CSF1R / CCR2 multispecific antibody

Abstract

Disclosed are molecules, such as multispecific molecules, that target CSF1R or CCR2, and methods of using the same.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 737,742, filed September 27, 2018, U.S. Provisional Application No. 62 / 750,163, filed October 24, 2018, and U.S. Provisional Application No. 62 / 776,820, filed December 7, 2018, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on September 27, 2019, is named E2070-7018WO_SL.txt, and is 711,029 bytes in size. Background Art

[0005] Disclosed are multispecific molecules that target tumor-associated macrophages (TAMs) or myeloid-derived suppressor cells (MDSCs) and methods of using the same. Summary of the Invention

[0006] The present disclosure relates, inter alia, to novel multispecific molecules comprising: (i) a first immunosuppressive myeloid cell (IMC) binding portion (e.g., a first tumor-associated macrophage (TAM) binding portion; or a first myeloid-derived suppressor cell (MDSC) binding portion) (e.g., an antibody molecule); and (ii) a second IMC binding portion (e.g., a first TAM binding portion; or a second MDSC binding portion) (e.g., an antibody molecule), wherein the first and second IMC (e.g., TAM or MDSC) binding portions are different. Without being bound by theory, it is expected that the multispecific molecules disclosed herein deplete TAMs and / or MDSCs. Thus, inter alia, provided herein are multispecific molecules (e.g., multispecific antibody molecules) comprising the above-mentioned portions, nucleic acids encoding the multispecific molecules, methods of producing the above-mentioned molecules, and methods of using the above-mentioned molecules to treat cancer.

[0007] In one aspect, provided herein are isolated multispecific molecules, e.g., bispecific molecules, comprising: (i) a first immunosuppressive myeloid cell (IMC) binding portion (e.g., a first tumor-associated macrophage (TAM) binding portion; or a first myeloid-derived suppressor cell (MDSC) binding portion) (e.g., an antibody molecule); and (ii) a second IMC binding portion (e.g., a second TAM binding portion; or a second MDSC binding portion) (e.g., an antibody molecule), wherein the first and second IMC (e.g., TAM or MDSC) binding portions are different. In some embodiments, the first and second IMC (e.g., TAM or MDSC) binding portions bind to different epitopes. In some embodiments, the first and second IMC (e.g., TAM or MDSC) binding portions bind to different antigens.

[0008] In some embodiments, the first IMC-binding moiety is a first MDSC-binding moiety; the second IMC-binding moiety is a second MDSC-binding moiety. In some embodiments, the first IMC-binding moiety is a first TAM-binding moiety; the second IMC-binding moiety is a second TAM-binding moiety. In some embodiments, the first TAM-binding moiety binds to CSF1R, CCR2, CXCR2, CD86, CD163, CX3CR1, MARCO, CD204, CD52, folate receptor beta, or PD-L1; and the second TAM-binding moiety binds to CCR2, CSF1R, CXCR2, CD86, CD163, CX3CR1, MARCO, CD204, CD52, folate receptor beta, or PD-L1. In some embodiments, the first TAM binding moiety binds to CSF1R, CCR2, CXCR2, or PD-L1 (e.g., human CSF1R, CCR2, CXCR2, or PD-L1), and the second TAM binding moiety binds to CCR2, CSF1R, CXCR2, or PD-L1 (e.g., human CCR2, CSF1R, CXCR2, or PD-L1). In some embodiments, the first TAM binding moiety binds to CSF1R, and the second TAM binding moiety binds to CCR2. In some embodiments, the first TAM binding moiety binds to CSF1R, and the second TAM binding moiety binds to CXCR2. In some embodiments, the first TAM binding moiety binds to CCR2, and the second TAM binding moiety binds to CXCR2. In some embodiments, the first TAM binding moiety binds to CSF1R, and the second TAM binding moiety binds to PD-L1. In some embodiments, the first TAM binding moiety binds to CCR2, and the second TAM binding moiety binds to PD-L1. In some embodiments, the first TAM binding moiety binds to CXCR2 and the second TAM binding moiety binds to PD-L1.

[0009] In some embodiments, the first TAM binding moiety binds to CSF1R, CCR2, CXCR2, or PD-L1 with a dissociation constant of less than about 10 nM, more typically 10-100 pM; the second TAM binding moiety binds to CCR2, CSF1R, CXCR2, or PD-L1 with a dissociation constant of less than about 10 nM, more typically 10-100 pM. In some embodiments, the first TAM binding moiety binds to a conformational or linear epitope on CSF1R, CCR2, CXCR2, or PD-L1; the second TAM binding moiety binds to a conformational or linear epitope on CCR2, CSF1R, CXCR2, or PD-L1.

[0010] In some embodiments, the multispecific molecule comprises at least two non-contiguous polypeptide chains. In some embodiments, the first IMC binding moiety comprises a first anti-IMC antibody molecule and / or the second IMC binding moiety comprises a second anti-IMC antibody molecule. In some embodiments, the first anti-IMC antibody molecule and the second anti-IMC antibody molecule are independently intact antibodies (e.g., comprising at least one, preferably two, complete heavy chains, and at least one, preferably two, complete light chains) or antigen-binding fragments (e.g., Fab, F(ab')2, Fv, scFv, single domain antibody, or diabody (dAb)).

[0011] In some embodiments, the first anti-IMC antibody molecule and / or the second anti-IMC antibody molecule comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, or IgG4, or a fragment thereof.

[0012] In some embodiments, the first anti-IMC antibody molecule and / or the second anti-IMC antibody molecule comprises a light chain constant region selected from κ or λ, or a fragment thereof. In some embodiments, the first anti-IMC antibody molecule comprises a κ light chain constant region or a fragment thereof, and the second anti-IMC antibody molecule comprises a λ light chain constant region or a fragment thereof. In some embodiments, the first anti-IMC antibody molecule comprises a λ light chain constant region or a fragment thereof, and the second anti-IMC antibody molecule comprises a κ light chain constant region or a fragment thereof. In some embodiments, the first anti-IMC antibody molecule comprises a λ light chain constant region or a fragment thereof, and the second anti-IMC antibody molecule comprises a κ light chain constant region or a fragment thereof. In some embodiments, the first anti-IMC antibody molecule and the second anti-IMC antibody molecule have a common light chain variable region.

[0013] In some embodiments, the multispecific molecule further comprises a heavy chain constant region (e.g., Fc region) selected from the group consisting of: IgG1, IgG2, and IgG4, more particularly, a heavy chain constant region of human IgG1, IgG2, or IgG4. In some embodiments, the heavy chain constant region (e.g., Fc region) is linked, e.g., covalently linked, to one or both of the first anti-IMC antibody molecule and the second anti-IMC antibody molecule. In some embodiments, the heavy chain constant region (e.g., Fc region) is altered, e.g., mutated, to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function. In some embodiments, the interface between the first heavy chain constant region and the second heavy chain constant region (e.g., Fc region) is altered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-engineered interface. In some embodiments, dimerization of the heavy chain constant region (e.g., Fc region) is enhanced by providing one or more of a paired cavity-protrusion ("knob-in-a-hole"), electrostatic interactions, or chain exchange to the Fc interface of the first and second Fc regions, thereby resulting in, for example, a greater ratio of heteromultimeric to homomultimeric forms relative to a non-engineered interface. In some embodiments, the heavy chain constant region (e.g., Fc region) comprises an amino acid replacement at one or more positions selected from, for example, 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 (numbered based on the Eu numbering system) of the Fc region of human IgG1. In some embodiments, the heavy chain constant region (e.g., Fc region) comprises an amino acid replacement selected from the group consisting of T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), or T366W (e.g., corresponding to a protrusion or knob), or a combination thereof, numbered based on the Eu numbering system.

[0014] In some embodiments, the heavy chain constant region (e.g., Fc region) comprises one or more mutations that increase or decrease one or more of the following relative to a naturally occurring heavy chain constant region: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function. In some embodiments, the first anti-IMC antibody molecule comprises a first heavy chain constant region (e.g., a first Fc region) and the second anti-IMC antibody molecule comprises a second heavy chain constant region (e.g., a second Fc region), wherein the first heavy chain constant region comprises one or more mutations that increase heterodimerization of the first heavy chain constant region and the second heavy chain constant region relative to a naturally occurring heavy chain constant region, and / or wherein the second heavy chain constant region comprises one or more mutations that increase heterodimerization of the second heavy chain constant region and the first heavy chain constant region relative to a naturally occurring heavy chain constant region. In some embodiments, the first heavy chain constant region and the second heavy chain constant region (e.g., the first Fc region and the second Fc region) comprise one or more of paired cavities and protrusions ("knob-in-hole structures"), electrostatic interactions, or chain exchange, such that, for example, a greater proportion of heteromultimeric forms is formed relative to naturally occurring heavy chain constant regions. In some embodiments, the first heavy chain constant region and / or the second heavy chain constant region (e.g., the first Fc region and / or the second Fc region, e.g., the first IgG1 Fc region and / or the second IgG1 Fc region) comprises an amino acid replacement at one or more positions selected from 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 (numbered based on the Eu numbering system). In some embodiments, the first heavy chain constant region and / or the second heavy chain constant region (e.g., the first Fc region and / or the second Fc region, e.g., the first IgG1 Fc region and / or the second IgG1 Fc region) comprises an amino acid replacement selected from the group consisting of T366S, L368A, Y407V, or Y349C (e.g., corresponding to a cavity or hole), or T366W or S354C (e.g., corresponding to a protrusion or knob), numbered based on the Eu numbering system, or a combination thereof.

[0015] In some embodiments, the multispecific molecule further comprises a linker, such as a linker between one or more of the following: a first anti-IMC antibody molecule and a second anti-IMC antibody molecule, a first anti-IMC antibody molecule and a heavy chain constant region (e.g., an Fc region), or a second anti-IMC antibody molecule and a heavy chain constant region. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises Gly and Ser.

[0016] In some embodiments, the heavy chain constant region (eg, Fc region) induces antibody-dependent cellular cytotoxicity (ADCC).

[0017] In some embodiments, the first or second TAM binding moiety antibody molecule binds to CSF1R and comprises one, two, or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 48, SEQ ID NO: 66, or SEQ ID NO: 69, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 48, SEQ ID NO: 66, or SEQ ID NO: 69; and / or comprises one, two, or three CDRs from the light chain variable region sequence of SEQ ID NO: 50, SEQ ID NO: 67, or SEQ ID NO: 70, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 50, SEQ ID NO: 67, or SEQ ID NO: 70. In some embodiments, the antibody molecule that binds to CSF1R comprises the following heavy chain variable region sequence: SEQ ID NO:48, SEQ ID NO:66 or SEQ ID NO:69, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO:48, SEQ ID NO:66 or SEQ ID NO:69); and / or comprises the following light chain variable region sequence: SEQ ID NO:50, SEQ ID NO:67 or SEQ ID NO:70, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO:50, SEQ ID NO:67 or SEQ ID NO:70).

[0018] In some embodiments, the first TAM binding moiety or the second TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two, or three CDRs from the heavy chain variable region sequence of SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64; and / or comprises one, two, or three CDRs from the light chain variable region sequence of SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:65, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64. The CDR of NO:65 has at least one amino acid change, but no more than two, three or four changes (eg, substitutions, deletions or insertions, such as conservative substitutions). In some embodiments, the antibody molecule that binds to CCR2 comprises the following heavy chain variable region sequence: SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64); and / or comprises the following light chain variable region sequence: SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:65, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64). The amino acid sequence of NO:65 has at least one amino acid alteration, but not more than five, ten or fifteen alterations (eg, substitutions, deletions or insertions, eg, conservative substitutions).

[0019] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 44, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 44; and / or comprises one, two or three CDRs from the light chain variable region sequence of SEQ ID NO: 45, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 45; and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 48, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: ID NO: 48 has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) in the CDR; and / or comprises one, two or three CDRs from the following light chain variable region sequence: SEQ ID NO: 50, or a closely related CDR, such as a CDR that has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the CDR of SEQ ID NO: 50.

[0020] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 54, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 54; and / or comprises one, two or three CDRs from the light chain variable region sequence of SEQ ID NO: 57, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 57; and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 66, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: ID NO: 66 has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) in the CDR; and / or comprises one, two or three CDRs from the following light chain variable region sequence: SEQ ID NO: 67, or a closely related CDR, such as a CDR that has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the CDR of SEQ ID NO: 67.

[0021] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 54, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 54; and / or comprises one, two or three CDRs from the light chain variable region sequence of SEQ ID NO: 57, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 57; and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 69, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: ID NO: 69 has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) in the CDR; and / or comprises one, two or three CDRs from the following light chain variable region sequence: SEQ ID NO: 70, or a closely related CDR, such as a CDR that has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the CDR of SEQ ID NO: 70.

[0022] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 59, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 59; and / or comprises one, two or three CDRs from the light chain variable region sequence of SEQ ID NO: 60, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 60; and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 66, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: ID NO: 66 has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) in the CDR; and / or comprises one, two or three CDRs from the following light chain variable region sequence: SEQ ID NO: 67, or a closely related CDR, such as a CDR that has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the CDR of SEQ ID NO: 67.

[0023] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 59, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 59; and / or comprises one, two or three CDRs from the light chain variable region sequence of SEQ ID NO: 60, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 60; and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 69, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: ID NO: 69 has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) in the CDR; and / or comprises one, two or three CDRs from the following light chain variable region sequence: SEQ ID NO: 70, or a closely related CDR, such as a CDR that has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the CDR of SEQ ID NO: 70.

[0024] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 62, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 62; and / or comprises one, two or three CDRs from the light chain variable region sequence of SEQ ID NO: 63, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 63; and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 66, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: ID NO: 66 has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) in the CDR; and / or comprises one, two or three CDRs from the following light chain variable region sequence: SEQ ID NO: 67, or a closely related CDR, such as a CDR that has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the CDR of SEQ ID NO: 67.

[0025] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 62, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 62; and / or comprises one, two or three CDRs from the light chain variable region sequence of SEQ ID NO: 63, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 63; and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 69, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: ID NO: 69 has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) in the CDR; and / or comprises one, two or three CDRs from the following light chain variable region sequence: SEQ ID NO: 70, or a closely related CDR, such as a CDR that has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the CDR of SEQ ID NO: 70.

[0026] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 64, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 64; and / or comprises one, two or three CDRs from the light chain variable region sequence of SEQ ID NO: 65, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 65; and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 66, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: ID NO: 66 has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) in the CDR; and / or comprises one, two or three CDRs from the following light chain variable region sequence: SEQ ID NO: 67, or a closely related CDR, such as a CDR that has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the CDR of SEQ ID NO: 67.

[0027] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 64, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 64; and / or comprises one, two or three CDRs from the light chain variable region sequence of SEQ ID NO: 65, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 65; and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises one, two or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 69, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three or four alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: ID NO: 69 has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) in the CDR; and / or comprises one, two or three CDRs from the following light chain variable region sequence: SEQ ID NO: 70, or a closely related CDR, such as a CDR that has at least one amino acid change, but not more than two, three or four changes (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the CDR of SEQ ID NO: 70.

[0028] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises the following heavy chain variable region sequence: SEQ ID NO: 44, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 44); and / or comprises the following light chain variable region sequence: SEQ ID NO: 45, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 45); and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises the following heavy chain variable region sequence: SEQ ID NO: 48, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: NO:48 has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions)); and / or comprises the following light chain variable region sequence: SEQ ID NO:50, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the amino acid sequence of SEQ ID NO:50).

[0029] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises the following heavy chain variable region sequence: SEQ ID NO: 54, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 54); and / or comprises the following light chain variable region sequence: SEQ ID NO: 57, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 57); and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises the following heavy chain variable region sequence: SEQ ID NO: 66, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: NO:66 has an amino acid sequence with at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions)); and / or comprises the following light chain variable region sequence: SEQ ID NO:67, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the amino acid sequence of SEQ ID NO:67).

[0030] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises the following heavy chain variable region sequence: SEQ ID NO: 54, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 54); and / or comprises the following light chain variable region sequence: SEQ ID NO: 57, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 57); and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises the following heavy chain variable region sequence: SEQ ID NO: 69, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: NO:69 has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions)); and / or comprises the following light chain variable region sequence: SEQ ID NO:70, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the amino acid sequence of SEQ ID NO:70).

[0031] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises the following heavy chain variable region sequence: SEQ ID NO: 59, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 59); and / or comprises the following light chain variable region sequence: SEQ ID NO: 60, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 60); and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises the following heavy chain variable region sequence: SEQ ID NO: 66, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: NO:66 has an amino acid sequence with at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions)); and / or comprises the following light chain variable region sequence: SEQ ID NO:67, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the amino acid sequence of SEQ ID NO:67).

[0032] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises the following heavy chain variable region sequence: SEQ ID NO: 59, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 59); and / or comprises the following light chain variable region sequence: SEQ ID NO: 60, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 60); and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises the following heavy chain variable region sequence: SEQ ID NO: 69, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: NO:69 has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions)); and / or comprises the following light chain variable region sequence: SEQ ID NO:70, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the amino acid sequence of SEQ ID NO:70).

[0033] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises the following heavy chain variable region sequence: SEQ ID NO: 62, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 62); and / or comprises the following light chain variable region sequence: SEQ ID NO: 63, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 63); and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises the following heavy chain variable region sequence: SEQ ID NO: 66, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: NO:66 has an amino acid sequence with at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions)); and / or comprises the following light chain variable region sequence: SEQ ID NO:67, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the amino acid sequence of SEQ ID NO:67).

[0034] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises the following heavy chain variable region sequence: SEQ ID NO: 62, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 62); and / or comprises the following light chain variable region sequence: SEQ ID NO: 63, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 63); and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises the following heavy chain variable region sequence: SEQ ID NO: 69, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: NO:69 has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions)); and / or comprises the following light chain variable region sequence: SEQ ID NO:70, an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the amino acid sequence of SEQ ID NO:70).

[0035] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises the following heavy chain variable region sequence: SEQ ID NO: 64, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 64); and / or comprises the following light chain variable region sequence: SEQ ID NO: 65, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 65); and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises the following heavy chain variable region sequence: SEQ ID NO: 66, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: NO:66 has an amino acid sequence with at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions)); and / or comprises the following light chain variable region sequence: SEQ ID NO:67, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the amino acid sequence of SEQ ID NO:67).

[0036] In one embodiment, the first TAM binding moiety is an antibody molecule that binds to CCR2 and comprises the following heavy chain variable region sequence: SEQ ID NO: 64, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 64); and / or comprises the following light chain variable region sequence: SEQ ID NO: 65, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 65); and the second TAM binding moiety is an antibody molecule that binds to CSF1R and comprises the following heavy chain variable region sequence: SEQ ID NO: 69, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten, or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: NO:69 has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions)); and / or comprises the following light chain variable region sequence: SEQ ID NO:70, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or has at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, such as conservative substitutions) relative to the amino acid sequence of SEQ ID NO:70).

[0037] In some embodiments, the first TAM binding moiety or the second TAM binding moiety is an antibody molecule that binds to PD-L1 and comprises one, two, or three CDRs from the heavy chain variable region sequence of SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 113, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 109, SEQ ID NO: 111, or SEQ ID NO: 113; and / or comprises one, two, or three CDRs from the light chain variable region sequence of SEQ ID NO: 110, SEQ ID NO: 112, or SEQ ID NO: 114, or closely related CDRs, e.g., CDRs that have at least one amino acid alteration but not more than two, three, or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to the CDRs of SEQ ID NO: 110, SEQ ID NO: 112, or SEQ ID NO: 114. In some embodiments, the antibody molecule that binds to PD-L1 comprises the following heavy chain variable region sequence: SEQ ID NO: 109, SEQ ID NO: 111 or SEQ ID NO: 113, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 109, SEQ ID NO: 111 or SEQ ID NO: 113); and / or comprises the following light chain variable region sequence: SEQ ID NO: 110, SEQ ID NO: 112 or SEQ ID NO: 114, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration but not more than five, ten or fifteen alterations (e.g., substitutions, deletions or insertions, e.g., conservative substitutions) relative to the amino acid sequence of SEQ ID NO: 110, SEQ ID NO: 112 or SEQ ID NO: 114).

[0038] In some embodiments, (i) the first IMC binding moiety monovalently binds a first antigen (e.g., CSF1R, CCR2, CXCR2, CD86, CD163, CX3CR1, MARCO, CD204, CD52, folate receptor beta, or PD-L1), and / or (ii) the second IMC binding moiety monovalently binds a second antigen (e.g., CCR2, CSF1R, CXCR2, CD86, CD163, CX3CR1, MARCO, CD204, CD52, folate receptor beta, or PD-L1), wherein the first antigen is different from the second antigen.

[0039] In some embodiments, (i) the multispecific molecule monovalently binds a first antigen (e.g., CSF1R, CCR2, CXCR2, CD86, CD163, CX3CR1, MARCO, CD204, CD52, folate receptor beta, or PD-L1), and / or (ii) the multispecific molecule monovalently binds a second antigen (e.g., CCR2, CSF1R, CXCR2, CD86, CD163, CX3CR1, MARCO, CD204, CD52, folate receptor beta, or PD-L1), wherein the first antigen is different from the second antigen.

[0040] In some embodiments, (i) the multispecific molecule inhibits a first antigen in the presence of a second antigen, optionally wherein the multispecific molecule reduces the activity of the first antigen in a cell when the cell expresses both the first and second antigens on the cell surface, e.g., by at least 40%, 50%, 60%, 70%, 80%, or 90%, and / or (ii) the multispecific molecule does not inhibit or does not substantially inhibit the first antigen in the absence of the second antigen, optionally wherein the multispecific molecule does not reduce the activity of the first antigen, or does not reduce the activity of the first antigen by more than 2%, 4%, 6%, 8%, 10%, or 15%, when the cell expresses the first antigen but does not express the second antigen on the cell surface.

[0041] In some embodiments, (i) the multispecific molecule inhibits the expression of a first antigen in the presence of a second antigen, optionally wherein the multispecific molecule reduces the activity of the second antigen in a cell when the cell expresses both the first and second antigens on the cell surface, e.g., by at least 40%, 50%, 60%, 70%, 80%, or 90%, and / or (ii) the multispecific molecule does not inhibit or does not substantially inhibit the expression of the second antigen in the absence of the first antigen, optionally wherein the multispecific molecule does not reduce the activity of the second antigen, or does not reduce the activity of the second antigen by more than 2%, 4%, 6%, 8%, 10%, or 15%, when the cell expresses the second antigen but does not express the first antigen on the cell surface.

[0042] In some embodiments, the multispecific molecule further comprises one or more additional binding moieties (e.g., a third binding moiety, a fourth binding moiety (e.g., a trispecific or tetraspecific molecule)). In some embodiments, the multispecific molecule further comprises one or more additional binding moieties (e.g., a third binding moiety, a fourth binding moiety (e.g., a trispecific or tetraspecific molecule)). In some embodiments, the multispecific molecule comprises a third TAM binding moiety (e.g., an antibody molecule), wherein the third TAM binding moiety is different from the first TAM binding moiety and the second TAM binding moiety. In some embodiments, the first TAM binding moiety binds to human CSF1R, the second TAM binding moiety binds to human CCR2, and the third TAM binding moiety binds to CXCR2.

[0043] In some embodiments, the multispecific molecule comprises a third binding moiety (eg, an antibody molecule) that is a tumor targeting moiety. In some embodiments, the tumor targeting moiety binds to PD-L1, mesothelin, CD47, ganglioside 2 (GD2), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PMSA), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), Ron kinase, c-Met, immature laminin receptor, TAG-72, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, SAP-1, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, p53, Ras, TGF-β receptor, AFP, ETA, MAGE, MUC-1, CA-125, BAGE, GAGE, NY-ESO-1, β-catenin, CDK4, CDC27, CD47, α-actinin-4, TRP1 / gp75, TRP2, gp100, Melan-A / MART1, gangliosides, WT1, EphA3, epidermal growth factor receptor (EGFR), CD20, MART-2, MART-1, MUC1, MUC2, MUM1, MUM2, MUM3, NA88-1, NPM, OA1, OGT, RCC, RUI1, RUI2, SAGE, TRG, TRP1, TSTA, folate receptor α, L1-CAM, CAIX, EGFRvIII, gpA33, GD3, GM2, VEGFR, integrin (integrin αVβ3, integrin α5βl), carbohydrate (Le), IGF1R, EPHA3, TRAILR1, TRAILR2, or RANKL.

[0044] In some embodiments, the multispecific molecule is a bispecific molecule comprising a first non-contiguous polypeptide and a second non-contiguous polypeptide, wherein: (i) the first polypeptide comprises, in the NC orientation, a first TAM binding moiety (e.g., an antibody molecule (e.g., a first portion of a first antigen domain, e.g., a first VH-CH1 of a Fab molecule)), e.g., that binds to, e.g., a first TAM antigen, e.g., CSF1R, CCR2, CXCR2, or PD-F1, optionally linked via a linker to a first domain (e.g., a first immunoglobulin constant domain) (e.g., a first Fc molecule described herein) that promotes association between the first polypeptide and the second polypeptide; (ii) the second polypeptide comprises, in the NC orientation, a second TAM binding moiety (e.g., an antibody molecule, e.g., that binds to, e.g., a second In some embodiments, the first polypeptide and the second polypeptide are a polypeptide comprising a first antigen domain that binds to a first TAM antigen, such as a CCR2, CSF1R, CXCR2, or PD-F1 antigen, optionally via a linker, connected to a second domain (e.g., a second immunoglobulin constant domain) (e.g., a second Fc molecule described herein); and (iii) the third polypeptide comprises, for example, a second portion of the first antigen domain that binds to the first TAM antigen in the NC direction, such as the first VF-CF of a Fab, e.g., wherein the third polypeptide is non-covalently bound to the first polypeptide; and (iv) the fourth polypeptide comprises, for example, a second portion of the second antigen domain that binds to the second TAM antigen in the NC direction, such as the second VF-CF of a Fab, e.g., wherein the fourth polypeptide is non-covalently associated with the second polypeptide. In some embodiments, the first polypeptide and the second polypeptide are homodimers or heterodimers.

[0045] In some embodiments, the multispecific molecule is a bispecific molecule, wherein: (i) the first TAM binding moiety (e.g., a binding moiety that binds to a first TAM antigen, such as CSF1R, CCR2, or CXCR2) comprises a first non-contiguous polypeptide and a second non-contiguous polypeptide, and (ii) the first TAM binding moiety (e.g., a binding moiety that binds to a second TAM antigen, such as CSF1R, CCR2, or CXCR2) comprises a third non-contiguous polypeptide and a fourth non-contiguous polypeptide, wherein:

[0046] (a) the first polypeptide comprises, for example, a first VH, a first CH1 in the NC direction, optionally connected via a linker to a first domain (e.g., a first Fc region) that promotes association between the first polypeptide and a third polypeptide,

[0047] (b) the second polypeptide comprises, for example, a first VL and a first CL in NC orientation,

[0048] (c) the third polypeptide comprises, for example, a second VH, a second CH1 in the NC orientation, optionally via a linker, a second domain (e.g., a second Fc region) that promotes association between the first polypeptide and the third polypeptide, and

[0049] (d) the fourth polypeptide comprises, for example, a second VL and a second CL in the NC orientation. In some embodiments, the first domain and the second domain (eg, the first Fc region and the second Fc region) form a homodimer or a heterodimer.

[0050] In one aspect, the present invention provides isolated multispecific molecules, e.g., bispecific molecules, comprising (i) an anti-CSF1R binding portion (e.g., an anti-CSF1R antibody molecule); and (ii) an anti-CCR2 binding portion (e.g., an anti-CCR2 antibody molecule). Without wishing to be bound by theory, the anti-CSF1R / anti-CCR2 multispecific molecule may preferentially bind to CSF1R-positive CCR2-positive cells relative to CSF1R-positive, CCR2-negative cells or CSF1R-negative, CCR2-positive cells. Exemplary CSF1R-positive, CCR2-positive cells include, but are not limited to, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Exemplary CSF1R-positive, CCR2-negative cells include, but are not limited to, tissue-resident macrophages (e.g., Kupffer cells) and Langerhans cells. Exemplary CSF1R-negative, CCR2-positive cells include, but are not limited to, T cells (e.g., activated T cells, e.g., activated CD4+ and / or CD8+ T cells), NK cells, and neutrophils. Without wishing to be bound by theory, anti-CSF1R / anti-CCR2 multispecific molecules may preferentially bind to CSF1R-positive, CCR2-positive cells (e.g., pro-tumorigenic TAMs or MDSCs), or CSF1R-negative, CCR2-positive cells (e.g., activated T cells or NK cells) relative to CSF1R-positive, CCR2-negative cells (e.g., tissue-resident macrophages (e.g., Kupffer cells) or Langerhans cells).

[0051] Without wishing to be bound by theory, protumorigenic tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) express CSF1R and CCR2. In contrast, Kupffer cells and other tissue macrophages express CSF1R but not CCR2. In one embodiment, the anti-CSF1R / anti-CCR2 multispecific antibody molecules disclosed herein selectively bind to intratumoral M-MDSCs and M2 macrophages. In one embodiment, the anti-CSF1R / anti-CCR2 multispecific antibody molecules disclosed herein reduce immunosuppressive myeloid cells and / or increase infiltration of cytotoxic T cells in tumors. In one embodiment, the anti-CSF1R / anti-CCR2 multispecific antibody molecules disclosed herein deplete TAMs but preserve healthy liver Kupffer cells. In one embodiment, the anti-CSF1R / anti-CCR2 multispecific antibody molecules disclosed herein selectively localize to tumors to reduce systemic immunotoxicity.

[0052] In some embodiments, anti-CSF1R / anti-CCR2 multispecific molecules, upon binding to target cells, can induce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) on target cells. In some embodiments, anti-CSF1R / anti-CCR2 multispecific molecules can preferentially bind to and reduce the number of immunosuppressive myeloid cells (e.g., TAMs or MDSCs) in the tumor microenvironment, while preserving homeostatic myeloid cells (e.g., tissue-resident macrophages (e.g., Kupffer cells)) and other anti-tumor immune cells (e.g., activated T cells and NK cells). Depletion of homeostatic myeloid cells in vivo may be partially responsible for the adverse effects seen in patients treated with anti-CSF1R antibodies.

[0053] In some embodiments, a multispecific molecule has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more) of the following properties:

[0054] (i) the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-positive, CCR2-negative cells, or CSF1R-negative, CCR2-positive cells, e.g., the binding of the multispecific molecule to CSF1R-positive, CCR2-positive cells is at least 2, 4, 6, 8, 10, 15, 20, or 25-fold stronger than the binding of the multispecific molecule to CSF1R-positive, CCR2-negative cells or CSF1R-negative, CCR2-positive cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 The method described was measured;

[0055] (ii) the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-positive, CCR2-negative cells, e.g., the EC50 for binding of the multispecific molecule to CSF1R-positive, CCR2-positive cells is no greater than 60%, 50%, 40%, 30%, 20%, or 10% of the EC50 for binding of the multispecific molecule to CSF1R-positive, CCR2-negative cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 The method described was measured;

[0056] (iii) the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-negative, CCR2-positive cells, e.g., the EC50 for binding of the multispecific molecule to CSF1R-positive, CCR2-positive cells is no greater than 50%, 40%, 30%, 20%, 10%, or 5% of the EC50 for binding of the multispecific molecule to CSF1R-negative, CCR2-positive cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 The method described was measured;

[0057] (iv) the multispecific molecule preferentially binds to tumor-associated macrophages (TAMs) or myeloid-derived suppressor cells (MDSCs) relative to T cells, NK cells, neutrophils, tissue-resident macrophages (e.g., Kupffer cells), or Langerhans cells, e.g., the binding of the multispecific molecule to TAMs or MDSCs is at least 2, 4, 6, 8, 10, 15, 20, or 25-fold stronger than the binding of the multispecific molecule to T cells, NK cells, neutrophils, tissue-resident macrophages (e.g., Kupffer cells), or Langerhans cells, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometry assay described in Example 6. Figure 5 The method described was measured;

[0058] (v) The multispecific molecule inhibits monocyte migration, e.g., monocyte chemoattractant protein 1 (MCP1)-induced monocyte migration, e.g., reduces MCP1-induced monocyte migration by at least 40%, 50%, 60%, or 70%, e.g., as measured using a transwell plate migration assay, e.g., using a reference assay as described in Example 3. Figure 2 The method described was measured;

[0059] (vi) The multispecific molecule inhibits the proliferation of macrophages, e.g., bone marrow-derived macrophages, e.g., CSF-1-induced proliferation of bone marrow-derived macrophages, e.g., reduces CSF-1-induced proliferation of bone marrow-derived macrophages by at least 50%, 60%, 70%, or 80%, e.g., as measured using a cell proliferation MTT assay, e.g., using the assay described in Example 4. Figure 3B The method described was measured;

[0060] (vii) the multispecific molecule does not inhibit or does not substantially inhibit the differentiation of monocytes, e.g., bone marrow-derived monocytes, e.g., CSF-1-induced differentiation of bone marrow-derived monocytes, e.g., does not reduce CSF-1-induced differentiation of bone marrow-derived monocytes by more than 2%, 4%, 6%, 8%, or 10%, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometry assay described in Example 5. Figure 4 The method described was measured;

[0061] (viii) the multispecific molecule depletes suppressive myeloid cells, such as TAMs or MDSCs, in vivo, e.g., reduces the number of suppressive myeloid cells, such as TAMs or MDSCs, by at least 80%, 85%, 90%, 95%, 99%, or 99.5%, e.g., as measured using flow cytometry analysis, e.g., using the methods described in Example 7. Figure 6 measured by the method described;

[0062] (ix) The multispecific molecule does not deplete or does not substantially deplete tissue-resident macrophages, such as Kupffer cells, in vivo, e.g., does not reduce the number of tissue-resident macrophages, such as Kupffer cells, by more than 4%, 6%, 8%, 10%, or 15%, e.g., as measured using an immunohistochemical assay, e.g., using the reference assay described in Example 8. Figure 7B and 7D The method described was measured;

[0063] (x) The multispecific molecule increases CD86 or MHC class II expression on TAMs, e.g., as measured using flow cytometry or immunohistochemistry, e.g., using a reference Figure 21 The method described was measured;

[0064] (xi) The multispecific molecule does not inhibit or does not substantially inhibit CSF-1-dependent cell survival of CSF1R-positive, CCR2-negative cells, e.g., does not reduce CSF-1-dependent cell survival of CSF1R-positive, CCR2-negative cells by more than 5%, 10%, or 15%, e.g., as measured using a cell viability MTT assay, e.g., using the assay described in Example 9. Figure 8A The method described was measured;

[0065] (xii) the multispecific molecule increases CD8+ T cell tumor infiltration in vivo, e.g., increases the percentage of CD8+ T cells among CD3+ T cells in the tumor by at least 1.5-fold, 2-fold, or 2.5-fold, e.g., as measured using flow cytometry analysis, e.g., using the method described in Example 10. Figure 9 The method described was measured;

[0066] (xiii) the multispecific molecule reduces Treg frequency in a tumor in vivo, e.g., reduces Treg frequency in a tumor by at least 15%, 20%, 25%, or 30%, e.g., as measured using flow cytometry analysis, e.g., as described in Example 11 with reference to 10A;

[0067] (xiv) the multispecific molecule increases the CD8+ T cell / Treg ratio in a tumor in vivo, for example, by increasing the CD8+ T cell / Treg ratio in a tumor by at least 2.5-fold, 3-fold, 3.5-fold, 4-fold, or 4.5-fold, for example, as measured using flow cytometry analysis, for example, using the method described in Example 11. Figure 10B measured by the method described; or

[0068] (xv) Multispecific molecules reduce tumor growth, increase survival of tumor-bearing animals, and / or enhance anti-tumor immune memory, for example, using the reference Figure 11A and 11B The method described above is used for measurement, or by using the method described in Example 19. Figure 23The method described was used for measurement.

[0069] In some embodiments, the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-positive, CCR2-negative cells, or CSF1R-negative, CCR2-positive cells, e.g., the binding of the multispecific molecule to CSF1R-positive, CCR2-positive cells is at least 2, 4, 6, 8, 10, 15, 20, or 25-fold stronger than the binding of the multispecific molecule to CSF1R-positive, CCR2-negative cells or CSF1R-negative, CCR2-positive cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 Describe the method measurements.

[0070] In some embodiments, the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-positive, CCR2-negative cells, e.g., the multispecific molecule binds to CSF1R-positive, CCR2-positive cells with an EC50 that is no greater than 60%, 50%, 40%, 30%, 20%, or 10% of the EC50 of the multispecific molecule binding to CSF1R-positive, CCR2-negative cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 The method described was used for measurement.

[0071] In some embodiments, the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-negative, CCR2-positive cells, e.g., the multispecific molecule binds to CSF1R-positive, CCR2-positive cells with an EC50 that is no greater than 50%, 40%, 30%, 20%, 10%, or 5% of the EC50 of the multispecific molecule binding to CSF1R-negative, CCR2-positive cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 The method described was used for measurement.

[0072] In some embodiments, the multispecific molecule preferentially binds to tumor-associated macrophages (TAMs) or myeloid-derived suppressor cells (MDSCs) relative to T cells, NK cells, neutrophils, tissue-resident macrophages (e.g., Kupffer cells), or Langerhans cells, e.g., binding of the multispecific molecule to TAMs or MDSCs is at least 2, 4, 6, 8, 10, 15, 20, or 25-fold stronger than binding of the multispecific molecule to T cells, NK cells, neutrophils, tissue-resident macrophages (e.g., Kupffer cells), or Langerhans cells, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometry assay described in Example 6. Figure 5 The method described was used for measurement.

[0073] In some embodiments, the multispecific molecule inhibits monocyte migration, e.g., monocyte chemoattractant protein 1 (MCP1)-induced monocyte migration, e.g., reduces MCP1-induced monocyte migration by at least 40%, 50%, 60%, or 70%, e.g., as measured using a transwell plate migration assay, e.g., using the reference assay described in Example 3. Figure 2 Describe the method measurements.

[0074] In some embodiments, the multispecific molecules inhibit the proliferation of macrophages, e.g., bone marrow-derived macrophages, e.g., CSF-1-induced proliferation of bone marrow-derived macrophages, e.g., reduce CSF-1-induced proliferation of bone marrow-derived macrophages by at least 50%, 60%, 70%, or 80%, e.g., as measured using an MTT assay for cell proliferation, e.g., using the assay described in Example 4. Figure 3B The method described was used for measurement.

[0075] In some embodiments, the multispecific molecule does not inhibit or does not substantially inhibit the differentiation of monocytes, e.g., bone marrow-derived monocytes, e.g., CSF-1-induced differentiation of bone marrow-derived monocytes, e.g., does not reduce CSF-1-induced differentiation of bone marrow-derived monocytes by more than 2%, 4%, 6%, 8%, or 10%, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometry assay described in Example 5. Figure 4 The method described was used for measurement.

[0076] In some embodiments, the multispecific molecules deplete suppressive myeloid cells, e.g., TAMs or MDSCs, in vivo, e.g., reduce the number of suppressive myeloid cells, e.g., TAMs or MDSCs, by at least 80%, 85%, 90%, 95%, 99%, or 99.5%, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometry assay described in Example 7. Figure 6 Measured by the method described.

[0077] In some embodiments, the multispecific molecules do not deplete or do not substantially deplete tissue-resident macrophages, such as Kupffer cells, in vivo, e.g., do not reduce the number of tissue-resident macrophages, such as Kupffer cells, by more than 4%, 6%, 8%, 10%, or 15%, e.g., as measured using immunohistochemical analysis, e.g., using the reference assays described in Example 8. Figure 7B and 7D The method described was used for measurement.

[0078] In some embodiments, the multispecific molecule increases CD86 or MHC class II expression on TAMs, e.g., as measured using flow cytometry or immunohistochemistry, e.g., using a reference Figure 21 The method described was used for measurement.

[0079] In some embodiments, the multispecific molecule does not inhibit or does not substantially inhibit CSF-1-dependent cell survival of CSF1R-positive, CCR2-negative cells, e.g., does not reduce CSF-1-dependent cell survival of CSF1R-positive, CCR2-negative cells by more than 5%, 10%, or 15%, e.g., as measured using a cell viability MTT assay, e.g., using the assay described in Example 9. Figure 8A The method described was used for measurement.

[0080] In some embodiments, the multispecific molecule increases CD8+ T cell tumor infiltration in vivo, e.g., increases the percentage of CD8+ T cells among CD3+ T cells in a tumor by at least 1.5-fold, 2-fold, or 2.5-fold, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometry assay described in Example 10. Figure 9 The method described was used for measurement.

[0081] In some embodiments, the multispecific molecule reduces Treg frequency in a tumor in vivo, e.g., reduces Treg frequency in a tumor by at least 15%, 20%, 25%, or 30%, e.g., as measured using flow cytometry analysis, e.g., as described in Example 11 with reference to 10A.

[0082] In some embodiments, the multispecific molecule increases the CD8+ T cell / Treg ratio in a tumor in vivo, for example, by at least 2.5-fold, 3-fold, 3.5-fold, 4-fold, or 4.5-fold, as measured using flow cytometry analysis, for example, using the method described in Example 11. Figure 10B The method described was used for measurement.

[0083] In some embodiments, the multispecific molecules reduce tumor growth, increase survival of tumor-bearing animals, and / or enhance anti-tumor immune memory, for example, using the reference proteins described in Example 12. Figure 11A and 11B The method described above is used for measurement, or by using the method described in Example 19. Figure 23 The method described was used for measurement.

[0084] In some embodiments, the anti-CSF1R binding portion and the anti-CCR2 binding portion are independently intact antibodies (e.g., comprising at least one, preferably two, complete heavy chains, and at least one, preferably two complete light chains) or antigen-binding fragments (e.g., Fab, F(ab')2, Fv, scFv, single domain antibody, or diabody (dAb)). In some embodiments, the anti-CSF1R binding portion and / or the anti-CCR2 binding portion comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, or IgG4, or a fragment thereof. In some embodiments, the anti-CSF1R binding portion and / or the anti-CCR2 binding portion comprises a heavy chain constant region that can mediate antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, the anti-CSF1R binding portion comprises a first heavy chain constant region (e.g., a first Fc region), and the anti-CCR2 binding portion comprises a second heavy chain constant region (e.g., a second Fc region), wherein the first heavy chain constant region comprises one or more mutations that increase heterodimerization of the first and second heavy chain constant regions relative to a naturally occurring heavy chain constant region, and / or wherein the second heavy chain constant region comprises one or more mutations that increase heterodimerization of the second and first heavy chain constant regions relative to a naturally occurring heavy chain constant region. In some embodiments, the anti-CSF1R binding portion and / or the anti-CCR2 binding portion comprises a light chain constant region or fragment thereof selected from κ or λ. In some embodiments, the anti-CSF1R binding portion comprises a κ light chain constant region or fragment thereof, and the anti-CCR2 binding portion comprises a λ light chain constant region or fragment thereof. In some embodiments, the anti-CSF1R binding portion comprises a λ light chain constant region or fragment thereof, and the anti-CCR2 binding portion comprises a κ light chain constant region or fragment thereof. In some embodiments, the anti-CSF1R binding portion and the anti-CCR2 binding portion have a common light chain variable region. In some embodiments, the multispecific molecule further comprises a heavy chain constant region selected from IgG1, IgG2, and IgG4, more particularly, a heavy chain constant region (e.g., Fc region) of a human IgG1, IgG2, or IgG4. In some embodiments, the multispecific molecule further comprises a heavy chain constant region that can mediate antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0085] In some embodiments, the multispecific molecule comprises an anti-CSF1R antibody molecule and an anti-CCR2 antibody molecule, wherein:

[0086] (i) the anti-CSF1R antibody molecule comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first light chain variable region (VL) and a first light chain constant region (CL), and the second polypeptide comprises a first heavy chain variable region (VH), a first heavy chain constant region 1 (CH1), and optionally a first CH2 and a first CH3, and

[0087] (ii) The anti-CCR2 antibody molecule comprises a third polypeptide and a fourth polypeptide, wherein the third polypeptide comprises a second VL and a second CL, and the fourth polypeptide comprises a second VH, a second CH1, and optionally a second CH2 and a second CH3.

[0088] In some embodiments, (i) the anti-CSF1R antibody molecule binds monovalently to CSF1R, and / or (ii) the anti-CCR2 antibody molecule binds monovalently to CCR2. In some embodiments, the multispecific molecule binds monovalently to CSF1R and / or monovalently to CCR2. In some embodiments, the multispecific molecule binds monovalently to CSF1R and monovalently to CCR2.

[0089] In some embodiments,

[0090] (i) the multispecific molecule inhibits CSF1R in the presence of CCR2, optionally wherein the multispecific molecule reduces CSF1R activity (e.g., CSF1R signaling, e.g., CSF1-induced CSF1R signaling) in the cell by, e.g., at least 40%, 50%, 60%, 70%, 80% or 90% when the cell expresses both CSF1R and CCR2 on the cell surface, and / or

[0091] (ii) the multispecific molecule does not inhibit or does not substantially inhibit CSF1R in the absence of CCR2, optionally wherein when the cell expresses CSF1R on the cell surface but does not express CCR2, the multispecific molecule does not reduce the activity of CSF1R (e.g., CSF1R signaling, e.g., CSF1-induced CSF1R signaling), or does not reduce the activity of CSF1R by more than 2%, 4%, 6%, 8%, 10% or 15%.

[0092] In some embodiments,

[0093] (i) the multispecific molecule inhibits CCR2 in the presence of CSF1R, optionally wherein the multispecific molecule reduces CCR2 activity in the cell by, for example, at least 40%, 50%, 60%, 70%, 80% or 90% when the cell expresses both CCR2 and CSF1R on the cell surface, and / or

[0094] (ii) the multispecific molecule does not inhibit or does not substantially inhibit CCR2 in the absence of CSF1R, optionally wherein the multispecific molecule does not reduce the activity of CCR2, or does not reduce the activity of CCR2 by more than 2%, 4%, 6%, 8%, 10% or 15%, when the cell expresses CCR2 but does not express CSF1R on the cell surface.

[0095] In one aspect, the invention provides an isolated multispecific molecule, e.g., a bispecific molecule, comprising:

[0096] (i) a first binding moiety that binds to a monocyte-mediated inflammatory monocyte, such as Ly6C 高 CCR2+CX3CR1 低 a molecule that binds to the trafficking of inflammatory monocytes, optionally wherein the first binding moiety binds to CCR2; and

[0097] (ii) a second binding moiety that binds to a molecule that mediates maturation and / or survival of monocytes and / or macrophages on inflamed tissue, optionally wherein the second binding moiety binds to CSF1R.

[0098] In one aspect, the invention provides an isolated multispecific molecule, e.g., a bispecific molecule, comprising:

[0099] (i) a first binding moiety that reduces recruitment of inflammatory monocytes to tumors, optionally wherein the first binding moiety binds to and / or inhibits CCR2; and

[0100] (ii) a second binding moiety that reduces maturation and / or survival of monocytes and / or macrophages in the tumor microenvironment, optionally wherein the second binding moiety binds to and / or inhibits CSF1R.

[0101] In one aspect, the invention provides an isolated multispecific molecule, e.g., a bispecific molecule, comprising (i) an anti-CSF1R binding portion (e.g., an anti-CSF1R antibody molecule); and (ii) an anti-PD-L1 binding portion (e.g., an anti-PD-L1 antibody molecule).

[0102] In some embodiments of the aforementioned aspects and embodiments, the multispecific molecule further comprises one or more additional binding moieties (e.g., a third binding moiety, a fourth binding moiety (e.g., a trispecific molecule or a tetraspecific molecule)), optionally wherein the third binding moiety is a third IMC binding moiety or a tumor targeting moiety. In some embodiments, the tumor targeting moiety is a tumor targeting moiety disclosed in WO2017165464, e.g., pages 108-118 of WO2017165464, the entire contents of which are incorporated herein by reference.

[0103] In some embodiments of the aforementioned aspects and embodiments, the multispecific molecule further comprises an immune cell connector selected from a T cell connector, an NK cell connector, a B cell connector, a dendritic cell connector, or a macrophage connector. In some embodiments, the immune cell connector is an immune cell connector disclosed in WO2017165464, e.g., pages 119-131 of WO2017165464, the entire contents of which are incorporated herein by reference. In some embodiments, the immune cell connector binds to and activates immune cells, e.g., effector cells. In some embodiments, the immune cell connector binds to but does not activate immune cells, e.g., effector cells.

[0104] In some embodiments, the immune cell engager is a T cell engager, for example, a T cell engager that mediates binding to and activating T cells, or a T cell engager that mediates binding to but not activating T cells. In some embodiments, the T cell engager binds to CD3, TCRα, TCRβ, TCRγ, TCRζ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 or CD226, for example, the T cell engager is an anti-CD3 antibody molecule.

[0105] In some embodiments, the immune cell engager is an NK cell engager, for example, a mediator that binds to NK cells and activates NK cells, or a mediator that binds to NK cells but does not activate T cells. In some embodiments, the NK cell engager is selected from an antibody molecule such as an antigen binding domain or a ligand that binds to (for example, activates) the following items: NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (for example, CD16a, CD16b or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E or CD160.

[0106] In some embodiments, the immune cell engager is a B cell engager, such as a CD40L, OX40L, or CD70 ligand, or an antibody molecule that binds to OX40, CD40, or CD70. In some embodiments, the immune cell engager is a macrophage engager, such as a CD2 agonist; CD40L; OX40L; an antibody molecule that binds to OX40, CD40, or CD70; an agonist of a Toll-like receptor (TLR) (e.g., TLR4, such as a constitutively active TLR4 (caTLR4) or TLR9 agonist); CD47; or a STING agonist.

[0107] In some embodiments, the immune cell engager is a dendritic cell engager, such as a CD2 agonist, an OX40 antibody, OX40L, a 41BB agonist, a Toll-like receptor agonist or fragment thereof (e.g., TLR4, such as constitutively active TLR4 (caTLR4)), a CD47 agonist, or a STING agonist.

[0108] In some embodiments of the aforementioned aspects and embodiments, the multispecific molecule further comprises a cytokine molecule. In some embodiments, the cytokine molecule is selected from interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 10 (IL-10), interleukin 15 (IL-15), interleukin -18 (IL-18), interleukin -21 (IL-21) or interferon gamma, or a fragment or variant thereof, or a combination of any of the above cytokines. In some embodiments, the cytokine molecule is a cytokine molecule disclosed in WO2017165464, for example, pages 108-118 of WO2017165464, the entire contents of which are incorporated herein by reference.

[0109] In some embodiments of the aforementioned aspects and embodiments, the multispecific molecule further comprises a matrix modification portion. In some embodiments, the matrix modification portion causes one or more of the following: reducing the level or production of matrix or extracellular matrix (ECM) components; reducing tumor fibrosis; increasing interstitial tumor transport; improving tumor perfusion; expanding tumor microvascular structure; reducing interstitial fluid pressure (IFP) in the tumor; or reducing or enhancing the penetration or diffusion of agents such as cancer therapy or cell therapy into the tumor or tumor vasculature. In some embodiments, the matrix modification portion is a matrix modification portion disclosed in WO02017165464, for example, pages 131-136 of WO2017165464, the entire contents of which are incorporated herein by reference.

[0110] In one aspect, the invention provides an isolated multispecific molecule, e.g., a bispecific molecule, comprising:

[0111] (i) an anti-CSF1R binding portion (e.g., an anti-CSF1R antibody molecule);

[0112] (ii) an anti-CCR2 binding portion (e.g., an anti-CCR2 antibody molecule); and one, two, or all of the following:

[0113] (iii) TGF-β inhibitors;

[0114] (iv) an anti-PDL1 binding portion (e.g., an anti-PDL1 antibody molecule); and

[0115] (v) Cytokine molecules, such as IL-2 molecules.

[0116] In one embodiment, the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-positive, CCR2-negative cells, or CSF1R-negative, CCR2-positive cells, e.g., the binding of the multispecific molecule to CSF1R-positive, CCR2-positive cells is at least 2, 4, 6, 8, 10, 15, 20, or 25-fold stronger than the binding of the multispecific molecule to CSF1R-positive, CCR2-negative cells or CSF1R-negative, CCR2-positive cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 In one embodiment, the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-positive, CCR2-negative cells, e.g., the multispecific molecule binds to CSF1R-positive, CCR2-positive cells with an EC50 that is no greater than 60%, 50%, 40%, 30%, 20%, or 10% of the EC50 of the multispecific molecule binding to CSF1R-positive, CCR2-negative cells, e.g., as measured using flow cytometry analysis, e.g., using the assay described in Example 2. Figure 1 In one embodiment, the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-negative, CCR2-positive cells, e.g., the multispecific molecule binds to CSF1R-positive, CCR2-positive cells with an EC50 that is no greater than 50%, 40%, 30%, 20%, 10%, or 5% of the EC50 of the multispecific molecule binding to CSF1R-negative, CCR2-positive cells, e.g., as measured using flow cytometry analysis, e.g., using the assay described in Example 2. Figure 1In one embodiment, the multispecific molecule preferentially binds to tumor-associated macrophages (TAMs) or myeloid-derived suppressor cells (MDSCs) relative to T cells, NK cells, neutrophils, tissue-resident macrophages (e.g., Kupffer cells), or Langerhans cells, e.g., binding of the multispecific molecule to TAMs or MDSCs is at least 2, 4, 6, 8, 10, 15, 20, or 25-fold stronger than binding of the multispecific molecule to T cells, NK cells, neutrophils, tissue-resident macrophages (e.g., Kupffer cells), or Langerhans cells, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometry assay described in Example 6. Figure 5 In one embodiment, the multispecific molecule inhibits monocyte migration, e.g., monocyte chemoattractant protein 1 (MCP1)-induced monocyte migration, e.g., reduces MCP1-induced monocyte migration by at least 40%, 50%, 60%, or 70%, e.g., as measured using a transwell plate migration assay, e.g., using the method described in Example 3. Figure 2 In one embodiment, the multispecific molecule inhibits the proliferation of macrophages, such as bone marrow-derived macrophages, such as CSF-1-induced proliferation of bone marrow-derived macrophages, for example, reduces CSF-1-induced proliferation of bone marrow-derived macrophages by at least 50%, 60%, 70% or 80%, for example, as measured using a cell proliferation MTT assay, for example, as described in Example 4. Figure 3B In one embodiment, the multispecific molecule does not inhibit or does not substantially inhibit the differentiation of monocytes, such as bone marrow-derived monocytes, e.g., CSF-1-induced differentiation of bone marrow-derived monocytes, e.g., does not reduce CSF-1-induced differentiation of bone marrow-derived monocytes by more than 2%, 4%, 6%, 8% or 10%, e.g., as measured using flow cytometry analysis, e.g., using the methods described in Example 5. Figure 4 In one embodiment, the multispecific molecule depletes suppressive myeloid cells, such as TAMs or MDSCs, in vivo, e.g., reduces the number of suppressive myeloid cells, such as TAMs or MDSCs, by at least 80%, 85%, 90%, 95%, 99%, or 99.5%, e.g., as measured using flow cytometry analysis, e.g., using the methods described in Example 7. Figure 6 In one embodiment, the multispecific molecule does not deplete or substantially deplete tissue-resident macrophages, such as Kupffer cells, in vivo, e.g., does not reduce the number of tissue-resident macrophages, such as Kupffer cells, by more than 4%, 6%, 8%, 10%, or 15%, e.g., as measured using immunohistochemical analysis, e.g., using the methods described in Example 8. Figure 7B and 7DIn one embodiment, the multispecific molecule increases CD86 or MHC class II expression on TAMs, e.g., as measured using flow cytometry or immunohistochemistry, e.g., using a reference Figure 21 In one embodiment, the multispecific molecule does not inhibit or does not substantially inhibit CSF-1-dependent cell survival of CSF1R-positive, CCR2-negative cells, e.g., does not reduce CSF-1-dependent cell survival of CSF1R-positive, CCR2-negative cells by more than 5%, 10%, or 15%, e.g., as measured using a cell viability MTT assay, e.g., using the assay described in Example 9. Figure 8A In one embodiment, the multispecific molecule increases CD8+ T cell tumor infiltration in vivo, e.g., increases the percentage of CD8+ T cells among CD3+ T cells in a tumor by at least 1.5-fold, 2-fold, or 2.5-fold, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometric assay described in Example 10. Figure 9 In one embodiment, the multispecific molecule reduces Treg frequency in a tumor in vivo, e.g., reduces Treg frequency in a tumor by at least 15%, 20%, 25%, or 30%, e.g., as measured using flow cytometry analysis, e.g., as measured using the method described in Example 11 with reference to 10A. In one embodiment, the multispecific molecule increases the CD8+ T cell / Treg ratio in a tumor in vivo, e.g., increases the CD8+ T cell / Treg ratio in a tumor by at least 2.5-fold, 3-fold, 3.5-fold, 4-fold, or 4.5-fold, e.g., as measured using flow cytometry analysis, e.g., as measured using the method described in Example 11 with reference to 10A. Figure 10B In one embodiment, the multispecific molecule reduces tumor growth, increases survival of tumor-bearing animals, and / or enhances anti-tumor immune memory, for example, using the methods described in Example 12. Figure 11A and 11B The method described above is used for measurement, or by using the method described in Example 19. Figure 23 The method described was used for measurement.

[0117] In one embodiment, the anti-CSF1R binding portion and the anti-CCR2 binding portion are independently intact antibodies (e.g., comprising at least one, preferably two, complete heavy chains, and at least one, preferably two complete light chains) or antigen-binding fragments (e.g., Fab, F(ab')2, Fv, scFv, single domain antibody, or diabody (dAb)). In one embodiment, the anti-CSF1R binding portion and / or the anti-CCR2 binding portion comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, or IgG4, or a fragment thereof. In one embodiment, the anti-CSF1R binding portion and / or the anti-CCR2 binding portion comprises a heavy chain constant region that can mediate antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In one embodiment, the anti-CSF1R binding portion comprises a first heavy chain constant region (e.g., a first Fc region), and the anti-CCR2 binding portion comprises a second heavy chain constant region (e.g., a second Fc region), wherein the first heavy chain constant region comprises one or more mutations that increase heterodimerization of the first and second heavy chain constant regions relative to a naturally occurring heavy chain constant region, and / or wherein the second heavy chain constant region comprises one or more mutations that increase heterodimerization of the second and first heavy chain constant regions relative to a naturally occurring heavy chain constant region. In one embodiment, the anti-CSF1R binding portion and / or the anti-CCR2 binding portion comprises a light chain constant region or fragment thereof selected from κ or λ. In one embodiment, the anti-CSF1R binding portion comprises a κ light chain constant region or fragment thereof, and the anti-CCR2 binding portion comprises a λ light chain constant region or fragment thereof. In one embodiment, the anti-CSF1R binding portion comprises a λ light chain constant region or fragment thereof, and the anti-CCR2 binding portion comprises a κ light chain constant region or fragment thereof. In one embodiment, the anti-CSF1R binding portion comprises a λ light chain constant region or fragment thereof, and the anti-CCR2 binding portion comprises a κ light chain constant region or fragment thereof. In one embodiment, the anti-CSF1R binding portion and the anti-CCR2 binding portion have a common light chain variable region. In one embodiment, the multispecific molecule further comprises a heavy chain constant region selected from IgG1, IgG2, and IgG4, more particularly, a heavy chain constant region (e.g., Fc region) of a human IgG1, IgG2, or IgG4. In one embodiment, the multispecific molecule further comprises a heavy chain constant region that can mediate antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).In one embodiment, a multispecific molecule comprises an anti-CSF1R antibody molecule and an anti-CCR2 antibody molecule, wherein: (i) the anti-CSF1R antibody molecule comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first light chain variable region (VL) and a first light chain constant region (CL), and the second polypeptide comprises a first heavy chain variable region (VH), a first heavy chain constant region 1 (CH1), and optionally a first CH2 and a first CH3, and (ii) the anti-CCR2 antibody molecule comprises a third polypeptide and a fourth polypeptide, wherein the third polypeptide comprises a second VL and a second CL, and the fourth polypeptide comprises a second VH, a second CH1, and optionally a second CH2 and a second CH3.

[0118] In one embodiment, the anti-CSF1R antibody molecule binds monovalently to CSF1R. In one embodiment, the anti-CCR2 antibody molecule binds monovalently to CCR2. In one embodiment, the multispecific molecule binds monovalently to CSF1R. In one embodiment, the multispecific molecule binds monovalently to CCR2. In one embodiment, the multispecific molecule binds monovalently to CSF1R and monovalently to CCR2.

[0119] In one embodiment, the multispecific molecule inhibits CSF1R in the presence of CCR2, optionally wherein the multispecific molecule reduces CSF1R activity (e.g., CSF1R signaling, e.g., CSF1-induced CSF1R signaling) in the cell by, for example, at least 40%, 50%, 60%, 70%, 80%, or 90% when the cell expresses both CSF1R and CCR2 on the cell surface. In one embodiment, the multispecific molecule does not inhibit or does not substantially inhibit CSF1R in the absence of CCR2, optionally wherein the multispecific molecule does not reduce CSF1R activity (e.g., CSF1R signaling, e.g., CSF1-induced CSF1R signaling) when the cell expresses CSF1R but does not express CCR2 on the cell surface, or does not reduce CSF1R activity by more than 2%, 4%, 6%, 8%, 10%, or 15%. In one embodiment, the multispecific molecule inhibits CCR2 in the presence of CSF1R, optionally wherein the multispecific molecule reduces CCR2 activity in the cell by, for example, at least 40%, 50%, 60%, 70%, 80%, or 90% when the cell expresses both CCR2 and CSF1R on the cell surface. In one embodiment, the multispecific molecule does not inhibit or does not substantially inhibit CCR2 in the absence of CSF1R, optionally wherein the multispecific molecule does not reduce CCR2 activity, or does not reduce CCR2 activity by more than 2%, 4%, 6%, 8%, 10%, or 15% when the cell expresses CCR2 but does not express CSF1R on the cell surface.

[0120] In one embodiment, the multispecific molecule comprises (i) an anti-CSF1R binding portion (e.g., an anti-CSF1R antibody molecule); (ii) an anti-CCR2 binding portion (e.g., an anti-CCR2 antibody molecule); and (iii) a TGF-β inhibitor. In one embodiment, the TGF-β inhibitor inhibits TGF-β1, TGF-β3, or both TGF-β1 and TGF-β3, for example, using the method described in Example 20. Figure 26B In one embodiment, the TGF-β inhibitor inhibits TGF-β1, TGF-β2, TGF-β3, both TGF-β1 and TGF-β3, or TGF-β1, TGF-β2 and TGF-β3, for example, using the method described in Example 20. Figure 26B In one embodiment, the multispecific molecule reduces tumor growth and / or increases survival of tumor-bearing animals, for example, by using the method described in Example 20. Figures 27A-27CIn one embodiment, the TGF-β inhibitor comprises a TGF-β receptor polypeptide (e.g., an extracellular domain of a TGF-β receptor or a functional variant thereof). In one embodiment, the TGF-β inhibitor comprises one, two, or all of the following: a TGFBR1 polypeptide (e.g., 1, 2, 3, or more TGFBR1 polypeptides), a TGFBR2 polypeptide (e.g., 1, 2, 3, or more TGFBR2 polypeptides), or a TGFBR3 polypeptide (e.g., 1, 2, 3, or more TGFBR3 polypeptides). In one embodiment, the TGF-β inhibitor comprises a TGFBR1 polypeptide. In one embodiment, the TGF-β inhibitor comprises an extracellular domain of TGFBR1, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises an extracellular domain of SEQ ID NO: 95, 96, 97, 120, 121, or 122, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 104 or 105, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises a TGFBR2 polypeptide. In one embodiment, the TGF-β inhibitor comprises the extracellular domain of TGFBR2, or a sequence substantially identical thereto (e.g., a sequence 80%, 85%, 90% or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises the extracellular domain of SEQ ID NO: 98, 99, 123 or 124, or a sequence substantially identical thereto (e.g., a sequence 80%, 85%, 90% or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises an amino acid sequence selected from SEQ ID NO: 100, 101, 102 or 103, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises a TGFBR3 polypeptide. In one embodiment, the TGF-β inhibitor comprises the extracellular domain of TGFBR3, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises the extracellular domain of SEQ ID NO: 106, 107, 125 or 126, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 108, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto).In one embodiment, the TGF-β inhibitor comprises two TGF-β receptor polypeptides that form a homodimer. In one embodiment, the TGF-β inhibitor comprises two TGFBR1 polypeptides that form a homodimer. In one embodiment, the TGF-β inhibitor comprises two TGFBR2 polypeptides that form a homodimer. In one embodiment, the TGF-β inhibitor comprises two TGFBR3 polypeptides that form a homodimer. In one embodiment, the TGF-β inhibitor comprises two TGF-β receptor polypeptides that form a heterodimer. In one embodiment, the TGF-β inhibitor comprises a TGFBR1 polypeptide and a TGFBR2 polypeptide that form a heterodimer. In one embodiment, the TGF-β inhibitor comprises a TGFBR1 polypeptide and a TGFBR3 polypeptide that form a heterodimer. In one embodiment, the TGF-β inhibitor comprises a TGFBR2 polypeptide and a TGFBR3 polypeptide that form a heterodimer.

[0121] In one embodiment, the TGF-β inhibitor comprises a first TGF-β receptor polypeptide and a second TGF-β receptor polypeptide. In one embodiment, the multispecific molecule comprises a first Fc region (e.g., a first CH1-Fc region) and a second Fc region (e.g., a second CH1-Fc region), optionally wherein: (i) the first TGF-β receptor polypeptide is linked to the first Fc region (e.g., a first CH1-Fc region), e.g., the C-terminus of the first Fc region (e.g., the first CH1-Fc region), e.g., via a linker, and (ii) the second TGF-β receptor polypeptide is linked to the second Fc region (e.g., a second CH1-Fc region), e.g., the C-terminus of the second Fc region (e.g., the second CH1-Fc region), e.g., via a linker. In one embodiment, the first TGF-β receptor polypeptide and the second TGF-β receptor polypeptide form a homodimer or a heterodimer, e.g., a homodimer. In one embodiment, the first TGF-β receptor polypeptide or the second TGF-β receptor polypeptide comprises an extracellular domain of TGFBR1, TGFBR2, or TGFBR3, e.g., an extracellular domain of TGFBR2. In one embodiment, the multispecific molecule has Figure 35Aor 35B configuration. In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 192 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 193 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 192 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 195 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 194 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 193 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 194 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 195 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto).

[0122] In one embodiment, the TGF-β inhibitor comprises a first TGF-β receptor polypeptide and a second TGF-β receptor polypeptide. In one embodiment, the multispecific molecule comprises a heavy chain constant region 1 (CH1) and a light chain constant region (CL), optionally wherein: (i) the first TGF-β receptor polypeptide is linked to CH1, e.g., the N-terminus of CH1, e.g., via a linker, and (ii) the second TGF-β receptor polypeptide is linked to CL, e.g., the N-terminus of CL, e.g., via a linker. In one embodiment, the first TGF-β receptor polypeptide and the second TGF-β receptor polypeptide form a homodimer or a heterodimer, e.g., a homodimer. In one embodiment, the first TGF-β receptor polypeptide or the second TGF-β receptor polypeptide comprises an extracellular domain of TGFBR1, TGFBR2, or TGFBR3, e.g., the extracellular domain of TGFBR2. In one embodiment, the multispecific molecule has Figure 35CIn one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 196 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 198 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 196 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 199 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 197 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 198 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 197 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 199 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto).

[0123] In one embodiment, the TGF inhibitor is linked to an anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule), e.g., via a linker. In one embodiment, the TGF inhibitor is linked to an anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule), e.g., via a linker. In one embodiment, the anti-CSF1R binding moiety and / or the anti-CCR2 binding moiety comprises an Fc region, wherein the TGF inhibitor is linked to the Fc region, e.g., the C-terminus of the Fc region, e.g., via a linker. In one embodiment, the multispecific molecule comprises a first TGF-β inhibitor and a second TGF-β inhibitor, wherein the first TGF-β inhibitor is linked to the anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule), e.g., via a linker and the second TGF-β inhibitor is linked to the anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule) via a linker. In one embodiment, the anti-CSF1R binding moiety comprises a first Fc region, the anti-CCR2 binding moiety comprises a second Fc region, and the multispecific molecule comprises a first TGF-β inhibitor and a second TGF-β inhibitor, wherein the first TGF-β inhibitor is linked to the first Fc region, e.g., via a linker, and the second TGF-β inhibitor is linked to the second Fc region, e.g., via a linker, e.g., via a linker, e.g., C-terminus of the second Fc region.

[0124] In one embodiment, the anti-CSF1R binding moiety comprises a first light chain and a first heavy chain, wherein the first heavy chain comprises a first Fc region, wherein the C-terminus of the first Fc region is linked to the extracellular domain of TGFBR2 or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In one embodiment, the anti-CCR2 binding moiety comprises a second light chain and a second heavy chain, wherein the second heavy chain comprises a second Fc region, wherein the C-terminus of the second Fc region is linked to the extracellular domain of TGFBR2 or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In one embodiment, the multispecific molecule has Figure 12B Configuration shown.

[0125] In one embodiment, the TGF inhibitor is linked to an anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule), e.g., via a linker. In one embodiment, the TGF inhibitor is linked to an anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule), e.g., via a linker. In one embodiment, the anti-CSF1R binding moiety and / or the anti-CCR2 binding moiety comprises an Fc region, wherein the TGF inhibitor is linked to the Fc region, e.g., via a linker, at the N-terminus of the Fc region.

[0126] In one embodiment, the multispecific molecule comprises (i) an anti-CSF1R binding portion (e.g., an anti-CSF1R antibody molecule); (ii) an anti-CCR2 binding portion (e.g., an anti-CCR2 antibody molecule); and (iii) an anti-PDL1 binding portion (e.g., an anti-PDL1 antibody molecule). In one embodiment, the anti-PDL1 binding portion inhibits PDL1. In one embodiment, the anti-PDL1 binding portion comprises a heavy chain variable domain (VH) comprising the HCDR1, HCDR2, and HCDR3 of any VH sequence in Table 10, or a sequence having at least one amino acid change but not more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In one embodiment, the anti-PDL1 binding portion comprises a VH comprising any VH sequence in Table 10, or a sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto) or having at least one amino acid change but not more than five, ten, or fifteen changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In one embodiment, the anti-PDL1 binding portion comprises a light chain variable domain (VL) comprising LCDR1, LCDR2, and LCDR3 of any VL sequence in Table 10, or a sequence having at least one amino acid change but not more than two, three, or four changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In one embodiment, the anti-PDL1 binding portion comprises a VL comprising any VL sequence in Table 10, or a sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto) or having at least one amino acid change but not more than five, ten, or fifteen changes (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions).

[0127] In one embodiment, the anti-PDL1 binding portion is linked to an anti-CSF1R binding portion (e.g., an anti-CSF1R antibody molecule) or an anti-CCR2 binding portion (e.g., an anti-CCR2 antibody molecule), e.g., via a linker. In one embodiment, the anti-CSF1R binding portion and / or the anti-CCR2 binding portion comprises an Fc region, wherein the anti-PDL1 binding portion is linked to the Fc region, e.g., the C-terminus of the Fc region, e.g., via a linker. In one embodiment, the PDL1 binding portion is an scFv. In one embodiment, the multispecific molecule has Figure 12C or Figure 12D Configuration shown.

[0128] In one embodiment, the multispecific molecule comprises (i) an anti-CSF1R binding portion (e.g., an anti-CSF1R antibody molecule); (ii) an anti-CCR2 binding portion (e.g., an anti-CCR2 antibody molecule); and (iii) an IL-2 molecule. In one embodiment, the IL-2 molecule is an IL-2 molecule disclosed herein, e.g., an IL-2 molecule comprising the amino acid sequence of SEQ ID NO: 237 or 238, or a sequence substantially identical thereto (e.g., a sequence 80%, 85%, 90%, or 95% identical thereto).

[0129] In one embodiment, the IL-2 molecule is linked to an anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule) or an anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule), e.g., via a linker. In one embodiment, the anti-CSF1R binding moiety and / or the anti-CCR2 binding moiety comprises a light chain constant region, wherein the IL-2 molecule is linked to the light chain constant region, e.g., the C-terminus of the light chain constant region, e.g., via a linker. In one embodiment, the multispecific molecule comprises a first IL-2 molecule and a second IL-2 molecule, wherein the first IL-2 molecule is linked to the anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule), e.g., via a linker, and the second IL-2 molecule is linked to the anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule) via a linker. In one embodiment, the anti-CSF1R binding moiety comprises a first light chain constant region, the anti-CCR2 binding moiety comprises a second light chain constant region, and the multispecific molecule comprises a first IL-2 molecule and a second IL-2 molecule, wherein the first IL-2 molecule is linked to the first light chain constant region, e.g., the C-terminus of the first light chain constant region, e.g., via a linker, and the second IL-2 molecule is linked to the second light chain constant region, e.g., the C-terminus of the second light chain constant region, e.g., via a linker. In one embodiment, the multispecific molecule has Figure 12E 、 Figure 12F or Figure 12G Configuration shown.

[0130] In one aspect, provided herein are antibody molecules (e.g., isolated antibody molecules) that bind to CSF1R (e.g., human CSF1R) comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 474, and 475, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the antibody molecule further comprises a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3. In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 432, 434, and 436, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 433, 435, and 437, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In one aspect, provided herein are antibody molecules (e.g., isolated antibody molecules) that bind to CSF1R (e.g., human CSF1R) comprising a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 432, 434, and 436, respectively, or comprise the amino acid sequences of SEQ ID NOs: 433, 435, and 437, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences.

[0131] In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequence of SEQ ID NO: 402, 476, and 477, respectively, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications.

[0132] In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the following amino acid sequences, respectively: SEQ ID NOs: 402, 404, and 413, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications; SEQ ID NOs: 402, 405, and 413, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications; SEQ ID NOs: 402, 406, and 413, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications; SEQ ID NOs: 402, 404, and 414, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications; SEQ ID NOs: 402, 407, and 413, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications; SEQ ID NOs: 402, 404, and 415, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications; NO: 402, 408 and 413, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5 or 6 modifications; SEQ ID NO: 402, 404 and 416, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5 or 6 modifications; SEQ ID NO: 402, 404 and 417, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5 or 6 modifications; SEQ ID NO: 402, 409 and 413, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5 or 6 modifications; SEQ ID NO: 402, 404 and 418, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5 or 6 modifications; SEQ ID NO: 402, 404 and 419, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5 or 6 modifications; SEQ ID NO: 402, 404 and 420, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5 or 6 modifications; SEQ ID NO: 402, 404 and 421, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5 or 6 modifications. In some embodiments, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NO: 402, 404 and 413, respectively. In some embodiments, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NO: 402, 405 and 413, respectively. In some embodiments, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NO: 402, 406 and 413, respectively. In some embodiments, the HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NO: 402, 404 and 414, respectively.In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 407, and 413, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, and 415, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 408, and 413, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, and 416, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, and 417, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 409, and 413, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, and 418, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, and 419, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, and 420, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, and 421, respectively.

[0133] In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs: 432, 434, and 436, respectively. In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs: 433, 435, and 437, respectively.

[0134] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 413, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 405, 413, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 406, 413, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 414, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 407, 413, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 415, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 408, 413, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 416, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 417, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 409, 413, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 418, 432, 434, and 436, respectively.In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 419, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 420, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 421, 432, 434, and 436, respectively.

[0135] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 323, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 324, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 325, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 326, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 327, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 328, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 329, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 330, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 331, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 332, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 333, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 334, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 335, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto.In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 336, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto.

[0136] In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 341, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 342, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto.

[0137] In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 323 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 324 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 325 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 326 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 327 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 328 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 329 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 330 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 331 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 332 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 333 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 334 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 335 and 341, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 336 and 341, respectively.

[0138] In one aspect, disclosed herein are antibody molecules (e.g., isolated antibody molecules) that bind to CSF1R (e.g., human CSF1R) comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 478, and 479, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the antibody molecule further comprises a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3. In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 432, 434, and 436, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 433, 435, and 437, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In one aspect, disclosed herein are antibody molecules (e.g., isolated antibody molecules) that bind to CSF1R (e.g., human CSF1R), comprising a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 432, 434, and 436, respectively, or comprise the amino acid sequences of SEQ ID NOs: 433, 435, and 437, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences.

[0139] In some embodiments, HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequence of SEQ ID NOs: 403, 410, and 422, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications; SEQ ID NOs: 403, 410, and 423, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications; SEQ ID NOs: 403, 411, and 422, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications; or SEQ ID NOs: 403, 412, and 422, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications.

[0140] In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs: 432, 434, and 436, respectively. In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NOs: 433, 435, and 437, respectively.

[0141] In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 410, 422, 433, 435, and 437, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 410, 423, 433, 435, and 437, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 411, 422, 433, 435, and 437, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 412, 422, 433, 435, and 437, respectively.

[0142] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 337, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 338, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 339, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 340, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto.

[0143] In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 341, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 342, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto.

[0144] In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 337 and 342, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 338 and 342, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 339 and 342, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 340 and 342, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 339 and 139, respectively.

[0145] In some embodiments, the antibody molecule binds to human and / or cynomolgus monkey CSF1R, for example, as measured using an ELISA assay, for example, using the assay described in Example 22. Figure 29A and 29B In some embodiments, the antibody molecule binds to human CSF1R cells, for example, as measured using flow cytometry analysis, for example, using the method described in Example 24. Figure 31 In some embodiments, the antibody molecule reduces MCP-1 secretion by monocytes in the presence of CSF-1, for example as measured using an electrochemiluminescence assay, for example as described in Example 23. Figure 30 In some embodiments, the antibody molecule reduces the activity of CSF1R.

[0146] In one aspect, provided herein are antibody molecules (e.g., isolated antibody molecules) that bind to CCR2 (e.g., human CCR2), comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region (VL) comprising a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequence of SEQ ID NO: 446, 447, 448, 454, 455, and 456, respectively, or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications, and wherein:

[0147] (i) VH does not comprise the amino acid sequence of SEQ ID NO: 480, and / or

[0148] (ii) VL does not comprise the amino acid sequence of SEQ ID NO: 481.

[0149] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 446, 447, 448, 454, 455, and 456, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 343, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 344, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 345, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 343 and 345, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 344 and 345, respectively.

[0150] In one aspect, provided herein are antibody molecules (e.g., isolated antibody molecules) that bind to PD-L1 (e.g., human PD-L1) comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 461, 462, and 463, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the antibody molecule further comprises a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 467, 468, and 469, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In one aspect, provided herein are antibody molecules (e.g., isolated antibody molecules) that bind to PD-L1 (e.g., human PD-L1) comprising a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 467, 468, and 469, respectively, or comprise no more than 1, 2, 3, 4, 5, or 6 modifications. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 461, 462, 463, 467, 468, and 469, respectively. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 346, or an amino acid sequence that is at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 347, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98% or 99% identity thereto. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NO: 346 and 347, respectively.

[0151] In some embodiments of the above aspects, the antibody molecule comprises a heavy chain constant region or a fragment thereof selected from IgG1, IgG2, IgG3 or IgG4. In some embodiments, the antibody molecule comprises a light chain constant region or a fragment thereof selected from a light chain constant region of κ or λ.

[0152] In one aspect, provided herein are multispecific molecules, e.g., bispecific molecules, comprising an anti-CSF1R binding portion and an anti-CCR2 binding portion. In some embodiments, the anti-CSF1R binding portion comprises an anti-CSF1R antibody molecule disclosed herein. In some embodiments, the anti-CCR2 binding portion comprises an anti-CCR2 antibody molecule disclosed herein. In some embodiments, the anti-CSF1R binding portion comprises an anti-CSF1R antibody molecule disclosed herein, and the anti-CCR2 binding portion comprises an anti-CCR2 antibody molecule disclosed herein.

[0153] In some embodiments, the anti-CSF1R binding portion comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein: (i) HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 474, and 475, respectively; and (ii) LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 432, 434, and 436 or SEQ ID NOs: 433, 435, and 437, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 476, and 477, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the following amino acid sequences, respectively: SEQ ID NOs: 402, 404, and 413; SEQ ID NOs: 402, 405, and 413; SEQ ID NOs: 402, 406, and 413; SEQ ID NOs: 402, 404, and 414; SEQ ID NOs: 402, 407, and 413; SEQ ID NOs: 402, 404, and 415; SEQ ID NOs: 402, 408, and 413; SEQ ID NOs: 402, 404, and 416; SEQ ID NOs: 402, 404, and 417; SEQ ID NOs: 402, 409, and 413; SEQ ID NOs: 402, 404, and 418; SEQ ID NOs: 402, 404, and 419; SEQ ID NOs: 402, 404, and 420; or SEQ ID NOs: NO: 402, 404 and 421. In some embodiments, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequence of SEQ ID NO: 432, 434 and 436, respectively. In some embodiments, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequence of SEQ ID NO: 433, 435 and 437, respectively.In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the following amino acid sequences, respectively: SEQ ID NOs: 402, 404, 413, 432, 434, and 436; SEQ ID NOs: 402, 405, 413, 432, 434, and 436; SEQ ID NOs: 402, 406, 413, 432, 434, and 436; SEQ ID NOs: 402, 404, 414, 432, 434, and 436; SEQ ID NOs: 402, 407, 413, 432, 434, and 436; SEQ ID NOs: 402, 404, 415, 432, 434, and 436; SEQ ID NOs: 402, 408, 413, 432, 434, and 436; NO:402, 404, 416, 432, 434 and 436; SEQ ID NO:402, 404, 417, 432, 434 and 436; SEQ ID NO:402, 409, 413, 432, 434 and 436; SEQ ID NO:402, 404, 418, 432, 434 and 436; SEQ ID NO:402, 404, 419, 432, 434 and 436; SEQ ID NO:402, 404, 420, 432, 434 and 436; or SEQ ID NO:402, 404, 421, 432, 434 and 436. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 323-336, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NOs: 341 or 342, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto. In some embodiments, VH and VL comprise the following amino acid sequences, respectively: SEQ ID NOs: 323 and 341; SEQ ID NOs: 324 and 341; SEQ ID NOs: 325 and 341; SEQ ID NOs: 326 and 341; SEQ ID NOs: 327 and 341; SEQ ID NOs: 328 and 341; SEQ ID NOs: 329 and 341; SEQ ID NOs: 330 and 341; SEQ ID NOs: 331 and 341; SEQ ID NOs: 332 and 341; SEQ ID NOs: 333 and 341; SEQ ID NOs: 334 and 341; SEQ ID NOs: 335 and 341; or SEQ ID NOs: 336 and 341.

[0154] In some embodiments, the anti-CSF1R binding portion comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein: (i) HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 478, and 479, respectively; and (ii) LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 432, 434, and 436 or SEQ ID NOs: 433, 435, and 437, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 410, and 422; SEQ ID NOs: 403, 410, and 423; SEQ ID NOs: 403, 411, and 422; or SEQ ID NOs: 403, 412, and 422, respectively. In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 432, 434, and 436, respectively. In some embodiments, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 433, 435, and 437, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 410, 422, 433, 435, and 437, respectively; SEQ ID NOs: 403, 410, 423, 433, 435, and 437; SEQ ID NOs: 403, 411, 422, 433, 435, and 437; or SEQ ID NOs: 403, 412, 422, 433, 435, and 437, respectively. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 337-340, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NOs: 341 or 342, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH and VL comprise the following amino acid sequences, respectively: SEQ ID NOs: 337 and 342; SEQ ID NOs: 338 and 342; SEQ ID NOs: 339 and 342; or SEQ ID NOs: 340 and 342.

[0155] In some embodiments, the anti-CCR2 binding portion comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 446, 447, 448, 454, 455, and 456, respectively, and wherein: (i) VH does not comprise the amino acid sequence of SEQ ID NO: 480, or (ii) VL does not comprise the amino acid sequence of SEQ ID NO: 481. In some embodiments, VH comprises the amino acid sequence of SEQ ID NOs: 343 or 344, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 345, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, the VH and VL comprise the following amino acid sequences: SEQ ID NO: 343 and 345; or SEQ ID NO: 344 and 345, respectively.

[0156] In some embodiments, the anti-CSF1R binding portion and the anti-CCR2 binding portion are independently intact antibodies (e.g., comprising at least one, preferably two, complete heavy chains, and at least one, preferably two complete light chains) or antigen-binding fragments (e.g., Fab, F(ab')2, Fv, scFv, single domain antibody, or diabody (dAb)). In some embodiments, the anti-CSF1R binding portion and / or the anti-CCR2 binding portion comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, or IgG4, or a fragment thereof. In some embodiments, the anti-CSF1R binding portion comprises a first heavy chain constant region (e.g., a first Fc region), and the anti-CCR2 binding portion comprises a second heavy chain constant region (e.g., a second Fc region), wherein the first heavy chain constant region comprises one or more mutations that increase heterodimerization of the first heavy chain constant region and the second heavy chain constant region relative to a naturally occurring heavy chain constant region, and / or wherein the second heavy chain constant region comprises one or more mutations that increase heterodimerization of the second heavy chain constant region and the first heavy chain constant region relative to a naturally occurring heavy chain constant region. In some embodiments, the anti-CSF1R binding portion and / or the anti-CCR2 binding portion comprises a light chain constant region or fragment thereof selected from kappa or lambda. In some embodiments, the anti-CSF1R binding portion comprises a kappa light chain constant region or fragment thereof, and the anti-CCR2 binding portion comprises a lambda light chain constant region or fragment thereof. In some embodiments, the anti-CSF1R binding portion comprises a lambda light chain constant region or fragment thereof, and the anti-CCR2 binding portion comprises a kappa light chain constant region or fragment thereof. In some embodiments, the anti-CSF1R binding portion and the anti-CCR2 binding portion have a common light chain variable region.

[0157] In some embodiments, the multispecific molecule monovalently binds CSF1R and / or the multispecific molecule monovalently binds CCR2, optionally wherein the multispecific molecule monovalently binds CSF1R and monovalently binds CCR2. In some embodiments, the multispecific molecule inhibits CSF1R in the presence of CCR2, optionally wherein the multispecific molecule reduces CSF1R activity (e.g., CSF1R signaling, e.g., CSF1-induced CSF1R signaling) in a cell by, e.g., at least 40%, 50%, 60%, 70%, 80%, or 90% when the cell expresses both CSF1R and CCR2 on the cell surface. In some embodiments, the multispecific molecule does not inhibit or does not substantially inhibit CSF1R in the absence of CCR2, optionally wherein when the cell expresses CSF1R but does not express CCR2 on the cell surface, the multispecific molecule does not reduce CSF1R activity (e.g., CSF1R signaling, e.g., CSF1-induced CSF1R signaling), or does not reduce CSF1R activity by more than 2%, 4%, 6%, 8%, 10%, or 15%. In some embodiments, the multispecific molecule inhibits CCR2 in the presence of CSF1R, optionally wherein when the cell expresses both CCR2 and CSF1R on the cell surface, the multispecific molecule reduces CCR2 activity in the cell by, e.g., at least 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the multispecific molecule does not inhibit or does not substantially inhibit CCR2 in the absence of CSF1R, optionally wherein the multispecific molecule does not reduce the activity of CCR2, or does not reduce the activity of CCR2 by more than 2%, 4%, 6%, 8%, 10% or 15% when the cell expresses CCR2 but does not express CSF1R on the cell surface.

[0158] In some embodiments, the multispecific molecule further comprises a TGFβ inhibitor. In some embodiments, the TGF-β inhibitor inhibits TGF-β1, TGF-β3, or both TGF-β1 and TGF-β3, for example, using the method described in Example 20. Figure 26B In one embodiment, the TGF-β inhibitor inhibits TGF-β1, TGF-β2, TGF-β3 / both TGF-β1 and TGF-β3, or TGF-β1, TGF-β2 and TGF-β3, for example, using the method described in Example 20. Figure 26BIn one embodiment, the TGF-β inhibitor comprises a TGF-β receptor polypeptide (e.g., an extracellular domain of a TGF-β receptor or a functional variant thereof). In one embodiment, the TGF-β inhibitor comprises one, two, or all of the following: a TGFBR1 polypeptide (e.g., 1, 2, 3, or more TGFBR1 polypeptides), a TGFBR2 polypeptide (e.g., 1, 2, 3, or more TGFBR2 polypeptides), or a TGFBR3 polypeptide (e.g., 1, 2, 3, or more TGFBR3 polypeptides). In one embodiment, the TGF-β inhibitor comprises a TGFBR1 polypeptide. In one embodiment, the TGF-β inhibitor comprises an extracellular domain of TGFBR1, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises an extracellular domain of SEQ ID NO: 95, 96, 97, 120, 121, or 122, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 104 or 105, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises a TGFBR2 polypeptide. In some embodiments, the TGF-β inhibitor comprises the extracellular domain of TGFBR2, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises the extracellular domain of SEQ ID NO: 98, 99, 123 or 124, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises an amino acid sequence selected from SEQ ID NO: 100, 101, 102 or 103, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises a TGFBR3 polypeptide. In one embodiment, the TGF-β inhibitor comprises the extracellular domain of TGFBR3, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises the extracellular domain of SEQ ID NO: 106, 107, 125 or 126, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto). In one embodiment, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 108, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto).In one embodiment, the TGF-β inhibitor comprises two TGF-β receptor polypeptides that form a homodimer. In one embodiment, the TGF-β inhibitor comprises two TGFBR1 polypeptides that form a homodimer. In one embodiment, the TGF-β inhibitor comprises two TGFBR2 polypeptides that form a homodimer. In one embodiment, the TGF-β inhibitor comprises two TGFBR3 polypeptides that form a homodimer. In one embodiment, the TGF-β inhibitor comprises two TGF-β receptor polypeptides that form a heterodimer. In one embodiment, the TGF-β inhibitor comprises a TGFBR1 polypeptide and a TGFBR2 polypeptide that form a heterodimer. In one embodiment, the TGF-β inhibitor comprises a TGFBR1 polypeptide and a TGFBR3 polypeptide that form a heterodimer. In one embodiment, the TGF-β inhibitor comprises a TGFBR2 polypeptide and a TGFBR3 polypeptide that form a heterodimer.

[0159] In one embodiment, the TGF-β inhibitor comprises a first TGF-β receptor polypeptide and a second TGF-β receptor polypeptide. In one embodiment, the multispecific molecule comprises a first Fc region (e.g., a first CH1-Fc region) and a second Fc region (e.g., a second CH1-Fc region), optionally wherein: (i) the first TGF-β receptor polypeptide is linked to the first Fc region (e.g., a first CH1-Fc region), e.g., the C-terminus of the first Fc region (e.g., the first CH1-Fc region), e.g., via a linker, and (ii) the second TGF-β receptor polypeptide is linked to the second Fc region (e.g., a second CH1-Fc region), e.g., the C-terminus of the second Fc region (e.g., the second CH1-Fc region), e.g., via a linker. In one embodiment, the first TGF-β receptor polypeptide and the second TGF-β receptor polypeptide form a homodimer or a heterodimer, e.g., a homodimer. In one embodiment, the first TGF-β receptor polypeptide or the second TGF-β receptor polypeptide comprises an extracellular domain of TGFBR1, TGFBR2, or TGFBR3, e.g., an extracellular domain of TGFBR2. In one embodiment, the multispecific molecule has Figure 35Aor 35B configuration. In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 192 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 193 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 192 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 195 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 194 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 193 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 194 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 195 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto).

[0160] In one embodiment, the TGF-β inhibitor comprises a first TGF-β receptor polypeptide and a second TGF-β receptor polypeptide. In one embodiment, the multispecific molecule comprises a heavy chain constant region 1 (CH1) and a light chain constant region (CL), optionally wherein: (i) the first TGF-β receptor polypeptide is linked to CH1, e.g., the N-terminus of CH1, e.g., via a linker, and (ii) the second TGF-β receptor polypeptide is linked to CL, e.g., the N-terminus of CL, e.g., via a linker. In one embodiment, the first TGF-β receptor polypeptide and the second TGF-β receptor polypeptide form a homodimer or a heterodimer, e.g., a homodimer. In one embodiment, the first TGF-β receptor polypeptide or the second TGF-β receptor polypeptide comprises an extracellular domain of TGFBR1, TGFBR2, or TGFBR3, e.g., the extracellular domain of TGFBR2. In one embodiment, the multispecific molecule has Figure 35CIn one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 196 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 198 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 196 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 199 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 197 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 198 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto). In one embodiment, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 197 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto) and the amino acid sequence of SEQ ID NO: 199 (or a sequence substantially identical thereto, such as a sequence at least 80%, 85%, 90%, or 95% identical thereto).

[0161] In some embodiments, the multispecific molecule further comprises an anti-PD-L1 binding portion. In some embodiments, the anti-PD-L1 binding portion comprises an anti-PD-L1 antibody molecule disclosed herein. In some embodiments, the anti-PD-L1 binding portion inhibits PD-L1. In some embodiments, the anti-PDL1 binding portion comprises a heavy chain variable region (VH) having a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region (VL) having a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 461, 462, 463, 467, 468, and 469, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 346, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 347, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98% or 99% identity thereto. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NO: 346 and 347, respectively.

[0162] In some embodiments, the multispecific molecule further comprises an IL-2 molecule. In some embodiments, the IL-2 molecule is an IL-2 molecule disclosed herein, such as an IL-2 molecule comprising the amino acid sequence of SEQ ID NO: 237 or 238, or a sequence substantially identical thereto (e.g., a sequence 80%, 85%, 90%, or 95% identical thereto).

[0163] In some embodiments, the multispecific molecule comprises one or more CDRs (e.g., 1, 2, or 3 CDRs), one or more (e.g., 1 or 2) heavy chain variable regions, one or more (e.g., 1 or 2) light chain variable regions, a heavy chain, or a light chain of an amino acid sequence disclosed in Table 25, or an amino acid sequence at least 90%, 92%, 94%, 96%, 98%, or 99% identical thereto. In some embodiments, the multispecific molecule comprises one or more CDRs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 CDRs), one or more (e.g., 1 or 2) heavy chain variable regions, one or more (e.g., 1 or 2) light chain variable regions, one or more (e.g., 1 or 2) heavy chains, and / or one or more (e.g., 1 or 2) light chains of a multispecific molecule disclosed in Table 34 or Table 28. In some embodiments, the multispecific molecule comprises a multispecific molecule disclosed in Table 34 or Table 28. In some embodiments, the multispecific molecule is or comprises any one of molecules 10-86 disclosed in Table 34. In some embodiments, the multispecific molecule is or comprises BIM0648 or BIM0652 disclosed in Table 34. In some embodiments, the multispecific molecule is or comprises any one of molecules 10-86 disclosed in Table 28. In some embodiments, the multispecific molecule is or comprises any one of the molecules disclosed in Table 28, BIM0204, BIM0205, BIM0206, BIM0207, BIM0208, BIM0209, BIM0210, BIM0211, BIM0542, BIM0543, BIM0544, BIM0545, BIM0546, BIM0547, BIM0548, BIM0549, BIM0550, BIM0551, BIM0552, BIM0553, BIM0554, BIM0555, BIM0556, BIM0566, and BIM0567.

[0164] In some embodiments, the multispecific molecule has Figures 12A-12J Configuration shown.

[0165] In one aspect, the present invention discloses a multispecific molecule comprising: (i) an anti-CSF1R binding portion (e.g., an anti-CSF1R antibody molecule); (ii) an anti-CCR2 binding portion (e.g., an anti-CCR2 antibody molecule); and (iii) a TGF-β inhibitor. In some embodiments, the multispecific molecule has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more) of the following properties:

[0166] (i) the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-positive, CCR2-negative cells, or CSF1R-negative, CCR2-positive cells, e.g., the binding of the multispecific molecule to CSF1R-positive, CCR2-positive cells is at least 2, 4, 6, 8, 10, 15, 20, or 25-fold stronger than the binding of the multispecific molecule to CSF1R-positive, CCR2-negative cells or CSF1R-negative, CCR2-positive cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 The method described was measured;

[0167] (ii) the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-positive, CCR2-negative cells, e.g., the EC50 for binding of the multispecific molecule to CSF1R-positive, CCR2-positive cells is no greater than 60%, 50%, 40%, 30%, 20%, or 10% of the EC50 for binding of the multispecific molecule to CSF1R-positive, CCR2-negative cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 The method described was measured;

[0168] (iii) the multispecific molecule preferentially binds to CSF1R-positive, CCR2-positive cells relative to CSF1R-negative, CCR2-positive cells, e.g., the EC50 for binding of the multispecific molecule to CSF1R-positive, CCR2-positive cells is no greater than 50%, 40%, 30%, 20%, 10%, or 5% of the EC50 for binding of the multispecific molecule to CSF1R-negative, CCR2-positive cells, e.g., as measured using flow cytometry analysis, e.g., using the reference assay described in Example 2. Figure 1 The method described was measured;

[0169] (iv) the multispecific molecule preferentially binds to tumor-associated macrophages (TAMs) or myeloid-derived suppressor cells (MDSCs) relative to T cells, NK cells, neutrophils, tissue-resident macrophages (e.g., Kupffer cells), or Langerhans cells, e.g., the binding of the multispecific molecule to TAMs or MDSCs is at least 2, 4, 6, 8, 10, 15, 20, or 25-fold stronger than the binding of the multispecific molecule to T cells, NK cells, neutrophils, tissue-resident macrophages (e.g., Kupffer cells), or Langerhans cells, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometry assay described in Example 6. Figure 5 The method described was measured;

[0170] (v) The multispecific molecule inhibits monocyte migration, e.g., monocyte chemoattractant protein 1 (MCP1)-induced monocyte migration, e.g., reduces MCP1-induced monocyte migration by at least 40%, 50%, 60%, or 70%, e.g., as measured using a transwell plate migration assay, e.g., using a reference assay as described in Example 3. Figure 2 The method described was measured;

[0171] (vi) The multispecific molecule inhibits the proliferation of macrophages, e.g., bone marrow-derived macrophages, e.g., CSF-1-induced proliferation of bone marrow-derived macrophages, e.g., reduces CSF-1-induced proliferation of bone marrow-derived macrophages by at least 50%, 60%, 70%, or 80%, e.g., as measured using a cell proliferation MTT assay, e.g., using the assay described in Example 4. Figure 3B The method described was measured;

[0172] (vii) the multispecific molecule does not inhibit or does not substantially inhibit the differentiation of monocytes, e.g., bone marrow-derived monocytes, e.g., CSF-1-induced differentiation of bone marrow-derived monocytes, e.g., does not reduce CSF-1-induced differentiation of bone marrow-derived monocytes by more than 2%, 4%, 6%, 8%, or 10%, e.g., as measured using flow cytometry analysis, e.g., using the flow cytometry assay described in Example 5. Figure 4 The method described was measured;

[0173] (viii) the multispecific molecule depletes suppressive myeloid cells, such as TAMs or MDSCs, in vivo, e.g., reduces the number of suppressive myeloid cells, such as TAMs or MDSCs, by at least 80%, 85%, 90%, 95%, 99%, or 99.5%, e.g., as measured using flow cytometry analysis, e.g., using the methods described in Example 7. Figure 6 measured by the method described;

[0174] (ix) The multispecific molecule does not deplete or does not substantially deplete tissue-resident macrophages, such as Kupffer cells, in vivo, e.g., does not reduce the number of tissue-resident macrophages, such as Kupffer cells, by more than 4%, 6%, 8%, 10%, or 15%, e.g., as measured using an immunohistochemical assay, e.g., using the reference assay described in Example 8. Figure 7B and 7D The method described was measured;

[0175] (x) The multispecific molecule increases CD86 or MHC class II expression on TAMs, e.g., as measured using flow cytometry or immunohistochemistry, e.g., using a reference Figure 21 The method described was measured;

[0176] (xi) The multispecific molecule does not inhibit or does not substantially inhibit CSF-1-dependent cell survival of CSF1R-positive, CCR2-negative cells, e.g., does not reduce CSF-1-dependent cell survival of CSF1R-positive, CCR2-negative cells by more than 5%, 10%, or 15%, e.g., as measured using a cell viability MTT assay, e.g., using the assay described in Example 9. Figure 8A The method described was measured;

[0177] (xii) the multispecific molecule increases CD8+ T cell tumor infiltration in vivo, e.g., increases the percentage of CD8+ T cells among CD3+ T cells in the tumor by at least 1.5-fold, 2-fold, or 2.5-fold, e.g., as measured using flow cytometry analysis, e.g., using the method described in Example 10. Figure 9 The method described was measured;

[0178] (xiii) the multispecific molecule reduces Treg frequency in a tumor in vivo, e.g., reduces Treg frequency in a tumor by at least 15%, 20%, 25%, or 30%, e.g., as measured using flow cytometry analysis, e.g., as described in Example 11 with reference to 10A;

[0179] (xiv) the multispecific molecule increases the CD8+ T cell / Treg ratio in a tumor in vivo, for example, by increasing the CD8+ T cell / Treg ratio in a tumor by at least 2.5-fold, 3-fold, 3.5-fold, 4-fold, or 4.5-fold, for example, as measured using flow cytometry analysis, for example, using the method described in Example 11. Figure 10B The method described was measured;

[0180] (xv) Multispecific molecules reduce tumor growth, increase survival of tumor-bearing animals, and / or enhance anti-tumor immune memory, for example, using the reference Figure 11A and 11B The method described in Example 19 was used to measure Figure 23 The method described was measured;

[0181] (xvi) The multispecific molecule preferentially binds to classical monocytes relative to intermediate monocytes or nonclassical monocytes, for example, using the reference molecule described in Example 28. Figure 39B and 39D measured by the method described; or

[0182] (xvii) The multispecific molecule reduces the activity of TGFβ, for example, by at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, for example, using the method described in Example 28. Figures 40A-40F The method described was used for measurement.

[0183] In some embodiments, the anti-CSF1R binding portion comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 474, 475, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 476, and 477, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 405, 413, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 413, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 406, 413, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 414, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 407, 413, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 415, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 408, 413, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 416, 432, 434, and 436, respectively.In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 417, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 409, 413, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 418, 432, 434, and 436, respectively. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 419, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 420, 432, 434, and 436, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 402, 404, 421, 432, 434, and 436, respectively.

[0184] In some embodiments, the VH comprises an amino acid sequence of any one of SEQ ID NOs: 323-336, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 324, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto. In some embodiments, the VH comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 129, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% identity thereto. In some embodiments, the VL comprises an amino acid sequence of SEQ ID NO: 341, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto. In some embodiments, the VL comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 130, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% identity thereto. In some embodiments, VH and VL comprise the following amino acid sequences, respectively: SEQ ID NOs: 324 and 341; SEQ ID NOs: 323 and 341; SEQ ID NOs: 325 and 341; SEQ ID NOs: 326 and 341; SEQ ID NOs: 327 and 341; SEQ ID NOs: 328 and 341; SEQ ID NOs: 329 and 341; SEQ ID NOs: 330 and 341; SEQ ID NOs: 331 and 341; SEQ ID NOs: 332 and 341; SEQ ID NOs: 333 and 341; SEQ ID NOs: 334 and 341; SEQ ID NOs: 335 and 341; or SEQ ID NOs: 336 and 341.

[0185] In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 127, or an amino acid sequence at least 80%, 85%, 90%, or 95% identical thereto. In some embodiments, the multispecific molecule comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 128, or a nucleotide sequence at least 80%, 85%, 90%, or 95% identical thereto.

[0186] In some embodiments, the anti-CSF1R binding portion comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 478, 479, 433, 435, and 437, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 411, 422, 433, 435, and 437, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 410, 422, 433, 435, and 437, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 410, 423, 433, 435, and 437, respectively. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 403, 412, 422, 433, 435, and 437, respectively.

[0187] In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: 337-340, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto, optionally wherein the VH comprises the amino acid sequence of SEQ ID NO: 339, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto. In some embodiments, the VH comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 138, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% identity thereto. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NOs: 139 or 342, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity thereto, optionally wherein the VL comprises the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence having at least 80%, 85%, 90%, or 95% identity thereto. In some embodiments, the VL comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 140, or a nucleotide sequence at least 80%, 85%, 90%, or 95% identical thereto. In some embodiments, the VH and VL comprise the following amino acid sequences, respectively: SEQ ID NOs: 339 and 139; SEQ ID NOs: 337 and 342; SEQ ID NOs: 338 and 342; SEQ ID NOs: 339 and 342; or SEQ ID NOs: 340 and 342.

[0188] In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 136, or an amino acid sequence at least 80%, 85%, 90%, or 95% identical thereto. In some embodiments, the multispecific molecule comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a nucleotide sequence at least 80%, 85%, 90%, or 95% identical thereto.

[0189] In some embodiments, the anti-CCR2 binding portion comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region (VL) comprising a light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 446, 447, 448, 454, 455, and 456, respectively. In some embodiments, the VH does not comprise the amino acid sequence of SEQ ID NO: 480, and in some embodiments, the VL does not comprise the amino acid sequence of SEQ ID NO: 481. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 480 and the VL does not comprise the amino acid sequence of SEQ ID NO: 481.

[0190] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 343 or 344, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98% or 99% identity thereto, optionally wherein the VH comprises the amino acid sequence of SEQ ID NO: 343, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98% or 99% identity thereto. In some embodiments, the VH comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 133, or a nucleotide sequence having at least 80%, 85%, 90% or 95% identity thereto. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 345, or an amino acid sequence having at least 90%, 92%, 94%, 96%, 98% or 99% identity thereto. In some embodiments, VL comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 272, or a nucleotide sequence at least 80%, 85%, 90%, or 95% identical thereto. In some embodiments, VH and VL comprise the following amino acid sequences: SEQ ID NO: 343 and 345, or SEQ ID NO: 344 and 345, respectively.

[0191] In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 131, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical thereto. In some embodiments, the multispecific molecule comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 132, or a nucleotide sequence that is at least 80%, 85%, 90%, or 95% identical thereto. In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 373, or an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical thereto. In some embodiments, the multispecific molecule comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 134, or a nucleotide sequence that is at least 80%, 85%, 90%, or 95% identical thereto.

[0192] In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 127, the amino acid sequence of SEQ ID NO: 131, the amino acid sequence of SEQ ID NO: 373. In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 136, the amino acid sequence of SEQ ID NO: 131, the amino acid sequence of SEQ ID NO: 373.

[0193] In some embodiments, the anti-CSF1R antibody molecule monovalently binds to CSF1R. In some embodiments, the anti-CCR2 antibody molecule monovalently binds to CCR2. In some embodiments, the multispecific molecule monovalently binds to CSF1R and monovalently binds to CCR2. In some embodiments, the multispecific molecule inhibits CSF1R in the presence of CCR2, optionally wherein the multispecific molecule reduces CSF1R activity (e.g., CSF1R signaling, e.g., CSF1-induced CSF1R signaling) in the cell by, for example, at least 40%, 50%, 60%, 70%, 80%, or 90% when the cell expresses both CSF1R and CCR2 on the cell surface. In some embodiments, the multispecific molecule does not inhibit or does not substantially inhibit CSF1R in the absence of CCR2, optionally wherein when the cell expresses CSF1R on the cell surface but does not express CCR2, the multispecific molecule does not reduce the activity of CSF1R (e.g., CSF1R signaling, e.g., CSF1-induced CSF1R signaling), or does not reduce the activity of CSF1R by more than 2%, 4%, 6%, 8%, 10% or 15%.

[0194] In some embodiments, the multispecific molecule inhibits CCR2 in the presence of CSF1R, optionally wherein the multispecific molecule reduces CCR2 activity in the cell by, for example, at least 40%, 50%, 60%, 70%, 80%, or 90% when the cell expresses both CCR2 and CSF1R on the cell surface. In some embodiments, the multispecific molecule does not inhibit or does not substantially inhibit CCR2 in the absence of CSF1R, optionally wherein the multispecific molecule does not reduce CCR2 activity, or does not reduce CCR2 activity by more than 2%, 4%, 6%, 8%, 10%, or 15% when the cell expresses CCR2 but does not express CSF1R on the cell surface.

[0195] In some embodiments, the TGF-β inhibitor inhibits TGF-β1, TGF-β2, TGF-β3, both TGF-β1 and TGF-β3, or all three of TGF-β1, TGF-β2, and TGF-β3, for example, using the method described in Example 20. Figure 26B In some embodiments, the TGFβ inhibitor comprises a TGF-β receptor polypeptide (e.g., an extracellular domain of a TGF-β receptor or a functional variant thereof). In some embodiments, the TGF-β inhibitor comprises one, two, or all of the following: a TGFBR1 polypeptide (e.g., 1, 2, 3, or more TGFBR1 polypeptides), a TGFBR2 polypeptide (e.g., 1, 2, 3, or more TGFBR2 polypeptides), or a TGFBR3 polypeptide (e.g., 1, 2, 3, or more TGFBR3 polypeptides). In some embodiments, the TGFβ inhibitor comprises a TGFBR1 polypeptide. In some embodiments, the TGFβ inhibitor comprises: (i) the extracellular domain of TGFBR1, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto), (ii) the extracellular domain of SEQ ID NO: 95, 96, 97, 120, 121 or 122, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto), or (iii) the amino acid sequence of SEQ ID NO: 104 or 108, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto).

[0196] In some embodiments, the TGFβ inhibitor comprises a TGFBR2 polypeptide. In some embodiments, the TGFβ inhibitor comprises: (i) the extracellular domain of TGFBR2, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto), (ii) the extracellular domain of SEQ ID NO: 98, 99, 123 or 124, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto), or (iii) an amino acid sequence selected from SEQ ID NO: 100, 101, 102 and 103, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90% or 95% identical thereto).

[0197] In some embodiments, the TGFβ inhibitor comprises a TGFBR3 polypeptide. In some embodiments, the TGFβ inhibitor comprises: (i) the extracellular domain of TGFBR3, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90%, or 95% identical thereto), (ii) the extracellular domain of SEQ ID NO: 106, 107, 125, or 126, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90%, or 95% identical thereto), or (iii) the amino acid sequence of SEQ ID NO: 108, or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90%, or 95% identical thereto).

[0198] In some embodiments, the TGF-β inhibitor comprises two TGF-β receptor polypeptides that form a homodimer, optionally wherein the TGF-β inhibitor comprises: (i) two TGFBR1 polypeptides that form a homodimer, (ii) two TGFBR2 polypeptides that form a homodimer, or (iii) two TGFBR3 polypeptides that form a homodimer.

[0199] In some embodiments, the TGF-β inhibitor comprises two TGF-β receptor polypeptides that form a heterodimer. In some embodiments, the TGFβ inhibitor comprises: (i) a TGFBR1 polypeptide and a TGFBR2 polypeptide that form a heterodimer, (ii) a TGFBR1 polypeptide and a TGFBR3 polypeptide that form a heterodimer, or (iii) a TGFBR2 polypeptide and a TGFBR3 polypeptide that form a heterodimer.

[0200] In some embodiments, the TGF-β inhibitor comprises a first TGF-β receptor polypeptide and a second TGF-β receptor polypeptide.

[0201] In some embodiments, the multispecific molecule comprises a first Fc region (e.g., a first CH1-Fc region) and a second Fc region (e.g., a second CH1-Fc region). In some embodiments, (i) the first TGF-beta receptor polypeptide is linked to the first Fc region (e.g., a first CH1-Fc region), e.g., the C-terminus of the first Fc region (e.g., the first CH1-Fc region), e.g., via a linker, and (ii) the second TGF-beta receptor polypeptide is linked to the second Fc region (e.g., a second CH1-Fc region), e.g., the C-terminus of the second Fc region (e.g., the second CH1-Fc region), e.g., via a linker. In some embodiments, the first TGF-beta receptor polypeptide and the second TGF-beta receptor polypeptide form a homodimer or a heterodimer, e.g., a homodimer. In some embodiments, the first TGF-beta receptor polypeptide or the second TGF-beta receptor polypeptide comprises the extracellular domain of TGFBR1, TGFBR2, or TGFBR3, e.g., the extracellular domain of TGFBR2. In some embodiments, the multispecific molecule has Figure 35A Or 35B configuration.

[0202] In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 192 and the amino acid sequence of SEQ ID NO: 193. In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 192 and the amino acid sequence of SEQ ID NO: 195. In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 194 and the amino acid sequence of SEQ ID NO: 193. In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 194 and the amino acid sequence of SEQ ID NO: 195.

[0203] In some embodiments, the multispecific molecule comprises a heavy chain constant region 1 (CH1) and a light chain constant region (CL). In some embodiments, (i) the first TGF-beta receptor polypeptide is linked to CH1, e.g., the N-terminus of CH1, e.g., via a linker, and (ii) the second TGF-beta receptor polypeptide is linked to CL, e.g., the N-terminus of CL, e.g., via a linker. In some embodiments, the first TGF-beta receptor polypeptide and the second TGF-beta receptor polypeptide form a homodimer or a heterodimer, e.g., a homodimer. In some embodiments, the first TGF-beta receptor polypeptide or the second TGF-beta receptor polypeptide comprises the extracellular domain of TGFBR1, TGFBR2, or TGFBR3, e.g., the extracellular domain of TGFBR2. In some embodiments, the multispecific molecule has Figure 35C Or 35D configuration.

[0204] In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 198. In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 199. In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 198. In some embodiments, the multispecific molecule comprises the amino acid sequence of SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 199.

[0205] In some embodiments, a TGF inhibitor is linked to an anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule) or an anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule), e.g., via a linker. In some embodiments, the anti-CSF1R binding moiety and / or the anti-CCR2 binding moiety comprises an Fc region, wherein the TGF inhibitor is linked to the Fc region, e.g., the C-terminus of the Fc region, e.g., via a linker. In some embodiments, a multispecific molecule comprises a first TGF-β inhibitor and a second TGF-β inhibitor, wherein the first TGF-β inhibitor is linked to the anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule), e.g., via a linker, and the second TGF-β inhibitor is linked to the anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule) via a linker. In some embodiments, the anti-CSF1R binding moiety comprises a first Fc region, the anti-CCR2 binding moiety comprises a second Fc region, and the multispecific molecule comprises a first TGF-β inhibitor and a second TGF-β inhibitor, wherein the first TGF-β inhibitor is linked to the first Fc region, e.g., via a linker, and the second TGF-β inhibitor is linked to the second Fc region, e.g., via a linker, and the second TGF-β inhibitor is linked to the second Fc region, e.g., via a linker, and the second TGF-β inhibitor is linked to the second Fc region, e.g., via a linker,

[0206] In some embodiments, (i) the anti-CSF1R binding moiety comprises a first light chain and a first heavy chain, wherein the first heavy chain comprises a first Fc region, wherein the C-terminus of the first Fc region is linked to the extracellular domain of TGFBR2 or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90%, or 95% identical thereto); and (ii) the anti-CCR2 binding moiety comprises a second light chain and a second heavy chain, wherein the second heavy chain comprises a second Fc region, wherein the C-terminus of the second Fc region is linked to the extracellular domain of TGFBR2 or a sequence substantially identical thereto (e.g., a sequence at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the multispecific molecule has Figure 12B Configuration shown.

[0207] In some embodiments, the multispecific molecule comprises a molecule disclosed in Table 34 or Table 28. In some embodiments, the multispecific molecule has or includes Figure 39F or Figures 12A-12J The configuration shown in any of the figures.

[0208] In some embodiments, the multispecific molecules disclosed herein induce antibody-dependent cellular cytotoxicity (ADCC).

[0209] In some embodiments, the multispecific molecules disclosed herein do not induce antibody-dependent cellular cytotoxicity (ADCC).

[0210] In another aspect, provided herein are isolated nucleic acid molecules encoding the multispecific molecules (eg, antibodies) disclosed herein.

[0211] In another aspect, provided herein are isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the multispecific molecules described herein, or a nucleotide sequence substantially homologous thereto (eg, at least 95% to 99.9% identical thereto).

[0212] In another aspect, provided herein are vectors, eg, expression vectors, comprising one or more nucleic acid molecules described herein.

[0213] In another aspect, provided herein are cells, eg, host cells, comprising a nucleic acid molecule described herein or a vector described herein.

[0214] In another aspect, provided herein are methods of making, e.g., producing, a multispecific molecule described herein, comprising culturing a cell described herein, e.g., a host cell, under suitable conditions, e.g., conditions suitable for gene expression and / or heterodimerization.

[0215] In another aspect, provided herein are pharmaceutical compositions comprising a multispecific molecule described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer.

[0216] On the other hand, provided herein is a method for treating hyperproliferative disorders, cancer, fibrotic disorders or the patient's condition, inflammatory disorders or the patient's condition or autoimmune disorders. In one embodiment, disease is a hyperproliferative disorder, for example, hyperproliferative connective tissue disorder (for example hyperproliferative fibrotic disease). In one embodiment, fibrosis (for example, hyperproliferative fibrosis) disease is multi-system or organ-specific. Exemplary fibrotic diseases include but are not limited to multi-system (for example systemic sclerosis, multifocal fibrosing sclerosis, scleroderma graft-versus-host disease in bone marrow transplant recipients, nephrogenic systemic fibrosis, scleroderma) and organ-specific disorders (for example, the fibrosis of lung, liver, heart, kidney, pancreas, skin and other organs). In other embodiments, fibrotic diseases are selected from hepatic fibrosis (for example, cirrhosis, NASH and other patient's conditions as described herein), pulmonary fibrosis (for example, IPF), renal fibrosis or myelofibrosis (for example, myelofibrosis).

[0217] In another aspect, provided herein are methods of treating cancer in a subject comprising administering to a subject in need thereof a multispecific molecule described herein, wherein the multispecific molecule is administered in an amount effective to treat the cancer.

[0218] In some embodiments, the cancer is a solid tumor or a metastatic lesion. In some embodiments, the solid tumor is one or more of pancreatic cancer (e.g., pancreatic adenocarcinoma), breast cancer, colorectal cancer, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), skin cancer (e.g., melanoma), ovarian cancer, liver cancer, brain cancer (e.g., glioma), bladder cancer, cervical cancer, head and neck cancer, kidney cancer, mesothelioma, thyroid cancer, or uterine cancer.

[0219] In some embodiments, the method further comprises administering a second therapy. In some embodiments, the second therapy comprises a therapeutic agent (e.g., a chemotherapeutic agent, a biological agent, a hormone therapy), radiation, or surgery. In some embodiments, the therapeutic agent is selected from: a chemotherapeutic agent or a biological agent. In some embodiments, the therapeutic agent is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from anti-CTLA4 antibody, anti-PD1 antibody (e.g., Nivolumab, Pembrolizumab or Pidilizumab), anti-PD-L1 antibody, anti-PD-L2 antibody, anti-TIM3 antibody, anti-LAG3 antibody, anti-CD 160 antibody, anti-2B4 antibody, anti-CD80 antibody, anti-CD86 antibody, anti-B7-H3 (CD276) antibody, anti-B7-H4 (VTCN1) antibody, anti-HVEM (TNFRSF14 or CD270) antibody, anti-BTLA antibody, anti-KIR antibody, anti-MHC class I antibody, anti-MHC class II antibody, anti-GAL9 antibody, anti-VISTA antibody, anti-BTLA antibody, anti-TIGIT antibody, anti-LAIR1 antibody and anti-A2aR antibody. In some embodiments, the checkpoint inhibitor is an anti-PD1 antibody.

[0220] In one aspect, provided herein are methods of treating cancer in a subject comprising administering to a subject in need thereof an effective amount of a combination of a multispecific molecule described herein (eg, an anti-CSF1R / anti-CCR2 multispecific molecule described herein) and an anti-PD1 antibody.

[0221] In one aspect, the invention relates to a multispecific molecule of the invention, an antibody molecule of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a pharmaceutical composition of the invention for use as a medicament.

[0222] In one aspect, the invention relates to a multispecific molecule of the invention, an antibody molecule of the invention, a nucleic acid molecule of the invention, a vector of the invention, a cell of the invention or a pharmaceutical composition of the invention for use as a medicament for treating cancer.

[0223] In one aspect, provided herein are methods of treating a fibrotic disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a multispecific molecule disclosed herein, thereby treating the fibrotic disease or condition. In some embodiments, the fibrotic disease or condition is a fibrotic disease or condition of the lungs, liver, heart or vasculature, kidneys, pancreas, skin, gastrointestinal tract, bone marrow or hematopoietic tissue, nervous system, eye, or a combination thereof. In some embodiments, the fibrotic disease or condition is pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis (IPF)) or liver fibrosis (e.g., non-alcoholic steatohepatitis (NASH)).

[0224] In some embodiments, treatment of a fibrotic condition comprises reducing or inhibiting one or more of: the formation or deposition of tissue fibrosis; reducing the size, cellularity (e.g., number of fibroblasts or immune cells), composition, or cellularity of fibrotic lesions; reducing the collagen or hydroxyproline content of fibrotic lesions; decreasing the expression or activity of fibrogenic proteins; reducing fibrosis associated with an inflammatory response; reducing weight loss associated with fibrosis; or increasing survival.

[0225] In some embodiments, the fibrotic disease or condition is primary fibrosis. In one embodiment, the fibrotic disease or condition is idiopathic. In other embodiments, the fibrotic disease or condition is associated with a disease (e.g., an infectious disease, an inflammatory disease, an autoimmune disease, a malignant or cancerous disease, and / or a connective disease); a toxin; an injury (e.g., an environmental hazard (e.g., asbestos, coal dust, polycyclic aromatic hydrocarbons), smoking, a wound); a medical treatment (e.g., a surgical incision, chemotherapy, or radiation), or a combination thereof (e.g., secondary to the above).

[0226] In one aspect, provided herein are methods for treating a liver disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a multispecific molecule disclosed herein, thereby treating the liver disease or condition. In some embodiments, the liver disease or condition is a fibrotic condition or connective tissue condition that affects liver function or physiology. In one embodiment, the fibrotic disease or connective tissue condition can be systemic (affecting the entire body), multi-organ, or organ-specific (e.g., liver-specific). Examples of liver fibrosis conditions include, but are not limited to, liver fibrosis (fibrosis of the liver), cirrhosis, and any condition associated with accumulation of extracellular matrix proteins (e.g., collagen) in the liver, liver scarring, and / or liver vascular abnormalities. In one embodiment, the liver disease or condition is cirrhosis. Cirrhosis is considered to be the end stage of liver fibrosis, involving regenerative nodules (due to the repair process), typically accompanied by deformation of the liver vasculature. In other embodiments, the liver disease or condition is liver cancer. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (HCC), primary hepatocellular carcinoma, hepatoma, fibrolamellar carcinoma, focal nodular hyperplasia, cholangiosarcoma, intrahepatic bile duct carcinoma, angiosarcoma (angiosarcoma or hemangiosarcoma), hepatic adenoma, hepatic hemangioma, hepatic hamartoma, hepatoblastoma, infantile hemangioendothelioma, mixed liver tumors, mesenchymal tumors, and hepatosarcoma. Liver cancer may also be associated with metastasis to non-liver cancers, such as breast cancer, colorectal cancer, esophageal cancer, kidney cancer (kidney cancer or renal cancer), lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer (e.g., melanoma), gastric cancer (gastric cancer or stomach cancer) (including gastrointestinal cancer), and uterine cancer. In one embodiment, the liver disease or disorder is HCC.In certain embodiments, the liver disease or disorder is caused by one or more insults, including but not limited to liver inflammation or damage; viral (e.g., chronic viral) infection (e.g., hepatitis B, hepatitis C virus, hepatitis A virus, hepatitis D virus (hepatitis delta virus), hepatitis E virus, Epstein-Barr adenovirus, or cytomegalovirus; or parasitic infection, such as schistosomiasis); alcoholism; fatty liver disease; metabolic disorders (e.g., hemochromatosis, diabetes, obesity, hypertension, dyslipidemia, galactosemia, or glycogen storage disease); autoimmune disorders (e.g., autoimmune hepatitis (AIH), autoimmune liver disease, lupus hepatitis, systemic lupus erythematosus, primary biliary cirrhosis ( PBC), scleroderma or systemic sclerosis); inflammatory liver disorders (e.g., steatohepatitis, primary sclerosing cholangitis (PSC), ulcerative colitis, Crohn's disease, inflammatory bowel disease); hereditary or congenital liver disease (e.g., Wilson's disease, Gilbert's disease, Byler syndrome, Greenland-Eskimo familial cholestasis, Zellweger syndrome, Alagille syndrome (ALGS), progressive familial intrahepatic cholestasis (PFIC), alpha 1 antitrypsin deficiency, cystic fibrosis, Indian childhood cirrhosis or hereditary hemochromatosis); and liver injury (e.g., drug toxicity, alcoholism, ischemia, malnutrition or physical trauma). In one embodiment, the liver disease or disorder is fatty liver (or FLD), alcoholic liver disease, non-alcoholic fatty liver disease (NAFFD), non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis, simple fatty liver, Reye's syndrome and any disorder associated with abnormal retention of lipids in liver cells.

[0227] In one aspect, provided herein are methods of treating an inflammatory disorder or condition in a subject in need thereof, comprising administering to the subject an effective amount of a multispecific molecule disclosed herein, thereby treating the inflammatory disorder or condition. In one embodiment, the inflammatory disorder or condition is an inflammatory disorder or condition in the kidney. In one embodiment, the inflammatory disorder or condition is lupus nephritis.

[0228] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Suitable methods and materials are described below, but methods and materials similar to or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification including definitions shall prevail. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

[0229] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0230] This patent or application file contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and the necessary fee.

[0231] Figure 1 In vitro binding of increasing concentrations of UniTI-01 to transiently transfected ExpiCHO cells expressing mCCR2 (circles), mCSF1R (squares), or both mCCR2 and mCSF1R (triangles), as determined by flow analysis. Figure 1 In Figure 5, the percentage (%) of fluorescence is plotted against the antibody concentration tested.

[0232] Figure 2 Effects of increasing concentrations of isotype control (mIgG2a), anti-mouse CCR2 monoclonal antibody (aCCR2), or UniTI-01 on MCP-1-dependent cell migration of bone marrow-derived monocytes in a transwell cell culture system.

[0233] Figure 3A and Figure 3B Effects of increasing concentrations of UniTI-01, anti-mouse CSF1R monoclonal antibody (aCSF1R), or isotype control (mIgG2a) on the mCSF-1-dependent in vitro differentiation and proliferation of bone marrow-derived monocytes into macrophages. Figure 3A Figure 2 is a pair of flow cytometric images showing staining of bone marrow cells with anti-CCR2 and anti-CSF1R antibodies on day 0 or day 4 of cell culture in the presence of mCSF1. Figure 3B The percentage of proliferation under each test condition is plotted. All antibodies were tested at 15 μg / ml.

[0234] Figure 4 Effects of UniTI-01, anti-mouse CSF1R monoclonal antibody (aCSF1R), or isotype control (mIgG2a) on mCSF-1-dependent differentiation and proliferation of bone marrow-derived monocytes. All antibodies were tested at 15 μg / ml.

[0235] Figure 5 In vitro binding of increasing concentrations of UniTI-01 to primary intratumoral M-MDSCs and M2-like macrophages as determined by flow cytometry. Figure 5 is a set of histograms showing the staining of CD206+ macrophages, M-MDSCs, neutrophils, or CD3+ T cells using UniTI-01.

[0236] Figure 6 Effects of in vivo administration of UniTI-01 on intratumoral myeloid cell populations in EMT6 and MC38 syngeneic tumor models.

[0237] Figures 7A-7D Effects of in vivo administration of UniTI-01 on Kupffer cells in the EMT6 syngeneic tumor model. Figure 7A and 7B Immunohistochemistry images of tumor and liver tissues stained with antibody F4 / 80, respectively. Figure 7C and 7D Graphs show the percentage of F4 / 80-positive area in tumor and liver tissues, respectively.

[0238] Figure 7E-7H Effects of in vivo administration of UniTI-01 on healthy tissue macrophages in the small intestine and kidney. Figure 7E and 7G Immunohistochemical images of the small intestine and kidney stained with antibody F4 / 80, respectively. Figure 7F and 7H Graphs show the percentages of F4 / 80-positive areas in the small intestine and kidney, respectively.

[0239] Figure 8A and 8B Effect of UniTI-01 on CSF-1-dependent cell survival of CCR2-negative NFS-60 cells in vitro. Figure 8A Cell viability is plotted against the antibody concentrations tested for the anti-CCR2 / anti-CSF1R bispecific antibody UniTI-01, the bivalent monospecific anti-CSF1R antibody (aCSF1R), or the monovalent monospecific anti-CSF1R antibody (mono-aCSF1R). Figure 8B is a pair of flow cytometry graphs showing staining of NFS-60 cells with anti-CCR2 antibody and anti-CSF1R antibody, or staining of NFS-60 cells with an isotype control antibody.

[0240] Figure 9 Effect of in vivo administration of UniTI-01 on CD8+ T cell infiltration in EMT6 model tumors. Figure 9 is a graph showing the percentage of CD8+ T cells among CD3+ T cells in tumors under the indicated treatments.

[0241] Figure 10A and 10B Effect of in vivo administration of UniTI-01 on Treg frequency in MC38 model tumors. Figure 10A is a graph showing the percentage of FOXP3+ cells among CD4+ T cells in tumors under the indicated treatments. Figure 10B In Figure 2, the CD8+ T cell / Treg ratio in tumors was plotted for each treatment.

[0242] Figure 11A 、 11B and 11C. Figure 11A and 11B : When combined with anti-PDL1 antibody in the EMT6 tumor model, UniTI-01 demonstrated anti-tumor efficacy, tumor regression, and improved survival. Figure 11A is a set of graphs showing tumor volume for each treatment. Figure 11B is a graph showing the percentage of survival under the indicated treatments. Figure 11C : In the MC38 colon model, UniTI-01 demonstrated superior anti-PDL1 combination benefit compared to anti-CSF1R antibodies. Figure 11C is a set of graphs showing tumor volume for each treatment.

[0243] Figures 12A-12J . Figure 12A Exemplary configurations of anti-CSF1R / anti-CCR2 bispecific antibodies are shown. In one embodiment, the anti-CSF1R / anti-CCR2 bispecific antibody comprises an anti-CSF1R binding portion and an anti-CCR2 binding portion. In one embodiment, the anti-CSF1R binding portion comprises a heavy chain and a kappa light chain, and the anti-CCR2 binding portion comprises a heavy chain and a lambda light chain. In one embodiment, the anti-CSF1R binding portion comprises a heavy chain and a lambda light chain, and the anti-CCR2 binding portion comprises a heavy chain and a kappa light chain. Figure 12B Exemplary configurations of anti-CSF1R / anti-CCR2 bispecific antibodies fused to a TGFβ trap (Trap) are shown. In one embodiment, the TGFβ trap comprises a homodimer of the two extracellular domains of the TGFβ receptor 2. Figure 12C and 12D Exemplary configurations of anti-CSF1R / anti-CCR2 bispecific antibodies fused to an anti-PDL1 binding moiety are shown. In one embodiment, the anti-PDL1 binding moiety is a scFv. Figures 12E-12G Exemplary configurations of anti-CSF1R / anti-CCR2 bispecific antibodies fused to one or more IL-2 molecules are shown. In one embodiment, one or more IL-2 molecules are fused to the C-terminus of the light chain of the anti-CSF1R binding portion or the anti-CCR2 binding portion. Figure 12H Exemplary configurations of anti-CSF1R / anti-CCR2 bispecific antibodies are shown. In one embodiment, the anti-CSF1R / anti-CCR2 bispecific antibody comprises an anti-CSF1R binding portion and an anti-CCR2 binding portion. In one embodiment, the anti-CSF1R binding portion comprises an anti-CSF1R scFv fused to an Fc. In one embodiment, the anti-CCR2 binding portion comprises an anti-CCR2 Fab fused to an Fc. Figure 12IExemplary configurations of anti-CSF1R / anti-CCR2 bispecific antibodies that further comprise a TGFβ trap are shown. In one embodiment, the anti-CSF1R / anti-CCR2 bispecific antibody comprises an anti-CSF1R binding portion and an anti-CCR2 binding portion. In one embodiment, the anti-CCR2 binding portion comprises an anti-CCR2 Fab fused to an Fc. In one embodiment, the anti-CSF1R binding portion comprises an anti-CSF1R scFv fused to the C-terminus of the Fc of the anti-CCR2 binding portion. In one embodiment, the TGFβ trap comprises a first TGFBR2 ECD fused to CH1 and Fc, and a second TGFBR2 ECD fused to CL. Figure 12J Exemplary configurations of anti-CSF1R / anti-CCR2 bispecific antibodies fused to a TGFβ trap are shown. In one embodiment, the anti-CSF1R / anti-CCR2 bispecific antibody comprises an anti-CSF1R binding portion and an anti-CCR2 binding portion. In one embodiment, the anti-CSF1R binding portion comprises an anti-CSF1R scFv fused to an Fc. In one embodiment, the anti-CCR2 binding portion comprises an anti-CCR2 Fab fused to an Fc. In one embodiment, the TGFβ trap comprises a first TGFBR2 ECD fused to the C-terminus of the Fc of the anti-CCR2 binding portion and a second TGFBR2 ECD fused to the C-terminus of the Fc of the anti-CSF1R binding portion.

[0244] Figure 13 . Figure 13 Schematic diagram showing that anti-CSF1R / anti-CCR2 antibody molecules can reduce immunosuppressive myeloid cells and increase the infiltration of cytotoxic T cells in tumors.

[0245] Figure 14 . Figure 14 is a set of graphs showing that UniTI-01 retains CSF1R-expressing osteoclasts when compared to anti-CSF1R treatment.

[0246] Figure 15A and Figure 15B . Figure 15A is a set of graphs showing the number of M-MDSCs per mg of tumor in mice bearing EMT6, MC38, or LLC1 tumors. Figure 15B is a set of graphs showing the number of TAMs per mg of tumor in mice bearing EMT6, MC38, or LLC1 tumors.

[0247] Figure 16 . Figure 16 is a set of figures showing immunohistochemical staining of tissues using anti-rat IgG antibody and anti-F4 / 80 antibody.

[0248] Figure 17 . Figure 17 are bar graphs showing the biodistribution of UniTI-01 in spleen, brain, lung, heart, colon, kidney, bone, muscle, or tumor at the indicated time points after injection.

[0249] Figure 18 . Figure 18 is a set of flow cytometry images showing the staining of tumor M-MDSCs or tumor G-MDSCs using anti-CSF1R antibody and anti-CCR2 antibody.

[0250] Figure 19A and Figure 19B . Figure 19A Figures show immunochemical staining of tumor tissues using anti-CSF1R bivalent monospecific antibody, anti-CCR2 bivalent monospecific antibody, or UniTI-01. Figure 19B is a graph showing the % CD3 positive area under each test condition.

[0251] Figure 20 . Figure 20 is a set of graphs showing the CD8-positive area %, FoxP3-positive area %, or CD8 / Treg ratio under each test condition.

[0252] Figure 21 . Figure 21 is a graph comparing the properties of an anti-CCR2 bivalent monospecific antibody, an anti-CSF1R bivalent monospecific antibody, and UniTI-01 based on in vivo studies using EMT6, MC38, and LLC1 syngeneic tumor models.

[0253] Figure 22A and Figure 22B UniTI-01 drives tumor regression and depletes suppressive myeloid cells. Figure 22A is a pair of graphs showing the % of M-MDSCs (upper panel) or the % of TAMs (lower panel) of CD45+ cells under each condition tested. Figure 22B is a set of graphs showing tumor volume in mice bearing EMT6 breast tumors under each condition tested. Similar results were observed with mice bearing CT26 and MC38 colon tumors.

[0254] Figure 23 The UniTI-01 / aPD-L1 combination resulted in durable responses and immune memory. Figure 23 The left panel is a pair of graphs showing tumor volume in mice bearing EMT or CT26 tumors. Figure 23 The right panel is a pair of graphs showing tumor volumes in mice that underwent EMT and were rechallenged with CT26 tumor cells.

[0255] Figure 24A 、 Figure 24Band Figure 24C UniTI-01 inhibits tumor growth in other mouse syngeneic cancer models. Figure 24A 、 24B 24C is a set of graphs showing tumor volume in mice bearing MC38, CT26, or EMT6 tumors under each condition tested.

[0256] Figure 25 . Figure 25 Schematic diagram showing that UniTI-102 reduces immunosuppressive myeloid cells and neutralizes TGFβ.

[0257] Figure 26A and Figure 26B . Figure 26A is a schematic diagram showing the configuration of UniTI-102. Figure 26B is a graph showing the level of TGFβ / Smad activation under each tested condition.

[0258] Figure 27A 、 Figure 27B and Figure 27C UniTI-102 demonstrated a strong monotherapy response in the EMT6 tumor model that was not enhanced with the addition of anti-PD-L1. Figures 27A-27C is a set of graphs showing tumor volume in mice bearing EMT6 tumors under each condition tested.

[0259] Figure 28 Shown are a pair of SDS gels using anti-CSF1R antibodies.

[0260] Figure 29A and Figure 29B is a pair of graphs showing the binding of anti-human CSF1R antibodies to human and cynomolgus monkey CSF1R-FcECD as measured by ELISA.

[0261] Figure 30 Figure 2 is a graph showing the levels of MCP-1 secretion by monocytes in the presence of various monovalent anti-CSF1R antibodies. The data shown are representative of one of two human donors.

[0262] Figure 31 is a graph showing the binding of monovalent anti-CSF1R antibodies to CSF1R-expressing cells measured by flow cytometry.

[0263] Figure 32 is a pair of SDS gels of exemplary CCR2×CSF1R bispecific antibodies.

[0264] Figure 33 Graph showing that the CCR2×CSF1R bispecific antibody blocks CCL2-dependent reporter gene activity in Tango CCR2-bla U2OS cells.

[0265] Figure 34 Figure 2 is a graph showing that a CCR2×CSF1R bispecific antibody blocks hCSF1-mediated CCL2 (MCP-1) secretion in purified human monocytes. Data shown are representative of 1 of 2 human donors.

[0266] Figures 35A-35D is a schematic diagram showing exemplary multispecific molecules comprising a TGFβ inhibitor. In some embodiments, the TGFβ inhibitor comprises a TGF-β receptor ECD homodimer. In some embodiments, the TGFβ inhibitor comprises a TGFBR2 ECD heterodimer. Figure 35A and Figure 35B In some embodiments, the two TGFBR ECD domains are connected to the C-termini of the two Fc regions. Figure 35A or Figure 35B The CH1-Fc-TGFBR ECD region shown in comprises the amino acid sequence of SEQ ID NO: 192 or 193. In some embodiments, Figure 35A The Fc-TGFBR ECD region shown in SEQ ID NO: 194 or 195 comprises the amino acid sequence of SEQ ID NO: 194 or 195. Figure 35C and 35D In some embodiments, the two TGFBR ECD domains are connected to CH1 and CL, respectively. Figure 35C or Figure 35D The TGFBR ECD-CH1-Fc region shown in comprises the amino acid sequence of SEQ ID NO: 196 or 197. In some embodiments, Figure 35C or Figure 35D The TGFBR ECD-CL region shown in comprises the amino acid sequence of SEQ ID NO: 198 or 199. In some embodiments, the multispecific molecule comprises a binding moiety A and a binding moiety B. In some embodiments, the binding moiety A or the binding moiety B is an anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule). In some embodiments, the binding moiety A or the binding moiety B is an anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule). In some embodiments, the binding moiety A is an anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule) and the binding moiety B is an anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule). In some embodiments, the binding moiety A is an anti-CCR2 binding moiety (e.g., an anti-CCR2 antibody molecule) and the binding moiety B is an anti-CSF1R binding moiety (e.g., an anti-CSF1R antibody molecule).

[0267] Figure 36TGFβ / Smad activation is plotted against TGFβ-trap concentration. Constructs tested in this study included: single TGFβ Fab-trap, anti-PDL1 x TGFβ-trap, UniTI-01 (anti-CCR2 x anti-CSF1R), and UniTI-102 (anti-CCR2 x anti-CSF1R x TGFβ-trap).

[0268] Figure 37 CSF1R and CCR2 are co-expressed on mononuclear myeloid-derived suppressor cells (Mo-MDSCs) and tumor-associated macrophages (TAMs) from cancer patients. Figure 37 Included is a panel of flow cytometry graphs showing staining of mo-MDSCs or TAMs from ovarian tumors (upper panel) or colorectal tumors (lower panel) using anti-CSF1R and anti-CCR2 antibodies. Figure 37 Also included are bar graphs showing the % CCR2+CSF1R+ cells in M-MDSCs, TAMs, granulocytic myeloid-derived suppressor cells (G-MDSCs), CD4+ T cells, CD8+ T cells, monocytes, or non-immune cells from ovarian tumors (upper panel) or colorectal tumors (lower panel). Co-expression of CSF1R and CCR2 was also detected on CD163+ TAMs.

[0269] Figures 38A-38D The therapeutic concept was demonstrated using the mouse surrogate, mUniTI-102, in a syngeneic tumor model resistant to PD-1 or TGF-β blockade. Figure 38A is a graph showing that mUniTI-102 depletes mo-MDSCs in EMT6 breast tumors. Figure 38B is a graph showing that mUniTI-102 preferentially depletes TAMs relative to tissue-resident macrophages. Figure 38C is a graph showing that mUniTI-102 demonstrates monotherapy anti-tumor activity in the EMT6 breast tumor model. Figure 38D This figure shows that mUniTI-102 improves the survival rate of EMT6 tumor-bearing mice.

[0270] Figures 39A-39F Human UniTI-102 preferentially binds to primary human classical monocytes in whole blood. Figure 39A is a cartoon image showing co-expression of CCR2 and CSF1R on classical monocytes. Figure 39B is a graph showing the normalized frequency of UniTI-102+ cells in classical monocytes at the indicated concentrations of BIM0648 ("648"), BIM0652 ("052"), or an isotype control antibody ("iso"). Figure 39C is a cartoon image showing CSF1R expression on nonclassical monocytes. Figure 39DFigure 2 shows the expression of nonclassical monocytes (CD14 - Figure 3 is a graph of the normalized frequency of UniTI-102+ cells in CD16+ cells. Figure 39E is a table showing the receptor density of CCR2 and CSF1R on different types of monocytes. Figure 39F The configurations of UniTI-102 molecules BIM0648 and BIM0652 are shown.

[0271] Figures 40A-40F The neutralization effect of human UniTI-102 on TGFβ was confirmed in vitro by various functional assays. Figure 40A In Figure 5, TGFβ / Smad activation is plotted against the concentrations of BIM0648, BIM0652, and human IgG1. Figure 40B is a graph showing the FOXP3+CD25+ % of CD4+ T cells for the following treatment groups: with TGFβ but without any construct (+TGFb), without TGFβ (No TGFb), the UniTI-102 molecule BIM0648, anti-PD-F1-TGFBR2 and human IgG1. Figure 40C is a graph showing specific killing for the following treatment groups: with TGFβ but without any construct (+TGFb), without TGFβ (No TGFb), the UniTI-102 molecule BIM0648, anti-PD-F1-TGFBR2 and human IgG1. Figure 40D It is a graph showing the amounts of TGFb1, TGFb2, TGFb3, IF-10, IF-4, and IF-6 secreted by SW840 cells. Figure 40E and 40F Figures are graphs showing the expression of CD206 (M2 marker) and HFA (M1 marker) in each treatment group, respectively. Data are representative of two donors. Comparable results were observed for BIM0652.

[0272] Figure 41A and Figure 41B Pharmacokinetic data of BIM0648 in healthy cynomolgus monkeys. Figure 41A is a graph showing the concentrations of BIM0648 at the indicated time points after administration of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 30 mg / kg to healthy cynomolgus monkeys. Figure 41B is a table summarizing the pharmacokinetic data of BIM0648.

[0273] Figure 42 .Evidence for target occupancy of BIM0648 in cynomolgus monkeys. Figure 42is a set of graphs showing serum levels of TGFβ1 (left), M-CSF (center), and MCP-1 (right) in cynomolgus monkeys after administration of BIM0648 at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 30 mg / kg.

[0274] Figures 43A-43D Biological response trends for BIM0648 were noted that were consistent with CSF1R blockade in monkeys and humans. Figures 43A-43D It is shown that when BIM0648 is 0.1mg / kg ( Figure 43A )、1mg / kg( Figure 43B )、10mg / kg( Figure 43C ) or 30 mg / kg ( Figure 43D ) are a panel of graphs showing non-classical monocytes (% of total monocytes) in cynomolgus monkeys at the indicated time points after administration of ).

[0275] Figure 44 Compared with anti-CSF1R mAb, the murine CCR2xCSF1R bispecific molecule (mUniTI-01) is more effective in depleting circulating Mo-MDSCs in EMT6 tumor-bearing mice. Figure 44 is a bar graph showing the % of M-MDSCs in EMT6 tumor-bearing mice after administration of mUniTI-01 or anti-CSF1R antibody ("CSF1R") combined with anti-PDL1 antibody.

[0276] Figure 45A and Figure 45B mUniTI-102 effectively binds to blood Ly6C in the EMT6 syngeneic tumor model 高 Monocytes. Figure 45A Included is a flow cytometry histogram showing the relationship between mUniTI-102 and Ly6C 高 Classical monocyte binding. Figure 45B TGFβRII+Ly6C detected using anti-human TGFβRII antibody 高 Graph of monocyte % .

[0277] Figure 46A and Figure 46B mUniTI-102 depletes blood Ly6C in the EMT6 syngeneic tumor model 高 Classical monocytes. Figure 46A and 46B Figure 3 shows Ly6C expression in single cells of EMT6 tumor-bearing mice after four doses of mUniTI-102. 高 Monocyte % and Ly6C of monocytes 低 Figure 2. Administration of mUniTI-102 resulted in the expression of Ly6C in the blood. 高Dose-dependent depletion of classical monocytes while preserving CCR2-negative Ly6C 低 Nonclassical monocytes. Little change was noted in whole blood CD3+%, Treg ratio, or CD4 / CD8 ratio.

[0278] Figure 47A and Figure 47B mUniTI-102 reduces serum TGFβ-1 levels in the EMT6 syngeneic tumor model. Figure 47A is a diagram showing the in vivo study design testing mUniTI-102 in the EMT6 syngeneic tumor model. Figure 47B is a bar graph showing TGF-β1 levels in acid-treated serum samples measured by ELISA.A similar decrease was also observed in the CT26 model.

[0279] Figure 48 Table 2 shows receptor densities for CCR2 and CSF1R in human and cynomolgus monkey whole blood. CSF1R expression is higher on non-classical monocytes in cynomolgus monkeys compared to humans. Consistent with this expression pattern, non-classical monocyte populations in cynomolgus monkey whole blood exhibit significant binding, unlike in human whole blood. No significant differences in the binding of BIM0648 and BIM0652 to monocyte populations in cynomolgus monkey whole blood were observed.

[0280] Figures 49A-49F UniTI-102 (BIM0648) exhibits excellent manufacturability. Figure 49A This section summarizes the product information for BIM0648. Figure 49B is a graph showing the results of a study testing the stability of BIM0648 in PBS at 40°C on days 0, 10, and 30. Figure 49C is a graph showing the high colloidal stability and low aggregation tendency of BIM0648. Figure 49D is a graph showing the results of a study testing the thermal stability of BIM0648. Figure 49E is a graph showing the results of a study testing the self-aggregation tendency of BIM0648. Figure 49F is a graph showing the results of a study testing the stability of BIM0648 at pH 3.5.

[0281] Figure 50 UniTI-102 (BIM0648) showed retained binding to Fc receptors. Figure 50 is a graph showing the binding of BIM0648 to Fc receptors assessed using Biacore.

[0282] Figure 51 UniTI-102 (BIM0648) showed negligible nonspecific binding tendency. Figure 51This is a bar graph showing the results of a study testing nonspecific binding of BIM0648. Polyreactivity was assessed by examining binding to baculovirus particles. hIgG1, bevacizumab, and pembrolizumab were used as benchmarks.

[0283] Figures 52A-52D Figure 1 shows the binding of BIM0648 to M1 macrophages, MDSCs, TAMs, and M2 macrophages. The bivalent anti-CSF1R antibody BHM1650 was used as a control. In vitro differentiated monocyte-derived M1 macrophages have the lowest expression of CSF1R and CCR2. TAMs and M2 macrophages have the highest expression. MDSCs have intermediate expression of CSF1R and similar levels of CCR2 expression to M1 macrophages. BIM0648 engagement correlates with CCR2 / CSF1R expression—highest in TAMs and M2 macrophages, intermediate in MDSCs, and no binding in M1 macrophages.

[0284] Figure 53A and Figure 53B The results showed that IgG, BIM0648 and BHM1650 (bivalent anti-CSF1R antibodies) were associated with classical monocytes ( Figure 53A ) or nonclassical monocytes ( Figure 53B BIM0648 binds to classical monocytes with greater affinity than nonclassical monocytes in PBMCs.

[0285] Figures 54A-54C It is a graph showing the results of anti-CSF1R functional assay: human monocyte survival assay. Figure 54A and 54B It is a graph showing the results from BIM0648, BIM0652, BHM1650 (bivalent anti-CSF1R antibody), human IgG1, a cytokine-free group, and a CSF1-only group. Figure 54C This is a table showing the inhibition of BIM0648, BIM0652, and BHM1650 on the survival / proliferation / IC50 (nM) of human monocytes.

[0286] Figures 55A-55B It is a graph showing the results of anti-CCR2 functional assay: human monocyte migration assay. Figure 55A It is a graph showing cell count (migration) versus antibody concentration for BIM0648, BIM0652, BHM0139 (bivalent anti-CCR2 antibody), human IgG1, a group without rhCCL2, and a group with only rhCCL2. Figure 55B This is a table showing the IC50 (nM) of BIM0648, BIM0652, and BHM0139 for inhibiting THP-1 migration.

[0287] Figure 56A and Figure 56B It is a diagram showing the results of TGFβ trap functional assay: reporter cell assay. Figure 56A Graphs showing TGFβ-induced Smad activation in BIM0648, BIM0652, BHM1603 (a bivalent anti-PDL1 antibody fused to a TGFβ trap at the C-terminus of the Fc region), human IgG1, a cell-only group, and a cell+human TGFβi group. Figure 56B is a table showing the IC50 (nM) of BIM0648, BIM0652, and BHM1603 for inhibiting TGFβ-induced Smad signaling.

[0288] Figure 57 It is a graph showing the results of TGFβ trap functional assay: human Treg differentiation assay. Figure 57 Included is a set of flow cytometry plots showing staining for CD25 and Foxp3. Antibodies tested in the human Treg differentiation assay included: BIM0648, BHM1603 (a bivalent anti-PDL1 antibody fused to the TGFβ trap at the C-terminus of the Fc region), BHM1583 (a C-terminal Fc fusion protein of the TGFβRII trap), and a human IgG1 isotype antibody.

[0289] Figure 58 .UniTI-102 (BIM0648) can effectively inhibit the differentiation of human Treg in vitro. Figure 58 Figure 2 is a graph showing the % FOXP3+CD25+ cells of CD4+ T cells for the following treatment groups: with TGFβ and no construct ("+TGFb"), no TGFβ, UniTI-102 (BIM0648), BHM1603 (a bivalent anti-PDL1 antibody fused to a TGFβ trap at the C-terminus of the Fc region), BHM1583 (a C-terminal Fc fusion protein of the TGFβRII trap), and human IgG1.

[0290] Figure 59 is a diagram showing TGFβ trap function assay: human NK cell killing assay.

[0291] Figures 60A-60C .UniTI-102 (BIM0648) reversed the inhibitory effect of TGFβ on primary human NK cell killing. Figure 60A is a set of flow cytometric graphs showing results from a human NK cell killing assay. Figure 60B and 60C are bar graphs showing K562 killing and NKp30 MFI for the indicated treatment groups, respectively. Antibodies tested in this study included: IgG isotype control antibody, BIM0648, and BIM1603 (a bivalent anti-PDL1 antibody fused to a TGFβ trap at the C-terminus of the Fc region).

[0292] Figures 61A-61C .UniTI-102 (BIM0648) reversed the inhibitory effect of TGFβ on primary human NK cell killing. Figure 61A is a graph showing the kill percentage. Figure 61B and 61C Figure 2 is a graph showing the percentage of CD56+NKp30+ cells. The following groups were tested in this study: human IgG, BIM0648, BHM1603 (a bivalent anti-PDL1 antibody fused to a TGFβ trap at the C-terminus of the Fc region), BHM1583 (a C-terminal Fc fusion protein of the TGFβRII trap), without TGFβ ("NK"), with TGFβ and without construct ("TGFb"). DETAILED DESCRIPTION

[0293] TAMs are derived from circulating monocytes, and their recruitment to tumors is driven by tumor-derived chemokines. TAMs can promote tumor cell proliferation and metastasis by causing reactions such as suppressing B and T cell activation, suppressing tumor-associated antigen presentation, suppressing cytotoxic granule release, increasing angiogenesis, and secreting various growth and angiogenic factors (see, for example, Liu et al., Cellular & Molecular Immunology (2015) 12, 1–4; and Noy, Roy et al., Immunity, Vol. 41, No. 1, 49-61; and Quatromoni et al., Am J Transl Res. 2012; 4 (4): 376–389). Therefore, many tumors with a large number of TAMs have increased tumor growth rate, local proliferation, and distant metastasis. Therefore, it would be useful to consume TAMs or inhibit their activity.

[0294] definition

[0295] As used herein, the term "transforming growth factor beta-1 (TGF-β1)" refers to a protein encoded by the gene TGFB1 or a homologous gene thereof in humans. Swiss-Prot Accession No. P01137 provides an exemplary human TGF-β1 amino acid sequence. SEQ ID NO:92 provides an exemplary immature human TGF-β1 amino acid sequence. SEQ ID NO:117 provides an exemplary mature human TGF-β1 amino acid sequence.

[0296] As used herein, the term "transforming growth factor beta-2 (TGF-β2)" refers to a protein encoded by the gene TGFB2 or a homologous gene thereof in humans. Swiss-Prot Accession No. P61812 provides an exemplary human TGF-β2 amino acid sequence. SEQ ID NO:93 provides an exemplary immature human TGF-β2 amino acid sequence. SEQ ID NO:118 provides an exemplary mature human TGF-β2 amino acid sequence.

[0297] As used herein, the term "transforming growth factor beta-3 (TGF-β3)" refers to a protein encoded by the gene TGFB3 or a homologous gene thereof in humans. Swiss-Prot Accession No. P10600 provides an exemplary human TGF-β3 amino acid sequence. SEQ ID NO:94 provides an exemplary immature human TGF-β3 amino acid sequence. SEQ ID NO:119 provides an exemplary mature human TGF-β3 amino acid sequence.

[0298] As used herein, "TGF-β receptor polypeptide" refers to a TGF-β receptor (eg, TGFBR1, TGFBR2, or TGFBR3) or a fragment or variant thereof.

[0299] As used herein, the term "transforming growth factor beta receptor type 1 (TGFBR1)" (also known as ALK-5 or SKR4) refers to a protein encoded by the gene TGFBR1 or a homologous gene thereof in humans. Swiss-Prot Accession No. P36897 provides an exemplary human TGFBR1 amino acid sequence. SEQ ID NOs: 95, 96, and 97 provide exemplary immature human TGFBR1 amino acid sequences. SEQ ID NOs: 120, 121, and 122 provide exemplary mature human TGFBR1 amino acid sequences. As used herein, "TGFBR1 polypeptide" refers to TGFBR1 or a fragment or variant thereof.

[0300] As used herein, the term "transforming growth factor beta receptor type 2 (TGFBR2)" refers to a protein encoded by the gene TGFBR2 or a homologous gene thereof in humans. Swiss-Prot Accession No. P37173 provides an exemplary human TGFBR2 amino acid sequence. SEQ ID NOs: 98 and 99 provide exemplary immature human TGFBR2 amino acid sequences. SEQ ID NOs: 123 and 124 provide exemplary mature human TGFBR2 amino acid sequences. As used herein, "TGFBR2 polypeptide" refers to TGFBR2 or a fragment or variant thereof.

[0301] As used herein, the term "transforming growth factor beta receptor type 3 (TGFBR3)" refers to a protein encoded by the gene TGFBR3 or a homologous gene thereof in humans. Swiss-Prot Accession No. Q03167 provides an exemplary human TGFBR3 amino acid sequence. SEQ ID NOs: 106 and 107 provide exemplary immature human TGFBR3 amino acid sequences. SEQ ID NOs: 125 and 126 provide exemplary mature human TGFBR3 amino acid sequences. As used herein, "TGFBR3 polypeptide" refers to TGFBR3 or a fragment thereof, or a variant thereof.

[0302] As used herein, the term "variant" of a parent sequence refers to a sequence having substantially the same amino acid sequence as the parent sequence, or a fragment thereof. In some embodiments, the variant is a functional variant.

[0303] As used herein, the articles "a" and "an" refer to one or more than one (e.g., at least one) of the grammatical object of the article. When used with the term "comprising," the use of the terms "a" or "an" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0304] As used herein, "about" and "approximately" generally refer to an acceptable degree of error for the quantity being measured, given the nature or precision of the measurements. Exemplary degrees of error are within 20%, typically within 10%, and more typically within 5% of a given value range.

[0305] As used herein, "antibody molecule" refers to a protein comprising at least one immunoglobulin variable region sequence, for example, an immunoglobulin chain or a fragment thereof. Antibody molecules include antibodies (e.g., full-length antibodies) and antibody fragments. In embodiments, antibody molecules include antigen binding or functional fragments of full-length antibodies, or full-length immunoglobulin chains. For example, full-length antibodies are immunoglobulin (Ig) molecules (e.g., IgG antibodies) that occur naturally or are formed by normal immunoglobulin gene fragment recombination processes. In embodiments, antibody molecules refer to the immunoactive antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. Antibody fragments, for example, functional fragments, are part of an antibody, for example, Fab, Fab', F(ab')2, F(ab)2, variable fragment (Fv), domain antibodies (dAb) or single-chain variable fragments (scFv). Functional antibody fragments bind to the same antigen recognized by complete (e.g., full-length) antibodies. The term "antibody fragment" or "functional fragment" also includes an isolated fragment consisting of a variable region (such as an "Fv" fragment consisting of the variable regions of a heavy chain and a light chain), or a recombinant single-chain polypeptide molecule in which the light chain and heavy chain variable regions are connected by a peptide linker ("scFv protein"). In some embodiments, the antibody fragment does not include an antibody portion without antigen binding activity, such as an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, for example, dAb (domain antibodies), single chains, Fab, Fab' and F(ab')2 fragments, and single-chain variable fragments (scFv).

[0306] As used herein, "immunoglobulin variable region sequence" refers to an amino acid sequence that can form the structure of an immunoglobulin variable region. For example, the sequence can include all or part of the amino acid sequence of a naturally occurring variable region. For example, the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other changes that are compatible with protein structure formation.

[0307] In an embodiment, the antibody molecule is monospecific, for example, it comprises binding specificity to a single epitope. In some embodiments, the antibody molecule is multispecific, for example, it comprises multiple immunoglobulin variable region sequences, wherein the first immunoglobulin variable region sequence has binding specificity to the first epitope, and the second immunoglobulin variable region sequence has binding specificity to the second epitope. In some embodiments, the antibody molecule is a bispecific antibody molecule. As used herein, a "bispecific antibody molecule" refers to an antibody molecule that is specific for more than one (e.g., two, three, four or more) epitopes and / or antigens.

[0308] As used herein, "antigen" (Ag) refers to a molecule that can cause an immune response (e.g., involving the activation of certain immune cells and / or antibody production). Any macromolecule, including almost all proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that can cause an immune response encodes an "antigen." In an embodiment, an antigen need not be encoded only by the full-length nucleotide sequence of a gene, nor need the antigen be encoded by a gene. In an embodiment, an antigen can be synthesized or can be derived from a biological sample, such as a tissue sample, a tumor sample, a cell, or a fluid with other biological components. As used herein, "tumor antigens" or, interchangeably, "cancer antigens" include any molecules present on or associated with cancer (e.g., cancer cells or tumor microenvironment that can cause an immune response). As used herein, "immune cell antigens" include any molecules present on or associated with immune cells that can cause an immune response.

[0309] The "antigen binding site" or "binding portion" of an antibody molecule refers to a part of an antibody molecule, such as an immunoglobulin (Ig) molecule, that participates in antigen binding. In embodiments, the antigen binding site is formed by amino acid residues in the variable (V) regions of heavy (H) and light (L) chains. The three highly divergent sequence segments within the variable regions of the heavy and light chains are called hypervariable regions, located between more conservative flanking sequence segments (called "framework regions" (FR)). FR is an amino acid sequence naturally present between and adjacent to the hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen binding surface that is complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions in each heavy and light chain are called "complementary determining regions" or "CDRs." The framework regions and CDRs are defined and described in, for example, Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 and Chothia, C. et al., (1987) J. Mol. Biol. 196: 901-917. Each variable chain (e.g., variable heavy chain and variable light chain) is typically composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following amino acid sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0310] As used herein, "cancer" can encompass all types of oncogenic processes and / or cancerous growths. In embodiments, cancer includes primary tumors as well as metastatic tissues or malignantly transformed cells, tissues, or organs. In embodiments, cancer encompasses all histopathologies and stages, such as the stage of invasiveness / severity of the cancer. In embodiments, cancer includes recurrent and / or drug-resistant cancers. The terms "cancer" and "tumor" can be used interchangeably. For example, both terms include solid tumors and liquid tumors. As used herein, the terms "cancer" or "tumor" include pre-malignant as well as malignant cancers and tumors.

[0311] The compositions and methods of the present invention include polypeptides and nucleic acids having a specified sequence or a sequence substantially identical or similar thereto (e.g., a sequence at least 85%, 90%, 95% identical or higher identical to a specified sequence). In the context of amino acid sequences, the term "substantially identical" as used herein refers to a first amino acid comprising a sufficient or minimum number of amino acid residues that are i) identical to the aligned amino acid residues in the second amino acid sequence, or ii) have conservative substitutions such that the first and second amino acid sequences can have a common domain and / or a common functional activity. For example, an amino acid sequence contains a common domain with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence (e.g., a sequence provided herein).

[0312] In the context of nucleotide sequences, the term "substantially identical" as used herein refers to a first nucleic acid sequence comprising a sufficient or minimum number of nucleotides that are identical to the aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode polypeptides having a common functional activity, or encoding a common structural polypeptide domain or a common functional polypeptide activity. For example, a nucleotide sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence (e.g., a sequence provided herein).

[0313] Calculations of homology or sequence identity (the terms are used interchangeably herein) between sequences are performed as follows.

[0314] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

[0315] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0316] Mathematical algorithms can be used to compare sequences and determine the percent identity between two sequences. In a preferred embodiment, the comparison of sequences and the determination of percent identity between two sequences are performed using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com)) using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. In another preferred embodiment, the GAP program in the GCG software package (available from http: / / www.gcg.com) uses the NWSgapdna.CMP matrix with a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred set of parameters (the set of parameters that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0317] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0318] The nucleic acid and protein sequences described herein can be used as a "query sequence" to search public databases to, for example, identify other family members or related sequences. Searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, wordlength = 12) to obtain nucleotide sequences homologous to nucleic acid molecules of the invention (e.g., SEQ ID NO: 1). BLAST protein searches can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402 can be utilized. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov .

[0319] It will be understood that the molecules of the invention may have additional conservative or non-essential amino acid substitutions that have no substantial effect on their function.

[0320] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that include both an amino and an acid functional group and that are capable of being included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D- or L- optical isomers and peptidomimetics.

[0321] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0322] The terms "polypeptide," "peptide," and "protein" (if single-chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or can be the product of synthetic methods.

[0323] The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence" and "polynucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide may be single-stranded or double-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. A nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin that does not occur naturally or is linked to another polynucleotide in a non-natural manner.

[0324] As used herein, the term "isolated" refers to material that is removed from its source or natural environment (e.g., the natural environment, if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system by human intervention is isolated. Such a polynucleotide can be part of a vector and / or such a polynucleotide or polypeptide can be part of a composition and still be isolated because such a vector or composition is not part of the environment in which it is found in nature.

[0325] As used herein, the term "immunosuppressive myeloid cells" or "IMCs" generally refers to myeloid lineage cells that promote immunosuppression (e.g., in the tumor microenvironment) (e.g., by inhibiting T cell activation, inhibiting T cell viability, promoting the induction and recruitment of T regulatory cells). Immunosuppressive myeloid cells include, for example, tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs).

[0326] As used herein, the term "tumor-associated macrophages" or "TAMs" generally refers to macrophages that are present in the microenvironment of a cancer, such as a tumor.

[0327] As used herein, the term "reduce TAMs" generally refers to reducing the number of TAMs. Reduction includes reducing the number of TAMs in or near a tumor (e.g., compared to the number of TAMs before administration of a multispecific molecule described herein (e.g., before administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times a multispecific molecule described herein)). Reduction includes reducing any number of TAMs (e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%, all or substantially) (e.g., compared to the number of TAMs before administration of a multispecific molecule described herein (e.g., before administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times a multispecific molecule described herein)).

[0328] As used herein, the term "myeloid-derived suppressor cell" or "MDSC" generally refers to cells of bone marrow origin that can promote immunosuppression and generally express CD33, CD11b, and CD45. Various subpopulations of MDSC have been defined, such as mononuclear MDSC (M-MDSC) that are generally associated with the expression of CD14 and CD124 and low expression of HLA-DR. In some embodiments, the MDSC colony is a MO-MDSC colony. Polymorphonuclear MDSC (PMN-MDSC) is associated with the expression of CD15, CD66b, and CD124, and is associated with the absence of HLA-DR expression. Immature MDSC (I-MDSC) is associated with the expression of CD117 and CD34, and is associated with the absence of LIN and HLA-DR expression. See, for example, Ugel et al., (2015) JCI Vol. 125 (9), p. 3365.

[0329] As used herein, the term "CSF1R-positive, CCR2-positive cells" refers to cells that express both CSF1R and CCR2 on the cell surface. The term "CSF1R-positive, CCR2-negative cells" refers to cells that express CSF1R but not CCR2 on the cell surface. The term "CSF1R-negative, CCR2-positive cells" refers to cells that express CCR2 but not CSF1R on the cell surface.

[0330] As used herein, a binding moiety (e.g., an antibody molecule) monovalently binds to a target when the binding moiety (e.g., an antibody molecule) binds to a single epitope on the target. In some embodiments, the binding moiety comprises only one antigen binding domain for the target. In some embodiments, one molecule of the binding moiety can only bind to one molecule of the target.

[0331] As used herein, when a binding moiety (e.g., an antibody molecule) binds to two epitopes on a target, the binding moiety (e.g., an antibody molecule) is bivalently bound to the target. In some embodiments, the two epitopes are identical. In some embodiments, the two epitopes are different. In some embodiments, the binding moiety comprises two antigen binding domains directed against the target. In some embodiments, one molecule of the binding molecule can bind to two molecules of the target.

[0332] Various aspects of the invention are described in further detail below. Additional definitions are set forth throughout the specification.

[0333] antigen

[0334] TAM-targeting antigens of the present disclosure include, for example, CSF1R, CCR2, CXCR2, CD68, CD163, CX3CR1, MARCO, CD204, CD52, and folate receptor beta. Exemplary amino acid sequences of TAM-targeting antigens are provided herein.

[0335] CSF1R

[0336] CSF1R (also known as macrophage colony-stimulating factor 1 receptor) is a tyrosine protein kinase that acts as a cell surface receptor for CSF1 and IL-34 and plays a crucial role in regulating the survival, proliferation, and differentiation of hematopoietic precursor cells, particularly mononuclear phagocytes such as macrophages and monocytes. CSF1R promotes the release of proinflammatory chemokines in response to IL-34 and CSF1, thereby playing an important role in innate immunity and inflammatory processes. SEQ ID NOs: 87 and 88 provide exemplary immature amino acid sequences of CSF1R.

[0337] CSF1R immature amino acid sequence isoform 1 (identifier: P07333-1):

[0338] MGPGVLLLLLVATAWHGQGIPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRVVESAYLNLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWLQCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSVGSGSWAFIPISAGAHTHPPDEFLFTPVVVACMSIMALLLLLLLLLLYKYKQKPKYQVRWKIIESYEGNSYTFIDPTQLPYNEKWEFPRNNLQFGKTLGAGAFGKVVEATAFGLGKEDAVLKVAVKMLKSTAHADEKEALMSELKIMSHLGQHENIVNLLGACTHGGPVLVITEYCCYGDLLNFLRRKAEAMLGPSLSPGQDPEGGVDYKNIHLEKKYVRRDSGFSSQGVDTYVEMRPVSTSSNDSFSEQDLDKEDGRPLELRDLLHFSSQVAQGMAFLASKNCIHRDVAARNVLLTNGHVAKIGDFGLARDIMNDSNYIVKGNARLPVKWMAPESIFDCVYTVQSDVWSYGILLWEIFSLGLNPYPGILVNSKFYKLVKDGYQMAQPAFAPKNIYSIMQACWALEPTHRPTFQQICSFLQEQAQEDRRERDYTNLPSSSRSGGSGSSSSELEEESSSEHLTCCEQGDIAQPLLQPNNYQFC

[0339] SEQ ID NO:87

[0340] CSF1R immature amino acid sequence isoform 2 (identifier: P07333-2):

[0341] MGPGVLLLLLVATAWHGQGIPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTN YSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRVVGTPSPSLCPA

[0342] SEQ ID NO:88

[0343] CCR2

[0344] CCR2 (also known as CC chemokine receptor type 2) is a G protein-coupled receptor for CCL2, CCL7, and CCL13 chemokines. CCR2 is known to play a role in the recruitment of monocytes / macrophages and T cells. CCR2 is expressed on monocytes and a small number of T cell subsets and exhibits almost identical expression patterns in mice and humans (Mack et al., J Immunol 2001; 166: 4697-4704). Exemplary CCR2 amino acid sequences are provided in SEQ ID NOs: 89 and 90.

[0345] CCR2 amino acid sequence isoform A (identifier: P41597-1):

[0346] MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRSLFHIALGCRIAPLQKPVCGGPGVRPGKNVKVTTQGLLDGRGKGKSIGRAPEASLQDKEGA

[0347] SEQ ID NO:89

[0348] CCR2 amino acid sequence isoform B (identifier: P41597-2):

[0349] MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL

[0350] SEQ ID NO:90

[0351] CXCR2

[0352] CXCR2 (also known as interleukin 8 receptor) is a G protein-coupled receptor for IL8, which is a chemotactic factor for neutrophils. The binding of IL8 to the receptor causes the activation of neutrophils. This reaction is mediated by G proteins that activate the phosphatidylinositol calcium second messenger system. CXCR2 binds to IL-8 with high affinity and also binds to CXCL3, GRO / MGSA and NAP-2 with high affinity. CXCR2 is expressed at high levels in circulating neutrophils and is crucial for guiding neutrophil migration to sites of inflammation (J Clin Invest. 2012; 122(9): 3127-3144). SEQ ID NO: 91 provides an exemplary CXCR2 amino acid sequence.

[0353] CXCR2 amino acid sequence (identifier: P25025-1):

[0354] MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPV LLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL

[0355] SEQ ID NO:91

[0356] Exemplary antibodies

[0357] Exemplary antibodies that bind to TAM antigens are provided throughout the specification and below. Exemplary anti-CSF1R antibodies are described herein and in WO2009026303A1; WO2011123381A1; WO2016207312A1; WO2016106180A1; US20160220669A1; US20160326254A1; WO2013169264A1; WO2013087699A1; WO2011140249A2; WO2011131407A1; WO2011123381A1; WO2011107553A1; and WO2011070024A1, all of which are incorporated herein by reference in their entirety. Exemplary CCR2 antibodies are described herein and in WO2013192596A2; WO2010021697A2; WO2001057226A1; and WO1997031949A1, all of which are herein incorporated by reference in their entirety. Exemplary CXCR2 antibodies are described in WO2014170317A1 and US20160060347 (see, e.g., a) SEQ ID NO: 14 (light chain) and SEQ ID NO: 15 (heavy chain); b) SEQ ID NO: 24 (light chain) and SEQ ID NO: 25 (heavy chain); c) SEQ ID NO: 34 (light chain) and SEQ ID NO: 35 (heavy chain); d) SEQ ID NO: 44 (light chain) and SEQ ID NO: 45 (heavy chain); e) SEQ ID NO: 54 (light chain) and SEQ ID NO: 55 (heavy chain); f) SEQ ID NO: 64 (light chain) and SEQ ID NO: 65 (heavy chain); g) SEQ ID NO: 74 (light chain) and SEQ ID NO: 75 (heavy chain); h) SEQ ID NO: 84 (light chain) and SEQ ID NO: 85 (heavy chain)), all of which are incorporated herein by reference in their entirety. Exemplary anti-CD163 antibodies are provided in US20120258107 (see, e.g., MAC2158, MAC2-48), which are incorporated herein by reference in their entirety. Exemplary anti-CD52 antibodies are described in US20050152898, which are incorporated herein by reference in their entirety. Exemplary anti-folate antibodies are described in US9522196, which are incorporated herein by reference in their entirety. Exemplary anti-CD52 antibodies are described in US20050152898, which are incorporated herein by reference in their entirety. Exemplary anti-MARCO antibodies are described in WO2016196612, which are incorporated herein by reference in their entirety.

[0358] Antibody molecules

[0359] In one embodiment, the multispecific molecule comprises an antibody molecule that binds to a first tumor-associated macrophage (TAM) antigen; and an antibody molecule that binds to a second TAM antigen. In some embodiments, the first TAM antigen and / or the second TAM antigen are, for example, mammalian, such as human. For example, the antibody molecule specifically binds to an epitope on a TAM antigen, such as a linear or conformational epitope.

[0360] In one embodiment, the multispecific molecule comprises an antibody molecule that binds to a first myeloid-derived suppressor cell (MDSC) antigen; and an antibody molecule that binds to a second MDSC antigen. In some embodiments, the first MDSC antigen and / or the second MDSC antigen are, for example, mammalian, such as human. For example, the antibody molecule specifically binds to an epitope on the MDSC antigen, such as a linear or conformational epitope.

[0361] In one embodiment, the antibody molecule is a monospecific antibody molecule and binds a single epitope. For example, a monospecific antibody molecule has multiple immunoglobulin variable region sequences, each sequence binding to the same epitope.

[0362] In one embodiment, antibody molecule is a multispecific antibody molecule, for example, it comprises multiple immunoglobulin variable region sequences, wherein the first immunoglobulin variable region sequence in the multiple has binding specificity to the first epitope, and the second immunoglobulin variable region sequence in the multiple has binding specificity to the second epitope. In one embodiment, the first and second epitopes are on the same antigen, for example, on the same protein (or the subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, for example, on different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody molecule comprises the third, fourth or fifth immunoglobulin variable region. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule or a tetraspecific antibody molecule.

[0363] In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for no more than two antigens. The bispecific antibody molecule is characterized in that the first immunoglobulin variable region sequence with binding specificity to the first epitope and the second immunoglobulin variable region sequence with binding specificity to the second epitope. In one embodiment, the first epitope and the second epitope are on the same antigen, for example, on the same protein (or subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap. In one embodiment, the first epitope and the second epitope do not overlap. In one embodiment, the first epitope and the second epitope are on different antigens, for example, on different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable region sequence and a light chain variable region sequence with binding specificity to the first epitope and a heavy chain variable region sequence and a light chain variable region sequence with binding specificity to the second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody with binding specificity to the first epitope and a half antibody with binding specificity to the second epitope. In one embodiment, the bispecific antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a scFv or Fab or fragment thereof having binding specificity for a first epitope and a scFv or Fab or fragment thereof having binding specificity for a second epitope.

[0364] In one embodiment, the antibody molecule comprises a diabody, a single-chain molecule, and an antigen-binding fragment of an antibody (e.g., Fab, F(ab')2, and Fv). For example, an antibody molecule can include a heavy (H) chain variable region sequence (abbreviated herein as VH)) and a light (L) chain variable region sequence (abbreviated herein as VL). In one embodiment, the antibody molecule comprises a heavy chain and a light chain or consists of them (referred to herein as a half antibody). In another example, the antibody molecule includes two heavy (H) chain variable region sequences and two light (L) chain variable region sequences, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single variable region antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modifying complete antibodies or synthesized de novo using recombinant DNA technology. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments can be from any kind of antibody, including but not limited to IgG, IgA, IgM, IgD and IgE, and from any subclass of antibody (such as IgG1, IgG2, IgG3 and IgG4). The preparation of antibody molecules can be monoclonal or polyclonal. Antibody molecules can also be people's, humanized, CDR-transplanted or in vitro produced antibodies. Antibodies can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3 or IgG4. Antibodies can also have a light chain selected from, for example, κ or λ. The term "immunoglobulin" (Ig) can be used interchangeably with the term "antibody" in this article.

[0365] Examples of antigen-binding fragments of antibody molecules include: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) diabody (dAb) fragments consisting of a VH domain; (vi) camelid or camelized variable regions; (vii) single-chain Fv (scFv), see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); and (viii) single-domain antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as intact antibodies.

[0366] Antibody molecules include complete molecules and functional fragments thereof. The constant region of an antibody molecule can be altered, for example, by mutation, to modify the properties of the antibody (e.g., to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function).

[0367] Antibody molecules can also be single-domain antibodies. Single-domain antibodies can include antibodies whose complementary determining regions are part of a single-domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that are naturally free of light chains, single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds that are not derived from antibodies. Single-domain antibodies can be any single-domain antibody in the art, or any future single-domain antibody. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, fish, sharks, goats, rabbits, and cattle. According to another aspect of the present invention, single-domain antibodies are naturally occurring single-domain antibodies, referred to as heavy chain antibodies lacking light chains. Such single-domain antibodies are disclosed, for example, in WO 9404678. For the sake of clarity, the variable region of such a heavy chain antibody derived from a natural lack of light chains is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camels, llamas, dromedaries, alpacas, and guanacos). Species other than Camelidae may also produce heavy chain antibodies that naturally lack light chains; such VHHs are within the scope of the present invention.

[0368] The VH and VL regions can be subdivided into regions of hypervariability, termed "complementarity determining regions" (CDRs), interspersed with regions that are more conserved, termed "framework regions" (FR or FW).

[0369] The extent of the framework regions and CDRs has been precisely defined by a variety of methods (see Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C., et al., (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM modeling software. See generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. This is in Antibody Engineering Lab Manual (Editors: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

[0370] As used herein, the terms "complementarity determining region" and "CDR" refer to the amino acid sequences within the variable region of an antibody that confer antigen specificity and binding affinity. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0371] The precise amino acid sequence boundaries of a given CDR can be determined using any of a variety of known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme). As used herein, CDRs defined according to the "Chothia" numbering scheme are sometimes also referred to as "hypervariable loops."

[0372] For example, under Kabat, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); while the CDR amino acid residues in the light chain variable region (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); while the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

[0373] Each VH and VL typically includes three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0374] Antibody molecules can be polyclonal or monoclonal.

[0375] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be prepared by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

[0376] Antibodies can be produced recombinantly, for example by phage display, or by combinatorial methods.

[0377] Phage display and combinatorial methods for generating antibodies are known in the art (e.g., in Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO 91 / 17271; Winter et al., International Publication No. WO 92 / 20791; Markland et al., International Publication No. WO 92 / 15679; Breitling et al., International Publication No. WO 93 / 01288; McCafferty et al., International Publication No. WO 92 / 01047; Garrard et al., International Publication No. WO 92 / 09690; Ladner et al., International Publication No. WO 90 / 02809; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al., (1992) Hum Antibod Hybridomas 3:81-85; Huse et al., (1989) Science 246:1275-1281; Griffths et al., (1993) EMBO J 12:725-734; Hawkins et al., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS 89:3576-3580; Garrad et al., (1991) Bio / Technology 9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982, the entire contents of which are incorporated herein by reference).

[0378] In one embodiment, the antibody is a fully human antibody (e.g., an antibody prepared in a mouse that has been genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, such as an antibody of a rodent (mouse or rat), goat, primate (e.g., monkey), or camel. Preferably, the non-human antibody is a rodent antibody (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0379] Human monoclonal antibodies can be produced using transgenic mice carrying human immunoglobulin genes rather than mouse systems. Splenocytes from these transgenic mice immunized with the antigen of interest are used to generate hybridomas that secrete human mAbs with specific affinity for epitopes of human proteins (see, e.g., Wood et al., International Application WO 91 / 00906, Kucherlapati et al., PCT Publication WO 91 / 10741; Lonberg et al., International Application WO 92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N et al., 1994 Nature 368:856-859; Green, LL et al., 1994 Nature Genet. 7:13-21; Morrison, SL et al., 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al., 1993 Year Immunol 7:33-40; Tuaillon et al., 1993 PNAS 90:3720-3724; Bruggeman et al., 1991 Eur J Immunol 21:1323-1326).

[0380] The antibody molecule can be an antibody molecule in which the variable region or a portion thereof, such as a CDR, is produced in a non-human organism, such as a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the present invention. Antibody molecules produced in a non-human organism, such as a rat or mouse, and then modified, for example, in the variable framework or constant region to reduce antigenicity in humans are within the present invention.

[0381] An "effectively human" protein is one that does not substantially elicit a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. In many cases, HAMA may be problematic if, for example, the antibody molecule is administered repeatedly, e.g., for the treatment of chronic or recurrent diseases. Due to increased clearance of antibodies from the serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32: 180-190 (1990)), and also due to potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5: 5117-5123 (1986)), the HAMA response may render repeated antibody administration ineffective.

[0382] Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication No. PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Application WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al., (1988 Science 240:1041-1043); Liu et al., (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0383] Humanized or CDR-grafted antibodies will have at least one or two, but typically all three, acceptor CDRs (of the immunoglobulin heavy and / or light chain) replaced by donor CDRs. The antibody may be replaced by at least a portion of the non-human CDRs, or only some of the CDRs may be replaced by non-human CDRs. Only the number of CDRs required for antigen binding need be replaced. Preferably, the donor will be a rodent antibody, such as a rat or mouse antibody, and the acceptor will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is referred to as the "donor," and the immunoglobulin providing the framework is referred to as the "acceptor." In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or a consensus framework, or a sequence having about 85% or more, preferably 90%, 95%, 99% or more identity thereto.

[0384] As used herein, the term "consensus sequence" refers to a sequence formed by the most frequently occurring amino acids (or nucleotides) in a family of related sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). Within a family of proteins, each position in the consensus sequence is occupied by the most frequently occurring amino acid at that position in the family. If two amino acids occur equally frequently, either one can be included in the consensus sequence. "Consensus framework" refers to the framework region in the consensus immunoglobulin sequence.

[0385] Antibody molecules can be humanized by methods known in the art (see, e.g., Morrison, SL, 1985, Science 229: 1202-1207; Oi et al., 1986, BioTechniques 4: 214 and Queen et al., US 5,585,089, US 5,693,761 and US 5,693,762, the entire contents of which are incorporated herein by reference).

[0386] Humanized or CDR-grafted antibody molecules can be produced by CDR grafting or CDR replacement, wherein one, two or all CDRs of an immunoglobulin chain can be replaced. See, for example, U.S. Patent No. 5,225,539; Jones et al., 1986 Nature 321:552-525; Verhoeyan et al., 1988 Science 239:1534; Beidler et al., 1988 J. Immunol. 141:4053-4060; Winter US 5,225,539, the entire contents of which are expressly incorporated herein by reference. Winter describes a CDR grafting method that can be used to prepare the humanized antibodies of the present invention (British patent application GB 2188638A filed March 26, 1987; Winter 5,225,539), the contents of which are expressly incorporated herein by reference.

[0387] Humanized antibody molecules are also within the scope of the present invention, wherein specific amino acids have been replaced, deleted or added. The standard for selecting amino acids from donors is described in US 5,585,089, for example, and US 5,585,089, for example, in US 5,585,089, the contents of which are incorporated herein by reference. Other technical descriptions for making antibody humanization are disclosed in Padlan et al., EP 519596 A1, disclosed on December 23, 1992.

[0388] The antibody molecule can be a single-chain antibody. Single-chain antibodies (scFV) can be engineered (e.g., see Colcher, D. et al., (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). Single-chain antibodies can be dimerized or multimerized to produce multivalent antibodies specific for different epitopes of the same target protein.

[0389] In other embodiments, the antibody molecule has a heavy chain constant region selected from, for example, the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; in particular, selected from, for example, the (e.g., human) heavy chain constant region of IgG1, IgG2, IgG3 and IgG4. In another embodiment, the antibody molecule has a light chain constant region selected from, for example, the (e.g., human) light chain constant region of κ or λ. The constant region can be changed, for example, by mutation, to modify the properties of the antibody (e.g., to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function and / or complement function). In one embodiment, the antibody has: effector function; and can repair complement. In other embodiments, the antibody does not: recruit effector cells; or repair complement. In another embodiment, the antibody has a reduced ability to bind to Fc receptors or does not have the ability to bind to Fc receptors. For example, it is an isoform or subtype, fragment or other mutant that does not support binding to an Fc receptor, eg, it has a mutagenized or deleted Fc receptor binding region.

[0390] Methods for changing the constant region of an antibody are known in the art. Antibodies with altered functions (e.g., changes in affinity for effector ligands (e.g., FcR on a cell or the C1 component of complement)) can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388,151 A1, U.S. Patent No. 5,624,821 and U.S. Patent No. 5,648,260, the entire contents of which are incorporated herein by reference). Similar types of changes can be described, and if applied to mice or other species, immunoglobulins will reduce or eliminate these functions.

[0391] Antibody molecules can be derived into or connected to another functional molecule (e.g., another peptide or protein). As used herein, "derived" antibody molecules are modified ones. Derivatization methods include but are not limited to adding fluorescent moieties, radionucleotides, toxins, enzymes or affinity ligands such as biotin. Therefore, the antibody molecules of the present invention are intended to include derivative forms and other modified forms of antibodies described herein, including immunoadhesion molecules. For example, antibody molecules can be functionally connected (by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody (e.g., bispecific antibody or double antibody), detectable reagent, cytotoxic agent, pharmaceutical agent, and / or can mediate the protein or peptide (e.g., streptavidin core region or polyhistidine tag) that binds to an antibody or antibody portion and another molecule.

[0392] One type of derivatized antibody molecule is produced by cross-linking two or more antibodies (of the same or different types, e.g., to produce bispecific antibodies). Suitable cross-linking agents include heterobifunctional cross-linking agents having two different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional cross-linking agents (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, 111.

[0393] Antibody molecules targeting CSF1R

[0394] In some embodiments, provided herein are antibody molecules (eg, monospecific or multispecific antibody molecules) that bind to CSF1R (eg, human CSF1R).

[0395] In some embodiments, the anti-CSF1R antibody molecule comprises one, two, or three heavy chain CDRs (e.g., HCDR1, HCDR2, and / or HCDR3) disclosed in Table 13 (e.g., a row of Table 13), or comprises no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the anti-CSF1R antibody molecule comprises one, two, or three heavy chain CDRs (e.g., HCDR1, HCDR2, and / or HCDR3) from a heavy chain variable region (VH) disclosed in Table 15, e.g., optionally, wherein the CDRs are defined according to Rabat, Chothia, or IMGT. In some embodiments, the anti-CSF1R antibody molecule comprises one, two, three, or four heavy chain framework regions (e.g., heavy chain FR1, FR2, FR3, and / or FR4) disclosed in Table 13 (e.g., a row of Table 13), or comprises no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the anti-CSF1R antibody molecule comprises one, two, three, or four heavy chain framework regions (e.g., heavy chain FR1, FR2, FR3, and / or FR4) from a VH disclosed in Table 15. In some embodiments, the anti-CSF1R antibody molecule comprises a VH disclosed in Table 15, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0396] In some embodiments, the anti-CSF1R antibody molecule comprises one, two, or three light chain CDRs (e.g., LCDR1, LCDR2, and / or LCDR3) disclosed in Table 14 (e.g., a row of Table 14), or comprises no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the anti-CSF1R antibody molecule comprises one, two, or three light chain CDRs (e.g., LCDR1, LCDR2, and / or LCDR3) from a light chain variable region (VL) disclosed in Table 16, e.g., optionally, wherein the CDRs are defined according to Rabat, Chothia, or IMGT. In some embodiments, the anti-CSF1R antibody molecule comprises one, two, three, or four light chain framework regions (e.g., light chain FR1, FR2, FR3, and / or FR4) disclosed in Table 14 (e.g., a row of Table 14), or comprises no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the anti-CSF1R antibody molecule comprises one, two, three, or four light chain framework regions (e.g., light chain FR1, FR2, FR3, and / or FR4) from a VL disclosed in Table 16. In some embodiments, the anti-CSF1R antibody molecule comprises a VL disclosed in Table 16, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0397] In some embodiments, the anti-CSF1R antibody molecule comprises a VH encoded by a nucleotide sequence disclosed in Table 15 (or a nucleotide sequence with at least 80%, 85%, 90%, or 95% identity thereof), and / or a VL encoded by a nucleotide sequence disclosed in Table 16 (or a nucleotide sequence with at least 80%, 85%, 90%, or 95% identity thereof).

[0398] Table 13. CDRs and framework regions (FRs) of exemplary heavy chain variable regions of anti-CSFIR antibody molecules.

[0399]

[0400]

[0401]

[0402] Table 29. Consensus CDRs of exemplary heavy chain variable regions of anti-CSF1R antibody molecules.

[0403]

[0404] Table 14. CDRs and framework regions (FRs) of exemplary light chain variable regions of anti-CSFIR antibody molecules.

[0405]

[0406] Table 15. Amino acid and nucleotide sequences of exemplary heavy chain variable regions of anti-CSF1R antibody molecules.

[0407]

[0408]

[0409]

[0410]

[0411]

[0412] Table 16. Amino acid and nucleotide sequences of exemplary light chain variable regions of anti-CSF1R antibody molecules.

[0413]

[0414] Antibody molecules targeting CCR2

[0415] In some embodiments, provided herein are antibody molecules (eg, monospecific or multispecific antibody molecules) that bind to CCR2 (eg, human CCR2).

[0416] In some embodiments, the anti-CCR2 antibody molecule comprises one, two, or three heavy chain CDRs (e.g., HCDR1, HCDR2, and / or HCDR3) disclosed in Table 17 (e.g., a row of Table 17), or comprises no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the anti-CCR2 antibody molecule comprises one, two, or three heavy chain CDRs (e.g., HCDR1, HCDR2, and / or HCDR3) from a heavy chain variable region (VH) disclosed in Table 19, e.g., optionally, wherein the CDRs are defined according to Rabat, Chothia, or IMGT. In some embodiments, the anti-CCR2 antibody molecule comprises one, two, three, or four heavy chain framework regions (e.g., heavy chain FR1, FR2, FR3, and / or FR4) disclosed in Table 17 (e.g., a row of Table 17), or comprises no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the anti-CCR2 antibody molecule comprises one, two, three, or four heavy chain framework regions (e.g., heavy chain FR1, FR2, FR3, and / or FR4) from a VH disclosed in Table 19. In some embodiments, the anti-CCR2 antibody molecule comprises a VH disclosed in Table 19, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0417] In some embodiments, the anti-CCR2 antibody molecule comprises one, two, or three light chain CDRs (e.g., LCDR1, LCDR2, and / or LCDR3) disclosed in Table 18 (e.g., a row of Table 18), or comprises no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the anti-CCR2 antibody molecule comprises one, two, or three light chain CDRs (e.g., LCDR1, LCDR2, and / or LCDR3) from a light chain variable region (VL) disclosed in Table 20, e.g., optionally, wherein the CDRs are defined according to Rabat, Chothia, or IMGT. In some embodiments, the anti-CCR2 antibody molecule comprises one, two, three, or four light chain framework regions (e.g., light chain FR1, FR2, FR3, and / or FR4) disclosed in Table 18 (e.g., a row of Table 18), or comprises no more than 1, 2, 3, 4, 5, or 6 modified amino acid sequences. In some embodiments, the anti-CCR2 antibody molecule comprises one, two, three, or four light chain framework regions (e.g., light chain FR1, FR2, FR3, and / or FR4) from a VL disclosed in Table 20. In some embodiments, the anti-CCR2 antibody molecule comprises a VL disclosed in Table 20, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the anti-CCR2 antibody molecule does not comprise a VH comprising the amino acid sequence: EVQLVESGGGLVKPGGSLRLSCAASGFSFNAYAMNWVRQAPGKGLEWVGRIRTKNNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTFYGNGVWGQGTLVTVSS (SEQ ID NO: 480). In some embodiments, the anti-CCR2 antibody molecule does not comprise a VL comprising the following amino acid sequence: DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTFLNWLLQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGGGTRLEIK (SEQ ID NO: 481).

[0418] In some embodiments, the anti-CCR2 antibody molecule comprises a VH encoded by a nucleotide sequence disclosed in Table 19 (or a nucleotide sequence with at least 80%, 85%, 90% or 95% identity thereof), and / or a VL encoded by a nucleotide sequence disclosed in Table 20 (or a nucleotide sequence with at least 80%, 85%, 90% or 95% identity thereof).

[0419] Table 17 CDRs and framework regions (FRs) of exemplary heavy chain variable regions of anti-CCR2 antibody molecules.

[0420]

[0421] Table 18. CDRs and framework regions (FRs) of exemplary light chain variable regions of anti-CCR2 antibody molecules.

[0422]

[0423] Table 19. Amino acid and nucleotide sequences of exemplary heavy chain variable regions of anti-CCR2 antibody molecules.

[0424]

[0425] Table 20. Amino acid and nucleotide sequences of exemplary light chain variable regions of anti-CCR2 antibody molecules.

[0426]

[0427] Antibody molecules targeting PD-L1

[0428] In some embodiments, provided herein are antibody molecules (e.g., monospecific or multispecific antibody molecules) that bind to PD-L1 (e.g., human PD-L1).

[0429] In some embodiments, the anti-PD-L1 antibody molecule comprises one, two, or three heavy chain CDRs (e.g., HCDR1, HCDR2, and / or HCDR3) disclosed in Table 21 (e.g., a row of Table 21), or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications. In some embodiments, the anti-PD-L1 antibody molecule comprises one, two, or three heavy chain CDRs (e.g., HCDR1, HCDR2, and / or HCDR3) from a heavy chain variable region (VH) disclosed in Table 23, e.g., optionally, wherein the CDRs are defined according to Rabat, Chothia, or IMGT. In some embodiments, the anti-PD-L1 antibody molecule comprises one, two, three, or four heavy chain framework regions (e.g., heavy chain FR1, FR2, FR3, and / or FR4) disclosed in Table 21 (e.g., a row of Table 21), or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications. In some embodiments, the anti-PD-L1 antibody molecule comprises one, two, three, or four heavy chain framework regions (e.g., heavy chain FR1, FR2, FR3, and / or FR4) from a VH disclosed in Table 23. In some embodiments, the anti-PD-L1 antibody molecule comprises a VH disclosed in Table 23, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0430] In some embodiments, the anti-PD-L1 antibody molecule comprises one, two, or three light chain CDRs (e.g., LCDR1, LCDR2, and / or LCDR3) disclosed in Table 22 (e.g., a row of Table 22) or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications. In some embodiments, the anti-PD-L1 antibody molecule comprises one, two, or three light chain CDRs (e.g., LCDR1, LCDR2, and / or LCDR3) from a light chain variable region (VL) disclosed in Table 24, e.g., optionally, wherein the CDRs are defined according to Rabat, Chothia, or IMGT. In some embodiments, the anti-PD-L1 antibody molecule comprises one, two, three, or four light chain framework regions (e.g., light chain FR1, FR2, FR3, and / or FR4) disclosed in Table 22 (e.g., a row of Table 22) or an amino acid sequence comprising no more than 1, 2, 3, 4, 5, or 6 modifications. In some embodiments, the anti-PD-L1 antibody molecule comprises one, two, three, or four light chain framework regions (e.g., light chain FR1, FR2, FR3, and / or FR4) from a VL disclosed in Table 24. In some embodiments, the anti-PD-L1 antibody molecule comprises a VL disclosed in Table 24, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0431] In some embodiments, the anti-PD-L1 antibody molecule comprises a VH encoded by a nucleotide sequence disclosed in Table 23 (or a nucleotide sequence with at least 80%, 85%, 90%, or 95% identity thereof), and / or a VL encoded by a nucleotide sequence disclosed in Table 24 (or a nucleotide sequence with at least 80%, 85%, 90%, or 95% identity thereof).

[0432] Table 21. CDRs and framework regions (FRs) of exemplary heavy chain variable regions of anti-PD-L1 antibody molecules.

[0433]

[0434] Table 22. CDRs and framework regions (FRs) of exemplary light chain variable regions of anti-PD-L1 antibody molecules.

[0435]

[0436] Table 23. Amino acid and nucleotide sequences of exemplary heavy chain variable regions of anti-PD-L1 antibody molecules.

[0437]

[0438] Table 24. Amino acid and nucleotide sequences of exemplary light chain variable regions of anti-PD-L1 antibody molecules.

[0439]

[0440] Multispecific antibody molecules

[0441] In an embodiment, the multispecific antibody molecule may include more than one antigen binding site, wherein different sites are specific for different antigens. In an embodiment, the multispecific antibody molecule can bind to more than one (e.g., two or more) epitopes on the same antigen. In an embodiment, the multispecific antibody molecule includes an antigen binding site that is specific for target cells (e.g., cancer cells) and different antigen binding sites that are specific for immune effector cells. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be divided into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with additional antigen binding moieties attached; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

[0442] BsIgG is a monovalent form for each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, common LC of a knob-in-hole structure, a knob-in-hole structure assembly, a charge pair, Fab arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, orthogonal Fab. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Exemplary BsIgG include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which comprises an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, BsIgG comprises a heavy chain engineered for heterodimerization. For example, the heavy chain can be engineered for heterodimerization using a "knobs-into-holes" strategy, a SEED platform, a common heavy chain (e.g., in a kλ body), and a heterodimer Fc region. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Strategies for avoiding heavy chain pairing of homodimers in BsIgG include knobs-into-holes, diabodies, azymetric, charge pairs, HA-TF, SEEDbody, and differential A protein affinity. See the previous document. BsIgG can be produced by expressing the component antibodies separately in different host cells and then purifying / assembling them into BsIgG. BsIgG can also be produced by expressing the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, for example, using A protein and continuous pH elution.

[0443] IgG with additional antigen-binding moiety is another form of bispecific antibody molecule.For example, it is possible to engineer monospecific IgG to have bispecificity by attaching additional antigen-binding unit on monospecific IgG (for example, at the N-terminus or C-terminus of heavy chain or light chain).Exemplary additional antigen-binding unit includes single domain antibody (for example, variable heavy chain or variable light chain), engineered protein scaffold and paired antibody variable region (for example, single chain variable fragment or variable fragment).Referring to the previous document.The example of additional IgG form includes dual variable domain IgG (DVD-Ig), IgG (H)-scFv, scFv-(H) IgG, IgG (L)-scFv, scFv-(L) IgG, IgG (L, H)-Fv, IgG (H)-V, V (H)-IgG, IgG (L)-V, V (L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody and D VI-IgG (four in one). See Spiess et al., Mol. Immunol. 67 (2015): 95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds to IGF-1R and HER3. Examples of DVD-Igs include ABT-981 (AbbVie), which binds to IL-1α and IL-1β; and ABT-122 (AbbVie), which binds to TNF and IL-17A.

[0444] Bispecific antibody fragment (BsAb) is the form of the bispecific antibody molecule lacking some or all of antibody constant domains.For example, some BsAb lack Fc district.In embodiments, bispecific antibody fragment comprises heavy chain and light chain district connected by pep...

Claims

1. A multispecific molecule comprising: (i) an anti-CSF1R binding portion; and (ii) anti-CCR2 binding moiety; in: (1) wherein the anti-CSF1R binding portion is an anti-CSF1R antibody or an antigen-binding fragment thereof, and the anti-CSF1R antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region (VH), comprising a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2), and a heavy chain complementary determining region 3 (HCDR3), and wherein the HCDR1, HCDR2, and HCDR3 are SEQ ID NOs: 402, 405, and 413, respectively; and a light chain variable region (VL), comprising a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2), and a light chain complementary determining region 3 (LCDR3), wherein LCDR1, LCDR2, and LCDR3 are SEQ ID NOs: 432, 434, and 436, respectively; and (2) wherein the anti-CCR2 binding portion is an anti-CCR2 antibody molecule or an antigen-binding fragment thereof comprising VH and VL, wherein the VH comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 446, 447 and 448, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 454, 455 and 456, respectively. 2 . The multispecific molecule of claim 1 , further comprising a TGF-β inhibitor, and wherein the TGF-β inhibitor is a TGFBR2 polypeptide. 3 . The multispecific molecule of claim 1 , wherein the anti-CSF1R binding portion comprises a VH comprising an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO:

324.

4. The multispecific molecule of claim 1, wherein the anti-CSF1R binding moiety comprises: A VL comprising a sequence that has at least 80% identity to SEQ ID NO:

341.

5. The multispecific molecule of claim 1, wherein the anti-CSF1R binding portion comprises VH and VL, wherein VH and VL each comprise the following amino acid sequences: SEQ ID NO:324 and 341.

6. The multispecific molecule of claim 1, wherein the anti-CCR2 binding moiety comprises: (i) VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 343 or 344, or an amino acid sequence encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 133; (ii) a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 345; or an amino acid sequence encoded by a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO: 272; or (iii) combinations thereof.

7. The multispecific molecule of claim 1, wherein the anti-CCR2 binding portion comprises VH and VL, wherein VH and VL each comprise the following amino acid sequences: SEQ ID NO:343 and 345, or SEQ ID NO:344 and 345.

8. The multispecific molecule of claim 1, wherein the anti-CCR2 binding fragment does not comprise a VH having an amino acid sequence of SEQ ID NO: 480, a VL having an amino acid sequence of SEQ ID NO: 481, or a combination thereof.

9. The multispecific molecule of claim 2, comprising an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 373, 131 and 127 or an amino acid sequence encoded by a nucleotide sequence that is at least 80% identical to any one of SEQ ID NOs: 134, 132 and 128.

10. The multispecific molecule of claim 2, comprising The amino acid sequence of SEQ ID NO:127, the amino acid sequence of SEQ ID NO:131, and the amino acid sequence of SEQ ID NO:

373.

11. The multispecific molecule of claim 2, wherein: The TGFBR2 polypeptide comprises: (i) the extracellular domain of TGFBR2, (ii) any one of SEQ ID NOs: 98, 99, 123 and 124, or (iii) any one sequence selected from SEQ ID NO: 100, 101, 102 and 103.

12. The multispecific molecule of claim 2, wherein the TGF-β inhibitor comprises a first TGFBR2 polypeptide and a second TGFBR2 polypeptide.

13. The multispecific molecule of claim 2, wherein the TGF-β inhibitor comprises a first TGFBR2 polypeptide and a second TGFBR2 polypeptide; wherein the multispecific molecule comprises a first Fc region and a second Fc region, wherein: (i) the first TGFBR2 polypeptide is linked to the first Fc region, and (ii) the second TGFBR2 polypeptide is linked to the second Fc region.

14. The multispecific molecule of claim 13, wherein (i) the first Fc region comprises a first CH1-Fc region; (ii) the second Fc region comprises a second CH1-Fc region; or (iii) a combination thereof.

15. The multispecific molecule of claim 13, wherein (i) the first TGFBR2 polypeptide is linked to the C-terminus or N-terminus of the first Fc region; (ii) the second TGFBR2 polypeptide is linked to the C-terminus or N-terminus of the second Fc region; or (iii) a combination thereof.

16. The multispecific molecule of claim 13, wherein (i) the first TGFBR2 polypeptide is linked to the first Fc region via a linker; (ii) the second TGFBR2 polypeptide is linked to the second Fc region via a linker; or (iii) a combination thereof.

17. The multispecific molecule of claim 13, wherein the multispecific molecule comprises: (i) the amino acid sequence of SEQ ID NO: 192 and the amino acid sequence of SEQ ID NO: 193, (ii) the amino acid sequence of SEQ ID NO: 192 and the amino acid sequence of SEQ ID NO: 195, (iii) the amino acid sequence of SEQ ID NO: 194 and the amino acid sequence of SEQ ID NO: 193, or (iv) the amino acid sequence of SEQ ID NO:194 and the amino acid sequence of SEQ ID NO:

195.

18. The multispecific molecule of claim 2, wherein the TGF-β inhibitor comprises a first TGFBR2 polypeptide and a second TGFBR2 polypeptide, wherein the multispecific molecule comprises a heavy chain constant region 1 (CH1) and a light chain constant region (CL), wherein: (i) the first TGFBR2 polypeptide is linked to the CH1, and (ii) the second TGFBR2 polypeptide is linked to the CL.

19. The multispecific molecule of claim 18, wherein (i) the first TGFBR2 polypeptide is linked to the N-terminus of the CH1; (ii) the second TGFBR2 polypeptide is linked to the N-terminus of the CL; or (iii) a combination thereof.

20. The multispecific molecule of claim 18, wherein (i) the first TGF-β receptor polypeptide is linked to the CH1 via a linker; (ii) the second TGF-β receptor polypeptide is linked to the CL via a linker; or (iii) a combination thereof.

21. The multispecific molecule of claim 18, wherein the multispecific molecule comprises: (i) the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 198, (ii) the amino acid sequence of SEQ ID NO: 196 and the amino acid sequence of SEQ ID NO: 199, (iii) the amino acid sequence of SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 198, or (iv) the amino acid sequence of SEQ ID NO:197 and the amino acid sequence of SEQ ID NO:

199.

22. The multispecific molecule of claim 12, wherein: (i) the TGF inhibitor is linked to the anti-CSF1R binding moiety or the anti-CCR2 binding moiety; (ii) the anti-CSF1R binding portion, the anti-CCR2 binding portion, or a combination thereof comprises an Fc region, wherein the TGF inhibitor is attached to the Fc region; (iii) the multispecific molecule comprises a first TGF-β inhibitor and a second TGF-β inhibitor, wherein the first TGF-β inhibitor is linked to the anti-CSF1R binding moiety and the second TGF-β inhibitor is linked to the anti-CCR2 binding moiety; (iv) the multispecific molecule comprises a first TGF-β inhibitor and a second TGF-β inhibitor, the anti-CSF1R binding moiety comprises a first Fc region, the anti-CCR2 binding moiety comprises a second Fc region, and wherein the first TGF-β inhibitor is linked to the first Fc region and the second TGF-β inhibitor is linked to the second Fc region; or (v) the multispecific molecule comprises a first TGF-β inhibitor and a second TGF-β inhibitor, the anti-CSF1R binding portion comprises a first light chain and a first heavy chain, wherein the first heavy chain comprises a first Fc region, wherein the first TGF-β inhibitor is linked to the first Fc region; and the anti-CCR2 binding portion comprises a second light chain and a second heavy chain, wherein the second heavy chain comprises a second Fc region, and wherein the second TGF-β inhibitor is linked to the second Fc region.

23. The multispecific molecule of claim 22, wherein: (i) wherein the TGF inhibitor is linked to the C-terminus or N-terminus of the Fc region of the anti-CSF1R binding moiety or the C-terminus or N-terminus of the Fc region of the anti-CCR2 binding moiety; (ii) the first TGF-β inhibitor is linked to the C-terminus or N-terminus of the first Fc region of the anti-CSF1R binding moiety, and the second TGF-β inhibitor is linked to the C-terminus or N-terminus of the second Fc region of the anti-CCR2 binding moiety, or a combination thereof; or (iii) the first TGF-β inhibitor is linked to the C-terminus or N-terminus of the first Fc region of the first heavy chain; and the second TGF-β inhibitor is linked to the C-terminus or N-terminus of the second Fc region of the second heavy chain, or a combination thereof.

24. The multispecific molecule of claim 22, wherein: (i) the TGF inhibitor is linked to the anti-CSF1R binding portion or the anti-CCR2 binding portion via a linker; (ii) wherein the TGF inhibitor is linked to the Fc region of the anti-CSF1R binding portion or the Fc region of the anti-CCR2 binding portion via a linker; (iii) the first TGF-β inhibitor is linked to the anti-CSF1R binding portion via a linker, and the second TGF-β inhibitor is linked to the anti-CCR2 binding portion via a linker, or a combination thereof; (iv) the first TGF-β inhibitor is linked to the first Fc region of the anti-CSF1R binding moiety via a linker, and the second TGF-β inhibitor is linked to the second Fc region of the anti-CCR2 binding moiety via a linker, or a combination thereof; or (v) the first TGF-β inhibitor is linked to the first Fc region of the first heavy chain via a linker; and the second TGF-β inhibitor is linked to the second Fc region of the second heavy chain via a linker, or a combination thereof.

25. A recombinant polynucleotide comprising a polynucleotide encoding the multispecific molecule of any one of claims 1 to 24.

26. A host cell comprising the recombinant polynucleotide of claim 25.

27. A method of preparing the multispecific molecule of any one of claims 1 to 24, comprising culturing the host cell of claim 26 under conditions suitable for gene expression and / or heterodimerization.

28. A medicament comprising the multispecific molecule of any one of claims 1 to 24 and a pharmaceutically acceptable carrier, excipient or stabilizer.

29. Use of the multispecific molecule of any one of claims 2 and 9-24 in the preparation of a medicament for treating cancer.

30. The use according to claim 29, wherein the cancer is a solid tumor cancer or a metastatic lesion.

31. The use according to claim 30, wherein the solid tumor cancer is one or more of the following: pancreatic cancer, breast cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, liver cancer, brain cancer, bladder cancer, cervical cancer, head and neck cancer, kidney cancer, mesothelioma, thyroid cancer or uterine cancer.

32. The use according to claim 29, wherein the cancer is a hematological cancer.

33. The use according to claim 32, wherein the hematological cancer is a myeloproliferative neoplasm.

34. The use according to claim 33, wherein the myeloproliferative neoplasm is primary or idiopathic myelofibrosis, essential thrombocythemia, polycythemia vera or chronic myeloid leukemia.

35. The use according to any one of claims 29 to 34, wherein the medicament is formulated for administration to a subject in combination with a second therapy.

36. The use of claim 35, wherein the second therapy comprises a therapeutic agent, radiation or surgery.

37. The use according to claim 36, wherein the therapeutic agent is selected from chemotherapeutic agents, biological agents, hormone therapy.

38. The use of claim 36, wherein the therapeutic agent is a checkpoint inhibitor.

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