EA039894B1 - Method of treating cancer, composition for treating cancer, use of the composition for preparation of a medicament for treating cancer characterized with presence of macrophages expressing csf1r - Google Patents

Abstract

The invention relates to a method of treating cancer in a subject, wherein the cancer is characterized with the presence of macrophages expressing CSF1R, the method comprising administering to the subject an anti-CSF1R antibody and at least one immune stimulating agent. The invention also relates to a composition for treating cancer characterized with the presence of macrophages expressing CSF1R, comprising an anti-CSF1R antibody and at least one immune stimulating agent, and to the use of the composition for the preparation of a medicament for treating cancer characterized with the presence of macrophages expressing CSF1R.

Description

Cross-reference to related applications

The present application claims priority to U.S. Provisional Application No. 62/146766, filed April 13, 2015; and US Interim Application No. 62/190945, filed July 10, 2015, all of which are incorporated herein by reference in their entirety for any purpose.

Technical field

Methods for treating cancer with antibodies that bind colony stimulating factor 1 receptor (CSF1R) in combination with one or more immunostimulatory agents.

State of the art

The colony stimulating factor 1 receptor (herein referred to as CSF1R; also referred to in the art as FMS receptor, FIM2 receptor, C-FMS receptor, MCSF receptor and CD115) is a singly membrane-passing receptor with an N-terminal extracellular domain (ECD) and a C-terminal intracellular domain with tyrosine kinase activity. Binding of CSF1 ligand or interleukin 34 ligand (herein referred to as IL34; Lin et al., Science 320: 807-11 (2008)) to CSF1R leads to receptor dimerization, activation of the tyrosine kinase activity of the CSF1R protein, phosphorylation of CSF1R tyrosine residues, and subsequent events signal transmission. Activation of CSF1R by CSF1 or IL-34 leads to the transport, survival, proliferation and differentiation of monocytes and macrophages, as well as other monocytic cell lineages such as osteoclasts, dendritic cells and microglia.

Many tumor cells or tumor stromal cells have been found to produce CSF1, which activates monocytes/macrophages via CSF1R. It has been shown that the level of CSF1 in tumors correlates with the level of tumor-associated macrophages (TAMs) in the tumor. Higher TAM levels have been found to correlate with worse patient prognosis for most cancers. In addition, CSF1 has been found to stimulate tumor growth and progression to metastasis, for example in human breast cancer xenografts in mice. See, for example, Paulus et al., Cancer Res. 66:4349-56 (2006). In addition, CSF1R plays a role in osteolytic bone destruction during bone metastasis. See, for example, Ohno et al., Mol. Cancer Ther. 5:2634-43 (2006). TAMs stimulate tumor growth, in part by suppressing the antitumor function of effector T cells through the release of immunosuppressive cytokines and the expression of T cell-inhibiting surface proteins. Thus, antibodies that bind to CSF1R may be useful in methods of treating cancer.

Some tumor cells may elude detection by the immune system, at least in part, by suppressing the immune response, such as by altering the expression of immunomodulatory genes. For example, the relative concentrations of both immunostimulatory and immune inhibitory molecules in the body can modulate the adaptive immune response. Relatively high levels of expression of inhibitory molecules and/or reduced expression of certain stimulatory molecules may create a control point or switch that suppresses the adaptive immune response. Agents that counteract this effect by activating the adaptive immune response or by stimulating the innate immune response are potential cancer treatments.

A combination regimen of agents that modulate the immune response in cancer, such as immunostimulatory agents, may increase the depth and duration of the response, and may also extend efficacy for patients who do not respond to a single agent alone.

The essence of the invention

In some embodiments, methods for treating cancer in a subject are provided, comprising administering to the subject an anti-CSF1R antibody and at least one immunostimulatory agent. In some embodiments, at least one immunostimulatory agent comprises an agonist of an immunostimulatory molecule, including a co-stimulatory molecule, while in some embodiments, at least one immunostimulatory agent comprises an antagonist of an immune inhibitory molecule, including a co-inhibitory molecule. In some embodiments, at least one immunostimulatory agent comprises an agonist of an immunostimulatory molecule, including a co-stimulatory molecule found on immune cells, such as T cells. In some embodiments, at least one immunostimulatory agent comprises an antagonist of an immune inhibitory molecule, including a co-inhibitory molecule found on immune cells, such as T cells. In some embodiments, at least one immunostimulatory agent comprises an agonist of an immunostimulatory molecule, including a costimulatory molecule found on cells involved in innate immunity, such as NK cells. In some embodiments, at least one immunostimulatory agent comprises an antagonist of an immune inhibitory molecule, including a co-inhibitory molecule found on cells involved in innate immunity, such as NK cells. In some embodiments, the combination enhances an antigen-specific T cell response in the individual being treated and/or enhances the innate immune response in the individual. In some embodiments, the combination results in an enhanced antitumor response in animal models of cancer, such as the xenograft model, compared to administration of either the anti-CSF1R antibody or the immunostimulatory agent alone. In some embodiments, the combination results in a synergistic response in an animal model of cancer, such as a xenograft model, compared to administration of either the anti-CSF1R antibody or the immunostimulatory agent alone.

In some embodiments, at least one immunostimulatory agent comprises a T cell activation inhibitor antagonist, while in some embodiments, at least one immunostimulatory agent comprises a T cell activation promoter agonist. In some embodiments, the at least one immunostimulatory agent comprises an antagonist of CTLA4, LAG-3, galectin 1, galectin 9, CEACAM-1, BTLA, CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM1, TIM3, TIM4, ILT4, IL-6, IL-10, TGFe, VEGF, KIR, LAG-3, A2A adenosine receptor, PI3K delta, or IDO. In some embodiments, at least one immunostimulatory agent comprises a B71, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD27, CD40, CD40L agonist , DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21, IFNa, STING, or a Toll-like receptor agonist such as a TLR2/4 agonist. In some embodiments, at least one immunostimulatory agent comprises an agent that binds to a member of the B7 family of membrane-bound proteins, such as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, B7 -H5 (VISTA) and B7-H6. In some embodiments, at least one immunostimulatory agent includes an agent that binds to a member of the TNF receptor family or a costimulatory or co-inhibitory molecule that binds to a member of the TNF receptor family, such as CD40, CD40L, OX40, OX40L, GITR, GITRL, CD70, CD27L , CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, EDA1, EDA2, TACI, APRIL, BCMA, SHI, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFe, TNFR2, TNFa, 1β2, FAS, FASL, RELT, DR6, TROY or NGFe. In some embodiments, at least one immunostimulatory agent includes an agent that is an antagonist or inhibitor of T cell activation, such as IL-6, IL-10, TGFe, VEGF. In some embodiments, at least one immunostimulatory agent comprises a chemokine antagonist, such as CXCR2, CXCR4, CCR2, or CCR4. In some embodiments, at least one immunostimulatory agent comprises a cytokine agonist that stimulates T cell activation, such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNa. In some embodiments, at least one immunostimulatory agent comprises an antibody. In some embodiments, the at least one immunostimulatory agent may include a vaccine, such as a mesothelin-targeted vaccine or an attenuated listeria cancer vaccine, such as CRS-207. Any one or more of the antagonists, agonists, and binding compounds described above may be combined with any one or more of the anti-CSF1R antibodies described herein.

In some embodiments, the implementation of at least one immunostimulatory agent includes a CD40 agonist, optionally in combination with at least one other immunostimulatory agent, as described above. In some embodiments, the CD40 agonist is an antibody. In some embodiments, the CD40 agonist is an anti-CD40 antibody. In some embodiments, an anti-CD40 antibody comprises a CDR of an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. In some embodiments, the CD40 antibody comprises the heavy chain and light chain variable regions of an antibody selected from CP-870.893; dacetuzumab; SeA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. In some embodiments, the anti-CD40 antibody is an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. In some embodiments, the CD40 agonist is recombinant CD40L. In some embodiments, at least one immunostimulatory agent comprises a CD40 agonist and at least one additional immunostimulatory agent as described above. For example, any one or more of the immunostimulatory agents described above may be combined with any one or more anti-CSF1R antibodies as well as a CD40 agonist such as a CD40 agonist antibody or recombinant CD40L such as any of the anti-CD40 antibodies described above.

In some embodiments, the anti-CSF1R antibody and at least one immunostimulatory agent are administered simultaneously or sequentially. In some embodiments, the anti-CSF1R antibody and at least one immunostimulatory agent are administered simultaneously. In some embodiments, one or more doses of at least one IM

- 2 039894 immunostimulating agent is administered prior to the introduction of antibodies against CSF1R. In some embodiments, the subject is given a full course of therapy with at least one immunostimulatory agent prior to administration of the anti-CSF1R antibody. In some embodiments, the anti-CSF1R antibody is administered during the second course of therapy with at least one immunostimulatory agent. In some embodiments, the individual is administered at least one, at least two, at least three, or at least four doses of at least one immunostimulatory agent prior to administration of the anti-CSF1R antibody. In some embodiments, at least one dose of at least one immunostimulatory agent is administered concurrently with the CSF1R inhibitor. In some embodiments, one or more doses of anti-CSF1R antibody are administered prior to administration of at least one immunostimulatory agent. In some embodiments, at least two, at least three, or at least four doses of an anti-CSF1R antibody are administered to the subject prior to administration of the at least one immunostimulatory agent. In some embodiments, at least one dose of an anti-CSF1R antibody is administered concurrently with at least one immunostimulatory agent.

In some embodiments, the cancer is selected from non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatic cell carcinoma, bladder cancer, endometrial cancer, Hodgkin's lymphoma, lung cancer, glioma, glioblastoma multiforme, colon cancer, breast cancer, bone cancer, skin cancer, uterine cancer, gastric cancer, gastric cancer, lymphoma, lymphocytic leukemia, multiple myeloma, prostate cancer, mesothelioma and kidney cancer. In some embodiments, the cancer is recurrent or progressive following therapy selected from surgery, chemotherapy, radiation therapy, or a combination thereof.

In some embodiments, compositions are provided comprising an anti-CSF1R antibody and at least one immunostimulatory agent. In some embodiments, at least one immunostimulatory agent comprises a T cell activation inhibitor antagonist, while in some embodiments, at least one immunostimulatory agent comprises a T cell activation promoter agonist. In some embodiments, at least one immunostimulatory agent comprises a antagonist of CTLA4, LAG3, galectin 1, galectin 9, CEACAM-1, BTLA, CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM1, TIM3, TIM4, ILT4, IL-6, IL-10, TGFe, VEGF, KIR, LAG-3, A2A adenosine receptor, PI3K delta, or IDO. In some embodiments, at least one immunostimulatory agent comprises a B7-1, B7-2, CD28, 4-1BB (CD137), 41BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD27, CD40, CD40L agonist , DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21, IFNa, STING, or a Toll-like receptor agonist such as a TLR2/4 agonist. In some embodiments, at least one immunostimulatory agent comprises an agent that binds to a member of the B7 family of membrane-bound proteins, such as B7-1, B72, B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA) and B7-H6. In some embodiments, at least one immunostimulatory agent includes an agent that binds to a member of the TNF receptor family or a co-stimulatory or co-inhibitory molecule that binds to a member of the TNF receptor family, such as CD40, CD40L, OX40, OX40L, GITR, GITRL, CD70, CD27L , CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, EDA1, EDA2, TACI, APRIL, BCMA, SHI, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFe, TNFR2, TNFa, 1β2, FAS, FASL, RELT, DR6, TROY or NGFe. In some embodiments, at least one immunostimulatory agent includes an agent that is an antagonist or inhibitor of a cytokine that inhibits T cell activation, such as IL-6, IL-10, TGFe, VEGF. In some embodiments, at least one immunostimulatory agent comprises a cytokine agonist that stimulates T cell activation, such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNa. In some embodiments, at least one immunostimulatory agent comprises a chemokine antagonist, such as CXCR2, CXCR4, CCR2, or CCR4. In some embodiments, at least one immunostimulatory agent comprises an antibody. In some embodiments, the at least one immunostimulatory agent may include a vaccine, such as a mesothelin-targeted vaccine or an attenuated listeria cancer vaccine, such as CRS-207.

In some embodiments, the compositions comprise any one or more of the antagonists, agonists, and binding compounds described above in combination with any one or more of the anti-CSF1R antibodies described herein. The compositions may include each drug in a separate container or compartment, or alternatively may include two or more drugs mixed together.

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In some embodiments, the compositions comprise an anti-CSF1R antibody and a CD40 agonist, optionally together with at least one other immunostimulatory agent as described above. In some embodiments, the CD40 agonist is an anti-CD40 antibody. In some embodiments, an anti-CD40 antibody comprises a CDR of an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. In some embodiments, the CD40 antibody comprises the heavy chain and light chain variable regions of an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. In some embodiments, the anti-CD40 antibody is an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. In some embodiments, the CD40 agonist is recombinant CD40L. In some embodiments, the compositions comprise any one or more of the immunostimulatory agents described above in combination with both any one or more of the anti-CSF1R antibodies described herein and a CD40 agonist, such as a CD40 agonist antibody or recombinant CD40L, such as any of the anti-CD40 antibodies described above. The compositions may include each drug in a separate container or compartment, or alternatively may include two or more drugs mixed together.

In any of the compositions or methods described herein, the heavy chain and/or light chain of an anti-CSF1R antibody may have the structure described below.

In any of the compositions or methods described herein, the heavy chain of an anti-CSF1R antibody may comprise a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to the sequence selected from SEQ ID NO: 9, 11, 13 and 39-45. In any of the methods described herein, the light chain of an anti-CSF1R antibody may comprise a sequence that is at least 90%, at least 95%, at least 97%, at least 99%, or 100% % is identical to the sequence selected from SEQ ID NOs: 10, 12, 14 and 46-52. In any of the compositions or methods described herein, the heavy chain of an anti-CSF1R antibody may comprise 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: 9, 11, 13, and 39-45, and the anti-CSF1R antibody light chain may contain 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 46-52.

In any of the compositions and methods described herein, CDR1 HC, CDR2 HC and CDR3 HC antibodies against CSF1R may contain a set of sequences selected from (a) SEQ ID NOS: 15, 16 and 17; (b) SEQ ID NOs: 21, 22 and 23; (c) SEQ ID NOs: 27, 28, and 29. In any of the compositions or methods described herein, CDR1 LC, CDR2 LC, and CDR3 LC of CSF1R antibodies may comprise a set of sequences selected from: (a) SEQ ID NO: 18, 19 and 20; (b) SEQ ID NOs: 24, 25 and 26; and (c) SEQ ID NOs: 30, 31 and 32.

In any of the compositions or methods described herein, the heavy chain of an anti-CSF1R antibody may be HC CDR1, HC CDR2, and HC CDR3, wherein HC CDR1, HC CDR2, and HC CDR3 comprise a set of sequences selected from (a) SEQ ID NO: 15 , 16 and 17; (b) SEQ ID NOs: 21, 22 and 23; (c) SEQ ID NOs: 27, 28 and 29; and the light chain may contain CDR1 LC, CDR2 LC and CDR3 LC, where CDR1 LC, CDR2 LC and CDR3 LC contain a set of sequences selected from (a) SEQ ID NO: 18, 19 and 20; (b) SEQ ID NOs: 24, 25 and 26; (c) SEQ ID NOs: 30, 31 and 32.

In any of the compositions or methods described herein, an anti-CSF1R antibody may comprise (a) a heavy chain containing 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 containing a sequence that is at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 10; (b) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 12; (c) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 14; (d) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 46; (e) a heavy chain containing a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 40, and a light chain containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 46; (f) a heavy chain containing a sequence that is at least

- 4 039894 is 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 41, and a light chain containing a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 46; (g) a heavy chain containing 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 containing 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 containing a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 40, and a light chain containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 47; (i) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 47; and (j) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 48; (k) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 49; (l) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 50; (m) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 48; (n) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 49; (o) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 50; (p) a heavy chain containing a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 44, and a light chain containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 51; (q) a heavy chain containing a sequence that is at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 44, and a light chain containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 52; (r) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 51; or (s) a heavy chain containing 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 containing a sequence that at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 52.

In any of the compositions or methods described herein, an anti-CSF1R antibody may comprise (a) a heavy chain comprising a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15, an HC CDR2 having the sequence of SEQ ID NO: 16, and a CDR3 HC having the sequence of SEQ ID NO: 17 and a light chain containing a light chain CDR1 (LC) having the sequence of SEQ ID NO: 18, a CDR2 LC having the sequence of SEQ ID NO: 19, and a CDR3 LC having the sequence of SEQ ID NO: 20; (b) a heavy chain containing a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 21, an HC CDR2 having the sequence of SEQ ID NO: 22, and an HC CDR3 having the sequence of SEQ ID NO: 23, and a light chain containing Light chain CDR1 (LC) having the sequence of SEQ ID NO: 24, CDR2 LC having the sequence of SEQ ID NO: 25, and CDR3 LC having the sequence of SEQ ID NO: 26; or (c) a heavy chain comprising a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 27, an HC CDR2 having the sequence of SEQ ID NO: 28, and an HC CDR3 having the sequence of SEQ ID NO: 29, and a light chain, containing a light chain CDR1 (LC) having the 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.

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In any of the compositions or methods described herein, an anti-CSF1R antibody may comprise (a) a heavy chain containing the sequence of SEQ ID NO: 53 and a light chain containing the sequence of SEQ ID NO: 60; (b) a heavy chain containing the sequence of SEQ ID NO: 53 and a light chain containing the sequence of SEQ ID NO: 61; or (c) a heavy chain comprising the sequence of SEQ ID NO: 58 and a light chain comprising the sequence of SEQ ID NO: 65. In some embodiments, the antibody comprises a heavy chain and a light chain, wherein the antibody comprises (a) a heavy chain consisting of sequence of SEQ ID NO: 53, and a light chain consisting of the sequence of SEQ ID NO: 60; (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: 65.

In any of the compositions or methods described herein, the anti-CSF1R antibody may be a humanized antibody. In any of the compositions or methods described herein, the anti-CSF1R antibody may be selected from Fab, Fv, scFv, Fab' and (Fab')2. In any of the compositions or methods described herein, the anti-CSF1R antibody may be a chimeric antibody. In any of the compositions or methods described herein, the anti-CSF1R antibody may be selected from IgA, IgG and IgD. In any of the compositions or methods described herein, the anti-CSF1R antibody may be an IgG. In any of the methods described herein, the antibody may be IgG1 or IgG2.

In any of the compositions or methods described herein, an anti-CSF1R antibody may bind to human CSF1R and/or may bind to cynomolgus monkey CSF1R. In any of the compositions or methods described herein, an anti-CSF1R antibody can block ligand binding to CSF1R. In any of the compositions or methods described herein, an anti-CSF1R antibody can block the binding of CSF1 and/or IL-34 to CSF1R. In any of the compositions or methods described herein, an anti-CSF1R antibody can block both CSF1 and IL-34 from binding to CSF1R. In any of the compositions or methods described herein, an anti-CSF1R antibody can inhibit ligand-induced phosphorylation of CSF1R. In any of the compositions or methods described herein, an anti-CSF1R antibody can inhibit CSF1 and/or IL-34 induced CSF1R phosphorylation. In any of the compositions or methods described herein, an anti-CSF1R antibody can bind to human CSF1R with an affinity (KD) of less than 1 nM. In any of the compositions or methods described herein, an anti-CSF1R antibody can inhibit monocyte proliferation and/or survival responses in the presence of CSF1 or IL-34.

Brief description of the drawings

In FIG. 1A-C show the alignment of the humanized heavy chain variable regions for each of the humanized huAb1-huAb16 antibodies as described in Example 1. The boxed residues are amino acids in the human acceptor sequence that have been changed back to the corresponding mouse residues.

In FIG. 2A-C show the alignment of the humanized light chain variable regions for each of the humanized huAb1-huAb16 antibodies as discussed in Example 1. The boxed amino acids are residues in the human acceptor sequence that have been changed back to the corresponding mouse residue.

In FIG. 3 shows that the combination of anti-CSF1R antibody and anti-CD40 antibody exhibits higher tumor growth suppression in the mouse MC38 tumor model than either of these therapies alone.

In FIG. 4A,B show tumor volume in individual mice (FIG. 4A) at day 11 and (FIG. 4B) at day 13. The combination of anti-CSF1R antibody and anti-CD40 antibody was significantly more effective than either of these therapies alone at both time points.

In FIG. 5 shows the body weight of the mice used in the study.

Detailed description

Tumor-associated macrophages (TAMs) are implicated in the pathogenesis of many malignant tumors and they correlate with poor prognosis. TAMs can suppress antitumor responses through a variety of mechanisms. TAMs express anti-inflammatory cytokines such as TGFe and IL-10, which act to inhibit the ability of intratumoral dendritic cells to stimulate cytotoxic T cell responses (Ruffell et al., 2014, Cancer Cell). TAMs also express chemokines that attract immunosuppressive regulatory T cells in tumors (Curiel et al., 2004, Nature Med.; Mizukami et al., 2008, Int. J. Cancer). Moreover, TAMs express ligands for inhibitory T- PD-1 and CTLA-4 receptor cells, which act to directly inhibit T cell activation and function. Inhibition of CSF1R can reduce the level of immunosuppressive TAMs in mouse models and in human tumors. See, for example, Ries et al., 2014, Cancer Cell, 25: 846-859; Pyontech et al., 2013, Nature Med., 19: 1264-1272; and Zhu et al., 2014, Cancer Res., 74: 5057-5069. In contrast to blockade of CSF1R, which acts by reducing immunosuppression, immunostimulatory agents act by stimulating the immune response.

The section headings used in this specification are for organizational purposes only and should not be construed as limiting the invention described.

Definitions

Unless otherwise defined, scientific and technical terms used in connection with the present invention have meanings that are commonly understood by those skilled in the art. In addition, unless the context otherwise requires, terms in the singular include the plural and terms in the plural include the singular.

Exemplary methods used in relation to recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (eg, electroporation, lipofection), enzymatic reactions, and purification methods are known in the art. Many of these methods and techniques are described, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), among others. In addition, exemplary methods for chemical synthesis, chemical analysis, preparation, formulation and delivery of pharmaceuticals, and treatment of patients are also known in the art.

In this application, the use of or means and/or unless otherwise indicated. In the context of a claim dependent on another dependent claim, the use of or, conversely, refers to more than one preceding independent or dependent claim only as an alternative. Also, terms such as element or component encompass both elements and components containing a single unit, and elements and components that contain more than one subunit, unless otherwise specifically indicated.

As used in accordance with the present invention, the following terms, unless otherwise indicated, should be understood as having the following meanings.

The terms nucleic acid molecule and polynucleotide can be used interchangeably and refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A nucleic acid sequence refers to a linear sequence of nucleotides that contains a nucleic acid molecule or polynucleotide.

The terms polypeptide and protein are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. The definition covers both full-length proteins and their fragments. The terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Moreover, for the purposes of the present invention, a polypeptide refers to a protein that includes modifications such as deletions, insertions and substitutions (generally of a conservative nature) in the native sequence, provided that the protein retains the desired activity. These modifications may be intentional, eg, through site-directed mutagenesis, or may be accidental, eg, due to mutation of the hosts that produce the proteins, or errors in amplification using PCR.

The term CSF1R refers herein to a full-length CSF1R that includes an N-terminal ECD, a transmembrane domain, and an intracellular tyrosine kinase domain, with or without an N-terminal leader sequence. In some embodiments, the CSF1R is a human CSF1R having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The term immunostimulatory agent, as used herein, refers to a molecule that stimulates the immune system, either by acting as an agonist of an immunostimulatory molecule, including a co-stimulatory molecule, or by acting as an antagonist of an immune inhibitory molecule, including a co-inhibitor molecule. The immunostimulatory agent may be a biological agent such as an antibody or antibody fragment, other protein or vaccine, or it may be a small molecule drug. An immunostimulatory molecule includes a receptor or ligand that acts to enhance, stimulate, induce or otherwise trigger an immune response. Immunostimulatory molecules, as defined herein, include costimulatory molecules. An immune inhibitory molecule includes a receptor or ligand that acts to reduce, inhibit, suppress, or otherwise turn off an immune response. Immune inhibitory molecules as defined herein include co-inhibitory molecules. Such immunostimulatory and immune inhibitory molecules may be, for example, receptors or ligands found on immune cells, such as T cells, or found on cells involved in innate immunity, such as NK cells.

The terms B-cell surface antigen CD40 and CD40 refer to full-length CD40, which includes an N-terminal ECD, a transmembrane domain and an intracellular domain, with or without an N-terminal leader sequence. In some embodiments, CD40

- 7 039894 is a human CD40 having the amino acid sequence of SEQ ID NO: 96 (precursor with signal sequence) or SEQ ID NO: 97 (mature form without signal sequence).

The term CD40 agonist refers to the part that interacts with CD40 and enhances CD40 activity. Non-limiting exemplary CD40 activities include signal transduction through CD40, enhancement of antigen presentation activity, induction of pro-inflammatory cytokines, and induction of tumoricidal activity. In some embodiments, the CD40 agonist is an anti-CD40 antibody.

The term anti-CD40 antibody refers to an antibody that specifically binds to CD40. Unless otherwise specified, the term anti-CD40 antibody, as used herein, refers to an anti-CD40 agonist antibody.

With respect to anti-CSF1R antibodies, the term blocks the binding of a ligand such as CSF1 and/or IL-34 and its grammatical variants are used to denote the ability to inhibit the interaction between CSF1R and a CSF1R ligand such as CSF1 and/or IL-34. Such inhibition can occur through any mechanism, including direct interference with ligand binding, for example, due to overlapping binding sites on CSF1R and/or conformational changes in CSF1R induced by an antibody that alter ligand affinity, etc. Antibodies and antibody fragments designated as functional are characterized by the presence of such properties.

The term antibody, as used herein, refers to a molecule containing at least a complementarity determining region (CDR) 1, a heavy chain CDR2 and CDR3 and at least a light chain CDR1, CDR2 and CDR3, wherein the molecule is capable of binding to an antigen. The term antibody includes, but is not limited to, fragments that are capable of binding antigen, such as Fv, single chain Fv (scFv), Fab, Fab' and (Fab') ₂ . The term antibody includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, cynomolgus monkey, etc.

In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the 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, the antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, wherein the light chain comprises a light chain variable region and at least a portion of a light chain constant region. chains. As used herein, a single chain Fv (scFv) or any other antibody that contains, for example, one polypeptide chain containing all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain and a light chain. In some such embodiments, a heavy chain is an antibody region that contains three heavy chain CDRs, and a light chain is an antibody region that contains three light chain CDRs.

The term heavy chain variable region, as used herein, refers to the region containing the heavy chain CDR1, framework region (FR)2, CDR2, FR3 and CDR3. In some embodiments, the heavy chain variable region also contains at least a portion of FR1 and/or at least a portion of FR4. In some embodiments, the heavy chain CDR1 corresponds to Kabat residues 26-35; Heavy chain CDR2 corresponds to Kabat residues 50-65; and the heavy chain CDR3 corresponds to Kabat residues 95-102. See, for example, Kabat, Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.); and fig. 1. In some embodiments, the heavy chain CDR1 corresponds to Kabat residues 31-35; Heavy chain CDR2 corresponds to Kabat residues 50-65; and the heavy chain CDR3 corresponds to Kabat residues 95-102. See ibid.

The term heavy chain constant region, as used herein, refers to a region containing at least three heavy chain constant domains: CH1, C _H 2 and C _H 3. Non-limiting examples of heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody containing constant region γ is an IgG antibody, an antibody containing constant region δ is an IgD antibody, and an antibody containing constant region α is an IgA antibody. In addition, the antibody containing the constant region μ is an IgM antibody, and the antibody containing the constant region ε is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing the γ ₁ constant region), IgG2 (containing the γ ₂ constant region), IgG3 (containing the γ ₃ constant region), and IgG4 (containing the γ ₄ constant region); IgA antibodies include, but are not limited to, IgA1 (containing the α1 constant region) and IgA2 (containing

- 8 039894 constant region α ₂ ); and IgM antibodies include, but are not limited to, IgM1 and IgM2.

In some embodiments, the heavy chain constant region contains one or more mutations (or substitutions), insertions, or deletions that confer desired characteristics on the antibody. A non-limiting exemplary mutation is the S241P mutation in the IgG4 hinge region (between CH1 and CH2 constant domains) that changes the IgG4 CPSCP motif to CPPCP, which is similar to the corresponding motif in IgG1. In some embodiments, this mutation results in a more stable IgG4 antibody. See, for example, 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 containing at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of the heavy chain constant region. The term full-length heavy chain, as used herein, refers to a polypeptide containing 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 the region containing the light chain CDR1, framework region (FR) 2, CDR2, FR3 and CDR3. In some embodiments, the implementation of the variable region of the light chain contains FR1 and/or FR4. In some embodiments, the light chain CDR1 corresponds to Kabat residues 24-34; Light chain CDR2 corresponds to Kabat residues 50-56; and the light chain CDR3 corresponds to Kabat residues 89-97. See, for example, Kabat, Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.); and fig. one.

The term light chain constant region, as used herein, refers to a region containing a C _L light chain constant domain. Non-limiting examples of the light chain constant region include λ and k.

The term light chain, as used herein, refers to a polypeptide containing at least a light chain variable region, with or without a leader sequence. In some embodiments, the implementation of the light chain contains at least part of the constant region of the light chain. The term full-length light chain, as used herein, refers to a polypeptide containing 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 containing at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such like humans, cynomolgus monkeys, etc.). In some embodiments, the chimeric antibody comprises at least one murine variable region and at least one human constant region. In some embodiments, the chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, the chimeric antibody comprises at least one rat variable region and at least one mouse constant region. In some embodiments, all of the variable regions of the chimeric antibody are from the first species and all of the constant regions of the chimeric antibody are from the second species.

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

A CDR-grafted antibody, as used herein, refers to a humanized antibody in which complementarity determining regions (CDRs) of a first (non-human) species are grafted into framework regions (FRs) of a second (human) species.

Human antibody as used herein refers to antibodies produced in humans, antibodies produced in non-human animals that contain 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-terminus of a polypeptide that facilitates secretion of the polypeptide from a mammalian cell. The leader sequence may be cleaved upon export of the polypeptide from a mammalian cell to form the mature protein. Leader sequences may be natural or synthetic, and they may be heterologous or homologous to the protein to which they are associated. Illustrative 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

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4, which correspond to the human light and heavy chain leader sequences, respectively. Non-limiting leader sequences also include leader sequences from heterologous proteins. In some embodiments, the antibody lacks a leader sequence. In some embodiments, the antibody contains at least one leader sequence, which may be selected from native leader sequences and heterologous leader sequences.

The term vector is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide or polynucleotides that can increase in number in a host cell. The vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (eg, such as promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (eg, such as an antibiotic resistance gene and genes that can be used in colorimetric assays, such as the β-galactosidase gene). 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 can be or is a recipient of a vector or an isolated polynucleotide. The host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate animal cells; fungal cells such as yeast; plant cells and insect cells. Non-limiting illustrative 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 is separated from at least some of the components with which it normally occurs in nature. For example, a polypeptide is said to be isolated when it has been separated from at least some of the components of the cell in which it is produced. When a polypeptide is secreted by a cell after expression, the physical separation of the supernatant containing the polypeptide from the cell that produced it is considered to be the release of the polypeptide. Similarly, a polynucleotide is said to be isolated when it is not part of a larger polynucleotide (such as genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it normally occurs in nature, or is separated from at least some of the components. the cell in which it is produced, for example in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector within a host cell may be designated isolated, provided that the polynucleotide does not naturally occur in the vector.

The term elevated level means a higher level of protein in a particular tissue of an individual relative to the same tissue in a control, such as an individual or individuals who do not suffer from a malignant tumor or other condition described in the present description. The elevated level can be the result of any mechanism such as increased expression, increased stability, reduced degradation, increased secretion, reduced excretion, and so on. squirrel.

The term reduce or reduces means a decrease in the level of a protein in a particular tissue of an individual by at least 10%. In some embodiments, an agent, such as an antibody that binds CSF1R, reduces the protein level in a particular tissue of an individual by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%. In some embodiments, the protein level is reduced relative to the protein level prior to contact with an agent, such as an antibody that binds CSF1R.

The term resistant, when used in the context of drug resistance, means a reduced response or no response to a standard dose of a drug, relative to the individual's response to a standard dose of a drug in the past, or relative to the expected response of a similar individual with a similar disorder to a standard dose of a drug. Thus, in some embodiments, the individual may be drug-resistant although the individual has not been previously administered the drug, or the individual may develop drug resistance after responding to the drug on one or more previous occasions.

The terms individual and patient are used interchangeably herein to refer to a person. In some embodiments, methods of treating mammals are also provided, including, but not limited to, rodents, monkeys, cats, dogs, horses, bulls, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and pets. -mammals.

- 10 039894

The term sample, as used herein, refers to a composition that is derived from or originates from an individual that contains a cellular and/or other molecular structure to be characterized, quantified and/or identified, for example based on physical, biochemical, chemical and/ or physiological characteristics. An illustrative image is a fabric sample.

The term tissue sample refers to a collection of similar cells obtained from an individual's tissue. The source of the tissue sample may be solid tissue, for example, from a fresh, frozen and/or preserved organ or tissue sample, or a biopsy or aspirate; blood or any blood components; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, synovial fluid or interstitial fluid; cells of any age of gestation or development of the individual. In some embodiments, the tissue sample is a synovial tissue biopsy and/or a synovial fluid sample. In some embodiments, the tissue sample is a synovial fluid sample. The tissue sample may be primary or cultured cells or cell lines. Optionally, the tissue sample is obtained from a diseased tissue/organ. The tissue sample may contain compounds that are not naturally mixed with tissue, such as preservatives, anticoagulants, buffers, fixers, nutrients, antibiotics, and the like. A control sample or control tissue, as used herein, refers to a sample, cell or tissue obtained from a known source or presumed to be free from the disease for which the individual is being treated.

For the purposes of this specification, a section of a tissue sample means a portion or fragment of a tissue sample, such as a section of tissue or cells, excised from a solid tissue sample.

The term cancer is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. The tumor can be benign (also referred to as a benign tumor), precancerous, or malignant.

The cancer cells may be solid cancer cells or leukemic cancer cells.

The term cancer growth is used herein to refer to the proliferation or growth of a cancer-forming cell or cells that results in a corresponding increase in the size or grade of the cancer.

Examples of a malignant tumor include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific, non-limiting examples of such cancers include squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), lung adenocarcinoma, squamous cell lung carcinoma, peritoneal cancer, hepatic cell carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, female vulva cancer, thyroid cancer, liver carcinoma, brain malignancy, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder carcinoma, cancer stomach, melanoma and various types of head and neck cancer (including squamous cell carcinoma of the head and neck).

The term recurrent cancer refers to a cancer that has returned after a treatment regimen after which there was a period of time during which the cancer could not be detected.

The term progressive cancer is cancer that has increased in size or spread since the start of a treatment regimen. In certain embodiments, a progressive cancer is a cancer that has grown in size, or a tumor that has spread by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% after the start of the treatment regimen.

A chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and Cytoxan® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamin; acetoginins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog of topotecan); bryostatin; callistatin; CC1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW2189 and CB1-TM1); eleutherobin; pankriastatin; sarcodictyin; spongistatin; nitrogen mustards, such

- 11 039894 as chlorambucil, chlornaphasine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembihin, phenesterin, prednemustine, trofosfamide, uracil mustard; nitrosourea compounds such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as endiine antibiotics (e.g. calicheamicin, especially calicheamycin gamma 1I and calicheamycin omega I1 (see, for example, Agnew, Speth Intl. Ed. Engl., 33: 183186 (1994)); dynemycin, including dynemycin A; bisphosphonates such as clodronate; esperamycin; as well as the chromophore neocarcinostatin and related chromoprotein chromophores based on endiine antibiotics), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, sactinomycin, carabicin, carminomycin, carcinophyllin, chromomycins, dactinomycin, daunorubicin, 6-diazorubicin -oxo-b-norleucine, doxorubicin Adriamycin® (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marsellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins , peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; adrenal depressants such as aminoglutethimide, mitotane, trilostane; a folate replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elfornitine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pyrarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anvidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel Taxol® (Bristol-Myers Squibb Oncology, Princeton, N.J.), a cremophor-free formulation of Abraxane® albumin engineered paclitaxel nanoparticles (American Pharmaceutical Partners, Schaumberg, Illinois), and Taxotere® doxetaxel (Rhone-Poulenc Rorer, Anthony, France); chloranbucil; Gemzar® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine® vinorelbine; novantron; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including an irinotecan treatment regimen with 5FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (eg erlotinib (Tarceva®)) and VEGF-A which reduces cell proliferation; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

The following non-limiting exemplary chemotherapeutic agents include anti-hormonal agents that act to regulate or inhibit the action of hormones on cancer, such as antiestrogen and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxy tamoxifen , trioxifene, keoxifene, LY117018, Fareston® onapristone and toremifene; aromatase inhibitors that inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, Megase® Magestrol acetate, Aromasin® exemestane, formestane, fadrozole, Rivisor® vorozol, Femara® lectrozole, and Arimidex anastrozole ®; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxcitabine (1,3-dioxolane nucleoside analogue of cytosine); antisense oligonucleotides, in particular oligonucleotides that inhibit the expression of genes in signal transduction cascades involved in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as an inhibitor of VEGF expression (eg Angiozyme® ribozyme) and an inhibitor of HER2 expression; vaccines such as gene therapy vaccines, eg Allovectin® vaccine, Leuvectin® vaccine and Vaxid® vaccine; rIL-2 Proleukin®; topoisomerase 1 inhibitor Lurtotecan®; Abarelix® rmRH and pharmaceutically acceptable salts, acids and derivatives of any of those described above.

An anti-angiogenic agent or an angiogenesis inhibitor refers to a small molecule, polynucleotide (including, for example, inhibitory RNA (RNA-i or siRNA)), polypeptide, isolated protein, recombinant protein, antibody, or conjugates or fusion proteins thereof, that inhibit angiogenesis, vascular formation, or unwanted vascular permeability, either directly,

- 12 039894 or indirectly. An anti-angiogenic agent should be understood to include agents that bind to and block the angiogenic activity of an angiogenic factor or its receptor. For example, an anti-angiogenic agent is an antibody or other angiogenic agent antagonist, such as anti-VEGF-A (eg, bevacizumab (Avastin®)) or VEGF-A receptor (eg, KDR receptor or Flt-1 receptor) antibodies, PDGFR inhibitors such as such as Gleevec® (imatinib mesylate), small molecule compounds that block VEGF signaling (e.g. PTK787/ZK2284, SU6668, Sutent®/SU11248 (sunitinib malate), AMG706 or the agents described, for example, in international patent application WO 2004/113304 ). Anti-angiogenic agents also include native inhibitors of angiogenesis, such as angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore (1991) Annu. Rev. physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179 (eg, Table 3 listing anti-angiogenic agents for malignant melanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (eg, Table 2 listing known anti-angiogenic factors); and Sato (2003) Int. J. Ciin. oncol. 8:200-206 (eg, Table 1 listing anti-angiogenic agents used in clinical trials).

A growth inhibitory agent, as used herein, refers to a compound or composition that inhibits the growth of a cell (such as a cell expressing VEGF), either in vitro or in vivo. Thus, the growth inhibitory agent may be an agent that significantly reduces the percentage of cells (such as a cell expressing VEGF) in S phase. Examples of growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (at a location other than S-phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include vinca alkaloids (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that effect G1 arrest also act on the S phase, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1 under the title Cell cycle regulation, oncogenes, and antineoplastic drugs by Murakami et al. (W.B. Saunders, Philadelphia, 1995), for example, p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs, both of which are derived from the yew tree. Docetaxel (Taxotere®, Rhone-Poulenc Rorer), derived from European yew, is a semi-synthetic analogue of paclitaxel (Taxol®, Bristol-Myers Squibb). Paclitaxel and docetaxel stimulate the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to inhibition of mitosis in cells.

The term antineoplastic composition refers to a composition suitable for the treatment of cancer, containing at least one active drug. Examples of drugs include, but are not limited to, for example, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, cancer immunotherapy agents, apoptotic agents, antitubulin agents, and other cancer treatment agents. such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonist (eg, tyrosine kinase inhibitor), HER1/EGFR inhibitor (eg, erlotinib (Tarceva®), platelet-derived growth factor inhibitors (eg, Gleevec® (imatinib mesylate)), COX-2 inhibitor (eg, celecoxib), interferons, CTLA4 inhibitors (eg, anti-CTLA antibody ipilimumab (YERVOY®)), TIM3 inhibitors (eg, anti-TIM3 antibodies), cytokines, antagonists (eg, neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, Er bB4, PDGFR-beta, BlyS, APRIL, BCMA, CTLA4, TIM3 or VEGF receptor(s), TRAIL/Apo2 and other bioactive and organic chemicals, etc. The invention also includes combinations thereof.

An agent is an antagonist of factor activity when the agent neutralizes, blocks, inhibits, eliminates, reduces and/or interferes with the activity of the factor, including its binding to one or more receptors, when the factor is a ligand.

Treatment, as used within the scope of the invention, refers to both therapeutic treatment and prophylactic or preventive measures, where the object is to prevent or slow down (reduce) the target pathological condition or disorder. In certain embodiments, the term treatment encompasses any administration or use of a therapeutic agent for a disease in a mammal, including a human, and includes inhibiting or slowing the disease or the progression of the disease; partial or complete mitigation of the disease, for example, by providing regression or restoration or repair of lost, missing or defective function; stimulating an ineffective process or providing a reduction in the severity of the plateau phase of the disease. The term treatment also includes reducing the severity of any phenotypic characteristic and/or reducing the occurrence, extent or likelihood of that characteristic. Individuals in need of treatment include individuals who already have a disorder, as well as individuals who are prone to having a disorder, or individuals in whom the disorder is intended to be prevented.

The term effective amount or therapeutically effective amount refers to an amount of a drug effective to treat a disease or disorder in an individual. In certain embodiments, an effective amount refers to an amount effective, at the required dosages and for the required periods of time, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an anti-CSF1R antibody and/or an immunostimulatory agent of the invention may vary depending on such factors as disease state, age, sex and body weight of the individual and the ability of the antibody or antibodies to induce the desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or deleterious effects of the antibody or antibodies are outweighed by the therapeutically beneficial effects. In some embodiments, an effective amount refers to an amount of an antibody that is effective in treating cancer.

A prophylactically effective amount refers to an amount effective, at the required dosages and for the required periods of time, to achieve the desired prophylactic result. Usually, but not necessarily, since a prophylactic dose is used in individuals before or at an early stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

The introduction in combination with one or more additional drugs includes simultaneous (joint) and sequential (alternate) administration in any order.

A pharmaceutically acceptable carrier refers to a non-toxic solid, semi-solid, or liquid excipient, diluent, encapsulating material, formulation aid, or carrier, generally accepted in the art for use with a drug, which together constitute a pharmaceutical composition for administration to an individual. The pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed, and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the composition used. For example, if the drug is administered orally, the carrier may be a gelatin capsule. If the drug is administered subcutaneously, the carrier ideally is not irritating to the skin and does not cause a reaction at the injection site.

Antibodies against CSF1R

Anti-CSF1R antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies containing the heavy chain and/or light chain CDRs described herein.

Illustrative humanized antibodies

In some embodiments, humanized antibodies are provided that bind CSF1R.

Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or abolish the human immune response to non-human antibodies (such as the human anti-mouse (HAMA) response), which can lead to an immune response to the antibody therapeutic agent and reduced efficacy. therapeutic agent.

Non-limiting illustrative humanized antibodies include huAb1 to huAb16 described herein. Non-limiting exemplary humanized antibodies also include antibodies comprising the heavy chain variable region of an antibody selected from huAb1-huAb16 and/or the light chain variable region of an antibody selected from huAb1-huAb16. Non-limiting exemplary humanized antibodies include antibodies comprising a heavy chain variable region selected from SEQ ID NOs: 39-45 and/or a light chain variable region selected from SEQ ID NOs: 46-52. Exemplary humanized antibodies also include, but are not limited to, humanized antibodies comprising the heavy chain CDR1, CDR2 and CDR3 and/or the light chain CDR1, CDR2 and CDR3 of an antibody selected from 0301, 0302 and 0311.

In some embodiments, a humanized anti-CSF1R antibody comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, and CDR3 of an antibody selected from 0301, 0302, and 0311. Non-limiting illustrative humanized anti-CSF1R antibodies include antibodies including CDR1, CDR2 sets and heavy chain CDR3 selected from SEQ ID NOs: 15, 16 and 17; SEQ ID NOs: 21, 22 and 23; and SEQ ID NOs: 27, 28, and 29. Non-limiting illustrative humanized anti-CSF1R antibodies also include antibodies containing sets of light chain CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25 and 26; and SEQ ID NOs: 30, 31 and 32.

Non-limiting illustrative humanized antibodies against CSF1R include antibodies containing sets of heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3, shown in table. 1 (indicated by SEQ ID NO; see Table 8 for sequences). Each row in the table one

- 14 039894 shows heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of an exemplary antibody.

Table 1. Heavy chain and light chain CDRs

heavy chain light chain Ab CDR1 SEQ ID CDR2 SEQ ID CDR3 SEQ ID CDR1 SEQ ID CDR2 SEQ ID CDR3 SEQ ID 0301 fifteen sixteen 17 eighteen nineteen 20 0302 21 22 23 24 25 26 0311 27 28 29 thirty 31 32

The following exemplary humanized antibodies In some embodiments, a humanized anti-CSF1R antibody comprises a heavy chain containing 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 selected from SEQ ID NO: 9, 11, 13 and 39-45 and wherein the antibody binds CSF1R. In some embodiments, a humanized anti-CSF1R antibody comprises a light chain containing 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 selected from SEQ ID NOs: 10, 12 , 14 and 46-52, where the antibody binds CSF1R. In some embodiments, a humanized anti-CSF1R antibody comprises a heavy chain containing 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 selected from SEQ ID NOs: 9, 11, 13 and 39-45; and a light chain containing 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: 10, 12, 14 and 46-52; where the antibody binds CSF1R.

As used within the scope of the invention, the presence of a particular polypeptide, for example, at least 95% identity with an amino acid sequence can be determined using, for example, a computer program. When determining whether a particular sequence is, for example, 95% identical to a reference sequence, percent identity is calculated over the entire length of the reference amino acid sequence.

In some embodiments, a humanized anti-CSF1R antibody comprises at least one of the CDRs described herein. Thus, in some embodiments, a humanized anti-CSF1R antibody comprises at least one CDR selected from a heavy chain CDR1 as described herein, a heavy chain CDR2 as described herein, a heavy chain CDR3 as described herein, a light chain CDR1 described in the present description, the CDR2 of the light chain described in the present description, and the CDR3 of the light chain described in the present description. In addition, in some embodiments, the humanized anti-CSF1R antibody comprises at least one mutant CDR based on the CDR described herein, wherein the mutant CDR contains 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. One skilled 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 predicted not to significantly alter the binding properties of an antibody containing the mutant CDR.

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

Exemplary 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 a heavy chain constant region of an isotype selected from IgA, IgG, and

- 15 039894

IgD. In some embodiments, the human light chain constant region is a light chain constant region selected from k and X. In some embodiments, the humanized antibody described herein comprises a human IgG constant region. In some embodiments, a humanized antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, the humanized antibody described herein contains the 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 light chain.

The choice of the heavy chain constant region may determine whether the antibody will have effector function in vivo. In some embodiments, such effector function includes antibody dependent cell mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) and may result in the killing of the cell to which the antibody has bound. In some treatments, including treatments for certain cancers, it may be desirable to kill cells, such as when an antibody binds to a cell that supports the existence or growth of a tumor. Exemplary cells that may support the existence or growth of a tumor include, but are not limited to, tumor cells themselves, cells that promote vessel recruitment to the tumor, and cells that provide ligands, growth factors, or counter-receptors that support or stimulate growth. tumor or tumor survival. In some embodiments, when an effector function is desired, an anti-CSF1R antibody comprising a human IgG1 heavy chain or a human IgG3 heavy chain is selected.

The antibody can be humanized in any way. Non-limiting exemplary humanization methods include those described in, for example, US Pat. Nos. 5,530,101; 5585089; 5693761; 5693762; 6180370; 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.

As noted above, a humanized antibody is an antibody in which at least one amino acid in a non-human variable region framework has been replaced with an amino acid from the corresponding position in the human framework. In some embodiments, 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 non-human variable framework regions are replaced by an amino acid from one or more corresponding positions in one or more human framework regions.

In some embodiments, some of the corresponding human amino acids used for replacement are from the framework regions of various human immunoglobulin genes. Thus, in some such embodiments, one or more of the non-human amino acids may be replaced by the corresponding amino acids from the human framework region of the first human antibody, or encoded by the first human immunoglobulin gene, one or more of the non-human amino acids may be replaced by the corresponding amino acids from the human framework. human region of the second human antibody or encoded by the second human immunoglobulin gene, one or more of the non-human amino acids may be replaced by corresponding amino acids from the human framework region of the third human antibody or encoded by the third human immunoglobulin gene, etc. Furthermore, in some embodiments, all of the corresponding human amino acids used for substitution in one framework, eg FR2, do not need to be from the same human framework. However, in some embodiments, all of the corresponding human amino acids used for replacement are from the same human antibody or are encoded by the same human immunoglobulin gene.

In some embodiments, the 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 with the non-human framework region being replaced. In some embodiments, such a humanized antibody is a CDR-grafted antibody.

In some embodiments, following CDR transplantation, one or more of the framework amino acids are replaced back with the corresponding amino acid in the mouse framework region. In some embodiments, such backmutations are carried out to retain one or more murine backbone amino acids that appear to contribute to the structure of one or more of the CDRs and/or that may be involved in antigenic contacts and/or appear to be involved in the overall structural integrity of the antibody. In some embodiments, ten or less, nine or less, eight or less, seven or less, six or less, five or less, four or less, three or less, two or less, one or zero are introduced into the framework regions of the antibody after CDR grafting.

- 16 039894 back mutations.

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

Illustrative chimeric antibodies

In some embodiments, the anti-CSF1R antibody is a chimeric antibody. In some embodiments, the anti-CSF1R antibody comprises at least one non-human variable region and at least one human constant region. In some such embodiments, all of the variable regions of the anti-CSF1R antibody are non-human variable regions and all of the constant regions of the anti-CSF1R antibody are human constant regions. In some embodiments, one or more of the variable regions of the chimeric antibody are mouse variable regions. The human constant region in the chimeric antibody must not be of the same isotype as the non-human constant region, if any, that it replaces. Chimeric antibodies are discussed, for example, in US patent No. 4816567 and Morrison et al. Proc. Natl. Acad. sci. USA 81: 6851-55 (1984).

Non-limiting illustrative chimeric antibodies include chimeric antibodies containing the heavy and/or light chain variable regions of an antibody selected from 0301, 0302, and 0311. Additional non-limiting illustrative chimeric antibodies include chimeric antibodies containing heavy chain CDR1, CDR2 and CDR3 and/or CDR1, CDR2 and a light chain CDR3 of an antibody selected from 0301, 0302 and 0311.

Non-limiting illustrative anti-CSF1R chimeric antibodies include those containing the following pairs of heavy and light chain variable regions: SEQ ID NOS: 9 and 10; SEQ ID NOs: 11 and 12; and SEQ ID NOs: 13 and 14.

Non-limiting exemplary anti-CSF1R antibodies include those containing the set of heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 shown in Table 1 above. one.

The following illustrative chimeric antibodies

In some embodiments, a chimeric anti-CSF1R antibody comprises a heavy chain containing 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 selected from SEQ ID NOs: 9, 11, 13 and 39-45 where the antibody binds CSF1R. In some embodiments, a chimeric anti-CSF1R antibody comprises a light chain containing 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 selected from SEQ ID NOs: 10, 12 , 14 and 4652, where the antibody binds CSF1R. In some embodiments, a chimeric anti-CSF1R antibody comprises a heavy chain containing 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 selected from SEQ ID NOs: 9, 11, 13 and 39-45; and a light chain containing 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: 10, 12, 14 and 46-52; where the antibody binds CSF1R.

In some embodiments, a chimeric anti-CSF1R antibody comprises at least one of the CDRs described herein. In other words, in some embodiments, the chimeric anti-CSF1R antibody comprises at least one CDR selected from a heavy chain CDR1 as described herein, a heavy chain CDR2 as described herein, a heavy chain CDR3 as described herein, a light chain CDR1 described in the present description, the CDR2 of the light chain described in the present description, and the CDR3 of the light chain described in the present description. In addition, in some embodiments, the chimeric anti-CSF1R antibody comprises at least one mutant CDR based on the CDR described herein, wherein the mutant CDR contains 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. One skilled 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 predicted not to significantly alter the binding properties of an antibody containing the mutant CDR.

Exemplary anti-CSF1R chimeric antibodies also include chimeric antibodies that compete for CSF1R binding to an antibody described herein. Thus, in

- 17 039894 some embodiments provide a chimeric anti-CSF1R antibody that competes for CSF1R binding with an antibody selected from Fab 0301, 0302 and 0311; and divalent (ie having two heavy chains and two light chains) versions of antibodies based on these Fabs.

Illustrative Chimeric Antibody Constant Regions

In some embodiments, a chimeric antibody described herein contains one or more human constant regions. In some embodiments, the human heavy chain constant region is a heavy chain constant region selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is a light chain constant region selected from k and λ. In some embodiments, the chimeric antibody described herein comprises a human IgG constant region. In some embodiments, a chimeric antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, the chimeric antibody described herein contains the S241P mutation in the human IgG4 constant region. In some embodiments, a chimeric antibody described herein comprises a human IgG4 constant region and a human light chain.

As noted above, whether an effector function is desired may depend on the particular method of treatment envisaged for the antibody. Thus, in some embodiments, when an effector function is desired, a chimeric anti-CSF1R antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when an effector function is not desired, a chimeric anti-CSF1R antibody containing a human IgG4 or IgG2 heavy chain constant region is selected.

Illustrative Human Antibodies

Human antibodies can be obtained by any suitable method. Non-limiting exemplary methods include generating human antibodies in transgenic mice that contain human immunoglobulin loci. See, for example, Jakobovits et al., Proc. Natl. 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 Pat. Nos. 5,545,807; 6713610; 6673986; 6162963; 5545807; 6300129; 6255458; 5877397; 5874299 and 5545806.

Non-limiting exemplary methods also include generating human antibodies using phage display libraries. See, for example, Hoogenboom et al., J. Mol. Biol. 227: 381-8 (1992); Marks et al., J. Mol. Biol. 222: 581-97 (1991) and PCT Publication No. WO 99/10494.

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

In some embodiments, a human anti-CSF1R antibody comprises one or more human constant regions. In some embodiments, the human heavy constant region is a heavy chain constant region of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is a light chain constant region selected from k and λ. In some embodiments, a human antibody described herein comprises a human IgG constant region. In some embodiments, a human antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, the human antibody described herein contains the S241P mutation in the human IgG4 constant region. In some embodiments, a human antibody described herein comprises a human IgG4 constant region and a human light chain.

In some embodiments, when desired, a human anti-CSF1R antibody is selected comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region. In some embodiments, when the effector function is undesired, a human anti-CSF1R antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.

Additional Illustrative Anti-CSF1R Antibodies

Illustrative CSF1R antibodies also include, but are not limited to, murine antibodies, humanized antibodies, human antibodies, chimeric antibodies, and engineered antibodies that contain, for example, one or more of the CDR sequences described herein. In some embodiments, an anti-CSF1R antibody comprises a heavy chain variable region as described herein. In some embodiments, an anti-CSF1R antibody comprises a light chain variable region as described herein. AT

- 18 039894 In some embodiments, an anti-CSF1R antibody comprises a heavy chain variable region as described herein and a light chain variable region as described herein. In some embodiments, the anti-CSF1R antibody comprises the heavy chain CDR1, CDR2, and CDR3 described herein. In some embodiments, the anti-CSF1R antibody comprises the light chain CDR1, CDR2, and CDR3 described herein. In some embodiments, an anti-CSF1R antibody comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3 as described herein.

In some embodiments, the anti-CSF1R antibody comprises the heavy chain variable region of an antibody selected from Fab 0301, 0302, and 0311. Non-limiting illustrative anti-CSF1R antibodies also include antibodies comprising the heavy chain variable region of an antibody selected from humanized huAb1-huAb16 antibodies. Non-limiting illustrative antibodies against CSF1R include antibodies containing a heavy chain variable region containing a sequence selected from SEQ ID NOS: 9, 11, 13 and 39-45.

In some embodiments, an anti-CSF1R antibody comprises a light chain variable region of an antibody selected from Fab 0301, 0302, and 0311. Non-limiting illustrative anti-CSF1R antibodies also include antibodies comprising a light chain variable region of an antibody selected from humanized huAb1-huAb16 antibodies. Non-limiting illustrative antibodies against CSF1R include antibodies containing a light chain variable region containing a sequence selected from SEQ ID NOS: 10, 12, 14 and 46-52.

In some embodiments, an anti-CSF1R antibody comprises a heavy chain variable region and a light chain variable region of an antibody selected from Fab 0301, 0302 and 0311. Non-limiting illustrative anti-CSF1R antibodies also include antibodies comprising a heavy chain variable region and a light chain variable region of an antibody selected from from humanized huAb1-huAb16 antibodies. Non-limiting illustrative antibodies against CSF1R include antibodies containing the following pairs of heavy and light chain variable regions: SEQ ID NOS: 9 and 10; SEQ ID NOs: 11 and 12; and 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; and SEQ ID NOs: 51 and 52. Non-limiting illustrative antibodies against CSF1R also include antibodies containing the following pairs of heavy and light chains: SEQ ID NOs: 33 and 34; SEQ ID NOs: 35 and 36; and SEQ ID NOs: 37 and 38.

In some embodiments, an anti-CSF1R antibody comprises heavy chain CDR1, CDR2, and CDR3 of an antibody selected from Fab 0301, 0302, and 0311. Non-limiting illustrative anti-CSF1R antibodies include antibodies comprising sets of heavy chain CDR1, CDR2, and CDR3 selected from: SEQ ID NO. : 15, 16 and 17; SEQ ID NOs: 21, 22 and 23; and SEQ ID NOs: 27, 28 and 29.

In some embodiments, an anti-CSF1R antibody comprises light chain CDR1, CDR2, and CDR3 of an antibody selected from Fab 0301, 0302, and 0311. Non-limiting illustrative anti-CSF1R antibodies include antibodies comprising sets of light chain CDR1, CDR2, and CDR3 selected from: SEQ ID NO. : 18, 19 and 20; SEQ ID NOs: 24, 25 and 26; and SEQ ID NOs: 30, 31 and 32.

In some embodiments, the anti-CSF1R antibody comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3 of an antibody selected from Fab 0301, 0302, and 0311.

Non-limiting illustrative examples of antibodies against CSF1R include antibodies containing sets of CDR1, CDR2 and CDR3 of the heavy chain and CDR1, CDR2 and CDR3 of the light chain, shown above in table. one.

The following exemplary antibodies

In some embodiments, the anti-CSF1R antibody comprises a heavy chain containing 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 NO: 9, 11, 13 and 39-45, where the antibody binds CSF1R. In some embodiments, the anti-CSF1R antibody comprises a light chain containing 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 NO: 10, 12, 14 and 46-52, where the antibody binds CSF1R. In some embodiments, the anti-CSF1R antibody comprises a heavy chain containing 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 NO: 9, 11, 13 and 39-45; and a light chain containing 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 selected from SEQ ID NOs: 10, 12, 14 and 46-19 039894

52; where the antibody binds CSF1R.

In some embodiments, an anti-CSF1R antibody comprises at least one of the CDRs described herein. In other words, in some embodiments, an anti-CSF1R antibody comprises at least one CDR selected from a heavy chain CDR1 as described herein, a heavy chain CDR2 as described herein, a heavy chain CDR3 as described herein, a light chain CDR1, described in the present description, the CDR2 of the light chain described in the present description, and the CDR3 of the light chain described in the present description. In addition, in some embodiments, the anti-CSF1R antibody comprises at least one mutant CDR based on the CDR described herein, wherein the mutant CDR contains 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. 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 predicted to not significantly alter the binding properties of an antibody containing the mutated CDR.

Exemplary anti-CSF1R antibodies also include antibodies that compete for CSF1R binding to an antibody described herein. Thus, in some embodiments, an anti-CSF1R antibody is provided that competes for CSF1R binding with an antibody selected from Fab 0301, 0302, and 0311, and bivalent (i.e., having two heavy chains and two light chains) versions of antibodies based on these Fab.

Exemplary 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 a heavy chain constant region of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is a light chain constant region selected from k 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 such embodiments, the antibody described herein contains the S241P mutation in the human IgG4 constant region. In some embodiments, an antibody described herein comprises a human IgG4 constant region and a human light chain.

As noted above, whether an effector function is desired may depend on the particular mode of treatment envisaged for the antibody. Thus, in some embodiments, when an effector function is desired, an anti-CSF1R antibody comprising a human IgG1 heavy chain constant region or a human IgG3 heavy chain constant region is selected. In some embodiments, when effector function is not desired, an anti-CSF1R antibody comprising a human IgG4 or IgG2 heavy chain constant region is selected.

Exemplary Variable Regions of Anti-CSF1R Antibody Heavy Chain

In some embodiments, heavy chain variable regions of an anti-CSF1R antibody are provided. In some embodiments, the heavy chain variable region of an anti-CSF1R antibody is a mouse variable region, a human variable region, or a humanized variable region.

The heavy chain variable region of an anti-CSF1R antibody contains CDR1, FR2, CDR2, FR3, and CDR3 of the heavy chain. In some embodiments, the heavy chain variable region of the anti-CSF1R antibody further comprises heavy chain FR1 and/or FR4. Non-limiting illustrative heavy chain variable regions include, but are not limited to, heavy chain variable regions having an amino acid sequence selected from SEQ ID NOs: 9, 11, 13, and 39-45.

In some embodiments, the heavy chain variable region of an anti-CSF1R antibody comprises a CDR1 containing a sequence selected from SEQ ID NOS: 15, 21, and 27.

In some embodiments, the heavy chain variable region of an anti-CSF1R antibody comprises a CDR2 containing a sequence selected from SEQ ID NOS: 16, 22, and 28.

In some embodiments, the heavy chain variable region of an anti-CSF1R antibody comprises a CDR3 containing a sequence selected from SEQ ID NOS: 17, 23, and 29.

Non-limiting exemplary heavy chain variable regions include, but are not limited to, heavy chain variable regions comprising sets of CDR1, CDR2, and CDR3 selected from: SEQ ID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22 and 23; and SEQ ID NOs: 27, 28 and 29.

In some embodiments, the heavy chain of an anti-CSF1R antibody comprises a variable region sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94% by at least 95%

- 20 039894 at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence selected from SEQ ID NO: 9, 11, 13 and 39-45, where heavy the chain, together with the light chain, is capable of forming an antibody that binds CSF1R.

In some embodiments, the heavy chain of an anti-CSF1R antibody contains at least one of the CDRs described herein. In other words, in some embodiments, the heavy chain of an anti-CSF1R antibody comprises at least one CDR selected from a heavy chain CDR1 as described herein, a heavy chain CDR2 as described herein, and a heavy chain CDR3 as described herein. In addition, in some embodiments, the anti-CSF1R antibody heavy chain comprises at least one mutant CDR based on the CDR described herein, wherein the mutant CDR contains 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. One skilled 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 predicted not to significantly alter the binding properties of the heavy chain containing the mutant CDR.

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

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

Exemplary Light Chain Variable Regions of an Anti-CSF1R Antibody

In some embodiments, light chain variable regions of an anti-CSF1R antibody are provided. In some embodiments, the light chain variable region of an anti-CSF1R antibody is a mouse variable region, a human variable region, or a humanized variable region.

The light chain variable region of an anti-CSF1R antibody contains the light chain CDR1, FR2, CDR2, FR3, and CDR3. In some embodiments, the light chain variable region of the anti-CSF1R antibody further comprises light chain FR1 and/or FR4. Non-limiting illustrative light chain variable regions include light chain variable regions having an amino acid sequence selected from SEQ ID NOs: 10, 12, 14, and 46-52.

In some embodiments, the light chain variable region of an anti-CSF1R antibody comprises a CDR1 containing a sequence selected from SEQ ID NOS: 18, 24, and 30.

In some embodiments, the light chain variable region of an anti-CSF1R antibody comprises a CDR2 containing a sequence selected from SEQ ID NOS: 19, 25, and 31.

In some embodiments, the light chain variable region of an anti-CSF1R antibody comprises a CDR3 containing a sequence selected from SEQ ID NOS: 20, 26, and 32.

Non-limiting exemplary light chain variable regions include, but are not limited to, light chain variable regions comprising sets of CDR1, CDR2, and CDR3 selected from: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25 and 26; and SEQ ID NOs: 30, 31 and 32.

In some embodiments, the anti-CSF1R 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 selected from SEQ ID NOs: 10, 12, 14 and 46 -52, where the light chain, together with the heavy chain, is able to form an antibody that binds CSF1R.

In some embodiments, the light chain of an anti-CSF1R antibody comprises at least one of the CDRs described herein. In other words, in some embodiments, the anti-CSF1R antibody light chain comprises at least one CDR selected from a light chain CDR1 as described herein, a light chain CDR2 as described herein, and a light chain CDR3 as described herein. In addition, in some embodiments, the anti-CSF1R antibody light chain comprises at least one mutant CDR based on the CDR described herein, wherein the mutant CDR contains 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.

- 21 039894

One skilled 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 predicted not to significantly alter the binding properties of the light chain containing the mutant CDR.

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

Illustrative additional CSF1R binding molecules

In some embodiments, additional molecules are provided that bind CSF1R. Such molecules include, but are not limited to, non-canonical scaffolds such as anticalins, adnectins, ankyrin repeats, etc. See, for example, Hosse et al., Prot. sci. 15:14 (2006); Fiedler, M. and Skerra, A., Non-Antibody Scaffolds, pp. 467-499, Handbook of Therapeutic Antibodies, Dubel, S., ed., Wiley-VCH, Weinheim, Germany, 2007.

Exemplary Properties of Anti-CSF1R Antibodies

In some embodiments, an antibody having the structure described above binds to CSF1R with a binding affinity (KD) of less than 1 nM, blocks binding of CSF1 and/or IL-34 to CSF1R, and inhibits CSF1R phosphorylation induced by CSF1 and/or IL-34.

In some embodiments, an anti-CSF1R antibody binds to the extracellular domain of CSF1R (CSF1R-ECD). In some embodiments, the anti-CSF1R antibody has a binding affinity (K _D ) for CSF1R of less than 1 nM, less than 0.5 nM, less than 0.1 nM, or less than 0.05 nM. In some embodiments, the anti-CSF1R antibody has a K _D of 0.01 to 1 nM, 0.01 to 0.5 nM, 0.01 to 0.1 nM, 0.01 to 0.05 nM, or 0 .02 to 0.05 nM.

In some embodiments, an anti-CSF1R antibody blocks ligand binding to CSF1R. In some embodiments, the anti-CSF1R antibody blocks the binding of CSF1 to CSF1R. In some embodiments, the anti-CSF1R antibody blocks the binding of IL-34 to CSF1R. In some embodiments, an anti-CSF1R antibody blocks both CSF1 and IL-34 from binding to CSF1R. In some embodiments, an antibody that blocks ligand binding binds to the extracellular domain of CSF1R. In some embodiments, an antibody blocks ligand binding to CSF1R when it reduces the level of detectable ligand binding to CSF1R by at least 50% using the assay described, for example, in US Pat. No. 8,206,715 B2, Example 7, which is incorporated herein in as a reference for any purpose. In some embodiments, the antibody reduces the level of detectable ligand binding to CSF1R by at least 60%, at least 70%, at least 80%, or at least 90%. In some such embodiments, the antibody is considered to block ligand binding by at least 50%, at least 60%, at least 70%, etc.

In some embodiments, the anti-CSF1R antibody inhibits ligand-induced phosphorylation of CSF1R. In some embodiments, the anti-CSF1R antibody inhibits CSF1-induced phosphorylation of CSF1R. In some embodiments, the anti-CSF1R antibody inhibits IL-34-induced phosphorylation of CSF1R. In some embodiments, the anti-CSF1R antibody inhibits both CSF1-induced and IL-34-induced CSF1R phosphorylation. In some embodiments, an antibody is considered to inhibit ligand-induced CSF1R phosphorylation when it reduces the level of detectable ligand-induced CSF1R phosphorylation by at least 50% using the assay described, e.g., in US Pat. No. 8,206,715 B2, Example 6, which is incorporated herein in as a link for any purpose. In some embodiments, the antibody reduces the level of detectable ligand-induced CSF1R phosphorylation by at least 60%, at least 70%, at least 80%, or at least 90%. In some such 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, the antibody inhibits monocyte proliferation and/or survival responses in the presence of CSF1 and/or IL-34. In some embodiments, an antibody is considered to be inhibitory of monocyte proliferation and/or survival responses when it reduces monocyte proliferation and/or survival responses in the presence of CSF1 and/or IL-34 by at least 50% using a described assay, e.g., US Patent No. 8206715 B2, example 10, which is included in the present description by reference for any purpose. In some embodiments, the antibody reduces monocyte proliferation and/or survival responses in the presence of CSF1 and/or IL-34 by at least 60%, at least 70%, at least 80%, or at least 90% . In some such embodiments, the antibody is considered to inhibit monocyte proliferation and/or survival responses by at least 50%, at least 60%, at least 70%, etc.

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Exemplary Immunostimulating Agents

Immunostimulatory agents may include, for example, a small molecule drug, an antibody or fragment thereof, or other biological or small molecule compounds. Examples of biological immunostimulatory agents include, but are not limited to, antibodies, antibody fragments, receptor or ligand polypeptide fragments, for example, that block receptor-ligand binding, vaccines, and cytokines. In one aspect, the antibody is a monoclonal antibody. In some aspects, the monoclonal antibody is a humanized or human antibody.

In some embodiments, at least one immunostimulatory agent comprises an agonist of an immunostimulatory molecule, including a co-stimulatory molecule, while in some embodiments, at least one immunostimulatory agent comprises an antagonist of an immune inhibitory molecule, including a co-inhibitory molecule. In some embodiments, at least one immunostimulatory agent comprises an agonist of an immunostimulatory molecule, including a costimulatory molecule found on immune cells, such as T cells. In some embodiments, at least one immunostimulatory agent comprises an antagonist of an immune inhibitory molecule, including a co-inhibitory molecule found on immune cells, such as T cells. In some embodiments, at least one immunostimulatory agent comprises an agonist of an immunostimulatory molecule, including a costimulatory molecule found on cells involved in innate immunity, such as NK cells. In some embodiments, at least one immunostimulatory agent comprises an antagonist of an immune inhibitory molecule, including a co-inhibitory molecule found on cells involved in innate immunity, such as NK cells. In some embodiments, the combination enhances an antigen-specific T cell response in the individual being treated and/or enhances the innate immune response in the individual. In some embodiments, the combination results in an enhanced antitumor response in an animal model of cancer, such as a xenograft model, as compared to administration of either an anti-CSF1R antibody or an immunostimulatory agent alone. In some embodiments, the combination results in a synergistic response in an animal model of cancer, such as a xenograft model, as compared to administration of either an anti-CSF1R antibody or an immunostimulatory agent alone.

In certain embodiments, the immunostimulatory agent targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin superfamily (IgSF). For example, an immunostimulatory agent may be an agent that targets (or specifically binds to) a member of the B7 family of membrane-bound ligands, which includes B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4 , B7-H5 (VISTA) and B7-H6, or a co-stimulatory or co-inhibitory receptor that binds specifically to a member of the B7 family. The immunostimulatory agent may be an agent that targets a member of the TNF family of membrane-bound ligands or a co-stimulatory or co-inhibitory receptor that binds specifically to a member of the TNF family. Exemplary members of the TNF and TNFR families that may be targeted by immunostimulatory agents include CD40 and CD40L, OX-40, OX-40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB) TRAIL/Apo2-L , VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α/TNFe, TNFR2, TNFa, LTeR, lymphotoxin α 1β2, FAS, FASL, RELT, DR6, TROY and NGFR. The immunostimulatory agent may be an agent, such as an antibody, targeted to an IgSF member, such as a member of the B7 family, a member of the B7 receptor family, a member of the TNF family, or a member of the TNFR family, such as those described above.

In some embodiments, the immunostimulatory agent may include (i) a protein antagonist that inhibits T cell activation (e.g., an immune checkpoint inhibitor), such as CTLA-4, LAG-3, TIM3, galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, TIM-4 and ILT4, and/or may include (ii) protein agonist that stimulates T cell activation, such as B7-1, B7-2, CD28, 41BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40 , CD40L, DR3 and CD28H.

In some embodiments, an immunostimulatory agent may include an agent that inhibits or antagonizes a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines), and in some embodiments, the immunostimulatory agent may include an agent that is an agonist of a cytokine such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNa (eg, the cytokine itself) that stimulates T cell activation. In some embodiments, immunostimulatory agents may

- 23 039894 include a chemokine antagonist such as CXCR2 (eg MK-7123), CXCR4 (eg AMD3100),

CCR2 or CCR4 (mogamulizumab).

In some embodiments, immunostimulatory agents may include inhibitory receptor antagonists on NK cells or activating receptor agonists on NK cells. For example, an anti-CSF1R antibody can be combined with a KIR antagonist, optionally together with at least one other immunostimulatory agent, such as a CD40 agonist.

Immunostimulatory agents may also include agents that inhibit TGF-β signaling, agents that enhance the presentation of tumor antigens, such as dendritic cell vaccines, GM-CSF secreting cell vaccines, CpG oligonucleotides, and imiquimod, or therapies that increase immunogenicity of tumor cells (eg anthracyclines).

Immunostimulatory agents may also include certain vaccines such as mesothelin-targeted vaccines or attenuated listeria cancer vaccines such as CRS-207.

Immunostimulatory agents may also include agents that deplete or block Treg cells, such as agents that specifically bind to CD25.

Immunostimulatory agents may also include agents that inhibit a metabolic enzyme such as indolamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase.

Immunostimulatory agents may also include agents that inhibit the formation of adenosine or inhibit the adenosine A2A receptor.

Immunostimulatory agents may also include agents that reverse/inhibit anergy or depletion of T cells and agents that trigger innate immune activation and/or inflammation at the tumor site.

An anti-CSF1R antibody can be combined with more than one immunostimulatory agent, for example a CD40 agonist and at least one additional immunostimulatory agent. An anti-CSF1R antibody, optionally together with a CD40 agonist, can be combined with a combinatorial approach that targets multiple elements of the immune cascade, such as one or more of the following: at least one agent that enhances the presentation of tumor antigens (e.g., a vaccine against based on dendritic cells, GM-CSF secreting cell vaccines, CpG oligonucleotides, imiquimod); at least one agent that inhibits immune downregulation, for example, by inhibiting the CTLA-4 cascade and/or depleting or blocking Treg or other immunosuppressive cells; therapy that stimulates positive immune regulation, for example, through agonists that stimulate the CD-137, OX-40 and/or GITR cascade and/or stimulate T cell effector function; at least one agent that systemically increases the frequency of antitumor T cells; therapy that depletes or inhibits Tregs, such as Tregs in tumors, eg using a CD25 antagonist (eg daclizumab) or via depletion using CD25-targeted beads ex vivo; at least one agent that affects the function of suppressor myeloid cells in the tumor; therapies that increase the immunogenicity of tumor cells (eg, anthracyclines); adoptive transfer of T cells or NK cells, including genetically modified cells, such as cells modified by chimeric antigen receptors (CAR-T therapy); at least one agent that inhibits a metabolic enzyme such as indolamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthase; at least one agent that reverses/inhibits anergy or depletion of T cells; therapy that triggers the activation of innate immunity and/or inflammation in the tumor area; administration of immunostimulatory cytokines or blocking of immunosuppressive cytokines.

For example, an anti-CSF1R antibody, optionally with a CD40 agonist, can be used with one or more agonist agents that ligate positive costimulatory receptors; one or more antagonists (blocking agents) that attenuate signal transduction through inhibitory receptors, such as antagonists that overcome various immunosuppressive cascades in the tumor microenvironment; one or more agents that systemically increase the frequency of antitumor immune cells, such as T cells, deplete or inhibit Treg (eg, through inhibition of CD25); one or more agents that inhibit metabolic enzymes such as IDO; one or more agents that reverse/inhibit anergy or T cell depletion; and one or more agents that trigger innate immune activation and/or inflammation in tumor regions.

In one embodiment, the at least one immunostimulatory agent comprises a CTLA-4 antagonist, such as a CTLA-4 antagonist antibody. Suitable anti-CTLA-4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.

In some embodiments, at least one immunostimulatory agent comprises a LAG-3 antagonist, such as a LAG-3 antagonist antibody. Suitable anti-LAG3 antibodies include, for example, BMS-986016 (WO10/19570, WO14/08218) or IMP-731 or IMP-321 (WO

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08/132601, WO 09/44273).

In some embodiments, at least one immunostimulatory agent comprises a CD137 (4-1BB) agonist, such as a CD137 agonist antibody. Suitable antibodies against

CD137 include, for example, urelumab or PF-05082566 (WO 12/32433).

In some embodiments, at least one immunostimulatory agent comprises a GITR agonist, such as a GITR agonist antibody. Suitable anti-GITR antibodies include, for example, TRX-518 (WO 06/105021, WO 09/009116), MK-4166 (WO 11/028683) or the anti-GITR antibody described in WO 2015/031667.

In some embodiments, at least one immunostimulatory agent comprises an OX40 agonist, such as an OX40 agonist antibody. Suitable anti-OX40 antibodies include, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888; WO 06/029879).

In some embodiments, at least one immunostimulatory agent comprises a CD27 agonist, such as a CD27 agonist antibody. Suitable anti-CD27 antibodies include, for example, varlilumab (CDX-112 7).

In some embodiments, the implementation of at least one immunostimulatory agent includes MGA271, which targets B7H3 (WO 11/109400).

In some embodiments, at least one immunostimulatory agent comprises a KIR antagonist, such as lirilumab.

In some embodiments, at least one immunostimulatory agent comprises an IDO antagonist.

IDO antagonists include, for example, INCB-024360 (WO 2006/122150, WO 07/75598, WO 08/36653, WO 08/36642), indoxymod, NLG-919 (WO 09/73620, WO 09/1156652, WO 11/ 56652, WO 12/142237) or F001287.

In some embodiments, at least one immunostimulatory agent comprises a Toll-like receptor agonist, eg, a TLR2/4 agonist (eg, Bacillus Calmette Guerin); a TLR7 agonist (eg hiltonol or imiquimod); a TLR7/8 agonist (eg resiquimod) or a TLR9 agonist (eg CpG7909).

In some embodiments, at least one immunostimulatory agent comprises a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197, or IMC-TR1.

CD40 agonists and exemplary CD40 agonist molecules

In some embodiments, at least one immunostimulatory agent comprises a CD40 agonist, optionally together with at least one additional immunostimulatory agent, as described herein. The cell surface molecule CD40 is a member of the tumor necrosis factor receptor superfamily and is expressed on antigen presenting cells such as dendritic cells, B cells, macrophages, and monocytes, and is also expressed on other cell types, including immune, hematopoietic, vascular, and epithelial cells, and also on various tumor cells. In antigen presenting cells, CD40 signaling leads to the activation and upregulation of costimulatory T cell molecules and other important immune mediators required to induce an immune response. CD40 agonists are potential agents for cancer therapy, causing tumor regression both through the activation of antitumor immunity and through a direct cytotoxic effect on tumor cells. CD40-targeting therapies have been clinically evaluated in phase 1 in patients with advanced cancer, and initial data have shown efficacy in the absence of significant toxicity.

With respect to CD40, for example, it has been shown in animal models that CD40 ligation on dendritic cells leads to the activation of cytotoxic T lymphocytes that mediate tumor destruction (Marzo et al., 2000, J. Immunol.; Todryk et al., 2001 , J. Immunol. Methods.) Activation of CD40 on macrophages leads to tumoricidal activity (Beatty et al., 2011, Science), and cytokines produced from stimulated CD40 antigen-presenting cells lead to the activation of natural killers important for tumor killing. Given the complex nature of the antitumor immune response, effective cancer therapy may require a combination of several immunotherapeutic agents. Accordingly, small molecule inhibition of CSF1R has been shown to act synergistically with blockade of the immune checkpoint by an anti-PD1 antibody in a pancreatic tumor model. See Zhu et al., 2014, Cancer Res., 74: 5057-5069. Thus, tumors that have CSF1R-expressing TAMs may be susceptible to combination therapy with an anti-CSF1R antibody and a CD40 agonist.

Exemplary CD40 agonists of the compositions and methods of the present invention include, for example, anti-CD40 antibodies that increase CD40 activity. Such antibodies can be humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies containing the heavy chain and/or light chain CDRs of the anti-CD40 antibody described herein.

Various CD40 agonist antibodies are known in the art. Non-limiting exemplary CD40 agonist antibodies include, but are not limited to, CP-870.893 (Pfizer and VLST; antibody 21.4.1 in EP 1476185 B1 and US Pat. No. 7338660; also see clinicaltrials.gov/ct2/show/ NCT02225002); dacetuzumab (Seattle Genetics; SEQ ID NOS: 98 and 99 herein; also see US Pat. No. 6,946,129 and US Pat. No. 8,303,955); RO7009789 (Roche; see, for example, clinicaltrials.gov/ct2/show/NCT 02304393); ADC-1013 (Alligator Bioscience; US Publication No. 2014/0348836; also see clinicaltrials.gov/ct2/show/NCT02379741); SEA-CD40 (Seattle Genetics; afucosylated form of the antibody containing SEQ ID NOS: 98 and 99; also see clinicaltrials.gov/ct2/show/NCT02376699); and Chi Lob 7/4 (Univ. Southampton; US Publication No. 2009/0074711; also see clinicaltrials.gov/ct2/show/NCT 01561911). See, for example, Vonderheide et al., 2013, Clin Cancer Res 19:1035.

Exemplary CD40 agonists also include recombinant CD40L.

Exemplary Antibody Conjugates

In some embodiments, the antibody is conjugated to a label and/or a cytotoxic agent. As used within the scope of the invention, a label is a portion that facilitates the detection of an antibody and/or facilitates the detection of a molecule to which the antibody binds. Non-limiting exemplary labels include, but are not limited to, radioisotopes, fluorescent groups, enzyme groups, chemiluminescent groups, biotin, epitope labels, metal binding labels, and the like. A person skilled in the art can select an appropriate label according to 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 a reduced proliferative capacity when the cell becomes less able to proliferate, for example, as the cell undergoes apoptosis or otherwise dies, the cell stops going through the cell cycle and/or does not divide, the cell differentiates, and so on. Non-limiting cytotoxic agents include, but are not limited to, radioisotopes, toxins, and chemotherapeutic agents. The person skilled in the art can select the appropriate cytotoxic agent according to the intended use.

In some embodiments, the label and/or cytotoxic agent is conjugated to the antibody using in vitro chemical methods. Non-limiting illustrative chemical conjugation methods are known in the art and include services, methods, and/or reagents commercially available from, for example, 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 the label and/or cytotoxic agent is a polypeptide, the label and/or cytotoxic agent can be expressed from the same expression vector with at least one antibody chain to form a polypeptide containing the label and/or cytotoxic agent, fused to the antibody chain. The person skilled in the art can select an appropriate method for conjugating the label and/or cytotoxic agent to the antibody according to the intended use.

Illustrative leader sequences

In order for certain secreted proteins to be expressed and secreted in large amounts, a leader sequence from a heterologous protein may be desirable. In some embodiments, the implementation of the leader sequence is selected from SEQ ID NO: 3 and 4, which are the leaders of the light chain and heavy chain, respectively. In some embodiments, the use of heterologous leader sequences may be advantageous because the resulting mature polypeptide may remain unchanged when the leader sequence is removed from the ER during the secretion process. The addition of a heterologous leader sequence may be required for the expression and secretion of certain proteins.

Certain illustrative leader sequences are described, for example, in the 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 antibodies

Provided are nucleic acid molecules containing polynucleotides that encode one or more antibody chains. In some embodiments, the nucleic acid molecule comprises a polynucleotide that encodes for the heavy chain or light chain of an antibody. In some embodiments, the nucleic acid molecule contains both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain of an antibody. In some embodiments, the first nucleic acid molecule contains a first polynucleotide that encodes for a heavy chain, and the second nucleic acid molecule contains a second polynucleotide that encodes for a light chain.

In some such embodiments, the heavy chain and light chain are expressed from a single nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, for example, when the antibody is a scFv, the single polynucleotide encodes for a single polypeptide containing both the heavy chain and

- 26 039894 and a light chain linked together.

In some embodiments, a polynucleotide encoding an antibody heavy chain or light chain contains a nucleotide sequence that encodes a leader sequence that, when translated, is located at the N-terminus of the heavy chain or light chain. As described above, the leader sequence may be a native heavy or light chain leader, or it may be another heterologous leader sequence.

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

Expression and production of antibodies

Vectors

Vectors are contemplated containing polynucleotides that encode heavy chains and/or light chains of antibodies. Also provided are vectors containing polynucleotides that encode heavy chains and/or light chains of antibodies. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, the 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 light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide, such as when the antibody is a scFv.

In some embodiments, the first vector contains a polynucleotide that encodes a heavy chain and the second vector contains 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 amounts by weight). In some embodiments, cells are transfected using a 5:1 to 1:5 molar or weight ratio of the first vector to the second vector. In some embodiments, a weight ratio of 1:1 to 1:5 is used for the heavy chain encoding vector and the light chain encoding vector. In some embodiments, a 1:2 weight ratio is used for the heavy chain encoding vector and the light chain encoding vector.

In some embodiments, a vector is selected that is optimized for polypeptide expression in CHO cells or cells derived from CHO or NSO cells. Illustrative such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

In some embodiments, the vector is selected for in vivo expression of antibody heavy chains and/or antibody light chains in animals, including humans. In some such embodiments, 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, for example, in PCT Publication No. WO 2006/076288.

Host cells

In various embodiments, the heavy chains and/or light chains of antibodies can be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells such as fungal (such as yeast), plant cells, insect cells and mammalian cells. Such expression can be carried out, for example, according to methods known in this field. Illustrative eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells including 2936E cells; CHO cells, including CHO-S and DG44 cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, heavy chains and/or light chains of antibodies can be expressed in yeast. See, for example, US Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make the desired post-translational modifications to antibody heavy chains and/or light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into the desired host cell can be carried out by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting illustrative methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ^ed . Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transfected into desired host cells transiently or stably, according to any suitable method.

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

Purification of antibodies

Antibodies can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include antigen and ligands that bind antibody constant regions. For example, a protein A, protein G, protein A/G, or antibody affinity column can be used to bind a constant region and purify an antibody. Also, hydrophobic interaction chromatography, such as a butyl or phenyl column, may be useful for purifying some polypeptides. Many methods for purifying polypeptides are known in the art.

Cell-free production of antibodies

In some embodiments, the antibody is produced in a cell-free system. Non-limiting illustrative cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695713 (2003).

Therapeutic compositions and methods Methods for treating cancer

In some embodiments, methods for treating cancer are provided, comprising administering an effective amount of an anti-CSF1R antibody and an effective amount of at least one immunostimulatory agent. In some embodiments, the anti-CSF1R antibody and at least one immunostimulatory agent are administered simultaneously. For example, the therapeutic agents may be administered by co-infusion or injected at approximately the same time. In some embodiments, the anti-CSF1R antibody and at least one immunostimulatory agent are administered sequentially. For example, in some embodiments, the anti-CSF1R antibody is administered sequentially before or after at least one immunostimulatory agent, such that the two therapeutic agents are administered 30, 60, 90, 120 minutes, 3, 6, 12, 24, 36, 48 hours apart. , 3, 5, 7 days or two weeks.

In some embodiments, at least one, at least two, at least three doses, at least five doses, or at least ten doses of anti-CSF1R antibody are administered prior to administration of at least one immunostimulatory agent. In some embodiments, at least one, at least two, at least three doses, at least five doses, or at least ten doses of at least one immunostimulatory agent are administered prior to administration of the CSF1R antibody. In some embodiments, the last dose of the immunostimulatory agent is at least one, two, three, five, or ten days, or one, two, three, five, twelve, or twenty-four weeks prior to the first dose of the CSFR1 inhibitor. In some embodiments, the last dose of the CSFR1 inhibitor is administered at least one, two, three, five, ten days, or one, two, three, five, twelve, or twenty-four weeks prior to the first dose doses. at least one immunostimulatory agent. In some embodiments, the individual has been or is being treated with at least one immunostimulatory agent and an anti-CSF1R antibody is added to the therapeutic regimen.

In some embodiments, a method is provided for selecting a patient for combination therapy with an anti-CSF1R antibody and at least one immunostimulatory agent, such as a CD40 agonist, comprising determining TAM and/or CD8+ T cell levels in the patient. In some embodiments, if the patient's TAM levels are high, the patient is selected for combination therapy. In some embodiments, if the patient's TAM and CD8+ T cell levels are high, the patient is selected for combination therapy. The level of TAM or CD8+ T cells is considered high if it is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75% or at least 100% higher than the level in an individual who does not have a malignant tumor. In some embodiments, the level of TAM or CD8+ T cells is considered high if it is above the average level found in individuals with cancer. In some embodiments, if the patient's TAM levels are high and CD8+ T cell levels are low, the patient is selected for combination therapy with an anti-CSF1R antibody and at least one immunostimulatory agent, such as a CD40 agonist. A CD8+ T cell level is considered low if it is at or below the average level found in individuals with cancer. In some embodiments, the level of CD8+ T cells is considered low if it is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, by at least 75% or at least 100% lower than the level in an individual who does not have a malignant tumor. In some embodiments, CSF1R expression on a patient's TAM is determined. In some embodiments, if the patient's TAMs express CSF1R, the patient is selected for combination therapy. In some embodiments, if the patient's TAMs express elevated levels of CSF1R, the patient is selected for combination therapy. In some embodiments, a TAM patient is considered to express elevated levels of CSF1R if the level of CSF1R is equal to or greater than the average level of expressed CSF1R detected on the TAM in individuals with cancer. In some embodiments, if a patient's CSF1R expression shows a high correlation with CD8+ T cell levels, the patient is selected for combination therapy. The correlation between expression levels is considered high if it is equal to or greater than the average level found in individuals with a malignant tumor.

TAM levels, CSF1R expression, CD8+ T cells and/or regulatory T cells can be measured by methods known in the art. Illustrative methods include immunohistochemistry (IHC), fluorescence activated cell sorting (FACS), protein arrays, and gene expression assays such as RNA sequencing, gene arrays, and quantitative PCR. In some embodiments, one or more markers selected from CSF1R, CD68, CD163, CD8, and FoxP3 can be detected by IHC, FACS, or gene expression analysis on tumor sections or dissociated cells from tumor sections.

In some embodiments, the cancer is selected from squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, peritoneal cancer, hepatic cell carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder carcinoma, gastric cancer, melanoma, and various types of head cancer and neck. In some embodiments, the lung cancer is non-small cell lung cancer or squamous cell lung carcinoma. In some embodiments, the leukemia is acute myeloid leukemia or chronic lymphocytic leukemia. In some embodiments, the breast cancer is an invasive breast carcinoma. In some embodiments, the ovarian cancer is a serous ovarian cystadenocarcinoma. In some embodiments, the kidney cancer is a clear cell carcinoma of the kidney. In some embodiments, the colon cancer is colon adenocarcinoma. In some embodiments, the bladder cancer is bladder urothelial carcinoma. In some embodiments, the cancer is selected from bladder cancer, cervical cancer (such as cervical squamous cell carcinoma), head and neck squamous cell carcinoma, rectal adenocarcinoma, non-small cell lung cancer, endometrial cancer, prostate adenocarcinoma, colon cancer, cancer ovary (such as serous epithelial ovarian cancer) and melanoma.

In some embodiments, the anti-CSF1R antibody blocks the binding of CSF1 and/or IL-34 to CSF1R and/or inhibits CSF1R phosphorylation induced by CSF1 and/or IL-34. In some embodiments, the anti-CSF1R antibody blocks the binding of CSF1 and IL-34 to CSF1R and/or inhibits CSF1R phosphorylation induced by CSF1 and/or IL-34. In some embodiments, the anti-CSF1R antibody comprises the CDRs or variable regions of an antibody selected from huAb1-huAb16 described herein. In some embodiments, the anti-CSF1R antibody comprises the CDRs or variable regions of huAb1.

In some embodiments, at least one immunostimulatory agent comprises an antagonist of a T cell activation inhibitor, while in some embodiments, at least one immunostimulatory agent comprises a T cell activation stimulator agonist. In some embodiments, at least one immunostimulatory agent comprises a CTLA4, LAG-3, galectin 1, galectin 9, CEACAM-1, BTLA, CD25, CD69, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM1, TIM3, TIM4, ILT4, IL-6, IL-10, TGFe, VEGF, KIR, LAG-3, A2A adenosine receptor, PI3K delta, or IDO. In some embodiments, at least one immunostimulatory agent comprises a B71, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD27, CD40, CD40L agonist , DR3, CD28H, IL-2, IL-7, IL-12, IL-15, IL-21, IFNa, STING, or a Toll-like receptor agonist such as a TLR2/4 agonist. In some embodiments, at least one immunostimulatory agent comprises an agent that binds to a member of the B7 family of membrane-bound proteins, such as B7-1, B7-2, B7-H2 (ICOS-L), B7-H3, B7-H4, B7 -H5 (VISTA) and B7-H6. In some embodiments, at least one immunostimulatory agent comprises an agent that binds to a member of the TNF receptor family or a co-stimulatory or co-inhibitory molecule that binds to a member of the TNF receptor family, such as CD40,

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CD40L, OX40, OX40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK , RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, EDA1, EDA2, TACI, APRIL, BCMA, LTeR, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFe, TNFR2, TNFa, 1β2 , FAS, FASL, RELT, DR6, TROY or NGFe. In some embodiments, at least one immunostimulatory agent includes an agent that is an antagonist or inhibitor of a cytokine that inhibits T cell activation, such as IL-6, IL-10, TGFe, VEGF. In some embodiments, at least one immunostimulatory agent comprises a cytokine agonist that stimulates T cell activation, such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNa. In some embodiments, at least one immunostimulatory agent comprises a chemokine antagonist such as CXCR2, CXCR4, CCR2, or CCR4. In some embodiments, at least one immunostimulatory agent comprises an antibody. In some embodiments, the at least one immunostimulatory agent may include a vaccine, such as a mesothelin-targeted vaccine or an attenuated listeria cancer vaccine, such as CRS-207.

In some embodiments, at least one immunostimulatory agent comprises a CD40 agonist, such as an anti-CD40 antibody. Non-limiting exemplary CD40 agonist antibodies include CP-870.893 (Pfizer and VLST); dacetuzumab (Seattle Genetics); RO7009789 (Roche); ACD-1013 (Alligator Bioscience); SEA-CD40 (Seattle Genetics) and Chi Lob 7/4 (Univ. Southampton). In some embodiments, the CD40 agonist is recombinant CD40L.

Routes of administration and carriers

In various embodiments, antibodies can be administered in vivo by a variety of 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 implantation or inhalation. The contemplated compositions may be formulated in solid, semi-solid, liquid or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injection forms, inhalation forms and aerosols. A nucleic acid molecule encoding an antibody can be applied to gold microparticles and delivered intradermally using a microparticle bombardment device or a gene gun as described in the literature (see, for example, Tang et al., Nature 356:152-154 (1992)) . The appropriate composition and route of administration may be selected depending on the intended use.

In various embodiments, compositions containing antibodies are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, for example, 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, 7th ed., Lippencott Williams and Wilkins (2004); ^Kibbe et al., Handbook of Pharmaceutical ^Excipients , 3rd ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers are available which include excipients, adjuvants and diluents. Moreover, various pharmaceutically acceptable adjuvants such as Ph adjusting and buffering agents, tonicity correctors, stabilizers, wetting agents, and the like are also available. Non-limiting illustrative carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

In various embodiments, compositions containing antibodies can be formulated for injection, including subcutaneous administration, by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and, if desired, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, 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. In various embodiments, the compositions can also be formulated as sustained release microcapsules, such as with biodegradable or non-biodegradable polymers. A non-limiting exemplary biodegradable formulation includes a lactic acid-glycolic acid polymer. A non-limiting exemplary non-biodegradable composition includes a glycerol fatty acid ester polymer. Certain methods for preparing such compositions are described, for example, in EP 1125584 A1.

Also contemplated are pharmaceutical packages and kits containing one or more containers, each containing one or more doses of an antibody or combination of antibodies. In some embodiments, a unit dosage is provided, where the unit dosage

- 30 039894 contains a given amount of a composition containing an antibody or a combination of antibodies with or without one or more additional agents. In some embodiments, such unit dosage is provided in a disposable pre-filled injection syringe. In various embodiments, the implementation of the composition contained in a single dosage may contain saline, sucrose, and the like; a buffer such as phosphate and the like; and/or may be formulated in a stable and effective Ph range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder, which may be reconstituted by the addition of an appropriate liquid, such as sterile water. In some embodiments, the composition contains one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, the implementation of the composition according to the invention contains heparin and/or proteoglycan.

The pharmaceutical compositions are administered in an amount effective to treat or prevent a particular indication. The therapeutically effective amount will generally depend on the weight of the individual being treated, his or her physical or health condition, the severity of the condition being treated, or the age of the individual being treated. Typically, antibodies can be administered in an amount ranging from about 10 μg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, antibodies can be administered in an amount ranging from about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, antibodies can be administered in an amount ranging from about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, antibodies can be administered in an amount ranging from about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, antibodies can be administered in an amount ranging from about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.

Antibody compositions can be administered to individuals as needed. The frequency of administration can be determined by those skilled in the art, such as the attending physician, taking into account the condition being treated, the age of the individual being treated, the severity of the condition being treated, the general health of the individual being treated, and the like. In some embodiments, an effective dose of the antibody is administered to the individual one or more times. In various embodiments, an effective dose of the antibody is administered to the individual once a month, less than once a month, such as every two months or every three months. In other embodiments, an effective dose of the antibody is administered more than once a month, such as every three weeks, every two weeks, or every week. In some embodiments, an effective dose of the antibody is administered once every 1, 2, 3, 4, or 5 weeks. In some embodiments, an effective dose of the antibody is administered two or three times a week. An effective dose of the antibody is administered to the subject at least once. In some embodiments, an effective dose of an antibody can be administered multiple times, including over periods of at least a month, at least six months, or at least a year.

Complementary Combination Therapy

The therapeutic combinations described above may be administered alone or with other treatments. They can be given before, substantially simultaneously with, or after other treatments, such as surgery, chemotherapy, radiation therapy, or administration of a biological agent such as another therapeutic antibody. In some embodiments, the cancer recurs or progresses after therapy selected from surgery, chemotherapy and radiation therapy, or a combination thereof.

For the treatment of cancer, the combinations may be co-administered with one or more additional cancer agents such as a chemotherapeutic agent, a growth inhibitory agent, a cancer vaccine such as a gene therapy vaccine, an anti-angiogenic agent, and/or an antineoplastic composition. Non-limiting examples of a chemotherapeutic agent, a growth inhibitory agent, a cancer vaccine, an anti-angiogenic agent, and an anti-neoplastic composition that can be used in combination with the antibodies of the present invention are provided herein in the Definitions section.

In some embodiments, with a combination of an anti-inflammatory drug, such as a steroidal or non-steroidal anti-inflammatory drug (NSAID).

Examples

The examples described below are intended solely to illustrate the invention and should not be considered as limiting the invention in any way. The examples do not reflect that the experiments described below are all and only experiments performed. Efforts have been made to ensure the accuracy of the quantities used (eg quantities, temperature, etc.), however, some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric.

Example 1 Humanized anti-CSF1R antibodies.

Various humanized antibodies against CSF1R have been developed in the past. See, for example, PCT Publication No. WO 2011/140249.

The sequences for all of the humanized heavy chain variable regions and the humanized light chain variable regions, aligned with the chimeric antibody parental variable region sequences and the human acceptor variable framework sequences, are shown in FIG. 1 (heavy chains) and 2 (light chains). Changes in the humanized variable region sequences relative to the acceptor variable framework sequences are boxed. Each of the CDRs for each of the variable regions is shown in a boxed region and is designated as CDR above the boxed sequences.

In table. 8 below shows the complete sequences for the humanized heavy chains and humanized light chains of the huAb1-huAb16 antibodies. The name and SEQ ID NO: humanized heavy chain and humanized light chain of each of these antibodies are presented in table. 3.

Table 3 Humanized huAb1-huAb16 heavy chains and light chains.

Humanized antibody humanized NA SEQ ID humanized LC SEQ ID NO NO huAbl h0301-H0 53 h0301-L0 60 huAb 2 h0301-hl 54 h0301-L0 60 huAb3 h0301-H2 55 h0301-L0 60 huAb 4 h0301-H0 53 h0301-Ll 61 huAb 5 h0301-hl 54 h0301-Ll 61 huAb 6 h0301-H2 55 h0301-Ll 61 huAb 7 h0302-hl 56 h0302-L0 62 huAb 8 h0302-hl 56 h0302-Ll 63 huAb 9 h0302-hl 56 h0302-L2 64 huAbl0 h0302-H2 57 h0302-L0 62 huAb11 h0302-H2 57 h0302-Ll 63 huAbl2 h0302-H2 57 h0302-L2 64 huAb 13 hO311-Hl 58 h0311-L0 65 huAbl4 hO311-Hl 58 hO311-Ll 66 huAb 15 hO311-H2 59 h0311-L0 65 huAbl 6 hO311-H2 59 hO311-Ll 66

Humanized antibodies were tested for binding to human, cynomolgus and mouse CSF1R ECDs as previously described. See, for example, PCT Publication No. WO 2011/140249. The antibodies were found to bind to both human and cynomolgus monkey CSF1R ECDs, but not to mouse CSF1R ECDs. Humanized antibodies have also been found to block CSF1 and IL-34 binding to both human and cynomolgus monkey CSF1R and inhibit CSF1-induced and IL-34-induced phosphorylation of human CSF1R expressed in CHO cells. See, for example, PCT Publication No. WO 2011/1402 49.

Ka, kd and K _D for binding to human CSF1R ECD have been previously determined and are presented in Table 1. 4. See, for example, PCT Publication No. WO 2011/140249.

Table 4. Humanized antibody binding affinity for human CSF1R

huAb k _a (M ^-1 s ^-1 ) Ka (CT ¹ ) K _D (nM) huAb 0301-L0H0 3.22x106 1.11x10-03 0.35

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huAb 0301-L0H1 3.56x106 1.22x10-03 0.34 huAb 0301-L0H2 2.32x106 6.60x10-04 0.28 huAb 0301-L1H0 3.29x106 1.15x10-03 0.35 huAb 0301-L1H1 2.87x106 9.21x10-04 0.32 huAb 0301-L1H2 2.95x106 7.42x10-04 0.25 huAb 0302-L0H1 3.54x106 3.69x10-03 1, 04 huAb 0302-L1H1 3.47x106 4.04x10-03 1, 17 huAb 0302-L2H1 1.60x106 9.14x10-04 0.57 huAb 0302-L0H2 3.40x106 1.79x10-03 0.53 huAb 0302-L1H2 2.71x106 1.53x10-03 0.56 huAb 0302-L2H2 1.84x106 8.40x10-04 0.46 huAb 0311-L0H1 1.22x106 5.40x10-04 0.44 huAb 0311-L1H1 1.32x106 6.64x10-04 0.50 huAb 0311-L0H2 1.34x106 4.73x10-04 0.35 huAb 0311-L1H2 1.51x106 6.09x10-04 0.40

Example 2 Enhancement of Antitumor Activity Using Combination Therapy.

Female C57BL/6 mice aged 6-8 weeks were kept 5 animals per cage with access to food and water without restrictions. After entering the vivarium, the mice were acclimatized for at least 3 days. Mice were weighed and their flanks were shaved prior to tumor cell line inoculation.

The mouse colon adenocarcinoma cell line MC38 is cultured in RPMI+10% FBS+2 mM L-glutamine+antibiotic/antifungal at 37°C with 5% CO ₂ . Cells are suspended in a solution of 50%/vol. DPBS and 50%/vol. matrigel at a concentration of 5 million cells/ml. 100 μl of cell solution (0.5 million cells) is implanted into the right block of each mouse using a 27G1/2 needle. Settling of cells to the bottom of the tube is prevented using a 18G needle and syringe and gentle shaking. Mice are anesthetized using isoflurane and to ensure more accurate implantation of tumor cells. The length (L) and width (W) of each tumor is measured using an electronic thickness gauge, and the tumor volume (V) is calculated using V=(LxW2)/2. After reaching an average tumor volume of approximately 105 mm ³ , mice are divided into groups and they are dosing, as discussed below.

Groups and dosing

Mice are assigned to the following groups and injected with a chimeric rat anti-mouse CSF1R antibody (mouse IgG1 referred to as cmFPA008) and/or an anti-CD40 FGK45 antibody (Bio X Cell; see Rolink et al., 1996, Immunity 5:319-330) at dosing regimens described below.

Control: mouse IgG, 30 mg/kg, ip 1x/wk starting on day 0. Rat IgG, 100 µg ip on day 0.

huAb1: anti-CSF1R antibody, 30 mg/kg, ip. 1x/wk starting on day 0. Rat IgG, 100 µg ip on day 0.

Anti-CD40 antibody (high): Anti-CD40 antibody, 100 µg ip on day 0. Mouse IgG, 30 mg/kg, ip, 1x/wk starting on day 0.

Combination (high): anti-CSF1R antibody 30mg/kg ip, 1x/wk starting on day 0, anti-CD40 antibody 100mcg ip on day 0.

Anti-CD40 antibody (low): Anti-CD40 antibody, 30 µg, ip on day 0. Mouse IgG, 30 mg/kg, ip, 1x/wk starting on day 0.

Combination (low): anti-CSF1R antibody 30mg/kg ip, 1x/wk starting on day 0, anti-CD40 antibody 30mcg ip on day 0.

The dosing scheme and distribution into groups are shown below in table. 5.

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Table 5. Dosing and allocation to groups in the study

Group Treatment #one: chimeric Abi Dosing (mg/kg, scheme) Treatment #2: antibody against CD40 Dosing (mg/kg, scheme) Mice (P) one mouse IgG 30, 1x per week (gw) Rat IgG 100 mcg per 0 day ten 2 Antibody against CSF1R ten 3 mouse IgG Antibody against CD40 100 mcg per 0 day ten 4 Antibody against CSF1R ten 5 mouse IgG 3 0 mcg per 0 day ten 6 Antibody against CSF1R ten

Animals are sacrificed before the end of the study if any of the following signs are observed:

weight loss equal to or greater than 15% of initial body weight;

see ulceration of the tumor;

mice look dying;

the individual tumor volume is equal to or greater than 10% of the initial body weight.

Plasma is taken for pharmacokinetic (RK) analysis. On the last day of the study, whole blood is taken by means of intracardiac sampling and plasma is isolated for PK analysis (bioanalytical group). At least five (5) tumors from each group are collected for the next analysis. Single tumor cell isolates are obtained by collagenase treatment and FACS is used to study immune cell infiltration into the tumor(s). Tumor sections are also snap frozen in liquid nitrogen and stored at -80° C. for protein and mRNA extraction. Tumor sections are embedded in Optimum Cutting Temperature (OST) compound and stored at -80°C. Tumor sections were also placed in 10% buffered formalin overnight and then transferred to 70% ethanol the following day.

The results of this experiment are shown in Fig. 3 and 4. As shown in FIG. 3, the combination of anti-CSF1R antibody and anti-CD40 antibody (high) resulted in early tumor regression followed by tumor stasis. The combination of anti-CSF1R antibody and anti-CD40 antibody (low) also showed higher efficacy than either of the treatments alone. In FIG. 4 shows individual tumor volumes on (A) Day 11 and (B) Day 13. Table 6 shows tumor volume statistics at day 11 and day 13 using one-way ANOVA. The combination of anti-CSF1R antibody and anti-CD40 antibody (high) showed significantly better efficacy than either of the treatments alone.

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Table 6 Tumor Volume Statistics

Group IgG/IgG 11 days Medium %TGI 0.00 p value Day 13 Average TV+SD 942.4± 278.3 %TGI 0.00 p value TV+SD 729.3 183.8 + Antibody against CSF1R /IgG against combination 579, 2 87, 87 + 20.58 0.0037 710.0 ± 80, 05 24, 66 0.0004 (low) IgG/anti-CD40 antibody (low) vs combination 452.5 111.9 + 37.95 0.5888 ns 645.2± 162.0 31.53 0.0139 (low) IgG/anti-CD40 antibody (high) vs combination (high) 450.8± 105.6 38.18 < 0.0001 650.6± 151.5 30, 96 < 0.0001 Combination (high) against anti-CSFIR/IgG antibody 157.4± 55.00 78.41 < 0.0001 223.3± 98.32 76, 31 < 0.0001

In FIG. 5 shows the body weight for all animals in the study measured at least twice a week. No significant difference in weight was observed for any of the groups relative to the control.

Sequence table

In table. 10 presents certain sequences described in the present description. All polypeptide and antibody sequences are shown without leader sequences unless otherwise indicated.

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Table 10. Sequences and descriptions

SEQ ID NO Description Subsequence one hCSFIR (full dimensional, without leader sequence) IPVIEPSVPE LWKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVWFE DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKF IQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEA AQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVD FQHAGNYSCV ASNVQGKHST SMFFRWESA YLNLSSEQNL IQEVTVGEGL NLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLP RLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLC AASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQ SLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHTHP pdEFLFTPW VACMSIMALL LLLLLLLLYK YKQKPKYQVR WKIIESYEGN SYTFIDPTQL PYNEKWEFPR NNLQFGKTLG AGAFGKWEA TAFGLGKEDA VLKVAVKMLK STAHADEKEA LMSELKIMSH LGQHENIVNL LGACTHGGPV LVITEYCCYG DLLNFLRRKA EAMLGPSLSP GQDPEGGVDY KNIHLEKKYV RRDSGFSSQG VDTYVEMRPV STSSNDSFSE QDLDKEDGRP LELRDLLHFS SQVAQGMAFL ASKNCIHRDV AARNVLLTNG HVAKIGDFGL ARDIMNDSNY IVKGNARLPV KWMAPESIFD CVYTVQSDVW SYGILLWEIF SLGLNPYPGI LVNSKFYKLV KDGYQMAQPA FAPKNIYSIM QACWALEPTH

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RPTFQQICSF LQEQAQEDRR ERDYTNLPSS SRSGGSGSSS SELEEESSSE HLTCCEQGDI AQPLLQPNNY QFC 2 hCSFIR (full dimensional + leader sequence ) MGPGVLLLLL VATAWHGQGI PVIEPSVPEL WKPGATVTL RCVGNGSVEW DGPPSPHWTL YSDGSSSILS TNNATFQNTG TYRCTEPGDP LGGSAAIHLY VKDPARPWNV LAQEVWFED QDALLPCLLT DPVLEAGVSL VRVRGRPLMR HTNYSFSPWH GFTIHRAKFI QSQDYQCSAL MGGRKVMSIS IRLKVQKVIP GPPALTLVPA ELVRIRGEAA QIVCSASSVD VNFDVFLQHN NTKLAIPQQS DFHNNRYQKV LTLNLDQVDF QHAGNYSCVA SNVQGKHSTS MFFRWESAY LNLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPK LANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRY PPEVSVIWTF INGSGTLLCA ASGYPQPNVT WLQCSGHTDR CDEAQVLQVW DDPYPEVLSQ EPFHKVTVQS LLTVETLEHN QTYECRAHNS VGSGSWAFIP ISAGAHTHPP DEFLFTPVW ACMSIMALLL LLLLLLLYKY KQKPKYQVRW KIIESYEGNS YTFIDPTQLP YNEKWEFPRN NLQFGKTLGA GAFGKWEAT AFGLGKEDAV LKVAVKMLKS TAHADEKEAL MSELKIMSHL GQHENIVNLL GACTHGGPVL VITEYCCYGD LLNFLRRKAE AMLGPSLSPG QDPEGGVDYK NIHLEKKYVR RDSGFSSQGV DTYVEMRPVS TSSNDSFSEQ DLDKEDGRPL ELRDLLHFSS QVAQGMAFLA SKNCIHRDVA ARNVLLTNGH VAKIGDFGLA RDIMNDSNYI VKGNARLPVK WMAPESIFDC VYTVQSDVWS YGILLWEIFS LGLNPYPGIL VNSKFYKLVK DGYQMAQPAF APKNIYSIMQ ACWALEPTHR PTFQQICSFL QEQA QEDRRE RDYTNLPSSS RSGGSGSSSS ELEEESSSEH LTCCEQGDIA QPLLQPNNYQ EU 5 hCSFIR ECD.506 IPVIEPSVPE LWKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFE DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKF IQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEA AQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVD FQHAGNYSCV ASNVQGKHST SMFFRWESA YLNLSSEQNL IQEVTVGEGL NLKVMVEAYP GLQGFNWTYL

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GPFSDHQPEP KLANATTKDT YRHTFTLSLP RLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLC AASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQ SLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAH 6 hCSFIR ECD.506-Fc IPVIEPSVPE LWKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVWFE DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKF IQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEA AQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVD FQHAGNYSCV ASNVQGKHST SMFFRWESA YLNLSSEQNL IQEVTVGEGL NLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLP RLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLC AASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQ SLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHEPK SSDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 7 cynoCSFIR ECD (c leader sequence) MGPGVLLLLL VVTAWHGQGI PVIEPSGPEL WKPGETVTL RCVGNGSVEW DGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY VKDPARPWNV LAKEVWFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIP GPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRS DFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MFFRWESAY LDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPK LANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRY PPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR

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CDEAQVLQVW VDPPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIP ISAGAR eight cynoCSFIR ECD-Fc (with leader sequence ) MGPGVLLLLL WTAWHGQGI PVIEPSGPEL WKPGETVTL RCVGNGSVEW DGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY VKDPARPWNV LAKEVWFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIP GPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRS DFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MEERWESAY LDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPK LANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRY PPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR CDEAQVLQVW VDPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIP ISAGARGSEP KSSDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 3 Light chain leader sequence METDTLLLWV LLLWVPGSTG 4 Heavy chain leader sequence MAVLGLLLCL VTFPSCVLS nine Fab Heavy Chain Variable Region EVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD INPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARES PYFSNLYVMD YWGQGTSVTV SS

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0301 ten Light chain variable region Fab 0301 NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL LIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLS TFGGGTKLEI K eleven Heavy chain variable region Fab 0302 EIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY INPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYF DGTFDYALDY WGQGTSITVS S 12 Light chain variable region Fab 0302 DVWTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL LIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPW TFGGGTRLEI K thirteen Heavy chain variable region Fab 0311 EIQLQQSGPD LMKPGASVKM SCKASGYIFT DYNMHWVKQN QGKSLEWMGE INPNNGVWY NQKFKGTTTL TVDKSSSTAY MDLHSLTTSED SAVYYCTRAL YHSNFGWYFD SWGKGTTLTV SS fourteen Light chain variable region Fab 0311 DIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL LIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPW TFGGGTRLEI K fifteen CDR1 heavy chain 0301 GYTFTDNYMI sixteen CDR2 heavy chain 0301 DINPYNGGTT FNQKFKG 17 CDR3 heavy chain 0301 ESPYFSNLYV MDY

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eighteen Light chain CDR1 0301 KASQSVDYDG DNYMN nineteen Light chain CDR2 0301 ASNLES 20 Light chain CDR3 0301 HLSNEDLST 21 CDR1 heavy chain 0302 GYTFSDFNIH 22 CDR2 heavy chain 0302 YINPYTDVTV YNEKFKG 23 CDR3 heavy chain 0302 YFDGTFDYAL DY 24 Light chain CDR1 0302 RASESVDNYG LSFMN 25 Light chain CDR2 0302 TASNLES 26 Light chain CDR3 0302 QQSKELPWT 27 CDR1 heavy chain 0311 GYIFTDYNMH 28 CDR2 heavy chain 0311 EINPNNGVW YNQKFKG 29 CDR3 severe ALYHSNFGWY FDS

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chains 0311 thirty Light chain CDR1 0311 KASQSVDYDG DSHMN 31 Light chain CDR2 0311 TASNLES 32 Light chain CDR3 0311 QQGNEDPWT 33 Heavy chain cAL 0301 EVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD INPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARES PYFSNLYVMD YWGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 34 Light chain cAL 0301 NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL LIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLS TFGGGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 35 Heavy chain cAL 0302 EIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY INPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYF DGTFDYALDY WGQGTSITVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV

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DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 36 Light chain cAL 0302 DVWTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL LIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPW TFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 37 Heavy chain cAL 0311 EIQLQQSGPD LMKPGASVKM SCKASGYTFT DYNMHWVKQN QGKSLEWMGE INPNNGVWY NQKFKGTTTL TVDKSSSTAY MDLHSLTSED SAVYYCTRAL YHSNFGWYFD SWGKGTTLTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 38 Light chain cAL 0311 DIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL LIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPW TFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 39 Heavy chain variable region h0301-H0 QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD INPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SS 40 Variable QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA

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th region of the heavy chain h0301-Hl PGQGLEWMGD INPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SS 41 Heavy chain variable region h0301-H2 QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD INPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SS 42 Heavy chain variable region H0302-H1 QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWMGY INPYTDVTVY NEKFKGRVTI TSDKSTSTAY MELSSLRSED TAVYYCASYF DGTFDYALDY WGQGTLVTVS S 43 Heavy chain variable region H0302-H2 QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY INPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYF DGTFDYALDY WGQGTLVTVS S 44 Heavy chain variable region H0311-H1 QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE INPNNGVWY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRAL YHSNFGWYFD SWGQGTLVTV SS 45 Heavy chain variable region H0311-H2 QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE INPNNGVWY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRAL YHSNFGWYFD SWGQGTLVTV SS 46 Light chain variable region h0301-L0 EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL LIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS TFGGGTKVEI K 47 Variable NIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY

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th region light chains h0301-Ll QQKPGQAPRL LIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS TFGGGTKVEI K 48 Light chain variable region H0302-L0 EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQSKELPW TFGQGTKVEI K 49 Light chain variable region H0302-L1 EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW TFGQGTKVEI K fifty Light chain variable region H0302-L2 EIWTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL LIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW TFGQGTKVEI K 51 Light chain variable region H0311-L0 EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPW TFGQGTKVEI K 52 Light chain variable region H0311-L1 DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPW TFGQGTKVEI K 53 heavy chain h0301-H0 QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD INPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP

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SVFLFPPKPK DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRSLWK EQ 54 heavy chain h0301-Hl QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD INPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 55 heavy chain h0301-h2 QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD INPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARES PYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 56 heavy chain H0302-H1 SGSLSLLY

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TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 57 heavy chain H0302-H2 QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY INPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYF DGTFDYALDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSW TVPSSSLGTK TYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKD TLMISRTPEV TCVWDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRWSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 58 heavy chain H0311-H1 QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE INPNNGVWY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRAL YHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 59 heavy chain H0311-H2 QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE INPNNGVWY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRAL YHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT

-47039894

VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK

Light EIVLTQSPAT QQKPGQAPRL SLEPEDFAVY LSLSPGERAT LIYAASNLES YCHLSNEDLS LSCKASQSVD GIPARFSGSG TFGGGTKVEI YDGDNYMNWY SGTDFTLTIS KRTVAAPSVF 60 chain IFPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS h0301-L0 GNSQESVTEQ DSKDSTYSLS STTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

Light NIVLTQSPAT QQKPGQAPRL SLEPEDFAVY LSLSPGERAT LIYAASNLES YCHLSNEDLS LSCKASQSVD GIPARFSGSG TFGGGTKVEI YDGDNYMNWY SGTDFTLTIS KRTVAAPSVF 61 chain IFPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS h0301-Ll GNSQESVTEQ DSKDSTYSLS STTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

Light EIVLTQSPAT QQKPGQAPRL SLEPEDFAVY LSLSPGERAT LIYTASNLES YCQQSKELPW LSCRASESVD GIPARFSGSG TFGQGTKVEI NYGLSFMNWY SGTDFTLTIS KRTVAAPSVF 62 chain IFPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS H0302-L0 GNSQESVTEQ DSKDSTYSLS STTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

63 Light chain H0302-L1 EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 64 Light chain H0302-L2 EIWTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL LIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW TFGQGTKVEI KRTVAAPSVF

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IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 65 Light chain H0311-L0 EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPW TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 66 Light chain H0311-L1 DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL LIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPW TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 67 Human CSF1 EEVSEYCSHM IGSGHLQSLQ RLIDSQMETS CQITFEFVDQ EQLKDPVCYL KKAFLLVQDI MEDTMRFRDN TPNAIAIVQL QELSLRLKSC FTKDYEEHDK ACVRTFYETP LQLLEKVKNV FNETKNLLDK DWNIFSKNCN NSFAECSSQG HERQSEGS 68 Human IL-34 NEPLEMWPLT QNEECTVTGF LRDKLQYRSR LQYMKHYFPI NYKISVPYEG VFRIANVTRL QRAQVSEREL RYLWVLVSLSATESVQDVLL EGHPSWKYLQ EVQTLLLNVQ QGLTDVEVSP KVESVLSLLN APGPNLKLVR PKALLDNCFR VMELLYCSCC KQSSVLNWQD CEVPSPQSCS PEPSLQYAAT QLYPPPPWSP SSPPHSTGSV RPVRAQGEGL LP 69 FR1 acceptor And a person QVQLVQSGAE VKKPGSSVKV SCKAS 70 FR2 acceptor And a person WVRQAPGQGL EWMG 71 FR3 acceptor And a person RVTITADKST STAYMELSSL RSEDTAVYYC AR 72 FR4 WGQGTLVTVS S

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acceptor And a person 73 FR1 acceptor In person QVQLVQSGAE VKKPGSSVKV SCKAS 74 FR2 acceptor In person WVRQAPGQGL EWMG 75 FR3 acceptor In person RVTITADKST STAYMELSSL RSEDTAVYYC AR 76 FR4 acceptor In person WGQGTLVTVSS 77 FR1 acceptor per person QVQLVQSGAE VKKPGSSVKV SCKAS 78 FR2 acceptor per person WVRQAPGQGL EWMG 79 FR3 acceptor per person RVTITADKST STAYMELSSL RSEDTAVYYC AR 80 FR4 acceptor per person WGQGTLVTVS S 81 FR1 acceptor D man EIVLTQSPAT LSLSPGERAT LSC 82 FR2 acceptor D man WYQQKPGQAP RLLIY 83 FR3 acceptor D man GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC

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84 FR4 acceptor D man FGGGTKVEIK 85 FR1 acceptor E man EIVLTQSPAT LSLSPGERAT LSC 86 FR2 acceptor E man WYQQKPGQAP RLLIY 87 FR3 acceptor E man GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC 88 FR4 acceptor E man FGQGTKVEIK 89 FR1 acceptor F man EIVLTQSPAT LSLSPGERAT LSC 90 FR2 acceptor F man WYQQKPGQAP RLLIY 91 FR3 acceptor F man GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC 92 FR4 acceptor F man FGQGTKVEIK 93 mCSFIR ECD-Fc APVIEPSGPE LWEPGETVT LRCVSNGSVE WDGPISPYWT LDPESPGSTL TTRNATFKNT GTYRCTELED PMAGSTTIHL YVKDPAHSWN LLAQEVTWE GQEAVLPCLI TDPALKDSVS LMREGGRQVL RKTVYFFSPW RGFIIRKAKV LDSNTYVCKT MVNGRESTST GIWLKVNRVH PEPPQIKLEP SKLVRIRGEA AQIVCSATNA EVGFNVILKR GDTKLEIPLN SDFQDNYYKK VRALSLNAVD FQDAGIYSCV ASNDVGTRTA TMNFQWESA YLNLTSEQSL LQEVSVGDSL ILTVHADAYP SIQHYNWTYL

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GPFFEDQRKL EFITQRAIYR YTFKLFLNRV KASEAGQYFL MAQNKAGWNN LTFELTLRYP PEVSVTWMPV NGSDVLFCDV SGYPQPSVTW MECRGHTDRC DEAQALQVWN DTHPEVLSQK PFDKVIIQSQ LPIGTLKHNM TYFCKTHNSV GNSSQYFRAV SLGQSKQEPK SSDKTHTCPP CPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVWDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRWSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 94 Human IgG4 S241P ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK 95 1dk human RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 96 Predecessor CD40 human (with signal sequence) UniProtKB/Swiss- Prot: P25942.1, 04-MAR2015 MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEK CHPWTSCETK DLWQQAGTN KTDWCGPQD RLRALWIPI IFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ

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97 CD40 human (mature, without signal sequences) EPPTACREKQ YLINSQCCSL CQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQH KYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCV LHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEK CHPWTSCETK DLVVQQAGTN KTDWCGPQD RLRALWIPI IFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPD DLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ 98 heavy chain dacetuzuma ba EVQLVESGGG LVQPGGSLRL SCAASGYSFT GYYIHWVRQA PGKGLEWVAR VIPNAGGTSY NQKFKGRFTL SVDNSKNTAY LQMNSLRAED TAVYYCAREG IYWWGQGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SWTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT NTSRRSPRARE LLGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK 99 Light chain dacetuzuma ba DIQMTQSPSS LSASVGDRVT ITCRSSQSLV HSNGNTFLHW YQQKPGKAPK LLIYTVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAT YFCSQTTHVP WTFGQGTKVE IKRTVAAPSV FIFPSDEQL KSGTASWCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

CLAIM

Claims (29)

CLAIM 1. A method of treating cancer in a subject, wherein the cancer is characterized by the presence of CSF1R-expressing macrophages, comprising administering to the subject an anti-CSF1R antibody and at least one immunostimulatory agent, wherein the anti-CSF1R antibody is selected from: a) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 39 and a light chain variable region comprising the sequence of SEQ ID NO: 46; b) an antibody containing a heavy chain containing a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15, an HC CDR2 having the sequence of SEQ ID NO: 16, and an HC CDR3 having the sequence of SEQ ID NO: 17, and a light chain containing a light chain (LC) CDR1 having the sequence of SEQ ID NO: 18, an LC CDR2 having the sequence of SEQ ID NO: 19, and an LC CDR3 having the sequence of SEQ ID NO: 20; and c) an antibody containing a heavy chain containing the sequence of SEQ ID NO: 53 and a light -53 039894 chain containing the sequence of SEQ ID NO: 60; and wherein at least one immunostimulatory agent comprises an antibody agonist against CD40. 2. The method of claim 1, wherein the at least one immunostimulatory agent further comprises one or more of: a) agonist B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD27, CD40, CD40L, DR3, CD28H, IL- 2, IL-7, IL-12, IL-15, IL-21, IFNa, STING, or a Toll-like receptor agonist such as a TLR2/4 agonist; and b) an agent that binds to a member of the TNF receptor family, or a costimulatory or co-inhibitory molecule that binds to a member of the TNF receptor family, such as CD40, CD40L, OX40, OX40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4- 1BBL, CD137 (41BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, EDA1, EDA2, TACI, APRIL , BCMA, LTaR, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFe, TNFR2, TNFa, 1β2, FAS, FASL, RELT, DR6, TROY or NGFe. 3. The method of claim 2, wherein the at least one additional immunostimulatory agent comprises recombinant CD40L. 4. The method of any one of the preceding claims, wherein the anti-CSF1R antibody and the anti-CD40 antibody agonist are administered simultaneously or sequentially. 5. The method of any one of the preceding claims, wherein the anti-CSF1R antibody and the anti-CD40 antibody agonist are administered simultaneously. 6. The method of claim 5, wherein one or more doses of the anti-CD40 antibody agonist are administered prior to administration of the anti-CSF1R antibody. 7. The method of claim 5 wherein one or more doses of the anti-CSF1R antibody is administered prior to administration of the anti-CD40 antibody agonist. 8. The method of any one of the preceding claims, wherein the cancer is selected from non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatic cell carcinoma, bladder cancer, endometrial cancer, lymphoma Hodgkin, lung cancer, glioma, glioblastoma multiforme, colon cancer, breast cancer, bone cancer, skin cancer, uterine cancer, stomach cancer, gastric cancer, lymphoma, lymphocytic leukemia, multiple myeloma, prostate cancer, mesothelioma, and kidney cancer. 9. The method of any one of the preceding claims, wherein the cancer is recurrent or progressive following therapy selected from surgery, chemotherapy, radiation therapy, or a combination thereof. 10. The method of any one of the preceding claims, wherein the anti-CSF1R antibody blocks the binding of human CSF1 and/or human IL-34 to human CSF1R. 11. The method of any one of the preceding claims, wherein the anti-CSF1R antibody inhibits ligand-induced phosphorylation of human CSF1R in vitro. 12. The method of any one of the preceding claims, wherein the anti-CSF1R antibody is a humanized antibody. 13. The method of any one of the preceding claims, wherein the anti-CSF1R antibody is selected from Fab, Fv, scFv, Fab' and (Fab') ₂ . 14. The method according to any one of the preceding paragraphs, where the anti-CD40 antibody agonist comprises a CDR of an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. 15. The method of claim 14, wherein the CD40 antibody agonist comprises the heavy chain and light chain variable regions of an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. 16. The method of claim 14, wherein the CD40 antibody agonist is an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. 17. Composition for the treatment of cancer characterized by the presence of macrophages expressing CSF1R, containing an antibody against CSF1R and at least one immunostimulatory agent, where the antibody against CSF1R is selected from: a) an antibody containing a heavy chain containing the sequence of SEQ ID NO: 39 and a light chain containing the sequence of SEQ ID NO: 46; b) an antibody containing a heavy chain containing a heavy chain (HC) CDR1 having the sequence of SEQ ID NO: 15, an HC CDR2 having the sequence of SEQ ID NO: 16, and an HC CDR3 having the sequence of SEQ ID NO: 17, and a light chain containing a light chain (LC) CDR1 having the sequence of SEQ ID NO: 18, an LC CDR2 having the sequence of SEQ ID NO: 19, and an LC CDR3 having the sequence of SEQ ID NO: 20; and c) an antibody containing a heavy chain containing the sequence of SEQ ID NO: 53 and a light - 54 039894 chain containing the sequence of SEQ ID NO: 60; and wherein at least one immunostimulatory agent comprises an antibody agonist against CD40. 18. The composition according to claim 17, where the composition further comprises: (a) B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD27, CD40, CD40L, DR3, CD28H, IL agonist -2, IL-7, IL-12, IL-15, IL-21, IFNa, STING, or a Toll-like receptor agonist such as a TLR2/4 agonist; and/or (b) an agent that binds to a member of the TNF receptor family, or a costimulatory or co-inhibitory molecule that binds to a member of the TNF receptor family, such as CD40, CD40L, OX40, OX40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (41BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, EDA1, EDA2 , TACI, APRIL, BCMA, LTeR, LIGHT, DeR3, HVEM, VEGL/TL1A, TRAMP/DR3, TNFR1, TNFe, TNFR2, TNFa, 1β2, FAS, FASL, RELT, DR6, TROY or NGFe. 19. The composition of claim 18, wherein the at least one additional immunostimulatory agent comprises recombinant CD40L. 20. The composition of any one of claims 17-19, wherein the anti-CSF1R antibody is a humanized antibody. 21. The composition according to any one of claims 17-20, wherein the anti-CSF1R antibody is selected from Fab, Fv, scFv, Fab' and (Fab') ₂ . 22. The composition according to any one of claims 17-21, where the anti-CD40 antibody agonist comprises a CDR of an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. 23. The composition of claim 22, wherein the CD40 antibody agonist comprises the heavy chain and light chain variable regions of an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. 24. The composition of claim 22, wherein the CD40 antibody agonist is an antibody selected from CP-870.893; dacetuzumab; SEA-CD40; ADC-1013; RO7009789 and Chi Lob 7/4. 25. A composition according to any one of claims 17-24, wherein each of the anti-CSF1R antibody and the anti-CD4 antibody agonist are in separate compartments or containers. 26. A composition according to any one of claims 17-25, wherein the anti-CSF1R antibody and the anti-CD4 antibody agonist are mixed or formulated together. 27. The composition according to any one of claims 17-26, wherein the cancer is selected from non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatic cell carcinoma, bladder cancer, endometrial cancer, Hodgkin's lymphoma, lung cancer, glioma, glioblastoma multiforme, colon cancer, breast cancer, bone cancer, skin cancer, uterine cancer, gastric cancer, gastric cancer, lymphoma, lymphocytic leukemia, multiple myeloma, prostate cancer, mesothelioma, and kidney cancer . 28. The use of a composition according to any one of claims 17 to 26 for the preparation of a medicament for the treatment of cancer characterized by the presence of macrophages expressing CSF1R. 29. Use according to claim 28, wherein the cancer is selected from non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, hepatic cell carcinoma, bladder cancer, endometrial cancer, Hodgkin's lymphoma , lung cancer, glioma, glioblastoma multiforme, colon cancer, breast cancer, bone cancer, skin cancer, uterine cancer, stomach cancer, gastric cancer, lymphoma, lymphocytic leukemia, multiple myeloma, prostate cancer, mesothelioma and kidney cancer.