WO2025232879A1

WO2025232879A1 - Anti-lilrb2 monospecific and bispecific antibody constructs and uses thereof

Info

Publication number: WO2025232879A1
Application number: PCT/CN2025/093785
Authority: WO
Inventors: Li Chen; Weiwei XIAN; Lili Yu
Original assignee: Cytocares (shanghai) Inc
Current assignee: Cytocares (shanghai) Inc
Priority date: 2024-05-10
Filing date: 2025-05-09
Publication date: 2025-11-13
Anticipated expiration: 2026-11-10

Abstract

The technology described herein is directed to monospecific anti-LILRB2 antibody constructs, as well as bispecific anti-LILRB2 antibody constructs that also inhibit targets such as PD-1 or VEGF. Such antibody constructs are capable of polarizing macrophages from an M2 phenotype to an M1 phenotype, activating T cells, and/or inhibiting growth of a cancer. Also described herein are nucleic acids and vector encoding such monospecific or bispecific anti-LILRB2 antibody constructs, cells expressing or in combination with such antibody constructs, and compositions comprising such antibody constructs, nucleic acids, vectors, or cells. The disclosure also relates to methods of modulating the immune or treating cancer using such monospecific or bispecific anti-LILRB2 antibody constructs.

Description

ANTI-LILRB2 MONOSPECIFIC AND BISPECIFIC ANTIBODY CONSTRUCTS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119 (b) of International Application No. PCT/CN2024/092263 filed May 10, 2024, the contents of which are incorporated herein by reference in their entirety.
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on April 29, 2025, is named [SPIE240380] . SEQ. xml and is 892, 203 bytes in size.

TECHNICAL FIELD

This technology relates generally to medicine, immunology, and cancer immunotherapy. More particularly, the present technology relates to anti-LILRB2 antibodies, bifunctional proteins and uses thereof.

BACKGROUND

Macrophages are abundant in the tumor microenvironment (TME) , and tumor-associated macrophages (TAMs) are typically the most abundant immune population within the TME. Given that TAMs are a common constituent of TME and because of their diverse arsenal of suppressive mechanisms, TAMs are a good therapeutic target, especially in combination with T-cell–targeted immunotherapy, such as checkpoint blockade.

Leukocyte immunoglobulin like receptor B2 (LILRB2) is a receptor expressed on immune cells, where it binds to MHC class I molecules on antigen-presenting cells and transduces a negative signal that inhibits stimulation of an immune response. Programmed cell death protein 1 (PD-1) is a cell surface receptor on immune cells that functions as a checkpoint protein and plays a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system. Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis and induces permeabilization of blood vessels. Inhibition of LILRB2, PD-1, and/or VEGF can modulate the immune system and/or cardiovascular system and can affect cancer outcomes. There is a great need for anti-cancer therapies that inhibit LILRB2, PD-1, and/or VEGF.

SUMMARY

The present technology provides antibodies or antigen-binding fragments thereof that bind to leukocyte immunoglobulin (Ig) -like receptor (LILR) B2 (LILRB2) ( "anti-LILRB2 antibodies" ) and interfere with its binding to its ligand, human leukocyte antigen G (HLA-G) . This technology also provides anti-PD-1 x anti-LILRB2 and anti-VEGF x anti-LILRB2 bifunctional proteins or bispecific antibodies. The anti-LILRB2 antibodies and bifunctional proteins described herein can modulate the transition of macrophages from M2 to M1 phenotype, and therefore, these antibodies can be used to treat cancer or other disorders where an increase in pro-inflammatory immune response is desired.

In some embodiments, the anti-LILRB2 antibodies are selected from a human Fab library.

In some embodiments, the anti-LILRB2 antibodies were generated by immunization of mice with human LILRB2 antigen, followed by Fab library construction and phage library panning, e.g., with immunotubes and magnetic bead methods.

In some embodiments, the anti-LILRB2 antibodies were generated by immunization of a camelid, e.g., an alpaca, with human LILRB2 antigen, followed by Fab library construction and phage library panning, e.g., with immunotubes and magnetic bead methods.

In one aspect, the anti-LILRB2 antibodies have a blocking effect on the binding of human LILRB2 receptor to its ligand, HLA-G. HLA-G is an immune checkpoint molecule. HLA-G is expressed on antigen-presenting cells (APCs) , such as myeloid-derived suppressor cells (MDSCs) or tolerogenic dendritic cells (DCs) , promotes T cell hyporesponsiveness and induces Treg differentiation. The anti-LILRB2 constructs described herein can be used to interfere with the binding of LILRB2 receptor and its ligand, HLA-G, and can prevent or decrease T-cell anergy or hyporesponsiveness.

In one aspect, provided herein are humanized anti-LILRB2 antibodies, including huB45, huB206, huC112, and huC68, among others.

In one aspect, provided herein are anti-LILRB2 antibodies that can modulate the polarization of macrophages from M2 to M1 phenotype and exhibit anti-tumor effect in a xenograft animal model.

In another aspect, provided herein are bispecific or bifunctional antibody constructs comprising an LILRB2-specific binding domain. In one embodiment, the bispecific or bifunctional antibody construct comprises an LILRB2-specific binding domain and a binding domain specific for PD-1 or VEGF. In some embodiments, bifunctional anti-PD-1 x anti-LILRB2 proteins were generated with five different antibody formats: HC-C (scFv) , HC-C (VHH) , LC-C (scFv) , LC-C (VHH) , and HC-N (VHH) (see e.g., Fig. 9A-9E) .

In some embodiments, bifunctional anti-VEGF x anti-LILRB2 proteins were generated with five different antibody formats: HC-C (scFv) , HC-C (VHH) , LC-C (scFv) , LC-C (VHH) , and HC-N (VHH) (see e.g., Fig. 10A-10E) .

In one aspect, bifunctional antibodies provided herein can modulate the polarization of macrophages from M2 to M1 phenotype. In one aspect, bifunctional antibodies provided herein exhibit an anti-tumor effect. In one embodiment, anti-tumor effect is as measured, for example, in a mixed lymphocyte reaction or a xenograft animal model.

In one aspect, bifunctional antibodies provided herein can induce the secretion of higher levels of inflammatory cytokine TNF-α and/or induce enhanced tumor cytotoxicity compared to monospecific antibodies alone. Administration of a bispecific antibodies can be clinically more efficient and less expensive than administering two monospecific antibodies separately or concurrently.

In one aspect, described herein is a composition comprising a binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) , wherein the composition is capable of: (a) polarizing macrophages from an M2 phenotype to an M1 phenotype; (b) activating T cells; and/or (c) inhibiting growth of a cancer.

In some embodiments of any of the aspects, the binding domain comprises an antigen binding domain of an antibody.

In some embodiments of any of the aspects, the binding domain is comprised by a human, humanized, affinity-matured, and/or chimeric antibody construct.

In some embodiments of any of the aspects, the binding domain is comprised by an anti-LILRB2 antibody construct derived from a primate, a rodent, or a camelid.

In some embodiments of any of the aspects, the binding domain is comprised by an anti-LILRB2 antibody construct derived from a human, a mouse, or an alpaca.

In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_H domain that specifically binds LILRB2.

In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_L domain that specifically binds LILRB2.

In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_H/V_L domain pair that specifically binds LILRB2.

In some embodiments of any of the aspects, the binding domain comprises the CDRs of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2.

In some embodiments of any of the aspects, LILRB2 comprises mammalian LILRB2.

In some embodiments of any of the aspects, LILRB2 comprises human LILRB2.

In some embodiments of any of the aspects, the binding domain specifically binds to a LILRB2 epitope or portion of a LILRB2 epitope comprising sequential and/or non-sequential residues of SEQ ID NO: 775.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H CDR1, a V_H CDR2, and a V_H CDR3 selected from Table 8; and (b) a V_L CDR1, a V_L CDR2, and a V_L CDR3 selected from Table 10.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H CDR1 comprising one of SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, or 118; (b) a V_H CDR2 comprising one of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, or 119; (c) a V_H CDR3 comprising one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, or 120; (d) a V_L CDR1 comprising one of SEQ ID NOs: 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, or 829; (e) a V_L CDR2 comprising one of SEQ ID NOs: 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 314, 317, or 320; and (f) a V_L CDR3 comprising one of SEQ ID NOs: 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, or 830.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H CDR1, a V_H CDR2, and a V_H CDR3 encoded by nucleic acid sequences selected from Table 9; and (b) a V_L CDR1, a V_L CDR2, and a V_L CDR3 encoded by nucleic acid sequences selected from Table 11.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, or 238; (b) a V_H CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, or 239; (c) a V_H CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, or 240; (d) a V_L CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361, 364, 367, 370, 373, 376, 379, 382, 385, 388, 391, 394, 397, 400, 403, or 406; (e) a V_L CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 368, 371, 374, 377, 380, 383, 386, 389, 392, 395, 398, 401, 404, or 407; and (f) a V_L CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, or 408.

In some embodiments of any of the aspects, the anti-LILRB2 antibody construct comprises:
(a) a V_H domain selected from Table 12 or a V_H domain that is at least 80%identical to an amino acid
sequence selected from Table 12; and (b) a V_L domain selected from Table 14 or a V_L domain that is at least 80%identical to an amino acid sequence selected from Table 14.

In some embodiments of any of the aspects, the anti-LILRB2 antibody construct comprises:
(a) a V_H domain comprising one of SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421,
422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, or 448, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, or 448; and (b) a V_L domain comprising one of SEQ ID NOs: 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, or 517.

In some embodiments of any of the aspects, the anti-LILRB2 antibody construct comprises:
(a) a V_H domain encoded by a nucleic acid sequence selected from Table 13 or a V_H domain that is at least
80%identical to a nucleic acid sequence selected from Table 13; and (b) a V_L domain encoded by a nucleic acid sequence selected from Table 15 or a V_L domain that is at least 80%identical to a nucleic acid sequence selected from Table 15.

In some embodiments of any of the aspects, the anti-LILRB2 antibody construct comprises:
(a) a V_H domain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 451, 452, 453, 454,
455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 571, 572, 573, 574, 579, 580, 581, 582, 583, 584, 590, 591, 592, 593, 594, 595, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 571, 572, 573, 574, 579, 580, 581, 582, 583, 584, 590, 591, 592, 593, 594, 595, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615; and (b) a V_L domain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 575, 576, 577, 578, 585, 586, 587, 588, 589, 596, 597, 598, 599, 600, 601, or 785, or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 575, 576, 577, 578, 585, 586, 587, 588, 589, 596, 597, 598, 599, 600, 601, or 785.

In some embodiments of any of the aspects, the anti-LILRB2 antibody construct comprises an antibody selected from the group consisting of: HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206.

In some embodiments of any of the aspects, the VHH that specifically binds LILRB2 comprises: (a) a V_H CDR1 comprising one of SEQ ID NOs: 82, 85, 88, 91, 94, 97, 100, 103, 106, 115, or 118; (b) a V_H CDR2 comprising one of SEQ ID NOs: 83, 86, 89, 92, 95, 98, 101, 104, 107, 116, or 119; and (c) a V_H CDR3 comprising one of SEQ ID NOs: 84, 87, 90, 93, 96, 99, 102, 105, 108, 117, or 120.

In some embodiments of any of the aspects, the VHH that specifically binds LILRB2 comprises: (a) a V_H CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 202, 205, 208, 211, 214, 217, 220, 223, 226, 235, or 238; (b) a V_H CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 203, 206, 209, 212, 215, 218, 221, 224, 227, 236, or 239; and (c) a V_H CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 204, 207, 210, 213, 216, 219, 222, 225, 228, 237, or 240.

In some embodiments of any of the aspects, the anti-LILRB2 antibody construct comprises: a VHH comprising one of SEQ ID NOs: 436, 437, 438, 439, 440, 441, 442, 443, 444, 447, or 448, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 436, 437, 438, 439, 440, 441, 442, 443, 444, 447, or 448.

In some embodiments of any of the aspects, the anti-LILRB2 antibody construct comprises: a VHH encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 476, 477, 478, 479, 480, 481, 482, 483, 484, 487, or 488, or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 476, 477, 478, 479, 480, 481, 482, 483, 484, 487, or 488.

In some embodiments of any of the aspects, the anti-LILRB2 antibody construct comprises an antibody selected from the group consisting of: AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huB45, and huB206.

In one aspect described herein is a composition comprising: (a) a first binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) ; and (b) a second binding domain that specifically binds: (i) programmed cell death protein 1 (PD-1) ; or (ii) vascular endothelial growth factor (VEGF) .

In some embodiments of any of the aspects, the composition is capable of: (a) polarizing macrophages from an M2 phenotype to an M1 phenotype; (b) activating T cells; and/or (c) inhibiting growth of a cancer.

In some embodiments of any of the aspects, the first and/or second binding domains comprise an antigen binding domain of an antibody.

In some embodiments of any of the aspects, the first and second binding domains each comprise an antigen binding domain of an antibody.

In some embodiments of any of the aspects, the first and second binding domains are comprised by a human, humanized, affinity-matured, and/or chimeric antibody construct.

In some embodiments of any of the aspects, the first and second binding domains are comprised by a bispecific antibody construct.

In some embodiments of any of the aspects, the first binding domain comprises the CDRs of a V_H domain that specifically binds LILRB2.

In some embodiments of any of the aspects, the first binding domain comprises the CDRs of a V_L domain that specifically binds LILRB2.

In some embodiments of any of the aspects, the bispecific antibody construct comprises: (a) the first binding domain comprising: (i) the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or (ii) the CDRs of a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2; and (b) the second binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds PD-1.

In some embodiments of any of the aspects, the bispecific antibody construct comprises: (a) the first binding domain comprising: (i) the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or (ii) the CDRs of a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2; and (b) the second binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds VEGF.

In some embodiments of any of the aspects, the bispecific antibody construct is selected from the group consisting of a tandem scFv (taFv or scFv₂) , diabody, dAb₂A/HH₂, knob-into-holes bispecific derivative, SEED-IgG, heteroFc-scFv, Fab-scFv, scFv-Jun/Fos, Fab'-Jun/Fos, tribody, DNL-F (ab) ₃, scFv₃-CH1/CL, Fab-scFv₂, IgG-scFab, IgG-scFv, scFv-IgG, IgG-VHH, VHH-IgG, scFv₂-Fc, F (ab') ₂-scFv₂, scDB-Fc, scDb-CH₃, Db-Fc, scFv₂-H/L, DVD-Ig, tandAb, scFv-dhlx-scFv, dAb2-IgG, dAb-IgG, and dAb-Fc-dAb constructs.

In some embodiments of any of the aspects, the bispecific antibody construct comprises: (a) the first binding domain comprising: (i) a single-chain fragment variable (scFv) comprising the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or (ii) a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) comprising the CDRs of a V_H domain that specifically binds LILRB2; and (b) the second binding domain comprising an immunoglobulin heavy chain and immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds PD-1.

In some embodiments of any of the aspects, the bispecific antibody construct comprises: (a) the first binding domain comprising: (i) a single-chain fragment variable (scFv) comprising the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or (ii) a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) comprising the CDRs of a V_H domain that specifically binds LILRB2; and (b) the second binding domain comprising an immunoglobulin heavy chain and immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds VEGF.

In some embodiments of any of the aspects, the scFv or the VHH is linked to the heavy chain.

In some embodiments of any of the aspects, the scFv or the VHH is linked to the C-terminus of the heavy chain.

In some embodiments of any of the aspects, the scFv or the VHH is linked to the N-terminus of the heavy chain.

In some embodiments of any of the aspects, the scFv or the VHH is linked to the light chain.

In some embodiments of any of the aspects, the scFv or the VHH is linked to the C-terminus of the light chain.

In some embodiments of any of the aspects, the scFv or the VHH is linked to the N-terminus of the light chain.

In some embodiments of any of the aspects, the LILRB2 comprises mammalian LILRB2; the PD-1 comprises mammalian PD-1; and/or the VEGF comprises mammalian VEGF.

In some embodiments of any of the aspects, the LILRB2 comprises human LILRB2; the PD-1 comprises human PD-1; and/or the VEGF comprises human VEGF.

In some embodiments of any of the aspects, the second binding domain specifically binds PD-1 and comprises nivolumab, pembrolizumab, toripalimab, sintilimab, cemiplimab, or dostarlimab, or functional fragments thereof.

In some embodiments of any of the aspects, the second binding domain specifically binds to at least one nivolumab PD-1 epitope or portion of a nivolumab PD-1 epitope comprising: residues 25 to 31 of SEQ ID NO: 776 (LDSPDRP, SEQ ID NO: 787) , residues 25 to 31 of SEQ ID NO: 776 (TSES, SEQ ID NO: 779) , and/or residues 127 to 132 of SEQ ID NO: 776 (SLAPKA, SEQ ID NO: 780) .

In some embodiments of any of the aspects, the second binding domain specifically binds to at least one pembrolizumab PD-1 epitope or portion of a pembrolizumab PD-1 epitope comprising: residues S60, S62, V64, N66, I126, L128, A129, K131, A132, and/or I134 of SEQ ID NO: 776, residues 75 to 78 of SEQ ID NO: 776 (QTDK, SEQ ID NO: 781) , and/or residues 81 to 90 of SEQ ID NO: 776 (AFPEDRSQPG, SEQ ID NO: 782) .

In some embodiments of any of the aspects, the second binding domain specifically binds VEGF and comprises bevacizumab, ranibizumab, or Aflibercept, or functional fragments thereof.

In some embodiments of any of the aspects, the second binding domain specifically binds to a bevacizumab VEGF epitope or portion of a bevacizumab VEGF epitope comprising residues 85 to 92 of SEQ ID NO: 777, residues 111 to 112 of SEQ ID NO: 778, residues 111 to 112 of SEQ ID NO: 786, or PHQGQHIG (SEQ ID NO: 783) .

In some embodiments of any of the aspects, the first binding domain that specifically binds LILRB2 comprises an antibody or functional fragment thereof selected from the group consisting of: HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206.

In some embodiments of any of the aspects, the first binding domain that specifically binds LILRB2 comprises an antibody or functional fragment thereof selected from the group consisting of: HB59, MIL-C68, huC68, MIL-C112, huC112, AIL-B45, huB45, AIL-B206, and huB206.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H CDR1 comprising one of SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, or 118; (b) a V_H CDR2 comprising one of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, or 119; (c) a V_H CDR3 comprising one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120; (d) a V_L CDR1 comprising one of SEQ ID NOs: 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, or 829; (e) a V_L CDR2 comprising one of SEQ ID NOs: 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 314, 317, or 320; and (f) a V_L CDR3 comprising one of SEQ ID NOs: 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, or 830.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H CDR1 comprising one of SEQ ID NOs: 19, 109, 112, 115, or 118; (b) a V_H CDR2 comprising one of SEQ ID NOs: 20, 110, 113, 116, or 119; (c) a V_H CDR3 comprising one of SEQ ID NOs: 21, 111, 114, 117, or 120; (d) a V_L CDR1 comprising one of SEQ ID NOs: 259, 316, or 319; (e) a V_L CDR2 comprising one of SEQ ID NOs: 260, 317, or 320; and (f) a V_L CDR3 comprising one of SEQ ID NOs: 261, 318, or 321.

In some embodiments of any of the aspects, the VHH that specifically binds LILRB2 comprises: (a) a V_H CDR1 comprising one of SEQ ID NOs: 82, 85, 88, 91, 94, 97, 100, 103, 106, 115, or 118; (b) a V_H CDR2 comprising one of SEQ ID NOs: 83, 86, 89, 92, 95, 98, 101, 104, 107, 116, or 119; and (c) a V_H CDR3 comprising one of SEQ ID NOs: 84, 87, 90, 93, 96, 99, 102, 105, 108, 117, 120.

In some embodiments of any of the aspects, the VHH that specifically binds LILRB2 comprises: (a) a V_H CDR1 comprising one of SEQ ID NOs: 115 or 118; (b) a V_H CDR2 comprising one of SEQ ID NOs: 116 or 119; and (c) a V_H CDR3 comprising one of SEQ ID NOs: 117 or 120.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H CDR1 comprising SEQ ID NO: 19; (b) a V_H CDR2 comprising SEQ ID NO: 20; (c) a V_H CDR3 comprising SEQ ID NO: 21; (d) a V_L CDR1 comprising SEQ ID NO: 259; (e) a V_L CDR2 comprising SEQ ID NO: 260; and (f) a V_L CDR3 comprising SEQ ID NO: 261.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises: (a) a V_H CDR1 comprising one of SEQ ID NOs: 547 or 550; (b) a V_H CDR2 comprising one of SEQ ID NOs: 548 or 551; (c) a V_H CDR3 comprising one of SEQ ID NOs: 549 or 552; (d) a V_L CDR1 comprising one of SEQ ID NOs: 556 or 559; (e) a V_L CDR2 comprising one of SEQ ID NOs: 557 or 560; and (f) a V_L CDR3 comprising one of SEQ ID NOs: 558 or 561.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises: (a) a V_H CDR1 comprising SEQ ID NO: 553; (b) a V_H CDR2 comprising SEQ ID NO: 554; (c) a V_H CDR3 comprising SEQ ID NO: 555; (d) a V_L CDR1 comprising SEQ ID NO: 562; (e) a V_L CDR2 comprising SEQ ID NO: 563; and (f) a V_L CDR3 comprising SEQ ID NO: 564.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H comprising one of SEQ ID NOs: 415, 432, 435, 445, 446, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 415, 432, 435, 445, 446; and (b) a V_L comprising one of SEQ ID NOs: 495, 512, 516, 515, 517 or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 495, 512, 516, 515, 517.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H comprising SEQ ID NO: 415 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 415; and (b) a V_L comprising SEQ ID NO: 495 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 495.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises: (a) a V_H comprising one of SEQ ID NOs: 565 or 567 or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 565 or 567; and (b) a V_L comprising one of SEQ ID NOs: 566 or 568 or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 566 or 568.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises: (a) a V_H comprising SEQ ID NO: 569 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 569; and (b) a V_L comprising SEQ ID NO: 570 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 570.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds LILRB2 comprises: (a) a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 455, 472, 475, 485, 486, 571-574, 579-584, 590-595 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 455, 472, 475, 485, 486, 579-584, 571-574, 590-595; and (b) a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 524, 541, 544, 545, 546, 575-578, 585-589, 596-601, 785, or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 524, 541, 544, 545, 546, 575-578, 585-589, 596-601, or 785.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises: (a) a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 616-618, 624-632 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 616-618, 624-632; and (b) a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 619-623, 633-638 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 619-623, 633-638.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises: (a) a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 639-647 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 639-647; and (b) a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 648-654 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 648-654.

In some embodiments of any of the aspects, the bispecific antibody construct comprises an anti-LILRB2 and anti-PD-1 bispecific antibody construct.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody construct comprises: (a) an immunoglobulin heavy chain comprising one of SEQ ID NOs: 655, 657, 659, 661, 671, 673, 675, 677, 683, 689, 691, 693, 695, 697, 703, 705, 707, 709, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 655, 657, 659, 661, 671, 673, 675, 677, 683, 689, 691, 693, 695, 697, 703, 705, 707, 709; and (b) an immunoglobulin light chain comprising one of SEQ ID NOs: 656, 658, 660, 662, 672, 674, 676, 678, 684, 690, 692, 694, 696, 698, 704, 706, 708, 710, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 656, 658, 660, 662, 672, 674, 676, 678, 684, 690, 692, 694, 696, 698, 704, 706, 708, 710.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody construct is selected from Bis-M23-1 and Bis-M28.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody is Bis-M23-1 comprising: (a) an immunoglobulin heavy chain comprising SEQ ID NO: 683 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 683; and (b) an immunoglobulin light chain comprising SEQ ID NO: 684, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 684.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody is Bis-M28 comprising: (a) an immunoglobulin heavy chain comprising SEQ ID NO: 689, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 689; and (b) an immunoglobulin light chain comprising SEQ ID NO: 690, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 690.

In some embodiments of any of the aspects, the bispecific antibody construct comprises an anti-LILRB2 and anti-VEGF bispecific antibody construct.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody construct comprises: (a) an immunoglobulin heavy chain comprising one of SEQ ID NOs: 663, 665, 667, 669, 679, 681, 685, 687, 699, 701, 711, 713, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 663, 665, 667, 669, 679, 681, 685, 687, 699, 701, 711, 713; and (b) an immunoglobulin light chain comprising one of SEQ ID NOs: 664, 666, 668, 670, 680, 682, 686, 688, 700, 702, 712, 714, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 664, 666, 668, 670, 680, 682, 686, 688, 700, 702, 712, 714.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody construct is selected from Bis-M24 and Bis-M27.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody is Bis-M24 comprising: (a) an immunoglobulin heavy chain comprising SEQ ID NO: 685 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 685; and (b) an immunoglobulin light chain comprising SEQ ID NO: 686 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 686.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody is Bis-M27 comprising: (a) an immunoglobulin heavy chain comprising SEQ ID NO: 687, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 687; and (b) an immunoglobulin light chain comprising SEQ ID NO: 688, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 688.

In one aspect, described herein is a nucleic acid encoding a polypeptide of a composition as described herein.

In one aspect, described herein is a vector comprising a nucleic acid as described herein.

In one aspect, described herein is a cell comprising a nucleic acid as described herein or a vector as described herein.

In some embodiments of any of the aspects, the cell is an antibody-producing cell.

In one aspect, described herein is a cell in combination with a composition as described herein.

In some embodiments of any of the aspects, the cell is an immune cell.

In some embodiments of any of the aspects, the cell is an M1 macrophage or an M2 macrophage.

In some embodiments of any of the aspects, the cell is a T lymphocyte.

In one aspect, described herein is a pharmaceutical composition comprising a composition as described herein, a nucleic acid as described herein, a vector as described herein, or a cell as described herein, and a pharmaceutically acceptable carrier.

In one aspect, described herein is a method of affinity maturing a composition as described herein, the method comprising: a) mutating at least one binding domain; b) determining the affinity of the mutated binding domain for its cognate ligand; and c) selecting the mutated binding domain that exhibits increased affinity for its cognate ligand compared to the unmutated binding domain.

In some embodiments of any of the aspects, the method further comprises producing a composition comprising the selected mutated binding domain.

In some embodiments of any of the aspects, the method further comprises producing an antibody construct comprising the selected mutated binding domain.

In one aspect, described herein is a composition produced by a method as described herein.

In one aspect, described herein is an antibody construct produced by a method as described herein.

In one aspect, described herein is a method for polarizing a macrophage from an M2 phenotype to an M1 phenotype, the method comprising contacting the macrophage with a composition as described herein.

In some embodiments of any of the aspects, after contacting, the macrophage exhibits decreased expression of CD163 and/or CD206.

In some embodiments of any of the aspects, after contacting, the macrophage exhibits increased expression of CD80.

In some embodiments of any of the aspects, after contacting, the macrophage exhibits increased secretion of TNF-α.

In some embodiments of any of the aspects, after contacting, the macrophage induces increased cancer cytotoxicity.

In some embodiments of any of the aspects, the macrophage is contacted for a sufficient amount of time.

In some embodiments of any of the aspects, the sufficient amount of time is at least 2 days.

In some embodiments of any of the aspects, the composition is an anti-LILRB2 antibody construct selected from the group consisting of HB59, AIL-B45, huB45, AIL-B206, huB206, MIL-C68, huC68, MIL-C112, and huC112.

In some embodiments of any of the aspects, the composition is an anti-LILRB2 and anti-PD-1 bispecific antibody construct selected from Bis-M23-1 or Bis-M28.

In some embodiments of any of the aspects, the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27.

In one aspect, described herein is a method for activating a T cell, the method comprising contacting the T cell with a composition as described herein.

In some embodiments of any of the aspects, after contacting, the T cell exhibits increased activation of NFAT signaling.

In some embodiments of any of the aspects, after contacting, the T cell exhibits increased cancer cytotoxicity.

In some embodiments of any of the aspects, the T cell is contacted for a sufficient amount of time.

In some embodiments of any of the aspects, the composition is an anti-LILRB2 antibody construct selected from the group consisting of HB74, HA23, HA31, HB59, MIL-C112, MILC18, MIL-C27, MIL-C3, MIL-C32, MIL-C68, MIL-C9, AIL-A1, AIL-B100, AIL-B124, AIL-B134, and AIL-B45.

In one aspect, described herein is a method of treating cancer, the method comprising administering an effective amount of a composition as described herein or a pharmaceutical composition as described herein to a subject in need thereof.

In some embodiments of any of the aspects, the composition is administered at a dose of 10 mg/kg to 20 mg/kg.

In some embodiments of any of the aspects, the composition is administered at least twice a week for at least three weeks.

In some embodiments of any of the aspects, the composition is administered intravenously.

In some embodiments of any of the aspects, the composition is an anti-LILRB2 antibody construct selected from MIL-C68, MIL-B206, or HB59.

In some embodiments of any of the aspects, the method has a tumor weight inhibition rate (TGI_TW) of at least 30%after 3 weeks of administration.

In some embodiments of any of the aspects, the method has a tumor volume inhibition rate (TGI_TV) of at least 35%after 3 weeks of administration.

In some embodiments of any of the aspects, the composition is the anti-LILRB2 and anti-VEGF bispecific antibody construct Bis-M24.

In some embodiments of any of the aspects, the composition is administered at a dose of about 20 mg/kg twice weekly for at least one week.

In some embodiments of any of the aspects, the composition is administered at a dose of about 4 mg/kg twice weekly for at least the fourth week after beginning treatment.

In some embodiments of any of the aspects, the method has a tumor weight inhibition rate (TGI_TW) of at least 50%after 4 weeks of administration.

In some embodiments of any of the aspects, the cancer is a blood cancer.

In some embodiments of any of the aspects, the cancer comprises at least one solid tumor.

In some embodiments of any of the aspects, the cancer is melanoma.

In some embodiments of any of the aspects, the cancer is breast cancer.

In some embodiments of any of the aspects, the cancer is colorectal adenocarcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A-1B show phage ELISA screening of anti-LILRB2 antibodies from a human Fab library. The phage library was subjected to three rounds of panning using magnetic beads and immunotube methods. Representative figures show the binding of each anti-LILRB2 antibody to rhLILRB2-His after second and third round of panning.

Fig. 2A-2B show phage ELISA screening of anti-LILRB2 antibodies from a mouse immune library. A mouse was immunized with recombinant human LILRB2-ECD antigen to generate anti-LILRB2 antibodies. RNA was extracted from spleen of immunized mice, and a Fab library was constructed. The phage library was subjected to three rounds of panning using magnetic beads and immunotube methods. Representative figures show the binding of each anti-LILRB2 antibody to rhLILRB2-His after first, second, and third round of panning.

Fig. 3A-3B show phage ELISA screening of anti-LILRB2 antibodies from an alpaca immune library. One alpaca was immunized with recombinant human LILRB2-ECD antigen to generate anti-LILRB2 antibodies. RNA was extracted from PBMCs of the immunized animal, and a Fab library was constructed. The phage library was subjected to three rounds of panning using magnetic beads and immunotube methods. Representative figures show the binding of each anti-LILRB2 antibody to rhLILRB2-His after first, second, and third round of panning.

Fig. 4A-4D show that the anti-LILRB2 antibodies effectively activated T cells in a LILRB2 overexpressing Jurkat-NFAT-Luc cell. Dose-dependent increase of the fluorescence intensity by anti-LILRB2 antibodies was observed in human LILRB2 overexpressing Jurkat-NFAT-luc cells in the presence of anti-CD3 antibody and HLA-G-A375 cells. Clones HB74, HA23, HA31, HB59, MIL-C112, MILC18, MIL-C27, MIL-C3, MIL-C32, MIL-C68, MIL-C9, AIL-A1, AIL-B100, AIL-B124, AIL-B134, and AIL-B45 displayed T cell activation effects in this assay system.

Fig. 5A-5D show binding of the humanized anti-LILRB2 antibodies to recombinant human LILRB2 antigen. Mouse antibodies MIL-C68 (C68-P) and MIL-C112 (C112-P) , as well as alpaca antibodies AIL-B45 (AIL-B45-P) and AIL-B206 (AIL-B206-P) , were humanized using the method of complementarity-determining region (CDR) grafting. The humanized antibodies with different humanness scores were designed and recombinantly expressed for ELISA binding analysis. Humanized antibody variants with comparable binding activities to their parental antibodies were obtained.

Fig. 6A-6H show the effect of anti-LILRB2 antibodies on polarization of macrophages. The effect of LILRB2 antibody-mediated polarization of macrophages from M2 to M1 was evaluated in human peripheral blood cell-derived macrophages by macrophage immunotyping and TNF-α level measurements. The anti-LILRB2 antibodies HB59, MIL-C68, huC68, MIL-C112, huC112, AIL-B45, huB45, AIL-B206, and huB206 induced the downregulation of the M2 phenotypes as indicated by the decreased expression of CD163 (Fig. 6B and 6F) and/or CD206 (Fig. 6A and 6E) ; and/or the upregulation of M1 phenotypes as indicated by the upregulation of CD80 expression (Fig. 6C and 6G) , accompanied with the secretion of inflammatory cytokine TNF-α (Fig. 6D and 6H) .

Fig. 7A-7B show the anti-tumor effect of LILRB2 antibody in a huPBMC-NOG mouse inoculated with HLA-G-A375 tumor model. Mouse anti-LILRB2 antibodies MIL-C68 and MIL-C112, and alpaca antibody AIL-B206, showed inhibitory effect to the growth of tumor, with TGI_TW of 26.50%, 24.16%, and 52.25%, respectively.

Fig. 8A-8B show the anti-tumor effect of LILRB2 antibody in a huPBMC-NOG mouse inoculated with HLA-G-A375 tumor model. Human anti-LILRB2 antibody HB59 and humanized antibodies huB206 and huC68 showed inhibitory effect to the growth of tumor, with TGI_TW of 34.96%, 23.54%, and 21.42%, respectively.

Fig. 9A-9E show non-limiting examples of anti-PD-1 x anti-LILRB2 bispecific antibody formats. Fig. 9A shows the HC-C (scFv) format, in which the N-terminus of an anti-LILRB2 (e.g., HB59) scFv (VL-linker-VH) is connected to the C-terminus of the heavy chain of the anti-PD-1 antibody through a polypeptide linker (e.g., Bis-M23-1) . Fig. 9B shows the HC-C (VHH) format, in which the N-terminus of an anti-LILRB2 camelid-derived VHH is connected to the C-terminus of the heavy chain of the anti-PD-1 antibody through a polypeptide linker. Fig. 9C shows the LC-C (scFv) format, in which the N-terminus of an anti-LILRB2 scFv is connected to the C-terminus of the light chain of the anti-PD-1 antibody through a polypeptide linker (e.g., Bis-M28) . Fig. 9D shows the LC-C (VHH) format, in which the N-terminus of an anti-LILRB2 VHH is connected to the C-terminus of the light chain of the anti-PD-1 antibody through a polypeptide linker. Fig. 9E shows the HC-N (VHH) format, in which the C-terminus of an anti-LILRB2 camelid-derived VHH is connected to the N-terminus of the heavy chain of the anti-PD-1 antibody through a polypeptide linker.

Fig. 10A-10E. show non-limiting examples of anti-VEGF x anti-LILRB2 bispecific antibody formats. Fig. 10A shows the HC-C (scFv) format, in which the N-terminus of an anti-LILRB2 (e.g., HB59) scFv (VL-linker-VH) is connected to the C-terminus of the heavy chain of the anti-VEGF antibody through a polypeptide linker (e.g., Bis-M24) . Fig. 10B shows the HC-C (VHH) format, in which the N-terminus of an anti-LILRB2 camelid-derived VHH is connected to the C-terminus of the heavy chain of the anti-VEGF antibody through a polypeptide linker. Fig. 10C shows the LC-C (scFv) format, in which the N-terminus of an anti-LILRB2 scFv is connected to the C-terminus of the light chain of the anti-VEGF antibody through a polypeptide linker (e.g., Bis-M27) . Fig. 10D shows the LC-C (VHH) format, in which the N-terminus of an anti-LILRB2 camelid-derived VHH is connected to the C-terminus of the light chain of the anti-VEGF antibody through a polypeptide linker. Fig. 10E shows the HC-N (VHH) format, in which the C-terminus of an anti-LILRB2 camelid-derived VHH is connected to the N-terminus of the heavy chain of the anti-VEGF antibody through a polypeptide linker.

Fig. 11A-11H. show the binding kinetics of antibodies towards recombinant human LILRB2-ECD-His (Fig. 11A, 11B, 11E, and 11F) , human PD-1-His (Fig. 11C and 11D) , huVEGF165-His (Fig. 11G and 11H) , as evaluated by Surface Plasmon Resonance (SPR) .

Fig. 12A-12B show that the antibodies interfered with HLA-G for its binding towards human LILRB2 protein. The antibodies were evaluated for their blocking effect on the binding of human LILRB2 protein to HLA-G overexpressing A375 cells (A375-HLA-G) using flow cytometry. The bispecific antibodies (Bis-M23-1 and Bis-M28 in Fig. 12A, Bis-M24 and Bis-M27 in Fig. 12B) interfered with HLA-G for its binding towards human LILRB2 protein with IC₅₀ ranging from 1.216 nM to 2.859 nM.

Fig. 13A-13D show the effect of anti-LILRB2 x anti-PD-1 bispecific antibodies on polarization of macrophages. The effect of antibody-mediated polarization of macrophages from M2 to M1 was evaluated in human peripheral blood cell-derived macrophages by macrophage immunotyping and TNF-α level measurements. The antibodies Bis-M23-1 and Bis-M28 induced the downregulation of the M2 phenotypes as indicated by the decreased expression of CD206 (Fig. 13A) and/or CD163 (Fig. 13B) ; and/or the upregulation of M1 phenotypes as indicated by the upregulation of CD80 expression (Fig. 13C) , accompanied with the secretion of inflammatory cytokine TNF-α (Fig. 13D) .

Fig. 14A-14D show the effect of anti-LILRB2 x anti-VEGF bispecific antibodies on polarization of macrophages. The effect of antibody-mediated polarization of macrophages from M2 to M1 was evaluated in human peripheral blood cell-derived macrophages by macrophage immunotyping and TNF-α level measurements. The antibodies Bis-M24 and Bis-M27 induced the downregulation of the M2 phenotype as indicated by the decreased expression of CD206 (Fig. 14A) and/or CD163 (Fig. 14B) ; and/or the upregulation of M1 phenotype as indicated by the upregulation of CD80 expression (Fig. 14C) , accompanied with the secretion of inflammatory cytokine TNF-α (Fig. 14D) .

Fig. 15A-15F show the effect of antibody-mediated polarization of tumor-associated macrophages (TAM) to M1-like macrophage, as evaluated in human peripheral blood cell-derived macrophages by macrophage immunotyping and TNF-α level measurements. The antibodies Bis-M23-1 and Bis-M28 induced the downregulation of CD206 (Fig. 15A) and/or CD163 (Fig. 15B) expression, and/or the upregulation of CD80 expression (Fig. 15C) , accompanied with the secretion of inflammatory cytokine TNF-α (Fig. 15D) . When co-cultured with CD3+ T cells from allogeneic donors and Raji-luc tumor cells, bispecific antibodies induced the secretion of higher levels of inflammatory cytokine TNF-α(Fig. 15E) with enhanced tumor cytotoxicity (Fig. 15F) than monospecific antibodies.

Fig. 16A-16F show the effect of antibody-mediated polarization of tumor-associated macrophages (TAM) to M1-like macrophage, as evaluated in human peripheral blood cell-derived macrophages by macrophage immunotyping and TNF-α level measurements. The antibodies Bis-M24 and Bis-M27 induced the downregulation of CD206 (Fig. 16A) and/or CD163 (Fig. 16B) expression, and/or the upregulation of CD80 expression (Fig. 16C) , accompanied with the secretion of inflammatory cytokine TNF-α (Fig. 16D) . When co-cultured with CD3+ T cells from allogeneic donors and Raji-luc tumor cells, bispecific antibodies induced the secretion of higher levels of inflammatory cytokine TNF-α(Fig. 16E) with enhanced tumor cytotoxicity (Fig. 16F) than monospecific antibodies.

Fig. 17A-17B show the effect of antibodies in a Mixed Lymphocyte Reaction, as evaluated in human peripheral blood cell-derived macrophages cocultured with T cells. Compared with monospecific antibodies, the bispecific antibodies Bis-M23-1 and Bis-M28 induced the secretion of higher level of inflammatory cytokine TNF-α (Fig. 17A) with enhanced tumor cytotoxicity (Fig. 17B) .

Fig. 18A-18B show the effect of antibodies in a Mixed Lymphocyte Reaction, as evaluated in human peripheral blood cell-derived macrophages coculturing with T cells. Compared with monospecific antibodies, the bispecific antibodies Bis-M24 and Bis-M27 induced the secretion of higher level of inflammatory cytokine TNF-α (Fig. 18A) with enhanced tumor cytotoxicity (Fig. 18B) .

Fig. 19A-19B show the anti-tumor effect of bispecific antibodies in an huPBMC-NOG mouse inoculated with MDA-MB-231 tumor model. Mice were randomized into 5 groups (6 mice/group) to receive intravenous dosing of: IgG4 isotype control (G1) ; HB59 (G2) ; Bevacizumab (anti-VEGF) (G3) ; HB59 (anti-LILRB2) + Bevacizumab (anti-VEGF) (G4) ; or Bis-M24 (anti-LILRB2 and anti-VEGF bispecific antibody) (G5) . Fig. 19A shows tumor volumes as measured over the course of the study. Fig. 19B shows tumor weight measured at the end of the study. At the study endpoint, TGI_TV of G2-G5 groups were 4.30%, 48.30%, 59.31%and 64.45%, respectively.

Fig. 20A-20B show the anti-tumor effect of anti-LILRB2/anti-PD-1 bispecific antibodies in PBMC humanized NPG mouse subcutaneously inoculated with HT-29 colorectal adenocarcinoma cells. Fig. 20A shows tumor volumes as measured over the course of the study. Fig. 20B shows tumor weight measured at the end of the study.

Fig. 21A-21B show the anti-tumor effect of anti-LILRB2/anti-VEGF bispecific antibodies in PBMC humanized NPG mouse subcutaneously inoculated with HT-29 colorectal adenocarcinoma cells. Fig. 21A shows tumor volumes as measured over the course of the study. Fig. 21B shows tumor weight measured at the end of the study.

DETAILED DESCRIPTION

Embodiments of the technology described herein are related to monospecific anti-LILRB2 antibody constructs, as well as bispecific anti-LILRB2 antibody constructs that also inhibit targets such as PD-1 or VEGF. Such antibody constructs are capable of polarizing macrophages from an M2 phenotype to an M1 phenotype, activating T cells, and/or inhibiting growth of a cancer. Also described herein are nucleic acids and vector encoding such monospecific or bispecific anti-LILRB2 antibody constructs, cells expressing or in combination with such antibody constructs, and compositions comprising such antibody constructs, nucleic acids, vectors, or cells. The disclosure also relates to methods of modulating the immune response or treating cancer using such monospecific or bispecific anti-LILRB2 antibody constructs.
Antibodies

In multiple aspects described herein are compositions comprising a binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) . In some embodiments of any of the aspects, the composition comprises a monospecific antibody construct comprising a binding domain that specifically binds LILRB2. In some embodiments of any of the aspects, the composition comprises a bispecific antibody construct comprising a first binding domain that specifically binds LILRB2 and a second binding domain that specifically binds programmed cell death protein 1 (PD-1) . In some embodiments of any of the aspects, the composition comprises a bispecific antibody construct comprising a first binding domain that specifically binds LILRB2 and a second binding domain that specifically binds vascular endothelial growth factor (VEGF) .

The term “antibody, ” as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or V_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C_H1, C_H2 and C_H3. As used herein, a “hinge region” of an antibody is the flexible amino acid stretch in the central part of the heavy chains of the IgG and IgA immunoglobulin classes, which links these 2 heavy chains by disulfide bonds. The hinge region is located in between the C_H1 and C_H2 domains. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and the like) or subclass. In some embodiments of any of the aspects, the Fc domain of a monospecific or bispecific antibody construct as described herein comprises the Fc domain of IgG4.

As described further herein, the compositions of the present disclosure comprise at least one binding domain comprising CDRs. As used herein, the term “Complementarity Determining Regions” (CDRs, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for specific antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region can comprise amino acid residues from a “complementarity determining region” as defined by Kabat (i.e., about residues 24-34 (L1) , 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1) , 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain) . Likewise, “frameworks” (FWs) comprise amino acids 1-23 (FW1) , 35-49 (FW2) , 57-88 (FW3) , and 98-107 (FW4) in the light chain variable domain and 1-30 (FW1) , 36-49 (FW2) , 66-94 (FW3) , and 103-113 (FW4) in the heavy chain variable domain taking into account the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1987, 1991) ) .

The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues. The actual linear amino acid sequence can contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR) , of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence.

Methods and computer programs for determining sequence similarity are publicly available, including, but not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12: 387, 1984) , BLASTP, BLASTN, FASTA (Altschul et al., J. Mol. Biol. 215: 403 (1990) , and the ALIGN program (version 2.0) . The Smith Waterman algorithm can also be used to determine similarity. The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, et al., NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0 at ncbi. nlm. nih. gov/blast/) . In comparing sequences, these methods account for various substitutions, deletions, and other modifications.

As used herein, “antibody variable domain” or “V_H/V_L domain pair” refers to the portions of the heavy and light chains of antibody molecules that include amino acid sequences of Complementarity Determining Regions (CDRs; i.e., CDR1, CDR2, and CDR3) , and Framework Regions (FRs) . V_H refers to the variable domain of the heavy chain. V_L refers to the variable domain of the light chain, which can be either a λ light chain or a κ light chain. As used herein, V_K refers to the variable domain (e.g., V_L) of a κlight chain. Together, a V_H/V_L domain pair can bind and preferably and specifically bind an epitope on a given antigen.

The binding domains of the compositions described herein can comprise at least one antigen binding domain of an antibody. The term “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” ) , as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments can also be bispecific, dual specific, or multi-specific formats, specifically binding to two or more different antigens via different antigen binding portions. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and C_H1 domains; (ii) a F (ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and C_H1 domains; (iv) an Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, and (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546, Winter et al., PCT publication WO 90/05144 A1 herein incorporated by reference) , which comprises a single variable domain. CDRs can also be displayed on a non-immunoglobulin scaffold. Non-limiting examples of non-immunoglobulin antigen-binding scaffolds include a DARPIN, an affibody, an affilin, an adnectin, an affitin, an Obody or Obodies, a repebody, a fynomer, an alphabody, an avimer, an atrimer, a centyrin, a pronectin, an anticalin, a kunitz domain, or an Armadillo repeat protein. Examples of non-immunoglobulin antigen binding scaffolds are described in WO 2017/172981 and the tables therein, which are incorporated herein by reference.

Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that permits them to be made as a single protein chain in which the V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) . Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which V_H and V_L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123) . Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-54041354-5) . In addition, single chain antibodies also include “linear antibodies” comprising a pair of tandem Fv segments (V_H-C_H1-V_H-C_H1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng. 8 (10) : 1057-1062 (1995) ; and U.S. Pat. No. 5,641,870) .

In some embodiments of any of the aspects, the compositions described herein comprise a single-domain antibody (sdAb) , also known as a Nanobody, which is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, the sdAb can bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, single-domain antibodies are much smaller than common antibodies (150–160 kDa, two heavy protein chains and two light chains) , and even smaller than Fab fragments (～50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (～25 kDa, two variable domains, one from a light and one from a heavy chain) . In some embodiments, the single-domain antibody is derived from the variable domain of a heavy-chain antibody found in camelids, termed a variable domain of the heavy chain of a heavy-chain antibody (VHH) . Camelid nanobodies have been shown to be just as specific as antibodies, and in some cases they are more robust. Camelid nanobodies can be isolated using the same phage panning procedure used for antibodies, allowing them to be cultured in vitro in large concentrations. The smaller size and single domain can make camelid nanobodies easier to transform into bacterial cells for bulk production.

In a further aspect of this embodiment, the antibody can be a recombinant antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody. As used herein, "recombinant" antibody means any antibody whose production involves expression of a non-native DNA sequence encoding the desired antibody structure in an organism.

The term "monoclonal antibody" , as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure can be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975) , or may be made by any of a wide variety of other recombinant DNA methods known to those of skill in the art (see e.g., U.S. Pat. No. 4,816,567) .

Additional types of antibodies include, but are not limited to, chimeric, humanized, and human antibodies. Human antibodies are derived from human samples, such as blood, plasma, or serum. In other embodiments, the antibody constructs described herein are derived from non-human samples, such as blood, plasma, or serum. For example, as described herein the binding domains comprised by an anti-LILRB2 antibody construct can be derived from a primate such as a human, or from a rodent or a camelid (e.g., a mouse or an alpaca) . For application in humans, it is often desirable to reduce immunogenicity of antibodies originally derived from other species. Reducing the immunogenicity of non-human-derived antibodies can be done by construction of chimeric antibodies, or by a process called "humanization" . In this context, a "chimeric antibody" is understood to be an antibody comprising a domain (e.g., a variable domain) derived from one species (e.g., mouse, alpaca) fused to a domain (e.g., the constant domains) derived from a different species (e.g., human) .

As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine, alpaca) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will ideally comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin (Jones et al., Nature 321 : 522-525 (1986) ; Riechmann et al., Nature 332: 323-329 (1988) ; and Presta, Curr. Op. Struct. Biol 2: 593-596 (1992) ) . The constant region, can if desired, include one or more modifications that modify or disrupt interaction of the human or humanized antibody with an Fc receptor. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321 : 522-525 (1986) ; Riechmann et al., Nature 332: 323-3'27 (1988) ; Verhoeyen et al., Science 239: 1534-1536 (1988) ) , by substituting non-human CDRs or CDR sequences (e.g., from a mouse or alpaca, as described herein) in place of the corresponding CDRs or CDR sequences of a human antibody, or in other words changing a non-human antibody to a human antibody framework comprising non-human CDRs or CDR sequences.

In some embodiments of any of the aspects, the antibody constructs described herein are affinity matured. As used herein, “affinity maturation” refers to the process by which antibodies are produced with increased affinity for antigen. With repeated exposures to the same antigen, a host or cell can produce antibodies of successively greater affinities.

Methods for designing and producing antibodies, including monoclonal, humanized, affinity-matured, or recombinant antibodies are well known in the art (see e.g., US 8, 663, 980, US 9, 683, 027, US 2018/0291101, WO 2011/015916, which are incorporated herein by reference in their entireties) . To generate antibodies, conventional hybridoma techniques have been used in which clones of hybrid cells expressing genes coding for the light and heavy chains of an antibody molecule are obtained by immunization with an antigen molecule. This technique necessitates the fusion of cells of lymphocytic origin, containing the genes for antibody formation and cells forming immortal lines. The cells carrying the genes in question are generally obtained by random creation of libraries of circulating cells, and screening of the hybridomas with an antigen-antibody reaction after the hybridoma clones are multiplied and cultured.

In addition, monoclonal antibodies and their fragments can be expressed in various host systems, such as E. coli, yeast, and mammalian host cells. In general, a mammalian expression vector can contain (1) regulatory elements, usually in the form of viral promoter or enhancer sequences and characterized by a broad host and tissue range; (2) a “polylinker” sequence, facilitating the insertion of a DNA fragment within the plasmid vector; and (3) the sequences responsible for intron splicing and polyadenylation of mRNA transcripts. This contiguous region of the promoter-polylinker-polyadenylation site is commonly referred to as the transcription unit. The vector will likely also contain (4) a selectable marker gene (s) (e.g., the beta-lactamase gene) , often conferring resistance to an antibiotic (such as ampicillin) , allowing selection of initial positive transformants in E. coli; and (5) sequences facilitating the replication of the vector in both bacterial and mammalian hosts. Non-limiting examples of a mammalian expression vector include CDM8, pCMX, pAd/CMV/V5-DEST^TM, pAd/PL-DEST^TM, pCEP4, pOptiVEC^TM-TOPO^TM, pTracer^TM-SV40, pcDNA^TM3.2-DEST, pCMV·SPORT-βgal, pcDNA^TM3.3-TOPO^TM, pcDNA^TM3.4 TOPO^TM, or pcDNA^TM4/HisMax TOPO^TM. Expression of monoclonal antibodies behind a strong promoter increases the chances of identifying high-producing cell lines and obtaining higher yields of monoclonal antibodies. Consequently, Ig vectors with strong promoters are highly desirable for expressing any monoclonal antibody of interest. In addition, vectors with unique DNA cloning sites downstream of strong promoters have an added convenience.

Antibodies can be produced in bacteria, yeast, fungi, protozoa, insect cells, plants, or mammalian cells (see e.g., Frenzel et al. (2013) Front Immunol. 4: 217) . A mammalian expression system is generally preferred for manufacturing most of therapeutic proteins, such as antibodies, as they require post-translational modifications. A variety of mammalian cell expression systems are now available for expression of antibodies, including but not limited to immortalized Chinese hamster ovary (CHO) cells, mouse myeloma (NSO) , mouse L-cells, myeloma cell lines like J558L and Sp2/0, baby hamster kidney (BHK) , or human embryo kidney (HEK-293) .
LILRB2

In multiple aspects described herein are compositions comprising a binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) . LILRB2 can also be referred to interchangeably as Leukocyte immunoglobulin-like receptor 2 (LIR-2) , LIRB2, CD85 antigen-like family member D (CD85d) , Immunoglobulin-like transcript 4 (ILT-4) , or Monocyte/macrophage immunoglobulin-like receptor 10 (MIR-10) . The LILRB2 gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family, which is found in a gene cluster at chromosomal region 19q13.4. The encoded LILRB2 protein belongs to the subfamily B class of LIR receptors which contain two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) . The LILRB2 receptor is expressed on immune cells, where it binds to MHC class I molecules, such as HLA-G, on antigen-presenting cells and transduces a negative signal that inhibits stimulation of an immune response. LILRB2 signaling can control inflammatory responses and cytotoxicity to help focus the immune response and limit autoreactivity.

In some embodiments of any of the aspects, the LILRB2, specifically bound by a binding domain as described herein, comprises mammalian LILRB2. In some embodiments of any of the aspects, the LILRB2, specifically bound by a binding domain as described herein, comprises human LILRB2. In some embodiments of any of the aspects, the LILRB2, specifically bound by a binding domain as described herein, comprises SEQ ID NO: 775 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 775, that maintains its function (e.g., HLA-G binding) .

SEQ ID NO: 775, LILRB2, Homo sapiens, 597 amino acids (aa) , UNIPROT Q8N423-2, LIRB2_HUMAN

The monospecific and bispecific antibodies described herein can comprise a binding domain specific for LILRB2 that interferes with the binding between LILRB2 and its ligand (s) , and thus polarizes macrophages from an M2 phenotype to an M1 phenotype, activates T cells, and/or inhibits growth of a cancer, as described further herein. In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 monospecific or bispecific antibody constructs as described herein) is capable of interfering with the interaction between the HLA-G and LILRB2 polypeptides. In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 monospecific or bispecific antibody constructs as described herein) is capable of decreasing cellular signaling downstream of LILRB2.

In some embodiments of any of the aspects, the composition comprises a monospecific antibody construct comprising a binding domain that specifically binds LILRB2. In some embodiments of any of the aspects, the composition comprises a bispecific antibody construct comprising a first binding domain that specifically binds LILRB2 and a second binding domain that specifically binds programmed cell death protein 1 (PD-1) . In some embodiments of any of the aspects, the composition comprises a bispecific antibody construct comprising a first binding domain that specifically binds LILRB2 and a second binding domain that specifically binds vascular endothelial growth factor (VEGF) .

In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_H domain that specifically binds LILRB2. In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_L domain that specifically binds LILRB2. In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_H/V_L domain pair that specifically binds LILRB2. In some embodiments of any of the aspects, the binding domain comprises the CDRs of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2.

In some embodiments of any of the aspects, the binding domain specifically binds to a LILRB2 epitope or portion of a LILRB2 epitope. In some embodiments of any of the aspects, the LILRB2 epitope or portion thereof comprises sequential and/or non-sequential residues of SEQ ID NO: 775, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, or more sequential and/or non-sequential residues of SEQ ID NO: 775. In some embodiments of any of the aspects, the anti-LILRB2 binding domain of at least one antibody construct as described herein specifically binds to an LILRB2 epitope or portion thereof that is distinct from the LILRB2 epitope or portion thereof to which an anti-LILRB2 binding domain of at least one other antibody construct as described here specifically binds (see e.g., Table 2 or Table 3) .

In some embodiments of any of the aspects, the anti-LILRB2 binding domain has an equilibrium dissociation constant (K_D) between about 2.0E-10 M to about 4.5E-8 M towards the extracellular domain of human LILRB2 (see e.g., Table 1) . In some embodiments of any of the aspects, the anti-LILRB2 binding domain has an equilibrium dissociation constant (K_D) between about 7.5E-12 M to about 1.5E-8 M towards the extracellular domain of human LILRB2 (see e.g., Table 18) . In some embodiments of any of the aspects, the anti-LILRB2 binding domain has an equilibrium dissociation constant (K_D) of about 7.5E-12 M, about 8.0E-12 M, about 8.5E-12 M, about 9.0E-12 M, about 9.5E-12 M, about 1.0E-11 M, about 1.5E-11 M, about 2.0E-11 M, about 2.5E-11 M, about 3.0E-11 M, about 3.5E-11 M, about 4.0E-11 M, about 4.5E-11 M, about 5.0E-11 M, about 5.5E-11 M, about 6.0E-11 M, about 6.5E-11 M, about 7.0E-11 M, about 7.5E-11 M, about 8.0E-11 M, about 8.5E-11 M, about 9.0E-11 M, about 9.5E-11 M, about 1.0E-10 M, about 1.5E-10 M, about 2.0E-10 M, about 2.5E-10 M, about 3.0E-10 M, about 3.5E-10 M, about 4.0E-10 M, about 4.5E-10 M, about 5.0E-10 M, about 5.5E-10 M, about 6.0E-10 M, about 6.5E-10 M, about 7.0E-10 M, about 7.5E-10 M, about 8.0E-10 M, about 8.5E-10 M, about 9.0E-10 M, about 9.5E-10 M, about 1.0E-9 M, about 1.5E-9 M, about 2.0E-9 M, about 2.5E-9 M, about 3.0E-9 M, about 3.5E-9 M, about 4.0E-9 M, about 4.5E-9 M, about 5.0E-9 M, about 5.5E-9 M, about 6.0E-9 M, about 6.5E-9 M, about 7.0E-9 M, about 7.5E-9 M, about 8.0E-9 M, about 8.5E-9 M, about 9.0E-9 M, about 9.5E-9 M, about 1.0E-8 M, about 1.5E-8 M, about 2.0E-8 M, about 2.5E-8 M, about 3.0E-8 M, about 3.5E-8 M, about 4.0E-8 M, about 4.5E-8 M, about 5.0E-8 M, about 5.5E-8 M, about 6.0E-8 M, about 6.5E-8 M, about 7.0E-8 M, about 7.5E-8 M, or about 8.0E-8 M, towards the extracellular domain of human LILRB2.

In some embodiments of any of the aspects, the anti-LILRB2 monospecific or bispecific antibody construct as described herein has an half-maximal inhibitory concentration (IC50) between at least 1.0 μg/mL to at most 6.715 μg/mL, or between at least 1.0 nM (e.g., about 0.2 μg/mL) to at most 3.5 nM (e.g., about 0.5 μg/mL) , to interfere with the binding between LILRB2 and its HLA-G ligand (see e.g., Table 4, Table 21) . In some embodiments of any of the aspects, the anti-LILRB2 monospecific or bispecific antibody construct as described herein has an IC50 of about 0.1 μg/mL, about 0.2 μg/mL, about 0.3 μg/mL, about 0.4 μg/mL, about 0.5 μg/mL, about 0.6 μg/mL, about 0.7 μg/mL, about 0.8 μg/mL, about 0.9 μg/mL, about 1.0 μg/mL, about 2.0 μg/mL, about 3.0 μg/mL, about 4.0 μg/mL, about 5.0 μg/mL, about 6.0 μg/mL, about 7.0 μg/mL, about 8.0 μg/mL, about 9.0 μg/mL, about 10.0 μg/mL, to interfere with the binding between LILRB2 and its HLA-G ligand. In some embodiments of any of the aspects, the anti-LILRB2 monospecific or bispecific antibody construct as described herein has an IC50 of about 1 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, about 1.6 nM, about 1.7 nM, about 1.8 nM, about 1.9 nM, about 2 nM, about 2.1 nM, about 2.2 nM, about 2.3 nM, about 2.4 nM, about 2.5 nM, about 2.6 nM, about 2.7 nM, about 2.8 nM, about 2.9 nM, about 3 nM, about 3.1 nM, about 3.2 nM, about 3.3 nM, about 3.4 nM, or about 3.5 nM to interfere with the binding between LILRB2 and its HLA-G ligand. For example, the molecular weight of Bis-M28 is approximately 200 kD and the molecular weight of HB59 is approximately 150 kD.

In some embodiments of any of the aspects, an antibody (e.g., monospecific or bispecific) construct specific for LILRB2 can comprise one or more (e.g., one, two, three, four, five, or six) CDRs of any one of the antibodies recited in Table 8, Table 10, or Table 30 or encoded by a nucleic acid recited in Table 9, Table 11, or Table 31. In some embodiments of any of the aspects, an antibody (e.g., monospecific or bispecific) construct specific for LILRB2 can comprise the six CDRs of any one of the antibodies recited in Table 8, Table 10, or Table 30 or encoded by a nucleic acid recited in Table 9, Table 11, or Table 31. In some embodiments of any of the aspects, an antibody (e.g., monospecific or bispecific) construct specific for LILRB2 can comprise the three heavy chain CDRs of any one of the antibodies recited in Table 8, or Table 30 or encoded by a nucleic acid recited in Table 9 or Table 31. In some embodiments of any of the aspects, an antibody (e.g., monospecific or bispecific) construct specific for LILRB2 can comprise the three light chain CDRs of any one of the antibodies recited in Table 10, or Table 30 or encoded by a nucleic acid recited in Table 11, or Table 31. In some embodiments of any of the aspects, an antibody (e.g., monospecific or bispecific) construct specific for LILRB2 can comprise the VH and/or VL domains of any one of the antibodies recited in Table 12, Table 14, or encoded by a nucleic acid recited in Table 13 or Table 15. In some embodiments of any of the aspects, an antibody (e.g., monospecific or bispecific) construct specific for LILRB2 can comprise the VH and VL domains of any one of the antibodies recited in Table 12, Table 14, or encoded by a nucleic acid recited in Table 13 or Table 15. Such antibody constructs are specifically contemplated for use in the methods and/or compositions described herein.

Table 32: Exemplary anti-LILBRB2 antibody CDR amino acid sequences; “#” refers to the corresponding SEQ ID NO. J-19. h1 and 1E1 (G4) are control anti-LILRB2 antibodies; 1E1 (G4) is an 1E1-Fc (IgG4) fusion protein (see e.g., patent publications WO2019/126514A2 and US2018/026160, the contents of which are incorporated herein by reference in their entireties) .

Table 33: Exemplary anti-LILBRB2 antibody CDR nucleic acid sequences; “#” refers to the corresponding SEQ ID NO.

Table 34: Exemplary anti-LILBRB2 antibody heavy and light chain amino acid and nucleic acid sequences; “#” refers to the corresponding SEQ ID NO.

In some embodiments of any of the aspects, the V_H/V_L domain pair or VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR1 comprising one of SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, or 118. In some embodiments of any of the aspects, the V_H/V_L domain pair or VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, or 238. In some embodiments of any of the aspects, the VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR1 comprising one of SEQ ID NOs: 82, 85, 88, 91, 94, 97, 100, 103, 106, 115, or 118. In some embodiments of any of the aspects, the VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 202, 205, 208, 211, 214, 217, 220, 223, 226, 235, or 238.

In some embodiments of any of the aspects, the V_H/V_L domain pair or VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR2 comprising one of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, or 119. In some embodiments of any of the aspects, the V_H/V_L domain pair or VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, or 239. In some embodiments of any of the aspects, the VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR2 comprising one of SEQ ID NOs: 83, 86, 89, 92, 95, 98, 101, 104, 107, 116, or 119. In some embodiments of any of the aspects, the VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 203, 206, 209, 212, 215, 218, 221, 224, 227, 236, or 239.

In some embodiments of any of the aspects, the V_H/V_L domain pair or VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR3 comprising one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, or 120. In some embodiments of any of the aspects, the V_H/V_L domain pair or VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, or 240. In some embodiments of any of the aspects, the VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR3 comprising one of SEQ ID NOs: 84, 87, 90, 93, 96, 99, 102, 105, 108, 117, or 120. In some embodiments of any of the aspects, the VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 204, 207, 210, 213, 216, 219, 222, 225, 228, 237, or 240.

In some embodiments of any of the aspects, the V_H/V_L domain pair of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_L CDR1 comprising one of SEQ ID NOs: 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, or 829. In some embodiments of any of the aspects, the V_H/V_L domain pair of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_L CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361, 364, 367, 370, 373, 376, 379, 382, 385, 388, 391, 394, 397, 400, 403, or 406.

In some embodiments of any of the aspects, the V_H/V_L domain pair of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_L CDR2 comprising one of SEQ ID NOs: 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 314, 317, or 320. In some embodiments of any of the aspects, the V_H/V_L domain pair of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_L CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 368, 371, 374, 377, 380, 383, 386, 389, 392, 395, 398, 401, 404, or 407.

In some embodiments of any of the aspects, the V_H/V_L domain pair of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_L CDR3 comprising one of SEQ ID NOs: 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, or 830. In some embodiments of any of the aspects, the V_H/V_L domain pair of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_L CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, or 408.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_H domain selected from Table 12 or a V_H domain that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to an amino acid sequence selected from Table 12, that maintains its function (e.g., LILRB2 binding) . In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_H domain encoded by a nucleic acid sequence selected from Table 13 or a V_H domain that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a nucleic acid sequence selected from Table 13, that maintains its function as a polypeptide (e.g., LILRB2 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_L domain selected from Table 14 or a V_L domain that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to an amino acid sequence selected from Table 14, that maintains its function (e.g., LILRB2 binding) . In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_L domain encoded by a nucleic acid sequence selected from Table 15 or a V_L domain that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a nucleic acid sequence selected from Table 15, that maintains its function as a polypeptide (e.g., LILRB2 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_H domain comprising one of SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, or 448, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, or 448, that maintains its function (e.g., LILRB2 binding) .

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_H domain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 571, 572, 573, 574, 579, 580, 581, 582, 583, 584, 590, 591, 592, 593, 594, 595, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 571, 572, 573, 574, 579, 580, 581, 582, 583, 584, 590, 591, 592, 593, 594, 595, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615, that maintains its function as a polypeptide (e.g., LILRB2 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a VHH comprising one of SEQ ID NOs: 436, 437, 438, 439, 440, 441, 442, 443, 444, 447, or 448, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 436, 437, 438, 439, 440, 441, 442, 443, 444, 447, or 448, that maintains its function (e.g., LILRB2 binding) .

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a VHH encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 476, 477, 478, 479, 480, 481, 482, 483, 484, 487, or 488, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 476, 477, 478, 479, 480, 481, 482, 483, 484, 487, or 488, that maintains its function as a polypeptide (e.g., LILRB2 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_L domain comprising one of SEQ ID NOs: 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, or 517, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, or 517, that maintains its function (e.g., LILRB2 binding) .

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_L domain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 575, 576, 577, 578, 585, 586, 587, 588, 589, 596, 597, 598, 599, 600, 601, or 785 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 575, 576, 577, 578, 585, 586, 587, 588, 589, 596, 597, 598, 599, 600, 601, or 785, that maintains its function as a polypeptide (e.g., LILRB2 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises an antibody or functional fragment thereof selected from the group consisting of: J-19. h1, 1E1 (G4) , HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises an antibody or functional fragment thereof selected from the group consisting of: AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huB45, and huB206.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises an antibody or functional fragment thereof selected from the group consisting of: HB59, MIL-C68, huC68, MIL-C112, huC112, AIL-B45, huB45, AIL-B206, and huB206.

In some embodiments of any of the aspects, the V_H/V_L domain pair or VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR1 comprising one of SEQ ID NOs: 19, 70, 109, 79, 112, 82, 115, 103 or 118. In some embodiments of any of the aspects, the V_H/V_L domain pair or VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR2 comprising one of SEQ ID NOs: 20, 71, 110, 80, 113, 83, 116, 104, or 119. In some embodiments of any of the aspects, the V_H/V_L domain pair or VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR3 comprising one of SEQ ID NOs: 21, 72, 111, 81, 114, 84, 117, 105, or 120.

In some embodiments of any of the aspects, the VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR1 comprising one of SEQ ID NOs: 82, 115, 103 or 118. In some embodiments of any of the aspects, the VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR2 comprising one of SEQ ID NOs: 83, 116, 104 or 119. In some embodiments of any of the aspects, the VHH of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_H CDR3 comprising one of SEQ ID NOs: 84, 117, 105 or 120.

In some embodiments of any of the aspects, the V_H/V_L domain pair of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_L CDR1 comprising one of SEQ ID NOs: 259, 310, 316, 313 or 319. In some embodiments of any of the aspects, the V_H/V_L domain pair of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_L CDR2 comprising one of SEQ ID NOs: 260, 311, 317, 314 or 320. In some embodiments of any of the aspects, the V_H/V_L domain pair of the (e.g., monospecific or bispecific) construct that specifically binds LILRB2 comprises a V_L CDR3 comprising one of SEQ ID NOs: 261, 312, 318, 315 or 321.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_H comprising one of SEQ ID NOs: 415, 432, 445, 435, 446, 447, 448 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 415, 432, 445, 435, 446, 447, 448, that maintains its function (e.g., LILRB2 binding) .

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_L comprising one of SEQ ID NOs: 495, 512, 516, 515, 517 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 495, 512, 516, 515, 517, that maintains its function as a polypeptide (e.g., LILRB2 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 445, 571-574, 472, 485, 579-584, 475, 590-595, 486, 476, 602-608, 487, 483, 488, 609-615 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 445, 571-574, 472, 485, 579-584, 475, 590-595, 486, 476, 602-608, 487, 483, 488, 609-615 that maintains its function (e.g., LILRB2 binding) .

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 524, 575-578, 541, 545, 585-589, 544, 546, 596-601, or 785, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 524, 575-578, 541, 545, 585-589, 544, 546, 596-601, or 785, that maintains its function as a polypeptide (e.g., LILRB2 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises an HB59 antibody or functional fragment thereof. In some embodiments of any of the aspects, the HB59-comprising anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises (a) a V_H CDR1 comprising SEQ ID NO: 19; (b) a V_H CDR2 comprising SEQ ID NO: 20; (c) a V_H CDR3 comprising SEQ ID NO: 21; (d) a V_L CDR1 comprising SEQ ID NO: 259; (e) a V_L CDR2 comprising SEQ ID NO: 260; and/or (f) a V_L CDR3 comprising SEQ ID NO: 261.

In some embodiments of any of the aspects, the HB59-comprising anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_H comprising SEQ ID NO: 415 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 415, that maintains its function (e.g., LILRB2 binding) .

In some embodiments of any of the aspects, the HB59-comprising anti-LILRB2 (e.g., monospecific or bispecific) antibody construct comprises a V_L comprising SEQ ID NO: 495 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 495.
PD-1 and VEGF

In multiple aspects described herein are compositions comprising a first binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) and a second binding domain that specifically binds programmed cell death protein 1 (PD-1) . PD-1 is encoded by the PDCD1 gene and can also be referred to interchangeably as CD279, Systemic Lupus Erythematosus Susceptibility 2, SLEB2, or HSLE1. PD-1 is an immune-inhibitory receptor expressed in activated T cells; PD-1 is involved in the regulation of T-cell functions, including those of effector CD8+ T cells. In addition, PD-1 can also promote the differentiation of CD4+ T cells into T regulatory cells. PDCD1 is expressed in many types of tumors including melanomas, and has been demonstrated to play a role in anti-tumor immunity. PD-1 is a cell surface receptor on T cells and B cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity, thus helping to prevent autoimmune diseases. However, PD-1 can also prevent the immune system from killing cancer or microbial cells. PD-1 is an immune checkpoint that guards against autoimmunity through two mechanisms. First, PD-1 promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, PD-1 reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells) . PD-1 binds two ligands, PD-L1 and PD-L2. PD-1 inhibitors, which interfere with the binding between PD-1 and its ligand (s) , can activate the immune system to attack tumors and can be used to treat certain types of cancer.

In some embodiments of any of the aspects, the PD-1, specifically bound by a binding domain as described herein, comprises mammalian PD-1. In some embodiments of any of the aspects, the PD-1, specifically bound by a binding domain as described herein, comprises human PD-1. In some embodiments of any of the aspects, the PD-1, specifically bound by a binding domain as described herein, comprises SEQ ID NO: 776 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 776, that maintains its function (e.g., immune checkpoint) .

SEQ ID NO: 776, PD-1, Homo sapiens, 288 amino acids (aa) , UNIPROT Q15116, PDCD1_HUMAN

The bispecific antibodies described herein can comprise a binding domain specific for PD-1 that interferes with the binding between PD-1 and its ligand (s) , and thus has an anti-cancer effect, such as checkpoint inhibition. In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 and anti-PD-1 bispecific antibody constructs as described herein) is capable of interfering with the interaction between PD-1 and PD-L1 and/or PD-L2 polypeptides. In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 and anti-PD-1 bispecific antibody constructs as described herein) is capable of decreasing cellular signaling downstream of PD-1.

In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_H domain that specifically binds PD-1. In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_L domain that specifically binds PD-1. In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_H/V_L domain pair that specifically binds PD-1. In some embodiments of any of the aspects, the binding domain comprises the CDRs of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds PD-1.

In some embodiments of any of the aspects, the binding domain specifically binds to a PD-1 epitope or portion of a PD-1 epitope. In some embodiments of any of the aspects, the PD-1 epitope or portion thereof comprises sequential and/or non-sequential residues of SEQ ID NO: 776, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, or more sequential and/or non-sequential residues of SEQ ID NO: 776. In some embodiments of any of the aspects, the anti-PD-1 binding domain comprises nivolumab, pembrolizumab, toripalimab, sintilimab, cemiplimab, or dostarlimab, or CDRs, VL domains, VH domains, or functional fragments thereof.

In some embodiments of any of the aspects, the binding domain that specifically binds PD-1 comprises nivolumab (OPDIVO) or CDRs, VL domains, VH domains, or functional fragments thereof. Nivolumab can be used to treat at least melanoma, lung cancer, kidney cancer, bladder cancer, head and neck cancer, and Hodgkin's lymphoma. In some embodiments of any of the aspects, the anti-PD1 binding domain specifically binds to at least one nivolumab PD-1 epitope or portion of a nivolumab PD-1 epitope comprising: the N-terminal loop of PD-1, residues 25 to 31 of SEQ ID NO: 776 (LDSPDRP, SEQ ID NO: 787) , residues 25 to 31 of SEQ ID NO: 776 (TSES, SEQ ID NO: 779) , and/or residues 127 to 132 of SEQ ID NO: 776 (SLAPKA, SEQ ID NO: 780) .

In some embodiments of any of the aspects, the binding domain that specifically binds PD-1 comprises pembrolizumab (KEYTRUDA) or CDRs, VL domains, VH domains, or functional fragments thereof. Pembrolizumab can be used to treat at least melanoma and lung cancer. In some embodiments of any of the aspects, the anti-PD1 binding domain specifically binds to at least one pembrolizumab PD-1 epitope or portion of a pembrolizumab PD-1 epitope comprising: the C’ D loop of PD-1, residues S60, S62, V64, N66, I126, L128, A129, K131, A132, and/or I134 of SEQ ID NO: 776, residues 75 to 78 of SEQ ID NO: 776 (QTDK, SEQ ID NO: 781) , and/or residues 81 to 90 of SEQ ID NO: 776 (AFPEDRSQPG, SEQ ID NO: 782) . See e.g., Fessas et al. "Amolecular and preclinical comparison of the PD-1–targeted T-cell checkpoint inhibitors nivolumab and pembrolizumab. " Semin Oncol. 2017 Apr; 44(2) : 136–140, the contents of which are incorporated herein by reference in their entirety.

In some embodiments of any of the aspects, the binding domain that specifically binds PD-1 comprises Toripalimab (Tuoyi^TM) or CDRs, VL domains, VH domains, or functional fragments thereof. Toripalimab can be used to treat at least melanoma, nasopharyngeal carcinoma, urothelial carcinoma, lung cancer, digestive tract tumors, hepatobiliary and pancreatic tumors, or neuroendocrine neoplasms. In some embodiments of any of the aspects, the anti-PD1 binding domain specifically binds to at least one Toripalimab PD-1 epitope or portion of a Toripalimab PD-1 epitope comprising: the FG loop of PD-1, residues P130, K131, A132, and/or I134 of SEQ ID NO: 776. In some embodiments of any of the aspects, Toripalimab comprises SEQ ID NO: 788 (heavy chain) and/or SEQ ID NO: 787 (light chain) . See e.g., Liu et al. “Glycosylation-independent binding of monoclonal antibody toripalimab to FG loop of PD-1 for tumor immune checkpoint therapy” MAbs. 2019 Jun; 11 (4) : 681–690, the contents of which are incorporated herein by reference in their entirety.

SEQ ID NO: 788, Toripalimab heavy chain, 452 aa, DRUGBANK Accession Number DB15043, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 817-819)

SEQ ID NO: 789, Toripalimab light chain, 219 aa, DRUGBANK Accession Number DB15043, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 820-822)

In some embodiments of any of the aspects, the binding domain that specifically binds PD-1 comprises Sintilimabor CDRs, VL domains, VH domains, or functional fragments thereof. Sintilimab can be used to treat at least classical Hodgkin’s lymphoma, hepatocellular carcinoma, squamous NSCLC, or non-squamous NSCLC. In some embodiments of any of the aspects, the anti-PD1 binding domain specifically binds to at least one Sintilimab PD-1 epitope or portion of a Sintilimab PD-1 epitope comprising: residues S127, L128, A129, P130, and/or A132 of SEQ ID NO: 776. In some embodiments of any of the aspects, Sintilimab comprises SEQ ID NO: 790 (heavy chain) and/or SEQ ID NO: 791 (light chain) . See e.g., Ma et al. “The binding epitope of sintilimab on PD-1 revealed by AbMap” , Acta Biochim Biophys Sin (Shanghai) , 2021 Apr 15; 53 (5) : 628-635, the contents of which are incorporated herein by reference in their entirety.

SEQ ID NO: 790, Sintilimab heavy chain, 447 aa, KEGG DRUG Database Entry D12119, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 823-825) :

SEQ ID NO: 791, Sintilimab light chain, 214 aa, KEGG DRUG Database Entry D12119, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 826-828) :

In some embodiments of any of the aspects, the binding domain that specifically binds PD-1 comprises cemiplimab (LIBTAYO) or CDRs, VL domains, VH domains, or functional fragments thereof. Cemiplimab can be used to treat at least basal cell carcinoma, myeloma, lung cancer, cervical cancer, or cutaneous squamous cell carcinoma. In some embodiments of any of the aspects, the anti-PD1 binding domain specifically binds to at least one cemiplimab PD-1 epitope or portion of a cemiplimab PD-1 epitope comprising: the BC loop of PD-1, the C’ D loop of PD-1, the FG loop of PD1; the CC’ FG strands of PD-1; or residues S60, E61, S62, F63, and/or N66 of the BC loop; or residues P83, E84, and/or R86 of the C’ D loop; or residues L128, A129, P130, K131, and/or A132 of the FG loop; or residues V64, N66, F82, I126, Q133, and/or I134 within the front β-sheet consisting of the CC’ FG strands; see e.g., SEQ ID NO: 776. In some embodiments of any of the aspects, cemiplimab comprises SEQ ID NO: 792 (heavy chain) and/or SEQ ID NO: 793 (light chain) . See e.g., Jeong et al., “The High-Resolution Structure Reveals Remarkable Similarity in PD-1 Binding of Cemiplimab and Dostarlimab, the FDA-Approved Antibodies for Cancer Immunotherapy, ” Biomedicines. 2022 Dec; 10 (12) : 3154, the contents of which are incorporated herein by reference in their entirety.

SEQ ID NO: 792, cemiplimab heavy chain, 444 aa, DRUGBANK Accession Number DB14707, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 796-798) :

SEQ ID NO: 793, cemiplimab light chain, 214 aa, DRUGBANK Accession Number DB14707, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 802-804) :

In some embodiments of any of the aspects, the binding domain that specifically binds PD-1 comprises dostarlimab (JEMPERLI) or CDRs, VL domains, VH domains, or functional fragments thereof. Dostarlimab can be used to treat at least mismatch repair deficient (dMMR) recurrent or endometrial cancer. In some embodiments of any of the aspects, the anti-PD1 binding domain specifically binds to at least one dostarlimab PD-1 epitope or portion of a dostarlimab PD-1 epitope comprising: the BC, C’ D and FG loops of PD-1, or at least residue R86 within the C’ D loop of PD-1 of SEQ ID NO: 776. In some embodiments of any of the aspects, dostarlimab comprises SEQ ID NO: 794 (heavy chain) and/or SEQ ID NO: 795 (light chain) . See e.g., Park et al. “Molecular basis of PD-1 blockade by dostarlimab, the FDA-approved antibody for cancer immunotherapy” , Biochemical and Biophysical Research Communications Volume 599, 9 April 2022, Pages 31-37, the contents of which are incorporated herein by reference in their entirety.

SEQ ID NO: 794, dostarlimab heavy chain, 443 aa, DRUGBANK Accession Number DB15627, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 799-801) :

SEQ ID NO: 795, dostarlimab light chain, 214 aa, DRUGBANK Accession Number DB15627, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 805-807) :

In some embodiments of any of the aspects, the anti-PD-1 binding domain has an equilibrium dissociation constant (K_D) between about 4.5E-9 M to about 8.0E-8 M towards human PD-1 (see e.g., Table 19) . In some embodiments of any of the aspects, the anti-PD-1 binding domain has an equilibrium dissociation constant (K_D) of about 1.0E-9 M, about 1.5E-9 M, about 2.0E-9 M, about 2.5E-9 M, about 3.0E-9 M, about 3.5E-9 M, about 4.0E-9 M, about 4.5E-9 M, about 5.0E-9 M, about 5.5E-9 M, about 6.0E-9 M, about 6.5E-9 M, about 7.0E-9 M, about 7.5E-9 M, about 8.0E-9 M, about 8.5E-9 M, about 9.0E-9 M, about 9.5E-9 M, about 1.0E-8 M, about 1.5E-8 M, about 2.0E-8 M, about 2.5E-8 M, about 3.0E-8 M, about 3.5E-8 M, about 4.0E-8 M, about 4.5E-8 M, about 5.0E-8 M, about 5.5E-8 M, about 6.0E-8 M, about 6.5E-8 M, about 7.0E-8 M, about 7.5E-8 M, or about 8.0E-8 M, towards human PD-1.

In some embodiments of any of the aspects, a bispecific antibody construct specific for PD-1 can comprise one or more (e.g., one, two, three, four, five, or six) CDRs of any one of the antibodies recited in Table 26 or Table 27. In some embodiments of any of the aspects, a bispecific antibody construct specific for PD-1 can comprise the six CDRs of any one of the antibodies recited in Table 26 or Table 27. In some embodiments of any of the aspects, a bispecific antibody construct specific for PD-1 can comprise the three heavy chain CDRs of any one of the antibodies recited in Table 26. In some embodiments of any of the aspects, a bispecific antibody construct specific for PD-1 can comprise the three light chain CDRs of any one of the antibodies recited in Table 27. In some embodiments of any of the aspects, a bispecific antibody construct specific for PD-1 can comprise the VH and/or VL domains of any one of the antibodies recited in Table 28 or encoded by a nucleic acid recited in Table 29. In some embodiments of any of the aspects, a bispecific antibody construct specific for PD-1 can comprise the VH and VL domains of any one of the antibodies recited in Table 28 or encoded by a nucleic acid recited in Table 29 or Table 36. Such antibody constructs are specifically contemplated for use in the methods and/or compositions described herein.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_H CDR1 comprising one of SEQ ID NOs: 547 or 550. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_H CDR2 comprising one of SEQ ID NOs: 548 or 551. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_H CDR3 comprising one of SEQ ID NOs: 549 or 552. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_L CDR1 comprising one of SEQ ID NOs: 556 or 559. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_L CDR2 comprising one of SEQ ID NOs: 557 or 560. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_L CDR3 comprising one of SEQ ID NOs: 558 or 561.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_H comprising one of SEQ ID NOs: 565 or 567 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 565 or 567, that maintains its function (e.g., PD-1 binding) .

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_L comprising one of SEQ ID NOs: 566 or 568 or an amino acid sequence that at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 566 or 568, that maintains its function (e.g., PD-1 binding) .

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 616-618, 624-632 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 616-618, 624-632, that maintains its function as a polypeptide (e.g., PD-1 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds PD-1 comprises a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 619-623, 633-638 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 619-623, 633-638, that maintains its function as a polypeptide (e.g., PD-1 binding) , or a codon-optimized version thereof.

In multiple aspects described herein are compositions comprising a first binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) and a second binding domain that specifically binds vascular endothelial growth factor (VEGF) . VEGF can also be referred to interchangeably as vascular permeability factor (VPF) . VEGF is a growth factor active in angiogenesis (the growth of blood vessels from pre-existing vasculature) , vasculogenesis (the de novo formation of the embryonic circulatory system) , and endothelial cell growth. VEGF induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis and induces permeabilization of blood vessels. VEGF binds to the FLT1/VEGFR1 and KDR/VEGFR2 receptors, heparan sulfate and heparin, and the NRP1/neuropilin-1 receptor. VEGF can contribute to disease. For example, solid cancers cannot grow beyond a limited size without an adequate blood supply; cancers that can express VEGF are able to grow and metastasize.

Anti-vascular endothelial growth factor therapy, also known as anti-VEGF therapy or medication, is the use of medications that block vascular endothelial growth factor. Anti-VEGF therapy can be done in the treatment of certain cancers and in age-related macular degeneration and can involve monoclonal antibodies such as bevacizumab.

The bispecific antibodies described herein can comprise a binding domain specific for VEGF that interferes with the binding between VEGF and its receptor (s) , and thus has anti-cancer effect, such as decreased angiogenesis to tumors. In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 and anti-VEGF bispecific antibody constructs as described herein) is capable of interfering with the interaction between VEGF and its receptor (s) , including but not limited to FLT1/VEGFR1 and KDR/VEGFR2 receptors, heparan sulfate and heparin, or the NRP1/neuropilin-1 receptor. In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 and anti-VEGF bispecific antibody constructs as described herein) is capable of decreasing cellular signaling downstream of at least one VEGF receptor.

In some embodiments of any of the aspects, the VEGF, specifically bound by a binding domain as described herein, comprises mammalian VEGF. In some embodiments of any of the aspects, the VEGF, specifically bound by a binding domain as described herein, comprises human VEGF. In some embodiments of any of the aspects, the VEGF, specifically bound by a binding domain as described herein, comprises human VEGF isoform A121 or human VEGF isoform A165, which are the two predominantly expressed VEGFA isoforms expressed in humans. In some embodiments of any of the aspects, the VEGF, specifically bound by a binding domain as described herein, comprises SEQ ID NO: 777, SEQ ID NO: 778, or SEQ ID NO: 786 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 777, SEQ ID NO: 778, or SEQ ID NO: 786 that maintains its function (e.g., angiogenesis, vasculogenesis, and/or endothelial cell growth) .

SEQ ID NO: 777, vascular endothelial growth factor (VEGF) A121, partial, Homo sapiens, 121 amino acids (aa) , GenBank: AMB36732.1; SEQ ID NO: 783 is bolded and double-underlined in residues 85 to 92 of SEQ ID NO: 777, which is a bevacizumab VEGF epitope:

SEQ ID NO: 778, vascular endothelial growth factor (VEGF) , isoform VEGF121, 147 amino acids (aa) , UNIPROT P15692-9, VEGFA_HUMAN; SEQ ID NO: 783 is bolded and double-underlined in residues 111 to 112 of SEQ ID NO: 778, which is a bevacizumab VEGF epitope:

SEQ ID NO: 786, vascular endothelial growth factor (VEGF) , isoform VEGF165, 191 amino acids (aa) , UNIPROT P15692-4, VEGFA_HUMAN; SEQ ID NO: 783 is bolded and double-underlined in residues 111 to 112 of SEQ ID NO: 786, which is a bevacizumab VEGF epitope:

In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_H domain that specifically binds VEGF. In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_L domain that specifically binds VEGF. In some embodiments of any of the aspects, the binding domain comprises the CDRs of a V_H/V_L domain pair that specifically binds VEGF. In some embodiments of any of the aspects, the binding domain comprises the CDRs of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds VEGF.

In some embodiments of any of the aspects, the binding domain specifically binds to a VEGF epitope or portion of a VEGF epitope. In some embodiments of any of the aspects, the VEGF epitope or portion thereof comprises sequential and/or non-sequential residues of SEQ ID NO: 777, SEQ ID NO: 778, or SEQ ID NO: 786, for example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30, or more sequential and/or non-sequential residues of SEQ ID NO: 777, SEQ ID NO: 778, or SEQ ID NO: 786. In some embodiments of any of the aspects, the anti-VEGF binding domain comprises bevacizumab, ranibizumab, or Aflibercept, or CDRs, VL domains, VH domains, or functional fragments thereof.

In some embodiments of any of the aspects, the binding domain that specifically binds VEGF comprises bevacizumab (AVASTIN) or CDRs, VL domains, VH domains, or functional fragments thereof. Bevacizumab can be used to treat at least colon cancer, lung cancer, ovarian cancer, glioblastoma, and renal-cell carcinoma. In some embodiments of any of the aspects, the anti-VEGF binding domain specifically binds to a bevacizumab VEGF epitope or portion of a bevacizumab VEGF epitope comprising the top of the β5–turn–β6 loop of VEGF or residues 85 to 92 of SEQ ID NO: 777 (PHQGQHIG, SEQ ID NO: 783) , which corresponds to residues 111 to 112 of SEQ ID NO: 778 or residues 111 to 112 of SEQ ID NO: 786; see e.g., Wentink et al. “Targeted vaccination against the bevacizumab binding site on VEGF using 3D-structured peptides elicits efficient antitumor activity” PNAS 113 (44) 12532-12537, the contents of which are incorporated herein by reference in its entirety.

In some embodiments of any of the aspects, the binding domain that specifically binds VEGF comprises ranibizumab (LUCENTIS) or CDRs, VL domains, VH domains, or functional fragments thereof. Ranibizumab can be used to treat at least macular degeneration, macular edema, diabetic macular edema, diabetic retinopathy, or myopic choroidal neovascularization. In some embodiments of any of the aspects, the anti-VEGF binding domain specifically binds to at least one ranibizumab VEGF epitope or portion of a ranibizumab VEGF epitope (see e.g., SEQ ID NO: 777, SEQ ID NO: 778, or SEQ ID NO: 786) . In some embodiments of any of the aspects, ranibizumab comprises SEQ ID NO: 808 (heavy chain) and/or SEQ ID NO: 809 (light chain) . See e.g., Magdelaine-Beuzelin et al. “Therapeutic antibodies in ophthalmology: old is new again. ” MAbs. 2010 Mar-Apr; 2 (2) : 176-80, the contents of which are incorporated herein by reference in their entirety.

SEQ ID NO: 808, ranibizumab heavy chain, 231 aa, DRUGBANK Accession Number DB01270, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 811-813) :

SEQ ID NO: 809, ranibizumab light chain, 214 aa, DRUGBANK Accession Number DB01270, CDRs bolded and double-underlined (see e.g., SEQ ID NOs: 814-816) :

In some embodiments of any of the aspects, the binding domain that specifically binds VEGF comprises Aflibercept (EYLEA) or functional fragments thereof. Aflibercept is a recombinant protein composed of the binding domains of two human vascular endothelial growth factor (VEGF) receptors, VEGFR1 and VEGFR2, fused with the Fc region of human immunoglobulin gamma 1 (IgG1) . Aflibercept can be used to treat at least colorectal cancer, diabetic macular edema, diabetic retinopathy, or retinopathy of prematurity. In some embodiments of any of the aspects, the anti-VEGF binding domain specifically binds to at least one Aflibercept VEGF epitope or portion of an Aflibercept VEGF epitope (see e.g., SEQ ID NO: 777, SEQ ID NO: 778, or SEQ ID NO: 786) . In some embodiments of any of the aspects, Aflibercept comprises SEQ ID NO: 810.

SEQ ID NO: 810, Aflibercept, 431 aa, DRUGBANK Accession Number DB08885

In some embodiments of any of the aspects, the anti-VEGF binding domain has an equilibrium dissociation constant (K_D) between about 2.5E-11 M to about 6.5E-11 M towards human VEGF (see e.g., Table 20) . In some embodiments of any of the aspects, the anti-VEGF binding domain has an equilibrium dissociation constant (K_D) of about 1.0E-11 M, about 1.5E-11 M, about 2.0E-11 M, about 2.5E-11 M, about 3.0E-11 M, about 3.5E-11 M, about 4.0E-11 M, about 4.5E-11 M, about 5.0E-11 M, about 5.5E-11 M, about 6.0E-11 M, about 6.5E-11 M, about 7.0E-11 M, about 7.5E-11 M, about 8.0E-11 M, about 8.5E-11 M, about 9.0E-11 M, or about 9.5E-11 M, towards human VEGF.

In some embodiments of any of the aspects, a bispecific antibody construct specific for VEGF can comprise one or more (e.g., one, two, three, four, five, or six) CDRs of any one of the antibodies recited in Table 26, Table 27, or Table 35. In some embodiments of any of the aspects, a bispecific antibody construct specific for VEGF can comprise the six CDRs of any one of the antibodies recited in Table 26, Table 27, or Table 35. In some embodiments of any of the aspects, a bispecific antibody construct specific for VEGF can comprise the three heavy chain CDRs of any one of the antibodies recited in Table 26 or Table 35. In some embodiments of any of the aspects, a bispecific antibody construct specific for VEGF can comprise the three light chain CDRs of any one of the antibodies recited in Table 27 or Table 35. In some embodiments of any of the aspects, a bispecific antibody construct specific for VEGF can comprise the VH and/or VL domains of any one of the antibodies recited in Table 28 or Table 36 or encoded by a nucleic acid recited in Table 29 or Table 36. In some embodiments of any of the aspects, a bispecific antibody construct specific for VEGF can comprise the VH and VL domains of any one of the antibodies recited in Table 28 or Table 36 or encoded by a nucleic acid recited in Table 29 or Table 36. Such antibody constructs are specifically contemplated for use in the methods and/or compositions described herein.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_H CDR1 comprising SEQ ID NO: 553. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_H CDR2 comprising SEQ ID NO: 554. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_H CDR3 comprising SEQ ID NO: 555. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_L CDR1 comprising SEQ ID NO: 562. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_L CDR2 comprising SEQ ID NO: 563. In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_L CDR3 comprising SEQ ID NO: 564.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_H comprising SEQ ID NO: 569 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 569, that maintains its function (e.g., VEGF binding) .

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_L comprising SEQ ID NO: 570 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 570, that maintains its function (e.g., VEGF binding) .

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 639-647 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 639-647, that maintains its function as a polypeptide (e.g., VEGF binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the V_H/V_L domain pair that specifically binds VEGF comprises a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 648-654 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 648-654, that maintains its function as a polypeptide (e.g., VEGF binding) , or a codon-optimized version thereof.

Table 35: Exemplary anti-PD-1 or anti-VEGF antibody CDR amino acid sequences; “#” refers to the corresponding SEQ ID NO.

Table 36: Exemplary anti-PD-1 or anti-VEGF antibody VH and VL amino acid and nucleic acid sequences; “#” refers to the corresponding SEQ ID NO.

Bispecific Antibodies

The compositions described can comprise a bispecific antibody construct, such as anti-LILRB2/anti-PD-1 or anti-LILRB2/anti-VEGF bispecific antibodies as described further herein. The term "bispecific antibody" or “bispecific antibody construct” refers to an antibody having the capacity to bind to two distinct epitopes either on a single antigen or two different antigens (see e.g., WO 2014/209804; Brinkmann and Kontermann (2017) MAbs 9 (2) : 182–212, especially Figure 2 “The zoo of bispecific antibody formats; ” incorporated herein by reference in their entireties) . As used herein, "epitope" or "antigenic determinant" refers to a site on an antigen to which an antibody specifically binds. Epitopes can be formed both from contiguous amino acids (linear epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (conformational epitopes) . Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3 amino acids, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996) . A preferred method for epitope mapping is surface plasmon resonance.

In one aspect, described herein is a composition comprising: (a) a first binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) ; and (b) a second binding domain that specifically binds programmed cell death protein 1 (PD-1) . In one aspect, described herein is a composition comprising: (a) a first binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) ; and (b) a second binding domain that specifically binds vascular endothelial growth factor (VEGF) .

In some embodiments, the bispecific antibody construct is selected from the group consisting of:tandem scFv (taFv or scFv₂) , diabody, dAb₂A/HH₂, knob-into-holes bispecific derivative, SEED-IgG, heteroFc-scFv, Fab-scFv, scFv-Jun/Fos, Fab'-Jun/Fos, tribody, DNL-F (ab) ₃, scFv₃-CH1/CL, Fab-scFv₂, IgG-scFab, IgG-scFv, scFv-IgG, IgG-VHH, VHH-IgG, scFv₂-Fc, F (ab') ₂-scFv₂, scDB-Fc, scDb-CH₃, Db-Fc, scFv₂-H/L, DVD-Ig, tandAb, scFv-dhlx-scFv, dAb2-IgG, dAb-IgG, and dAb-Fc-dAb constructs. In some embodiments, the bispecific antibody construct contains more than one antigen-binding domain for each antigen. For example, additional V_H and/or V_L domains can be fused to the N-terminus of the V_H and/or V_L domains of an existing antibody, effectively arranging the antigen-binding sites in tandem.

In one aspect, described herein is a bispecific antibody construct comprising: (a) a first binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; and (b) a second binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds PD-1.

In one aspect, described herein is a bispecific antibody construct comprising: (a) a first binding domain comprising the CDRs of a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2; and (b) a second binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds PD-1.

In one aspect, described herein is a bispecific antibody construct comprising: (a) a first binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; and (b) a second binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds VEGF.

In one aspect, described herein is a bispecific antibody construct comprising: (a) a first binding domain comprising the CDRs of a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2; and (b) a second binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds VEGF.

In one aspect, described herein is a bispecific antibody construct comprises: (a) a first binding domain comprising a single-chain fragment variable (scFv) comprising the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; and (b) a second binding domain comprising an immunoglobulin heavy chain and immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds PD-1.

In one aspect, described herein is a bispecific antibody construct comprises: (a) a first binding domain comprising a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) comprising the CDRs of a V_H domain that specifically binds LILRB2; and (b) a second binding domain comprising an immunoglobulin heavy chain and immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds PD-1.

In one aspect, described herein is a bispecific antibody construct comprises: (a) a first binding domain comprising a single-chain fragment variable (scFv) comprising the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; and (b) a second binding domain comprising an immunoglobulin heavy chain and immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds VEGF.

In one aspect, described herein is a bispecific antibody construct comprises: (a) a first binding domain comprising a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) comprising the CDRs of a V_H domain that specifically binds LILRB2; and (b) a second binding domain comprising an immunoglobulin heavy chain and immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds VEGF.

In some embodiments, the bispecific antibody construct comprises an scFv specific to a first epitope linked to the C-terminus of a heavy chain of an antibody specific to a second epitope (i.e., “HC-C (scFv) ” ) . In some embodiments, the bispecific antibody construct comprises a VHH specific to a first epitope linked to the C-terminus of the heavy chain of an antibody specific to a second epitope (i.e., “HC-C (VHH) ” ) . In some embodiments, the bispecific antibody construct comprises an scFv specific to a first epitope linked to the C-terminus of the light chain of an antibody specific to a second epitope (i.e., “LC-C (scFv) ” ) . In some embodiments, the bispecific antibody construct comprises a VHH specific to a first epitope linked to the C-terminus of the light chain of an antibody specific to a second epitope (i.e., “LC-C (VHH) ” ) . In some embodiments, the bispecific antibody construct comprises an scFv specific to a first epitope linked to the N-terminus of the heavy chain of an antibody specific to a second epitope (i.e., “HC-N (scFv) ” ) . In some embodiments, the bispecific antibody construct comprises a VHH specific to a first epitope linked to the N-terminus of the heavy chain of an antibody specific to a second epitope (i.e., “HC-N (VHH) ” ) . In some embodiments, the bispecific antibody construct comprises an scFv specific to a first epitope linked to the N-terminus of the light chain of an antibody specific to a second epitope (i.e., “LC-N (scFv) ” ) . In some embodiments, the bispecific antibody construct comprises a VHH specific to a first epitope linked to the N-terminus of the light chain of an antibody specific to a second epitope (i.e., “LC-N (VHH) ” ) .

In some embodiments, the bispecific antibody construct comprises an anti-LILRB2 scFv linked to the C-terminus of the heavy chain (i.e., “HC-C (scFv) ” ) of an anti-PD-1 (see e.g., Fig. 9A) or anti-VEGF (see e.g., Fig. 10A) antibody. Non-limiting examples of the anti-LILRB2/anti-PD-1 HC-C (scFv) bispecific format include Bis-M17, Bis-M17-1, Bis-M23, Bis-M29, and Bis-M23-1, as described further herein (see e.g., Fig. 9A) . Non-limiting examples of the anti-LILRB2/anti-VEGF HC-C (scFv) bispecific format include Bis-M18, Bis-M18-1, and Bis-M24, as described further herein (see e.g., Fig. 10A) .

In some embodiments, the bispecific antibody construct comprises an anti-LILRB2 VHH linked to the C-terminus of the heavy chain (i.e., “HC-C (VHH) ” ) of an anti-PD-1 (see e.g., Fig. 9B) or anti-VEGF (see e.g., Fig. 10B) antibody. Non-limiting examples of the anti-LILRB2/anti-PD-1 HC-C (VHH) bispecific format include Bis-M13 and Bis-M13-1, as described further herein (see e.g., Fig. 9B) . Non-limiting examples of the anti-LILRB2/anti-VEGF HC-C (VHH) bispecific format include Bis-M14 and Bis-M14-1, as described further herein (see e.g., Fig. 10B) .

In some embodiments, the bispecific antibody construct comprises an anti-LILRB2 scFv linked to the C-terminus of the light chain (i.e., “LC-C (scFv) ” ) of an anti-PD-1 (see e.g., Fig. 9C) or anti-VEGF (see e.g., Fig. 10C) antibody. Non-limiting examples of the anti-LILRB2/anti-PD-1 LC-C (scFv) bispecific format include Bis-M28, Bis-M30, Bis-M30-1, Bis-M31, and Bis-M31-1, as described further herein (see e.g., Fig. 9C) . Non-limiting examples of the anti-LILRB2/anti-VEGF LC-C (scFv) bispecific format include Bis-M27, Bis-M32, and Bis-M32-1, as described further herein (see e.g., Fig. 10C) .

In some embodiments, the bispecific antibody construct comprises an anti-LILRB2 VHH linked to the C-terminus of the light chain (i.e., “LC-C (VHH) ” ) of an anti-PD-1 (see e.g., Fig. 9D) or anti-VEGF (see e.g., Fig. 10D) antibody. Non-limiting examples of the anti-LILRB2/anti-PD-1 LC-C (VHH) bispecific format include Bis-M33, Bis-M33-1, Bis-M34, and Bis-M34-1, as described further herein (see e.g., Fig. 9D) . Non-limiting examples of the anti-LILRB2/anti-VEGF LC-C (VHH) bispecific format include Bis-M35 and Bis-M35-1, as described further herein (see e.g., Fig. 10D) .

In some embodiments, the bispecific antibody construct comprises an anti-LILRB2 VHH linked to the N-terminus of the heavy chain (i.e., “HC-N (VHH) ” ) of an anti-PD-1 (see e.g., Fig. 9E) or anti-VEGF (see e.g., Fig. 10E) antibody. Non-limiting examples of the anti-LILRB2/anti-PD-1 HC-N (VHH) bispecific format include Bis-M16 and Bis-M16-1, as described further herein (see e.g., Fig. 9E) . Non-limiting examples of the anti-LILRB2/anti-VEGF HC-N (VHH) bispecific format include Bis-M15 and Bis-M15-1, as described further herein (see e.g., Fig. 10E) .

In some embodiments, the bispecific antibody construct comprises an anti-LILRB2 VHH linked to the N-terminus of the light chain (i.e., “LC-N (VHH) ” ) of an anti-PD-1 or anti-VEGF antibody. In some embodiments, the bispecific antibody construct comprises an anti-LILRB2 scFv linked to the N-terminus of the light chain (i.e., “LC-N (scFv) ” ) of an anti-PD-1 or anti-VEGF antibody. In some embodiments, the bispecific antibody construct comprises an anti-LILRB2 scFv linked to the N-terminus of the heavy chain (i.e., “HC-N (scFv) ” ) of an anti-PD-1 or anti-VEGF antibody.

In some embodiments, the bispecific antibody construct comprises an anti-PD-1 or anti-VEGF scFv linked to the C-terminus of the heavy chain (i.e., “HC-C (scFv) ” ) of an anti-LILRB2 antibody. In some embodiments, the bispecific antibody construct comprises an anti-PD-1 or anti-VEGF VHH linked to the C-terminus of the heavy chain (i.e., “HC-C (VHH) ” ) of an anti-LILRB2 antibody. In some embodiments, the bispecific antibody construct comprises an anti-PD-1 or anti-VEGF scFv linked to the C-terminus of the light chain (i.e., “LC-C (scFv) ” ) of an anti-LILRB2 antibody. In some embodiments, the bispecific antibody construct comprises an anti-PD-1 or anti-VEGF VHH linked to the C-terminus of the light chain (i.e., “LC-C (VHH) ” ) of an anti-LILRB2 antibody. In some embodiments, the bispecific antibody construct comprises an anti-PD-1 or anti-VEGF VHH linked to the N-terminus of the heavy chain (i.e., “HC-N (VHH) ” ) of an anti-LILRB2 antibody. In some embodiments, the bispecific antibody construct comprises an anti-PD-1 or anti-VEGF VHH linked to the N-terminus of the light chain (i.e., “LC-N (VHH) ” ) of an anti-LILRB2 antibody. In some embodiments, the bispecific antibody construct comprises an anti-PD-1 or anti-VEGF scFv linked to the N-terminus of the light chain (i.e., “LC-N (scFv) ” ) of an anti-LILRB2 antibody. In some embodiments, the bispecific antibody construct comprises an anti-PD-1 or anti-VEGF scFv linked to the N-terminus of the heavy chain (i.e., “HC-N (scFv) ” ) of an anti-LILRB2 antibody.

In some embodiments, the bispecific antibody construct comprises a heavy chain selected from Table 37A. The exemplary heavy chain components in Table 37A can be in any order from N to C terminus. For example, in a two-component heavy chain, the bispecific heavy chain can comprise from N to C terminus: heavy chain, scFV; heavy chain, VHH; scFV, heavy chain; or VHH, heavy chain. In some embodiments, the bispecific antibody construct comprises a light chain selected from Table 37B. The exemplary light chain components in Table 37B can be in any order from N to C terminus. For example, in a two-component light chain, the bispecific light chain can comprise from N to C terminus: light chain, scFV; light chain, VHH; scFV, light chain; or VHH, light chain. In some embodiments, the bispecific antibody construct comprises any combination of a heavy chain selected from Table 37A and a light chain selected from Table 37B. In some embodiments, the bispecific antibody construct comprises any combination of two heavy chains selected from Table 37A, which can be the same or different, and two light chains selected from Table 37B, which can be the same or different.

Exemplary anti-LILRB2 x anti-PD-1 bispecific antibody sequences are provided in Table 38. In some embodiments of any of the aspects, a bispecific antibody construct as described herein comprises a heavy chain selected from Table 38. In some embodiments of any of the aspects, a bispecific antibody construct as described herein comprises a light chain selected from Table 38. In some embodiments of any of the aspects, a bispecific antibody construct as described herein comprises a heavy chain and a light chain selected from Table 38. In some embodiments of any of the aspects, a bispecific antibody construct as described herein comprises a pair of heavy chains and a pair of light chains selected from Table 38.

Table 38: Exemplary anti-LILRB2 x anti-PD-1 bispecific antibody heavy and light chain amino acid and nucleic acid sequences; “#” refers to the corresponding SEQ ID NO.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody construct comprises an immunoglobulin heavy chain comprising one of SEQ ID NOs: 655, 657, 659, 661, 671, 673, 675, 677, 683, 689, 691, 693, 695, 697, 703, 705, 707, 709, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 655, 657, 659, 661, 671, 673, 675, 677, 683, 689, 691, 693, 695, 697, 703, 705, 707, 709, that maintains its function (e.g., LILRB2 and/or PD-1 binding) .

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody construct comprises an immunoglobulin light chain comprising one of SEQ ID NOs: 656, 658, 660, 662, 672, 674, 676, 678, 684, 690, 692, 694, 696, 698, 704, 706, 708, 710, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 656, 658, 660, 662, 672, 674, 676, 678, 684, 690, 692, 694, 696, 698, 704, 706, 708, 710, that maintains its function (e.g., LILRB2 and/or PD-1 binding) .

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody construct comprises an immunoglobulin heavy chain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 715, 717, 719, 721, 731, 733, 735, 737, 743, 749, 751, 753, 755, 757, 763, 765, 767, 769 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 715, 717, 719, 721, 731, 733, 735, 737, 743, 749, 751, 753, 755, 757, 763, 765, 767, 769, that maintains its function as a polypeptide (e.g., LILRB2 and/or PD-1 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody construct comprises an immunoglobulin light chain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 716, 718, 720, 722, 732, 734, 736, 738, 744, 750, 752, 754, 756, 758, 764, 766, 768, 770 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 716, 718, 720, 722, 732, 734, 736, 738, 744, 750, 752, 754, 756, 758, 764, 766, 768, 770, that maintains its function as a polypeptide (e.g., LILRB2 and/or PD-1 binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody construct is selected from the group consisting of: Bis-M13, Bis-M13-1, Bis-M16, Bis-M16-1, Bis-M17, Bis-M17-1, Bis-M23, Bis-M29, Bis-M23-1, Bis-M28, Bis-M30, Bis-M30-1, Bis-M31, Bis-M31-1, Bis-M33, Bis-M33-1, Bis-M34, and Bis-M34-1. In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody construct is selected from Bis-M23-1 and Bis-M28.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody is Bis-M23-1 comprising: (a) an immunoglobulin heavy chain comprising SEQ ID NO: 683 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 683, that maintains its function (e.g., LILRB2 and/or PD-1 binding) ; and (b) an immunoglobulin light chain comprising SEQ ID NO: 684, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 684, that maintains its function (e.g., LILRB2 and/or PD-1 binding) .

In some embodiments of any of the aspects, the anti-LILRB2 and anti-PD-1 bispecific antibody is Bis-M28 comprising: (a) an immunoglobulin heavy chain comprising SEQ ID NO: 689, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 689, that maintains its function (e.g., LILRB2 and/or PD-1 binding) ; and (b) an immunoglobulin light chain comprising SEQ ID NO: 690, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 690, that maintains its function (e.g., LILRB2 and/or PD-1 binding) .

Exemplary anti-LILRB2 x anti-VEGF bispecific antibody sequences are provided in Table 39. In some embodiments of any of the aspects, a bispecific antibody construct as described herein comprises a heavy chain selected from Table 39. In some embodiments of any of the aspects, a bispecific antibody construct as described herein comprises a light chain selected from Table 39. In some embodiments of any of the aspects, a bispecific antibody construct as described herein comprises a heavy chain and a light chain selected from Table 39. In some embodiments of any of the aspects, a bispecific antibody construct as described herein comprises a pair of heavy chains and a pair of light chains selected from Table 39.

Table 39: Exemplary anti-LILRB2 x anti-VEGF bispecific antibody heavy and light chain amino acid and nucleic acid sequences; “#” refers to the corresponding SEQ ID NO.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody construct comprises an immunoglobulin heavy chain comprising one of SEQ ID NOs: 663, 665, 667, 669, 679, 681, 685, 687, 699, 701, 711, 713, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 663, 665, 667, 669, 679, 681, 685, 687, 699, 701, 711, 713, that maintains its function (e.g., LILRB2 and/or VEGF binding) .

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody construct comprises an immunoglobulin light chain comprising one of SEQ ID NOs: 664, 666, 668, 670, 680, 682, 686, 688, 700, 702, 712, 714, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 664, 666, 668, 670, 680, 682, 686, 688, 700, 702, 712, 714, that maintains its function (e.g., LILRB2 and/or VEGF binding) .

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody construct comprises an immunoglobulin heavy chain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 723, 725, 727, 729, 739, 741, 745, 747, 759, 761, 771, 773 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 723, 725, 727, 729, 739, 741, 745, 747, 759, 761, 771, 773, that maintains its function as a polypeptide (e.g., LILRB2 and/or VEGF binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody construct comprises an immunoglobulin light chain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 724, 726, 728, 730, 740, 742, 746, 748, 760, 762, 772, 774 or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to one of SEQ ID NOs: 724, 726, 728, 730, 740, 742, 746, 748, 760, 762, 772, 774, that maintains its function as a polypeptide (e.g., LILRB2 and/or VEGF binding) , or a codon-optimized version thereof.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody construct is selected from the group consisting of: Bis-M14, Bis-M14-1, Bis-M15, Bis-M15-1, Bis-M18, Bis-M18-1, Bis-M24, Bis-M27, Bis-M32, Bis-M32-1, Bis-M35, and Bis-M35-1. In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody construct is selected from Bis-M24 and Bis-M27.

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody is Bis-M24 comprising: (a) an immunoglobulin heavy chain comprising SEQ ID NO: 685 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 685, that maintains its function (e.g., LILRB2 and/or VEGF binding) ; and (b) an immunoglobulin light chain comprising SEQ ID NO: 686 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 686, that maintains its function (e.g., LILRB2 and/or VEGF binding) .

In some embodiments of any of the aspects, the anti-LILRB2 and anti-VEGF bispecific antibody is Bis-M27 comprising: (a) an immunoglobulin heavy chain comprising SEQ ID NO: 687, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 687, that maintains its function (e.g., LILRB2 and/or VEGF binding) ; and (b) an immunoglobulin light chain comprising SEQ ID NO: 688, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to SEQ ID NO: 688, that maintains its function (e.g., LILRB2 and/or VEGF binding) .

In some embodiments of any of the aspects, the bispecific antibody construct comprises a dual-variable-domain antibody (DvD-Ig) (see e.g., Tarcsa, E. et al. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 171 -185 (2011) ) . One advantage of the DvD-Ig format is that the respective V_H/V_L domain pairs can only associate with their cognate partners, as opposed to the random assortment of V_H and V_L domains that can occur in some other bispecific formats. In the DvD-Ig format, only cognate V_H/V_L pairs will form, and all such pairs will be competent to bind their respective antigens. DvD-Ig design and production is well known in the art (see e.g., US Patent No. 7,612,181, which is incorporated herein by reference in its entirety) .

In embodiments including DvD-Ig bispecific antibodies, the V_H of the first V_H/V_L domain pair is joined to the V_H of the second V_H/V_L domain pair by a linker (e.g., V_H1-V_H2) and the V_L of the first V_H/V_L domain pair is joined to the V_L of the second V_H/V_L domain pair by a linker (e.g., V_L1-V_L2) . The linker can be a chemical linker or a polypeptide linker. The linker can be a “short linker” or a “long linker” , non-limiting examples of which are known in the art (see e.g., US20210139582A1, the contents of which are incorporated herein by reference in their entirety) . The linker chosen for joining the V_H of the first V_H/V_L domain pair to the V_H of the second V_H/V_L domain pair can be the same or different as the linker chosen for joining the V_L of the first V_H/V_L domain pair to the V_L of the second V_H/V_L domain pair. In some embodiments, the first V_H/V_L domain pair is on the amino terminus of the bispecific antibody construct. In other embodiments, the second V_H/V_L domain pair is on the amino terminus of the bispecific antibody construct.

Bispecific antibodies can be produced via biological methods, such as somatic hybridization; or genetic methods, such as the expression of a non-native DNA sequence encoding the desired antibody structure in an organism; chemical methods, such as chemical conjugation of two antibodies; or a combination thereof (see e.g., Kontermann, R. E. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 1 -28 (2011) ) .

Chemically conjugated bispecific antibodies arise from the chemical coupling of two existing antibodies or antibody fragments. Typical couplings include cross-linking two different full-length antibodies, cross-linking two different Fab'fragments to produce a bispecific F (ab') 2, and cross-linking a F (ab') 2 fragment with a different Fab'fragment to produce a bispecific F (ab') 3. For chemical conjugation, oxidative reassociation strategies can be used. Current methodologies include the use of the homo-or heterobifunctional cross-linking reagents.

Heterobifunctional cross-linking reagents have reactivity toward two distinct reactive groups on, for example, antibody molecules. Examples of heterobifunctional cross-linking reagents include SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate) , SATA (succinimidyl acetylthioacetate) , SMCC (succinimidyl trans-4- (maleimidylmethyl) cyclohexane-1 -carboxylate) , EDAC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) , PEAS (N- ( (2-pyridyldithio) ethyl) -4-azidosalicylamide) , ATFB, SE (4-azido-2, 3, 5, 6-tetrafluorobenzoic acid, succinimidyl ester) , benzophenone-4-maleimide, benzophenone-4-isothiocyanate, 4-benzoylbenzoic acid, succinimidyl ester, iodoacetamide azide, iodoacetamide alkyne, Click-iT maleimide DIBO alkyne, azido (PEO) 4 propionic acid, succinimidyl ester, alkyne, succinimidyl ester, Click-iT succinimidyl ester DIBO alkyne, Sulfo-SBED (Sulfo-N-hydroxysuccinimidyl-2- (6- [biotinamido] -2- (p-azido benzamido) -hexanoamido) ethyl-1 , 3'-dithioproprionate) , photoreactive amino acids {e.g., L-Photo-Leucine and L-Photo-Methionine) , NHS-haloacetyl crosslinkers such as, for example, Sulfo-SIAB, SIAB, SBAP, SIA, NHS-maleimide crosslinkers such as, for example, Sulfo-SMCC, SM (PEG) n series crosslinkers, SMCC, LC-SMCC, Sulfo-EMCS, EMCS, Sulfo-GMBS, GMBS, Sulfo-KMUS, Sulfo-MBS, MBS, Sulfo-SMPB, SMPB, AMAS, BMPS, SMPH, PEG12-SPDP, PEG4-SPDP, Sulfo-LC-SPDP, LC-SPDP, SMPT, DCC (N, N'-Dicyclohexylcarbodiimide) , EDC (1 -Ethyl-3- (3-dimethylaminopropyl) carbodiimide) , NHS (N-hydroxysuccinimide) , Sulfo-NHS (N-hydroxysulfosuccinimide) , BMPH, EMCH, KMUH, MPBH, PDPH, and PMPI.

Homobifunctional cross-linking reagents have reactivity toward the same reactive group on a molecule, for example, an antibody. Examples of homobifunctional cross-linking reagents include DTNB (5, 5'-dithiobis (2-nitrobenzoic acid) , o-PDM (o-phenylenedimaleimide) , DMA (dimethyl adipimidate) , DMP (dimethyl pimelimidate) , DMS (dimethyl suberimidate) , DTBP (dithiobispropionimidate) , BS (PEG) 5, BS (PEG) 9, BS3, BSOCOES, DSG, DSP, DSS, DST, DTSSP, EGS, Sulfo-EGS, TSAT, DFDNB, BM (PEG) n crosslinkers, BMB, BMDB, BMH, BMOE, DTME, and TMEA.

Somatic hybridization is the fusion of two distinct hybridoma (afusion of B cells that produce a specific antibody and myeloma cells) cell lines, producing a quadroma capable of generating two different antibody heavy (VHA and VHB) and light chains (VLA and VLB) . (Kontermann, R. E. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 1 -28 (2011) ) . These heavy and light chains combine randomly within the cell, resulting in bispecific antibodies (a VHA combined with a VLA and a VHB combined with a VLB) , as well as some nonfunctional (e.g., two VHAs combined with two VLBs) and monospecific (two VHAs combined with two VLAs) antibodies. The bispecific antibodies can then be purified using, for example, two different affinity chromatography columns. Similar to monospecific antibodies, bispecific antibodies can also contain an Fc region that elicits Fc-mediated effects downstream of antigen binding. These effects can be reduced by, for example, proteolytically cleaving the Fc region from the bispecific antibody by pepsin digestion, resulting in bispecific F (ab') 2 molecules.

Bispecific antibodies can also be generated via genetic means, e.g., in vitro expression of a plasmid containing a DNA sequence corresponding to the desired antibody structure. See, e.g., Kontermann, R. E. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 1 -28 (2011) . Such bispecific antibodies are discussed in greater detail herein.

A bispecific antibody can be bivalent, trivalent, or tetravalent. As used herein, "valent" , "valence" , "valencies" , or other grammatical variations thereof, mean the number of antigen binding sites in an antibody molecule or construct. These antigen recognition sites can recognize the same epitope or different epitopes. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J Immunol 148: 1547, Pack and Pluckthun (1992) Biochemistry 31 : 1579, Hollinger et al., 1993, Gruber et al. (1994) J lmmunol: 5368, Zhu et al. (1997) Protein Sci 6: 781, Hu et al. (1996) Cancer Res. 56: 3055, Adams et al. (1993) Cancer Res. 53: 4026, and McCartney, et al. (1995) Protein Eng. 8: 301. Trivalent bispecific antibodies and tetravalent bispecific antibodies are also known in the art. See, e.g., Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 199-216 (2011) . A bispecific antibody can also have valencies higher than 4. Such antibodies can be generated by, for example, dock and lock conjugation method. (Chang, C. -H. et al. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 199-216 (2011) ) .

Variable regions of antibodies can be isolated as single-chain Fv (scFv) or Fab fragments. ScFv fragments are composed of VH and VL domains linked by a short, e.g., 10-25 amino acid, linker. Once isolated, scFv fragments can be genetically linked with a flexible peptide linker such as, for example, one or more repeats of Ala-Ala-Ala, Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 784) , etc. The resultant peptide, a tandem scFv (taFv or scFv2) can be arranged in various ways, with V_H-V_L or V_L-V_H ordering for each scFv of the taFv. (Kontermann, R. E. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 1 -28 (2011) ) .

Bispecific diabodies are another form of bispecific antibody construct. In contrast to tandem scFvs (taFvs) , diabodies are composed of two separate polypeptide chains from, for example, antibodies A and B, each chain bearing two variable domains (V_HA-V_LB and V_HB-V_LA or V_LA-V_HB and V_LB-V_HA) . The linkers joining the variable domains are short (e.g., about five amino acids) , preventing the association of V_H and V_L domains on the same chain, and promoting the association of V_H and V_L domains on different chains. Heterodimers that form are functional against both target antigens, (such as, e.g., V_HA-V_LB with V_HB-V_LA or V_LA-V_HB with V_LB-V_HA) ; however, homodimers can also form (such as, e.g., V_HA-V_LB with V_HA-V_LB, V_HB-V_LA with V_HB-V_LA, etc. ) , leading to nonfunctional molecules. Several strategies exist to prevent homodimerization, including the introduction of disulfide bonds to covalently join the two polypeptide chains, modification of the polypeptide chains to include large amino acids on one chain and small amino acids on the other (e.g., knobs-into-holes structures, discussed below) , and addition of cysteine residues at C-terminal extensions. Another strategy is to join the two polypeptide chains by a linker sequence, producing a single-chain diabody molecule (scDb) that exhibits a more compact structure than a taFv. ScDbs or Dbs can also be fused to the IgG1 C_H3 domain or the Fc region, producing di-diabodies. Examples of di-diabodies include, but are not limited to, scDb-Fc, Db-Fc, scDb-Chi3, and Db-Chi3. Additionally, scDbs can be used to make tetravalent bispecific molecules. By shortening the linker sequence of scDbs from about 15 amino acids to about 5 amino acids, dimeric single-chain diabody molecules result, known as TandAbs (Muller, D. and Kontermann, R. E. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 83-100 (2011) ) .

Yet another strategy for generating a bispecific antibody includes fusing heterodimerizing zinc peptides to the C-termini of the antibody molecules (scFvs or Fabs) . A non-limiting example of this strategy is the use of antibody fragments linked to jun-fos leucine zippers (e.g. scFv-Jun/Fos and Fab'-Jun/Fos) .

An additional method for generating a bispecific antibody molecule includes derivatizing two antibodies with different antigen binding moieties with biotin and then linking the two antibodies via streptavidin, followed by purification and isolation of the resultant bispecific antibody.

Constant immunoglobulin domains can also be used to promote heterodimerization of two polypeptide chains (IgG-like antibodies, discussed below) . Non-limiting examples of this type of approach to making a bispecific antibody include the introduction of knobs-into-holes structures into the two polypeptides and utilization of the naturally occurring heterodimerization of the C_L and C_H domains (Kontermann, R. E. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 1 -28 (2011) ) .

Additional types of bispecific antibodies include those that contain more than one antigen-binding site for each antigen. As described previously, additional V_H and V_L domains can be fused to the N-terminus of the V_H and V_L domains of an existing antibody, effectively arranging the antigen-binding sites in tandem. The HC-C (scFv) , HC-C (VHH) , LC-C (scFv) , LC-C (VHH) , HC-N (scFv) , HC-N (VHH) , LC-N (scFv) , LC-N (VHH) , and DvD-Ig formats discussed above are non-limiting examples of bispecific antibody constructs that position two different antigen-binding domains on each arm of an Ig construct. If so desired, additional binding domains can be added to the N-terminal or C-terminal ends of the constructs. (see e.g., Tarcsa, E. et al. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 171 -185 (2011) ) .

Because many antibodies approved for therapy have been IgG or IgG-like, one embodiment the bispecific construct as described herein can be engineered to be IgG-like, to the extent that they can have an Fc domain. Similar to diabodies that require heterodimerization of engineered polypeptide chains, IgG-like antibodies can also require heterodimerization to prevent the interaction of like heavy chains or heavy chains and light chains from two antibodies of different specificity (see e.g., Jin, P. and Zhu, Z. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 151 -169 (2011) ) .

So-called “knobs-into-holes” structures facilitate heterodimerization of polypeptide chains by introducing large amino acids (knobs) into one chain of a desired heterodimer and small amino acids (holes) into the other chain of the desired heterodimer. Steric interactions will favor the interaction of the knobs with holes, rather than knobs with knobs or holes with holes. In the context of bispecific IgG-like antibodies, like heavy chains can be prevented from homodimerizing by the introduction of knobs-into-holes structures into the C_H3 domain of the Fc region. Similarly, promoting the interaction of heavy chains and light chains specific to the same antigen can be accomplished by engineering knobs-into-holes structures at the V_H-V_L interface. Other examples of knobs-into-holes structures exist and the examples discussed above should not be construed to be limiting. Other methods to promote heterodimerization of Fc regions include engineering charge polarity into Fc domains (see e.g., Gunasekaran et al., 2010) and SEED technology (SEED-IgG) (see e.g., Davis et al, 2010) .

Additional heterodimerized IgG-like antibodies include, but are not limited to, heteroFc-scFvs, Fab-scFvs, IgG-scFv, and scFv-IgG. HeteroFc-scFvs link two distinct scFvs to heterodimerizable Fc domains while Fab-scFvs contain a Fab domain specific to one epitope linked to an scFv specific to a different epitope. IgG-scFv and scFv-IgG are Ig-like antibodies that have scFvs linked to their C-termini and N-termini, respectively (Kontermann, R. E. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 151 -169 (2011) ) .

Though most naturally occurring antibodies are composed of heavy chains and light chains, camelids (e.g. camels, dromedaries, llamas, and alpacas) and some sharks and other cartilaginous fish produce antibodies that consist only of heavy chains. These antibodies bind antigenic epitopes using a single variable domain known as V_HH. When produced in Escherichia coli, these molecules are termed single domain antibodies (dAbs) . One application of dAbs in bispecific antibodies is to link two different dAbs together to form dAb2S (V_HH2s) . dAbs can also be applied to IgG-like bispecific antibodies. Examples of this include, but are not limited to, dAb2-IgGs, dAb-IgGs, and dAb-Fc-dAbs. dAb2-IgGs have a similar structure to intact antibodies, but with dAbs linked to the N-terminal end of the molecule. dAb-IgGs are intact antibodies specific for one epitope with a single dAb specific for another epitope linked to the N-termini or C-termini of the heavy chains. Lastly, dAb-Fc-dAbs are Fc domains with dAbs specific for one epitope linked to the N-termini and dAbs specific for another epitope linked to the C-termini (Chames, P. and Baty, D. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 101 -1 14 (2011) ) .

Several types of trivalent antibodies have been developed. Tribodies are composed of three distinct scFv regions joined by linker sequences approximately 20 amino acids in length. Tribodies utilize the natural in vivo heterodimerization of the heavy chain (C_H1 domain) and light chain (C_L domain) to form a scaffold on which multiple scFvs can be added. For example, a scFv specific to one antigen can be linked to a C_H1 domain, which is also linked to a scFv specific to another antigen and this chain can interact with another chain containing an scFv specific to either antigen linked to a C_L domain (SCFV3-C_H1/C_L) . Another example of a trivalent construction involves the use of a Fab fragment specific to one epitope C-terminally linked to two scFvs specific to another epitope, one on each chain (Fab-scFv2) . Yet another example of a trivalent molecule consists of an intact antibody molecule specific to one antigen with a single chain Fab (scFab) linked to the C-terminal end of the molecule (IgG-scFab) . The dock-and-lock (DNL) approach has also been used to generate trivalent antibodies (DNL-F (ab) 3) (Chang, C. -H. et al. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 199-216 (2011) ) .

Tetravalent antibodies have also been constructed. Examples of tetravalent antibodies include, but are not limited to, scFv2-Fc, F (ab') 2-scFv2, scFv2-H/L, and scFv-dhlx-scFv molecules. Bispecific scFv2-Fc constructs have an Fc domain with two scFvs specific to one molecule linked to the N-termini of the Fc chains and another two scFvs specific to another molecule linked to the C-termini of the Fc chain. Bispecific F (ab') 2-scFv2 constructs include scFv fragments linked to the C-terminal end of an F (ab') 2 fragment. scFv2-H/L constructs have scFvs specific to one molecule linked to the heavy chains while scFvs specific to another molecule are linked to the light chains. Finally, scFv-dhlx-scFv constructs contain one type of scFv linked to a helical dimerization domain followed by another type of scFv. Two chains of this type can dimerize, generating a tetravalent antibody (Kontermann, R. E. In: Bispecific Antibodies. Kontermann RE (ed. ) , Springer Heidelberg Dordrecht London New York, pp. 1 -28 (2011) ) .
Antibody Modifications

The antibody constructs described herein can be modified, for example to reduce effector function of the Fc region, to increase the antibody circulating half-life, to stabilize the hinge region, and/or to increase affinity. The term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, which may be generated by papain digestion of an intact antibody. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a C_H2 domain and a C_H3 domain, and optionally comprises a C_H4 domain. Replacements of amino acid residues in the Fc portion to alter antibody effector function are known in the art (Winter, et al. U.S. Pat. Nos. 5,648,260; 5,624,821) . The Fc portion of an antibody mediates several important effector functions e.g., cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life/clearance rate of antibody and antigen-antibody complexes. In some cases, these effector functions are desirable for therapeutic antibodies but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives.

In some embodiments, the antibody compositions herein, as well as the antibodies used in the methods and uses described herein, can be “effector-deficient. ” As used herein, an “effector-deficient” antibody is defined as an antibody having an Fc region that has been altered so as to reduce or eliminate Fc-binding to CD16, CD16a, CD16a^V158, CD16a^F158, CD16b, CD32, CD32a, CD32b, CD32c, CD23, DC-SIGN, and/or FcRn Fc receptors. A non-limiting example of mutations that reduce Fc-binding to CD16, CD32, and CD64 include E233P, L234A, L235A, G237M, D265A, D265N, E269R, D270A, D270N, N297A, N297Q, N297D, N297R, S298N, T299A, or any combinations thereof (numbering is EU index of Kabat) . A non-limiting example of mutations that reduce Fc-binding to FcRn include I253A, H310A, H435A, or any combinations thereof (numbering is EU index of Kabat) . An effector-deficient antibody can have one or more of the aforementioned mutations, or any combinations thereof. In some embodiments, the antibody compositions herein, as well as the antibodies used in the methods and uses described herein, are not “effector-deficient” and do not comprise the mutations described above.

The antibody compositions herein, as well as the antibodies used in the methods and uses described herein, can be mutated to increase their circulating half-life. In some embodiments, the Fc region can comprise mutations that enhance FcRn binding to the Fc region, in order to extend the half-life of these medications. Non-limiting examples of half-life-enhancing mutations include M252Y, S254T, T256E, ΔE294, G385D, Q386P, N389S, M428L, H433K, N434F, N434S, Y436H, or any combination thereof (see e.g., US 8, 323, 962; Zalevsky et al. (2010) Nat. Biotechnol. 28 (2) : 157-159; Bas et al. (2019 Jan 25) J. Immunol., “Fc Sialylation Prolongs Serum Half-Life of Therapeutic Antibodies” ) . An antibody as described herein may have one or more of the aforementioned half-life-enhancing mutations, or any combinations thereof.

In one embodiment, the reduction in Fc-binding to Fc receptors is a complete reduction as compared to an effector-competent control. In other aspects, the reduction in about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%, or more, as compared to an effector-competent antibody control. Methods for determining whether an antibody has a reduced Fc-binding to CD16, CD32, CD64 and/or FcRn are well known in the art (see e.g., US 2011/0212087 A1, WO 2013/165690, US 9, 382, 321 B2, US 2018/0291101 A1, and Vafa O. et al. “An engineered Fc variant of an IgG eliminates all immune effector functions via structural perturbations” (January 2014) Methods 65: 114; PubMed ID: 23872058) .

In some embodiments of any of the aspects, the immunoglobulin constant region can include a C_H3 C-terminal lysine deletion (ΔK445) (Lys0) and or an S226P mutation to stabilize the hinge region.

In some embodiments of any of the aspects, the anti-LILRB2 monospecific or bispecific antibody construct as described herein can be affinity matured. Affinity maturation is the process by which antibodies gain increased affinity, avidity, and anti-pathogen activity. Affinity maturation can be the result of somatic hypermutation (SHM) of immunoglobulin genes in B cells, coupled to selection for antigen binding. In one aspect, described herein is a method of affinity maturing an antibody composition as described herein, the method comprising: a) mutating at least one binding domain; b) determining the affinity of the mutated binding domain for its cognate ligand; and c) selecting the mutated binding domain that exhibits increased affinity for its cognate ligand compared to the unmutated binding domain. In one aspect, described herein is a method of affinity maturing an anti-LILRB2 antibody composition as described herein, the method comprising: a) mutating at least one anti-LILRB2 binding domain; b) determining the affinity of the mutated anti-LILRB2 binding domain for its LILRB2; and c) selecting the mutated anti-LILRB2 binding domain that exhibits increased affinity for LILRB2 compared to the unmutated anti-LILRB2 binding domain.

In some embodiments, the method further comprises producing a composition comprising the selected mutated binding domain (e.g., a mutated anti-LILRB2 binding domain) . In some embodiments, the method further comprises producing an antibody construct comprising the selected mutated binding domain (e.g., a mutated anti-LILRB2 binding domain) . In one aspect, described herein is a composition produced by the affinity maturation method as described herein. In one aspect, described herein is an antibody construct produced by the affinity maturation method as described herein.
Nucleic Acids and Vectors

The compositions described herein can be encoded and/or expressed by nucleic acids and/or vectors. Accordingly, in one aspect described herein is a nucleic acid encoding an anti-LILRB2 monospecific antibody construct as described herein. In another aspect described herein is a nucleic acid encoding an anti-LILRB2 and anti-PD-1 bispecific antibody construct as described herein. In another aspect described herein is a nucleic acid encoding an anti-LILRB2 and anti-VEGF bispecific antibody construct as described herein.

In one aspect described herein is a vector comprising nucleic acid encoding for an anti-LILRB2 monospecific antibody construct as described herein. In another aspect described herein is a vector comprising nucleic acid encoding for an anti-LILRB2 and anti-PD-1 bispecific antibody construct as described herein. In another aspect described herein is a vector comprising nucleic acid encoding for an anti-LILRB2 and anti-VEGF bispecific antibody construct as described herein.

In some embodiments of any of the aspects, the nucleic acid encoding an anti-LILRB2 monospecific or bispecific antibody construct described herein comprises DNA. In some embodiments of any of the aspects, the nucleic acid encoding an anti-LILRB2 monospecific or bispecific antibody construct described herein consists essentially of DNA. In some embodiments of any of the aspects, the nucleic acid encoding an anti-LILRB2 monospecific or bispecific antibody construct described herein consists of DNA.

In some embodiments of any of the aspects, a DNA molecule encoding an anti-LILRB2 monospecific or bispecific antibody construct as described herein comprises at least one regulatory sequence upstream of the encoded antibody. In some embodiments of any of the aspects, a DNA molecule encoding an anti-LILRB2 monospecific or bispecific antibody construct as described herein comprises a promoter for transcription of the antibody using an RNA polymerase. In some embodiments of any of the aspects, a DNA molecule encoding an anti-LILRB2 monospecific or bispecific antibody construct as described herein comprises a T7 promoter.

When the nucleic acid molecule that encodes any of the anti-LILRB2 monospecific or bispecific antibodies described herein is expressed in a cell, a variety of transcription control sequences (e.g., promoter/enhancer sequences) can be used to direct its expression. The promoter can be a native promoter, e.g., the promoter of the at least one antibody in its endogenous context, which provides normal regulation of expression of the antibody. In some embodiments the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of the antibody. A variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule.

The precise nature of the regulatory sequences needed for expression can vary between species or cell types, but in general can include, as necessary, 5′non-transcribed and 5′non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5′non-transcribed regulatory sequences can include a promoter region which includes a promoter sequence for transcriptional control of the encoded anti-LILRB2 monospecific or bispecific antibody. Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired.

As used herein, an antibody-encoding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the antibody-encoding sequence under the influence or control of the regulatory sequences. If it is desired that at least one antibody encoded in the nucleic or vector be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5′regulatory sequences results in the transcription of the antibody and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the antibody, or (3) interfere with the ability of the antibody be translated into a protein.

A nucleic acid molecule that encodes an antibody as described herein can be introduced into a cell or cells using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule encoding an antibody as described herein can also be accomplished by integrating the nucleic acid molecule into the genome.

In some embodiments, one or more of the anti-LILRB2 monospecific or bispecific antibodies described herein is expressed in a recombinant expression vector or plasmid. In some embodiments, the vector is the pcDNA3.1 expression vector.

As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring nucleic acids (e.g., DNA encoding anti-LILRB2 monospecific or bispecific antibodies as described herein) into a host cell. The vector can encompass any genetic element that is capable of replication when associated with the proper control elements and that can transfer nucleic acid sequences to cells. The term “vector” includes a plasmid, a cloning vector, an expression vector, naked DNA, a mini-chromosome, a chromosome, a transposon, a cosmid, a virus, virion, phage, and the like. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989) . One type of vector is a "plasmid, " which refers to a circular double stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, wherein additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) . Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors" . In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" is used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) , which serve equivalent functions.

In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like) .

In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism) . In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector can be cut in a determinable fashion and into which a desired DNA sequence (e.g., a DNA template for an anti-LILRB2 monospecific or bispecific antibody construct as described herein) can be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.

An expression vector is one into which a desired DNA sequence (e.g., a DNA template for an anti-LILRB2 monospecific or bispecific antibody construct as described herein) can be inserted by restriction and ligation such that it is operably joined to regulatory sequences and can be expressed as an antibody transcript. Vectors can further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds (e.g., ampicillin resistance) , genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase, luciferase or alkaline phosphatase) , and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein) . In certain embodiments, the vectors used herein are capable of autonomous replication and expression of the antibodies present in the DNA segments to which they are operably joined. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (or RNA) . That heterologous DNA (or RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.

As used herein, the term “viral vector"refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Non-limiting examples of a viral vector of this invention include an AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a herpesvirus vector, an alphavirus vector, a poxvirus vector, a baculovirus vector, and a chimeric virus vector.

It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleic acid of interest (e.g., encoding an anti-LILRB2 monospecific or bispecific antibody construct as described herein) in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
Compositions and Administration

The anti-LILRB2 monospecific or bispecific antibody constructs described herein can be comprised by compositions, such as pharmaceutical compositions. In one aspect, described herein is a pharmaceutical composition comprising an anti-LILRB2 monospecific or bispecific antibody construct as described herein and a pharmaceutically acceptable carrier. In one aspect, described herein is a pharmaceutical composition comprising a nucleic acid or vector encoding an anti-LILRB2 monospecific or bispecific antibody construct as described herein and a pharmaceutically acceptable carrier. In one aspect, described herein is a pharmaceutical composition comprising a cell comprising or expressing an anti-LILRB2 monospecific or bispecific antibody construct as described herein and a pharmaceutically acceptable carrier.

In some embodiments, the anti-LILRB2 monospecific or bispecific antibody constructs described herein can be comprised by cells, such as eukaryotic cells. In one aspect, described herein is a cell comprising a nucleic acid or vector encoding an anti-LILRB2 monospecific or bispecific antibody construct as described herein. In one aspect, described herein is a cell expressing an anti-LILRB2 monospecific or bispecific antibody construct as described herein. In one aspect, described herein is a cell in combination with an anti-LILRB2 monospecific or bispecific antibody construct as described herein.

In some embodiments of any of the aspects, the cell is an immune cell. In some embodiments of any of the aspects, the cell is an antibody-producing cell, such as a plasma B cell. In some embodiments of any of the aspects, the cell is a macrophage. In some embodiments of any of the aspects, the cell is a tumor-associated macrophage. In some embodiments of any of the aspects, the cell is an M0 macrophage. In some embodiments of any of the aspects, the cell is an M1 macrophage. In some embodiments of any of the aspects, the cell is an M2 macrophage. In some embodiments of any of the aspects, the cell is a T lymphocyte. In some embodiments of any of the aspects, the cell is a CD3+ T cell. In some embodiments of any of the aspects, the cell is a CD4+ T cell. In some embodiments of any of the aspects, the cell is a CD8+ T cell.
Formulations

In some embodiments, the technology described herein relates to a pharmaceutical composition comprising an anti-LILRB2 monospecific or bispecific antibody construct, as described herein, or nucleic acids or vectors encoding such antibodies, or cells comprising or in combination with such antibodies, nucleic acids, or vectors, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise the anti-LILRB2 monospecific or bispecific antibody construct as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of the anti-LILRB2 monospecific or bispecific antibody construct as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of the anti-LILRB2 monospecific or bispecific antibody construct as described herein.

Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG) ; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum component, such as serum albumin, HDL and LDL; (24) C₂-C₁₂ alcohols; (25) surfactants, including but not limited to Polysorbate 20 (PS20) or Polysorbate 80 (PS80) ; and (26) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient" , " carrier" , "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g., the anti-LILRB2 monospecific or bispecific antibody construct as described herein.

In some embodiments, the pharmaceutical composition comprising the anti-LILRB2 monospecific or bispecific antibody construct as described herein can be a parenteral dose form (i.e., administered or occurring elsewhere in the body than the mouth and alimentary canal) . Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms of the anti-LILRB2 monospecific or bispecific antibody construct as disclosed within are well known to those skilled in the art. Non-limiting examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Pharmaceutical compositions comprising the anti-LILRB2 monospecific or bispecific antibody construct can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets) , pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005) .
Dosing

In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. the anti-LILRB2 monospecific or bispecific antibody construct to a subject in order to alleviate a symptom of a disease or disorder, such as a cancer. As used herein, "alleviating a symptom of a cancer" is ameliorating any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art.

For systemic administration, subjects can be administered a therapeutic amount of a composition comprising the anti-LILRB2 monospecific or bispecific antibody construct as described herein, such as, e.g., 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.

In some embodiments, the anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered in a unit dose of about 3 mg/kg to about 20 mg/kg, about 10 mg/kg to about 20 mg/kg, or about 15 mg/kg to about 20 mg/kg. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered in a unit dose of about 3 mg/kg. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered in a unit dose of about 9 mg/kg. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered in a unit dose of about 10 mg/kg. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered in a unit dose of about 15 mg/kg. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered in a unit dose of about 20 mg/kg.

In some embodiments, the anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered at different dosages according to the time of treatment. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered at a unit dose of about 20 mg/kg of the antibody construct during week 1 of treatment and a unit dose of about 4 mg/kg of the antibody construct after beginning treatment.

The term “effective amount" as used herein refers to the amount of the anti-LILRB2 monospecific or bispecific antibody construct as described herein needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of the anti-LILRB2 monospecific or bispecific antibody construct as described herein that is sufficient to provide a particular anti-cancer effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease) , or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount" . However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g., a cancer by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 %or at least 90%or more.

Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the ED50 (the dose therapeutically effective in 50%of the population) . The dosage can vary depending upon the dosage form employed and the route of administration utilized. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the anti-LILRB2 monospecific or bispecific antibody construct as described herein, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by Enzyme linked immunosorbent assay (ELISA) . The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assays, such as flow cytometry or the mixed lymphocyte reaction, as described herein. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

The dosage ranges for the administration of the anti-LILRB2 monospecific or bispecific antibody construct as described herein, according to the methods described herein depend upon, for example, the form of the antibody, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for tumor volume and/or tumor weight. The dosage should not be so large as to cause adverse side effects, such as autoimmunity, allergy, nausea, diarrhea, and/or rashes. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

The efficacy of the anti-LILRB2 monospecific or bispecific antibody construct as described herein in, e.g., the treatment of a cancer, can be determined by the skilled clinician. However, a treatment is considered “effective treatment, " as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10%following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g., cancer symptoms, tumor volume, tumor weight, etc. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted) . Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g., pain, inflammation, fatigue, etc. ) ; or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g., decreased cancer symptoms, tumor volume, tumor weight) . It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters.

In vitro and animal model assays are provided herein which allow the assessment of a given dose of the anti-LILRB2 monospecific or bispecific antibody construct as described herein. By way of non-limiting example, the properties and/or effects of the anti-LILRB2 monospecific or bispecific antibody construct as described herein can be assessed by screening assays (e.g., Fab library construction and phage library panning with immunotubes and magnetic bead methods; see e.g., Example 2, Example 3) ; binding and/or blocking assays (e.g., ELISA, SPR, flow cytometry; see e.g., Example 4, Example 5, Example 11, Example 12) ; flow cytometry and/or the mixed lymphocyte reaction, e.g., for measuring immune cell differentiation and/or activation, macrophage polarization and/or activation (see e.g., Example 8, Example 13) , T cell differentiation and/or activation (see e.g., Example 6, Example 14) , amongst others.

Efficacy can be assessed in animal models of a condition described herein, for example treatment of melanoma (e.g., huPBMC-NOG mouse inoculated with HLA-G-A375 tumor model, see e.g., Example 9) , breast cancer (e.g., huPBMC-NOG mouse inoculated with MDA-MB-231 tumor model, see e.g., Example 15) , colorectal adenocarcinoma (e.g., huPBMC-NPG mouse inoculated with HT-29 tumor model, see e.g., Example 16) . When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g., tumor volume and/or tumor weight.

With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.

In certain embodiments, an effective dose of a composition comprising an anti-LILRB2 monospecific or bispecific antibody construct as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising an anti-LILRB2 monospecific or bispecific antibody construct as described herein can be administered to a patient repeatedly.

The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the antibody. The desired dose or amount can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising an anti-LILRB2 monospecific or bispecific antibody construct as described herein can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, 25 minute, 30 minute, 35 minute, 40 minute, 45 minute, 50 minute, 55 minute, or 60 minute period.

In some embodiments, an anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered biweekly (twice a week, “BIW” ) for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or more. In some embodiments, an anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered at least once during week 1 and at least once during week 4 of treatment. In some embodiments, an anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered biweekly (twice a week, “BIW” ) during week 1 and week 4 of treatment.

In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer.
Administration

A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous (IV) , intramuscular (IM) , subcutaneous (SC) , transdermal, airway (aerosol) , pulmonary, cutaneous, topical, injection, intraosseous (IO) , intraperitoneal (IP) , intrarectal, intravaginal, intraarticular (IA) , or intratumoral administration; see e.g., Tashima, “Delivery of Orally Administered Digestible Antibodies Using Nanoparticles” , Int J Mol Sci 22: 3349 (2021) on the subject of orally administered antibodies. Administration can be local or systemic.

In some embodiments of any of the aspects, the anti-LILRB2 monospecific or bispecific antibody construct as described herein is administered as a monotherapy, e.g., another treatment for the cancer is not administered to the subject.

In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g., as part of a combinatorial therapy. Non-limiting examples of a second agent and/or treatment can include a cancer therapy selected from the group consisting of: radiation therapy, surgery, gemcitabine, cisplatin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa andcyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide and trimethylol melamine; acetogenins (especially bullatacin and bullatacinone) ; a camptothecin (including the synthetic analogue topotecan) ; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues) ; cryptophycins (particularly cryptophycin 1 and cryptophycin 8) ; dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1) ; eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994) ) ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores) , aclacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin) , epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU) ; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, Oreg. ) ; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2” -trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) ; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( "Ara-C" ) ; cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N. J. ) , Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill. ) , anddoxetaxel (Rhone-Poulenc Rorer, Antony, France) ; chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin) ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO) ; retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV) ; oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX) ; lapatinibinhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib) and VEGF-A (e.g., pazopanib, sunitinib, sorafenib, regorafenib, cabozantinib, lenvatinib, ponatinib, ziv-aflibercept, axitinib, tivozanib, vandetanib, ramucirumab, bevacizumab) that reduce cell proliferation; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In some embodiments of any of the aspects, the cancer treatment method further comprises administering an immune checkpoint inhibitor. In some embodiments of any of the aspects, the immune checkpoint inhibitor comprises an immune checkpoint inhibitor antibody. In some embodiments of any of the aspects, the checkpoint inhibitor immunotherapy is an inhibitor of a checkpoint molecule selected from the group consisting of: programmed cell death 1 (PD-l) , programmed death-ligand 1 (PD-L1) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Adenosine A2A receptor (A2AR) , CD276, CD39, CD73, B7 family immune checkpoint molecules, V-set domain-containing T-cell activation inhibitor 1 (B7H4) , B and T Lymphocyte Attenuator (BTLA) , Indoleamine 2, 3-dioxygenase (IDO) , Killer-cell Immunoglobulin-like Receptor (KIR) , Lymphocyte Activation Gene-3 (LAG-3) , nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2) , T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) , T cell immunoreceptor with Ig and ITIM domains (TIGIT) , V-domain Ig suppressor of T cell activation (VISTA) , and Sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7) .

Non-limiting examples of immune checkpoint inhibitors (ICIs) include: pembrolizumab nivolumabcemiplimabspartalizumab, camrelizumab (AiRuiKa^TM) , sintilimabtislelizumab, toripalimab (Tuoyi^TM) , dostarlimab (JEMPERLI) , INCMGA00012, AMP-224, AMP-514 (MEDI0608) , atezolizumabavelumabenvafolimab (KN035) , cosibelimab (CK-301) , AUNP12, CA-170, BMS-986189, BMS-936559 (MDX-1105) , durvalumabtremelimumab, and ipilimumabSee e.g., US Patents US5811097, US5855887, US6051227, US6682736, US6984720, US7595048, US7605238, US7943743, US8008449, US8217149, US8354509, US8383796, US8728474, US8735553, US8779105, US8779108, US8907053, US8900587, US8952136, US9067999, US9073994, US9683048, US9987500, US10160736, US10316089, US10441655, US10590199, US11225522, US Patent Publication US2014341917; Storz et al., MAbs. 2016 Jan; 8 (1) : 10–26; the contents of each of which are incorporated herein by reference in their entireties.

One of skill in the art can readily identify a chemotherapeutic agent of use (e.g., see Physicians'Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones &Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff’s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed) : The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003) .

In addition, the methods of treatment can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.

The methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g., as part of a combinatorial therapy. By way of non-limiting example, if a subject is to be treated for pain or inflammation according to the methods described herein, the subject can also be administered a second agent and/or treatment known to be beneficial for subjects suffering from pain or inflammation. Examples of such agents and/or treatments include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs -such as aspirin, ibuprofen, or naproxen) ; corticosteroids, including glucocorticoids (e.g. cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclometasone) ; methotrexate; sulfasalazine; leflunomide; anti-TNF medications; cyclophosphamide; pro-resolving drugs; mycophenolate; or opiates (e.g. endorphins, enkephalins, and dynorphin) , steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.
Immune Modulation

The anti-LILRB2 monospecific or bispecific antibody constructs as described herein are capable of modulating the immune system. For example, such antibodies can polarize macrophages from an M2 phenotype to an M1 phenotype; activate T cells; and/or inhibit growth of a cancer.
Macrophages

In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 monospecific or bispecific antibody constructs as described herein) is capable of polarizing macrophages from an M2 phenotype to an M1 phenotype. M1 macrophages are classically activated, such as by IFN-γ or lipopolysaccharide (LPS) , and produce proinflammatory cytokines, phagocytize microbes, and initiate an immune response. M1 macrophages produce nitric oxide (NO) or reactive oxygen intermediates (ROI) to protect against bacteria and viruses. M1 macrophages have anti-tumor effects and can distinguish tumor cells from normal cells. M1 macrophages are positively associated with longer survival times and most positive clinical outcomes in many cancers. M1 macrophages are associated with decreased expression of CD163 and/or mannose receptor (CD206) ; increased expression of CD80, MHC class II, CD86, and/or CD40; and increased secretion of TNF-α, IL-12, IL-1β, and/or IL-6, as compared to M2 macrophages. M1 macrophages can also be referred to as inflammatory macrophages. M1 macrophages secrete pro-inflammatory/T helper 1 (Th1) -promoting cytokines such as IL-12, IL-1β, IL-6, and/or TNF-α. Macrophages, including M1 macrophages, can interact with T cells in order to bring about T cell activation in target organs, and the macrophages themselves can be activated by inflammatory messenger molecules (cytokines) produced by the T cells.

M2 macrophages are alternatively activated by exposure to certain cytokines such as IL-4, IL-10, or IL-13. M2 macrophages produce either polyamines to induce proliferation or proline to induce collagen production. M2 macrophages are associated with wound healing and tissue repair. M2 macrophages are associated with increased expression of CD163 and/or CD206, decreased expression of CD80, and decreased secretion of TNF-α as compared to M1 macrophages. M2 macrophages can be pro-tumorigenic, whereas M1 macrophages can be anti-tumorigenic.

M0 macrophages are undifferentiated macrophages with the potential to polarize into specific macrophage subtypes, such as tumor-associated macrophages (TAMs) , including M1 or M2 macrophages. As a non-limiting example, exposure to IL-4 can induce an M0 macrophage to differentiate into an M2 macrophage. As another example, exposure of an M0 macrophage to supernatant from cancer cells can induce its differentiation into a TAM. Tumor-associated macrophages (TAMs) are some of the most abundant immune cells within tumors and can be categorized into M1 and M2 macrophages. TAMs can adopt an M1-like pro-inflammatory phenotype at the early phases of oncogenesis and mediate immune response that inhibits tumor growth. As tumors progress, anabatic hypoxia of the TME gradually induces the M2-like functional transformation of TAMs. The compositions described herein (e.g., anti-LILRB2 monospecific or bispecific antibody constructs as described herein) can polarize macrophages, including tumor-associated macrophages, from an M2 phenotype to an M1 phenotype (see e.g., Example 8, Example 13) .

Accordingly, in one aspect described herein is a method for polarizing a macrophage from an M2 phenotype to an M1 phenotype. In some embodiments, the method comprises contacting the macrophage with an anti-LILRB2 monospecific or bispecific antibody construct as described herein.

In some embodiments, after contacting the macrophage with the antibody, the macrophage exhibits decreased expression of CD163 and/or CD206. Such contacting can be, for example, in an amount and for an amount of time sufficient to induce such change in CD163 and/or CD206. In some embodiments, after contacting the macrophage with the antibody, e.g., for a sufficient amount of time, the macrophage exhibits expression of CD163 and/or CD206 decreased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a macrophage not contacted with the antibody or not contacted with the antibody, e.g., for a sufficient amount of time.

In some embodiments, after contacting the macrophage with the antibody, the macrophage exhibits increased expression of CD80. Such contacting can be, for example, in an amount and for an amount of time sufficient to induce such change in CD80. In some embodiments, after contacting the macrophage with the antibody, e.g., for a sufficient amount of time, the macrophage exhibits expression of CD80 increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a macrophage not contacted with the antibody or not contacted with the antibody, e.g., for a sufficient amount of time.

In some embodiments, after contacting the macrophage with the antibody, the macrophage exhibits increased secretion of TNF-α. Such contacting can be, for example, in an amount and for an amount of time sufficient to induce such change in TNF-α. In some embodiments, after contacting the macrophage with the antibody, e.g., for a sufficient amount of time, the macrophage exhibits secretion of TNF-α increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a macrophage not contacted with the antibody or not contacted with the antibody, e.g., for a sufficient amount of time.

In some embodiments of any of the aspects, the macrophage directly or indirectly (e.g., through an activated T cell) increased cancer cytotoxicity. In some embodiments of any of the aspects, cancer cytotoxicity can be measured by using a tumor cell killing assay and determining the number or percentage of tumor cells killed by the macrophage. In some embodiments of any of the aspects, the macrophage induces directly or indirectly cancer cytotoxicity, which is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a macrophage not contacted with the antibody or not contacted with the antibody for a sufficient amount of time.

In some embodiments, the macrophage is contacted with the antibody at an effective dosage for a sufficient amount of time, e.g., in an amount and for an amount of time sufficient to induce a desired change (e.g., decreased expression of CD163 and/or CD206, increased expression of CD80, increased secretion of TNF-α, and/or increased cancer cytotoxicity) . In some embodiments, the sufficient amount of time for contacting the macrophage with the antibody is at least 2 days. In some embodiments, the sufficient amount of time for contacting the macrophage with the antibody is at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, or more. In some embodiments, the sufficient amount of time for contacting the macrophage with the antibody is about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, or more. In some embodiments, the sufficient amount of time for contacting the macrophage with the antibody is at most 2 days, at most 2.5 days, at most 3 days, at most 3.5 days, at most 4 days, at most 4.5 days, at most 5 days, or more.

In some embodiments, the composition is an anti-LILRB2 antibody construct selected from the group consisting of J-19. h1, 1E1 (G4) , HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206. In some embodiments, the composition is an anti-LILRB2 antibody construct selected from the group consisting of HB59, AIL-B45, huB45, AIL-B206, huB206, MIL-C68, huC68, MIL-C112 and huC112.

In some embodiments, the composition is an anti-LILRB2 and anti-PD-1 bispecific antibody construct selected from the group consisting of: Bis-M13, Bis-M13-1, Bis-M16, Bis-M16-1, Bis-M17, Bis-M17-1, Bis-M23, Bis-M29, Bis-M23-1, Bis-M28, Bis-M30, Bis-M30-1, Bis-M31, Bis-M31-1, Bis-M33, Bis-M33-1, Bis-M34, and Bis-M34-1. In some embodiments, the composition is an anti-LILRB2 and anti-PD-1 bispecific antibody construct selected from Bis-M23-1 or Bis-M28.

In some embodiments, the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from the group consisting of: Bis-M14, Bis-M14-1, Bis-M15, Bis-M15-1, Bis-M18, Bis-M18-1, Bis-M24, Bis-M27, Bis-M32, Bis-M32-1, Bis-M35, and Bis-M35-1. In some embodiments, the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27.
T cells

In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 monospecific or bispecific antibody constructs as described herein) is capable of activating CD3+ T cells. For example, pro-inflammatory cytokines secreted by M1 macrophages, such as IL-12, IL-1β, IL-6, and/or TNF-α, can activate T cells. As another example, macrophages engulfing foreign materials, displaying the foreign peptides on MHC-II, and presenting the MHC-peptide complex at the surface of the cell to T cells can activate T cells. M1 macrophages can promote differentiation of naive CD4 T cells into Th1 or Th17 phenotypes via production of Th1-promoting or Th17-promoting cytokines. M1 macrophages can recruit CD8+ T cells to the tumor microenvironment through antigen presentation to the T-cell receptor (TCR) and chemokine secretion. In some embodiments, T cell activation is associated with increased expression of pro-inflammatory cytokines, such as IL-1, IL-2, IL-6, IL-12, IL-17, IL-18, IFN-γ, and TNF-α (see e.g., Example 6, Example 13, Example 14) . In some embodiments, T cell activation is associated with increased cytotoxicity against cancer cells.

Accordingly, in one aspect described herein is a method for activating a T cell. In some embodiments, the method comprises contacting the T cell with an anti-LILRB2 monospecific or bispecific antibody construct as described herein. In some embodiments, the method comprising contacting a macrophage with an anti-LILRB2 monospecific or bispecific antibody construct as described herein, thus polarizing the macrophage from an M2 phenotype to an M1 phenotype, and the M1-polarized macrophage activates a T cell.

In some embodiments of any of the aspects, the T cell exhibits increased activation of Nuclear Factor of Activated T cell (NFAT) signaling (see e.g., Example 6, Fig. 4A-4D) . In some embodiments, the T cell exhibits increased activation of NFAT signaling by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a T cell not contacted with the antibody or not contacted with the antibody for a sufficient amount of time.

In some embodiments of any of the aspects, the activated T cell exhibits increased cancer cytotoxicity. In some embodiments of any of the aspects, cancer cytotoxicity can be measured by using a tumor cell killing assay and determining the number or percentage of tumor cells killed by the activated T cells. In some embodiments of any of the aspects, the activated T cell exhibits cancer cytotoxicity that is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a T cell not contacted with the antibody or not contacted with the antibody for a sufficient amount of time, or as compared to a T cell not exposed to a polarized macrophage (e.g., to the M1 phenotype) .

In some embodiments, the T cell is contacted with the antibody, or exposed to a polarized macrophage after contacting with the antibody, at an effective dosage for a sufficient amount of time, e.g., in an amount and for an amount of time sufficient to induce a desired change (e.g., increased secretion of TNF-α, and/or increased cancer cytotoxicity) . In some embodiments, the sufficient amount of time for contacting the T cell with the antibody, or for exposing the T cell to a polarized macrophage after contacting with the antibody, is at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, or more. In some embodiments, the sufficient amount of time for contacting the T cell with the antibody, or for exposing the T cell to a polarized macrophage after contacting with the antibody, is about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, or more. In some embodiments, the sufficient amount of time for contacting the T cell with the antibody, or for exposing the T cell to a polarized macrophage after contacting with the antibody, is at most 2 days, at most 2.5 days, at most 3 days, at most 3.5 days, at most 4 days, at most 4.5 days, at most 5 days, or more.

In some embodiments, the composition is an anti-LILRB2 antibody construct selected from the group consisting of J-19. h1, 1E1 (G4) , HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206. In some embodiments, the composition is an anti-LILRB2 antibody construct selected from the group consisting of HB74, HA23, HA31, HB59, MIL-C112, MILC18, MIL-C27, MIL-C3, MIL-C32, MIL-C68, MIL-C9, AIL-A1, AIL-B100, AIL-B124, AIL-B134, and AIL-B45.

In some embodiments, the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from the group consisting of: Bis-M14, Bis-M14-1, Bis-M15, Bis-M15-1, Bis-M18, Bis-M18-1, Bis-M24, Bis-M27, Bis-M32, Bis-M32-1, Bis-M35, and Bis-M35-1. In some embodiments, the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27.
Anti-Cancer Immune Response

In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 monospecific or bispecific antibody constructs as described herein) is capable of inhibiting growth of a cancer, such as a tumor. The compositions produce an anti-cancer effect in cancers including but not limited to melanoma, breast cancer, and colorectal adenocarcinoma. The compositions described herein produce increased numbers and/or activation of M1 macrophages and/or T cells in the tumor microenvironment, which exhibit increased cytotoxicity against tumor cells. Inhibition of the cancer be measured using the volume and/or weight of the tumor (see e.g., Example 9, Example 15, Example 16) .

In some embodiments of any of the aspects, administering an anti-LILRB2 monospecific or bispecific antibody construct as described herein to a subject in need thereof decreases tumor volume by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a subject not administered an effective amount of the antibody.

In some embodiments of any of the aspects, administering an effective amount of an anti-LILRB2 monospecific or bispecific antibody construct as described herein to a subject in need thereof decreases tumor weight by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a subject not administered an effective amount of the antibody.

In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 and anti-PD-1 bispecific antibody constructs as described herein) is capable of inhibiting a checkpoint protein. In some embodiments of any of the aspects, administering an effective amount of an anti-LILRB2 and anti-PD-1 bispecific antibody construct as described herein to a subject in need thereof decreases checkpoint activation (e.g., PD-1 signaling) by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a subject not administered an effective amount of the antibody.

In some embodiments of any of the aspects, the composition (e.g., anti-LILRB2 and anti-VEGF bispecific antibody constructs as described herein) is capable of inhibiting angiogenesis. In some embodiments of any of the aspects, administering an effective amount of an anti-LILRB2 and anti-VEGF bispecific antibody construct as described herein to a subject in need thereof decreases angiogenesis (e.g., VEGF signaling, blood vessel growth towards a tumor) by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, as compared to a subject not administered an effective amount of the antibody.

In some embodiments, the composition is an anti-LILRB2 antibody construct selected from the group consisting of J-19. h1, 1E1 (G4) , HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206. In some embodiments, the composition is an anti-LILRB2 antibody construct selected from MIL-C68, huC68, MIL-C112, huB45, AIL-B206, huB206, or HB59. In some embodiments, the composition is an anti-LILRB2 antibody construct selected from MIL-C68, AIL-B206, or HB59.

In some embodiments, the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from the group consisting of: Bis-M14, Bis-M14-1, Bis-M15, Bis-M15-1, Bis-M18, Bis-M18-1, Bis-M24, Bis-M27, Bis-M32, Bis-M32-1, Bis-M35, and Bis-M35-1. In some embodiments, the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27. In some embodiments, the composition is the anti-LILRB2 and anti-VEGF bispecific antibody construct Bis-M24.
Treatment Methods

In multiple aspects, described herein are methods of treating a subject in need of an antibody-based therapy comprising administering to the subject a therapeutically effective amount of an anti-LILRB2 monospecific or bispecific antibody construct as described herein, selected from the group consisting of: an anti-LILRB2 monospecific antibody construct as described herein, an anti-LILRB2 and anti-PD-1 bispecific antibody construct as described herein, an anti-LILRB2 and anti-VEGF bispecific antibody construct as described herein, a nucleic acid or vector encoding an anti-LILRB2 monospecific or bispecific antibody construct as described herein, a cell or population thereof expressing or in combination with an anti-LILRB2 monospecific or bispecific antibody construct as described herein, or a pharmaceutical composition as described herein.

In multiple aspects, the methods described herein relate to treating a subject having or diagnosed as having cancer with an anti-LILRB2 monospecific or bispecific antibody construct as described herein. Subjects having a cancer can be identified by a physician using current methods of diagnosing the cancer. Symptoms and/or complications of cancer which characterize these conditions and aid in diagnosis are well known in the art and depend on the specific cancer and can include but are not limited to, breast changes (e.g., lump or firm feeling in your breast or under the arm; nipple changes or discharge; skin that is itchy, red, scaly, dimpled, or puckered) ; bladder changes (e.g., difficulty urinating, pain when urinating, blood in the urine) ; bleeding or bruising for no known reason; bowel changes (e.g., blood in the stools, changes in bowel habits) ; persistent cough or hoarseness; eating problems (e.g., pain after eating; persistent heartburn or indigestion; difficulty swallowing; stomach pain; nausea and/or vomiting; appetite changes) ; severe and/or persistent fatigue; fever or night sweats for no known reason; mouth changes (e.g., white or red patches on the tongue or in the mouth; bleeding, pain, or numbness in the lip or mouth) ; neurological problems (e.g., headaches, seizures, vision changes, hearing changes, drooping of the face) ; skin changes (e.g., a flesh-colored lump that bleeds or turns scaly; a new mole or a change in an existing mole; a sore that does not heal; jaundice, which includes yellowing of the skin and whites of the eyes) ; swelling or lumps, anywhere such as in the neck, underarm, stomach, or groin; and/or weight gain or weight loss for no known reason.

Tests that may aid in a diagnosis of, e.g., cancer depend on the specific cancer and include, but are not limited to, blood chemistry test; complete blood count (CBC) ; cytogenetic analysis; immunophenotyping; liquid biopsy; sputum cytology; tumor marker tests; urinalysis; urine cytology; imaging tests (e.g., CT scan, MRI, nuclear scan, bone scan, PET scan, ultrasound, X-rays, colonoscopy, mammography) ; biopsy (e.g., endoscopy, colonoscopy, bronchoscopy, surgical biopsy such a nevus biopsy) .

A family history of a cancer, or exposure to risk factors for a cancer (e.g., tobacco use, infectious agents, unhealthy diet, excess body weight, physical inactivity, alcohol consumption, unprotected and/or extended sun exposure, age, reproductive history, hormone replacement therapy, inflammatory bowel disease, diabetes, etc. ) can also aid in determining if a subject is likely to have a cancer or in making a diagnosis of a cancer.

The compositions described herein can be administered to a subject having or diagnosed as having cancer. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. an anti-LILRB2 monospecific or bispecific antibody construct as described herein, to a subject in order to alleviate a symptom of a cancer. As used herein, "alleviating a symptom of a cancer" is ameliorating any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%or more as measured by any standard technique.

In one aspect, described herein is a method of treating cancer. In some embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising an anti-LILRB2 monospecific or bispecific antibody construct as described herein to a subject in need thereof. In some embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising an anti-LILRB2 monospecific or bispecific antibody construct as described herein to a subject in need thereof.

In some embodiments, the composition is administered at a dose of about 10 mg/kg to about 20 mg/kg. In some embodiments, the composition is administered at a dose of about 0.1 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5 mg/kg, about 4.0 mg/kg, about 4.5 mg/kg, about 5.0 mg/kg, about 5.5 mg/kg, about 6.0 mg/kg, about 6.5 mg/kg, about 7.0 mg/kg, about 7.5 mg/kg, about 8.0 mg/kg, about 8.5 mg/kg, about 9.0 mg/kg, about 9.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, or more.

In some embodiments, the composition is administered at least twice a week. In some embodiments, the composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times a week. In some embodiments, the composition is administered for at least three weeks. In some embodiments, the composition is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more week. In some embodiments, the composition is administered at a dose of about 20 mg/kg twice weekly for at least one week. In some embodiments, the composition is administered at a dose of about 4 mg/kg twice weekly for at least the fourth week after beginning treatment. In some embodiments, the composition is administered intravenously.

In some embodiments, the method has a tumor weight inhibition rate (TGI_TW) of at least 30%after a sufficient amount of time. In some embodiments, the method has a tumor weight inhibition rate (TGI_TW) of at least 50%after a sufficient amount of time. In some embodiments, the method has a tumor weight inhibition rate (TGI_TW) of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more after a sufficient amount of time.

In some embodiments, the method has a tumor volume inhibition rate (TGI_TV) of at least 35%after a sufficient amount of time. In some embodiments, the method has a tumor volume inhibition rate (TGI_TV) of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more after a sufficient amount of time.

In some embodiments, the sufficient amount of time for the treatment (e.g., to result in a desired TGI_TW and/or TGI_TV) is at least 3 weeks of administration. In some embodiments, the sufficient amount of time for the treatment (e.g., to result in a desired TGI_TW and/or TGI_TV) is at least 4 weeks of administration. In some embodiments, the sufficient amount of time for the treatment (e.g., to result in a desired TGI_TW and/or TGI_TV) is at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, or more of administration.

In some embodiments, the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from the group consisting of: Bis-M14, Bis-M14-1, Bis-M15, Bis-M15-1, Bis-M18, Bis-M18-1, Bis-M24, Bis-M27, Bis-M32, Bis-M32-1, Bis-M35, and Bis-M35-1. In some embodiments, the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27. In some embodiments, the composition is the anti-LILRB2 and anti-VEGF bispecific antibody construct Bis-M24.

In some embodiments, the method of treatment can comprise first diagnosing a subject or patient who can benefit from treatment by a composition described herein. In some embodiments, such diagnosis comprises detecting or measuring an abnormal level of a marker (e.g., tumor) in a sample from the subject or patient. In some embodiments, the method further comprises administering to the patient an anti-LILRB2 monospecific or bispecific antibody construct as described herein.

In some embodiments, the subject has previously been determined to have an abnormal level of an analyte described herein relative to a reference. In some embodiments, the reference level can be the level in a sample of similar cell type, sample type, sample processing, and/or obtained from a subject of similar age, sex and other demographic parameters as the sample/subject. In some embodiments, the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g., the same number and type of cells.

The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood or plasma sample from a subject. In some embodiments of any of the aspects, the technology described herein encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, or bodily fluid. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject.

In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise performing or having performed an assay on a sample obtained from the subject to determine/measure the level of analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise receiving the results of an assay on a sample obtained from the subject to determine/measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise receiving a report, results, or other means of identifying the subject as a subject with an increased or decreased level of the analyte.

In one aspect of any of the embodiments, described herein is a method of treating cancer in a subject in need thereof, the method comprising: a) determining if the subject has an abnormal level of an analyte (e.g., a tumoral antigen) ; and b) instructing or directing that the subject be administered an anti-LILRB2 monospecific or bispecific antibody construct as described herein, a nucleic acid or vector as described herein, a cell as described herein, or a pharmaceutical composition as described herein, if the level of the analyte is increased or otherwise abnormal relative to a reference. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and/or treatment recommendations in view of the assay results.
Cancer

In multiple aspects, described herein are methods of treating cancer, e.g., using the anti-LILRB2 monospecific or bispecific antibody constructs as described herein. As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.

In some embodiments, the anti-LILRB2 monospecific or bispecific antibody constructs as described herein are used to treat a blood cancer, such as a leukemia or a lymphoma. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody constructs as described herein are used to treat a cancer comprising at least one solid tumor. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody constructs as described herein are used to treat melanoma. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody constructs as described herein are used to treat breast cancer. In some embodiments, the anti-LILRB2 monospecific or bispecific antibody constructs as described herein are used to treat colorectal adenocarcinoma.

In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits) , invasion (i.e., intrusion on and destruction of adjacent tissues) , and metastasis (i.e., spread to other locations in the body via lymph or blood) . As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis. ” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the term “benign” or “non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

As used herein the term "neoplasm" refers to any new and abnormal growth of tissue, e.g., an abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues. Thus, a neoplasm can be a benign neoplasm, premalignant neoplasm, or a malignant neoplasm.

A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastases. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.

Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer) ; glioblastoma (GBM) ; hepatic carcinoma; hepatoma; intra-epithelial neoplasm. ; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung) ; lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx) ; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL) ; small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia) ; chronic lymphocytic leukemia (CLL) ; acute lymphoblastic leukemia (ALL) ; Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD) , as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors) , and Meigs’s yndrome.

A “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.
Definitions

For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

The terms “decrease” , “reduced” , “reduction” , or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce, ” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10%as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100%inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal, e.g., for an individual without a given disorder.

The terms “increased” , “increase” , “enhance” , or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased” , “increase” , “enhance” , or “activate” can mean an increase of at least 10%as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%or up to and including a 100%increase or any increase between 10-100%as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual, ” “patient” and “subject” are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer, including but not limited to melanoma, breast cancer, or colorectal adenocarcinoma. A subject can be male or female.

A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., cancer, including but not limited to melanoma, breast cancer, or colorectal adenocarcinoma) or one or more complications related to such a condition, and optionally, have already undergone treatment for cancer or the one or more complications related to cancer. Alternatively, a subject can also be one who has not been previously diagnosed as having cancer (including but not limited to melanoma, breast cancer, or colorectal adenocarcinoma) or one or more complications related to such a cancer. For example, a subject can be one who exhibits one or more risk factors for a cancer or one or more complications related to a cancer or a subject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

As used herein, the terms “protein" and “polypeptide" are used interchangeably to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein" , and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc. ) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise) , alleles, homologs, conservatively modified variants, and/or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another) , or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn) . Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the binding assays or immunoassays described herein to confirm that a desired activity, e.g., specific binding to LILBR2, PD-1, and/or VEGF, e.g., immune activation, of the native or reference polypeptide is retained.

Amino acids can be grouped according to similarities in the properties of their side chains (in A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975) ) : (1) non-polar: Ala (A) , Val (V) , Leu (L) , Ile (I) , Pro (P) , Phe (F) , Trp (W) , Met (M) ; (2) uncharged polar: Gly (G) , Ser (S) , Thr (T) , Cys (C) , Tyr (Y) , Asn (N) , Gln (Q) ; (3) acidic: Asp (D) , Glu (E) ; (4) basic: Lys (K) , Arg (R) , His (H) . Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a polypeptide which retains at least 50%of the wild-type reference polypeptide’s activity (e.g., specific binding to LILBR2, PD-1, and/or VEGF) according to the assays described herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

In some embodiments, the polypeptide described herein can be a variant of a polypeptide sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant, " as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions, or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a protein or fragment thereof that retains activity of the native or reference polypeptide. A wide variety of, for example, PCR-based, site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan to generate and test artificial variants.

A variant amino acid or DNA sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTp or BLASTn with default settings) .

A variant amino acid sequence can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to a native or reference sequence. As used herein, “similarity” refers to an identical amino acid or a conservatively substituted amino acid, as described herein. Accordingly, the percentage of “sequence similarity” is the percentage of amino acids which is either identical or conservatively changed; e.g., “sequence similarity” = (%sequence identity) + (%conservative changes) . It should be understood that a sequence that has a specified percent similarity to a reference sequence necessarily encompasses a sequence with the same specified percent identity to that reference sequence. The skilled person will be aware of various computer programs, using different mathematical algorithms, that are available to determine the identity or similarity between two sequences. For instance, use can be made of a computer program employing the Needleman and Wunsch algorithm (Needleman et al. (1970) ) ; the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego U.S.A. ) ; the algorithm of E. Meyers and W. Miller (Meyers et al. (1989) ) which has been incorporated into the ALIGN program (version 2.0) ; or more preferably the BLAST (Basic Local Alignment Tool using default parameters) ; see e.g., US Patent 10, 023, 890, the content of which is incorporated by reference herein in its entirety.

As used herein, the phrase “maintains the same function” , when used in reference to an antibody construct, refers to a polypeptide variant that specifically binds to the same epitope (s) of LILBR2, PD-1, and/or VEGF.

Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. A wide variety of, site-specific mutagenesis approaches, e.g., Kunkel’s method, cassette mutagenesis, PCR site-directed mutagenesis (e.g., traditional PCR, primer extension, or inverse PCR) , whole plasmid mutagenesis, in vivo site-directed mutagenesis, CRISPR/Cas-guided mutagenesis, are known in the art and can be applied by the ordinarily skilled artisan to introduce mutations into specific nucleic acid loci. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42: 133, 1986) ; Bauer et al. (Gene 37: 73, 1985) ; Craik (BioTechniques, January 1985, 12-19) ; Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981) ; Braman, Jeff, ed. (2002) In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol. 182 (2nd ed. ) ; Khudyakov and Fields (2002) , Artificial DNA: Methods and Applications, CRC Press; Hsu et al. (2014) , Cell 157 (6) : 1262–78; Cerchione et al. (2020) PLOS ONE 15 (4) : e0231716; and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond (s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., a plasmid.

The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (e.g., mRNA) or antisense RNA derived from a nucleic acid fragment or fragments and/or to the translation of mRNA into a polypeptide.

As used herein, the terms "treat, ” "treatment, " "treating, ” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g., cancer (including but not limited to melanoma, breast cancer, or colorectal adenocarcinoma) . The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a cancer. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom (s) , diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total) , and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment) .

As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g., a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in or within nature.

As used herein, the term "administering, " refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.

As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, transfection, transduction, perfusion, injection, or other delivery method known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine. A cell in a subject can be contacted with any of the antibodies described herein following administration of a composition as described herein to the subject. “Contacting” of a cell can be performed in vitro, ex vivo, or in vivo.

In some embodiments of any of the aspects, cells can be maintained in culture. As used herein, “maintaining” refers to continuing the viability of a cell or population of cells. A maintained population of cells will have at least a subpopulation of metabolically active cells.

As used herein, the term “specific binding” refers to a chemical or physical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity (e.g., the binding domain (s) of an antibody as described herein) for the second target entity (e.g., its cognate epitope (s) on LILRB2, PD-1, and/or VEGF) which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third non-target entity (e.g., a protein other than LILRB2, PD-1, or VEGF) . A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized. In some embodiments, “specific binding” includes a maximum equilibrium dissociation constant (K_D) between an antigen-binding domain and the antigen, for example at most 10E-6 M, e.g., at most 10E-7, at most 10E-8, at most 10E-9, at most 10E-10 M or less.

The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference or a p-value of less than 0.05.

Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about. ” The term “about” when used in connection with percentages can mean ±1%.

As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic (s) of that embodiment of the invention.

The singular terms "a, " "an, " and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g. " is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g. " is synonymous with the term "for example. "

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in cell biology, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp &Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421) ; Robert S. Porter et al. (eds. ) , The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908) ; and Robert A. Meyers (ed. ) , Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) ; Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds. ) , W.W. Norton &Company, 2016 (ISBN 0815345054, 978-0815345053) ; Lewin's Genes XI, published by Jones &Bartlett Publishers, 2014 (ISBN-1449659055) ; Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414) ; Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X) ; Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed. ) Elsevier, 2013 (ISBN 0124199542) ; Current Protocols in Molecular Biology (CPMB) , Frederick M. Ausubel (ed. ) , John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385) , Current Protocols in Protein Science (CPPS) , John E. Coligan (ed. ) , John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds. ) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737) , the contents of which are all incorporated by reference herein in their entireties.

Other terms are defined herein within the description of the various aspects of the invention.

All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A composition comprising a binding domain that specifically binds leukocyte immunoglobulin-
like receptor subfamily B member 2 (LILRB2) , wherein the composition is capable of:
(a) polarizing macrophages from an M2 phenotype to an M1 phenotype;
(b) activating T cells; and/or
(c) inhibiting growth of a cancer.
2. The composition of paragraph 1, wherein the binding domain comprises an antigen binding domain
of an antibody.
3. The composition of paragraph 1 or 2, wherein the binding domain is comprised by a human,
humanized, affinity-matured, and/or chimeric antibody construct.
4. The composition of any one of paragraphs 1-3, wherein the binding domain is comprised by an anti-
LILRB2 antibody construct derived from a primate, a rodent, or a camelid.
5. The composition of any one of paragraphs 1-4, wherein the binding domain is comprised by an anti-
LILRB2 antibody construct derived from a human, a mouse, or an alpaca.
6. The composition of any one of paragraphs 1-5, wherein the binding domain comprises the CDRs of
a V_H domain that specifically binds LILRB2.
7. The composition of any one of paragraphs 1-6, wherein the binding domain comprises the CDRs of
a V_L domain that specifically binds LILRB2.
8. The composition of any one of paragraphs 1-7, wherein the binding domain comprises the CDRs of
a V_H/V_L domain pair that specifically binds LILRB2.
9. The composition of any one of paragraphs 1-8, wherein the binding domain comprises the CDRs of
a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2.
10. The composition of any one of paragraphs 1-9, wherein LILRB2 comprises mammalian LILRB2.
11. The composition of any one of paragraphs 1-10, wherein LILRB2 comprises human LILRB2.
12. The composition of any one of paragraphs 1-11, wherein the binding domain specifically binds to
a LILRB2 epitope or portion of a LILRB2 epitope comprising sequential and/or non-sequential residues of SEQ ID NO: 775.
13. The composition of any one of paragraphs 1-12, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H CDR1, a V_H CDR2, and a V_H CDR3 selected from Table 8; and
(b) a V_L CDR1, a V_L CDR2, and a V_L CDR3 selected from Table 10.
14. The composition of any one of paragraphs 1-13, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H CDR1 comprising one of SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46,
49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, or 118;
(b) a V_H CDR2 comprising one of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47,
50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, or 119;
(c) a V_H CDR3 comprising one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48,
51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, or 120;
(d) a V_L CDR1 comprising one of SEQ ID NOs: 247, 250, 253, 256, 259, 262, 265, 268, 271, 274,
277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, or 829;
(e) a V_L CDR2 comprising one of SEQ ID NOs: 248, 251, 254, 257, 260, 263, 266, 269, 272, 275,
278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 314, 317, or 320; and
(f) a V_L CDR3 comprising one of SEQ ID NOs: 249, 252, 255, 258, 261, 264, 267, 270, 273, 276,
279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, or 830.
15. The composition of any one of paragraphs 8-14, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H CDR1, a V_H CDR2, and a V_H CDR3 encoded by nucleic acid sequences selected from
Table 9; and
(b) a V_L CDR1, a V_L CDR2, and a V_L CDR3 encoded by nucleic acid sequences selected from Table
11.
16. The composition of any one of paragraphs 8-15, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 127, 130, 133,
136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, or 238;
(b) a V_H CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 128, 131, 134,
137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, or 239;
(c) a V_H CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 129, 132, 135,
138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, or 240;
(d) a V_L CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 328, 331, 334,
337, 340, 343, 346, 349, 352, 355, 358, 361, 364, 367, 370, 373, 376, 379, 382, 385, 388, 391, 394, 397, 400, 403, or 406;
(e) a V_L CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 329, 332, 335,
338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 368, 371, 374, 377, 380, 383, 386, 389, 392, 395, 398, 401, 404, or 407; and
(f) a V_L CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 330, 333, 336,
339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, or 408.
17. The composition of any one of paragraphs 4-16, wherein the anti-LILRB2 antibody construct
comprises:
(a) a V_H domain selected from Table 12 or a V_H domain that is at least 80%identical to an amino
acid sequence selected from Table 12; and
(b) a V_L domain selected from Table 14 or a V_L domain that is at least 80%identical to an amino
acid sequence selected from Table 14.
18. The composition of any one of paragraphs 4-17, wherein the anti-LILRB2 antibody construct
comprises:
(a) a V_H domain comprising one of SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 418, 419,
420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, or 448, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, or 448; and
(b) a V_L domain comprising one of SEQ ID NOs: 491, 492, 493, 494, 495, 496, 497, 498, 499,
500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, or 517.
19. The composition of any one of paragraphs 4-18, wherein the anti-LILRB2 antibody construct
comprises:
(a) a V_H domain encoded by a nucleic acid sequence selected from Table 13 or a V_H domain that
is at least 80%identical to a nucleic acid sequence selected from Table 13; and
(b) a V_L domain encoded by a nucleic acid sequence selected from Table 15 or a V_L domain that is
at least 80%identical to a nucleic acid sequence selected from Table 15.
20. The composition of any one of paragraphs 4-19, wherein the anti-LILRB2 antibody construct
comprises:
(a) a V_H domain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 451, 452,
453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 571, 572, 573, 574, 579, 580, 581, 582, 583, 584, 590, 591, 592, 593, 594, 595, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 571, 572, 573, 574, 579, 580, 581, 582, 583, 584, 590, 591, 592, 593, 594, 595, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615; and
(b) a V_L domain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 520, 521,
522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 575, 576, 577, 578, 585, 586, 587, 588, 589, 596, 597, 598, 599, 600, 601, or 785, or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 575, 576, 577, 578, 585, 586, 587, 588, 589, 596, 597, 598, 599, 600, 601, or 785.
21. The composition of any one of paragraphs 4-20, wherein the anti-LILRB2 antibody construct
comprises an antibody selected from the group consisting of: HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206.
22. The composition of any one of paragraphs 9-21, wherein the VHH that specifically binds LILRB2
comprises:
(a) a V_H CDR1 comprising one of SEQ ID NOs: 82, 85, 88, 91, 94, 97, 100, 103, 106, 115, or 118;
(b) a V_H CDR2 comprising one of SEQ ID NOs: 83, 86, 89, 92, 95, 98, 101, 104, 107, 116, or 119;
and
(c) a V_H CDR3 comprising one of SEQ ID NOs: 84, 87, 90, 93, 96, 99, 102, 105, 108, 117, or 120.
23. The composition of any one of paragraphs 9-22, wherein the VHH that specifically binds LILRB2
comprises:
(a) a V_H CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 202, 205, 208,
211, 214, 217, 220, 223, 226, 235, or 238;
(b) a V_H CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 203, 206, 209,
212, 215, 218, 221, 224, 227, 236, or 239; and
(c) a V_H CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 204, 207, 210,
213, 216, 219, 222, 225, 228, 237, or 240.
24. The composition of any one of paragraphs 4-23, wherein the anti-LILRB2 antibody construct
comprises: a VHH comprising one of SEQ ID NOs: 436, 437, 438, 439, 440, 441, 442, 443, 444, 447, or 448, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 436, 437, 438, 439, 440, 441, 442, 443, 444, 447, or 448.
25. The composition of any one of paragraphs 4-24, wherein the anti-LILRB2 antibody construct
comprises: a VHH encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 476, 477, 478, 479, 480, 481, 482, 483, 484, 487, or 488, or a nucleic acid sequence that is at least 80% identical to one of SEQ ID NOs: 476, 477, 478, 479, 480, 481, 482, 483, 484, 487, or 488.
26. The composition of any one of paragraphs 4-25, wherein the anti-LILRB2 antibody construct
comprises an antibody selected from the group consisting of: AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huB45, and huB206.
27. A composition comprising:
(a) a first binding domain that specifically binds leukocyte immunoglobulin-like receptor
subfamily B member 2 (LILRB2) ; and
(b) a second binding domain that specifically binds:
(i) programmed cell death protein 1 (PD-1) ; or
(ii) vascular endothelial growth factor (VEGF) .
28. The composition of paragraph 27, wherein the composition is capable of:
(a) polarizing macrophages from an M2 phenotype to an M1 phenotype;
(b) activating T cells; and/or
(c) inhibiting growth of a cancer.
29. The composition of paragraph 27 or 28, wherein the first and/or second binding domains comprise
an antigen binding domain of an antibody.
30. The composition of any one of paragraphs 27-29, wherein the first and second binding domains
each comprise an antigen binding domain of an antibody.
31. The composition of any one of paragraphs 27-30, wherein the first and second binding domains are
comprised by a human, humanized, affinity-matured, and/or chimeric antibody construct.
32. The composition of any one of paragraphs 27-31, wherein the first and second binding domains are
comprised by a bispecific antibody construct.
33. The composition of paragraph 27-32, wherein the first binding domain comprises the CDRs of a V_H
domain that specifically binds LILRB2.
34. The composition of paragraph 27-33, wherein the first binding domain comprises the CDRs of a V_L
domain that specifically binds LILRB2.
35. The composition of paragraph 32-34, wherein the bispecific antibody construct comprises:
(a) the first binding domain comprising:
(i) the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or
(ii) the CDRs of a V_H domain of a variable domain of the heavy chain of a heavy-
chain antibody (VHH) that specifically binds LILRB2; and
(b) the second binding domain comprising the CDRs of a V_H/V_L domain pair that
specifically binds PD-1.
36. The composition of paragraph 32-35, wherein the bispecific antibody construct comprises:
(a) the first binding domain comprising:
(i) the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or
(ii) the CDRs of a V_H domain of a variable domain of the heavy chain of a heavy-
chain antibody (VHH) that specifically binds LILRB2; and
(b) the second binding domain comprising the CDRs of a V_H/V_L domain pair that
specifically binds VEGF.
37. The composition of paragraph 32-36, wherein the bispecific antibody construct is selected from
the group consisting of a tandem scFv (taFv or scFv₂) , diabody, dAb₂A/HH₂, knob-into-holes bispecific derivative, SEED-IgG, heteroFc-scFv, Fab-scFv, scFv-Jun/Fos, Fab'-Jun/Fos, tribody, DNL-F (ab) ₃, scFv₃-CH1/CL, Fab-scFv₂, IgG-scFab, IgG-scFv, scFv-IgG, IgG-VHH, VHH-IgG, scFv₂-Fc, F (ab') ₂-scFv₂, scDB-Fc, scDb-CH₃, Db-Fc, scFv₂-H/L, DVD-Ig, tandAb, scFv-dhlx-scFv, dAb2-IgG, dAb-IgG, and dAb-Fc-dAb constructs.
38. The composition of paragraph 32-37, wherein the bispecific antibody construct comprises:
(a) the first binding domain comprising:
(i) a single-chain fragment variable (scFv) comprising the CDRs of a V_H/V_L domain
pair that specifically binds LILRB2; or
(ii) a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody
(VHH) comprising the CDRs of a V_H domain that specifically binds LILRB2; and
(b) the second binding domain comprising an immunoglobulin heavy chain and
immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds PD-1.
39. The composition of paragraph 32-38, wherein the bispecific antibody construct comprises:
(a) the first binding domain comprising:
(i) a single-chain fragment variable (scFv) comprising the CDRs of a V_H/V_L domain
pair that specifically binds LILRB2; or
(ii) a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody
(VHH) comprising the CDRs of a V_H domain that specifically binds LILRB2; and
(b) the second binding domain comprising an immunoglobulin heavy chain and
immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds VEGF.
40. The composition of paragraph 38 or 39, wherein the scFv or the VHH is linked to the heavy chain.
41. The composition of any one of paragraphs 38-40, wherein the scFv or the VHH is linked to the C-
terminus of the heavy chain.
42. The composition of any one of paragraphs 38-41, wherein the scFv or the VHH is linked to the N-
terminus of the heavy chain.
43. The composition of any one of paragraphs 38-42, wherein the scFv or the VHH is linked to the light
chain.
44. The composition of any one of paragraphs 38-43, wherein the scFv or the VHH is linked to the C-
terminus of the light chain.
45. The composition of any one of paragraphs 38-44, wherein the scFv or the VHH is linked to the N-
terminus of the light chain.
46. The composition of any one of paragraphs 27-45, wherein the LILRB2 comprises mammalian
LILRB2; the PD-1 comprises mammalian PD-1; and/or the VEGF comprises mammalian VEGF.
47. The composition of any one of paragraphs 27-46, wherein the LILRB2 comprises human
LILRB2; the PD-1 comprises human PD-1; and/or the VEGF comprises human VEGF.
48. The composition of any one of paragraphs 27-47, wherein the second binding domain specifically
binds PD-1 and comprises nivolumab, pembrolizumab, toripalimab, sintilimab, cemiplimab, or dostarlimab, or functional fragments thereof.
49. The composition of any one of paragraphs 27-48, wherein the second binding domain specifically
binds to at least one nivolumab PD-1 epitope or portion of a nivolumab PD-1 epitope comprising: residues 25 to 31 of SEQ ID NO: 776 (LDSPDRP, SEQ ID NO: 787) , residues 25 to 31 of SEQ ID NO: 776 (TSES, SEQ ID NO: 779) , and/or residues 127 to 132 of SEQ ID NO: 776 (SLAPKA, SEQ ID NO: 780) .
50. The composition of any one of paragraphs 27-49, wherein the second binding domain specifically
binds to at least one pembrolizumab PD-1 epitope or portion of a pembrolizumab PD-1 epitope comprising: residues S60, S62, V64, N66, I126, L128, A129, K131, A132, and/or I134 of SEQ ID NO: 776, residues 75 to 78 of SEQ ID NO: 776 (QTDK, SEQ ID NO: 781) , and/or residues 81 to 90 of SEQ ID NO: 776 (AFPEDRSQPG, SEQ ID NO: 782) .
51. The composition of any one of paragraphs 27-47, wherein the second binding domain specifically
binds VEGF and comprises bevacizumab, ranibizumab, or Aflibercept, or functional fragments thereof.
52. The composition of any one of paragraphs 27-47 or 51, wherein the second binding domain
specifically binds to a bevacizumab VEGF epitope or portion of a bevacizumab VEGF epitope comprising residues 85 to 92 of SEQ ID NO: 777, residues 111 to 112 of SEQ ID NO: 778, residues 111 to 112 of SEQ ID NO: 786, or PHQGQHIG (SEQ ID NO: 783) .
53. The composition of any one of paragraphs 27-47 or 51-52, wherein the first binding domain that
specifically binds LILRB2 comprises an antibody or functional fragment thereof selected from the group consisting of: HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206.
54. The composition of any one of paragraphs 27-53, wherein the first binding domain that
specifically binds LILRB2 comprises an antibody or functional fragment thereof selected from the group consisting of: HB59, MIL-C68, huC68, MIL-C112, huC112, AIL-B45, huB45, AIL-B206, and huB206.
55. The composition of any one of paragraphs 35-54, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H CDR1 comprising one of SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46,
49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, or 118;
(b) a V_H CDR2 comprising one of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47,
50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, or 119;
(c) a V_H CDR3 comprising one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48,
51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120;
(d) a V_L CDR1 comprising one of SEQ ID NOs: 247, 250, 253, 256, 259, 262, 265, 268, 271, 274,
277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, or 829;
(e) a V_L CDR2 comprising one of SEQ ID NOs: 248, 251, 254, 257, 260, 263, 266, 269, 272, 275,
278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 314, 317, or 320; and
(f) a V_L CDR3 comprising one of SEQ ID NOs: 249, 252, 255, 258, 261, 264, 267, 270, 273, 276,
279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, or 830.
56. The composition of any one of paragraphs 35-54, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H CDR1 comprising one of SEQ ID NOs: 19, 109, 112, 115, or 118;
(b) a V_H CDR2 comprising one of SEQ ID NOs: 20, 110, 113, 116, or 119;
(c) a V_H CDR3 comprising one of SEQ ID NOs: 21, 111, 114, 117, or 120;
(d) a V_L CDR1 comprising one of SEQ ID NOs: 259, 316, or 319;
(e) a V_L CDR2 comprising one of SEQ ID NOs: 260, 317, or 320; and
(f) a V_L CDR3 comprising one of SEQ ID NOs: 261, 318, or 321.
57. The composition of any one of paragraphs 35-56, wherein the VHH that specifically binds LILRB2
comprises:
(a) a V_H CDR1 comprising one of SEQ ID NOs: 82, 85, 88, 91, 94, 97, 100, 103, 106, 115, or 118;
(b) a V_H CDR2 comprising one of SEQ ID NOs: 83, 86, 89, 92, 95, 98, 101, 104, 107, 116, or 119;
and
(c) a V_H CDR3 comprising one of SEQ ID NOs: 84, 87, 90, 93, 96, 99, 102, 105, 108, 117, 120.
58. The composition of any one of paragraphs 35-57, wherein the VHH that specifically binds LILRB2
comprises:
(a) a V_H CDR1 comprising one of SEQ ID NOs: 115 or 118;
(b) a V_H CDR2 comprising one of SEQ ID NOs: 116 or 119; and
(c) a V_H CDR3 comprising one of SEQ ID NOs: 117 or 120.
59. The composition of any one of paragraphs 35-58, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H CDR1 comprising SEQ ID NO: 19;
(b) a V_H CDR2 comprising SEQ ID NO: 20;
(c) a V_H CDR3 comprising SEQ ID NO: 21;
(d) a V_L CDR1 comprising SEQ ID NO: 259;
(e) a V_L CDR2 comprising SEQ ID NO: 260; and
(f) a V_L CDR3 comprising SEQ ID NO: 261.
60. The composition of any one of paragraphs 35-59, wherein the V_H/V_L domain pair that specifically
binds PD-1 comprises:
(a) a V_H CDR1 comprising one of SEQ ID NOs: 547 or 550;
(b) a V_H CDR2 comprising one of SEQ ID NOs: 548 or 551;
(c) a V_H CDR3 comprising one of SEQ ID NOs: 549 or 552;
(d) a V_L CDR1 comprising one of SEQ ID NOs: 556 or 559;
(e) a V_L CDR2 comprising one of SEQ ID NOs: 557 or 560; and
(f) a V_L CDR3 comprising one of SEQ ID NOs: 558 or 561.
61. The composition of any one of paragraphs 35-59, wherein the V_H/V_L domain pair that specifically
binds VEGF comprises:
(a) a V_H CDR1 comprising SEQ ID NO: 553;
(b) a V_H CDR2 comprising SEQ ID NO: 554;
(c) a V_H CDR3 comprising SEQ ID NO: 555;
(d) a V_L CDR1 comprising SEQ ID NO: 562;
(e) a V_L CDR2 comprising SEQ ID NO: 563; and
(f) a V_L CDR3 comprising SEQ ID NO: 564.
62. The composition of any one of paragraphs 35-61, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H comprising one of SEQ ID NOs: 415, 432, 435, 445, 446, or an amino acid sequence that
is at least 80%identical to one of SEQ ID NOs: 415, 432, 435, 445, 446; and
(b) a V_L comprising one of SEQ ID NOs: 495, 512, 516, 515, 517 or an amino acid sequence that
is at least 80%identical to one of SEQ ID NOs: 495, 512, 516, 515, 517.
63. The composition of any one of paragraphs 35-62, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H comprising SEQ ID NO: 415 or an amino acid sequence that is at least 80%identical to
SEQ ID NO: 415; and
(b) a V_L comprising SEQ ID NO: 495 or an amino acid sequence that is at least 80%identical to
SEQ ID NO: 495.
64. The composition of any one of paragraphs 35-62, wherein the V_H/V_L domain pair that specifically
binds PD-1 comprises:
(a) a V_H comprising one of SEQ ID NOs: 565 or 567 or an amino acid sequence that is at least 80%
identical to one of SEQ ID NOs: 565 or 567; and
(b) a V_L comprising one of SEQ ID NOs: 566 or 568 or an amino acid sequence that is at least 80%
identical to one of SEQ ID NOs: 566 or 568.
65. The composition of any one of paragraphs 35-62, wherein the V_H/V_L domain pair that specifically
binds VEGF comprises:
(a) a V_H comprising SEQ ID NO: 569 or an amino acid sequence that is at least 80%identical to
SEQ ID NO: 569; and
(b) a V_L comprising SEQ ID NO: 570 or an amino acid sequence that is at least 80%identical to
SEQ ID NO: 570.
66. The composition of any one of paragraphs 35-65, wherein the V_H/V_L domain pair that specifically
binds LILRB2 comprises:
(a) a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 455, 472, 475, 485,
486, 571-574, 579-584, 590-595 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 455, 472, 475, 485, 486, 579-584, 571-574, 590-595; and
(b) a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 524, 541, 544, 545,
546, 575-578, 585-589, 596-601, 785, or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 524, 541, 544, 545, 546, 575-578, 585-589, 596-601, or 785.
67. The composition of any one of paragraphs 35-66, wherein the V_H/V_L domain pair that specifically
binds PD-1 comprises:
(a) a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 616-618, 624-632 or
a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 616-618, 624-632; and
(b) a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 619-623, 633-638 or
a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 619-623, 633-638.
68. The composition of any one of paragraphs 35-66, wherein the V_H/V_L domain pair that specifically
binds VEGF comprises:
(a) a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 639-647 or a nucleic
acid sequence that is at least 80%identical to one of SEQ ID NOs: 639-647; and
(b) a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 648-654 or a nucleic
acid sequence that is at least 80%identical to one of SEQ ID NOs: 648-654.
69. The composition of any one of paragraphs 32-68, wherein the bispecific antibody construct
comprises an anti-LILRB2 and anti-PD-1 bispecific antibody construct.
70. The composition of paragraph 69, wherein the anti-LILRB2 and anti-PD-1 bispecific antibody
construct comprises:
(a) an immunoglobulin heavy chain comprising one of SEQ ID NOs: 655, 657, 659, 661, 671,
673, 675, 677, 683, 689, 691, 693, 695, 697, 703, 705, 707, 709, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 655, 657, 659, 661, 671, 673, 675, 677, 683, 689, 691, 693, 695, 697, 703, 705, 707, 709; and
(b) an immunoglobulin light chain comprising one of SEQ ID NOs: 656, 658, 660, 662, 672, 674,
676, 678, 684, 690, 692, 694, 696, 698, 704, 706, 708, 710, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 656, 658, 660, 662, 672, 674, 676, 678, 684, 690, 692, 694, 696, 698, 704, 706, 708, 710.
71. The composition of paragraph 69 or 70, wherein the anti-LILRB2 and anti-PD-1 bispecific
antibody construct is selected from Bis-M23-1 and Bis-M28.
72. The composition of any one of paragraphs 69-71, wherein the anti-LILRB2 and anti-PD-1
bispecific antibody is Bis-M23-1 comprising:
(a) an immunoglobulin heavy chain comprising SEQ ID NO: 683 or an amino acid sequence that
is at least 80%identical to SEQ ID NO: 683; and
(b) an immunoglobulin light chain comprising SEQ ID NO: 684, or an amino acid sequence that is
at least 80%identical to SEQ ID NO: 684.
73. The composition of any one of paragraphs 69-71, wherein the anti-LILRB2 and anti-PD-1
bispecific antibody is Bis-M28 comprising:
(a) an immunoglobulin heavy chain comprising SEQ ID NO: 689, or an amino acid sequence that
is at least 80%identical to SEQ ID NO: 689; and
(b) an immunoglobulin light chain comprising SEQ ID NO: 690, or an amino acid sequence that is
at least 80%identical to SEQ ID NO: 690.
74. The composition of any one of paragraphs 32-68, wherein the bispecific antibody construct
comprises an anti-LILRB2 and anti-VEGF bispecific antibody construct.
75. The composition of paragraph 74, wherein the anti-LILRB2 and anti-VEGF bispecific antibody
construct comprises:
(a) an immunoglobulin heavy chain comprising one of SEQ ID NOs: 663, 665, 667, 669, 679,
681, 685, 687, 699, 701, 711, 713, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 663, 665, 667, 669, 679, 681, 685, 687, 699, 701, 711, 713; and
(b) an immunoglobulin light chain comprising one of SEQ ID NOs: 664, 666, 668, 670, 680, 682,
686, 688, 700, 702, 712, 714, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 664, 666, 668, 670, 680, 682, 686, 688, 700, 702, 712, 714.
76. The composition of paragraph 74 or 75, wherein the anti-LILRB2 and anti-VEGF bispecific
antibody construct is selected from Bis-M24 and Bis-M27.
77. The composition of any one of paragraphs 74-76, wherein the anti-LILRB2 and anti-VEGF
bispecific antibody is Bis-M24 comprising:
(a) an immunoglobulin heavy chain comprising SEQ ID NO: 685 or an amino acid sequence that
is at least 80%identical to SEQ ID NO: 685; and
(b) an immunoglobulin light chain comprising SEQ ID NO: 686 or an amino acid sequence that is
at least 80%identical to SEQ ID NO: 686.
78. The composition of any one of paragraphs 74-76, wherein the anti-LILRB2 and anti-VEGF
bispecific antibody is Bis-M27 comprising:
(a) an immunoglobulin heavy chain comprising SEQ ID NO: 687, or an amino acid sequence that
is at least 80%identical to SEQ ID NO: 687; and
(b) an immunoglobulin light chain comprising SEQ ID NO: 688, or an amino acid sequence that is
at least 80%identical to SEQ ID NO: 688.
79. A nucleic acid encoding a polypeptide of the composition of any one of paragraphs 1-78.
80. A vector comprising the nucleic acid of paragraph 79.
81. A cell comprising the nucleic acid of paragraph 79 or the vector of paragraph 80.
82. The cell of paragraph 81, wherein the cell is an antibody-producing cell.
83. A cell in combination with the composition of any one of paragraphs 1-78.
84. The cell of paragraph 83, wherein the cell is an immune cell.
85. The cell of paragraph 83 or 84, wherein the cell is an M1 macrophage or an M2 macrophage.
86. The cell of paragraph 83 or 84, wherein the cell is a T lymphocyte.
87. A pharmaceutical composition comprising the composition of any one of paragraphs 1-78, the
nucleic acid of paragraph 79, the vector of paragraph 80, or the cell of any one of paragraphs 81-86, and a pharmaceutically acceptable carrier.
88. A method of affinity maturing the composition of any one of paragraphs 1-78, the method
comprising:
a) mutating at least one binding domain;
b) determining the affinity of the mutated binding domain for its cognate ligand; and
c) selecting the mutated binding domain that exhibits increased affinity for its cognate
ligand compared to the unmutated binding domain.
89. The method of paragraph 88, further comprising producing a composition comprising the selected
mutated binding domain.
90. The method of paragraph 88 or 89, further comprising producing an antibody construct
comprising the selected mutated binding domain.
91. A composition produced by the method of any one of paragraphs 88-90.
92. An antibody construct produced by the method of any one of paragraphs 88-90.
93. A method for polarizing a macrophage from an M2 phenotype to an M1 phenotype, the method
comprising contacting the macrophage with the composition of any one of paragraphs 1-78.
94. The method of paragraph 93, wherein after contacting, the macrophage exhibits decreased
expression of CD163 and/or CD206.
95. The method of paragraph 93 or 94, wherein after contacting, the macrophage exhibits increased
expression of CD80.
96. The method of any one of paragraphs 93-95, wherein after contacting, the macrophage exhibits
increased secretion of TNF-α.
97. The method of any one of paragraph 93-96, wherein after contacting, the macrophage induces
increased cancer cytotoxicity.
98. The method of any one of paragraphs 93-98, wherein the macrophage is contacted for a sufficient
amount of time.
99. The method of paragraph 98, wherein the sufficient amount of time is at least 2 days.
100. The method of any one of paragraphs 93-99, wherein the composition is an anti-LILRB2
antibody construct selected from the group consisting of HB59, AIL-B45, huB45, AIL-B206, huB206, MIL-C68, huC68, MIL-C112, and huC112.
101. The method of any one of paragraphs 93-99, wherein the composition is an anti-LILRB2
and anti-PD-1 bispecific antibody construct selected from Bis-M23-1 or Bis-M28.
102. The method of any one of paragraphs 93-99, wherein the composition is an anti-LILRB2
and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27.
103. A method for activating a T cell, the method comprising contacting the T cell with the
composition of any one of paragraphs 1-78.
104. The method of paragraph 103, wherein after contacting, the T cell exhibits increased
activation of NFAT signaling.
105. The method of paragraph 103 or 104, wherein after contacting, the T cell exhibits
increased cancer cytotoxicity.
106. The method of any one of paragraphs 103-105, wherein the T cell is contacted for a
sufficient amount of time.
107. The method of paragraph 106, wherein the sufficient amount of time is at least 2 days.
108. The method of any one of paragraphs 103-107, wherein the composition is an anti-
LILRB2 antibody construct selected from the group consisting of HB74, HA23, HA31, HB59, MIL-C112, MILC18, MIL-C27, MIL-C3, MIL-C32, MIL-C68, MIL-C9, AIL-A1, AIL-B100, AIL-B124, AIL-B134, and AIL-B45.
109. The method of any one of paragraphs 103-107, wherein the composition is an anti-
LILRB2 and anti-PD-1 bispecific antibody construct selected from Bis-M23-1 or Bis-M28.
110. The method of any one of paragraphs 103-107, wherein the composition is an anti-
LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27.
111. A method of treating cancer, the method comprising administering an effective amount of
the composition of any one of paragraphs 1-78 or the pharmaceutical composition of paragraph 87 to a subject in need thereof.
112. The method of paragraph 111, wherein the composition is administered at a dose of 10
mg/kg to 20 mg/kg.
113. The method of paragraph 111 or 112, wherein the composition is administered at least
twice a week for at least three weeks.
114. The method of any one of paragraphs 111-113, wherein the composition is administered
intravenously.
115. The method of any one of paragraphs 111-114, wherein the composition is an anti-
LILRB2 antibody construct selected from MIL-C68, MIL-B206, or HB59.
116. The method of any one of paragraphs 111-115, wherein the method has a tumor weight
inhibition rate (TGI_TW) of at least 30%after 3 weeks of administration.
117. The method of any one of paragraphs 111-116, wherein the method has a tumor volume
inhibition rate (TGI_TV) of at least 35%after 3 weeks of administration.
118. The method of any one of paragraphs 111-117, wherein the composition is an anti-
LILRB2 and anti-PD-1 bispecific antibody construct selected from Bis-M23-1 or Bis-M28.
119. The method of any one of paragraphs 111-117, wherein the composition is an anti-
LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27.
120. The method of any one of paragraphs 111-117, wherein the composition is the anti-
LILRB2 and anti-VEGF bispecific antibody construct Bis-M24.
121. The method of any one of paragraphs 111-120, wherein the composition is administered
at a dose of about 20 mg/kg twice weekly for at least one week.
122. The method of any one of paragraphs 111-120, wherein the composition is administered
at a dose of about 4 mg/kg twice weekly for at least the fourth week after beginning treatment.
123. The method of any one of paragraphs 111-122, wherein the method has a tumor weight
inhibition rate (TGI_TW) of at least 50%after 4 weeks of administration.
124. The method of any one of paragraphs 111-123, wherein the cancer is a blood cancer.
125. The method of any one of paragraphs 111-123, wherein the cancer comprises at least one
solid tumor.
126. The method of paragraph 125, wherein the cancer is melanoma.
127. The method of paragraph 125, wherein the cancer is breast cancer.
128. The method of paragraph 125, wherein the cancer is colorectal adenocarcinoma.

The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
EXAMPLES
Example 1: Construction of humanFab library and anti-LILRB2 antibody screening
1.1 Construction of humanFab library

In the following example, a humanFab library was constructed and used for anti-LILRB2 antibody panning. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers by FICOLL-PAQUE (GE, Cat#17144003S) density gradient centrifugation. Total RNA was extracted and reverse-transcribed into cDNA using PRIMESCRIPT^TM II 1st Strand cDNA Synthesis Kit (TAKARA, Cat#6210A) . Degenerate primers were designed at the front end of the V region and the back end of the first constant region of the heavy and light chain, respectively. The DNA fragments encoding for heavy and light chain variable regions were obtained by polymerase chain reaction (PCR) , then amplified and ligated by overlapping PCR to obtain fragments that contained both the heavy and light chain variable regions. They were then cloned into phagemid vectors (pMID21, NOVOPROTEIN, Cat#V004730) , followed by transformation into the E. coli competent cell SS320 (LUCIGEN, MC1061F) by electroporation (BIO-RAD, MICROPULSER) . The transformed E. coli SS320 cells were spread on 2-YT agar plates supplied with 100 μg/ml ampicillin, followed by inoculation into 2-YT liquid medium for bacterial library stock preparation. The obtained Fab library had a library size of 3 x 10¹¹cfu/mL. Based on the size of the library, bacteria with 50 optical density (OD) (1OD equals 5 x 10⁸cfu) was inoculated into 2-YT liquid medium and allowed to grow into exponential phase. The VSCM13 helper phage (STRATAGENE) with a multiplicity of infection (MOI) of 50 was then added and co-cultured with the bacteria for phage stock preparation. Finally, the phage particles were precipitated with polyethylene glycol (PEG) and NaCl from the culture supernatant and preserved for subsequent antibody panning.
1.2 Phage library panning

The preserved phage library was screened for anti-LILRB2 antibodies with magnetic beads or using immunotube methods. Briefly, the phage suspension was first diluted and blocked with 2.5%bovine serum albumin (BSA) , followed by incubation with DYNABEADS M-280 (INVITROGEN) to remove non-specific binding. The magnetic beads were coated with the biotinylated extracellular domain (ECD) of human LILRB2, blocked with 2.5%BSA, and then incubated with the phage library. Panning was performed with a magnetic bead processor KINGFISHER (THERMOELECTRON) , following manufacturer’s instruction for binding and washing. For library panning with the immunotube method, the preserved phage suspension was first blocked by 5%PBSM (PBS+5%milk) . Then the immunotube was coated with recombinant human LILRB2 ECD at 4 ℃ overnight and blocked with 5%PBSM. The phage suspension was incubated with the antigen-coated and blocked immunotube. Binding and washing was performed according to the immunotube method, using Trypsin for phage elution.

The eluate containing phages was mixed thoroughly with E. coli SS320 cells and incubated at 37℃ overnight for phage amplification. The panning was repeated for 3 rounds to obtain antibody clones with high affinity.

The collected supernatants from the 3 rounds of panning were 1: 5 serial diluted with phosphate buffered saline (PBS) and subjected to screening and quantification by ELISA. After primary ELISA screening, the first batch of 646 clones were picked, and 184 positive clones were obtained, including 96 sequence-unique molecules, of which 17 sequence-unique molecules were selected for full-length construction. A total of 180 clones were picked in the second batch, 71 positive clones were obtained, and 23 molecules with unique sequences were selected for the full-length construct. Results are shown in Fig. 1A-1B.
Example 2. Screening of anti-LILRB2 antibody from mouse immune library

In this example, anti-LILRB2 antibodies were generated by immunization of mice with human LILRB2-ECD antigen, followed by Fab library construction and phage library panning with immunotube and magnetic bead methods.

Briefly, mice were first immunized with recombinant human LILRB2-ECD antigen in complete Freund's adjuvant by intraperitoneal plus subcutaneous multi-site injection, and subsequently boosted twice with LILRB2-ECD protein in incomplete Freund adjuvant. Serum of the immunized mice was collected, and antibody titer was measured by ELISA. Then RNA was extracted from spleen of the immunized mice usingHP Total RNA kit (OMEGA) , and reverse transcribed to obtain cDNA by PRIMESCRIPT^TM II 1st Strand cDNA Synthesis Kit (TAKARA, Cat#6210A) . Construction of Fab library and phage panning was performed using the same method as detailed in Example 1.

After the initial ELISA screening, a total of 1486 clones were selected, and 94 sequence-unique molecules were obtained. Based on sequence diversity analysis, a total of 49 molecules were selected for full-length construction. Results are shown in Fig. 2A-2B.
Example 3. Screening of anti-LILRB2 antibody from Alpaca immune library

In this example, anti-LILRB2 antibodies were generated by immunization of alpacas with human LILRB2-ECD antigen, followed by Fab library construction and phage library panning with immunotube and magnetic bead methods.

One alpaca (NM002) was immunized with recombinant human LILRB2-ECD-FC (IgG1) antigen every two weeks for a total of four times, and the other alpaca (NSY051) was cross-immunized with human LILRB2-ECD-FC (IgG1) and cynomolgus LILRB2-ECD-FC antigen for six times in nine weeks. Two weeks after the last immunization, blood was collected for serum titer testing. Then PBMCs were isolated from blood of the immunized alpacas. RNA was extracted and reverse transcribed into cDNA by PRIMESCRIPT^TM II 1st Strand cDNA Synthesis Kit (TAKARA, Cat#6210A) . Library sizes of 1.73 x 10⁹ colony-forming units (cfu) and 1.36 x 10⁹ cfu were constructed from alpaca NSY051 and
NM002, respectively.

The steps for alpaca immune library panning are the same as detailed in Example 1.

After primary ELISA screening, a total of 1214 clones were selected, and 781 positive clones with cross-reactivity with cynomolgus LILRB2-ECD protein were obtained. After sequence analysis, 49 sequence-unique molecules were obtained, and 37 molecules were selected for full-length construction. Results are shown in Fig. 3A-3B.
Example 4. Binding activity measurements by ELISA and SPR

The binding activity of the anti-LILRB2 antibodies to recombinant human LILRB2 antigen was evaluated by ELISA methods. Briefly, 96-well plates were coated with recombinant human LILRB2 antigen at 2℃-8℃ overnight, and subsequently blocked with 200 μl of 1%Casein + 3%BSA blocking solution. Various anti-LILRB2 antibodies were serially diluted with PBS, added to the coated wells of the 96-well plates at 100μL/well, and incubated at 37℃ for 1 hour. Plate-bound anti-LILRB2 antibodies were detected with horseradish peroxidase (HRP) conjugated Goat anti-human antibody and TMB (3, 3', 5, 5'tetramethylbenzidine dihydrochloride) substrate. Results showed that the binding EC₅₀ values of antibodies HA14, HA17, HA119, HA23, HA31, HA58, HA9, HB3, HB37, HB5, HB50, HB59, HB68, HB70, HB71, HB72 and HB74 to LILRB2 antigen were 99.7ng/ml, 262.5ng/ml, 1048ng/ml, 18.7ng/ml, 27.4ng/ml, 24.3ng/ml, 17.4ng/ml, 23.3ng/ml, 15.5ng/ml, 80.7ng/ml, 52.8ng/ml, 38.6ng/ml, 63.7ng/ml, 58.0ng/ml, 70.2ng/ml, 161.4ng/ml and 60.3ng/ml, respectively.

The kinetics of anti-LILRB2 antibodies towards recombinant human LILRB2-ECD-His was evaluated by Surface Plasmon Resonance (SPR) . Results are shown in Table 1. J-19. h1 is an anti-LILRB2 antibody with sequence from international patent publication WO2019/126514A2 and used as a control antibody in this example.

Table 1 Binding kinetics of Anti-LILRB2 antibodies towards rhLILRB2-ECD-His

Epitope binning of anti-LILRB2 antibodies was performed for 19 antibodies by the Bio-Layer Interferometry (BLI) method using a sandwich format. Each antibody was cross-binned with the rest of antibodies to determine whether they could interfere with each other. The binding signal ratio (%) of the second antibody over first antibody was calculated and results were shown in Table 2 and Table 3. Antibody clones HB50, HB59, HB68, HB72, B45, B134, C112, and C77 exhibited a different antigen-binding epitope from control antibody J-19. h1; clones B45, B206, C68, and C112 may share the same antigen-binding epitope with another control antibody 1E1 (G4) . 1E1 (G4) is also an anti-LILRB2 antibody with sequence from U.S. patent publication US2018/026160.

Table 2 Epitope binning of anti-LILRB2 antibodies

Table 3 Epitope binning of anti-LILRB2 antibodies

Example 5. In vitro binding assay to detect the blocking effect of anti-LILRB2 antibodies

In this example, the anti-LILRB2 antibodies were evaluated for their blocking effect on the binding of human LILRB2 protein to HLA-G overexpressing A375 cells (A375-HLA-G) using flow cytometry. As shown in Table 4, the anti-LILRB2 antibodies exhibited various degrees of blocking effect as compared to IgG4 isotype control with IC₅₀ ranged from 1.606 μg/ml to 6.715 μg/ml.

Table 4 Anti-LILRB2 antibodies interfere with HLA-G ligand binding to the human LILRB2 protein

Example 6. Effect of anti-LILRB2 antibodies on T-cell activation

The effect of anti-LILRB2 antibodies on T cell-activation was evaluated in LILRB2 overexpressing Jurkat-NFAT-Luc system. Briefly, cell culture plates were coated with anti-CD3 antibodies at 4 ℃ overnight. Human LILRB2 overexpressing Jurkat-NFAT-luc cells and HLA-G-A375 melanoma cells were mixed at a ratio of 1: 2 (2×10⁵ cells/mL and 4×10⁵ cells/mL, respectively) and added to the anti-CD3 antibody coated plates together with the different anti-LILRB2 antibodies. BRIGHT-LITE ASSAY solution (VAZYME, DD1204-03) containing luciferin was added to the cell plates and measured for mean fluorescence intensity. As shown in Fig. 4A-4D, clones HB74, HA23, HA31, HB59, MIL-C112, MILC18, MIL-C27, MIL-C3, MIL-C32, MIL-C68, MIL-C9, AIL-A1, AIL-B100, AIL-B124, AIL-B134, and AIL-B45 displayed T cell activation effects in this assay system.
Example 7. Humanization of mouse and alpaca anti-LILRB2 antibodies

Mouse antibodies MIL-C68 and MIL-C112, as well as alpaca antibodies AIL-B45 and AIL-B206, were humanized using the method of CDR grafting in which the CDRs of non-human antibodies were grafted onto the human frameworks. Briefly, the sequences of the V region of the parental antibody were checked and compared with the database (IGBLAST) to determine the human Germline with the highest homology to the framework regions of non-human antibody. After the desired human frameworks were chosen, some Vernier zone residues that were critical to affinities of antibodies were back-mutated to the original parental residues. The humanized antibodies with different humanness scores were designed and recombinantly expressed for affinity evaluation. Binding of the humanized antibodies to recombinant human LILRB2 was evaluated with ELISA and results shown in Fig. 5A-5D. Most of the humanized antibodies demonstrated comparable binding activities to the parental antibodies.
Example 8. The effect of anti-LILRB2 antibodies on polarization of peripheral blood mononuclear cell-
derived macrophages

In this example, the effect of LILRB2 antibody-mediated polarization of macrophages from M2 to M1 was evaluated in human peripheral blood cell-derived macrophages by macrophage immunotyping and TNF-α level measurements.

Specifically, monocyte-derived macrophages (MDM) were isolated from human peripheral blood mononuclear cells (PBMC) using STEMCELL EASYSEP (19059, STEMCELL) and induced to differentiate into M0 macrophages by macrophage colony-stimulating factor (M-CSF) (MCF-H5218, ACRO) , followed by polarization into M2 macrophages under IL-4 stimulation. Subsequently, M2 macrophages were treated with 10μg/mL anti-LILRB2 antibodies at 37 ℃ for 2 days. Then the cluster of differentiation (CD) marker expression on the surface of macrophages were evaluated by flow cytometry, and changes in the level of inflammatory cytokines in the supernatant were measured. As shown in Fig. 6, anti-LILRB2 antibodies HB59, AIL-B45, huB45, AIL-B206, huB206, MIL-C68, huC68, MIL-C112, and huC112 induced the downregulation of the M2 phenotypes as indicated by the decreased expression of CD163 (see e.g., Fig. 6B and 6F) and/or CD206 (see e.g., Fig. 6A and 6E) ; and/or the upregulation of M1 phenotypes as indicated by the upregulation of CD80 expression (see e.g., Fig. 6C and 6G) , accompanied with the secretion of inflammatory cytokine TNF-α (see e.g., Fig. 6D and 6H) .
Example 9. Anti-tumor effect of LILRB2 antibody in huPBMC-NOG mouse inoculated with HLA-G-
A375 tumor model

The anti-tumor effect of LILRB2 antibodies were evaluated in PBMC humanized NOG (NOD/Shi-scid, IL-2Rγnull) mouse subcutaneously inoculated with HLA-G-A375 cells, a human melanoma cell line. All animal handling procedures were approved by a Biomedical Laboratory Animal Management and Use Committee (IACUC) . Briefly, female NOG mice (BEIJING VITALSTAR BIOTECHNOLOGY) were inoculated subcutaneously with 5×10⁶ HLA-G-A375 cells on the right flank. When tumor volume reached at about 70 mm³, mice were randomized into 5 groups (6 mice/group) , including vehicle control group (G1) and 4 different anti-LILRB2 antibody groups (G2: MIL-C68; G3: MIL-C112; G4: AIL-B45; G5: AIL-B206) . The mice were humanized with 5×10⁶ PBMC via caudal vein injection on the day of grouping and 10 days after grouping, respectively. Two days after the first PBMC injection, vehicle control or different anti-LILRB2 antibodies were administrated intravenously at dose level of 10 mg/kg twice a week (BIW) for 3 weeks. Body weight and tumor volume were measured and recorded twice a week for each mouse. The anti-tumor efficacy was evaluated by tumor weight or volume inhibition (TGI_TV and TGI_TW) .

TGI_TV was calculated as shown in Formula I below:
Formula I:

V_nt: tumor volume on day T in mice numbered n;
V_n0: tumor volume on day 0 in mice numbered n;
RTV_n: relative tumor volume of mice numbered n on day T;
meanRTV_treat: the mean RTV of the treatment groups;
meanRTV_vehicle: the mean RTV of the control group.

TGI_TW was calculated as shown in Formula II below:
Formula II:

meanTW_treat: the mean tumor weight in the treatment group;
meanTW_vehicle: the mean tumor weight of the vehicle control group.

The mean tumor volume of mice of each group during the study period is shown in Fig. 7A. At the end of the study, TGI_TV of G2-G5 groups were 22.51%, 27.57%, -10.52%and 47.58%, respectively.

The tumors were collected at the study endpoint, weighed and the tumor weight inhibition rate (TGI_TW) was calculated. Results are shown in Table 5 and Fig. 7B.

Table 5. Tumor weight and tumor weight inhibition rate of each group at study endpoint

Note: Data are shown as mean± standard error of the mean (SEM) ; Statistical analysis was performed
using Independent Samples T-test vs G1 (*: P<0.05, ***: P<0.001) .

In another experiment, anti-LILRB2 antibodies HB59 and humanized anti-LILRB2 antibodies huB45, huB206, huC112, and huC68 were tested for their anti-tumor effects using the same animal model. The anti-LILRB2 antibodies were administrated intravenously at dose level of 10 mg/kg (for alpaca-derived antibodies, huB45 and huB206) or 20 mg/kg (for HB59, huC112, and huC68) twice a week (BIW) for 3 weeks.

The mean tumor volume of mice of each group is shown Fig. 8A, and changes of tumor volume inhibition rate (TGI_TV) in each group during administration are shown in Table 6.

Table 6. Changes in tumor volume inhibition rate (TGI_TV) in different groups during the administration period (D0-D23)

The tumors were collected at the study endpoint, weighed and the tumor weight inhibition rate (TGI_TW) was calculated. Results are shown in Table 7 and Fig. 8B.

Table 7. Tumor weight and tumor weight inhibition rate of each group at study endpoint

Note: Data are shown as mean± SEM; Statistical analysis was performed using Independent Samples T-
test vs G1 (*: P<0.05) .

During the administration period, no abnormal findings were noticed in the ear temperature or food and water intake of the mice in each group. No significant difference in mouse body weight was observed between each treatment group and the vehicle control group.
Example 10. Exemplary anti-LILRB2 antibody sequences

In Tables 8-15, “#” refers to the SEQ ID NO of the corresponding sequence.

Table 8: Amino acid sequence of CDRs for anti-LILRB2 antibodies (heavy chain) .

Table 9: DNA sequence of CDRs for anti-LILRB2 antibodies (heavy chain) .

Table 10: Amino acid sequence of CDRs for anti-LILRB2 antibodies (light chain) .

Table 11: DNA sequence of CDRs for anti-LILRB2 antibodies (light chain) .

Table 12: Amino acid sequence of anti-LILRB2 antibodies (VH domains, heavy chain) .

Table 13: DNA sequence of anti-LILRB2 antibodies (VH domains, heavy chain) .

Table 14: Amino acid sequence of anti-LILRB2 antibodies (VL domains, Light chain) .

Table 15: DNA sequence of anti-LILRB2 antibodies (VL domains, light chain) .

Example 11. Generation, expression and purification of anti-PD1 x anti-LILRB2 bispecific antibodies
and anti-VEGF x anti-LILRB2 bispecific antibodies

Described herein are bispecific antibodies, including anti-PD1 x anti-LILRB2 bispecific antibodies and anti-VEGF x anti-LILRB2 bispecific antibodies.

The anti-PD-1 x anti-LILRB2 bispecific antibodies were developed in five different formats as depicted in Fig. 9A to 9E. Briefly, the N-terminus of anti-LILRB2 antibody scFv (VL-linker-VH) or VHH was connected to the C-terminus of the heavy chain or light chain of the anti-PD-1 antibody through a polypeptide linker to form 4 different formats (HC-C (scFv) , LC-C (scFv) , HC-C (VHH) , LC-C (VHH) ) of anti-PD-1 x anti-LILRB2 bispecific antibodies, including Bis-M17, Bis-M17-1, Bis-M23, Bis-M29, Bis-M23-1, Bis-M28, Bis-M13, Bis-M13-1, Bis-M30, Bis-M30-1, Bis-M31, Bis-M31-1, Bis-M33, Bis-M33-1, Bis-M34, Bis-M34-1. Alternatively, the C-terminus of anti-LILRB2 antibody VHH was connected to the N-terminus of the heavy chain of the anti-PD-1 antibody through a polypeptide linker to form the HC-N(VHH) format of anti-PD-1 x anti-LILRB2 bispecific antibodies (Bis-M16 and Bis-M16-1) .

Table 16. anti-LILRB2 x anti-PD-1 bispecific antibodies

Similarly, the anti-VEGF x anti-LILRB2 bispecific antibodies were also developed in four different formats as depicted in Fig. 10A to 10E. Briefly, the N-terminus of anti-LILRB2 antibody scFv (VL-linker-VH) or VHH was connected to the C-terminus of the heavy chain or light chain of the anti-VEGF antibody through a polypeptide linker to form 4 different formats (HC-C (scFv) , LC-C (scFv) , HC-C (VHH) , LC-C (VHH) ) of anti-PD-1 x anti-LILRB2 bispecific antibodies, including Bis-M18, Bis-M18-1, Bis-M24, Bis-M27, Bis-M14, Bis-M14-1, Bis-M32, Bis-M32-1, Bis-M35, and Bis-M35-1. Alternatively, the C-terminus of anti-LILRB2 antibody VHH was connected to the N-terminus of the heavy chain of the anti-VEGF antibody through a polypeptide linker to form the HC-N (VHH) format of anti-VEGF x anti-LILRB2 bispecific antibodies (Bis-M15 and Bis-M15-1) .

Table 17. anti-LILRB2 x anti-VEGF bispecific antibodies

The bispecific antibody variants were constructed into the pcDNA3.1 expression vector and produced by transient expression in expi293F cells (GIBCO) . Seven days after transfection, the supernatant from transfected cells were collected and quantified for antibody titer.

The bispecific antibodies were purified using protein A affinity chromatography (BESTCHROM) , followed by cation exchange chromatography (SP BESTAROSE HP, BESTCHROM) to remove aggregates. The purified antibodies were dialyzed against 20mM citrate buffer containing 125mM Lys-HCl (pH 6.0) overnight. Reduced and non-reduced sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the monomeric and aggregates fraction of the bispecific antibodies.

The binding kinetics of the anti-LILRB2 x anti-PD-1 and anti-LILRB2 x anti-VEGF bispecific antibodies towards recombinant human LILRB2-ECD-His or LILRB2-ECD-mFc, human PD-1-His, and huVEGF165-His was evaluated by Bio-Layer Interferometry (BLI) or Surface Plasmon Resonance (SPR) . Results were shown in Tables 18-20. The kinetic binding curves of Bis-M23-1, Bis-M24, Bis-M27, Bis-M28 are shown in Fig. 11A-11H.

Table 18. Binding kinetics of antibodies towards hLILRB2-ECD

Table 19. Binding kinetics of antibodies towards human PD-1 (C-6His)

Table 20. Binding kinetics of antibodies towards huVEGF165-His

Example 12. In vitro binding assay to detect the blocking effect of bispecific antibodies.

In this example, the bispecific antibodies were evaluated for their blocking effect on the binding of human LILRB2 protein to HLA-G overexpressing A375 cells (A375-HLA-G) . Briefly, the bispecific antibodies were co-incubated with recombinant human LILRB2 protein (huLILRB2-Fc (mIgG) ) at 4℃ for 1 hour. Subsequently, A375-HLA-G cells were added and incubated for another 1 hour to allow the binding of human LILRB2 protein with membrane-bound HLA-G. Then PE-anti mouse IgG Fc antibody (405307, BIOLEGEND) was added and used for the detection of cell-bound huLILRB2-Fc (mIgG) with flow cytometry. Results are shown in Fig. 12 and Table 21, the bispecific antibodies (Bis-M23-1, Bis-M24, Bis-M27, and Bis-28) interfered with HLA-G for its binding towards human LILRB2 protein with IC₅₀ ranged from 1.216 nM to 2.859 nM.

Table 21. Antibodies interfered with HLA-G for its binding towards human LILRB2 protein

Example 13. The effect of bispecific antibodies on polarization of peripheral blood mononuclear cell-
derived macrophages

In this example, the effect of antibody-mediated polarization of macrophages from M2 to M1 was evaluated in human peripheral blood cell-derived macrophages.

Specifically, monocytes (MDM) were isolated from human peripheral blood mononuclear cells (PBMC) using STEMCELL EASYSEP (19059, STEMCELL) and induced to differentiate into M0 macrophages by M-CSF (MCF-H5218, ACRO) , followed by polarization into M2 macrophages under IL-4 stimulation for 2 days. Subsequently, M2 macrophages were treated with 68 nM monospecific or bispecific antibodies at 37 ℃ for another 2 days. Then the CD marker expression on the surface of macrophages were evaluated by flow cytometry, and changes in the level of inflammatory cytokines in the supernatant were measured. Results are shown in Fig. 13 and Fig. 14. The bispecific antibodies exhibited extraordinary effect in macrophage reprogramming, much higher than monoclonal anti-LILRB2 antibody HB59 alone. Some bispecific antibodies had even better effects than HB59 in combination with anti-VEGF antibody or anti-PD-1 antibody, especially in the secretion of inflammatory cytokine TNF-α (see e.g., Fig. 13D and 14D) .

In another experiment, the M0 macrophages were treated with supernatant from cultured human pancreatic cancer cell line AsPC-1 (CRL-1682, ATCC) for 3 days to induce the tumor-associated macrophage (TAM) phenotype. Subsequently the TAMs were co-incubated with 68 nM monospecific or bispecific antibodies at 37 ℃ for another 3 days, followed by macrophage phenotyping and cytokine measurements to evaluate the effect of antibody-mediated polarization of TAMs to M1-like phenotype. Results are shown in Fig. 15 and 16. As compared with monospecific antibodies, the 4 bispecific antibodies (Bis-M23-1, Bis-M24, Bis-M27, and Bis-28) exhibited higher activity in reversing of the inhibitory phenotype of TAM towards M1-like phenotype as indicated by the decreased expression of CD163 and/or CD206 (see e.g., Fig. 15A-15B; Fig. 16A-16B) , and/or the upregulation of CD80 expression (see e.g., Fig. 15C and 16C) , accompanied with the secretion of inflammatory cytokine TNF-α(see e.g., Fig. 15D and 16D) . To further test the inflammatory macrophage induced tumor cytotoxicity effect, CD3⁺ T cells from allogeneic donors were added and co-cultured with the macrophages for 3 days. Then Raji-luc cells were added to the macrophage-T cell culture mix for 2 days, and tumor cytotoxicity was measured. Results are shown in Fig. 15E-15F and 16E-16F. Bispecific antibodies induced the secretion of higher levels of inflammatory cytokine TNF-α (see e.g., Fig. 15E and 16E) with enhanced tumor cytotoxicity (see e.g., Fig. 15F and 16F) as compared to monospecific antibodies.
Example 14. The effect of bispecific antibodies in Mixed Lymphocyte Reaction

In this example, the effect of bispecific antibodies in a Mixed Lymphocyte Reaction was evaluated. Specifically, monocytes (MDM) were isolated from human peripheral blood mononuclear cells (PBMC) using STEMCELL EASYSEP (19059, STEMCELL) and seeded in a 48-well plate at 4 x 10⁵ cells/well. Recombinant M-CSF (25 ng/mL) and 68 nM antibodies were added to co-incubate with the MDM for 7 days to induce M0 phenotype. Subsequently, lipopolysaccharide (LPS) was added to the culture medium and co-cultured with the macrophage for 1 day. Then CD3⁺ T cells from allogeneic donors were added and co-cultured for 5 days. Changes in the level of inflammatory cytokines in the supernatant were measured. To further test the tumor cytotoxicity capability, Raji-luc cells were added to the macrophage-T cell culture mix for 2 days, and tumor cytotoxicity was measured. Results are shown in Fig. 17 and Fig. 18. Bispecific antibodies induced the secretion of higher levels of inflammatory cytokine TNF-α (see e.g., Fig. 17A and 18A) with enhanced tumor cytotoxicity (see e.g., Fig. 17B and 18B) as compared to monospecific antibodies.
Example 15. Anti-tumor effect of anti-LILRB2 x anti-VEGF bispecific antibodies in huPBMC-NOG
mouse inoculated with MDA-MB-231 tumor model.

The anti-tumor effects of antibodies were evaluated in a PBMC humanized NOG mouse subcutaneously inoculated with MDA-MB-231 cells, a model for late-stage breast cancer as the cell line was isolated from a pleural effusion of a patient with invasive ductal carcinoma. All animal handling procedures were approved by a Therapeutics Laboratory Animal Management and Use Committee (IACUC) . Briefly, female NOG mice (BEIJING VITALSTAR BIOTECHNOLOGY) were inoculated subcutaneously with 1.5×10⁷ MDA-MB-231 cells on the right flank. When tumor volume reached about 95 mm³, mice were randomized into 5 groups (6 mice/group) , including IgG4 isotype control (G1) , HB59 (G2) , Bevacizumab (G3) , HB59 + Bevacizumab (G4) and Bis-M24 (G5) . Dose level and frequency of each group was listed as below in Table 22.

Table 22: Dose level and frequency

The mean tumor volume of mice of each group during the study period are shown in Fig. 19A. At the end of the study, TGI_TV of G2-G5 groups were 4.30%, 48.30%, 59.31%and 64.45%, respectively.

The tumors were collected at the study endpoint and weighed, and the tumor weight inhibition rate (TGI_TW) was calculated. Results are shown in Table 23 and Fig. 19B. Both the bispecific antibody Bis-M24 and the anti-LILRB2 antibody HB59 in combination with Bevacizumab exhibited higher tumor inhibition rate than the monospecific antibodies.

Table 23. Tumor weight and tumor weight inhibition rate of each group at study endpoint

Note: Data are shown as mean± SEM; Statistical analysis was performed using Independent Samples T-test vs G1 (*: P<0.05, ***: P<0.001) .
Example 16. Anti-tumor effect of bispecific antibodies in huPBMC-NPG mouse inoculated with HT-29
tumor model

The anti-tumor effects of antibodies were evaluated in PBMC humanized NPG mouse subcutaneously inoculated with HT-29 cells, which is a cell line from a colorectal adenocarcinoma patient. All animal handling procedures were approved by a Laboratory Animal Management and Use Committee (IACUC) . Briefly, female NPG mice (BEIJING VITALSTAR BIOTECHNOLOGY) were inoculated subcutaneously with 5×10⁶ HT-29 cells on the right flank. When the tumor volume reached about 65 mm³, mice were randomized into 10 groups (5 mice/group) to receive test article treatments twice a week (BIW) for 4 weeks, including PBS control (G1) , HB59 (G2) , 1E1 (G4) (G3) , Nivolumab (G4) , Bevacizumab (G5) , HB59 + Nivolumab (G6) , HB59 + Bevacizumab (G7) , Bis-M23-1 (G8) , Bis-M24 (G9) and Bis-M27 (G10) . Dose level and frequency of each group was listed as below in Table 24.

Table 24 Test article, dose level and frequency of each group in huPBMC-NPG mouse model

The mean tumor volume of mice of each group during the study period are shown in Fig. 20A and 21A. At the end of the study, TGI_TV of anti-LILRB2 antibody groups (G2-G3) were 14.71%and 21.89%, respectively; and TGI_TV of nivolumab and bevacizumab groups (G4-G5) were 22.85%and 47.91%, respectively. The anti-LILRB2 antibody in combination with anti-PD-1 antibody treatment (G6) and Bis-M23-1 bispecific antibody (G8) exhibited higher tumor inhibition rate than each of the monospecific antibodies, with TGI_TV of 46.62% (G6) and 47.62% (G8) , respectively. Similarly, the anti-LILRB2 antibody in combination with anti-VEGF antibody treatment (G7) and anti-LILRB2 x anti-VEGF bispecific antibody treatment (G9-G10) also exhibited higher tumor inhibition rate than each of the monospecific antibodies, with TGI_TV of 69.84% (G7) , 70.98% (G9) and 54.32% (G10) , respectively.

The tumors were collected at the study endpoint, weighed and the tumor weight inhibition rate (TGI_TW) was calculated. Results are shown in Table 25, Fig. 20B and 21B; the bispecific antibody Bis-M23-1, Bis-M24, Bis-M27 and the anti-LILRB2 antibody HB59 in combination with Nivolumab or Bevacizumab all exhibited higher tumor inhibition rate than each of the monospecific antibodies.

Table 25. Tumor weight and tumor weight inhibition rate of each group at study endpoint

Note: Data are shown as mean± SEM; Statistical analysis was performed using Independent Samples T-test vs. G1 (*: P<0.05) .
Example 17: Exemplary bispecific antibody sequences

In Tables 26-31, “#” refers to the SEQ ID NO of the corresponding sequence.

Table 26: Amino acid sequence of CDRs for anti-PD-1 x anti-LILRB2 and anti-VEGF x anti-LILRB2 bispecific antibodies (heavy chain) .

Table 27: Amino acid sequence of CDRs for anti-PD-1 x anti-LILRB2 and anti-VEGF x anti-LILRB2 bispecific antibodies (light chain) .

Table 28: The VH and VL amino acid sequence of anti-LILRB2, anti-PD-1 and anti-VEGF antibodies for the construction of anti-PD-1 x anti-LILRB2 and anti-VEGF x anti-LILRB2 bispecific antibodies.

Table 29: The VH and VL DNA sequence of anti-LILRB2, anti-PD-1 and anti-VEGF antibodies for the construction of anti-PD-1 x anti-LILRB2 and anti-VEGF x anti-LILRB2 bispecific antibodies. The nucleic acid sequences in Table 29 are codon-optimized in the context of the entire specific antibody of which they are a part.

Table 30: Amino acid sequence of heavy chain and light chain of the anti-PD-1 x anti-LILRB2 and anti-VEGF x anti-LILRB2 bispecific antibodies.

Table 31: DNA sequence of heavy chain and light chain of the anti-PD-1 x anti-LILRB2 and anti-VEGF x anti-LILRB2 bispecific antibodies.

Claims

A composition comprising a binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) , wherein the composition is capable of:

(a) polarizing macrophages from an M2 phenotype to an M1 phenotype;

(b) activating T cells; and/or

(c) inhibiting growth of a cancer.
The composition of claim 1, wherein the binding domain comprises an antigen binding domain of an antibody.
The composition of claim 1 or 2, wherein the binding domain is comprised by a human, humanized, affinity-matured, and/or chimeric antibody construct.
The composition of any one of claims 1-3, wherein the binding domain is comprised by an anti-LILRB2 antibody construct derived from a primate, a rodent, or a camelid.
The composition of any one of claims 1-4, wherein the binding domain is comprised by an anti-LILRB2 antibody construct derived from a human, a mouse, or an alpaca.
The composition of any one of claims 1-5, wherein the binding domain comprises the CDRs of a V_H domain that specifically binds LILRB2.
The composition of any one of claims 1-6, wherein the binding domain comprises the CDRs of a V_L domain that specifically binds LILRB2.
The composition of any one of claims 1-7, wherein the binding domain comprises the CDRs of a V_H/V_L domain pair that specifically binds LILRB2.
The composition of any one of claims 1-8, wherein the binding domain comprises the CDRs of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2.
The composition of any one of claims 1-9, wherein LILRB2 comprises mammalian LILRB2.
The composition of any one of claims 1-10, wherein LILRB2 comprises human LILRB2.
The composition of any one of claims 1-11, wherein the binding domain specifically binds to a LILRB2 epitope or portion of a LILRB2 epitope comprising sequential and/or non-sequential residues of SEQ ID NO: 775.
The composition of any one of claims 1-12, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H CDR1, a V_H CDR2, and a V_H CDR3 selected from Table 8; and

(b) a V_L CDR1, a V_L CDR2, and a V_L CDR3 selected from Table 10.
The composition of any one of claims 1-13, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H CDR1 comprising one of SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, or 118;

(b) a V_H CDR2 comprising one of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, or 119;

(c) a V_H CDR3 comprising one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, or 120;

(d) a V_L CDR1 comprising one of SEQ ID NOs: 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, or 829;

(e) a V_L CDR2 comprising one of SEQ ID NOs: 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 314, 317, or 320; and

(f) a V_L CDR3 comprising one of SEQ ID NOs: 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, or 830.
The composition of any one of claims 8-14, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H CDR1, a V_H CDR2, and a V_H CDR3 encoded by nucleic acid sequences selected from Table 9; and

(b) a V_L CDR1, a V_L CDR2, and a V_L CDR3 encoded by nucleic acid sequences selected from Table 11.
The composition of any one of claims 8-15, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 208, 211, 214, 217, 220, 223, 226, 229, 232, 235, or 238;

(b) a V_H CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224, 227, 230, 233, 236, or 239;

(c) a V_H CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 198, 201, 204, 207, 210, 213, 216, 219, 222, 225, 228, 231, 234, 237, or 240;

(d) a V_L CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 328, 331, 334, 337, 340, 343, 346, 349, 352, 355, 358, 361, 364, 367, 370, 373, 376, 379, 382, 385, 388, 391, 394, 397, 400, 403, or 406;

(e) a V_L CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 329, 332, 335, 338, 341, 344, 347, 350, 353, 356, 359, 362, 365, 368, 371, 374, 377, 380, 383, 386, 389, 392, 395, 398, 401, 404, or 407; and

(f) a V_L CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 330, 333, 336, 339, 342, 345, 348, 351, 354, 357, 360, 363, 366, 369, 372, 375, 378, 381, 384, 387, 390, 393, 396, 399, 402, 405, or 408.
The composition of any one of claims 4-16, wherein the anti-LILRB2 antibody construct comprises:

(a) a V_H domain selected from Table 12 or a V_H domain that is at least 80%identical to an amino acid sequence selected from Table 12; and

(b) a V_L domain selected from Table 14 or a V_L domain that is at least 80%identical to an amino acid sequence selected from Table 14.
The composition of any one of claims 4-17, wherein the anti-LILRB2 antibody construct comprises:

(a) a V_H domain comprising one of SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, or 448, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, or 448; and

(b) a V_L domain comprising one of SEQ ID NOs: 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, or 517.
The composition of any one of claims 4-18, wherein the anti-LILRB2 antibody construct comprises:

(a) a V_H domain encoded by a nucleic acid sequence selected from Table 13 or a V_H domain that is at least 80%identical to a nucleic acid sequence selected from Table 13; and

(b) a V_L domain encoded by a nucleic acid sequence selected from Table 15 or a V_L domain that is at least 80%identical to a nucleic acid sequence selected from Table 15.
The composition of any one of claims 4-19, wherein the anti-LILRB2 antibody construct comprises:

(a) a V_H domain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 571, 572, 573, 574, 579, 580, 581, 582, 583, 584, 590, 591, 592, 593, 594, 595, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 571, 572, 573, 574, 579, 580, 581, 582, 583, 584, 590, 591, 592, 593, 594, 595, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615; and

(b) a V_L domain encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 575, 576, 577, 578, 585, 586, 587, 588, 589, 596, 597, 598, 599, 600, 601, or 785, or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 575, 576, 577, 578, 585, 586, 587, 588, 589, 596, 597, 598, 599, 600, 601, or 785.
The composition of any one of claims 4-20, wherein the anti-LILRB2 antibody construct comprises an antibody selected from the group consisting of: HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206.
The composition of any one of claims 9-21, wherein the VHH that specifically binds LILRB2 comprises:

(a) a V_H CDR1 comprising one of SEQ ID NOs: 82, 85, 88, 91, 94, 97, 100, 103, 106, 115, or 118;

(b) a V_H CDR2 comprising one of SEQ ID NOs: 83, 86, 89, 92, 95, 98, 101, 104, 107, 116, or 119; and

(c) a V_H CDR3 comprising one of SEQ ID NOs: 84, 87, 90, 93, 96, 99, 102, 105, 108, 117, or 120.
The composition of any one of claims 9-22, wherein the VHH that specifically binds LILRB2 comprises:

(a) a V_H CDR1 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 202, 205, 208, 211, 214, 217, 220, 223, 226, 235, or 238;

(b) a V_H CDR2 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 203, 206, 209, 212, 215, 218, 221, 224, 227, 236, or 239; and

(c) a V_H CDR3 encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 204, 207, 210, 213, 216, 219, 222, 225, 228, 237, or 240.
The composition of any one of claims 4-23, wherein the anti-LILRB2 antibody construct comprises: a VHH comprising one of SEQ ID NOs: 436, 437, 438, 439, 440, 441, 442, 443, 444, 447, or 448, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 436, 437, 438, 439, 440, 441, 442, 443, 444, 447, or 448.
The composition of any one of claims 4-24, wherein the anti-LILRB2 antibody construct comprises: a VHH encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 476, 477, 478, 479, 480, 481, 482, 483, 484, 487, or 488, or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 476, 477, 478, 479, 480, 481, 482, 483, 484, 487, or 488.
The composition of any one of claims 4-25, wherein the anti-LILRB2 antibody construct comprises an antibody selected from the group consisting of: AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huB45, and huB206.
A composition comprising:

(a) a first binding domain that specifically binds leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) ; and

(b) a second binding domain that specifically binds:

(i) programmed cell death protein 1 (PD-1) ; or

(ii) vascular endothelial growth factor (VEGF) .
The composition of claim 27, wherein the composition is capable of:

(a) polarizing macrophages from an M2 phenotype to an M1 phenotype;

(b) activating T cells; and/or

(c) inhibiting growth of a cancer.
The composition of claim 27 or 28, wherein the first and/or second binding domains comprise an antigen binding domain of an antibody.
The composition of any one of claims 27-29, wherein the first and second binding domains each comprise an antigen binding domain of an antibody.
The composition of any one of claims 27-30, wherein the first and second binding domains are comprised by a human, humanized, affinity-matured, and/or chimeric antibody construct.
The composition of any one of claims 27-31, wherein the first and second binding domains are comprised by a bispecific antibody construct.
The composition of claim 27-32, wherein the first binding domain comprises the CDRs of a V_H domain that specifically binds LILRB2.
The composition of claim 27-33, wherein the first binding domain comprises the CDRs of a V_L domain that specifically binds LILRB2.
The composition of claim 32-34, wherein the bispecific antibody construct comprises:

(a) the first binding domain comprising:

(i) the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or

(ii) the CDRs of a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2; and

(b) the second binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds PD-1.
The composition of claim 32-35, wherein the bispecific antibody construct comprises:

(a) the first binding domain comprising:

(i) the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or

(ii) the CDRs of a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) that specifically binds LILRB2; and

(b) the second binding domain comprising the CDRs of a V_H/V_L domain pair that specifically binds VEGF.
The composition of claim 32-36, wherein the bispecific antibody construct is selected from the group consisting of a tandem scFv (taFv or scFv₂) , diabody, dAb₂A/HH₂, knob-into-holes bispecific derivative, SEED-IgG, heteroFc-scFv, Fab-scFv, scFv-Jun/Fos, Fab'-Jun/Fos, tribody, DNL-F (ab) ₃, scFv₃-CH1/CL, Fab-scFv₂, IgG-scFab, IgG-scFv, scFv-IgG, IgG-VHH, VHH-IgG, scFv₂-Fc, F (ab') ₂-scFv₂, scDB-Fc, scDb-CH₃, Db-Fc, scFv₂-H/L, DVD-Ig, tandAb, scFv-dhlx-scFv, dAb2-IgG, dAb-IgG, and dAb-Fc-dAb constructs.
The composition of claim 32-37, wherein the bispecific antibody construct comprises:

(a) the first binding domain comprising:

(i) a single-chain fragment variable (scFv) comprising the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or

(ii) a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) comprising the CDRs of a V_H domain that specifically binds LILRB2; and

(b) the second binding domain comprising an immunoglobulin heavy chain and immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds PD-1.
The composition of claim 32-38, wherein the bispecific antibody construct comprises:

(a) the first binding domain comprising:

(i) a single-chain fragment variable (scFv) comprising the CDRs of a V_H/V_L domain pair that specifically binds LILRB2; or

(ii) a V_H domain of a variable domain of the heavy chain of a heavy-chain antibody (VHH) comprising the CDRs of a V_H domain that specifically binds LILRB2; and

(b) the second binding domain comprising an immunoglobulin heavy chain and immunoglobulin light chain, wherein the heavy and light chains comprise the CDRs of a V_H/V_L domain pair that specifically binds VEGF.
The composition of claim 38 or 39, wherein the scFv or the VHH is linked to the heavy chain.
The composition of any one of claims 38-40, wherein the scFv or the VHH is linked to the C-terminus of the heavy chain.
The composition of any one of claims 38-41, wherein the scFv or the VHH is linked to the N-terminus of the heavy chain.
The composition of any one of claims 38-42, wherein the scFv or the VHH is linked to the light chain.
The composition of any one of claims 38-43, wherein the scFv or the VHH is linked to the C-terminus of the light chain.
The composition of any one of claims 38-44, wherein the scFv or the VHH is linked to the N-terminus of the light chain.
The composition of any one of claims 27-45, wherein the LILRB2 comprises mammalian LILRB2; the PD-1 comprises mammalian PD-1; and/or the VEGF comprises mammalian VEGF.
The composition of any one of claims 27-46, wherein the LILRB2 comprises human LILRB2; the PD-1 comprises human PD-1; and/or the VEGF comprises human VEGF.
The composition of any one of claims 27-47, wherein the second binding domain specifically binds PD-1 and comprises nivolumab, pembrolizumab, toripalimab, sintilimab, cemiplimab, or dostarlimab, or functional fragments thereof.
The composition of any one of claims 27-48, wherein the second binding domain specifically binds to at least one nivolumab PD-1 epitope or portion of a nivolumab PD-1 epitope comprising: residues 25 to 31 of SEQ ID NO: 776 (LDSPDRP, SEQ ID NO: 787) , residues 25 to 31 of SEQ ID NO: 776 (TSES, SEQ ID NO: 779) , and/or residues 127 to 132 of SEQ ID NO: 776 (SLAPKA, SEQ ID NO: 780) .
The composition of any one of claims 27-49, wherein the second binding domain specifically binds to at least one pembrolizumab PD-1 epitope or portion of a pembrolizumab PD-1 epitope comprising: residues S60, S62, V64, N66, I126, L128, A129, K131, A132, and/or I134 of SEQ ID NO: 776, residues 75 to 78 of SEQ ID NO: 776 (QTDK, SEQ ID NO: 781) , and/or residues 81 to 90 of SEQ ID NO: 776 (AFPEDRSQPG, SEQ ID NO: 782) .
The composition of any one of claims 27-47, wherein the second binding domain specifically binds VEGF and comprises bevacizumab, ranibizumab, or Aflibercept, or functional fragments thereof.
The composition of any one of claims 27-47 or 51, wherein the second binding domain specifically binds to a bevacizumab VEGF epitope or portion of a bevacizumab VEGF epitope comprising residues 85 to 92 of SEQ ID NO: 777, residues 111 to 112 of SEQ ID NO: 778, residues 111 to 112 of SEQ ID NO: 786, or PHQGQHIG (SEQ ID NO: 783) .
The composition of any one of claims 27-47 or 51-52, wherein the first binding domain that specifically binds LILRB2 comprises an antibody or functional fragment thereof selected from the group consisting of: HA23, HB5, HB37, HB50, HB59, HB68, HB70, HB72, HB74, MIL-C3, MIL-C5, MIL-C8, MIL-C9, MIL-C11, MIL-C25, MIL-C26, MIL-C28, MIL-C32, MIL-C42, MIL-C43, MIL-C45, MIL-C68, MIL-C77, MIL-C104, MIL-C112, AIL-B45, AIL-B82, AIL-B94, AIL-B100, AIL-B110, AIL-B120, AIL-B134, AIL-B206, AIL-B260, huC68, huC112, huB45, and huB206.
The composition of any one of claims 27-53, wherein the first binding domain that specifically binds LILRB2 comprises an antibody or functional fragment thereof selected from the group consisting of: HB59, MIL-C68, huC68, MIL-C112, huC112, AIL-B45, huB45, AIL-B206, and huB206.
The composition of any one of claims 35-54, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H CDR1 comprising one of SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, or 118;

(b) a V_H CDR2 comprising one of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, or 119;

(c) a V_H CDR3 comprising one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120;

(d) a V_L CDR1 comprising one of SEQ ID NOs: 247, 250, 253, 256, 259, 262, 265, 268, 271, 274, 277, 280, 283, 286, 289, 292, 295, 298, 301, 304, 307, 310, 313, 316, 319, or 829;

(e) a V_L CDR2 comprising one of SEQ ID NOs: 248, 251, 254, 257, 260, 263, 266, 269, 272, 275, 278, 281, 284, 287, 290, 293, 296, 299, 302, 305, 308, 311, 314, 317, 314, 317, or 320; and

(f) a V_L CDR3 comprising one of SEQ ID NOs: 249, 252, 255, 258, 261, 264, 267, 270, 273, 276, 279, 282, 285, 288, 291, 294, 297, 300, 303, 306, 309, 312, 315, 318, 321, or 830.
The composition of any one of claims 35-54, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H CDR1 comprising one of SEQ ID NOs: 19, 109, 112, 115, or 118;

(b) a V_H CDR2 comprising one of SEQ ID NOs: 20, 110, 113, 116, or 119;

(c) a V_H CDR3 comprising one of SEQ ID NOs: 21, 111, 114, 117, or 120;

(d) a V_L CDR1 comprising one of SEQ ID NOs: 259, 316, or 319;

(e) a V_L CDR2 comprising one of SEQ ID NOs: 260, 317, or 320; and

(f) a V_L CDR3 comprising one of SEQ ID NOs: 261, 318, or 321.
The composition of any one of claims 35-56, wherein the VHH that specifically binds LILRB2

comprises:

(a) a V_H CDR1 comprising one of SEQ ID NOs: 82, 85, 88, 91, 94, 97, 100, 103, 106, 115, or 118;

(b) a V_H CDR2 comprising one of SEQ ID NOs: 83, 86, 89, 92, 95, 98, 101, 104, 107, 116, or 119; and

(c) a V_H CDR3 comprising one of SEQ ID NOs: 84, 87, 90, 93, 96, 99, 102, 105, 108, 117, 120.
The composition of any one of claims 35-57, wherein the VHH that specifically binds LILRB2 comprises:

(a) a V_H CDR1 comprising one of SEQ ID NOs: 115 or 118;

(b) a V_H CDR2 comprising one of SEQ ID NOs: 116 or 119; and

(c) a V_H CDR3 comprising one of SEQ ID NOs: 117 or 120.
The composition of any one of claims 35-58, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H CDR1 comprising SEQ ID NO: 19;

(b) a V_H CDR2 comprising SEQ ID NO: 20;

(c) a V_H CDR3 comprising SEQ ID NO: 21;

(d) a V_L CDR1 comprising SEQ ID NO: 259;

(e) a V_L CDR2 comprising SEQ ID NO: 260; and

(f) a V_L CDR3 comprising SEQ ID NO: 261.
The composition of any one of claims 35-59, wherein the V_H/V_L domain pair that specifically binds PD-1 comprises:

(a) a V_H CDR1 comprising one of SEQ ID NOs: 547 or 550;

(b) a V_H CDR2 comprising one of SEQ ID NOs: 548 or 551;

(c) a V_H CDR3 comprising one of SEQ ID NOs: 549 or 552;

(d) a V_L CDR1 comprising one of SEQ ID NOs: 556 or 559;

(e) a V_L CDR2 comprising one of SEQ ID NOs: 557 or 560; and

(f) a V_L CDR3 comprising one of SEQ ID NOs: 558 or 561.
The composition of any one of claims 35-59, wherein the V_H/V_L domain pair that specifically binds VEGF comprises:

(a) a V_H CDR1 comprising SEQ ID NO: 553;

(b) a V_H CDR2 comprising SEQ ID NO: 554;

(c) a V_H CDR3 comprising SEQ ID NO: 555;

(d) a V_L CDR1 comprising SEQ ID NO: 562;

(e) a V_L CDR2 comprising SEQ ID NO: 563; and

(f) a V_L CDR3 comprising SEQ ID NO: 564.
The composition of any one of claims 35-61, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H comprising one of SEQ ID NOs: 415, 432, 435, 445, 446, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 415, 432, 435, 445, 446; and

(b) a V_L comprising one of SEQ ID NOs: 495, 512, 516, 515, 517 or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 495, 512, 516, 515, 517.
The composition of any one of claims 35-62, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H comprising SEQ ID NO: 415 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 415; and

(b) a V_L comprising SEQ ID NO: 495 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 495.
The composition of any one of claims 35-62, wherein the V_H/V_L domain pair that specifically binds PD-1 comprises:

(a) a V_H comprising one of SEQ ID NOs: 565 or 567 or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 565 or 567; and

(b) a V_L comprising one of SEQ ID NOs: 566 or 568 or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 566 or 568.
The composition of any one of claims 35-62, wherein the V_H/V_L domain pair that specifically binds VEGF comprises:

(a) a V_H comprising SEQ ID NO: 569 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 569; and

(b) a V_L comprising SEQ ID NO: 570 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 570.
The composition of any one of claims 35-65, wherein the V_H/V_L domain pair that specifically binds LILRB2 comprises:

(a) a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 455, 472, 475, 485, 486, 571-574, 579-584, 590-595 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 455, 472, 475, 485, 486, 579-584, 571-574, 590-595; and

(b) a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 524, 541, 544, 545, 546, 575-578, 585-589, 596-601, 785, or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 524, 541, 544, 545, 546, 575-578, 585-589, 596-601, or 785.
The composition of any one of claims 35-66, wherein the V_H/V_L domain pair that specifically binds PD-1 comprises:

(a) a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 616-618, 624-632 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 616-618, 624-632; and

(b) a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 619-623, 633-638 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 619-623, 633-638.
The composition of any one of claims 35-66, wherein the V_H/V_L domain pair that specifically binds VEGF comprises:

(a) a V_H encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 639-647 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 639-647; and

(b) a V_L encoded by a nucleic acid sequence comprising one of SEQ ID NOs: 648-654 or a nucleic acid sequence that is at least 80%identical to one of SEQ ID NOs: 648-654.
The composition of any one of claims 32-68, wherein the bispecific antibody construct comprises an anti-LILRB2 and anti-PD-1 bispecific antibody construct.
The composition of claim 69, wherein the anti-LILRB2 and anti-PD-1 bispecific antibody construct comprises:

(a) an immunoglobulin heavy chain comprising one of SEQ ID NOs: 655, 657, 659, 661, 671, 673, 675, 677, 683, 689, 691, 693, 695, 697, 703, 705, 707, 709, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 655, 657, 659, 661, 671, 673, 675, 677, 683, 689, 691, 693, 695, 697, 703, 705, 707, 709; and

(b) an immunoglobulin light chain comprising one of SEQ ID NOs: 656, 658, 660, 662, 672, 674, 676, 678, 684, 690, 692, 694, 696, 698, 704, 706, 708, 710, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 656, 658, 660, 662, 672, 674, 676, 678, 684, 690, 692, 694, 696, 698, 704, 706, 708, 710.
The composition of claim 69 or 70, wherein the anti-LILRB2 and anti-PD-1 bispecific antibody construct is selected from Bis-M23-1 and Bis-M28.
The composition of any one of claims 69-71, wherein the anti-LILRB2 and anti-PD-1 bispecific antibody is Bis-M23-1 comprising:

(a) an immunoglobulin heavy chain comprising SEQ ID NO: 683 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 683; and

(b) an immunoglobulin light chain comprising SEQ ID NO: 684, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 684.
The composition of any one of claims 69-71, wherein the anti-LILRB2 and anti-PD-1 bispecific antibody is Bis-M28 comprising:

(a) an immunoglobulin heavy chain comprising SEQ ID NO: 689, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 689; and

(b) an immunoglobulin light chain comprising SEQ ID NO: 690, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 690.
The composition of any one of claims 32-68, wherein the bispecific antibody construct comprises an anti-LILRB2 and anti-VEGF bispecific antibody construct.
The composition of claim 74, wherein the anti-LILRB2 and anti-VEGF bispecific antibody construct comprises:

(a) an immunoglobulin heavy chain comprising one of SEQ ID NOs: 663, 665, 667, 669, 679, 681, 685, 687, 699, 701, 711, 713, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 663, 665, 667, 669, 679, 681, 685, 687, 699, 701, 711, 713; and

(b) an immunoglobulin light chain comprising one of SEQ ID NOs: 664, 666, 668, 670, 680, 682, 686, 688, 700, 702, 712, 714, or an amino acid sequence that is at least 80%identical to one of SEQ ID NOs: 664, 666, 668, 670, 680, 682, 686, 688, 700, 702, 712, 714.
The composition of claim 74 or 75, wherein the anti-LILRB2 and anti-VEGF bispecific antibody construct is selected from Bis-M24 and Bis-M27.
The composition of any one of claims 74-76, wherein the anti-LILRB2 and anti-VEGF bispecific antibody is Bis-M24 comprising:

(a) an immunoglobulin heavy chain comprising SEQ ID NO: 685 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 685; and

(b) an immunoglobulin light chain comprising SEQ ID NO: 686 or an amino acid sequence that is at least 80%identical to SEQ ID NO: 686.
The composition of any one of claims 74-76, wherein the anti-LILRB2 and anti-VEGF bispecific antibody is Bis-M27 comprising:

(a) an immunoglobulin heavy chain comprising SEQ ID NO: 687, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 687; and

(b) an immunoglobulin light chain comprising SEQ ID NO: 688, or an amino acid sequence that is at least 80%identical to SEQ ID NO: 688.
A nucleic acid encoding a polypeptide of the composition of any one of claims 1-78.
A vector comprising the nucleic acid of claim 79.
A cell comprising the nucleic acid of claim 79 or the vector of claim 80.
The cell of claim 81, wherein the cell is an antibody-producing cell.
A cell in combination with the composition of any one of claims 1-78.
The cell of claim 83, wherein the cell is an immune cell.
The cell of claim 83 or 84, wherein the cell is an M1 macrophage or an M2 macrophage.
The cell of claim 83 or 84, wherein the cell is a T lymphocyte.
A pharmaceutical composition comprising the composition of any one of claims 1-78, the nucleic acid of claim 79, the vector of claim 80, or the cell of any one of claims 81-86, and a pharmaceutically acceptable carrier.
A method of affinity maturing the composition of any one of claims 1-78, the method comprising:

a) mutating at least one binding domain;

b) determining the affinity of the mutated binding domain for its cognate ligand; and

c) selecting the mutated binding domain that exhibits increased affinity for its cognate ligand compared to the unmutated binding domain.
The method of claim 88, further comprising producing a composition comprising the selected mutated binding domain.
The method of claim 88 or 89, further comprising producing an antibody construct comprising the selected mutated binding domain.
A composition produced by the method of any one of claims 88-90.
An antibody construct produced by the method of any one of claims 88-90.
A method for polarizing a macrophage from an M2 phenotype to an M1 phenotype, the method comprising contacting the macrophage with the composition of any one of claims 1-78.
The method of claim 93, wherein after contacting, the macrophage exhibits decreased expression of CD163 and/or CD206.
The method of claim 93 or 94, wherein after contacting, the macrophage exhibits increased expression of CD80.
The method of any one of claims 93-95, wherein after contacting, the macrophage exhibits increased secretion of TNF-α.
The method of any one of claim 93-96, wherein after contacting, the macrophage induces increased cancer cytotoxicity.
The method of any one of claims 93-98, wherein the macrophage is contacted for a sufficient amount of time.
The method of claim 98, wherein the sufficient amount of time is at least 2 days.
The method of any one of claims 93-99, wherein the composition is an anti-LILRB2 antibody construct selected from the group consisting of HB59, AIL-B45, huB45, AIL-B206, huB206, MIL-C68, huC68, MIL-C112, and huC112.
The method of any one of claims 93-99, wherein the composition is an anti-LILRB2 and anti-PD-1 bispecific antibody construct selected from Bis-M23-1 or Bis-M28.
The method of any one of claims 93-99, wherein the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27.
A method for activating a T cell, the method comprising contacting the T cell with the composition of any one of claims 1-78.
The method of claim 103, wherein after contacting, the T cell exhibits increased activation of NFAT signaling.
The method of claim 103 or 104, wherein after contacting, the T cell exhibits increased cancer cytotoxicity.
The method of any one of claims 103-105, wherein the T cell is contacted for a sufficient amount of time.
The method of claim 106, wherein the sufficient amount of time is at least 2 days.
The method of any one of claims 103-107, wherein the composition is an anti-LILRB2 antibody construct selected from the group consisting of HB74, HA23, HA31, HB59, MIL-C112, MILC18, MIL-C27, MIL-C3, MIL-C32, MIL-C68, MIL-C9, AIL-A1, AIL-B100, AIL-B124, AIL-B134, and AIL-B45.
The method of any one of claims 103-107, wherein the composition is an anti-LILRB2 and anti-PD-1 bispecific antibody construct selected from Bis-M23-1 or Bis-M28.
The method of any one of claims 103-107, wherein the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27.
A method of treating cancer, the method comprising administering an effective amount of the composition of any one of claims 1-78 or the pharmaceutical composition of claim 87 to a subject in need thereof.
The method of claim 111, wherein the composition is administered at a dose of 10 mg/kg to 20 mg/kg.
The method of claim 111 or 112, wherein the composition is administered at least twice a week for at least three weeks.
The method of any one of claims 111-113, wherein the composition is administered intravenously.
The method of any one of claims 111-114, wherein the composition is an anti-LILRB2 antibody construct selected from MIL-C68, MIL-B206, or HB59.
The method of any one of claims 111-115, wherein the method has a tumor weight inhibition rate (TGI_TW) of at least 30%after 3 weeks of administration.
The method of any one of claims 111-116, wherein the method has a tumor volume inhibition rate (TGI_TV) of at least 35%after 3 weeks of administration.
The method of any one of claims 111-117, wherein the composition is an anti-LILRB2 and anti-PD-1 bispecific antibody construct selected from Bis-M23-1 or Bis-M28.
The method of any one of claims 111-117, wherein the composition is an anti-LILRB2 and anti-VEGF bispecific antibody construct selected from Bis-M24 or Bis-M27.
The method of any one of claims 111-117, wherein the composition is the anti-LILRB2 and anti-VEGF bispecific antibody construct Bis-M24.
The method of any one of claims 111-120, wherein the composition is administered at a dose of about 20 mg/kg twice weekly for at least one week.
The method of any one of claims 111-120, wherein the composition is administered at a dose of about 4 mg/kg twice weekly for at least the fourth week after beginning treatment.
The method of any one of claims 111-122, wherein the method has a tumor weight inhibition rate (TGI_TW) of at least 50%after 4 weeks of administration.
The method of any one of claims 111-123, wherein the cancer is a blood cancer.
The method of any one of claims 111-123, wherein the cancer comprises at least one solid tumor.
The method of claim 125, wherein the cancer is melanoma.
The method of claim 125, wherein the cancer is breast cancer.
The method of claim 125, wherein the cancer is colorectal adenocarcinoma.