contenido en las siguientes reivindicaciones:1. Un ácido nucleico aislado, caracterizado porque comprende: a) una secuencia de polinucleótidos que codifica una cadena pesada que comprende un factor determinante de complementariedad de cadena pesada (CDR)1 (HC) que tiene la secuencia de la SEQ ID NO: 15, una CDR2 HC que tiene la secuencia de la SEQ ID NO: 16, ;γ una CDR3 HC que tiene la secuencia de la SEQ ID NO: 17;o una secuencia de polinucleótidos que codifica una cadena ligera que comprende una CDR1 de cadena ligera (LC) que tiene la secuencia de la SEQ ID NO: 18, una CDR2 LC que tiene la secuencia de la SEQ ID NO: 19, : y una CDR3 LC que tiene la secuencia de la SEQ ID NO: 20;o una primera secuencia de polinucleótidos que codifica una cadena pesada que comprende una CDR1 de cadena pesada (HC) que tiene la secuencia de la SEQ ID NO : 15, una CDR2 HC que tiene la secuencia de la SEQ ID NO: 16, y una CDR3 HC que tiene la secuencia de la SEQ ID 125 IMPI INSTITUTO MEXICANO ne la monto* d INDUSTRIAL NO: 17, y una segunda secuencia de polinucleótidos que codifica una cadena ligera que comprende una CDR1 de cadena ligera (LC) que tiene la secuencia de la SEQ ID NO: 18, una CDR2 LC que tiene la secuencia de la SEQ ID NO: 19, y una CDR3 LC que tiene la secuencia de la SEQ ID NO: 20.
2
El ácido nucleico aislado de conformidad con la reivindicación 1, caracterizado porque la cadena pesada es humanizada, o la cadena ligera es humanizada, o tanto la cadena pesada como la cadena ligera son humanizadas.
3
El ácido nucleico aislado de conformidad con la reivindicación 1, caracterizado porque comprende:a) una secuencia de polinucleótidos que codifica una cadena pesada que comprende una secuencia de la SEQ ID NO: 39;o b) una secuencia de polinucleótidos que codifica una cadena ligera que comprende una secuencia de la SEQ ID NO: 46;o c) una primera secuencia de polinucleótidos que codifica una cadena pesada que comprende una secuencia de la SEQ ID NO: 39 y una segunda secuencia de polinucleótidos que codifica una cadena ligera que comprende una secuencia de la SEQ ID NO: 46. 126 INSTITUTO MfXJON.) Of ‘.A «0*1(0* O ‘N ‘l!$TRlAL
4
El ácido nucleico aislado de conformidad con la reivindicación 3, caracterizado porque comprende:a) una secuencia de polinucleótidos que codifica una cadena pesada que comprende una secuencia de la SEQ ID NO: 53;o b) una secuencia de polinucleótidos que codifica una cadena ligera que comprende una secuencia de la SEQ ID NO: 60;o c) una primera secuencia de polinucleótidos que codifica una cadena pesada que comprende una secuencia de la SEQ ID NO: 53 y una segunda secuencia de polinucleótidos que codifica una cadena ligera que comprende una secuencia de la SEQ ID NO: 60.
5
El ácido nucleico aislado de conformidad con la reivindicación 3, caracterizado porque comprende:a) una secuencia de polinucleótidos que codifica una cadena pesada que consiste de una secuencia de la SEQ ID NO: 53;o b) una secuencia de polinucleótidos que codifica una cadena ligera que consiste de una secuencia de la SEQ ID NO: 60;o c) una primera secuencia de polinucleótidos que codifica una cadena pesada que consiste de una secuencia de la SEQ ID NO: 53 y una segunda IMPIOS 127 INSTITUTO MFKlC*N oe ι-a ♦’RORfOAO iFHXhTMAt secuencia de polinucleótidos que codifica una cadena ligera que consiste de una secuencia de la SEQ ID NO: 60.
6
Una célula hospedadora aislada, caracterizada porque comprende:a) un ácido nucleico que comprende una primera secuencia de polinucleótidos que codifica una cadena pesada que comprende una CDR1 de cadena pesada (HC) que tiene la secuencia de la SEQ ID NO: 15, una CDR2 HC que tiene la secuencia de la SEQ ID NO: 16, y una CDR3 HC que tiene la secuencia de la SEQ ID NO: 17, y una segunda secuencia de polinucleótidos que codifica una cadena ligera que comprende una CDR1 de cadena ligera (LC) que tiene la secuencia de la SEQ ID NO: 18, una CDR2 LC que tiene la secuencia de la SEQ ID NO: 19, y una CDR3 LC que tiene la secuencia de la SEQ ID NO: 20;o b) un primer ácido nucleico que comprende una primera secuencia de polinucleótidos que codifica una cadena pesada que comprende una CDR1 de cadena pesada (HC) que tiene la secuencia de la SEQ ID NO: 15, una CDR2 HC que tiene la secuencia de la SEQ ID NO: 16, y una 128 IMPI INSTITUTO MEXICANO Of I.A »*O*lfO*T· CDR3 HC que tiene la secuencia de la SEQ ID NO: 17, y un segundo ácido nucleico que comprende una segunda secuencia de polinucleótidos que codifica una cadena ligera 5 que comprende una CDR1 de cadena ligera (LC) que tiene la secuencia de la SEQ ID NO: 18, una CDR2 LC que tiene la secuencia de la SEQ ID NO: 19, y una CDR3 LC que tiene la secuencia de la SEQ ID NO: 20. 10
7
La célula hospedadora de conformidad con la reivindicación 6, caracterizada porque la cadena pesada y la cadena ligera son humanizadas.
8
La célula hospedadora de conformidad con la reivindicación 6, caracterizada porque:15 a) el ácido nucleico comprende una primera secuencia de polinucleótidos que codifica una cadena pesada que comprende una secuencia de la SEQ ID NO: 39 y una segunda secuencia de polinucleótidos que codifica una cadena ligera 20 que comprende una secuencia de la SEQ ID NO: 46;o b) el primer ácido nucleico comprende una primera secuencia de polinucleótidos que codifica una cadena pesada que comprende una secuencia de 25 la SEQ ID NO: 39 y el segundo ácido nucleico 129 comprende una segunda secuencia de polinucleótidos que codifica una cadena ligera que comprende una secuencia de la SEQ ID NO: IMPIág INSTITUTO MEXICAN Dt LA PHOWfOAC 1NÍXISTWAI 5
9
La célula hospedadora de conformidad con la reivindicación 8, caracterizada porque:a) el ácido nucleico comprende una primera secuencia de polinucleótidos que codifica una cadena pesada que comprende una secuencia de 10 la SEQ ID NO: 53 y una segunda secuencia de polinucleótidos que codifica una cadena ligera que comprende una secuencia de la SEQ ID NO: 60;o b) el primer ácido nucleico comprende una primera 15 secuencia de polinucleótidos que codifica una cadena pesada que comprende una secuencia de la SEQ ID NO: 53 y el segundo ácido nucleico comprende una segunda secuencia de polinucleótidos que codifica una cadena ligera 20 que comprende una secuencia de la SEQ ID NO: 60.
10
La célula hospedadora de conformidad con la reivindicación 8, caracterizada porque:a) el ácido nucleico comprende una primera secuencia de polinucleótidos que codifica una 130 IMPI INSTITUTO MEXICANO Ot LA MOHCOaO INPUSTXMt cadena pesada que consiste de ' una''sécuéncla^de la SEQ ID NO: 53 y una segunda secuencia de polinucleótidos que codifica una cadena ligera que consiste de una secuencia de la SEQ ID NO: 5 60;o b) el primer ácido nucleico comprende una primera secuencia de polinucleótidos que codifica una cadena pesada que consiste de una secuencia de la SEQ ID NO: 53 y el segundo ácido nucleico 10 comprende una segunda secuencia de polinucleótidos que codifica una cadena ligera que consiste de una secuencia de la SEQ ID NO: 60.
11
La célula hospedadora de conformidad con la 15 reivindicación 6, caracterizada porque es una célula CHO o una célula 293.
12
La célula hospedadora de conformidad con la reivindicación 7, caracterizada porque es una célula CHO o una célula 293. 20
13
La célula hospedadora de conformidad con la reivindicación 8, caracterizada porque es una célula CHO o una célula 293.
14
La célula hospedadora de conformidad con la reivindicación 9, caracterizada porque es una célula CHO o 25 una célula 293. 131 IMPI ¡NSTiTtrro mexicano DE LA fKOKIEDAO INOUSTKIAI
15
La célula hospedadora de conformidad con la reivindicación 10, caracterizada porque es una célula CHO o una célula 293.
16
Un método in vitro para producir un anticuerpo que se une al receptor 1 del factor estimulante de la colonia humana (CSF1R), caracterizado porque comprende cultivar la célula hospedadora de conformidad con la reivindicación 6 bajo condiciones suficientes para producir el anticuerpo.
17
Un método in vitro para producir un anticuerpo que se une al receptor 1 del factor estimulante de la colonia humana (CSF1R), caracterizado porque comprende cultivar la célula hospedadora de conformidad con la reivindicación 7 bajo condiciones suficientes para producir el anticuerpo.
18
Un método in vitro para producir un anticuerpo que se une al receptor 1 del factor estimulante de la colonia humana (CSF1R), caracterizado porque comprende cultivar la célula hospedadora de conformidad con la reivindicación 8 bajo condiciones suficientes para producir el anticuerpo.
19
Un método in vitro para producir un anticuerpo que se une al receptor 1 del factor estimulante de la colonia humana (CSF1R), caracterizado porque comprende cultivar la célula hospedadora de conformidad con la reivindicación 9 bajo condiciones suficientes para producir el anticuerpo.
20
Un método in vitro para producir un anticuerpo que se une al receptor 1 del factor estimulante de la colonia 132 IMPIAS J NXTITVTO Μ EX ICAN ) r OI LA f «ORfDAO C¿*W l*tnU T4M humana (CSFIR), caracterizado porque comprende cultivar la célula hospedadora de conformidad con la reivindicación 10 bajo condiciones suficientes para producir el anticuerpo.
21
El método de conformidad con la reivindicación 16, caracterizado porque el anticuerpo se selecciona de un Fab, un Fv, un scFv, un Fab' y un (Fab')2.
22
El método de conformidad con la reivindicación 17, caracterizado porque el anticuerpo se selecciona de un Fab, un Fv, un scFv, un Fab' y un (Fab')2.
23
El método de conformidad con la reivindicación 18, caracterizado porque el anticuerpo se selecciona de un Fab, un Fv, un scFv, un Fab' y un (Fab')2.

Independent claims23

1,013 paragraphs in 838 sections, as filed

In accordance with the as of the date of presentation

I pjgojteadSnftk ^ fa'rjjaaOíier current Itefrdteechos.

Who subscribes this title lot ^ PgMi fundHta | g¡p en lotSsjkiest ^ pw, MM¡ltidíilefe K_fracc «pg¡¿ III and 7 ° bis 2 of the Industrial Property Law (Official Gazette of the Federation 4 &) Sp27 / 9en99r '' l * fcirmáa on Ϊ2Β8 ^ 1994, »/ 10 ^ 996, 12/26/1897 (05/47/1999, 01/26/2004, 06/16/2005, 01/25/2006, 05/06 / 2009.06 / 01/2010, ™ · ρ / 2Μ0, ιβ8 / 06 / 201Ι «7 /» 2012ΐ (»» / 2012? ArtjSil®. 3 ° fraaitón Vlncijte a), 4 ° and 12 ”fractions I and III of Regulations of the Mexican Institute Sfcgropleíted laiteltnal ^ ifef. 1 999, ¿y | omadΪΈΙ 0lS) 7 / W2f IS / 07/2004, 28/07/2004 and 7/09/2007);

Articles 1, 3 ", 4", 5 fraction V Subsection a), (Otefcclortgsl jjdll y 30l! R »etete« te'Orgán¡co ^ (tCJostitÚ9ífMeitteeTÍo of Industrial Property (DOF 12/27/1999, amended on 10/10/2002, 07/29 / ^ 04 ^ 06/44/2904 ^^ 09/13/2007), 10^ ,. 3 ° and 5. ^ Jncí ^ aÓdet Actiardo that delegates powers to the Deputy General Directors, Coordinator, Directors ^ Ott ^ MteamTtererMtrieDivisional Deputy Directors, Departmental Coordinators and other subordinates of the MextctefojteS LaProperty Institute | π31η

08/04/2004 and 09/13/2007).

^ É * 1 © i2 / 1999, amended 02/04/2000, 07/29/2004,

This document is signed with an advanced electronic signature (FIEL), based on articles 7 BIS 2 of the Industrial Property Law; 3 of its Regulations, and 1 section III, 2 section V, 26 BIS and 26 TER of the Agreement establishing the guidelines for the use of the Payment and Electronic Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated,

THE DIVISIONAL DIRECTOR OF PATENTS

NAHANNY CANAL REYES

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55ΗΙ8

ANTIBODIES BINDING STIMULATING FACTOR (CSF1R) _

FIELD OF THE INVENTION

Antibodies are provided that bind to CSF1R. Also provided are antibody heavy chains and light chains that are capable of forming antibodies that bind to CSF1R. In addition, antibodies, heavy chains, and light chains comprising one or more particular complementarity determining regions (CDRs) are provided. Polynucleotides encoding antibodies to CSF1R are provided. Polynucleotides encoding antibody heavy chains or light chains are also provided. Treatment methods using antibodies to CSF1R are provided. The methods include, but are not limited to, methods for treating rheumatoid arthritis, bone loss, and multiple sclerosis.

BACKGROUND OF THE INVENTION

Colony Stimulating Factor 1 Receptor (referred to herein as CSF1R; also referred to in the art as FMS, FIM2, C-FMS, and CD115) is a single-pass transmembrane receptor with an extracellular domain (ECD). ) of N-Terminal and an intracellular domain of C-Terminal with tyrosine kinase activity. Binding of CSF1 Ligands or Interleukin 34 Ligand

REF. 257472

(referred to herein as IL34; Lin et al. Ga-i & ni-nri 3 20: 807-11 (2008)) a CSF1R causes receptor dimerization events, up-regulation of CSF1R tyrosine kinase activity, phosphorylation of residues of CSF1R tyrosine and downstream signaling. Both CSF1 and IL34 stimulate the survival, proliferation, and differentiation of monocytes in macrophages.

Various tumor cells have been found to secrete CSF1, which activates monocytes / macrophage cells by CSF1R. The level of CSF1 in tumors has been shown to correlate with the level of tumor associated macrophages (TAMs) in the tumor. Higher TAM levels have been found to correlate with less accurate patient prognosis. Furthermore, CSF1 has been found to promote tumor growth and metastasis progression in, for example, human breast cancer xenografts in mice. See eg. , Paulus et al. , Cancer Res. 66: 4349-56 (2006). Furthermore, CSF1R appears to play a role in osteolytic bone destruction in bone metastases, as a small molecule inhibitor of receptor tyrosine kinase activity suppresses this destruction. See eg. , Ohno et al. , Mol.

Cancer Ther. 5: 2634-43 (2006).

It was also found that CSF1 and its receptor are involved in various inflammatory diseases and

IMPI

INSTITUTO MEXICANO Gi LA PROPIEI.'AG

INOC? LH! AL

autoimmune. See eg. , Hamilton,,: 533 44 (2008). For example, synovial endothelial cells from joints affected with rheumatoid arthritis were found to produce CSF1, suggesting the role of CSF1 and its receptor in disease. Blocking CSF1R activity with an antibody causes positive clinical effects in mouse models of arthritis, including a reduction in bone and cartilage destruction and a reduction in macrophage numbers. See eg. , Kitaura et al., J. Clin. Invest. 115: 3418-3427 (2005).

Mature cells of the differentiated myeloid lineage such as macrophages, microglial cells, and osteoclasts contribute to the pathology of various diseases such as rheumatoid arthritis, multiple sclerosis, and bone loss diseases. Differentiated myeloid lineage cells are derived from peripheral blood monocyte intermediates. CSF1R stimulation contributes to the development of monocytes from bone marrow precursors, the proliferation and survival of monocytes, and the differentiation of peripheral blood monocytes into differentiated myeloid lineage cells such as macrophages, microglial cells, and osteoclasts. The stimulation of CSF1R thus contributes to the proliferation, survival, activation and maturation of cells of the differentiated myeloid lineage, and in the pathological context, the stimulation of CSF1R contributes to the capacity of cells of the myeloid lineage.

differentiated to mediate disease pathologySes'T * ™ '' -—- Therefore, other antagonists of

CSF1R would be useful for the treatment of various CSF1R-related diseases, such as cancer, inflammatory conditions, and autonomic diseases.

BRIEF DESCRIPTION OF THE INVENTION

The present inventors have invented a new set of antibodies, including humanized antibodies, directed against the extracellular domain of human CSF1R (CSF1R ECD). A Fab phage display library was made from spleens of mice that were immunized with a human CSF1R ECD-Fc fusion protein. 1056 Fabs expressing phage clones that bind to CSF-1R ECD-Fc were isolated by affinity selection of this library. When all 1056 Fabs were expressed as purified protein, 668 were found to bind to the CSF1R ECD. Of those 668 binding Fabs, only 121 Fabs blocked the binding of CSF1 and / or IL34 to CSF1R. Only 33 of those Fabs blocked the binding of CSF1 and IL34 to CSF1R. After sequencing, the 33 Fabs represented 19 unique sets of sequences. Eleven Fabs with subnanomolar affinity for human CSF1R ECD were chosen to make chimeric antibodies for further study. Based on macaque monkey and human CSF1R binding affinities, blocking CSF1 and IL34 binding to CSF1R, and

INSTITUTO MEXICANO ΤΓ «* · ιη DE LA PROPERTY

IN OUSTRIAI. ^ * 7 ™ inhibition of CSF1R ligand-induced phosphorylation, three chimeric antibodies were selected for humanization and sixteen humanized antibodies were made based on those three chimeric antibodies.

Fourteen of the sixteen humanized antibodies retained subnanomolar binding affinities for human CSF1R ECD. See, eg, Table 5. These humanized antibodies block the binding of CSF1 and IL34 ligands to human CSF1R, and many also block the binding of CSF1 and IL34 to macaque monkey CSF1R. See, e.g., Table 4.

For the therapeutic development of the drug, it is beneficial to have antibodies that bind to human and macaque antigens with similar affinity. The three chimeric antibodies chosen for humanization were selected in part because they had similar ECD binding affinities of human and macaque monkey CSF1R. Most of the humanized versions of one of the chimeric antibodies, 0302, however, lost considerable ECD-binding affinity of macaque monkey CSF1R upon humanization, although they retained strong ECD-binding affinity of human CSF1R. See, eg, Table 3. The humanized versions of 0301 and 0311 similarly retained strong ECD binding of human and macaque monkey CSF1R, with binding affinity differences for the two species of less than about 2-fold.

Depending on the binding affinities to CSF1R,

Ligand inhibition and possibility of inrtlUIlOgSfllCíÜad ^ 'T were selected for three humanized antibodies for further study. The three humanized antibodies were derived from the two chimeric antibodies that did not significantly lose binding affinity to macaque monkey CSF1R upon humanization. Those three humanized antibodies inhibit ligand-induced phosphorylation of human CSF1R, and also block ligand-induced proliferation and survival responses in major human monocytes. See, eg, Tables 6 and 7, and Figures 10 and 11. Therefore, these antibodies are useful for treating diseases involving, for example, ligand-induced proliferation and survival responses in major human monocytes. .

Blocking CSF1R-induced responses with an anti-CSFIR antibody should inhibit proliferation, survival, activation, maturation of differentiated myeloid lineage cells and attenuate their ability to mediate disease pathology. Furthermore, blocking CSF1R-induced responses with an anti-CSFIR antibody should inhibit the differentiation of peripheral blood monocyte intermediates into differentiated myeloid lineage cells, decreasing the number of pathology-mediating differentiated myeloid lineage cells.

Therefore, humanized anti-CSFIR antibodies

described herein can be used to treat chronic uiifUiiiiududuy 'with existing symptoms by inhibiting the ability of differentiated myeloid lineage cells to mediate disease pathology. Humanized antibodies can also be used to treat chronic diseases that are relapsing and remitting in nature by inhibiting the development of new pathology-mediating cells of the myeloid lineage differentiated from peripheral blood monocytes during the remission phase of the disease, thereby attenuating the amount of existing and new formation of pathology mediating cells.

In some embodiments, an isolated antibody comprising a heavy chain and a light chain is provided, where the antibody binds to CSF1R. In some embodiments, the heavy chain and / or light chain have the following structure.

In some embodiments, the heavy chain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence that is selected from SEQ ID NOs: 9, 11 , 13 and 39-45. In some embodiments, the light chain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to a sequence that is selected from the SEQ ID NOs: 10, 12, 14 and 46 to 52. In some embodiments, the heavy chain comprises a sequence that is at least 90%, at least

95%, at least 97%, at least 99% or 100% identical to a

sequence that is selected from SEQ ID NOs': y, 11, 13, and 39 to 45, and the light chain comprises a sequence that is at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to a sequence selected from SEQ ID NOs:

10, 12, 14 and 46 to 52.

In some embodiments, CDR1 HC, CDR2 HC, and CDR3 HC comprise a set of sequences that are selected from: (a) SEQ ID NOs: 15, 16, and 17; (b) SEQ ID NOs: 21, 22 and 23; and (c) SEQ ID NOs: 27, 28 and 29. In some embodiments, CDR1 LC, CDR2 LC and CDR3 LC comprise a set of sequences that are selected from: (a) SEQ ID NOs: 18, 19 and twenty; (b) SEQ ID NOs: 24, 25 and 26; and (c) the

SEQ ID NOs: 30, 31 and 32.

In some embodiments, the heavy chain comprises a CDR1 HC, CDR2 HC and CDR3 HC, where CDR1 HC, CDR2 HC and CDR3 HC comprise a set of sequences that are selected from: (a) SEQ ID NOs: 15, 16 and 17; (b) SEQ ID NOS: 21, and 23; and (c) SEQ ID NOs: 27, 28 and 29; and the light chain comprises a CDR1 LC, CDR2 LC and CDR3 LC, where CDR1 LC, CDR2 LC and CDR3 LC comprise a set of sequences that are selected from: (a) SEQ ID NOs: 18, 19 and 20; (b) SEQ ID NOs: 24, 25 and 26; and (c) SEQ ID NOs:

30, 31 and 32.

In some embodiments, an isolated antibody is provided, where the antibody comprises a heavy chain and

a light chain, where the antibody comprises the heavy chain comprises a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 9 and a light chain that comprises a sequence what is at least

95%, at least 97%, at least 99% or 100% identical to SEQ ID

NO: 10; (b) a heavy chain that comprises a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 11 and a light chain that comprises a sequence that is at least 95 %, at least 97%, at least 99% or 100% identical to SEQ ID NO: 12; (c) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 13 and a light chain comprising a sequence that is at least 95 %, at least 97%, at least 99% or 100% identical to SEQ ID NO: 14; (d) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to that

SEQ ID NO: 39 and a light chain comprising a sequence

<td>what is al</td><td>minus 95%, at</td><td>less</td><td> 97%,</td><td>at least</td><td>99% or 100%</td>

<td>identical to</td><td>SEQ ID NO:</td><td> 46;</td><td colspan="2">(e) a string</td><td>heavy that</td>

<td colspan="2">comprises a sequence that</td><td>is to</td><td>less</td><td colspan="2">95%, at least 97%, at</td>

<td>minus 99% or</td><td>100% identical</td><td>to</td><td>SEQ ID</td><td>NO: 40 and</td><td>a chain</td>

lightweight comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 46;

(f) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to that

SEQ ID NO: 41 and a light chain comprising 'binds

which is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 46; (g) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 39 and a light chain comprising a sequence that is at least 95 %, at least 97%, at least 99% or 100% identical to SEQ ID NO: 47; (h) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to that

SEQ ID NO: 40 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 47; (i) a heavy chain that comprises a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 41 and a light chain that comprises a sequence that is at least 95 %, at least 97%, at least 99% or 100% identical to SEQ ID NO: 47; and (j) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to that

SEQ ID NO: 42 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 48; (k) a heavy chain that comprises a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 42 and a light chain that comprises a sequence that is at least 95 %, at least 97%, at least 99% or 100% identical to SEQ ID NO: 49;

ΙΜΡϊ

Mexican PWJTnrro is THE INDtisnu ^ i PROPERTY

(1) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to that

SEQ ID NO: 42 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 50; (m) a heavy chain that comprises a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 43 and a light chain that comprises a sequence that is at least 95 %, at least 97%, at least 99% or 100% identical to SEQ ID NO: 48;

(n) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to that

SEQ ID NO: 43 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 49; (o) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 43 and a light chain comprising a sequence that is at least 95 %, at least 97%, at least 99% or 100% identical to SEQ ID NO: 50;

(p) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to that

SEQ ID NO: 44 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 51; (q) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 44 and a chain

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lightweight comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 52;

(r) a heavy chain comprising a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to that

SEQ ID NO: 45 and a light chain comprising a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 51; or (s) a heavy chain that comprises a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 45 and a light chain that comprises a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 52.

In some embodiments, an antibody is provided, where the antibody comprises a heavy chain and a light chain, where the antibody comprises: (a) a heavy chain comprising a heavy chain (HC) CDR1 having a sequence of SEQ ID NO: 15; a CDR2 HC having the sequence of SEQ ID NO: 16; and a CDR3 HC having the sequence of SEQ ID NO: 17, and a light chain comprising a CDR1 light chain (LC) having a sequence of SEQ ID NO: 18, a CDR2 LC

<td>what's wrong with it</td><td>the</td><td>sequence of</td><td>I KNOW THAT</td><td>ID</td><td>NOT:</td><td> 19,</td><td>and a CDR3 LC</td><td>what</td>

<td>has the</td><td colspan="2">SEQ sequence</td><td>ID</td><td>NOT:</td><td> 20;</td><td>(b)</td><td colspan="2">a heavy chain</td>

<td colspan="2">that understands</td><td>a CDR1 of</td><td colspan="2">chain</td><td colspan="2">heavy</td><td>(HC) you have</td><td>a</td>

<td>sequence</td><td>from</td><td>SEQ ID NO:</td><td> 21</td><td colspan="2">; a</td><td>CDR2</td><td>HC you have</td><td>the</td>

<td>sequence</td><td>from</td><td>SEQ ID NO:</td><td> 22 ;</td><td>Y</td><td>a</td><td>CDR3</td><td>HC you have</td><td>the</td>

1? Τ

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INDUSTS1AL sequence of SEQ ID NO: 23, and a light chain comprising a CDR1 light chain (LC) having a sequence of SEQ ID NO: 24, a CDR2 LC having the sequence of SEQ ID NO:

25, and a CDR3 LC having the sequence of SEQ ID NO: 26; or (c) a heavy chain comprising a heavy chain CDR1 (HC) having a sequence of SEQ ID NO: 27; a CDR2 HC having the sequence of SEQ ID NO: 28; and a CDR3 HC having the sequence of SEQ ID NO: 29, and a light chain comprising a CDR1 light chain (LC) having a sequence of SEQ ID NO: 30, a CDR2 LC having the sequence of SEQ ID NO: 31, and a CDR3 LC having the sequence of SEQ ID NO: 32.

In some embodiments, an antibody comprises a heavy chain and a light chain, where the antibody comprises: (a) a heavy chain comprising a sequence of SEQ ID NO: 53 and a light chain comprising a sequence of SEQ ID NO: 60 ; (b) a heavy chain comprising a sequence of SEQ ID NO: 53 and a light chain comprising a sequence of SEQ ID NO: 61; or (c) a heavy chain comprising a sequence of SEQ ID NO: 58 and a light chain comprising a sequence of SEQ ID NO: 65. In some embodiments, an antibody comprises a heavy chain and a light chain, where the antibody comprises: (a) a heavy chain consisting of the sequence of SEQ ID NO: 53 and a light chain consisting of the

I JMt Ρ1

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sequence of SEQ ID NO: SO; (b) a heavy chain consisting of the sequence of SEQ ID NO: 53 and a light chain consisting of the sequence of SEQ ID NO: 61; or (c) a heavy chain consisting of the sequence of SEQ ID NO: 58 and a light chain consisting of the sequence of SEQ ID NO:

In some embodiments, an antibody is a humanized antibody. In some embodiments, an antibody is selected from a Fab, a Fv, a scFv, a Fab ', and a (Fab')2. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is selected from IgA, IgG, and IgD. In some embodiments, an antibody is an IgG. In some embodiments, an antibody is an IgG4. In some embodiments, an antibody is an IgG4 comprising an S241P mutation in at least one heavy chain constant region of IgG4.

In some embodiments, an antibody binds to human CSF1R and / or binds to macaque monkey CSF1R. In some embodiments, an antibody blocks the binding of the ligand to CSF1R. In some embodiments, an antibody blocks the binding of CSF1 and / or IL34 to CSF1R. In some embodiments, an antibody inhibits ligand-induced phosphorylation of CSF1R. In some embodiments, an antibody inhibits CSF1-induced and / or IL34-induced phosphorylation of CSF1R. In some embodiments, an antibody binds to CSF1R

human with an affinity (KD) of less than 1 nM. In some embodiments, the antibody inhibits monocyte survival and / or proliferation responses in the presence of

CSF1 or IL34.

In some embodiments, a pharmaceutical composition is provided comprising an antibody that binds to CSF1R.

In some embodiments, an isolated nucleic acid is provided, where the isolated nucleic acid comprises a polynucleotide sequence encoding a heavy chain described above. In some embodiments, an isolated nucleic acid encodes a light chain described above. In some embodiments, an isolated nucleic acid encodes a heavy chain described above and a light chain described above. In some embodiments, a composition is provided, wherein the composition comprises a first nucleic acid comprising a polynucleotide sequence encoding a heavy chain described above, and a second nucleic acid comprising a polynucleotide sequence encoding a light chain described above. In some embodiments, a host cell is provided comprising a nucleic acid or composition described above. In some embodiments, a host cell is a eukaryotic host cell. In some embodiments, a host cell is a mammalian host cell.

In some embodiments, a host cell is selected from a CHO cell, a 293 cell, an NSO cell, and a PER.C6 cell. In some embodiments, a host cell is a 293-6E cell or a DG44 cell.

In some embodiments, methods of treating disease are provided which comprise administering to a patient a pharmaceutical composition comprising an antibody that binds to CSF1R. In some embodiments, a method of treating multiple sclerosis is provided which comprises administering to a patient a pharmaceutical composition comprising an antibody that binds to CSF1R. In some embodiments, a method of treating rheumatoid arthritis is provided which comprises administering to a patient a pharmaceutical composition comprising an antibody that binds to CSF1R. In some embodiments, a method of treating bone loss is provided which comprises administering to a patient a pharmaceutical composition comprising an antibody that binds to CSF1R. In some embodiments, osteolytic bone loss is selected from osteoporosis, metastasis-induced osteolytic bone loss, and rheumatoid arthritis-induced bone loss. In some embodiments, a method of treating cancer is provided which comprises administering to a patient a pharmaceutical composition comprising an antibody that binds to CSF1R. In some modalities, the cancer is

<td colspan="3">select from cancer</td><td>breast,</td><td>Cancer</td><td>from</td><td>prostate,</td><td>Cancer</td>

<td colspan="2">endometrial cancer</td><td>from</td><td>bladder,</td><td>Cancer</td><td>from</td><td colspan="2">kidney cancer</td>

<td>esophagus,</td><td>carcinoma</td><td>from</td><td>cells</td><td colspan="2">flaky,</td><td>melanoma</td><td>uveal,</td>

<td>lymphoma</td><td>follicular,</td><td colspan="2">carcinoma</td><td colspan="2">of cells</td><td>kidney,</td><td>Cancer</td>

<td>cervical,</td><td>Cancer</td><td>from</td><td>ovary,</td><td>Cancer</td><td>from</td><td>lung,</td><td>Cancer</td>

colorectal, brain cancer, pancreatic cancer, cancer of

<td>head and</td><td>neck,</td><td>Cancer</td><td>liver,</td><td>leukemia,</td><td>lymphoma,</td>

<td>disease</td><td>from</td><td>Hodgkin,</td><td>myelorna</td><td>multiple,</td><td>melanoma,</td>

astrocytoma, stomach cancer and lung adenocarcinoma.

In some embodiments, a method of treating an inflammatory condition is provided which comprises administering to a patient a pharmaceutical composition comprising an antibody that binds to CSF1R.

In some embodiments, CSF1R-binding antibodies and compositions comprising CSF1R-binding antibodies are provided for use in methods of treating humans or animals. In some embodiments, CSF1R-binding antibodies and compositions comprising CSF1R-binding antibodies are provided for use in a method of treating rheumatoid arthritis in a human or animal. In some embodiments, CSF1R-binding antibodies and compositions comprising CSF1R-binding antibodies are provided for use in a method of treating multiple sclerosis in a human or animal. In some embodiments, antibodies are provided that bind to

CSF1R and compositions comprising antibodies that bind to CSF1R for use in a method of treating cancer in a human or animal. In some embodiments, CSF1R-binding antibodies and compositions comprising CSF1R-binding antibodies are provided for use in a method of treating an inflammatory condition in a human or animal.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show an alignment of the humanized heavy chain variable regions for each of the humanized antibodies Abl to Abl6, as described in Example 4. The boxed residues are amino acids of the human acceptor sequence that were re-switched upon corresponding mouse residue.

FIGS. 2A-2C show an alignment of the humanized light chain variable regions for each of the humanized antibodies Abl to Ab 16, as described in Example 4. The boxed amino acids are residues of the human acceptor sequence that were changed back to the corresponding mouse residue.

FIGS. 3A-3C show binding curves for certain humanized antibodies that bind to the human CSF1R ECD, as described in Example 5. Figure 3A shows binding curves for parental chimeric antibodies (cAb) 0301 and humanized antibodies (huAb) 0301.1, 0301.2, 0302.3, 0301.4, 0301.5 and 0301.6 (h0301-L0H0, h030119

L0H1, hO301-L0H2, h0301-LlH0, h0301-LlHl and h0301-LlH2, respectively). Figure 3B shows binding curves for parental cAb 0302 and humanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5 and 0302.6 (h0302-L0Hl, h0302L1H1, h0302-L2Hl, h0302-LOH02-Ll2-Ll2-Ll2-Ll2 , respectively). Figure 3C shows binding curves for parental cAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3, and 0311.4 (h0311-L0Hl, hO311-LlHl, h0311-L0H2, and hO311-LlH2, respectively).

FIGS. 4A-4C show binding curves for certain humanized antibodies that bind to the macaque CSF1R ECD, as described in Example 5. Figure 4A shows binding curves for parental cAbs 0301 and humanized antibodies (huAb) 0301.1, 0301.2 , 0302.3, 0301.4, 0301.5 and 0301.6 (hO301-LOHO, h0301-L0Hl, hO3Ol-L0H2, h0301-LlH0, h0301-LlHl and h0301-LlH2, respectively). Figure 4B shows binding curves for parental cAb 0302 and humanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5 and 0302.6 (h0302-L0Hl, h0302-LlHl, h0302-L2Hl, h0302-L02-Ll2, h0302-Ll2 -L2H2, respectively). Figure 4C shows binding curves for parental cAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3, and 0311.4 (h0311-L0Hl, hO311-LlHl, h0311-L0H2, and hO311-LlH2, respectively).

FIGS. 5A-5C show junction curves for

certain humanized antibodies that bind to the mouse CSF1R ECD, as described in Example 5. Figure 5A shows binding curves for parental cAbs 0301 and humanized antibodies (huAb) 0301.1, 0301.2, 0302.3,

0301.4, 0301.5 and 0301.6 (h0301-L0H0, h0301-L0Hl, hO301-LOH2, h0301-LlH0, h0301-LlHl and h0301-LlH2, respectively). Figure 5B shows binding curves for parental cAb 0302 and humanized antibodies (huAb) 0302.1, 0302.2, 0302.3,

0302.4, 0302.5 and 0302.6 (hO3O2-L0Hl, h0302-LlHl, h0302-L2Hl, h0302-L0H2, h0302-LlH2 and h0302-L2H2, respectively). Figure 5C shows binding curves for parental cAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3 and

0311.4 (h0311-L0Hl, hO311-LlHl, h0311-L0H2 and h0311-LlH2, respectively).

FIGS. 6A-6C show the inhibition of CSF1R-induced phosphorylation by CSF1 by certain humanized antibodies, as described in Example

6. Figure 6A shows blocking curves for parental cAb 0301 and humanized antibodies (huAb) 0301.1, 0301.2, 0302.3, 0301.4, 0301.5, and 0301.6 (hO3Ol-L0H0, h0301-L0Hl, h0301-LOH2, h0301-LlH0, h030 -LlH2, respectively). Figure 6B shows blocking curves for parental cAb 0302 and humanized antibodies (huAb) 0302.1, 0302.2, 0302.3,

0302.4, 0302.5 and 0302.6 (h0302-L0Hl, h0302-LlHl, h0302-L2Hl, h0302-L0H2, h0302-LlH2 and h0302-L2H2, respectively). The

Figure 6C shows blocking curves for cAb 0311 páreríTalés * and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3 and 0311.4 (h0311-L0Hl, hO311-LlHl, h0311-L0H2 and h0311-LlH2, respectively).

FIGS. 7A-7C show inhibition of IL34-induced CSF1R phosphorylation by certain humanized antibodies, as described in Example 6. Figure 7A shows blocking curves for parental cAbs 0301 and humanized antibodies (huAb) 0301.1, 0301.2, 0302.3 ,

0301.4, 0301.5 and 0301.6 (hO301-L0H0, hO301-LOHl, h0301-L0H2, h0301-LlH0, h030l-LlHl and h030l-LlH2, respectively). Figure 7B shows blocking curves for parental cAb 0302 and humanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5, and 0302.6 (h0302-L0Hl, h0302-LlHl, h0302-L2Hl, h0302-L02-Ll2, h0302-Ll2, h0302-Ll2 -L2H2, respectively). Figure 7C shows blocking curves for parental cAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3 and 0311.4 (h0311-L0Hl, hO311-LlHl, h0311-L0H2 and h0311-LlH2, respectively).

FIGS. 8A-8C show the blocking of the binding of human CSF1 to the macaque CSF1R ECD by certain humanized antibodies, as described in Example 7. Figure 8A shows blocking curves for parental cAbs 0301 and humanized antibodies (huAb) 0301.1 , 0301.2, 0302.3, 0301.4, 0301.5 and 0301.6 (h0301-L0H0, h0301-L0Hl, h0301-L0H2,

*W,'™TO MEXiCAM> DE la r »opi;: cAi) industrial h0301-LlH0, h0301-LlHl and h0301-LlH2, re spect iváilfSíTie ^ -r— ^ réu Figure 8B shows blocking curves for parental cAb 0302 and humanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5 (0302.5 and 0302.4, 0302.5 ( hO302-LOHl, h0302-LlHl, hO3O2-L2Hl, h0302-L0H2, h0302-LlH2 and hO3O2-L2H2, respectively). Figure 8C shows blocking curves for parental cAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3 and 0311.4 (h0311-L0Hl, hO311-LlHl, h0311-L0H2 and h0311-LlH2, respectively).

FIGS. 9A-9C show blocking of human IL34 binding to macaque CSF1R ECD by certain humanized antibodies, as described in Example 7. Figure 9A shows blocking curves for parental 0301 cAbs and humanized antibodies (huAb) 0301.1 , 0301.2, 0302.3,

0301.4, 0301.5 and 0301.6 (hO301-LOHO, h0301-L0Hl, h0301-LOH2, hO301-LlHO, h0301-LlHl and h0301-LlH2, respectively). Figure 9B shows blocking curves for parental cAb 0302 and humanized antibodies (huAb) 0302.1, 0302.2, 0302.3,

0302.4, 0302.5 and 0302.6 (hO302-LOHl, h0302-LlHl, h0302-L2Hl, h0302-LOH2, h0302-LlH2 and h0302-L2H2, respectively). Figure 9C shows blocking curves for parental cAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3 and

0311.4 (h0311-L0Hl, h0311-LlHl, h0311-L0H2 and h0311-LlH2, respectively).

The

FIGS.

10A-10B show the blocking of the «>

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CSF1- (FIG. 10A) and IL34 (FIG. 10B) -induced phosphorylation of CSF1R in CHO cells expressing human CSF1R by humanized antibodies 0301-L0H0, 0301-L1H0 and 0311-L0H1, as described in Example 9.

FIGS. 11A-11B show the blocking of the survival / proliferation responses of monocytes induced by CSF1- (FIG. 11A) and IL34- (FIG. 11B) by humanized antibodies 0301-L0H0, 0301-L1H0 and 0311-LOH1, as shown described in Example 10.

FIGS. 12A-12C show that the humanized antibodies 0301-LOHO, 0301-L1HO and 0311-L0H1 do not stimulate the survival or proliferation of major monocytes, using monocytes from three different donors, as described in Example 11.

DETAILED DESCRIPTION OF THE INVENTION

Methods for treating diseases are provided which comprise administering novel antibodies to CSF1R. All of the antibodies have binding affinities for human CSF1R ECD of less than 2 nM, and all but two of the humanized antibodies have sub-nanomolar binding affinities for human CSF1R ECD. Furthermore, the new antibodies block the binding of CSF1 and IL34 to human CSF1R, and inhibit ligand-induced phosphorylation of human CSF1R. Several of the new antibodies also block the binding of CSF1 and IL34 to macaque CSF1R, making it easier for the

INSTITU Y MEXICANO OE LA PROPERTY

INL'L'Í Γ'ίΙΛΙ in vivo experiments favor the development of anti-CSFIR antibody therapeutics. The new antibodies are thus highly suitable for therapeutic use in human diseases, including, but not limited to, cancer, autonomic diseases, and inflammatory conditions.

The section titles used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

Definitions

Unless defined otherwise, scientific and technical terms used in connection with the present invention will have meanings commonly accepted by those skilled in the art. Also, unless the context requires otherwise, terms in the singular will include plurals and plurals will include the singular.

Examples of techniques used in connection with recombinant DNA techniques, oligonucleotide synthesis, transformation and cell culture (eg, electroporation, lipofection), enzymatic reactions, and purification are known in the art. Several of the techniques and procedures are described, eg, in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd edition, Coid Spring Harbor Laboratory Press, Coid Spring Harbor, NY (1989)), among others. In addition, examples of techniques of

Chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and administration and treatment of patients are also known in the art.

In the present application, the use of or means and / or unless otherwise specified. In the context of a claim that depends on many, the use of or refers to more than one previous dependent or independent claim only in the alternative. Also, terms such as element or component comprise both elements and components that comprise one unit and elements and components that comprise more than one subunit unless otherwise specified.

As used in accordance with the present description, the following terms, unless otherwise indicated, will be understood to have the following meanings:

The terms nucleic acid molecule and polynucleotide can be used interchangeably and refer to a polymer of nucleotides. Nucleotide polymers can contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA.

Nucleic acid sequence refers to the linear sequence of nucleotides that the nucleic acid or polynucleotide molecule comprises.

The terms polypeptide and protein are used interchangeably

IMPI ^

INSTITUTO MRXíCANO Dt LA PkOpifcDAL · INDIJSTklAl interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polymers of amino acid residues can contain natural or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are within the definition. The terms also include post-expression modifications of the polypeptide, eg, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of the present invention, a polypeptide refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, as long as the protein maintains the desired activity. These modifications can be premeditated, such as by site-directed mutagenesis, or they can be accidental, such as by host mutations that produce the proteins or errors due to PCR amplification.

The term "CSF1R" refers herein to the full-length CSF1R, which includes the N-Terminal ECD, the transmembrane domain, and the intracellular tyrosine kinase domain, with or without an N-Terminal leader sequence. In some embodiments, the CSF1R is a human CSF1R that has the? Ί

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amino acid sequence of SEQ ID NO: l or SEQ

The term CSF1R extracellular domain (ECD of

CSF1R) as used herein refers to a CSF1R polypeptide lacking transmembrane and intracellular domains. CSF1R ECDs include the full-length CSF1R ECD and CSF1R ECD fragments that are capable of binding to CSF1R and / or IL34. The human full-length CSF1R ECD defined herein comprises either amino acids 1 to 512 (ie, including the leader sequence) or amino acids 20 to 512 (ie, lacking the leader sequence) of SEQ ID NO: 2. In some embodiments, a human CSF1R ECD fragment comprises amino acids 20 to 506 of SEQ ID NO: 2 (see SEQ ID

NO: 5). In some embodiments, a human CSF1R fragment ends at amino acid 507, 508, 509, 510, or 511. In some embodiments, a cynoCSFIR ECD comprises the sequence of the

SEQ ID NO: 7 (with leader sequence) or amino acids 20 to 506 of SEQ ID NO: 7 (without leader sequence).

The term "antibody" as used herein refers to a molecule comprising at least one complementarity determining region (CDR) 1, CDR2 and CDR3 of a heavy chain and at least CDR1, CDR2 and CDR3 of a light chain, where the molecule is capable of binding to an antigen. The term antibody includes, but is not limited to, fragments that are capable of binding to an antigen, such as

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Fv, single chain Fv (scFv), Fab, Fab 'and (Fab') 2 · 'ET term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, macaque monkey, etc.

In some embodiments, an antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, an antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, an antibody comprises at least two heavy chains, where each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, where each light chain comprises a variable region light chain and at least a portion of a light chain constant region. As used herein, a single chain Fv (scFv), or any other antibody comprising, for example, a single chain of polypeptides comprising all six CDRs (three heavy chain CDRs and three CDRs from light chain) have a heavy chain and a light chain. In some

IÍJ.ÍTI MEXICAN VOTE Di LA Ai.OHWAD INDUSTRIAL

III h! Ml In embodiments, the heavy chain is the region of the antibody that comprises the three heavy chain CDRs and the light chain in the region of the antibody that comprises the three light chain CDRs.

The term "heavy chain variable region" as used herein refers to a region comprising heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a heavy chain variable region also comprises at least a portion of an FR1 and / or at least a portion of an FR4. In some embodiments, a heavy chain CDR1 corresponds to Kabat residues 26 to 35; a heavy chain CDR2 corresponds to Kabat residues 50 to 65; and a heavy chain CDR3 corresponds to Kabat residues 95-102. See, eg, Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.); and Figures 1A-1C. In some embodiments, a heavy chain CDR1 corresponds to Kabat residues 31 to 35; a light chain CDR2 corresponds to Kabat residues 50 to 65; and a heavy chain CDR3 corresponds to Kabat residues 95-102. See id.

The term "heavy chain constant region" as used herein refers to a region comprising at least three heavy chain constant domains, CH1 CHtwo and CH3. Non-exhaustive examples of heavy chain constant regions include γ, δ, and oí. Non-exhaustive examples of heavy chain constant regions also include ε and μ Each heavy constant region corresponds to an isotype of

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antibody. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising a constant region a is an IgA antibody. Furthermore, an antibody comprising a µ constant region is an IgM antibody, and an antibody comprising an ε constant region is an igE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 antibodies (comprising a constant region YJ, IgG2 (comprising a constant region γ2), IgG3 (comprising a constant region γ3), and IgG4 (comprising a constant region γ4); IgA antibodies include, but are not limited to, IgAl antibodies (comprising a constant region ax) and IgA2 (comprising a constant region a2)? and IgM antibodies include, but are not limited to, IgMl and IgM2.

In some embodiments, a heavy chain constant region comprises one or more mutations (or substitutions), additions, or deletions that confer a desired characteristic on the antibody. An example of a non-exhaustive mutation is the S241P mutation in the hinge region of IgG4 (between the constant domains CH1 and CH2), which alters the IgG4 CPSCP motif to CPPCP, which is similar to the corresponding motif in IgGl. This mutation, in some modalities, causes a

more stable IgG4 antibody. See eg. , Angal et al. ,

Mol. Immunol. 30: 105-108 (1993); Bloom et al., Prot. Sci. 6:

407-415 (1997); Schuurman et al. , Mol. Immunol. 38: 1-8 (2001).

The term "heavy chain" as used herein refers to a polypeptide comprising at least one heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy, chain constant region. The term "full length heavy chain" as used herein refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

The term "light chain variable region" as used herein refers to a region comprising

Light chain CDR1, framework (FR) 2, CDR2, FR3 and CDR3. In some embodiments, a light chain variable region also comprises an FRI and / or an FR4. In some embodiments, a light chain CDR1 corresponds to Kabat residues 24 to 34; a light chain CDR2 corresponds to Kabat residues 50 to 56; and a light chain CDR3 corresponds to Kabat residues 89 to 97. See, eg. , Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.); and Figures 1A-1C.

The term light chain constant region such as

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used herein refers to a region that comprises a light chain constant domain, CL. Non-exhaustive examples of light chain constant regions include λ and K.

The term "light chain" as used herein refers to a polypeptide comprising at least one light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full length light chain" as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

A chimeric antibody as used herein refers to an antibody that comprises at least one variable region from a first species (such as mouse, rat, macaque monkey, etc.) and at least one constant region from a second species ( such as human, macaque monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one macaque variable region and at least one human constant region. In some embodiments, all the variable regions of a chimeric antibody are of a first species and all the constant regions of the

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chimeric antibody are of a second species.

A humanized antibody as used herein refers to an antibody where at least one amino acid in a framework region of a non-human variable region was replaced with the corresponding amino acid of a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is Fab, scFv, (Fab ')2, etc.

A CDR-grafted antibody as used herein refers to a humanized antibody where the complementarity determining regions (CDRs) from a first species (non-human) were grafted into the framework regions (FR) from a second species (human). ).

A human antibody as used herein refers to antibodies produced in humans, antibodies produced in non-human animals comprising human immunoglobulin genes, such as XenoMouse®, and antibodies selected using in vitro methods, such as phage display, where the antibody repertoire is based on human immunoglobulin sequences.

The term "leader sequence" refers to a sequence of amino acid residues located at the N-Terminal of a polypeptide that facilitates the secretion of a polypeptide from a mammalian cell. A leader sequence can be excised

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upon export of the polypeptide from the mammalian cell, forming a mature protein. Leader sequences can be natural or synthetic, and can be heterologous or homologous to the protein to which they are attached. Examples of leader sequences include, but are not limited to, antibody leader sequences, such as, for example, the amino acid sequences of SEQ ID NO: 3 and 4, which correspond to human heavy and light chain leader sequences, respectively. . Non-exhaustive examples of leader sequences also include heterologous protein leader sequences. In some embodiments, an antibody lacks a leader sequence. In some embodiments, an antibody comprises at least one leader sequence, which can be selected from native antibody leader sequences and heterologous leader sequences.

The term "vector" is used to describe a polynucleotide that can be modified to contain a cloned polynucleotide or polynucleotides that can be propagated into a host cell. A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and / or enhancers) that regulate the expression of the polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistant genes and genes that can be used in

colorimetric assays, eg, β-galactosidase). The term "expression vector" refers to a vector that is used to express a polypeptide of interest in a host cell.

A "host cell" refers to a cell that may be or has been a recipient of an isolated vector or polynucleotide. The host cells can be prokaryotic cells or eukaryotic cells. Examples of eukaryotic cells include mammalian cells, such as cells from primate or non-primate animals; fungal cells, such as yeast; plant cells and insect cells. Non-exhaustive examples of mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively.

The term "isolated" as used herein refers to a molecule that has been separated from at least some of the components generally found in nature. For example, a polypeptide is called "isolated" when it is separated from at least some of the components of the cell where it was produced. When a cell secretes a polypeptide upon expression, physically separating the supernatant containing the polypeptide from the cell it produced, the polypeptide is considered to be isolating. Similarly, a polynucleotide is called isolated when it is not part of the larger polynucleotide (such as,

INSTITUTO MEXICANO D £ LA PROPERTY INDUSTRIAL for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) where it is generally found in nature, or is separated from at least some of the components of the cell where it was produced, eg. , in the case of an RNA polynucleotide. Therefore, a DNA polynucleotide that is found on a vector within a host cell can be termed "isolated" as long as that polynucleotide is not found on that vector in nature.

The terms subject and patient are used interchangeably herein to refer to a human. In some embodiments, methods are also provided for treating other mammals, including, but not limited to, rodents, apes, felines, canines, equines, bovines, swine, sheep, goats, laboratory mammals, farm mammals, animals. sport mammals and mammalian pets.

The term rheumatoid arthritis (RA) refers to a chronic autoimmune disease characterized primarily by inflammation of the lining (synovium) of the joints, which can lead to joint damage, resulting in chronic pain, loss of function and disability. Because RA can affect multiple organs in the body, including the skin, lungs, and eyes, it is called a systemic disease.

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Say LA FROi'IETAD t ~ ·· '. ·' RtS 'v· Woustuía !. '®3ί £? ΪΓ. you'. The term multiple sclerosis (MS) refers to the chronic autoimmune demyelinating disease of the CNS where the body generates antibodies and white blood cells against the cells that produce the myelin sheath. Demyelination occurs when the myelin sheath becomes inflamed, damaged, and detaches from the nerve fiber.

The term cancer refers to a proliferative disorder associated with uncontrolled cell proliferation, excessive cell growth, and less cell apoptosis. Cancer includes, but is not limited to, breast cancer, prostate cancer, lung cancer, kidney cancer, thyroid cancer, esophageal cancer, melanoma, follicular lymphomas, uveal melanoma, brain cancer, head and neck cancer , lung adenocarcinoma, including, but not limited to, colon cancer, heart tumors, pancreatic cancer, retinoblastoma, glioblastoma, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, Kaposi's sarcoma, ovarian cancer, leukemia (including acute leukemias (eg, acute lymphocytic leukemia, acute myelocytic leukemia, including myeloblastic, promyelocytic, myelomonocytic, monocytic), and erythroleukemia) chronic leukemias (for example, leukemia

IMPIAS

MEXICAN INSTITUTE OF THE FKOWEPAD

INDUSTRIAL chronic myeloid (granulocytic) and chronic lymphocytic leukemia), polycythemia vera myelodysplastic syndrome, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain diseases, and solid tumors including, of Non-exhaustive mode, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary carcinoma, adenocarcinoma, papillary carcinoma, papillary carcinoma, papillary carcinoma, adenocarcinoma bronchogenic, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, endometrial cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, and menangioma. The terms cancer metastasis and metastasis are used interchangeably herein to refer to the ability of a cancer cell to spread to other tissues. For example, metastasis in the ^ ίύ

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<td>bone</td><td>It refers to the</td><td>ability</td><td>from</td><td colspan="2">determined</td><td>types</td><td>from</td><td></td>

<td>Cancer</td><td>which include, of</td><td>mode no</td><td colspan="2">restrictive,</td><td>Mommy,</td><td colspan="2">prostate,</td><td></td>

<td>lung,</td><td>kidney, thyroid and</td><td>melanoma,</td><td>from</td><td>make</td><td colspan="2">metastasis in</td><td>the</td><td></td>

The term osteolytic disorders is used herein to refer to any condition caused by an increase in the activity of osteoclasts, which are cells responsible for bone resorption. The terms osteolysis and osteolytic bone loss can be used interchangeably to refer to osteoclast-mediated bone resorption or bone loss associated with an osteolytic disorder. Osteolytic disorders can occur in subjects with a predisposition to develop an osteolytic disorder, or they can occur in subjects with a disease that leads to or contributes to an osteolytic disorder by stimulating osteoclast activity. In examples of embodiments of the present invention, the osteolytic disorder can include osteolytic bone loss and osteolytic bone loss induced by cancer metastasis. In examples of further embodiments of the present invention, osteolytic bone disorder includes metabolic bone disease, including endocrinopathies, such as hypercortisolism, hypogonadism, primary or secondary hyperparathyroidism, and hyperthyroidism; dietary deficiency, including rickets, osteomalacia, scurvy, and y μ ρ τ

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malnutrition; osteoporosis; use of drugs, including glucocorticoids (glucocorticoid-induced osteoporosis), heparin, and alcohol; chronic disease, including malabsorption syndromes; chronic kidney failure, including renal osteodystrophy; chronic liver disease, including hepatic osteodystrophy; inherited disease, including osteogenesis imperfecta and homocystinuria; and bone inflammation associated with arthritis, rheumatoid arthritis, psoriatic arthritis, fibrous dysplasia, periodontal disease, and Paget's disease.

The terms "metastasis-induced osteolytic bone loss" and cancer metastasis-induced osteolytic bone loss are used interchangeably herein to refer to osteolysis or osteolytic bone loss caused by cancer cell metastases to bone. . The term "cancer metastasis-induced osteoclast activation" is used herein to refer to the ability of cancer cells that metastasized to bone to induce osteoclast activation.

The term tumor is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. A tumor can be benign, pre-malignant, or malignant; malignant tumor cells are cancerous. Tumor cells can be cells

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IN solid tumor or leukemi tumor cells? S ^ TTTíT '^ íTitr ± rfCT ·' tumor growth is used herein to refer to the proliferation or growth of a cell or cells comprising a tumor causing a corresponding increase in tumor size. The term "CSFIR-dependent tumor growth" is used herein to refer to the requirement on the part of a tumor cell or cells for CSFIR-mediated function (s) in order for the tumor cell (s) to proliferate or grow.

Treatment, as used herein, covers any administration or application of a therapeutic agent for a disease in a mammal, including a human, and includes inhibiting the disease or disease progression, inhibiting or slowing the disease or its progression. , slow its development, partially or completely alleviating the disease, or curing the disease, for example, causing regression or restoring or repairing a lost, missing or defective function; or by stimulating an ineffective process.

The terms inhibition or inhibit refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation of the incidence, degree or probability of that characteristic.

A pharmaceutically acceptable carrier refers to a non-toxic solid, semi-solid or liquid filler, diluent,

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INSTITUTE MEBCajA DI LA PROPERTY INDUSTRIAL

encapsulating material, formulation aid or carrier conventional in the art for use with a therapeutic agent which together comprise a pharmaceutical composition for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the doses and concentrations employed and is compatible with other ingredients in the formulation. The pharmaceutically acceptable carrier is suitable for the formulation used. For example, if the therapeutic agent is to be administered orally, the carrier can be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally does not irritate the skin and does not cause a reaction at the injection site.

Anti-CSFIR antibodies

The present inventors invented a new set of antibodies directed against CSF1R. Anti-CSFIR antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and / or light chain CDRs described herein.

Examples of humanized antibodies

In some embodiments, humanized antibodies are provided that bind to CSF1R. Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or eliminate the response.

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INSTITUTO MEXICANO DS LA PROPERTY INDUSTRIAL

immune response to non-human antibodies (such as the human anti-mouse antibody (HAMA) response), which can elicit an immune response to an antibody therapeutic and decrease the effectiveness of the therapeutic agent.

Non-exhaustive examples of humanized antibodies include Abl through Abl6, described herein. Non-exhaustive examples of humanized antibodies also include antibodies that comprise a heavy chain variable region of an antibody that is selected from Abl to Abl6 and / or a light chain variable region of an antibody that is selected from Abl to Abl6. Non-exhaustive examples of humanized antibodies include antibodies comprising a heavy chain variable region that is selected from SEQ ID NOs: 39 to 45 and / or a light chain variable region that is selected from SEQ ID NOs: 46 to 52 . Examples of humanized antibodies also include, but are not limited to, humanized antibodies comprising CDR1, CDR2 and CDR3 heavy chain and / or CDR1, CDR2 and CDR3 light chain of an antibody that is selected from 0301, 0302 and 0311.

In some embodiments, a humanized anti-CSFIR antibody comprises heavy chain CDR1, CDR2 and CDR3 and / or light chain CDR1, CDR2 and CDR3 of an antibody that is selected from 0301, 0302 and 0311. Non-limiting examples of anti -Humanized CSFIR include antibodies that

They comprise sets of chain CDR1, CDR2 and CDR3, 'pe'sadar which are selected from: SEQ ID NO: 15, 16 and 17; SEQ ID NOs: 21, 22 and 23; SEQ ID NOs: 27, 28 and 29. Non-exhaustive examples of humanized anti-CSFIR antibodies also include antibodies comprising sets of light chain CDR1, CDR2 and CDR3 that are selected from: SEQ ID NO: 18, 19 and twenty; SEQ ID NOs: 24, 25 and 26; SEQ ID NOs: 30, and 32.

Non-exhaustive examples of humanized anti-CSFIR antibodies include antibodies comprising the sets of CDR1, CDR2 and CDR3 heavy chain and CDR1, CDR2 and CDR3 light chain of Table 1 (SEQ ID NOs shown; see Table 8 for the sequences ). Each row in Table 1 shows the CDR1, CDR2 and CDR3 heavy chain and CDR1, CDR2 and CDR3 light chain of an example antibody.

Table 1: Heavy chain and light chain CDRs

<td colspan="3">Heavy chain</td><td colspan="3">Light chain</td>

IMPI

Additional examples of humanized antibodies

In some embodiments, a humanized anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence that is selected from SEQ ID NOs: 9, 11, 13 and 39 to 45, and where the antibody is binds CSF1R. In some embodiments, a humanized anti-CSFIR antibody comprises a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence that is selected from SEQ ID NOs: 10, 12, 14 and 46 to 52, where the antibody binds to CSF1R. In some embodiments, a humanized anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least

99% identical to the sequence selected from SEQ ID NOs: 9, 11, 13 and 39 to 45; and a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence that is selected from SEQ ID NOs: 10, 12, 14 and 46 to 52; where he

antibody binds to CSF1R. ~

As used herein, whether a particular polypeptide is, for example, at least 95% identical to an amino acid sequence, it can be determined using, eg, a computer program. In determining whether a particular sequence is, for example, 95% identical to a reference sequence, percent identity is calculated over the full length of the reference amino acid sequence.

In some embodiments, a humanized anti-CSFIR antibody comprises at least one of the CDRs described herein. That is, in some embodiments, a humanized anti-CSFIR antibody comprises at least one CDR that is selected from a heavy chain CDR1 described herein, a heavy chain CDR2 described herein, a heavy chain CDR3 described herein. present, a light chain CDR1 described herein, a light chain CDR2 described herein, and a light chain CDR3 described herein. Furthermore, in some embodiments, a humanized anti-CSFIR antibody comprises at least one CDR mutated based on a CDR described herein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acid substitutions relative to the CDR described in the present. In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions.

MEXICAN INSTITUTE. . )

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INDUSTRY!

One of skill in the art can select one or more suitable conservative amino acid substitutions for a particular CDR sequence, where suitable conservative amino acid substitutions are not expected to significantly alter the binding properties of the antibody comprising the mutated CDR.

Examples of humanized anti-CSFIR antibodies also include antibodies that compete to bind CSF1R with an antibody described herein. Thus, in some embodiments, a humanized anti-CSFIR antibody that competes for binding to CSF1R is provided with an antibody that is selected from Fabs 0301, 0302, and 0311; and bivalent antibody versions (ie, with two heavy chains and two light chains) of these Fabs.

Examples of Humanized Antibody Constant Regions

In some embodiments, a humanized antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype that is selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype that is selected from κ and λ. In some embodiments, a humanized antibody described herein comprises a constant region of human IgG. In some embodiments, a humanized antibody described herein comprises a τ

INSTITUTO MEXICANO D3 LA ΓΚΟ ', ΤΕΪ'ΛΟ

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human IgG4 heavy chain constant region In some embodiments, a humanized antibody described herein comprises an S241P mutation in the human IgG4 constant region. In some embodiments, a humanized antibody described herein comprises a human IgG4 constant region and a human κ light chain.

The choice of heavy chain constant region can determine whether an antibody will have effector function in vivo or not. Effector function, in some embodiments, includes antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), and can lead to destruction of cells to which the antibody is bound . In some treatment methods, including methods for treating some cancers, killing of cells may be desired, for example, when the antibody binds to a cell that promotes tumor maintenance or growth. Examples of cells that can promote the maintenance or growth of a tumor include, but are not limited to, tumor cells themselves, cells that aid in the recruitment of vasculature to the tumor, and cells that provide ligands, growth factors or counterreceptors that favor or promote tumor growth or tumor survival. In some embodiments, when effector function is desired, an anti49 antibody is selected.

CSF1R comprising a human IgGl heavy chain - ^^ a * ·· human IgG3 heavy chain.

In some treatment methods, the effector function may not be desired. For example, in some embodiments, it may be desired that the antibodies used in the treatment of MS and / or RA and / or osteolysis have no effector function. Therefore, in some embodiments, anti-CSFIR antibodies developed for the treatment of cancer may not be suitable for use in the treatment of MS and / or RA and / or osteolysis. Accordingly, in some embodiments, an anti-CSFIR antibody lacking significant effector function is used in the treatment of MS and / or RA and / or osteolysis. In some embodiments, an anti-CSFIR antibody for the treatment of MS and / or RA and / or osteolysis comprises a human IgG4 or IgG2 heavy chain constant region. In some embodiments, the IgG4 constant region comprises an S241P mutation.

An antibody can be humanized by any method. Examples of non-exhaustive methods of humanization include methods described in, eg, US Pat.

Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370;

Jones et al. , Nature 321: 522-525 (1986); Riechmann et al. , Nature 332: 323-27 (1988); Verhoeyen et al., Science 239: 1534-36 (1988); and US Publication No. US 2009/0136500.

IMPI

As noted above, a humanized antibody is an antibody where at least one amino acid in a framework region of a non-human variable region was replaced with the amino acid in the corresponding locus of a human variable region. In some modalities, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 15 or at least 20 amino acids in the framework regions of a non-human variable region are replaced by an amino acid from one or more of the corresponding locations in one or more human framework regions.

In some embodiments, some of the corresponding human amino acids used for substitution are from the framework regions of different human immunoglobulin genes. That is, in some of the embodiments, one or more non-human amino acids can be replaced with corresponding amino acids from a human framework region of a first human antibody or encoded by a first human immunoglobulin gene, or one or more non-human amino acids can be replaced. by the corresponding amino acids of a human framework region of a second human antibody or encoded by a second immunoglobulin gene, or one or more non-human amino acids can be replaced by corresponding amino acids from a human framework region of a third human antibody or encoded by a third gene from

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human immunoglobulin, etc. Furthermore, in some embodiments, it is not necessary that all of the corresponding human amino acids being used for substitution in a single framework region, eg, FR2, are from the same human framework. In some embodiments, however, all of the corresponding human amino acids that are being used for substitution are from the same human antibody or are encoded by the same human immunoglobulin gene.

In some embodiments, an antibody is humanized by replacing one or more entire framework regions with corresponding human framework regions. In some embodiments, a human framework region is selected that has the highest level of homology to the non-human framework that is being replaced. In some embodiments, the humanized antibody is a CDR-grafted antibody.

In some embodiments, after CDR grafting, one or more framework amino acids change back to the corresponding amino acid in a mouse framework region. Reverse mutations are made, in some embodiments, to retain one or more mouse framework amino acids that appear to contribute to the structure of one or more of the CDRs and / or that may be involved in antigen contacts and / or appear to be involved in the integrity of the overall structure of the antibody. In some modalities, ten or less, nine or less, eight or less, seven or less, six or less are performed,

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five or less, four or less, three or less, two or less, one or zero reverse mutations to the framework regions of an antibody after CDR grafting.

In some embodiments, a humanized antibody also comprises a human heavy chain constant region and a human light chain constant region.

Examples of chimeric antibodies

In some embodiments, an anti-CSFIR antibody is a chimeric antibody. In some embodiments, an anti-CSFIR antibody comprises at least one non-human variable region and at least one human constant region. In some of the embodiments, all of the variable regions of an anti-CSFIR antibody are non-human variable regions, and all of the

<td>constant regions</td><td>from</td><td>a</td><td>antibody</td><td>anti-CSFIR</td><td>They are</td><td>regions</td>

<td>human constants</td><td colspan="2">On</td><td colspan="2">some modalities,</td><td colspan="2">one or more</td>

<td>variable regions</td><td>from</td><td>a</td><td>antibody</td><td>chimerical</td><td>They are</td><td>regions</td>

<td>mouse variables.</td><td>Not</td><td>it is</td><td>necessary</td><td colspan="2">that the region</td><td>constant</td>

chimeric antibody is of the same isotype as the non-human constant region, if any, that it replaces. Chimeric antibodies are described, eg. , in US Patent No. 4,816,567; and Morrison et al.

Proc. Nati. Acad. Sci. USA 81: 6851-55 (1984).

Non-limiting examples of chimeric antibodies include chimeric antibodies that comprise light and / or heavy chain variable regions of an antibody that are

IMPI

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INSTITUTO MEXICANO DE UA INDUSTRIAL PROPERTY

selects from 0301, 0302 and 0311. Additional non-limiting examples of chimeric antibodies include chimeric antibodies comprising CDR1, CDR2 and CDR3 heavy chain and / or CDR1, CDR2 and CDR3 light chain of an antibody that is selected from 0301, 0302 and 0311.

Non-exhaustive examples of chimeric anti-CSFIR antibodies include antibodies comprising the following pairs of heavy and light chain variable regions: SEQ ID NO: 9 and 10; SEQ ID NOs: 11 and 12; SEQ ID NOs:

and 14.

Non-exhaustive examples of anti-CSFIR antibodies include antibodies comprising a set of CDR1, CDR2 and CDR3 heavy chain and CDR1, CDR2 and CDR3 light chain shown in Table 1.

Additional examples of chimeric antibodies

In some embodiments, a chimeric anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least

91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence that is selected from SEQ ID NOs: 9, 11, 13 and 39 to 45, where the antibody binds to CSF1R. In some embodiments, a chimeric anti-CSFIR antibody comprises a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least

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92%, at least 93%, at least 94%, at least 95%, to TtiéTOB at least 97%, at least 98% or at least 99% identical to the sequence that is selected from SEQ ID NOs: 10, 12 , 14 and 52, where the antibody binds to CSF1R. In some embodiments, a chimeric anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least

93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence that is selected from SEQ ID NOs: 9, 11, 13 and 39 to 45; and a light chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least

93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence that is selected from SEQ ID NOs: 10, 12, 14 and 46 to 52; where the antibody binds CSF1R.

In some embodiments, a chimeric anti-CSFIR antibody comprises at least one of the CDRs described herein. That is, in some embodiments, a chimeric anti-CSFIR antibody comprises at least one CDR that is selected from a heavy chain CDR1 described herein, a heavy chain CDR2 described herein, a heavy chain CDR3 described herein. present, a light chain CDR1 described herein, a light chain CDR2 described herein, and a light chain CDR3

described herein. Furthermore, in some embodiments, a chimeric anti-CSFIR antibody comprises at least one CDR mutated based on a CDR described herein, where

<td>the mutated CDR comprises</td><td> 1,</td><td> 2,</td><td> 3</td><td>or 4</td><td>substitutions</td><td>from</td>

<td>amino acids relative to</td><td>the</td><td>CDR</td><td colspan="2">described</td><td>at the moment.</td><td>On</td>

<td>some modalities, one</td><td>or</td><td>more</td><td>from</td><td>the</td><td>substitutions</td><td>from</td>

amino acids are conservative amino acid substitutions.

Examples of chimeric anti-CSFIR antibodies also include chimeric antibodies that compete to bind CSF1R with an antibody described herein. Thus, in some embodiments, a chimeric anti-CSFIR antibody that competes for binding to CSF1R is provided with an antibody that is selected from Fabs 0301, 0302, and 0311; and bivalent antibody versions (ie, with two heavy chains and two light chains) of these Fabs. Examples of Chimeric Antibody Constant Regions

In some embodiments, a chimeric antibody described herein comprises one or more human constant regions. In some modalities, the region

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INSTITUTO MEXICANO iC'-Síw.'TÍ *: Λ Dt THE PROPERTY

INDUSTRIAL human heavy chain constant is of an isotype that is selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype that is selected from κ and λ. In some embodiments, a chimeric antibody described herein comprises a constant region of human IgG. In some embodiments, a chimeric antibody described herein comprises a human IgG4 heavy chain constant region. In some embodiments, a chimeric antibody described herein comprises an S241P mutation in the constant region of human IgG4. In some embodiments, a chimeric antibody described herein comprises a human IgG4 constant region and a human κ light chain.

As noted above, whether or not an effector function is desired may depend on the particular method or treatment intended for an antibody. Thus, in some embodiments, when effector function is desired, a chimeric anti-CSFIR antibody is selected that comprises a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, when effector function is not desired, a chimeric anti-CSFIR antibody is selected that comprises a human IgG2 or IgG4 heavy chain constant region.

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Examples of human antibodies

Human antibodies can be made by any suitable method. Non-exhaustive examples of methods include making human antibodies in transgenic mice that comprise human immunoglobulin sites. See eg. , Jakobovits et al., Proc. Nati. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al., Nature 362: 255-8 (1993); Lonberg et al., Nature 368: 856-9 (1994); and US Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129;

6,255,458; 5,877,397; 5,874,299 and 5,545,806.

Non-exhaustive examples of methods also include making human antibodies using phage display libraries. See eg. , Hoogenboom et al. , J. Mol. Biol. 227: 381: -8 (1992); Marks et al. , J. Mol. Biol. 222: 581-97 (1991); and PCT Publication No. US 99/10494.

In some embodiments, a human anti-CSFIR antibody binds to a polypeptide having the sequence of SEQ ID NO: 1. Examples of human anti-CSFIR antibodies also include antibodies that compete for binding to CSF1R with an antibody described in Present. Thus, in some embodiments, an anti-human CSFIR antibody that competes for binding to CSF1R is provided with an antibody that is selected from Fabs 0301, 0302, and 0311; and bivalent antibody versions (ie, with two heavy chains and two light chains) of these Fabs.

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In some embodiments, an antibody comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype that is selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype that is selected from κ and λ. In some embodiments, a human antibody described herein comprises a constant region of human IgG. In some embodiments, a human antibody described herein comprises a human IgG4 heavy chain constant region. In some embodiments, a human antibody described herein comprises an S241P mutation in the constant region of human IgG4. In some embodiments, a human antibody described herein comprises a human IgG4 constant region and a human κ light chain.

In some embodiments, when effector function is desired, an anti-human CSFIR antibody is selected that comprises a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, when effector function is not desired, an anti-human CSFIR antibody is selected that comprises a human IgG2 or IgG4 heavy chain constant region.

Additional examples of anti-CSFIR antibodies

Examples of anti-CSFIR antibodies also include, but are not limited to, mouse antibodies, fV Ρ Τ '* τ · 1 l'ZÜL Ji jj. ÍyNÁ '

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they comprise, for example, one or more CDR sequences described herein. In some embodiments, an anti-CSFIR antibody comprises a heavy chain variable region described herein. In some embodiments, an anti-CSFIR antibody comprises a light chain variable region described herein. In some embodiments, an anti-CSFIR antibody comprises a heavy chain variable region described herein and a light chain variable region described herein. In some embodiments, an anti-CSFIR antibody comprises heavy chain CDR1, CDR2, and CDR3 described herein. In some embodiments, an anti-CSFIR antibody comprises CDR1, CDR2, and

Light chain CDR3s described herein. In some embodiments, an anti-CSFIR antibody comprises heavy chain CDR1, CDR2 and CDR3 described herein and CDR1, CDR2 and

Light chain CDR3 described herein.

In some embodiments, an anti-CSFIR antibody comprises a heavy chain variable region of an antibody that is selected from Fabs 0301, 0302, and 0311. Non-limiting examples of anti-CSFIR antibodies also include antibodies that comprise a heavy chain variable region of an antibody that is selected from humanized Abl to Abl6 antibodies. Non-exhaustive examples of anti-CSFIR antibodies include antibodies that comprise a heavy chain variable region that sequence that is selected from SEQ ID NOs:

to 45.

9, 11, 13 and 39

In some embodiments, an anti-CSFIR antibody comprises a light chain variable region of an antibody that is selected from Fabs 0301, 0302, and 311. Non-limiting examples of anti-CSFIR antibodies also include antibodies that comprise a light chain variable region. of an antibody that is selected from humanized Abl to Abl6 antibodies. Non-exhaustive examples of anti-CSFIR antibodies include antibodies that comprise a light chain variable region comprising a sequence that is selected from SEQ ID NOs: 10, 12, 14 and 52.

In some embodiments, an anti-CSFIR antibody comprises a heavy chain variable region and a light chain variable region of an antibody that is selected from Fabs 0301, 0302, and 0311. Non-limiting examples of anti-CSFIR antibodies also include antibodies that They comprise a heavy chain variable region and light chain variable region of an antibody that is selected from humanized antibodies Abl to Abl6. Non-exhaustive examples of anti-CSFIR antibodies include antibodies comprising the following pairs of heavy and light chain variable regions: SEQ ID NO: 9 and 10; SEQ ID • wasji, - * '"

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NOS: 11 and 12; SEQ ID NOs: 13 and 14; SEQ ID NOs: 39 and 40; SEQ ID NOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ ID NOS: 47 and 48; SEQ ID NOs: 49 and 50; SEQ ID NOs: 51 and 52. Non-limiting examples of anti-CSFIR antibodies also include antibodies comprising the following pairs of heavy and light chains: SEQ ID NO: 33 and 34; SEQ ID NOs: 35 and 36; SEQ ID

NOs: 37 and 38.

In some embodiments, an anti-CSFIR antibody comprises heavy chain CDR1, CDR2, and CDR3 of an antibody that is selected from Fabs 0301, 0302, and 0311. Non-limiting examples of anti-CSFIR antibodies include antibodies that comprise sets of CDR1, CDR2 and heavy chain CDR3 which are selected from: SEQ ID NO: 15, 16 and 17; SEQ ID NOs: 21, 22 and 23; SEQ ID NOs: 27, 28 and 29.

In some embodiments, an anti-CSFIR antibody comprises light chain CDR1, CDR2, and CDR3 of an antibody that is selected from Fabs 0301, 0302, and 0311. Non-limiting examples of anti-CSFIR antibodies include antibodies that comprise sets of CDR1, CDR2 and light chain CDR3 which are selected from: SEQ ID NO: 18, 19 and 20; SEQ ID

NOs: 24, 25 and 26; SEQ ID NOs: 30, 31 and 32.

In some embodiments, an anti-CSFIR antibody comprises CDR1, CDR2, and CDR3 heavy chain and CDR1, CDR2, and CDR3 light chain of an antibody that is selected from

Fabs 0301, 0302 and 0311. --------.

Non-exhaustive examples of anti-CSFIR antibodies include antibodies comprising the sets of CDR1, CDR2 and CDR3 heavy chain and CDR1, CDR2 and CDR3 light chain shown in Table 1.

Additional examples of antibodies

In some embodiments, an anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 %, at least 96%,

<td>at least 97%,</td><td colspan="2">, at least 98% or at</td><td colspan="3">less 99% identical</td><td>The nail</td>

<td>sequence that</td><td>I know</td><td>select from the</td><td>I KNOW THAT</td><td>ID NOs: 9,</td><td> 11,</td><td>13 and 39</td>

<td>to 45, where</td><td>the</td><td>antibody is</td><td>joins</td><td>to CSF1R.</td><td>On</td><td>some</td>

<td>modalities,</td><td>a</td><td colspan="2">anti-CSFIR antibody</td><td>understands</td><td>a</td><td>chain</td>

light comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

98% or at least 99% identical to the sequence that is selected from SEQ ID NOs: 10, 12, 14 and 46 to 52, where the antibody binds to CSF1R. In some embodiments, an anti-CSFIR antibody comprises a heavy chain comprising a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

96%, at least 97%, at least 98% or at least 99% identical to the sequence that is selected from SEQ ID NOs: 9, 11, 13 and 39 tW. ΡI

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variable region that is at least 90%, at least 91%, at least

92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence that is selected from SEQ ID NOs: 10, 12, 14 and 46 to 52; where the antibody binds CSF1R.

In some embodiments, an anti-CSFIR antibody comprises at least one of the CDRs described herein.

That is, in some embodiments, an anti-CSFIR antibody comprises at least one CDR that is selected from a heavy chain CDR1 described herein, a heavy chain CDR2 described herein, a heavy chain CDR3 described herein. , a light chain CDR1 described herein, a light chain CDR2 described herein, and a light chain CDR3 described herein. Furthermore, in some embodiments, an anti-CSFIR antibody comprises at least one CDR mutated based on a CDR described herein, wherein the mutated CDR comprises 1, 2, or 4 amino acid substitutions relative to the CDR described herein. . In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions. One skilled in the art can select one or more conservative amino acid substitutions suitable for a particular CDR sequence, where the amino acid substitutions are not anticipated. "

Suitable preservatives significantly alter the binding properties of the antibody comprising the mutated CDR.

Examples of anti-CSFIR antibodies also include antibodies that compete to bind CSF1R with an antibody described herein. Thus, in some embodiments, an anti-CSFIR antibody that competes for binding to CSF1R is provided with an antibody that is selected from Fabs 0301, 0302, and 0311; and bivalent antibody versions (ie, with two heavy chains and two light chains) of these Fabs.

Examples of antibody constant regions

In some embodiments, an antibody described herein comprises one or more human constant regions. In some embodiments, the human heavy chain constant region is of an isotype that is selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype that is selected from κ and λ. In some embodiments, an antibody described herein comprises a human IgG constant region. In some embodiments, an antibody described herein comprises a human IgG4 heavy chain constant region. In some of the embodiments, an antibody described herein comprises an S241P mutation in the constant region of human IgG4. In some embodiments, an antibody

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Human IgG4 and a human κ light chain.

As noted above, whether or not an effector function is desired may depend on the particular method of treatment intended for an antibody. Therefore, in some embodiments, when effector function is desired, an anti-CSFIR antibody is selected that comprises a human IgGl heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, when effector function is not desired, an anti-CSFIR antibody comprising a human IgG2 or IgG4 heavy chain constant region is selected.

Examples of anti-CSFIR heavy chain variable regions

In some embodiments, anti-CSFIR antibody heavy chain variable regions are provided. In some embodiments, an anti-CSFIR antibody heavy chain variable region is a mouse variable region, a human variable region, or a humanized variable region.

An anti-CSFIR antibody heavy chain variable region comprises heavy chain CDR1, FR2, CDR2, FR3 and CDR3. In some embodiments, an anti-CSFIR antibody heavy chain variable region further comprises heavy chain FR1 and / or FR4. Non-exhaustive examples of heavy chain variable regions include, but are not limited to, heavy chain variable regions that have a

amino acid sequence that is selected from · -6EQ — íe — NOí—— 9, 11, 13 and 39 to 45.

In some embodiments, an anti-CSFIR antibody heavy chain variable region comprises a CDR1 comprising a sequence that is selected from SEQ ID NOs:

15, 21 and 27.

In some embodiments, an anti-CSFIR antibody heavy chain variable region comprises a CDR2 comprising a sequence that is selected from SEQ ID NOs:

16, 22 and 28.

In some embodiments, an anti-CSFIR antibody heavy chain variable region comprises a CDR3 comprising a sequence that is selected from SEQ ID NOs:

17, 23 and 29.

Non-exhaustive examples of heavy chain variable regions include, but are not limited to, heavy chain variable regions comprising sets of CDR1, CDR2 and CDR3 which are selected from: SEQ ID NO: 15, 16 and 17; SEQ ID NOs: 21, 22 and 23; SEQ ID NOs: 27, 28 and 29.

In some embodiments, an anti-CSFIR antibody heavy chain comprises a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to a sequence that is selected from SEQ ID NOs: 9, 11, 13 and 3 9 to 45, where the chain

Heavy together with a light chain is capable of forming an antibody that binds to CSF1R.

In some embodiments, an anti-CSFIR antibody heavy chain comprises at least one of the CDRs described herein. That is, in some embodiments, an anti-CSFIR antibody heavy chain comprises at least one CDR that is

<td>select from</td><td>a</td><td>CDR1</td><td>from</td><td>chain</td><td>heavy</td><td>described</td>

<td>previously,</td><td>a</td><td>CDR2</td><td>from</td><td>chain</td><td>heavy</td><td>described</td>

<td>previously</td><td>and one</td><td colspan="2">CDR3 of</td><td>chain</td><td>heavy</td><td>described</td>

<td>previously.</td><td>What's more,</td><td>on</td><td>some</td><td colspan="3">modalities, a chain</td>

Anti-CSFIR antibody weight comprises at least one CDR mutated based on a CDR described herein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acid substitutions relative to the CDR described herein. In some embodiments, one or more of the amino acid substitutions are conservative amino acid substitutions.

In some embodiments, a heavy chain comprises a heavy chain constant region. In some embodiments, a heavy chain comprises a chain constant region

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MEXICAN INSTITUTE OF INDUSTRY PROPERTY !.

heavy human. In some embodiments, the human heavy chain 'ConsEanfce region is of an isotype that is selected from IgA, IgG, and IgD. In some embodiments, the human heavy chain constant region is an IgG constant region. In some embodiments, a heavy chain comprises a human IgG4 heavy chain constant region. In some embodiments, the human IgG4 heavy chain constant region comprises an S241P mutation.

In some embodiments, when effector function is desired, a heavy chain comprises a human IgG1 or IgG3 heavy chain constant region. In some embodiments, where effector function is less desirable, a heavy chain comprises a human IgG4 or IgG2 heavy chain constant region.

Examples of anti-CSFIR light chain variable regions

In some embodiments, anti-CSFIR antibody light chain variable regions are provided. In some embodiments, an anti-CSFIR antibody light chain variable region is a mouse variable region, a human variable region, or a humanized variable region.

An anti-CSFIR antibody light chain variable region comprises light chain CDR1, FR2, CDR2, FR3 and CDR3. In some embodiments, an anti-CSFIR antibody light chain variable region further comprises light chain FR1 and / or FR4. Non-exhaustive examples of

Light chain variable regions include, light chain variable regions having an amino acid sequence that is selected from SEQ ID NO: 10, 12, 14 and 52.

In some embodiments, an anti-CSFIR antibody light chain variable region comprises a CDR1 comprising a sequence that is selected from SEQ ID NOs:

18, 24 and 30.

In some embodiments, an anti-CSFIR antibody light chain variable region comprises a CDR2 comprising a sequence that is selected from SEQ ID NOs:

19, 25 and 31.

In some embodiments, an anti-CSFIR antibody light chain variable region comprises a CDR3 comprising a sequence that is selected from SEQ ID NOs:

20, 26 and 32.

Non-exhaustive examples of light chain variable regions include, but are not limited to, light chain variable regions comprising sets of CDR1, CDR2 and CDR3 which are selected from: SEQ ID NO: 18, 19 and

twenty; SEQ ID NOs: 24, 25 and 26; SEQ ID NOs: 30, 31 and 32.

In some embodiments, an anti-CSFIR antibody light chain comprises a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

98% or at least 99% identical to a sequence that is selected from SEQ ID NOs: 10, 12, 14 and 46 to 52, where the light chain together with a heavy chain is capable of forming an antibody that binds to CSF1R .

In some embodiments, an anti-CSFIR antibody light chain comprises at least one of the CDRs described herein. That is, in some embodiments, an anti-CSFIR antibody light chain comprises at least one CDR that is

<td>select from</td><td>a</td><td>CDR1</td><td>from</td><td>chain</td><td>light</td><td>described</td>

<td>previously,</td><td>a</td><td>CDR2</td><td>from</td><td>chain</td><td>light</td><td>described</td>

<td>previously</td><td>and one</td><td colspan="2">CDR3 of</td><td>chain</td><td>heavy</td><td>described</td>

<td>previously.</td><td>What's more,</td><td>on</td><td>some</td><td colspan="3">modalities, a chain</td>

anti-CSFIR antibody light comprises at least one CDR mutated based on a CDR described herein, where

<td>the mutated CDR comprises</td><td> 1,</td><td> 2,</td><td> 3</td><td>or 4</td><td>substitutions</td><td>from</td>

<td>amino acids relative to</td><td>the</td><td>CDR</td><td colspan="2">described</td><td>at the moment.</td><td>On</td>

<td>some modalities, one</td><td>or</td><td>more</td><td>from</td><td>the</td><td>substitutions</td><td>from</td>

amino acids are conservative amino acid substitutions.

In some embodiments, a light chain comprises a

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human light chain constant region. In some embodiments, a human light chain constant region is selected from a human κ and human λ light chain constant region.

Additional examples of CSF1R binding molecules

In some embodiments, additional molecules are provided that bind to CSF1R. Molecules include, but are not limited to, non-canonical structures, such as anticalins, adnectins, ankyrin repeats, etc. See, eg, Hosse et al., Prot. Sci. 15:14 (2006);

Fíedler, M. and Skerra, A., Non-Antibody Scaffolds, pp. 467499 in Handbook of Therapeutic Antibodies, Dubel, S., ed., Wiley-VCH, Weinheim, Germany, 2007.

Examples of anti-CSFIR antibody properties

In some embodiments, an antibody having a structure described above binds CSF1R with a binding affinity (KD) of less than 1 nM, blocks the binding of CSF1 and / or IL34 to CSF1R, and inhibits CSF1 and / or IL34-induced phosphorylation of CSF1R.

In some embodiments, an anti-CSFIR antibody binds to the extracellular domain of CSF1R (CSF1R ECD). In some embodiments, an anti-CSFIR antibody has a binding affinity (KD) by CSF1R less than 1 nM, less than 0.5 nM, less than 0.1 nM or less than 0.0 5 nM. In some embodiments, an anti-CSFIR antibody has a KD between 0.01 and 1 nM,

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between 0.01 and 0.5 nM, between 0.01 and 0.1 nM, between 0.01 and 0.05 nM or between 0.02 and 0.05 nM,

In some embodiments, an anti-CSFIR antibody blocks the binding of the ligand to CSFIR. In some embodiments, an anti-CSFIR antibody blocks the binding of CSF1 to CSFIR. In some embodiments, an anti-CSFIR antibody blocks the binding of IL34 to CSFIR. In some embodiments, an anti-CSFIR antibody blocks the binding of CSF1 and IL34 to CSFIR. In some embodiments, an antibody that blocks the binding of the ligand binds to the extracellular domain of CSFIR. An antibody is considered to block the binding of ligand to CSFIR when it reduces the amount of detectable binding of a ligand to

CSFIR by at least 50%, using the assay described in

Example 7. In some embodiments, an antibody reduces the amount of detectable binding of a ligand to CSFIR by at least 60%, at least 70%, at least 80%, or at least 90%, using the assay described in Example 7. In some of the embodiments, the antibody is said to block ligand binding by at least 50%, at least 60%, at least 70%, etc.

In some embodiments, an anti-CSFIR antibody inhibits ligand-induced phosphorylation of CSFIR. In some embodiments, an anti-CSFIR antibody inhibits CSF1-induced phosphorylation of CSFIR. In some embodiments, an anti-CSFIR antibody inhibits IL34-induced phosphorylation of CSFIR. In some embodiments, an antibody

anti-CSFIR inhibits IL34-induced and CSF1-induced phosphorylation of CSF1R. An antibody is considered to inhibit ligand-induced CSF1R phosphorylation when it reduces the amount of detectable ligand-induced CSF1R phosphorylation by at least 50%, using the assay described in Example 6. In some embodiments, an antibody reduces the amount of detectable CSF1R phosphorylation induced by the ligand by at least 60%, at least 70%, at least 80%, or at least 90%, using the assay described in Example 6. In some Of the embodiments, the antibody is said to inhibit ligand-induced CSF1R phosphorylation by at least 50%, at least 60%, at least 70%, etc.

In some embodiments, an antibody inhibits monocyte survival and / or proliferation responses in the presence of CSF1 and / or IL34. An antibody is considered to inhibit monocyte proliferation and / or survival responses when it reduces the amount of monocyte proliferation and / or survival responses in the presence of CSF1 and / or IL34 by at least 50%, using the assay described in Example 10. In some embodiments, an antibody reduces the amount of monocyte proliferation and / or survival responses in the presence of CSF1 and / or IL34 by at least 60%, at least 70%, at least 80%, or at least 90%, using The assay described in Example 10. In some of the embodiments, the antibody is said to inhibit phosphorylation responses' ^ Tmn-OMRlCANE OF INDUSTRIAL FRGl'JEDAD

and / or monocyte survival by at least 50%, ”* a ± ~ at least 70%, etc.

Examples of Antibody Conjugates

In some embodiments, an anti-CSFIR antibody is conjugated to a marker and / or cytotoxic agent. As used herein, a marker is a moiety that facilitates the detection of the antibody and / or facilitates the detection of a molecule to which the antibody binds. Non-exhaustive examples of markers include, but are not limited to, radioisotopes, fluorescent groups, enzyme groups, chemiluminescent groups, biotin, epitope tags, metal-binding tags, etc. One skilled in the art can select a suitable marker in accordance with the intended application.

As used herein, a cytotoxic agent is a moiety that reduces the proliferative capacity of one or more cells. A cell has reduced proliferative capacity when the cell becomes less able to proliferate, for example, because the cell undergoes apoptosis or otherwise dies, the cell fails to complete the cell cycle and / or fails to divide, the cell differentiates, etc. Non-exhaustive examples of cytotoxic agents include, but are not limited to, radioisotopes, toxins, and chemotherapeutic agents. One skilled in the art can select a suitable cytotoxic agent in accordance with the

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intended application. ............. .......—

In some embodiments, a marker and / or cytotoxic agent is conjugated to an antibody using in vitro chemical methods. Non-exhaustive examples of chemical conjugation methods are known in the art and include commercially available services, methods and / or reagents from, eg,

Thermo Scientific Life Science Research Produces (formerly Pierce; Rockford, IL), Prozyme (Hayward, CA), SACRI Antibody Services (Calgary, Canada), AbD Serotec (Raleigh, NC), etc. In some embodiments, when a marker and / or cytotoxic agent is a polypeptide, the marker and / or cytotoxic agent can be expressed from the same expression vector with at least one chain of antibodies to produce a polypeptide comprising the marker and / or agent. cytotoxic fused to an antibody chain. One skilled in the art can select a suitable method for conjugating a marker and / or cytotoxic agent to an antibody according to the intended application.

Examples of leader sequences

For some secreted proteins to express and secrete in large amounts, a leader sequence from a heterologous protein may be desired. In some embodiments, a leader sequence is selected from SEQ ID NOs: 3 and 4, which are light chain and heavy chain leader sequences, respectively. In some modalities, use sequences

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Heterologous leaders may be advantageous in that a resulting mature perbipepfe-ide ^ may remain unaltered when the leader sequence is removed from ER during the secretion process. The addition of a heterologous leader sequence may be required to express and secrete some proteins.

Certain examples of leader sequence are described, eg, in the Online Leader Sequence Database maintained by the Department of Biochemistry, National University of Singapore. See Choo et al., BMC Bioinformatics, 6: 249 (2005); and PCT publication No. WO

2006/081430.

• Nucleic acid molecules encoding anti-CSFIR antibodies

Nucleic acid molecules are provided comprising polynucleotides that encode one or more chains of anti-CSFIR antibodies. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an anti-CSFIR antibody. In some embodiments, a nucleic acid molecule comprises both a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of an anti-CSFIR antibody. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule that comprises a second

polynucleotide encoding a light chain. —.....

In some embodiments, the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when an antibody is a scFv, a single polynucleotide encodes a single polypeptide comprising a heavy chain and a light chain linked together.

In some embodiments, a polynucleotide encoding a heavy chain or light chain of an anti-CSFIR antibody comprises a sequence of nucleotides encoding a leader sequence, which, when translated, are located at the N-terminal of the heavy chain or light chain. As described above, the leader sequence may be the native heavy or light chain leader sequence, or it may be another heterologous leader sequence.

Nucleic acid molecules can be constructed using recombinant DNA techniques standard in the art. In some embodiments, a nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

Expression and production of anti-CSFIR antibodies

Cartoon vector

Vectors are provided comprising polynucleotides encoding anti-CSFIR heavy chains and / or light chains.

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anti-CSFIR. Vectors comprising polynucleotides encoding anti-CSFIR heavy chains and / or anti-CSFIR light chains are also provided. Vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and the light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and the light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.

In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain and a second vector that comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts). In some embodiments, a molar or mass ratio of between 5: 1 and 1: 5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1: 1 and 1: 5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a

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1: 2 mass ratio for the vector that cqdjfica 1a rarlpna-heavy and the vector that encodes the light chain.

In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells or in NSO cells. Examples of the vectors are described in, eg. , Running Deer et al. ,

Biotechnol. Prog. 20: 880-889 (2004).

In some embodiments, a vector is chosen for in vivo expression of anti-CSFIR heavy chains and / or anti-CSFIR light chains in animals, including humans. In some embodiments, the expression of the polypeptide is under the control of a promoter that functions in a tissue-specific manner. For example, liver-specific promoters are described in, eg. , PCT Publication No. WO

2006/076288.

• Host cells

In various embodiments, anti-CSFIR heavy chains and / or anti-CSFIR light chains can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Examples of eukaryotic cells that can be used to express polypeptides include, but are not limited to,

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COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, anti-CSFIR heavy chains and / or anti-CSFIR light chains can be expressed in yeast. See, eg, US Publication No. US 2006/0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to anti-CSFIR heavy chains and / or anti-CSFIR light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in cells.

293 .

Introduction of one or more nucleic acids into a desired host cell can be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection , etc. Examples of non-exhaustive methods are described in, eg, Sambrook et al. , Molecular Cloning, A Laboratory Manual, 3it was ed. Coid Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cells, according to any

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proper method.

In some embodiments, one or more polypeptides can be produced in vivo in an animal that has been genetically modified or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method.

Purification of anti-CSFIR antibodies

Anti-CSFIR antibodies can be purified by any suitable method. The methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include CSF1R ECDs and ligands that bind antibody constant regions. For example, a Protein A,

Protein G, Protein A / G or an antibody affinity column can be used to bind the constant region and to purify an anti-CSFIR antibody. Hydrophobic interactive chromatography, for example a butyl or phenyl column, may also be suitable for purifying some polypeptides. Several suitable polypeptide purification methods are known in the art.

Cell-free production of anti-CSFIR antibodies

In some embodiments, an anti-CSFIR antibody is produced in a cell-free system. Non-exhaustive examples of cell-free systems are described, eg, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends

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Biotechnol. 22: 538-45 (2004); Endo et al. , 'Blotéchnol. AcJv.

21: 695-713 (2003).

Compositions and therapeutic methods • Methods for treating diseases using anti-CSFIR antibodies

Antibodies of the invention and compositions comprising antibodies of the invention are provided for use in methods of treatment for humans or animals. Also provided are methods of treating a disease comprising administering anti-CSFIR antibodies. Non-exhaustive examples of diseases that can be treated with anti-CSFIR antibodies include, but are not limited to, RA, MS, cancer, metastasis-induced osteolytic bone loss, osteolytic disorders, and hypercalcemia-induced bone loss.

In some embodiments, methods of treating inflammatory conditions are provided which comprise administering an anti-CSFIR antibody. In some embodiments, an inflammatory condition is selected from psoriasis, SLE (lupus), COPD, atopic dermatitis and atherosclerosis, macrophage activation syndrome, and histiocytosis X.

In some embodiments, methods are provided for treating an inflammatory condition comprising administering an anti-CSFIR antibody, wherein the inflammatory condition is selected from: proliferative vascular disease, syndrome of

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INDUSTRIAL N~ * acute respiratory distress, goY ~ 'trit'crine-mediated toxicity, interleukin-2 toxicity, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous, acute and ischemic, diverticulitis, epiglottitis, achachitis , cholecystitis, hepatitis, inflammatory bowel disease, Crohn's disease, enteritis, Whipple's disease, asthma, allergy, anaphylactic shock, complex immune disease, organ ischemia, Reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis, cystic fibrosis pneumonitis, alvealitis, bronchiolitis, pharyngitis, pleurisy, sinusitis, influenza, respiratory syncytial virus infection, herpes infection, HIV infection, Hepatitis B virus infection, Hepatitis C virus infection, disseminated bacteremia, dengue fever, candidiasis, malaria, filariasis, amoebiasis, hydatic cyst, burning, dermatitis, dermatomyositis, sunburn, urticaria, warts, hives, vasulitis, angitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease, disease

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celiac, congestive heart failure, meningitis, encephalitis, stroke, cerebral embolism, Guillain-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritides, arthralgia, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, synovitis, myasthenia gravis, thyroiditis, systemic lupus erythematosus, Goodpasture syndrome, Behcets syndrome, allograft rejection, graft-versus-host disease, ankylosing spondylitis, Berger's disease, type 1 diabetes, type 2 diabetes, Berger's disease, Retier's syndrome, and Hodgkins disease or to treat inflammation associated with these conditions.

In some embodiments, methods of treating cancer are provided which comprise administering an anti-CSFIR antibody. In some embodiments, the cancer is a CSF1-secreting cancer. In some modalities, the cancer is one or more cancers that are selected from breast cancer, prostate cancer, endometrial cancer, bladder cancer, kidney cancer, esophageal cancer, squamous cell carcinoma, uveal melanoma, follicular lymphoma, carcinoma of kidney cells, cervical cancer and ovarian cancer. In some embodiments, an anti-CSFIR antibody is useful to treat one or more cancers that are selected from lung cancer, colorectal cancer, brain cancer, cancer

IMPI

INSTITUTO MEXICANO DE LA PROPERTY t NDUSTt IA L pancreatic, head and neck cancer, liver cancer, leukemia, lymphoma, Hodgkin's disease, multiple myeloma, melanoma, astrocytoma, stomach cancer and lung adenocarcinoma.

Routes of administration and carriers

In various embodiments, anti-CSFIR antibodies can be administered in vivo by various routes, including, but not limited to, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal. , topical, transdermal and intrathecal, or otherwise by implant or inhalation. The subject compositions can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers and aerosols. A nucleic acid molecule encoding an anti-CSFIR antibody can be coated with gold microparticles and administered intradermally by a delivery device.

<td>bombardment of</td><td>particles, or</td><td>gun</td><td>of genes,</td><td>such</td><td>how I know</td>

<td>describes in</td><td>the bibliography</td><td>(see,</td><td>eg. ,</td><td>Tang</td><td>et al.,</td>

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Appropriate administration can be selected according to the intended request.

In various embodiments, compositions comprising

IMPI anti-CSFIR antibodies are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, eg, Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7to ed. , Lippencott Williams and Wilkins (2004); Kibbe et al. , Handbook of Pharmaceutical Excipients, 3to ed. , Pharmaceutical Press (2000)). There are a number of pharmaceutically acceptable carriers available, including vehicles, adjuvants, and diluents. In addition, various pharmaceutically acceptable auxiliary substances are also available, such as buffering and pH adjusting agents, tonicity adjusting agents, stabilizers, wetting agents, and the like. Non-limiting examples of carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

In various embodiments, compositions comprising anti-CSFIR antibodies can be formulated for injection, including subcutaneous administration, dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents,

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suspending agents, emulsifying agents, stabilizers and preservatives. In various embodiments, the compositions can be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. The compositions can also be formulated, in various embodiments, into sustained release microcapsules, such as biodegradable or non-biodegradable polymers. A non-exhaustive example of a biodegradable formulation includes polylactic acid-glycolic acid polymer. A non-exhaustive example of a non-biodegradable formulation includes a polyglycerin fatty acid ester. Certain methods for making such formulations are described, for example, in EP 1 125 584

To the.

Pharmaceutical packs and kits are also provided comprising one or more containers, each containing one or more doses of an anti-CSFIR antibody. In some embodiments, a unit dosage is provided where the unit dosage contains a predetermined amount of a composition comprising an anti-CSFIR antibody, with or without one or more additional agents. In some embodiments, such a unit dosage is delivered by single use prefilled injection syringe. In various embodiments, the composition contained in the unit dosage may comprise saline, sucrose

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Similar; A buffer, such as phosphophosphoresis, -γ / ρ can be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition can be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid, eg, sterile water. In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, a composition of the invention comprises heparin and / or proteoglycan.

The pharmaceutical compositions are administered in a treatment or prophylaxis effective amount of the specific indication. The therapeutically effective amount generally depends on the weight of the subject being treated, his physical or health condition, the extent of the condition to be treated or the age of the subject being treated. In general, anti-CSFIR antibodies can be administered in an amount ranging from about 10 pg / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, the anti-CSFIR antibodies can be administered in an amount ranging from about 50 pg / kg of body weight to about 5 mg / kg of body weight per dose. In some embodiments, the anti-CSFIR antibodies can be administered in an amount ranging from about 100 pg / kg in body weight to about 10 mg / kg.

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in body weight per dose. In some embodiments, the anti-CSFIR antibodies can be administered in an amount ranging from about 100 pg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the anti-CSFIR antibodies can be administered in an amount ranging from about 0.5 mg / kg of body weight to about 20 mg / kg of body weight per dose.

Anti-CSFIR antibody compositions can be administered to subjects as needed. Determination of the frequency of administration can be made by experts in the art, such as a treating physician based on considerations of the condition being treated, age of the subject being treated, severity of the condition being treated, general state of health of the subject being treated and the like. In some embodiments, an effective dose of an anti-CSFIR antibody is administered to a subject one or more times. In various embodiments, an effective dose of an anti-CSFIR antibody is administered to the subject once a month, with an interval greater than once a month, such as, for example, every two months or every three months. In other embodiments, an effective dose of an anti-CSFIR antibody is administered at an interval of less than once a month, such as, for example, every two weeks or every week. An effective dose of an anti-CSFIR antibody is administered to the subject by

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INBUSTRIAL

least once. In some embodiments, the effective dose of an anti-CSFIR antibody can be administered multiple times, including periods of at least one month, at least six months, or at least one year.

Combination therapy

Anti-CSFIR antibodies can be administered alone or with other modes of treatment. They can be provided before, substantially contemporaneously with or after other modes of treatment, for example, surgery, chemotherapy, radiation therapy, or the administration of a biological agent, such as another therapeutic antibody. For the treatment of rheumatoid arthritis, anti-CSFIR antibodies can be administered with other therapeutic agents, for example, methotrexate, anti-TNF agents such as Remicade, Humira, Simponi and Enbrel; glucocorticoids such as prednisone;

Leflunomide; Azothioprine; JAK inhibitors such as CP 590690; SYK inhibitors such as R788; anti-IL-6 antibodies; anti-IL-6R antibodies; anti-CD-20 antibodies; anti-CD19 antibodies; anti-GM-CSF antibodies and anti-GM-CSF-R antibodies. For the treatment of multiple sclerosis, anti-CSFIR antibodies can be administered with other therapeutic agents, eg, interferon alpha; interferon beta; prednisone, anti-alpha4 integrin antibodies such as Tysabri; antibodies

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anti-CD20 such as Rituxan; FTY72Ó (Fingolimod) and Cladribine (Leustatin).

EXAMPLES

The examples described below are intended to be purely examples of the invention and should not be construed as limiting the invention in any way. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (eg quantities, temperature, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is or near atmospheric.

Example 1: Selection of Fabs that bind to the extracellular domain (ECD) of CSF1R

Mice were immunized with a human CSF1R extracellular domain Fe fusion, ECD.506-hCSFIR Fc (SEQ ID NO: 6). Spleens from immunized mice were isolated and a Fab phage display library was created from splenocytes. A phage expressing Fab was selected to bind to the human CSF1R ECD. Positive binding phage Fabs were expressed and purified from bacteria. A total of 1056 Fab clones were selected for analysis

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Fabs were tested for the ability to bind human CSF1R ECD, human CSF1R blocking binding to human CSF1R ECD, and human IL34 blocking binding to human CSF1R ECD. Sequence analysis and clustering of Fabs that were selected from that analysis were then performed and certain unique Fabs were selected.

Unique Fabs were further tested for the ability to bind the human CSF1R ECD, the ability to bind the macaque CSF1R ECD, and the ability to bind the mouse CSF1R ECD. Fabs were also tested for the ability to block the binding of human CSF1 to the human CSF1R ECD and the ability to block the binding of human IL34 to the human CSF1R ECD and the ability to inhibit ligand-induced phosphorylation of CSF1R in the presence of CSF1 or IL34. (Data not shown).

• Example 2: Reformatting of anti-CSFIR Fabs to make chimeric antibodies

After Fab characterization, eleven of the Fabs were selected to reformat into chimeric antibodies. Each Fab was reformatted into a chimeric antibody comprising a human IgG4 heavy chain constant region with the S241P mutation, and a human κ light chain constant region. Briefly, the VH regions of Fab are

IMPI _

INSTITUTO MEXICANO • E LA PROPERTY INDUSTRIAL cloned and expressed the vector pTT5 (Biotechnology Research Institute, Montreal, Canada; and National Research Research Council of Cañada, Ottawa, Canada) modified to contain a mouse IgH leader sequence (SEQ ID NO: 4 ) and a human IgG4 heavy chain constant region with the S241P mutation (SEQ ID NO: 94). Fab VL regions were cloned and expressed from the modified pTT5 vector to contain a mouse IgK leader sequence (SEQ ID NO: 3) and a human IgK light chain constant region (SEQ ID NO: 95). The Fab V regions were inserted in such a way as not to introduce non-antibody derived amino acid sequences into the final proteins.

• Example 3: Expression and characterization of chimeric antibodies

Chimeric antibodies were transiently expressed and substantially purified as described in Example 5 below.

All 11 chimeric antibodies were assayed for ECD binding of human, macaque and mouse CSF1R. Chimeric antibodies were also tested for the ability to block the binding of human CSF1 to the human CSF1R ECD, the ability to block the binding of human IL34 to the human CSF1R ECD, the ability to block the binding

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from human CSF1 to CSF1R ECD ÜS 'riTáS ^ o-7-Ua ability to inhibit ligand-induced phosphorylation of CSF1R in the presence of CSF1 or IL34.

Chimeric antibodies were further tested for binding of CSF1R to the surface of cells. Finally, the chimeric antibodies were tested to confirm that they do not induce phosphorylation of CSF1R in the absence of the ligand. (Data not shown).

• Example 4: Humanization of anti-CSFIR antibodies

From the assays described above, chimeric anti-CSFIR antibodies 0301, 0302, and 0311 were selected for humanization. The antibodies were humanized by changing certain amino acid residues in the framework regions of the heavy and light chain variable regions. The criteria used for humanization were as previously described, eg, in US Publication No. US 2009/0136500.

For cAb 0301, three humanized heavy chain variable regions and two humanized light chain variable regions were designed, for a total of six humanized antibodies, Abl to Abé. For cAb 0302, two humanized variable regions were designed

IMPI

heavy chain and three humanized light chain variable regions, for a total of six humanized antibodies, Ab7 to Abl2. For cAb 0311, two humanized heavy chain variable regions and two humanized light chain variable regions were designed, for a total of four humanized antibodies, Abl3 to Abl6.

The sequences for each of the humanized heavy chain variable regions and humanized light chain variable regions, aligned with the sequences of the parental chimeric antibody variable regions and the sequences of the human acceptor variable framework regions are shown in Figures 1A- 1C (heavy chains) and 2 (light chains). Changes in humanized variable region sequences relative to human acceptor variable framework sequences are boxed. Each of the CDRs for each of the variable regions is shown in a boxed region, and is marked as CDRs on the boxed sequences.

Table 8, below, shows the complete sequences for the humanized heavy chains and humanized light chains of the Abl to Abl6 antibodies. The name and SEQ ID NOs of the

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Humanized heavy chain and humanized light chain of each of those antibodies are shown in the

Table 2.

Table 2: Heavy chains and humanized light chains from Abl to Abl6

<td>Antibody humanized</td><td>Humanized HC</td><td>SEQ ID NO</td><td>Lnunanized LC</td><td>SEQ ID NO</td>

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INSTiTUTC) MEXICAN OF INDUSTRY PROPERTY!

• Example 5: Humanized anti-CSFIR antibodies bind to the human and macaque CSF1R ECD, but not to the mouse CSF1R ECD.

The 16 humanized antibodies were transiently expressed in CHO cells, as follows. CHO-3E7 cells were co-transfected with individual heavy and light chain expression plasmids at a molar ratio of 1 heavy chain plasmid to 2 light chain plasmids using polyethyleneinimine (PEI) at a DNA: PEI ratio of 1: 5. The total DNA used per transfection was 1.5 pg / ml of cells.

Humanized antibodies were purified from transiected cell supernatants using HiTrap Protein A HP columns (GE Healthcare) followed by further purification using Phenyl HP columns (GE Healthcare). Antibody-containing supernatants were loaded onto HiTrap Protein A HP columns pre-equilibrated with PBS / 0.5M NaCl. Columns loaded with antibody were washed with 10 column volumes of PBS / 0.5M NaCl, and eluted with a mixed linear step gradient of 0.1 M glycine, pH 2.7 / 0.5 M NaCl directly in 100 ul Tris IM buffer, pH 8.0 . The antibody-containing eluates were dialyzed against PBS, after which (NH4)2SW4 2.4 M (Sigma) to achieve conductivity equal to that of potassium phosphate

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MlMIO MtXICAN ns LA MOHEDA INPDSTtlAL lOmM pH7.0 / (NH4) 2SW4 1.2 M. Antibodies were then loaded onto 1 ml Phenyl HP columns (GE Healthcare) pre-equilibrated with 10 mM potassium phosphate pH7.0 / (NH4) 2S04 1.2 M. Antibody loaded columns were washed with 15 column volumes of 10 mM potassium phosphate pH7. 0 / (NH4) 2SW4 1.2 M, and were eluted with a gradient of 20 column volumes of 10 mM potassium phosphate, pH 7.0. Fractions containing antibodies were pooled and dialyzed against PBS.

Humanized antibodies, along with their parental chimeric antibodies (cAbs), were assayed for ECD binding of human, macaque, and mouse CSF1R, as follows.

Human CSF1R binding activity

Ninety-six well clear bottom ELISA plates were covered overnight with 1 pg / ml recombinant hCSFIR ECD.506-Fc (SEQ ID NO: 6; FivePrime Therapeutics) or human M-CSF R Fe chimera ( R&D Systems) on PBS. The next morning, the wells were washed four times with 0.05% Tween20 in PBS (PBST) and blocked with Blocker-Blotto (Pierce). fifty 0.5x μΐ of serial dilutions of the parental humanized antibody or chimeric antibody, starting with 2000 ng / ml, diluted 1: 1 in Blocker-Blotto were added to the wells coated with CSF1R. After

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incubated at room temperature (RT) for 90 min, the wells were washed four times with PBST, and a 1: 5000 dilution of peroxidase-conjugated goat anti-human kappa light chain antibody (Sigma) was added in Blocker-Blotto to each well. After incubation at RT for 60 min, the wells were washed four times with PBST, and 50 µΐ of o-phenylenediamine dihydrochloride from the peroxidase substrate (Sigma) was added to each well. After incubation at RT for 30 min, the A450 values of each well were read directly on a SpectraMaxPlus spectrophotometer with SoftMaxPro software (Molecular Devices).

The results of that experiment are shown in Figures 3A-3C. All humanized antibodies bound to the human CSF1R ECD within the concentration range.

Macaque CSF1R binding curve

The binding curve for each humanized antibody that binds to the macaque CSF1R ECD was determined as described above for human CSF1R, except for wells of clear bottom ELISA plates which were coated overnight with 2 pg / ml of Recombinant cynoCSFIR ECD-Fc (FivePrime Therapeutics, SEQ ID NO: 8, but without the 19 amino acid leader sequence).

The results of that experiment are shown in the iMPir ^

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Figures 4A-4C. All humanized antibodies bound to the macaque CSF1R ECD within the concentration range.

Mouse CSF1R binding curve

The binding curve for each humanized antibody that binds to the mouse CSF1R ECD was determined as described above for human CSF1R, except for wells of clear bottom ELISA plates which were coated overnight with 2 pg / ml ECD -Recombinant mCSFIR (FivePrime Therapeutics, SEQ ID NO: 93).

The results of that experiment are shown in Figures 5A-5C. None of the humanized antibodies or parental chimeric antibodies detectably bound to the mouse CSF1R ECD over the range of concentrations tested.

Calculation of EC50

Table 3 shows the EC50, calculated using the nonlinear regression analysis algorithm (curve fitted) of the GraphPad Prism software (GraphPad Software) for each humanized antibody binding to the human CSF1R ECD and the macaque CSF1R ECD. Because none of the chimeric antibodies detectably bound to the mouse CSF1R ECD, an EC50 could not be calculated from these data. Table 3 also includes the calculated EC50s for the parental chimeric antibodies.

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Table 3: Binding activity of humanized anti-CSFIR antibodies

<td>Antibody humanized</td><td>Human CSFIR ECD EC50 (ng / ml)</td><td>Macaque CSFIR ECD EC50 (ng / ml)</td>

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• Example 6: Humanized anti-CSFIR antibodies inhibit ligand-induced phosphorylation of CSF1R

CSF1R is phosphorylated in the presence of the ligands CSF1 or IL34. Humanized antibodies, along with their parental chimeric antibodies (cAbs), were tested for their ability to inhibit the phosphorylation of CSF1R induced by any ligand, as follows. Inhibition of CSF1-induced phosphorylation

CHO cells transfected with CSF1R (SEQ ID NO: 2) were incubated with serial dilutions of each humanized antibody or a parental chimeric antibody, starting at 8 pg / ml, for 60 min on ice, after which 3.3 nM of Human CSF1 (M-CSF, R&D Systems) to cells. (For the 0301 series of humanized antibodies, serial dilutions starting at 2 pg / ml of humanized antibody and parental chimeric antibody were used.) Cells were incubated for 3 minutes at 37 ° C, and then used by addition of 1 / 10x 10 volume of cell lysis buffer (Cell Signaling Technology). The amount of

Cellular Uses phosphorylated CSF1R was quantitated using a human phospho-M-CSF R ELISA kit (R&D Systems) according to the manufacturer's instructions.

The results of that experiment are shown in Figures 6A to 6C. All humanized antibodies were able to inhibit the human CSF1-induced phosphorylation of the ECD of

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Human CSF1R within the range of. ,, conccntfQgrones · tested.

Inhibition of IL-34-induced phosphorylation

CHO cells transfected with CSF1R (SEQ ID NO: 2) were incubated with 0.002 to 8 pg / ml of each humanized antibody or a parental chimeric antibody for 60 min on ice, after which 3.3 nM human IL34 (FivePrime Therapeutics; SEQ ID NO: 68) to cells. Cells were incubated for 3 minutes at 37 ° C, and then lysed by adding 1/10 volume of 10 cell lysis buffer (Cell Signaling Technology). The amount of

Cellular Uses phosphorylated CSF1R was quantitated using a human phospho-M-CSF R ELISA kit (R&D Systems) according to the manufacturer's instructions.

The results of that experiment are shown in Figures 7A to 7C. All humanized antibodies were able to inhibit human IL34-induced phosphorylation of human CSF1R within the range of concentrations tested.

• Example 7: Humanized anti-CSFIR antibodies block human CSF1 and human IL34 that bind to human and macaque CSF1R.

Blocking activity of human CSF1R / CSF1

Humanized antibodies, along with parental chimeric antibodies (cAbs), were tested for their

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104 ability to block the binding of human CSFl to human and macaque prn CSF1R, as follows.

Recombinant human CSFl (M-CSF; R&D Systems) was biotinylated using an NH2-Biotin marker kit (Dojindo Molecular Technologies). One hundred μΐ of 1 pg / ml biotinylated CSFl in PBST / 0.1% BSA was added to the wells of Streptavidin Reacti-Bind (Pierce) coated plates pre-blocked with SuperBlock (Pierce) blocking buffer according to the manufacturer's instructions. fifty 0.5x μΐ of serial dilutions of the parental humanized antibody or chimeric antibody, starting at 2,000 ng / ml, were incubated with 50 ng / ml of hCSFIR ECD.506-Fc (SEQ ID NO: 6; FivePrime Therapeutics) or 50 ng / ml cynoCSFIR ECD-Fc (FivePrime Therapeutics, SEQ ID NO: 8, but without amino acid leader sequence 19) in 100 μΐ PBST / 0.1% BSA for 90 min at RT, after which the mixture transferred to one or more wells of a ligand-coated plate. After 90 min at RT, the wells were washed with PBST, and a 1: 5000 dilution of a Fe fragment specific peroxidase-conjugated goat anti-human IgG (Jackson Immuno Research) in PBST / 0.1% BSA was added to each well. . After incubation at RT for 60 min, the wells were washed with PBST / 0.1% BSA, and o-phenylenediamine dihydrochloride from the peroxidase substrate (Sigma) was added to each well. After incubation at RT for 30 min, the A450 values of each well were read

105 directly on a SpectraMáxPlus spectrophotometer with software

SoftMaxPro (Molecular Devices).

The results of that experiment for macaque CSF1R are shown in Figures 8Ά to 8C. All humanized antibodies based on Fabs 0301 and 0311 were able to block the binding of human CSF1 to the macaque CSF1R ECD within the range of concentrations tested. None of the humanized Fab 0302-based antibodies showed similar blocking activity in that experiment compared to the blocking activity of cAb 0302.

Human CSF1R / IL34 blocking activity

Humanized antibodies were tested for their ability to block the binding of human IL34 to the human CSF1R ECD. The blocking activity of each humanized antibody was determined as described above for blocking CSF1, except recombinant human IL34 (FivePrime Therapeutics; SEQ ID NO: 68) that was biotinylated using an NH2-Biotin marker kit (Dojindo Molecular Technologies), and then 100 μΐ of 1 pg / ml of recombinant biotinylated IL34 in PBST / 0.1% BSA was added to the wells of plates. coated with Streptavidin Reacti-Bind (Pierce) pre-locked with SuperBlock (Pierce) locking buffer according to manufacturer's instructions.

The results of that experiment for macaque CSF1R are shown in Figures 9A to 9C. All humanized antibodies based on Fabs 0301 and 0311 were able to block the

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Binding of human IL34 to the macaque CSF1R ECD within the range of concentrations tested. None of the humanized Fab 0302-based antibodies showed similar blocking activity in that experiment compared to the blocking activity of cAb 0302.

Calculation of ICSO

Table 4 shows the IC50, calculated using the nonlinear regression analysis algorithm (curve fitted) of the GraphPad Prism software (GraphPad Software) for the inhibition of ligand-induced CSF1R phosphorylation by each humanized antibody. Table 4 also shows the IC50, calculated using the nonlinear regression analysis algorithm (curve fitted) of the GraphPad Prism software (GraphPad Software) to block the binding of the ligand to the CSF1R ECD by each humanized antibody. Finally, Table 4 shows the amount of amino acids in the heavy and light chain framework regions of each humanized antibody that back-mutated at the corresponding mouse amino acid residue. For example, the humanized antibody h0301LlHl has one amino acid in a light chain framework region that was back-mutated at the mouse amino acid, and one amino acid in the heavy chain framework regions that was back-mutated at the mouse amino acid. Referring to Figures 1A-1C and 2A-2C, the back-mutated amino acid in the light chain framework is at position 1 in framework 1, and the back-mutated amino acid in the

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Table 4: Blocking activity of humanized anti-CSFIR antibodies

<td>Antibody humanized</td><td>Luunan CSF1R ECD IC50 CSF1 Luunan (nginl)</td><td>ECD IC50 human CSF1R / IL34 Luunan (ng / ml)</td><td>CyiioCSFIR / C'SFl luunan (nginl) ECD IC'50</td><td>CynoCSF IR / IL34 luunan ECD IC50 (ng / ml)</td><td>Mouse residues re-trmuted in FR (L + H)</td>

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• Example 8: Blocking constants of humanized anti-CSFIR antibodies

The kto, kd and KD ECD binding of human CSF1R was determined for each of the humanized antibodies as follows.

The binding kinetics of humanized anti-CSFIR antibodies to the CSF1R ECD was determined using Biacore T100

Surface Plasmon Resononance (SPR) (GE Healthcare Life

Sciences, Piscataway, NY). Each of the humanized anti-CSFIR antibodies was captured on a CM5 sensor chip immobilized with anti-human IgG antibody using the Human Antibody Capture Kit (GE Healthcare Life Sciences, Piscataway, NY) at 15 0RU so that the Rmax value for binding of ECD. 506 of hCSFIR (SEQ ID NO: 5) was 100RU. Rmax values less than 150RU are recommended to accurately determine kinetic values. 10 mM Hepes saline, pH 7.4, with 0.05% Tween20 (HPS-P; GE Healthcare Life Sciences, Piscataway, NY) was used as the dilution and run buffer. HCSFIR ECD.506 was injected at six concentrations (90 nM, 30 nM, 10 nM, 3.33 nM, 1.11 nM, and 0 nM) for 2 minutes and dissociation was observed for 5 minutes to determine humanized antibody / ECD binding kinetic parameters. by hCSFIR. The association constant, dissociation constant, affinity and binding capacity of each of the Fabs for ECD of the human CSF1R are

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calculated using the Biacore 'ΪΓ00 Évaluation software package using the 1: 1 binding model.

The results of the kinetic determinations are shown in Table 5.

Table 5: Humanized antibody binding affinity for

Human CSF1R

<td>huAbAb</td><td>k, (mV)</td><td>I <d «')</td><td>K »(hM)</td>

All but two humanized antibodies showed sub-nanomolar binding affinities for human CSF1R ECD, and the remaining two humanized antibodies showed binding affinities for human CSF1R ECD less than 2 nM.

110

INSTITUTO MEXICANO DE LA FROPIF.DA »INDUSTRIAL • Example 9: Humanized anti-CSFIR antibodiesblock ligand-induced phosphorylation

Based on the above data, including CSF1R binding and ligand inhibition, and the probability of immunogenicity for each humanized antibody, three humanized antibodies were selected for further study: 0301-L0H0, 0301-L1H0, and 0311-L0H1.

After confirming that each O3O1-LOH0, 0301-L1H0, and 0311-L0H1 binds CSF1R on the surface of cells (data not shown), each of the antibodies was tested for the ability to block phosphorylation. of CSF1R induced by the ligand in CHO cells, as described in Example 6.

The results of that experiment are shown in Figures 10A-10B. All three humanized antibodies tested blocked both CSF1 (A) -induced and IL34 (B) -induced phosphorylation of CSF1R in CHO cells. Table 6 shows the IC50 to block ligand-induced phosphorylation of CSF1R for each antibody.

Table 6: IC50 Blocking Ligand-Induced Phosphorylation for Humanized Antibodies

J Antibody Blocking IC50 \ Blocking IC50

<td>humanized</td><td>CSF1 (ng / ml)</td><td>IL34 (ng / ml)</td>

<td>0301-PARROT</td><td> 305.4</td><td> 340.8</td>

111 • Example 10: Anti-ΰθ · Ρ ± Κ · - human.-gados— antibodies block proliferation / survival responses induced by major human monocyte ligand

Humanized antibodies O3O1-L0HO, 0301-L1H0, and 0311-L0H1 were tested for their ability to block ligand-induced proliferation / survival responses of monocytes, as follows.

Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donor blood by centrifugation on a Ficoll-Paque pad (GE Healthcare Bio-Sciences) according to the manufacturer's instructions. Peripheral blood monocytes were subsequently isolated from the recovered PBMC fraction by centrifugation on a 48.5% Percoll ™ pad (GE Healthcare Bio-Sciences). Following recovery from the Percoll ™ cushion, purified peripheral blood monocytes were stimulated with 162 pM recombinant human CSF1 or 1.6 nM recombinant human IL34 (both from R&D Systems) in the presence or absence of serial dilutions of humanized antibody 0301-L0H0 , humanized antibody 0301-L1H0 or humanized antibody 0311-L0H1. After incubation at 37 ° C for hours, the relative cellular ATP content of each individual culture was evaluated using CellTiter Glo® reagent (Promega) according to the manufacturer's instructions. In this test, the relative cellular ATP content is ¿<ÜL jí 1 fW:

112

OF THE PROPERTY

MEXICAN INSTITUTE **** ΤΧ >>

INDUSTRIAL directly proportional to the number of viable cells in culture, and therefore reflects the survival / proliferation responses of monocytes.

The results of that experiment are shown in Figures 11A-11B. All three humanized antibodies tested were able to block monocyte survival / proliferation responses after CSF1 (A) or IL34 (B) stimulation. Table 7 shows the

IC50 to block ligand-induced monocyte survival / proliferation responses for each antibody. The values shown in the

Table 7 represents the range observed from three different major donors tested.

Table 7: Monocyte Survival / Proliferation Blocking IC50 for Humanized Antibodies

<td colspan="2">Antibody (IC50 blocking humanized CSF1 (ng / ml)</td><td>IL34 blocking IC50 (ng / num)</td>

• Example 11: Humanized anti-CSFIR antibodies do not directly stimulate the survival or proliferation responses of major human monocytes.

The humanized antibodies O3O1-L0HO, 0301-L1H0 and

113

0311-LOH1 were tested for their ability to directly stimulate the proliferation and / or survival of major monocytes, as follows.

Monocytes from human peripheral blood were isolated as described in Example 10. Serial dilutions of humanized antibody 0301-L0H0, humanized antibody 0301-L1H0, or humanized antibody 0311-L0H1 were added to monocytes in the absence of stimulation either by Exogenous CSF1 or exogenous IL34. After incubation at 37 ° C for 48 hours, the relative ATP content of each individual culture was evaluated using CellTiter Glo® reagent (Promega) as in Example 10. The experiment was carried out on peripheral blood monocytes from three different donors.

The results of that experiment are shown in Figures 12A-12C. None of the humanized antibodies stimulated the proliferation or survival of major monocytes of any of the major monocyte preparations tested.

• Sequence table

Table 8 provides certain sequences discussed herein. All sequences of

114

IMPI

INSTITUTO MEXICANt O € LA PRONEOAL> INDUSTTIAl

Antibodies and polypeptides are shown - s-rrr - i-crs · leader sequences, unless otherwise indicated.

Table 8: Brief description of the sequences

I KNOW THAT

ID

ΛΌ

Description

Sequence

IPVIEPSVFE LWKPGATVT

S1WATFQNT GTYRCT3PGD

FQDALl-PCLL TDPVLEAGVS

IQSQDYQCSA I. MGGRKVMS X

AQIVCSASSV DVNFDVFLQH

FQHAGNYSCV ASHVQGXHST

NLKVMVEAYP GLCGPNWTYL

RLKPS3AC-RY SFLARNFCGW llCSFIR, AASGYPQPNV TWLQCSGKTD (length i SLLTVETLEH NQTY3CRAHN full, VACKSIMALL LLLLLLItiYK no sequence 'PYNEKWEFFR NNLQFGXTIKEA.GKTDLLSAMP iLLSAM0LIMAGLIME 2 lead)' ' VDTYVBMRFV STSSNDSFSE

ASKNCIHRDV AARNVLLTNG 'KWMAPBSIFD CVYTVQSDVW

KDGYQMAQPA FAPKNIYSIK

ERDYTNLPSS SKSGGSGSSS

QFC

LRCVGNG5V3 WDGPPSPHKT LYSDGSSSIL ¡PLGGSAATKL YVKDPARPWN VLAQEVWFE | LVRVRGRFLM RKTNYSPSPW HGFTIHRAX? ¡SIRLXVQKVI PGPPALTLVP ASLVRIRGSA NNTKLAIPQC SDFHNNRYQK VLTLNLDQVD j SMFFRWESA YLNLSSEQNL IQEVTVGEGL ¡GPPSDHQP3? KLANATTKDT YRHTFTLSL? | RALTF3LTLR YFPEVSVIWT FINGSGTLLC RCD3AQVLQV WDDPXPEVLS Q3PFKKVTVQ ί SVGSCSWAFI PZSAGAKTHF PDEFLFTPW 'YKQKPKYQVR WXIIESYEGK SYTFIPPTQL i agafgkwea tafglgkeda vlkvavkklk LGQHEKIVNL LGACTHGGPV LV3TEYCCYG j GODPEGGVDY KNIELEKKYV RRDSGFSSQG; QDLDK3DGRP L-ELRDLLHFS SQVAQGXAFL 'HVAKI3DFGL ARDIMNDSNY ZVKGNARLPV ! SYGILLWFIF SLGLNPYPGX LVNSKFYXLV; QACWALEPTE RPTFQQICS? LQEQAQSDRR SELEEESSSE HLTCCEQGDI AQPLLQPNNY hCSFIR (full length + leader sequence)

MGPGVLLLLL VATAWHGQGI PVIEPSVP2L WXPGATVTL SCV3NGSV3W DGPPSPHWTL YSDGSSSILS TNNATFQNTG rYRCTEPGDl? LGGSAAZKLY VKDFARPWNV LAQ3WVFEC QDALLPCLLT DPVLEAGVSL VRVRGRPLMR HTHYSFSPWK GFTIHSAKFI. QSQDYQCSAL MGGRKVMSIS IRLKVQKVIP GFPALTIVPA SLVRIRG3AA QIVCSASSVD VNFDVFLQHN NTKLAIPQQS DFHNNRYQKV LTLNLDQVDF QHAGNYSCVA SNVQGKHSTS MFFRWSSAY LKLSSEQNLI QEVTVGSGLN L.KVMVEAYPG LQGFNWTYLG FFSDHQPEPK LANATTKDTY RETFTLSLPR LKPSEAGKY3 FLARNPGGWR ALTFELTLRY PP3VSVIWTF INGSGTLLCA ASGYPQPNVT WLQCSGHTDR CDEAQVLQVK DDPYPEVLSO KPFHXVTVQS LLTV3TL3HN QTYECRAHNS VGSGSWAFIP ISAGAHTHFP DEFLFTFVVV ACM3IMALLL I.LLLLLLYKY KQKFKYQVRW! KIIESYE3KS YTFIDPTQLP YNEKWSFPRN KLQFGKTLGA GAF3KWEAT I AFGLGKEDAV LXVAVKMLKS TAHADSKEAL MSELKIMSHL GQHENIVt -. 'LL' GACTHGGFVL VITEYCCYGD LLNFLRRKAE AML-GVSLSFG QDPEGGVDYK; NIHLEKKYVR RDSGFSSQGV DTYVEMRPVS TSSNDSFSBQ PLDODGRPL

115

I EDRDLDKFSS

RDIMNDSNYI

LGLNPYFGIL

PTFOQICSFL! LTCCEQGDIA

QVAQGaAFLA.

VKGNARLPVK

VKSKFYKLVK

QEQAQ3DKRE

QPLLQPKNYQ

SXNCIHRDVA

WMAPESIFDC

DGYQMAQPAF

RDYTNLPSSS

FC

ARNVLLTNGH

VYTVQSDVWS

APKNIYSIMQ

RSGGSGSSSS

VAKIGDFGLA

YCILLWEIFS

ACWALEPTHR

ELSEESS3EH

ECD.?06 of bCSFlR: IPVIEFSVP3 LWKPGATVC LECVGNGSVE WDGPPSPHWT STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSFW IQSQDYQCSA LJÍGGRKVMSI SIRLKVQKVI PGPPALTLVP AQIVCSASSV DVNFDVFLQH NNTKLAIFQ5 SDFHNNRYQK FQHAGNYSCV ASNVQGKKST SMFFRVVESA YLNLSSEQNL N'LKVMVEAYP GLQC-FKWTYL GPPSDEQPEP KLANATTKDT RLKFSEAGRY SFÍjARNPGGW RALTF3LTLR YPPEVSVIWT AASGYFQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS SLLTVSTLHH HQTYECSAKN SVGSGSWAFI

PTSAGAH

LYSDGSSSIL

VLAQEWVFE

HC-FTIHRAKF

AELVRIRGEA

VLTLXLDQVD

IQEVTVGEGL

YRHTFTLSL?

F7NGSGTLLC

QEPFKKVTVQ

ECD.506-R by JiCSFlK

I PVIEPSVP3! STNNATFQNT DQDALLPCLL IQSQDYQCSA AQIVCSASSV FQHAGNYSCV NLK.VMVEAYF RLKFSEAGRY AAS3YPQPNV SLLTVETLEH

LWXFGATVT

GTYRCT3PGD

TDPVLEAGVS

LMGGRKVM3I

DVNFDVFLQH

A3NVQGXHST

CLQGFN'WTYL

SFLARNPGGW

TWLQCSGHTD

NQTY3CRAHN

LRCVGNGSVK PLGGSAAIHL I.VRVRGRPLM SIRLKVQKVI NNTKLAIPQQ SMFFRWESA GPFSDKQPEP raltfeltlr RCDEAQVLQV SVGSGSWAFI

WDGPPSPHWT

YVKDPARPWN

RHTNYSFSFW

PGPPAL-TLVP

SDFHNNRYQK

YLNLSSEQNL

KLANAITKDT

YPPEVSVIWT

WDDPYPEVLS

PISAGAHSFK

LYSDGSSSIL

VLAQ3WVFE

HGFTIHRAKF

AELVRIRGEA

VLTLHLDQVD

IQEVTVGSGL

YRKTFTLSLP

FIWGSGTLLC

QEPFKKVTVQ

SSDKTHTCPP

CPAPELLGGF SVFLFPPKPK DTLMISRTPE VTCVVVDVSH BDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRW3VLTV LHQDWLNGKE YKCXVSNXAL PAPIEK7J.sk akgqprepqv YTLPPSRDEL tknqvsltcl VKGFYPSDIA VSWESNGQPE KNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK

CynoCSFIR ECD (with leader sequence)

MGPC-VLLLI.L

DGFISFHWTL

VKDPARPWNV

HTNYSFSPWH

GPPALTLVPA

DFKDNRYQKV

LDLSSEQNLI

LANATTKDTY

FPEVSVIWTS

VDPHPEVLSQ i ISACAR

WTAWHGQGI

YSDGPSSVLT

I.AKEVWFED

GFTIKRAXFI

ELVRIRGEAA

LTI.SLGCVDF

QEVTVGEGLN

RETFTLSLPR

INGSG7LLCA

SPFQKVTVQS

PVIEPSGPEL

TTNATFQNTR

QDALLPCLLT

QGQDYQCSAL

QIVCSASKID

QHAGNYSCVA

LKVMVEAYPG

LKPSEAGRYS

ASGYPQFNVT

LLTA3TLEHN

WKPGETVTL

TYRCTEFC-DP

DPVLEAGVSL

MGSRKVM3IS

VDFDVFLQHN

SNVQC-KHS7S

I.QGFKWTYLG

FLARNP3GWR

WLQCAGHTDR

QTYECRAHNS

RCVGNGSVEW

LGGSAAIHLY

VRLRGRPLLR

IRLKVQKVIP

TTKLAIPQRS

MFFRVVESAY

PFSDHQPEPK

ALTF3DTLRY

CDEAQVLQVW

VGSGSWAFI?

CvuoCSFlR ECD-Fc (with leader sequence)

[MGPGVLLLLL í DGPISPMWTL I VKDPARPWNV

HTNYSFSPWH

C-PPALTLVPA

DFKDNRYQKV

LDLSSEQNLI

LANATTKDTY

PPEVSVIWTS

VDPHPEVLSQ

ISAGARGSE?

EVTCWVDV3

VLHQDWLNGK

LTKNQVSLTC

SXI.TVDKSRW

WTAWHGQGI

YSDGPSSVLT

LAKEWVFED

GFTIHRAKFI

ELVRIRGSAA

LTLSLGQVDF

QEVTVGEGLN

KHTFTLSLPR

IKGSGTLLCA

BPFQKVTVQS

KSSDKTHTCP

HEDPEVKFNW

EYKCKVSNKA

LVKGFYPSDI

QQGNVFSCSV

PVIEPSGPEL

TTNATFQNTR

QDALLPCLLT

QGQDYQCSAL

QIVC3ASNID

QHAGNYSCVA

LKVMVEAYPG

LKPSEAGRYS

ASGYPQFNVT

LLTAETLEKN

PCPAP5LLGG

YVDGVEVHNA

LPAPIEKTIS

AVEWESNGQP

MH3ALKNKYT

WKPGETVTL

TYRCTEPGDP

DPVLEAGVSL

KGSEKVMSIS

VDFDVFLQKN

SKVCGKESTS

LQGFNKTYLG

FLARKPGGKR

WLQCAGHTDR

QTYECRAHNS

PSVFLFPFKP

K'IKPREEQYN

KAKGQPREPQ

ENNYXTTPPV

QKSLSLSPGK

RCVGNGSVEK 'LGGSAAIHLY j VRLRGRPLLR i IRLKVQKVIP ¡TTKLAIPQRS' MFFRVVESAY I J'FSDHQPEPK · ALTFELTLRY ! CDEAQVLQVW 1 VGSGSWAFIP XDTLMISRTP STYRWSVLT i VYTLPPSKDE j LDSDGSFFLY i

116

IM í * i

<td> 5</td><td>Smooth chain leader sequence</td><td>mstdtlllkv lllwvfgstg</td>

<td> 4</td><td>Heavy chain leader sequence</td><td>MAVLGLLLCL VTFF3CVLS</td>

<td> 9</td><td>Fab 0301 heavy chain variable region</td><td>EVQLQQSGPE LVRPGASVKH SCKASGYTFT DNYMIWVKQS HGKSLEWTGD INPYNGGTTF KQKFKGKATL TVBKSSSTAY MQLNSLTS3D SAVYYCARSS PYFSNLYVMD YWGQC-TSVTV SS</td>

<td> 10</td><td>Smooth chain variable region Fab 0301</td><td>NIVLTCSPAS IAVSLGQRAT ISCKASQSVD YDGCKYMNWY QQKFGQPPKI, LIYAASNLES GIPASFSGSG SGTDFTLNTH FVEESDAATY YCHLSNEDLS TFC-GGTKLEI K</td>

<td> 11</td><td>• - 1 Fab 0302 heavy chain variable region</td><td>SIQLQQSGFE LVKPGASVKK SCKASGYTFS DF'NI'HWVKQK PGQGLEWJ.GY INPYTDVTVY N3KFK3KATL TSDRSSSTAY MDL5SLTSBD SAVYYCASYF DGTFDYALDY WGQGTSITVS £ WGQGTSITVS</td>

<td> 12</td><td>Fab 0302 light chain variable region</td><td>DVWTQIPAS LAVSLGQRAT ISCRASESVC NYGI.SFMNW? QQKFGQPPKL LIYTASNLES C-IPARPSGGG SRTDl'TLTID PVEADDAATY 7CQQ3KELPW YFGGGTRLE.IX</td>

<td> 1.7</td><td>Regioti variable heavy chain Fab 0311</td><td>EZQLQQSGPD LMKPGASVKM SCKASGY2FT DYNMHWVKQN QGKSLEWMGE INPSNGVWY NQKFKGTTTLTVDKSSSTAY MDIKSLTSED SAVYYCTRAL YHSNPGWYFD SWGKGTTLTV SS</td>

<td> 14</td><td>Smooth chain variable region Fab 0311</td><td>DIVLTQSPAS IAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKFGQPPKL LIYTASNLES GIPARFSGSG SGADFTLTIH FVEE2DAATY YCQQGNEDPW TFG3GTRL3I K</td>

<td> 15</td><td>CDR1 heavy chain 0301</td><td>GYTFTDNYI4I</td>

<td> 16</td><td>CDR2 heavy chain 0301</td><td>DIlvFYNGGTT FNQXFKG {</td>

<td> 17</td><td>CDR3 heavy chain 0301</td><td>HSPYr3NLYV MDY</td>

<td>IS</td><td>CDR1 smooth chain 0301</td><td>KASCSVDYDG DKYYN</td>

<td> 19</td><td>CDR2 smooth chain 0301</td><td>AASNLES</td>

<td> 20</td><td>CDR3 figera chain 0301</td><td>HLSNSDLS?</td>

<td> 21</td><td>CDR1 heavy chain 0302</td><td>GYTFSDFNIH I</td>

<td> 22</td><td>CDR2 heavy chain 0302</td><td>YZNPYTDVTV YÍJEKFKG</td>

<td> 2?</td><td>CDR3 heavy chain 0302</td><td>YFSGTFDYAL DY i</td>

<td> 24</td><td>CDR1 light chain 0302</td><td>KASESVDKYG LSFMN | i</td>

117 smooth chain t'DR2 0302

IMPI

Mexican Institute of Industrial Property

TASNLES

Light chain CDR3 0302

OCSKELPWT

27 heavy chain CDR1 0311

GYIFTDYNMH

Heavy chain CDR2 0311

H INPNNGVW YNQKFKG

Heavy chain CDR3 0311

ALYHSNFGWY SDS

Light Chain L'DRl 0311

KASQSVDYDG DSHMN

31 chain CDR2 (light 0311

TASNLES

K3DR3 chain! 32 | lrgeraÜ311

QQGNGDPWT heavy chain cAb 0301

EVQLQQSGPñ '

INPYNGGTTF

PYFSNLYVMD

VKDYFFEPVT

KTYTCNVDHK

DTLMISRTPE

TYRWSVLTV

YTLPPSQEEM

DSDGSFFLYS

LVRPGASVKM

NQKFXGKATL

YWGQGTSVTV

VSWKSGALTS

PSNTKVDKRV

VTCVWDVSQ

LHQDWLNGKE

KNQVSbTCL

RLTVDKSRKQ

SCKASGYTFT

TVEKSSSTAY

SSASTKGPSV

GVHTFPAVLQ

ESKYGPPCPP

EDF2VQFNWY

YKCKVSNKGL

VKGFYPSDIA

EGNVFSCSVM

DNYMIWVKQS M01.NSLTSKD FPLAPCSRST SSGLYSLSSV CPAPEFLGGP VDGVSVHHAK PSSIEKTTSK VEWE SNGQPE HEALHNHYTQ

HGKSLEWIGD

SAVYYCASFS

SESTAALGCL

VTVPSSSLGT

SVFLFPPKPK

TKFREEQFNS

AKGQPRBPQV

NNYKTI'PPVL

KSLSLSLGK light chain CAb 0301, NIVLTQSPAS; LIYAASNLES I tfggc-tklei iQWKVDNALQS! TIIQGLSSPV'Í '4LAVSLGQRAT

GIPARFSGSG

KRTVAAPSVF

GKSQESVTEQ

KSFMRGEC

ISCKASQSVD YEGDNYMNWY SGTDFTLNIH PVEEEDAATY IFPPSDEGLK SGTASWCLL DSKDSTYSLS STLTLSKADY

QQKPGQPPKL> YCHLSNEDLS i NNFYFRBAKV i EKHXVYACEV I heavy chain cAb 0302

RIQLQQSGPE

INPYTDVTVY

DC-TFDYALDY

KDYFPEPVTV

TYTCNVDHK?

TLMISRTPEV

YRWSVLTVL

TLPPSQEEMT

SDGSFFLYSP.

LVKPGASVKM

NEK7KGKATL

WCQGTSITVS

SWNSGALTSG

3NTKVDSRVE

TCVWDVSCE

HQDWLNGKEY

KNQVSLTCLV

LTVDKSRWQS

SCKASGYTFS

TSDRSSSTAY

SASTKGPSVF

VHTFPAVLQS

SKYGPPCPPC

DPEVQFNWYV

KCKVSNKGL?

KGFYFSDIAV

GNVFSCSVMH

DFNIHWVKQK

MDLSSLTSED

PLAPCSRSTS

SGL.YSLSSW

PAPEFLGGP3

DGVEVHNAKT

SSIEKTISKA

EWESNGQPEN

EALHNHYTQK

FGQGLEWIGY

SAVYYCASYF

FSTAALGCLV

TVPSSSLGTK

VFLFPPKPKD

KPREEOFNST

KGQPREPQVY

NYKTTFPVLD

SLSLSLGK string

Osera cAb 0302

DVWTQTPAS

LIYTASNLES

TFGGGTRLEI

QWKVDNALQS

THQGL3SPVT

LAVSLGQRAT

GIPARFSGGG

XSTVAAPSVF

GNSQESVTEQ

KSFNRG3C

ISCRASESVD

SRTDFTLTID

IFPPSDEQL.K

DSKDSTYSLS

NYGLSFKNWF

PVEADDAATY

SGTASWCLL

STLTLSKADY

QQKr'GQPPKL

FCQQSKELFK

NNFYFREAXV

EKHKVYACEV heavy chain cAb 0311

EIQLQQSGPD INPNWGWVY YHSNFGWYFD VKDYFFEPVT ΚΤΥΊ ΟΛΤΗΧ DTLMISRTPÍS TYRWSVLTV YTLPPSQEEM DSDGSFFLYS

LMKPGASVKK

NQXFKGTTTL

SWGKGTTLTV

VSWNSGALTS

PSNTKVDKRV

VTCVWDVSQ

LHQDWLNGKE

TKNQVSLTCL

RLTVDKSRWQ

SCKASGYIFT

TVDKSSSTAY

SSASTKGPSV

GVHTFPAVLQ

ESKYGPPCPP

EDPEVQFNWY

YKCKVSKKGL

VKGFYPSDIA

EGNVFSCSVM

DYNMHWVXQN

MDLHSLTSED

FPLAPCSRST

SS3LYSLSSV

C? A? EFLGG?

VD3VEVHNAK

PSSIEKTISK

VEWESNGQPE

HEALHNHYTQ

QGKSLEWMGE

SAVYYCTRAL

SESTAALGCL

VTVPSSSLGT

SVFLFPPXPK

TKPREECFNS

AKGQPREFQV

NNYKTTPFVL

KSLSLSLGK

118

<td>3S</td><td>light chain cAbtnil</td><td>DIVLTQSPAS LIYTASNLES TFGGGTSLEI QWKVDNALQS THQGLSSPVT</td><td>LAVSLGQRAT GIPARPSGSG KRTVAAPSVP GNSQESVTEQ KSFNRGEC</td><td>ISCKASQSVD YDGDSHMNWY SC-ADFTLTTK PVESEDAATY IFPPSDEQLX SC-TASWCLL DSKDSTYSLS STLTLSKADY</td><td>QQKPGQPPKL I YCQQGÑEDPW i NNFYPREAKV EKHKVYAC3V |</td>

<td> 39</td><td>IO301-H0 heavy chain variable cleavage</td><td>QVQLVQSGAE IKPYNGGTTF PYF'SNLYVMD</td><td>VKKPGSSVKV NQKRKGRVTI YWGQGTLVTV</td><td>SCKASGYTFT DNYMIWVRQA TADKSTSTAY MELSSLRSED H.H</td><td>PGQGLHWMGD i TAVYYCARES j</td>

<td> 40</td><td>Hü301-H heavy chain variable region 1</td><td>QVQLVQSGÁ3 IKFYNGGTTF PYFSNLYVMD</td><td>VKKPGSSVKV NQKFKGRVTI YWGQGTLVTV</td><td>SCKASGYTFT DNYMIWVRQA TVDKSTSTAY MELSSLRSED H.H</td><td>FGQGLEWMGD TAVYYCARES</td>

<td> 41</td><td>HO3O1-H2 heavy chain variable region</td><td>QVQLVQSGAE IKPYNGC-TTF PYFSNLYVMD</td><td>VKKPGSSVKV NQKFKGRATL YWGQGTLVTV</td><td>SCKASC-YTFT DNYMIWVRQA TVDKSTSTAY MELSSLRSED SS</td><td>"T FGQGLEWIGD TAVYYCARES i</td>

<td> 42</td><td>Heavy chain variable region 110302-111</td><td>QVQLVQSGAE INPYTDVTVY DGTFDYALDY</td><td>VKKPGSSVKV NEKFKGRVTI WGQGT1.VTVS</td><td>3CKASGYTFS DFNIHWVRQA TSDKSTSTAY MELSSLRSED Ξ</td><td>PGQGLEVJMGY 'TAVYYCASYF</td>

<td> 43</td><td>H0302-H2 heavy chain variable region</td><td>QVQLVQSGAE INPYTDVTVY DGTFDYALDY</td><td>VKKPGSSVKV NEKFKGRATL WGQGTLVTV3</td><td>SCKASGYTIrS DFNIHWVRQA TSDKSTSTAY MELSSLRSED s</td><td>PGQGL2WIGY TAVYYCASYF</td>

<td> 44</td><td>Heavy chain variable region 11031 lH 1</td><td>--..... QVQLVQSGAE INPNN3WVY YHSNFGWYFD</td><td>VKKPGSSVKV NQKFKGRVTI SWGQGTLVTV</td><td>SCKASGYIFT DYNMHWVRQA TVDKSTSTAY MELSSLRSED SS</td><td>PGQGLEWMGE TAVYYCTRAL</td>

<td> 43</td><td>H0311-H2 heavy chain variable region</td><td>QVQLVCSGAS .1NPNNGVWY YHSNFGWYFD</td><td>VKKPGSSVKV NQKF'KGTTTL SWGQGTLVTV</td><td>SCKASGYIFT DYNMHWVRQA TVDKSTSTAY MELSSLRSED H.H</td><td>PGQGLSWKGE TAVYYCTRAL</td>

<td> 46</td><td>Smooth chain variable region 1) 0301-1.0</td><td>EIVLTQSPAT LIYAASNLSS TFGGGTKVEI</td><td>LSLSPGERAT GTPARFSGSC K</td><td>LSCKASQSVD YDGDNYMKWY SGTDFTLTIS SLEPEDFAVY</td><td>QQKFGQAPRL YCHLSNEDLS</td>

<td> 47</td><td colspan="2">Variable region! NIVLTQSPAT chain LIYfJiSNLES light TFGGGTKVEI h0301-Ll</td><td>LSLSFGSRAT GIFARFSGSG K</td><td>I.SCKASQSVD YDGDNYMKWY SGTDFTLTIS SLEPEDFAVY</td><td>CQKPGQAPRL YCHLSNEDLS</td>

<td> 4?</td><td>HO3O2-IO light chain variable region</td><td>EIVLTQSPAT LIYTASNLES TFGQGTKVEI</td><td>LSLSPGERAT GIFARFSGSG K</td><td>LSCRASESVD WYGLSFMNWY SGTDFTLTIS SLEPEDFAVY</td><td>QQKFGQAPRL YCQQSKELPW</td>

<td> 49</td><td>Smooth chain variable region HO3O2-L1</td><td>EIVLTQSPAT LIYTASKLES TFGQGTKVEI</td><td>LSLSPGERAT CI PARF3GSG K</td><td>LSCRASESVD MYGLSFMNWY SRTDFTLTIS SLEPEDFAVY</td><td>QQKFGQAPRL YCQQSKELPW</td>

<td> — 50</td><td>Smooth chain variable region H0302-L2</td><td>EIWTQSPAT LIYTASKLES TFGQGTKVEI</td><td>LSLSPGERAT GIPARFSC-SG X</td><td>LSCRASESVD NYGLSFMNWF SRTDFTLTIS SLEPEDFAVY</td><td>QQKFGQAPRL YCQQSKELPW _</td>

119

IMPIOUS

MEXICAN INSTITUTE

<td>1 variable legion</td><td>EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL</td>

<td>:chain</td><td>LIYTASNLES GIPARPSGSG SGTDFTLTIS SLEFEDFAVY YCQQGNSDPW</td>

<td>-1 i light</td><td>TFGQGTKVEI K</td>

Variable regionI DTVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL String LIYTASNLES GIPARFSGSG 3GADFTLTIS SLEPEDFAVY YGQQGNEDPW

- light! TFGQGTKV3I K, 110311-Lt;

QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD

INPYNGGTTF NQKFKGRVTI TADXSTSTAY MELSSLRSED TAVYYCARES

PYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL

VKDYPPEPVT VSWKSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT heavy chain! KTYTCKVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVPLFPPKPK ll03ül-110 I DTLMISRTPS VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKFR3EQFNS iRKGVLVIEPKTISKYRWCNG PSNGTISKTISPQDWV AKQREKQVTISKTISPKP

YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA. VEWESNGQPS NNYKTTPPVL

DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK nesada chain W1.301-H1

QVQLVQSGAE

INPYNGGTTF

PYFSNLYVKD

VKDYFPEPVT

K7YTCNVDHK

DTLMISRTPE

TYRWSVLTV

YTLPPSQEEM

DSDGSFFLYS

VKKPGSSVKV

NQKFKGRVTI

YWGQGTLVTV

VSWNSGALTS

PSNTKVDKRV

VTCVWDVSQ

LHQDWLNC-XE

TKNQVSLTCL

RLTVDKSRWQ

SCKASGYTFT

TVDKSTSTAY

SSASTKGPSV

GVHTFPAVLQ

ESKYGPPCPP

EDPEVQFNWY

YKCKVSKKGL

VKGFYPSDIA

EGNVFSCSVM

DNYMIWVRQA

MELSSLRSED

FPLAPCSRST

SSGLYSLSSV

CPAPEFLGGF

VDGVEVHNAK

PSSIEKTISK

VEWESNGQPS

HEALHNHYTQ

PGQGLEWMGD

TAVYYCARES

SESTAALGCL

VTVPSSSLGT

SVFLFPPKPK

TKPREEQFNS

AKGQPREPQV

NNYKTTPPVL

KSLSLSLGK heavy chain h030I-IJ2

QVQLVQSGAE

INPYNGGTTF

PYFSNLYVMD

VKDYFPEPVT

KTYTCNVDKK

DTLMISRTPE

TYRWSVLTV

YTLPPSQEEM

DSDGSFFLYS

VKKPGSSVKV

NQKFKGRATL

YWGQGTLVTV

VSWNSGALTS

PSNTKVDKRV

VTCVWDVSQ

LHQDWLNGKE

TKNQVSLTCL

RLTVDKSRWQ

SCKASGYTFT

TVDKSTSTAY

SSASTKGPSV

GVHTFPAVLQ

ESKYGPPCPP

EDPSVQFNWY

YKCKVSKKGL

VKGFYPSDIA

EGNVFSCSVM

DNYMIWVRQA MELSSLRSED FPLAPCSRST SSGLYSLSSV CPAPEFLGGP VDGVEVHNAK PSSIEKTISK V3WESKGQPE HEALHNHYTQ

PGQGLEWIGD

TAVYYCASES

SESTAALGCL

VTVPSSSLGT

SVFLFPPKPK

TKPREEQFNS

AKGQPREPQV

NNYKTTPPVL

KSLSLSLGK heavy chain 110302-H1

QVQLVQSGAE

INPYTDVTVY

DGTFDYALDY

KDYFPEPVTV

TYTCNVDHKP

TLMISRTFEV

YRWSVLTVL

TLPPSQEEMT

SDGSFFLYSR

VKKPGSSVXV

NEKFKGRVTI

WGQGTLVTVS

SWNSGALTSG

SNTKVDKRVE

TCWVDVSQE

HQDWLNGKEY

KNQVSLTCLV

LTVDKSRWQE

SCKASGYTFS

TSDKSTSTAY

SASTKGPSVF

VHTFPA.VLQS

SKYGPPCFPC

DPEVQFKWYV

KCKVSNKGLP

KGFYPSDIAV

GNVFSGSVMH

DFMIHWVRQA

MELSSLRSED

PLAPCSRSTS

SGLYSLSSW

PAPEFLGGPS

DGVEVHNAKT

SSIEKTISKA

EWESNGQPEN

EALHNHYTQK

PC-QGLEWMGY

TAVYYCASYF

ESTAALGCLV

TVPSSSLGTK

VFLFPPKFKD

KPREEQFKST

KGQPREPQVY

NYKTTPPVLD

SLSLSLGK heavy chain

H03O2-H2

QVQLVQSGAE

INPYTDVTVY

DGTFDYALDY

KDYFPEPVTV

TYTCNVDHKP

TLMISRTPEV

YRWSVLTVL

TLPPSQE3MT

SDGSFFLYSR

VKKPGSSVKV

NEKFKGRATL

WGQGTLVTVS

SWNSGALTSG

SNTKVDKRVE

TCWVDVSQE

HQDWLNGKEY

KNQVSLTCLV

LTVDKSRWQE

SCKASGYTFS

TSDKSTSTAY

SASTKGPSVF

VHTFPAVLQS

SKYGPPCFPC

DPEVQFNWYV

KCKVSNKGLP

KGFYPSDIAV

GNVFSCSVMH

DFNIHWVRQA

MELSSLRSED

PLAPCSRSTS

SGLYSLSSW

PAPEFLGGPS

DGVEVHNAKT

SSIEKTISKA

EWESNGQPEN

EALHNHYTQK

FC-QGLEWIGY TAVYYCASYF ESTAALGCLV TVPSS SLGTK VFLFPPKPKD KFREEQFNST KGQPREPQVY NYKTTPPVLD SLSLSLGK ii QVQLVQSGAE • heavy chain ¡INPNNGWVY ¡110311Fί I YHSDFP i YHSDFP

VKKPGSSVKV SCKASGYTFT DYNMHWVRQA I'GQGLEWMGE

NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRAL

SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL

VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT

120

IΜ Ρ1

ESRYGPPCPP CPAP'3TO? 3P '3VFLFF3? KPX7

INSTITUTO MF.XICAK.J UF IA FR-.WPAP

IN '' ilSTKIA !.

KTYTCKVDEK

DTLMISRTPE

TYRWSVLTV

YTLPPSQEEM

DSDG3FFLYS

QVQLVQSGAE

INPNNGWVY

YHSNFGWYFD

VKDYFPEFVT heavy chain H0311-H2

KTYICKVDHK

DTLMISRTPE

TYRWSVLTV

Y7LPPSQEEM

DSDGSFFLYS

EIVLTQSPAT light chain h0301-l_0

LIYAASKLES

TFGGGTKVEI

QWKVDNALQS

THQGLSSPVT

NIVLTQSPAT light chain ί hÚ301-lJ!

LIYAASNLBS

TFGGGTKVEI

QWKVDNALQS

THQGLSSPVT

PSNTKVDKRV

VTCWVDVSQ bHQSWLNGKE

TKNQVSLTCL

ELTVDKSRWQ

VKKPGSSVXV

NQKFKGTTTL

SWGQGTLVTV

VSWNSGALTS

PSNTKVDKRV

VTCWVDVSQ

LKQDWLNGKE

TXNQVSLTCL

RLTVDXSRWQ

LSLSPGERAT

GIPARFSGSG

KRTVAAPSVF

GNSQESVTEQ

KSFNRGEC

LSLSPGERAT

GIFARFSGSG

KRTVAAPSVF

GNSQESVTEQ

KSFNRGEC

EDPEVQFNWY

YKCKVSNKGL

VKGFYPSDIA VEWESNGQPE E3NVFSCSVM HEA.LHNI-JYTQ sckasgyif't DYÑMHWVRQA

NNYKTTPPVL

KSLSLSLGK

PGQGLEWMGE

TVDKS7STAY

SSASTKGPSV

GVHTFFAVLQ

ESKYGPFCPP

EDPEVQFNWY

YKCKVSNKGL

VKGFYPSDIA

EGNVFSCSVN

LSCKASQSVD

SGTDFTLTIS

IFPPSDEQLK

DSKDSTYSLS

LSCKASQSVD

SGTDFTLTIS

IFPPSDEQLK

DSKDSTYSLS

VDGVEVHKAK

PSSIEKTISK

MELSSLRSBD

FPLAFCSRST

SSGLYSL5SV VTVPSSSLGT CPAFSFLGGP SVFLFPPKPX VDGVEVHKAK TKPREEQFNS PSSIEKTISK AKGQPREPQV VEWESNGQPE NNYKTTPPVL HEALKNHYTQ KSLSLSLGK

YDGDNYKKWY

SLEPEDFAVY

SGTASVVCLL

STLTLSKADY

YDGDNYKKWY

SLEFEDFAVY

SGTASVVCLL

STLTLSKADY

TKPREEQFNS

AKGQPREPQV

TAVYYCTRAL

SESTAALGCL

QQKPGQAPRL

YCHLSNEDLS

NNFYPREAKV

EKHXVYACEV

QQKPGQAPRL

YCHLSNEDLS

NNFYPREAKV

EKHXVYACEV light chain

110302-L0

EIVLTQSPAT

LIYTASNLES

TFGQGTKVEI

QWKVDNALQS

THQGLSSPVT

LSLSPGERAT

GIPARFSGSG

KRTVAAPSVF

GNSQESVTEQ

KSFNRGEC

LSCRASESVD

SGTDFTLTIS

IFPPSDEQLK

DSKDSTYSLS

NYGLSFMNWY

SLEFEDFAVY

SGTASVVCLL

STLTLSKADY

QQKPGQAPRL

YCQQSKELPW

NNFYPREAKV

EKHKVYACEV light chain H0302-L1

EIVLTQSPAT

LIYTASNLES

TFGQGTKVEI

QWKVDNALQS

THQGLSSPVT

LSLSPGERAT

GIPARFSGSG

KRTVAAPSVF

GNSQESVTEQ

KSFNRGEC

LSCRASESVD

SRTDFTLTIS

IFPPSDEQLK

DSKDSTYSLS

NYGLSFMNWY

SLEFEDFAVY

SGTASVVCLL

STLTLSKADY

QQKPGQAPRL

YCQQSKELPW

NNFYPREAKV

EKHKVYACEV light chain i H0302-L2

3IWTQSFAT LSLSPGERAT LIYTASNLES GIPARFSGSG TFGQGTKVEI KRTVAAPSVF; QWKVDNALQS GNSQESVTEQ THQGLSSPVT KSFNRGEC

LSCRASESVD

SRTDFTLTÍS

IFPPSDEQLK

DSKDSTYSLS

NYGLSFKNWF

SLEFEDFAVY

SGTA3WCLL

STLTLSKADY

QQKPGQAPRL

YCQQSKELPW

NNFYPREAKV

EKHKVYACEV

EIVLTQSPAT LSLSPGERAT

LIYTASNLES GIPARFSGSG light chain; TFGQGTKVEI KRTVAAPSVF HÜ311-L0 i QWKVDNALQS GNSQESVTEQ i THQGLSSPVT KSFNRGEC

LSCKASQSVD

SGTDFTLTIS

IFPPSDEQLK

DSXDSTYSLS

YDGDSHMNWY

SLEPEDFAVY

SGTASVVCLL

STLTLSKADY

QQKPGQAPRL

YCQQGKEDFW

NNFYPREAKV

EKHKVYACEV smooth chain I H031I-L1!

DIVLTQSPAT

LIYTASNLES

TFGQGTKVEI

QWKVDNALQS

LSLSPGERAT

GIPARFSGSG

KRTVAAPSVF

GNSQESVTEQ

LSCKASQSVD

SGADFTLTIS

IFPPSDEQLK

DSKDSTYSLS

YDGDSEMNWY

SLEPEDFA.VY

SGTASVVCLL

STLTLSKADY

QQKPGQAPRL

YCQQGNEDPW

NNFYPREAKV

EKHKVYACEV

THQGLSSPVT KSFNRGEC; CSF1 67! human

EEVSEY'CSHM IGSGHLQSLQ KKAFLLVQDI HEDTMRFRDK ACVRTFYETP LQLLEKVKNV HEEQSEGS

RLIDSQMETS

TFNAIAIVQL

FNETKNLLDK

CQITFSFVDQ

QELSL-RLKSC

DWNIFSKNCN

EQLKDPVCYL

ETKDYEEHDK i NSFAECS3QG i

1L34 human

NEPLEMWPLT QNEECTVTGF VFR1ANVTRL QRAQVSERSL

LRDKLQYRSR LQYMKHYFPI MYKISVPYEGRYLKVLVSLSA.TE5VQDVLL SGHPSWKYLQ ¡

121

ΙΜ

<td></td><td></td><td>2VQTLÍ.LNVQ QGLTDVEVSP KVSSVLSLLN APGPNLKLVR PKALLONCFR VMELLYCSCC KQ3SVLNWQD CEVPSPQSC5 PEFSLQYAAT QLYPPPPWS? SSPFH5TG3V RFVRAQGSGL LP</td>

<td> 60</td><td>FRl accepted human A</td><td>QVQLVQSGAE VKKPGSSVKV SCKAS</td>

<td> 70</td><td>FR2 human acceptor A</td><td>WVRQAPGQGL EKMG</td>

<td> 71</td><td>FR3 accepted human A</td><td>RVTITADKST STAYMELSSL RSEDTAVYYC AR</td>

<td> 72</td><td>FR4 human acceptor A</td><td>WGQGTLVTVS S</td>

<td></td><td>FRl accepted human B</td><td>QVQLVQSGAE VKKPGSSVKV SCKAS</td>

<td> 74</td><td>FR2 accepted human B</td><td>WVRQAPGQGL EWI4G</td>

<td> 75</td><td>FR3 human acceptor B</td><td>RVTXTADKST STAYMELSSL RSEDTAVYYC AR</td>

<td> 76</td><td>FR4 human acceptor B</td><td>WGQGTLVTVSS</td>

<td> 77</td><td>FRl human acceptor C</td><td>QVQLVQSGAE VKKPGSSVKV SCKAS</td>

<td>7S</td><td>FR2 human acceptor C</td><td>WVRQAPGQGL EWKG</td>

<td> 79</td><td>FR3 human acceptor C</td><td>RVTXTADKST STAYMELSSL RSEDTAVYYC AR</td>

<td>SW</td><td>FR4 accept human C</td><td>WGQGTLVTVS S ..</td>

<td>YES</td><td>FRl acceptor human D</td><td>- EIVLTQSPAT LSLSPGSSAT L3C i</td>

<td>S2</td><td>FR2 acceptor human D</td><td>WYQQKPGQ'AP SLLIY</td>

<td>S3</td><td>FR3 acceptor luuuanaD</td><td>GIPARFSGSG S0TDR7LTIS SLEPEDFAVY YC</td>

<td> 84</td><td>FR4 human acceptor D</td><td>FGGC-TKVEIK:</td>

122

Χ'.ΐΧ, ρ

ΙΜΡΪ

INSTITUTE MtMC-MN 'DE LA FU ΉΗ-ΛΙ) INDUSTRIAL

<td>yes?</td><td>FR1 accepted human E</td><td>EZVLTQSPAT LSLSPGERAT LSC;</td>

<td>S6</td><td>FR.2 accepted it human E</td><td>WYQQKPGQAP RLLZY;</td>

<td colspan="2">i FR3 accepted it j human E</td><td>GIPARFS3SG SGTDFTLTIS SLEPEDFAVY YC;</td>

<td colspan="2">! FR4 accepted it</td><td>--------1 FGQGTKVEIK</td>

human E

S9

FR1 accepted human F

EIVLTQSFAT LSLSPGERAT LSC

I FR2

9θ accepted human F

WYQCXPGQA? RLLZY

FR3 accepted human F

GZPARFSGSG SGTDFTLTIS SLSPEDFAVY YC

FR4 accepted human F

FGQGTKVEZK

NiCSFIR ECD-Fc

APVZEPSGPE

TTRNATFKNT

GQEAVLFCLI

L23NTYVCRT

AQIVCSATNA

FQDAGIYSCV

ILTVHAZIAYP

KASEAGQYFL

SGYPQFSVTN

LPIGTLKHKM

CPAPELLGGF

VDGVEVHNAX

PAFISKTI8K

VEWESNGQPE

KEALKNHYTQ

LWEPGETVT

GTYRCTELSD

TDPALXDSVS

MVNGREST'ST

EVGFNVILXR

ASNDVGTRTA

SZQHYNWTYL

MAQNKAGWNN

MECRGH7DRC

TYFCKTHNSV

SVFLFPPKFK

TKPREEQYNS

AKGQPRSFQV

NNYKTTPFVL

KSLSLSPGK

LRCVSNGSVE

PKAGSTTIHL

LMREGGRQVL

GIWLKVNRVE

GDTKLEIPLN

TMNFQWESA

GPI'FEDQRKL

I.rFELTLRYP

DEAQALQVWN

GNSSQYFRAV

DTLMZSRTFE

TYRWSVLTV

YTLPPSRDEL

DSDGSFFLYS

WDGPISPYWT

YVKDPA.HSWN

RKTVYFFSPW

PEPPQIKLEP

SDFQDNYYKX

YLNLTSEQSL

EFZTQRAIYR

PEVSVTWMPV

DTHPEVLSQK

SLGQSKQEPK

VTCVWDVSH

LHQDWLNGKE

TKNQVSLTCL

KLTVDKSRWQ

LDPESPGSTL

LLAQEVTWE RGFZIRKAKV SKLVXZRGEA VRALSLNAVD LQEVSV3DSL I YTFKLFLNSV! NC-SDVLFCDV 'PFDKVIIQSQ! SSDKTHTCPF i EDPEVKFNWY; YKCKVSNKAL j VKGFYPSDIA) QGNVFSCSVM ¡

Human IgG4 S241P

ASTKGPSVFP

HTFPAVLQSS

KYGPPCPPCP

P3VQFNWYVD

CKVSNKGLPS

GFYPSDIAVE

NVFSCSVMHE

LAPCSRSTSE

GLYSLSSWT

APEFLGGPSV

GVEVHNAKTK

SIEXTISKAK

WESNGQPENN

ALHNHYTQKS

S'TAALGCLVK

VPSSSLGTKT

FLFPFKPKDT

PREEQFNS'TY

GQPREPQVYT

YKTTPPVLDS

LSLSLGK

Z5YFPEPVTVS ytcnvdhkps

LMISRTPEVT

RWSVLTVLH bPFSQEEMTK

DGSFFLYSR.L

WNSGALTSGV

NTKVDKRVSS

CVWDVSQED

QDWLNGKEYK

KQVSLTCLVK

TVDKSRWQEG ltíK Human

RTVAAPSVFI FFPSDEQI.KS GTASWCLLN NPYPRBAKVQ KKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC livii

INSTO UTO MEXICANO r / J

123 ι i ··, npiki) Ao

It is stated that in relation to it, the best method known to the applicant for putting the aforementioned invention into practice is the one that is clear from the present description of the invention.

Contents838

101 members in 26 offices

This recordMember, by publication datePriority claim16 more offices are in the list below

Priority claims9

Priority claims
Document	Office	Kind	Date
33117710	United States of America	P	2010-05-04
33117710	United States of America	P	2010-05-04
61331177	United States of America	–	2010-05-04
2011035231	United States of America	W	2011-05-04
2011035231	United States of America	W	2011-05-04
61331177	–	–	–
PCTUS2011035231	–	–	–
US20100331177P	–	–	–
WO2011US35231	–	–	–

Members101

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CA2797399A1	Canada	A1	2011-11-10
US2011274683A1	United States of America	A1	2011-11-10
WO2011140249A2	World Intellectual Property Organization (WIPO)	A2	2011-11-10
TW201206467A	Taiwan Province of China	A	2012-02-16
US8206715B2	United States of America	B2	2012-06-26
US2012219524A1	United States of America	A1	2012-08-30
AU2011248083A1	Australia	A1	2012-11-15
SG185035A1	Singapore	A1	2012-11-29
IL222629A0	Israel	A0	2012-12-31
IL222629D0	Israel	D0	2012-12-31
MX2012012790A	Mexico	A	2013-02-15
EP2566517A2	European Patent Office (EPO)	A2	2013-03-13
EA201291161A2	Eurasian Patent Organization (EAPO)	A2	2013-04-30
CN103179985A	China	A	2013-06-26
WO2011140249A3	World Intellectual Property Organization (WIPO)	A3	2013-08-15
JP2013535952A	Japan	A	2013-09-19
KR20130108078A	Republic of Korea	A	2013-10-02
EA201291161A3	Eurasian Patent Organization (EAPO)	A3	2013-10-30
US8747845B2	United States of America	B2	2014-06-10
NZ603193A	New Zealand	A	2014-07-25
US2014322757A1	United States of America	A1	2014-10-30
EP2566517A4	European Patent Office (EPO)	A4	2015-05-20
AU2015202477A1	Australia	A1	2015-05-28
AU2011248083B2	Australia	B2	2015-09-10
NZ626610A	New Zealand	A	2015-11-27
US9200075B2	United States of America	B2	2015-12-01
US2016046719A1	United States of America	A1	2016-02-18
TWI542361B	Taiwan Province of China	B	2016-07-21
SG10201604798SA	Singapore	A	2016-07-28
JP6008842B2	Japan	B2	2016-10-19
CN103179985B	China	B	2016-10-26
JP2016193898A	Japan	A	2016-11-17
TW201641517A	Taiwan Province of China	A	2016-12-01
AU2016262662A1	Australia	A1	2016-12-08
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AU2015202477B2	Australia	B2	2017-03-09
BR112012027994A2	Brazil	A2	2017-03-21
US2017081415A1	United States of America	A1	2017-03-23
US9695243B2	United States of America	B2	2017-07-04
CN106977603A	China	A	2017-07-25
CN107011438A	China	A	2017-08-04
TWI595008B	Taiwan Province of China	B	2017-08-11
JP6196347B2	Japan	B2	2017-09-13
AU2015202477C1	Australia	C1	2017-11-16
JP2017221215A	Japan	A	2017-12-21
IL255524D0	Israel	D0	2018-01-31
KR101838698B1	Republic of Korea	B1	2018-03-16
KR20180028560A	Republic of Korea	A	2018-03-16
TW201809002A	Taiwan Province of China	A	2018-03-16
MX355418BThis record	Mexico	B	2018-04-18
IL257712D0	Israel	D0	2018-04-30
US9957327B2	United States of America	B2	2018-05-01
US2018215830A1	United States of America	A1	2018-08-02
EP3357510A1	European Patent Office (EPO)	A1	2018-08-08
EP2566517B1	European Patent Office (EPO)	B1	2018-10-24
IL255524A	Israel	A	2018-10-31
IL255524B	Israel	B	2018-10-31
AU2016262662B2	Australia	B2	2018-11-01
DK2566517T3	Denmark	T3	2019-01-07
LT2566517T	Lithuania	T	2019-01-10
PT2566517T	Portugal	T	2019-01-24
SI2566517T1	Slovenia	T1	2019-01-31
AU2019200691A1	Australia	A1	2019-02-21
TR2019000368T4	Türkiye	T4	2019-02-21
TR201900368T4	Türkiye	T4	2019-02-21
TWI651331B	Taiwan Province of China	B	2019-02-21
HRP20190047T1	Croatia	T1	2019-02-22
ES2706412T3	Spain	T3	2019-03-28
RS58211B1	Serbia	B1	2019-03-29
PL2566517T3	Poland	T3	2019-04-30
JP6529552B2	Japan	B2	2019-06-12
TW201925232A	Taiwan Province of China	A	2019-07-01
HUE042898T2	Hungary	T2	2019-07-29
JP2019180413A	Japan	A	2019-10-24
CY1121102T1	Cyprus	T1	2019-12-11
SG10201911345WA	Singapore	A	2020-01-30
US10562970B2	United States of America	B2	2020-02-18
KR102105776B1	Republic of Korea	B1	2020-04-28
KR20200044999A	Republic of Korea	A	2020-04-29
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IL257712A	Israel	A	2020-07-30
IL257712B	Israel	B	2020-07-30
EP3357510B1	European Patent Office (EPO)	B1	2020-08-05
EA036336B1	Eurasian Patent Organization (EAPO)	B1	2020-10-28
IL222629A	Israel	A	2020-10-29
IL222629B	Israel	B	2020-10-29
CN107011438B	China	B	2020-11-20
CN106977603B	China	B	2020-11-27
TWI713942B	Taiwan Province of China	B	2020-12-21
AU2019200691B2	Australia	B2	2021-01-21
CA2797399C	Canada	C	2021-03-02
CN112480256A	China	A	2021-03-12
TW202112827A	Taiwan Province of China	A	2021-04-01
ES2820517T3	Spain	T3	2021-04-21
BR112012027994B1	Brazil	B1	2021-10-13
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JP2022008382A	Japan	A	2022-01-13
EP3943154A1	European Patent Office (EPO)	A1	2022-01-26
US2022153851A1	United States of America	A1	2022-05-19
NZ757754A	New Zealand	A	2023-03-31

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