Compositions and Methods Comprising Anti-NRP2 Antibodies

Abstract

Affinity-matured, humanized antibodies and antigen-binding fragments thereof that specifically bind to human neuropilin-2 (NRP2) polypeptides are provided, including those that modulate the binding interaction between human NRP2 and at least one NRP2 ligand, thereby modulating subsequent NRP2-mediated downstream signaling events, including related therapeutic compositions and methods for modulating NRP2 activity and treating diseases, such as NRP2-associated diseases.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims the benefit of 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 024,960, filed May 14, 2020, and U.S. Provisional Application No. 62 / 910,042, filed October 3, 2019, each of which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding the Sequence Listing The sequence listing for this application is provided in text format instead of a paper copy, and is hereby incorporated by reference in its entirety. The name of the text file containing the sequence listing is ATYR_136_02WO_ST25.txt. The text file is approximately 261 KB, was created on October 2, 2020, and was electronically submitted via EFS - Web.

Background Art

[0003] Background Technical Field Embodiments of the present disclosure relate to affinity - matured, humanized antibodies and antigen - binding fragments thereof that specifically bind to human neuropilin - 2 (NRP2) polypeptide, modulate the binding interaction between human NRP2 and at least one NRP2 ligand, thereby modulating subsequent NRP2 - mediated downstream signaling events, and include related therapeutic compositions and methods for modulating NRP2 activity and treating diseases such as NRP2 - related diseases.

[0004] Description of Related Fields Recent research and development has suggested that tRNA synthetases play important roles in cellular responses beyond their well-characterized roles in protein synthesis. In particular, there is a growing recognition that tRNA synthetases are involved in a range of yet unrecognized roles in response to cellular stress and tissue homeostasis, both in intracellular and extracellular environments.

[0005] There has been significant progress in elucidating the role of extracellular HARS-induced proteins, including the identification of neuropilin-2 (NRP2 or NRP-2), a putative cell receptor. The interaction between HARS and NRP2 is thought to be mediated by the N-terminal region of HARS and can lead to important changes in the cellular functions of NRP2.

[0006] Thus, the recent discovery of this new regulatory pathway represents an unknown mechanism that acts as a central regulator of cellular processes, directly related to, for example, axon guidance, endocytosis, cell migration, proliferation, survival, apoptosis, lymphangiogenesis, cell differentiation and cancer induction, growth, metastasis and chemoresistance, as well as cell adhesion including muscle, vascular, nerve, bone and immune homeostasis. Dysregulation of any of these processes can lead to a variety of diseases, which can be addressed by the development of anti-NRP2 antibodies that selectively target the neuropilin-2 axis. The present disclosure provides such antibodies and related embodiments. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] BRIEF SUMMARY Embodiments of the present disclosure include antibodies or antigen-binding fragments thereof (anti-NRP2 antibodies) that specifically bind to human neuropilin-2 (NRP2) polypeptides.

[0008] One particular embodiment is a therapeutic composition comprising at least one antibody or antigen-binding fragment thereof that specifically binds to a human neuropilin-2 (NRP2) polypeptide (anti-NRP2 antibody), wherein the at least one antibody or antigen-binding fragment thereof specifically binds to a human NRP2 polypeptide and comprises a complementarity-determining region V selected from Table A1 or Table A3 H CDR1, V H CDR2, and V H CDR3 sequences, and variants thereof, of the heavy-chain variable region (V H ); and a complementarity-determining region V selected from Table A1 or Table A3 that specifically binds to a human NRP2 polypeptide L CDR1, V L CDR2, and V L CDR3 sequences, and variants thereof, of the light-chain variable region (V L ). The therapeutic composition is included

[0009] In some embodiments V H CDR1, V H CDR2, and V H CDR3 sequences each comprise SEQ ID NOs: 1-3 and variants thereof, and V L CDR1, V L CDR2, and V L CDR3 sequences each comprise SEQ ID NOs: 4-6 and variants thereof; or V H CDR1, V H CDR2, and V H CDR3 sequences each comprise SEQ ID NOs: 7-9 and variants thereof, and V L CDR1, V L CDR2, and V L CDR3 sequences each comprise SEQ ID NOs: 10-12 and variants thereof; or V H CDR1, V H CDR2, and V H CDR3 sequences each comprise SEQ ID NOs: 13-15 and variants thereof, and V L CDR1, V LCDR2 and V L The CDR3 sequences each contain SEQ ID NOs: 16 to 18 and their variants; V H CDR1, V H CDR2 and V H The CDR3 sequences each contain SEQ ID NOs: 19 to 21 and their variants, and V L CDR1, V L CDR2 and V L The CDR3 sequences each contain SEQ ID NOs: 22 to 24 and their variants; V H CDR1, V H CDR2 and V H The CDR3 sequences each contain SEQ ID NOs: 25 to 27 and their variants, and V L CDR1, V L CDR2 and V L The CDR3 sequences each contain SEQ ID NOs: 28 to 30 and their variants; V H CDR1, V H CDR2 and V H The CDR3 sequences each contain SEQ ID NOs: 31 to 33 and their variants, and V L CDR1, V L CDR2 and V L The CDR3 sequences each contain SEQ ID NOs: 34 to 36 and their variants; V H CDR1, V H CDR2 and V H The CDR3 sequences each contain SEQ ID NOs: 34 to 39 and their variants, and V L CDR1, V L CDR2 and V L The CDR3 sequences each contain SEQ ID NOs: 40 to 42 and their variants; V H CDR1, V H CDR2 and V H The CDR3 sequences each contain SEQ ID NOs: 57 to 59 and their variants, and V L CDR1, V L CDR2 and VL Each CDR3 sequence either includes SEQ ID NOs: 60 - 62 and their variants; or V H CDR1, V H CDR2 and V H Each CDR3 sequence includes SEQ ID NOs: 63 - 65 and their variants, and V L CDR1, V L CDR2 and V L Each CDR3 sequence includes SEQ ID NOs: 66 - 68 and their variants.

[0010] In some embodiments, the V H sequence is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to a sequence selected from Table A2, and optionally, the V H sequence has 1, 2, 3, 4 or 5 modifications in the framework region. In some embodiments, the V L sequence is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to a sequence selected from Table A2, and optionally, the V L sequence has 1, 2, 3, 4 or 5 modifications in the framework region.

[0011] In some embodiments, the V H sequence includes a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO: 43, and the V L sequence includes a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO: 44; or the V H sequence includes a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO: 45, and the V L sequence includes a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO: 46; or the V H sequence includes a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO: 47, and the V LDoes the array contain a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 48? V H The array contains a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 49, and V L Does the array contain a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 50? V H The array contains a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 51, and V L Does the array contain a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 52? V H The array contains a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 53, and V L Does the array contain a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 54? V H The array contains a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 55, and V L Does the array contain a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 56? V H The array contains a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 69, and V L Does the array contain a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 70? V H The array contains a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 71, and V L Does the array contain a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to sequence number 72; or V HThe array contains a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO: 73, V L The array contains a sequence that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO: 74.

[0012] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to a human NRP2 polypeptide selected from the full-length human NRP2 polypeptide or Table N1, optionally with an affinity of about 10 pM to about 500 pM or about 10 pM to about 50 nM, or about, at least about, or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or optionally about 10 pM to about 500 pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM, and optionally, at least one antibody or antigen-binding fragment thereof specifically binds to a human NRP2 polypeptide in its native form but does not substantially bind to a human NRP2 polypeptide in its denatured form.

[0013] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds, optionally, with an affinity of about 10 pM to about 500 pM or about 10 pM to about 50 nM, or about, at least about, or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, to at least one epitope in a neuropilin domain selected from one or more of a neuropilin b1 domain, a neuropilin a1 domain, a neuropilin a2 domain, a neuropilin b2 domain, a neuropilin c domain, a neuropilin a1 / a2 composite domain, a neuropilin b1 / b2 composite domain, a neuropilin a2 / b1 composite domain, a neuropilin b2 / c composite domain, a neuropilin a2 / b1 / b2 composite domain, a neuropilin a2 / b1 / b2 / c composite domain, a neuropilin a1 / a2 / b1 composite domain, a neuropilin a1 / a2 / b1 / b2 composite domain, a neuropilin a1 / a2 / b1 / b2 / c composite domain, and a neuropilin b1 / b2 / c composite domain, or optionally, with an affinity in the range of about 10 pM to about 500 pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

[0014] In some embodiments, at least one antibody or antigen-binding fragment thereof binds to at least one epitope in the neuropilin a1 domain, neuropilin a2 domain, and / or neuropilin a1a2 composite domain, and adjacent linker regions, optionally, (Neuropilin a1 domain) residues 20-148, 30-141, 40-141, 50-141, 60-141, 70-141, 80-141, 90-141, 100-141, 110-141, 120-141, 130-141; 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30, as defined by the human NRP2 precursor sequence (see Table N1); (Neuropilin a2 domain) residues 142-280, 150-265, 160-265, 170-265, 180-265, 190-265, 200-265, 210-265, 220-265, 230-265, 240-265, 250-265, 260-265, 141-270, 141-260, 141-250, 141-240, 141-230, 141-220, 141-210, 141-200, 141-190, 141-180, 141-170, 141-160, 141-150, 200-250, 210-250, 220-250, 230-250, 200-240, 210-240, 220-240, 230-240, 227-247, 228-247, 229-247, 230-247, 231-247, 232-247, 233-247, 234-247, 235-247, 236-247; 227-246, 227-245, 227-244, 227-243, 227-242, 227-241, 227-240, 227-239, 227-238; 235-240, 236-239, 236-238, or residue 237; or (Composite a1a2 domain) Residues 20-280, 30-280, 40-280, 50-280, 60-280, 70-280, 80-280, 90-280, 100-280, 110-280, 120-280, 130-280, 140-280, 150-280, 160-280, 170-280, 180-280, 190-280, 200-280, 210-280, 220-280, 230-280, 240-280, 260-280, 270-280, 20-270, 20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-200, 20-190, 20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30, defined by the human NRP2 precursor sequence (see Table N1), specifically binds in the vicinity of.

[0015] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin b1 domain, neuropilin b2 domain, and / or neuropilin b1 / b2 composite domain, and adjacent linker regions, optionally (Neuropilin b1 domain) Residues 299-420, 266-426, 280-426, 290-426, 300-426, 310-426, 320-426, 330-426, 340-426, 350-426, 360-426, 370-426, 380-426, 390-426, 400-426, 410-426, 420-426, 280-420, 280-410, 280-400, 280-390, 280-380, 280-370, 280-360, 280-350, 280-340, 280-330, 280-320, 280-310, 280-300, or 280-290, defined by the human NRP2 precursor sequence (see Table N1), optionally, the epitope is a discontinuous epitope comprising 1, 2, or 3 of residues 299Y, 354N, and / or 416S defined by the human NRP2 precursor sequence; (Neuropilin b2 domain) Residues 438 - 591, 450 - 591, 460 - 591, 470 - 591, 480 - 591, 490 - 591, 500 - 591, 510 - 591, 520 - 591, 530 - 591, 540 - 591, 550 - 591, 560 - 591, 570 - 591, 580 - 591, 438 - 590, 438 - 580, 438 - 570, 438 - 560, 438 - 550, 438 - 540, 438 - 530, 438 - 520, 438 - 510, 438 - 500, 438 - 490, 438 - 480, 438 - 470, 438 - 460, or 438 - 450, defined by the human NRP2 precursor sequence (see Table N1); or (Neuropilin b1 / b2 composite domain) Residues 266 - 591, 276 - 591, 286 - 591, 296 - 591, 306 - 591, 316 - 591, 326 - 591, 336 - 591, 346 - 591, 356 - 591, 366 - 591, 376 - 591, 386 - 591, 396 - 591, 406 - 591, 416 - 591, 426 - 591, 436 - 591, 446 - 591, 456 - 591, 466 - 591, 476 - 591, 486 - 591, 498 - 591, 508 - 591, 518 - 591, 528 - 591, 538 - 591, 548 - 591, 558 - 591, 568 - 591, 578 - 591, 588 - 591, 266 - 581, 266 - 571, 266 - 561, 266 - 551, 266 - 541, 266 - 531, 266 - 521, 266 - 511, 266 - 501, 266 - 491, 266 - 481, 266 - 471, 266 - 461, 266 - 451, 266 - 441, 266 - 431, 266 - 421, 266 - 411, 266 - 401, 266 - 391, 266 - 381, 266 - 371, 266 - 361, 266 - 351, 266 - 341, 266 - 331, 266 - 321, 266 - 311, 266 - 301, 266 - 291, 266 - 281, or 266 - 271, defined by the human NRP2 precursor sequence (see Table N1) binds specifically in the vicinity of.

[0016] In some embodiments, at least one antibody or antigen-binding fragment thereof binds to at least one epitope in the neuropilin a2 / b1 complex domain and / or the neuropilin b2c complex domain, and the adjacent linker region, optionally, (Neuropilin a2b1 complex domain) residues 149 - 437, 159 - 426, 169 - 426, 179 - 426, 189 - 426, 199 - 426, 209 - 426, 219 - 426, 229 - 426, 239 - 426, 249 - 426, 259 - 426, 269 - 426, 279 - 426, 289 - 426, 299 - 426, 309 - 426, 319 - 426, 329 - 426, 339 - 426, 349 - 426, 359 - 426, 369 - 426, 379 - 426, 389 - 426, 399 - 426, 409 - 426, 419 - 426, 149 - 436, 149 - 426, 149 - 416, 149 - 406, 149 - 396, 149 - 386, 149 - 376, 149 - 366, 149 - 356, 149 - 346, 149 - 336, 149 - 326, 149 - 316, 149 - 306, 149 - 296, 149 - 286, 149 - 276, 149 - 266, 149 - 256, 149 - 246, 149 - 236, 149 - 226, 149 - 216, 149 - 206, 149 - 196, 146 - 186, 146 - 176, 146 - 166, or 146 - 155; as defined by the human NRP2 precursor sequence (see Table N1); or (Neuropilin b2c composite domain) Residues 438 - 794, 448 - 794, 458 - 794, 468 - 794, 478 - 794, 487 - 794, 497 - 794, 507 - 794, 517 - 794, 527 - 794, 537 - 794, 547 - 794, 557 - 794, 567 - 794, 587 - 794, 597 - 794, 607 - 794, 617 - 794, 627 - 794, 637 - 794, 647 - 794, 657 - 794, 667 - 794, 677 - 794, 687 - 794, 697 - 794, 707 - 794, 717 - 794, 727 - 794, 737 - 794, 747 - 794, 757 - 794, 767 - 794, 777 - 794, 787 - 794, 427 - 794, 438 - 784, 438 - 774, 438 - 764, 438 - 754, 438 - 744, 438 - 734, 438 - 728, 438 - 714, 438 - 704, 438 - 694, 438 - 684, 438 - 674, 438 - 664, 438 - 654, 438 - 644, 438 - 634, 438 - 624, 438 - 614, 438 - 604, 438 - 596, 438 - 586, 438 - 576, 438 - 566, 438 - 556, 438 - 546, 438 - 536, 438 - 526, 438 - 516, 438 - 506, 438 - 494, 438 - 484, 438 - 474, 438 - 464, 438 - 454, 438 - 444, defined by the human NRP2 precursor sequence (see Table N1). Binds specifically near

[0017] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin c domain and adjacent linker region, optionally in the vicinity of residues 591-794, 600-794, 610-794, 620-794, 630-794, 640-794, 650-794, 660-794, 670-794, 680-794, 690-794, 700-794, 710-794, 720-794, 730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 591-790, 591-780, 591-770, 591-760, 591-750, 591-740, 591-730, 591-720, 591-710, 591-700, 591-690, 591-680, 591-670, 591-660, 591-650, 591-640, 591-630, 591-620, 591-610, or 591-600, as defined by the human NRP2 precursor sequence (see Table N1).

[0018] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin b1 / b2 / c complex domain and adjacent linker region, optionally in the vicinity of residues 276 - 794, 286 - 794, 296 - 794, 306 - 794, 316 - 794, 326 - 794, 336 - 794, 346 - 794, 356 - 794, 366 - 794, 376 - 794, 387 - 794, 396 - 794, 406 - 794, 416 - 794, 426 - 794, 436 - 794, 446 - 794, 456 - 794, 466 - 794, 476 - 794, 486 - 794, 496 - 794, 506 - 794, 516 - 794, 526 - 794, 536 - 794, 546 - 794, 556 - 794, 566 - 794, 576 - 794, 586 - 794, 596 - 794, 606 - 794, 616 - 794, 626 - 794, 636 - 794, 646 - 794, 656 - 794, 666 - 794, 676 - 794, 686 - 794, 696 - 794, 706 - 794, 716 - 794, 726 - 794, 736 - 794, 746 - 794, 756 - 794, 766 - 794, 776 - 794, 786 - 794, 266 - 794, 276 - 784, 276 - 774, 276 - 764, 276 - 754, 276 - 744, 276 - 734, 276 - 724, 276 - 714, 276 - 704, 276 - 694, 276 - 684, 276 - 674, 276 - 664, 276 - 654, 276 - 644, 276 - 634, 276 - 624, 276 - 614, 276 - 604, 276 - 594, 276 - 584, 276 - 574, 276 - 564, 276 - 554, 276 - 544, 276 - 534, 276 - 524, 276 - 514, 276 - 504, 276 - 594, 276 - 584, 276 - 574, 276 - 564, 276 - 554, 276 - 544, 276 - 534, 276 - 524, 276 - 514, 276 - 504, or 276 - 496, as defined by the human NRP2 precursor sequence (see Table N1).

[0019] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in a juxtamembrane domain selected from one or more of the juxtamembrane domain of NRP2a (variant 1), the juxtamembrane domain of NRP2a (variant 2), the juxtamembrane domain of NRP2a (variant 3), the juxtamembrane domain of NRP2b (variant 4) and the juxtamembrane domain of NRP2b (variant 5), and combinations thereof (see Table N1).

[0020] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to a conformational epitope composed of two or more discontinuous epitope regions, and optionally, the conformational epitope is (a) a first epitope region within the a1 domain and a second epitope region within the a2 domain of the human NPR2 polypeptide; (b) a first epitope region within the a1 domain and a second epitope region within the b1 domain of the human NPR2 polypeptide; (c) a first epitope region within the a1 domain and a second epitope region within the b2 domain of the human NPR2 polypeptide; (d) a first epitope region within the a1 domain and a second epitope region within the c domain of the human NPR2 polypeptide; (e) a first epitope region within the a1 domain and a second epitope region within the juxtamembrane domain of the human NPR2 polypeptide selected from variants 1, 2, 3, 4 and 5; (f) a first epitope region within the a2 domain and a second epitope region within the b1 domain of the human NPR2 polypeptide; (g) a first epitope region within the a2 domain and a second epitope region within the b2 domain of the human NPR2 polypeptide; (h) a first epitope region within the a2 domain and a second epitope region within the c domain of the human NPR2 polypeptide; (i) The first epitope region within the a2 domain and the second epitope region within the membrane-proximal domain of a human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; (j) The first epitope region within the b1 domain and the second epitope region within the b2 domain of a human NPR2 polypeptide; (k) The first epitope region within the b1 domain and the second epitope region within the c domain of a human NPR2 polypeptide; (l) The first epitope region within the b1 domain and the second epitope region within the membrane-proximal domain of a human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; (m) The first epitope region within the b2 domain and the second epitope region within the c domain of a human NPR2 polypeptide; (n) The first epitope region within the b2 domain and the second epitope region within the membrane-proximal domain of a human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; or (o) The first epitope region within the c domain and the second epitope region within the membrane-proximal domain of a human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5 comprising or consisting of.

[0021] In some embodiments, at least one antibody or antigen-binding fragment thereof modulates the binding to at least one NRP2 ligand of a human NRP2 polypeptide (optionally, an NRP2 ligand selected from Table N2 or Table N3, and / or a human histidyl tRNA synthetase (HRS) polypeptide selected from Table H1, optionally, one or more HRS splice variants selected from SV9 (HRS(1 - 60)), SV11 (HRS(1 - 60)+(399 - 509)), and SV14 (HRS(1 - 100)+(399 - 509))).

[0022] In some embodiments, at least one antibody or antigen-binding fragment thereof is a blocking antibody that inhibits about or at least about 80-100%, optionally about or at least about 80, 85, 90, 95, or 100% of the theoretical maximum binding between a human NRP2 polypeptide and at least one NRP2 ligand after pre-incubation with a human NRP2 polypeptide at stoichiometric equivalence. In some embodiments, at least one antibody or antigen-binding fragment thereof is a partial blocking antibody that inhibits about or at least about 20-80%, optionally about or at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% of the theoretical maximum binding between a human NRP2 polypeptide and at least one NRP2 ligand after pre-incubation with a human NRP2 polypeptide at stoichiometric equivalence. In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to the HRS polypeptide interaction region of the NRP2 polypeptide and mimics or agonizes one or more signaling activities of an HRS polypeptide that binds to the NRP2 polypeptide. In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to the HRS polypeptide interaction region of the NRP2 polypeptide and modulates the binding activity / signaling activity between the NRP2 polypeptide and at least one NRP2 ligand. In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signaling activity between the NRP2 polypeptide and at least one NRP2 ligand.

[0023] In some embodiments, at least one antibody or antigen-binding fragment thereof agonizes or enhances the binding activity / signaling activity between the NRP2 polypeptide and at least one NRP2 ligand. In some embodiments, at least one NRP2 ligand is - VEGF selected from one or more of VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, and PIGF-2; - A VEGF receptor (VEGFR) selected from VEGFR2 and VEGFR3; - A semaphorin selected from one or more of SEMA3-A, SEMA-3B, SEMA-3C, SEMA-3D, SEMA-3F, and SEMA-3G; - A plexin selected from one or more of plexin A1, A2, A3, A4, and D1; - A growth factor selected from one or more of fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF); - A growth factor receptor selected from one or more of fibroblast growth factor receptor (FGFR), hepatocyte growth factor receptor (HGFR), and platelet-derived growth factor receptor (PDGF); - A galectin or a galectin receptor; - A transcription factor selected from FAC1 and bromoprotein PHD finger transcription factor; - An adapter protein selected from one or more of GIPC1, GIPC2, and GIPC3; - Table N3, optionally, α V β1, α V β3, α V β5, α V β6, α V β8, α6β1, and α6β4; - Transforming growth factor beta selected from one or more of TGFβ1, TGFβ2, TGFβ3, and their corresponding TGFβ receptors; and - An HRS polypeptide selected from Table H1, optionally, HisRS N1 、HisRS N2 、HisRS N3 、HisRS N4 (SV9), HisRS N5 、HisRS C1 、HisRS C2 、HisRS C3 、HisRS C4 、HisRS C5 、HisRS C6 、HisRSC7 、HisRS C8 (SV11) and HisRS C9 An HRS splice variant selected from one or more of selected from

[0024] In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between the NRP2 polypeptide and the plexin receptor and / or semaphorin, without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and VEGFR2 or VEGFR3 or VEGF-C. In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between the NRP2 polypeptide and the plexin receptor and / or semaphorin, without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and the HRS polypeptide. In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between the NRP2 polypeptide and the plexin receptor and / or semaphorin, without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and VEGFR2 or VEGFR3 or VEGF-C, and without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and the HRS polypeptide. In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between the NRP2 polypeptide and the plexin receptor and / or semaphorin, without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and VEGFR3. In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between the NRP2 polypeptide and VEGFR3 or VEGF-C, without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and a different ligand, and optionally the HRS polypeptide.

[0025] In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between NRP2 polypeptide and plexin receptor without substantially modulating the ligand binding of semaphorin 3 to NRP2. In some embodiments, the plexin receptor is selected from plexin A1, A2, A3, A4 and D1. In some embodiments, the semaphorin is selected from semaphorin 3B, 3C, 3D, 3F and 3G.

[0026] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope of at least 5 contiguous amino acids within the a2 domain of human NRP2, and at least one antibody or antigen-binding fragment thereof selectively inhibits receptor dimerization between NRP2 and plexin A1 without substantially inhibiting dimerization between NRP2 and FLT4 (VEGFR3). In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within amino acids 232-242 of the human NRP2 precursor (see Table N1). In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to a discontinuous epitope contained within amino acids 299-416 of the b1 domain of human NRP2, and at least one antibody or antigen-binding fragment thereof selectively inhibits receptor dimerization between NRP2 and plexin A1 without substantially inhibiting dimerization between NRP2 and FLT4 (VEGFR3) and KDR (VEGFR2).

[0027] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope of at least 5 contiguous amino acids within the b2 domain of human NRP2, and at least one antibody or antigen-binding fragment thereof inhibits receptor dimerization between NRP2 and FLT4 (VEGFR3) and inhibits dimerization between NRP2 and plexin A1. In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope of at least 5 contiguous amino acids within the c domain of human NRP2, and at least one antibody or antigen-binding fragment thereof inhibits receptor dimerization between NRP2 and plexin A1 and partially inhibits dimerization between NRP2 and FLT4 (VEGFR3).

[0028] In some embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ) for each of the corresponding regions of (i) a human NRP2 polypeptide and (ii) a cynomolgus NRP2 polypeptide, and the affinities for (i) and (ii) are in the range of about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 0.4 to about 1.2 nM, about 0.9 to about 5.5 nM, about 0.9 to about 5 nM, or about 1 nM to about 10 nM. In some embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ) for each of the corresponding regions of (i) a human NRP2 polypeptide and (ii) a mouse NRP2 polypeptide, and the affinities for (i) and (ii) are in the range of about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, or about 1 nM to about 10 nM.

[0029] In some embodiments, at least one antibody or antigen-binding fragment thereof selectively binds to the NRP2a isoform of NRP2 (optionally, variants 1, 2, and / or 3 of Table N1) and does not substantially bind to the NRP2b isoform (optionally, variants 4 and / or 5 of Table N1). In some embodiments, at least one antibody or antigen-binding fragment thereof selectively binds to the NRP2b isoform (optionally, variants 4 and / or 5 of Table N1) and does not substantially bind to the NRP2a isoform (optionally, variants 1, 2, and / or 3 of Table N1). In some embodiments, at least one antibody or antigen-binding fragment thereof, after pre-incubation with an NRP2 polypeptide in substantially stoichiometric equivalence of the anti-NRP2 antibody, optionally in the presence of an NRP2 ligand, reduces homoor heterodimerization between NRP2 polypeptides, optionally by about or at least about 20-100% (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100%).

[0030] In some embodiments, at least one antibody or antigen-binding fragment thereof enhances homodimerization or heterodimerization between NRP2 polypeptides, optionally by pre-incubating with NRP2 polypeptide in substantially stoichiometric equivalents of the anti-NRP2 antibody, optionally in the presence of an NRP2 ligand, by about or at least about 20 to 100% (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100%). In some embodiments, at least one antibody or antigen-binding fragment thereof selectively binds to a human NRP2 polypeptide (see Table N1) as compared to a mouse NRP2 polypeptide, and optionally, its affinity for the human NRP2 polypeptide is about or at least about 2, 5, 10, 20, 30, 40, 50, 100, 500, or 1000 times, or higher, or significantly stronger than its affinity for the mouse NRP2 polypeptide. In some embodiments, at least one antibody or antigen-binding fragment thereof binds to a human NRP2 polypeptide and does not substantially bind to a mouse NRP2 polypeptide, and optionally, the mouse NRP2 polypeptide is the NRP2 polypeptide of Mus musculus. In some embodiments, at least one antibody or antigen-binding fragment thereof binds to an epitope in the b1 domain that includes residues 299Y, 354N, and 416S, as defined by the human NRP2 precursor sequence (see Table N1).

[0031] In some embodiments, at least one antibody or antigen-binding fragment thereof comprises an Fc domain of IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4) or IgM, optionally including a human Fc domain or hybrids and / or variants thereof. In some embodiments, at least one antibody or antigen-binding fragment thereof comprises an Fc domain of IgG having high effector function in humans, optionally including the Fc domain of IgG1 or IgG3. In some embodiments, at least one antibody or antigen-binding fragment thereof comprises an Fc domain of IgG having low effector function in humans, optionally including the Fc domain of IgG2 or IgG4. In some embodiments, at least one antibody or antigen-binding fragment thereof optionally comprises an Fc domain of IgG1 or IgG4 selected from Table F1. In some embodiments, at least one antibody or antigen-binding fragment thereof comprises a modified Fc domain of IgG1 or IgG4 with altered binding to FcRn, optionally the modified Fc domain of IgG1 or IgG4 comprises any one or more of the mutations YD (M252Y / T256D), DQ (T256D / T307Q), DW (T256D / T307W), YTE (M252Y / S254T / T256E), AAA (T307A / E380A / N434A), LS (M428L / N434S), M252Y, T256D / E, K288D / N, T307Q / W, E380C, N434FY and / or Y436H / N / W (EU numbering), and combinations thereof.

[0032] In some embodiments, at least one antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, at least one antibody or antigen-binding fragment thereof is a humanized antibody. In some embodiments, at least one antibody or antigen-binding fragment thereof is an Fv fragment, a single-chain Fv (scFv) polypeptide, an adnectin, an anticalin, an aptamer, an affimer, a camelid antibody, a designed ankyrin repeat protein (DARPin), a minibody, a nanobody or a unibody.

[0033] In some embodiments, the therapeutic composition has a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% on a protein basis with respect to at least one antibody or antigen-binding fragment and is substantially non-aggregated. In some embodiments, the therapeutic composition is substantially endotoxin-free. In some embodiments, the therapeutic composition is a sterile injectable solution, optionally a sterile injectable solution suitable for intravenous, intramuscular, subcutaneous or intraperitoneal administration.

[0034] In some embodiments, the therapeutic composition further comprises at least one additional agent selected from one or more of a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent and a kinase inhibitor. In some embodiments, the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint modulating agent, a cancer vaccine, an oncolytic virus, a cytokine and a cell-based immunotherapy. In some embodiments, the immune checkpoint modulating agent is a polypeptide, optionally an antibody or antigen-binding fragment thereof or a ligand or a small molecule. In some embodiments, the immune checkpoint modulating agent is (a) an antagonist of an inhibitory immune checkpoint molecule; or (b) an agonist of a stimulatory immune checkpoint molecule and, optionally, the immune checkpoint modulating agent specifically binds to an immune checkpoint molecule.

[0035] In some embodiments, the inhibitory immune checkpoint molecule is selected from one or more of programmed death ligand 1 (PD-L1), programmed death 1 (PD-1), programmed death ligand 2 (PD-L2), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), V domain Ig suppressor of T cell activation (VISTA), B and T lymphocyte attenuator (BTLA), CD160, herpesvirus entry mediator (HVEM), and T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0036] In some embodiments, the antagonist is an antagonist of PD-L1 and / or PD-L2, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L1 and / or PD-L2, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736); the antagonist is an antagonist of PD-1, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514, PDR001, and pidilizumab; the antagonist is an antagonist of CTLA-4, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, and tremelimumab; The antagonist is an antagonist of IDO selected as needed from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to IDO, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat; The antagonist is an antagonist of TDO selected as needed from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91, and LM10; The antagonist is an antagonist of TIM-3 selected as needed from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TIM-3; The antagonist is an antagonist of LAG-3 selected as needed from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3 and BMS-986016; The antagonist is an antagonist of VISTA selected as needed from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to VISTA; The antagonist is an antagonist of BTLA, CD160, and / or HVEM selected as needed from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to BTLA, CD160, and / or HVEM; and / or The antagonist is an antagonist of TIGIT selected as needed from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TIGIT.

[0037] In some embodiments, the stimulatory immune checkpoint molecule is selected from one or more of OX40, CD40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0038] In some embodiments, the agonist is an agonist of OX40 optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40, OX86, Fc-OX40L, and GSK3174998; the agonist is an agonist of CD40 optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40, CP-870,893, dacetuzumab, Chi Lob7 / 4, ADC-1013, and rhCD40L; the agonist is an agonist of GITR optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1, and MEDI1873; the agonist is an agonist of CD137 optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD137, utomilumab, and 4-1BB ligand; the agonist is an agonist of CD27 optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27, varlilumab, and CDX-1127 (1F5); the agonist is an agonist of CD28 optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28 and TAB08; and / or the agonist is an agonist of HVEM optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to HVEM.

[0039] In some embodiments, the cancer vaccine is selected from one or more of oncolytic phage, human papillomavirus (HPV) vaccine, optionally Gardasil or Cervarix, hepatitis B vaccine, optionally Engerix-B, Recombivax HB or Twinrix, and sipuleucel-T (Provenge), or one or more cancer antigens selected from human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), phosphatase of regenerating liver 3 (PRL-3), prostate acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

[0040] In some embodiments, the oncolytic virus is selected from one or more of talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Seneca virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0041] In some embodiments, the cytokine is selected from one or more of interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte macrophage colony-stimulating factor (GM-CSF).

[0042] In some embodiments, the cell-based immunotherapeutic agent includes cancer antigen-specific T cells, optionally including ex vivo-derived T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TIL), and peptide-induced T cells.

[0043] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or type II), and antimicrotubule agents.

[0044] In some embodiments, The alkylating agent is selected from one or more of nitrogen mustard (if necessary, mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide and busulfan), nitrosourea (if necessary, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine and streptozotocin), tetrazine (if necessary, dacarbazine, mitozolomide and temozolomide), aziridine (if necessary, thiotepa, mitomycin and diaziquone (AZQ)), cisplatin and its derivatives (if necessary, carboplatin and oxaliplatin) and non-classical alkylating agents (if necessary, procarbazine and hexamethylmelamine); The antimetabolite is selected from one or more of folic acid antimetabolites (if necessary, methotrexate and pemetrexed), fluoropyrimidines (if necessary, 5-fluorouracil and capecitabine), deoxynucleoside analogs (if necessary, ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine and pentostatin) and thiopurines (if necessary, thioguanine and mercaptopurine); The cytotoxic antibiotic is selected from one or more of anthracyclines (if necessary, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin; The topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, melvalonate and aclarubicin; and / or The antimicrotubule agent is selected from one or more of taxanes (if necessary, paclitaxel and docetaxel) and vinca alkaloids (if necessary, vinblastine, vincristine, vindesine, vinorelbine).

[0045] In some embodiments, at least one hormonal therapeutic agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of progestogen (progestin), corticosteroid (prednisolone, methylprednisolone or dexamethasone as needed), insulin-like growth factor, VEGF-derived angiogenic factor and lymphangiogenic factor (VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2 as needed), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgen, estrogen, and somatostatin analog. In some embodiments, the hormone antagonist is a hormone synthesis inhibitor, an aromatase inhibitor or a gonadotropin-releasing hormone (GnRH) or its analog as needed, and a hormone receptor antagonist, a selective estrogen receptor modulator (SERM) or an anti-androgen agent as needed, or an antibody against a hormone receptor, cetuximab, daratumumab, figitumumab, ganitumab, istiratumab, robatumumab, aratuzumab pegol, bevacizumab, icrucumab, ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, imigatuzumab, laprituximab emtansine, matuzumab, modotuximab, nesitumumab, nimotuzumab, panitumumab, tomezumab, zalutumumab, apulizumab ixadotin, bemarituzumab, orlatumumab or tovetumab as needed, selected from one or more of them.

[0046] In some embodiments, the kinase inhibitor is selected from one or more of adabosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, enzootinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, luxitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0047] A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutic composition comprising at least one antibody or antigen-binding fragment thereof that specifically binds to a human neuropilin-2 (NRP2) polypeptide, optionally as a therapeutic composition described herein, wherein the at least one antibody or antigen-binding fragment thereof modulates (e.g., inhibits) the binding of the human NRP2 polypeptide to a human histidyl tRNA synthetase (HRS) polypeptide is also included.

[0048] In some embodiments, the disease or condition is an NRP2-related disease or condition. In some embodiments, the disease or condition is cancer, and diseases and pathways associated with cancer, including cancer cell growth, induction, migration, adhesion, invasion, chemoresistance, and / or metastasis; diseases associated with inappropriate activation or migration of immune cells, such as graft-versus-host disease (GVHD), including diseases associated with inflammation, autoimmunity, and related inflammatory diseases; diseases associated with lymphangiogenesis, lymphangioma, lymphovenous angiogenesis, and lymphatic vessel injury, including, for example, edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability; diseases associated with infection, including latent infection; allergic disorders / diseases, diseases associated with allergic reactions, including, for example, skin-related neutrophil-mediated diseases such as chronic obstructive pulmonary disease (COPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, systemic lupus erythematosus, rheumatoid arthritis, inflammasome-related diseases, and pyoderma gangrenosum; diseases associated with granulomatous inflammatory diseases, including sarcoidosis and granuloma; diseases associated with fibrosis, including fibrosis, endothelial-mesenchymal transition (EMT), and wound healing; diseases associated with inappropriate smooth muscle contractility, smooth muscle compensation and decompensation, and inappropriate migration and adhesion of vascular smooth muscle cells; diseases associated with inappropriate autophagy, phagocytosis, and efferocytosis; diseases associated with inappropriate migratory cell movement; neurological diseases, diseases associated with peripheral nervous system remodeling and pain sensation; and diseases associated with bone development and bone remodeling, selected from one or more of the foregoing.

[0049] In some embodiments, the disease is cancer, and optionally, the cancer expresses or overexpresses NRP2, and optionally, the cancer exhibits NRP2-dependent growth, NRP2-dependent adhesion, NRP2-dependent migration, and / or NRP2-dependent invasion. In some embodiments, the cancer expresses or overexpresses NRP2 but does not substantially express neuropilin-1 (NRP1). Certain methods are directed to reducing or preventing cancer recurrence in a subject in need thereof, and administration of the therapeutic composition enables the generation of immune memory against the cancer. In some embodiments, the subject has lymphedema.

[0050] Certain embodiments involve administering to a subject at least one additional agent selected from one or more of a cancer immunotherapy agent, a chemotherapeutic agent, a hormonal therapy agent, and a kinase inhibitor. In some embodiments, the at least one anti-NRP2 antibody or antigen-binding fragment thereof and the at least one agent are administered separately as separate compositions. In some embodiments, the at least one anti-NRP2 antibody and the at least one agent are administered together as part of the same therapeutic composition, optionally as a therapeutic composition described herein. In some embodiments, the cancer immunotherapy agent is selected from one or more of an immune checkpoint modulating agent, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapy.

[0051] In some embodiments, the immune checkpoint modulating agent is a polypeptide, optionally an antibody or antigen-binding fragment thereof or a ligand or small molecule. In some embodiments, the immune checkpoint modulating agent is (a) an antagonist of an inhibitory immune checkpoint molecule; or (b) an agonist of a stimulatory immune checkpoint molecule and, optionally, the immune checkpoint modulating agent specifically binds to an immune checkpoint molecule.

[0052] In some embodiments, the inhibitory immune checkpoint molecule is selected from one or more of programmed death ligand 1 (PD-L1), programmed death 1 (PD-1), programmed death ligand 2 (PD-L2), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), V domain Ig suppressor of T cell activation (VISTA), B and T lymphocyte attenuator (BTLA), CD160, herpesvirus entry mediator (HVEM), and T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0053] In some embodiments, the antagonist is an antagonist of PD-L1 and / or PD-L2 that specifically binds to PD-L1 and / or PD-L2, which is optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L1 and / or PD-L2, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), and optionally, the cancer is selected from one or more of colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer, and renal cell carcinoma; the antagonist is an antagonist of PD-1 that specifically binds to PD-1, which is optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514, PDR001, and pidilizumab, and optionally, the PD-1 antagonist is nivolumab and the cancer is selected from one or more of Hodgkin lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer; the PD-1 antagonist is pembrolizumab and the cancer is optionally selected from one or more of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial cancer; The antagonist is an antagonist of CTLA-4 that is optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, tremelimumab, and optionally, the cancer is selected from one or more of melanoma, prostate cancer, lung cancer, and bladder cancer; The antagonist is an antagonist of IDO that is optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to IDO, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, and optionally, the cancer is metastatic breast cancer, and brain cancer, and optionally, is selected from one or more of glioblastoma multiforme, glioma, sarcoma, or malignant brain tumor; Is the antagonist an antagonist of TDO that is optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91, and LM10? Is the antagonist an antagonist of TIM-3 that is optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TIM-3? Is the antagonist an antagonist of LAG-3 that is optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3 and BMS-986016? Is the antagonist an antagonist of VISTA that is optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to VISTA? Is the antagonist an antagonist of BTLA, CD160, and / or HVEM that is optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to BTLA, CD160, and / or HVEM? The antagonist is an antagonist of TIGIT selected as needed from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TIGIT.

[0054] In some embodiments, the stimulatory immune checkpoint molecule is selected from one or more of OX40, CD40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpesvirus entry mediator (HVEM).

[0055] In some embodiments, is the agonist an agonist of OX40 selected as needed from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40, OX86, Fc-OX40L, and GSK3174998; is the agonist an agonist of CD40 selected as needed from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40, CP-870,893, dacetuzumab, Chi Lob7 / 4, ADC-1013, and rhCD40L, and the cancer is selected from one or more of melanoma, pancreatic cancer, mesothelioma, and blood cancers, and optionally one or more of lymphomas such as non-Hodgkin lymphoma as needed; is the agonist an agonist of GITR selected as needed from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1, and MEDI1873; is the agonist an agonist of CD137 selected as needed from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD137, utomilumab, and 4-1BB ligand; Is the agonist an agonist of CD27, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27, balstilimab, and CDX-1127 (1F5); Is the agonist an agonist of CD28, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28, and TAB08; and / or Is the agonist an agonist of HVEM, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to HVEM.

[0056] In some embodiments, the cancer vaccine is an oncolytic phage, a human papillomavirus (HPV) vaccine, such as Gardasil or Cervarix as needed, a hepatitis B vaccine, such as Engerix-B or Recombivax as needed.Selected from one or more of HB or Twinrix, and sipuleucel-T (Provenge), or cancer antigens selected from one or more of human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin alpha v beta 3, integrin alpha 5 beta 1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostate acid phosphatase, Lewis-Y antigen, GD2 (disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin, and optionally, the subject has or is at risk of having cancer that includes the corresponding cancer antigen.

[0057] In some embodiments, the oncolytic virus is selected from one or more of talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Seneca virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0058] In some embodiments, the cytokine is selected from one or more of interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte macrophage colony-stimulating factor (GM-CSF).

[0059] In some embodiments, the cell-based immunotherapeutic agent includes cancer antigen-specific T cells, optionally including ex vivo-derived T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells, T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TIL), and peptide-induced T cells.

[0060] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or II), and antimicrotubule agents.

[0061] In some embodiments, The alkylating agent is selected from one or more of nitrogen mustard (if necessary, mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide and busulfan), nitrosourea (if necessary, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine and streptozotocin), tetrazine (if necessary, dacarbazine, mitozolomide and temozolomide), aziridine (if necessary, thiotepa, mitomycin and diaziquone (AZQ)), cisplatin and its derivatives (if necessary, carboplatin and oxaliplatin) and non-classical alkylating agents (if necessary, procarbazine and hexamethylmelamine); The antimetabolite is selected from one or more of folic acid antimetabolites (if necessary, methotrexate and pemetrexed), fluoropyrimidines (if necessary, 5-fluorouracil and capecitabine), deoxynucleoside analogs (if necessary, ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine and pentostatin) and thiopurines (if necessary, thioguanine and mercaptopurine); The cytotoxic antibiotic is selected from one or more of anthracyclines (if necessary, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin; The topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mevalonic acid and aclarubicin; and / or The antimicrotubule agent is selected from one or more of taxanes (if necessary, paclitaxel and docetaxel) and vinca alkaloids (if necessary, vinblastine, vincristine, vindesine, vinorelbine).

[0062] In some embodiments, at least one hormonal therapeutic agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of progestogen (progestin), corticosteroid (optionally, prednisone, methylprednisolone or dexamethasone), insulin-like growth factor, VEGF-derived angiogenic factor and lymphangiogenic factor (optionally, VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgen, estrogen and somatostatin analog. In some embodiments, the hormone antagonist is a hormone synthesis inhibitor, optionally, an aromatase inhibitor or gonadotropin-releasing hormone (GnRH) or its analog, and a hormone receptor antagonist, optionally, a selective estrogen receptor modulator (SERM) or an anti-androgen agent, or an antibody against a hormone receptor, optionally, cetuximab, daratumumab, figitumumab, ganitumab, istiratumab, robatumumab, alacizumab pegol, bevacizumab, icrucumab, ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, imigatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomozyxumab, zalutumumab, apulizumab ixadotin, bemariizumab, olaparitumumab or toveremab.

[0063] In some embodiments, the kinase inhibitor is selected from one or more of adabosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, enzastaurtinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, luxitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0064] In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer, optionally a metastatic cancer that expresses NRP2a and / or NRP2b. In some embodiments, the cancer is selected from one or more of melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, relapsed acute myelogenous leukemia), lymphoma, hepatocellular carcinoma (hepatocellular cancer), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer. In some embodiments, the metastatic cancer is (a) bladder cancer that has metastasized to bone, liver, and / or lung; (b) breast cancer that has metastasized to bone, brain, liver, and / or lung; (c) colorectal cancer that has metastasized to liver, lung, and / or peritoneum; (d) kidney cancer that has metastasized to adrenal gland, bone, brain, liver, and / or lung; (e) lung cancer that has metastasized to adrenal gland, bone, brain, liver, and / or other lung sites; (f) melanoma that has metastasized to bone, brain, liver, lung, and / or skin / muscle; (g) Ovarian cancer that has metastasized to the liver, lung, and / or peritoneum; (h) Pancreatic cancer that has metastasized to the liver, lung, and / or peritoneum; (i) Prostate cancer that has metastasized to the adrenal gland, bone, liver, and / or lung; (j) Gastric cancer that has metastasized to the liver, lung, and / or peritoneum; (l) Thyroid cancer that has metastasized to the bone, liver, and / or lung; and (m) Uterine cancer that has metastasized to the bone, liver, lung, peritoneum, and / or vagina is selected from one or more of.

[0065] In some embodiments, the subject has, in either bound or free form, an increased circulating or serum level of at least one NRP2 ligand (optionally, an NRP2 ligand from Table N2 or Table N3, and / or an HRS polypeptide from Table H1), optionally about or at least about 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, or 5000 pM of at least one NRP2 ligand, or about or at least about 30 - 100, 40 - 100, 50 - 100, 30 - 2000, 40 - 2000, 50 - 2000, 60 - 2000, 70 - 2000, 80 - 2000, 90 - 2000, 100 - 2000, 200 - 2000, 300 - 2000, 400 - 2000, 500 - 2000, 600 - 2000, 700 - 2000, 800 - 2000, 900 - 2000, 1000 - 2000, 2000 - 3000, 3000 - 4000, or 4000 - 5000 pM of at least one NRP2 ligand compared to a healthy control or the standard of care control or the level of a population of subjects, and / or is selected for treatment based on having it.

[0066] In some embodiments, the subject has a disease associated with increased levels or expression of at least one NRP2 ligand (optionally, an NRP2 ligand from Table N2 or Table N3, and / or an HRS polypeptide from Table H1) and / or its coding mRNA, compared to a healthy control or a standard of care control or a population of subjects, and / or is selected for treatment based thereon. Optionally, the cancer has increased levels or expression of at least one NRP2 ligand and / or its coding mRNA, compared to non-cancerous cells or tissues of the same type as the cancer, optionally compared to non-cancerous cells or tissues of the same type as the cancer compared to non-cancerous controls. Optionally, the HRS polypeptide is HisRS N1 HisRS N2 HisRS N3 HisRS N4 HisRS N5 HisRS C1 HisRS C2 HisRS C3 HisRS C4 HisRS C5 HisRS C6 HisRS C7 HisRS C8 and HisRS C9 is a splice variant selected from.

[0067] In some embodiments, the subject is either in bound or free form and has an increased circulating or serum level of soluble neuropilin 2 (NRP2) polypeptide (optionally selected from Table N1), compared to the level of a healthy control or a standard of a corresponding control or the population of subjects, optionally a circulating or serum level of soluble NRP2 polypeptide of about or at least about 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000 pM, or optionally a circulating or serum level of soluble NRP2 polypeptide of about 30 - 50, 50 - 100, 100 - 2000, 200 - 2000, 300 - 2000, 400 - 2000, 500 - 2000, 600 - 2000, 700 - 2000, 800 - 2000, 900 - 2000, 1000 - 2000, 2000 - 3000, 3000 - 4000, 4000 - 5000 pM, and / or is selected for treatment based on having it.

[0068] In some embodiments, the subject has a disease associated with an increased level or expression of NRP2 polypeptide (optionally selected from Table N1) and / or its coding mRNA, compared to a healthy control or a standard of a corresponding control or the population of subjects, and / or is selected for treatment based on having it, and optionally, the cancer has an increased level or expression of NRP2 polypeptide (optionally selected from Table N1) and / or its coding mRNA, compared to non-cancerous control cells or tissues, optionally compared to non-cancerous cells or tissues of the same type as the cancer.

[0069] In some embodiments, a subject is selected for treatment based on having a disease associated with increased levels or expression of NRP2a and / or NRP2b, or an altered ratio of NRP2a:NRP2b expression, compared to a healthy control or a standard of care control or a population of subjects, and / or based on having it. In some embodiments, the level of NRP2b is increased by about or at least about 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% compared to a healthy control or a standard of care control or a population of subjects. In some embodiments, the healthy control or a standard of care control or a population of subjects includes an age-matched sample of cancerous or non-cancerous cells or tissues of the same type as the cancer, including specific characteristics such as drug resistance, metastatic potential, aggressiveness, gene signature (optionally, p53 mutation, PTEN deletion, IGFR expression) and / or expression pattern. In some embodiments, a subject is selected for treatment based on having increased circulating levels of the HRS:NRP2 complex, compared to a healthy or a standard of care control or a population of subjects, and / or based on having it.

[0070] Certain embodiments include administering at least one anti-NRP2 antibody in an amount and frequency sufficient to achieve an average sustained serum or circulating level of soluble NRP2 polypeptide of about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM or 10 pM, or less than about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM or 10 pM. Certain embodiments include administering at least one anti-NRP2 antibody in an amount and frequency sufficient to achieve a reduction in the circulating level of the HRS:NRP2 complex, optionally by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, 99 or 100% reduction.

[0071] In some embodiments, at least one anti-NRP2 antibody enhances the immune response against cancer by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more as compared to a control. In some embodiments, at least one anti-NRP2 antibody reduces the rate of in vitro growth of cancer by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more as compared to an untreated control. In some embodiments, at least one anti-NRP2 antibody reduces the in vitro adhesion of cancer to a substrate by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more as compared to an untreated control, and optionally, the substrate comprises laminin.

[0072] In some embodiments, at least one anti-NRP2 antibody reduces the invasiveness of cancer by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more as compared to an untreated control. In some embodiments, at least one anti-NRP2 antibody inhibits the migration or motility rate of cancer by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more as compared to an untreated control. In some embodiments, at least one anti-NRP2 antibody inhibits the rate of autophagy or endosomal mutations (endosomal acidification, if desired) of cancer or related immune cells by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more as compared to an untreated control. In some embodiments, at least one anti-NRP2 antibody enhances the sensitivity of cancer to one or more additional agents selected from one or more of chemotherapeutic agents, hormonal therapeutic agents, and kinase inhibitors by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more as compared to the additional agent alone. In some embodiments, at least one anti-NRP2 antibody enhances the antitumor activity and / or immunostimulatory activity of a cancer immunotherapeutic agent by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more as compared to the cancer immunotherapeutic agent alone.

[0073] Certain embodiments include administering at least one anti-NRP2 antibody in an amount and frequency sufficient to achieve a steady state concentration or average circulating concentration of the at least one anti-NRP2 antibody of from about 1 nM to about 1 μM, from about 1 nM to about 100 nM, from about 1 nM to about 10 nM, or from about 1 nM to about 3 μM.

[0074] A patient care kit comprising (a) at least one antibody or antigen-binding fragment thereof that specifically binds to a human neuropilin-2 (NRP2) polypeptide; and optionally (b) at least one additional agent selected from a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent, and a kinase inhibitor is also included.

[0075] In some embodiments, (a) and (b) are in separate therapeutic compositions. In some embodiments, (a) and (b) are in the same therapeutic composition. In some embodiments, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type I or II), and an antimicrotubule agent.

[0076] In some embodiments, the alkylating agent is selected from one or more of nitrogen mustard (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosourea (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazine (optionally dacarbazine, mitozolomide, and temozolomide), aziridine (optionally thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and its derivatives (optionally carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine); The metabolic antagonist is selected from one or more of folic acid metabolic antagonists (if necessary, methotrexate and pemetrexed), fluoropyrimidines (if necessary, 5-fluorouracil and capecitabine), deoxynucleoside analogs (if necessary, ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine and pentostatin) and thiopurines (if necessary, thioguanine and mercaptopurine); The cytotoxic antibiotic is selected from one or more of anthracyclines (if necessary, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin; The topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, melvalonate and aclarubicin; and / or The antimicrotubule agent is selected from one or more of taxanes (if necessary, paclitaxel and docetaxel) and vinca alkaloids (if necessary, vinblastine, vincristine, vindesine, vinorelbine).

[0077] In some embodiments, at least one hormonal therapeutic agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of progesterone (progestin), corticosteroids (prednisolone, methylprednisolone or dexamethasone as required), insulin-like growth factor, VEGF-derived angiogenic factors and lymphangiogenic factors (VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2 as required), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgen, estrogen and somatostatin analog. In some embodiments, the hormone antagonist is a hormone synthesis inhibitor, an aromatase inhibitor or a gonadotropin-releasing hormone (GnRH) or its analog as required, and a hormone receptor antagonist, a selective estrogen receptor modulator (SERM) or an anti-androgen agent as required, or an antibody against a hormone receptor, trastuzumab, daratumumab, figitumumab, ganitumab, istiratumab, robatumumab, alacizumab pegol, bevacizumab, icrucumab, ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, imigatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomozyxizumab, zalutumumab, apulizumab ixadotin, bemarituzumab, olaparitumumab or tovetumab as required, selected from one or more of them.

[0078] In some embodiments, the kinase inhibitor is selected from one or more of adabosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, enzotrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, luxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0079] Also included is a bioassay system comprising a host cell line that expresses a substantially pure anti-NRP2 antibody or an antigen-binding fragment thereof, as defined herein if necessary, and a human NRP2 polypeptide on the cell surface. In some embodiments, the NRP2 polypeptide is labeled with a detectable label. In some embodiments, the anti-NRP2 antibody is labeled with a detectable label. In some embodiments, the NRP2 polypeptide is operably linked to a readout or indicator such as a fluorescent or luminescent indicator of the biological activity of the NRP2 polypeptide. In some embodiments, the NRP2 polypeptide is selected from Table N1. Certain bioassay systems include at least one NRP2 ligand (an NRP2 ligand selected from Table N2 or Table N3, and / or a human histidyl tRNA synthetase (HRS) polypeptide selected from Table H1, if necessary), and optionally, the host cell expresses at least one NRP2 ligand. In some embodiments, the HRS polypeptide is selected from Table H1, and optionally, the HRS polypeptide is an HRS splice variant, and optionally, HisRS N1 HisRS N2 HisRS N3 HisRS N4 HisRS N5 HisRS C1 HisRS C2 HisRS C3, HisRS C4 , HisRS C5 , HisRS C6 , HisRS C7 , HisRS C8 and HisRS C9 comprising an HRS splice variant selected from the group consisting of HisRS C4 , HisRS C5 , HisRS C6 , HisRS C7 , HisRS C8 , HisRS C9 , HisRS N1 , HisRS N2 , HisRS N3 , HisRS N4 , HisRS N5 , HisRS C1 , HisRS C2 , HisRS C3 , HisRS C4 , HisRS C5 , HisRS C6 , HisRS C7 , HisRS C8 , HisRS C9 . In some embodiments, at least one NRP2 ligand is selected from Table N2 or Table N3.

[0080] A detection system comprising a cell expressing a human neuropilin 2 (NRP2) polypeptide, at least one NRP2 ligand (optionally a recombinant NRP2 ligand selected from Table N2 or Table N3 and / or a human histidyl tRNA synthetase (HRS) polypeptide selected from Table H1), and optionally a human or humanized anti-NRP2 antibody or antigen-binding fragment thereof as defined herein, and modulating the interaction between the NRP2 polypeptide and the at least one NRP2 ligand. Also included is a detection system. In some embodiments, the anti-NRP2 antibody is labeled with a detectable label. In some embodiments, the NRP2 polypeptide is selected from Table N1. In some embodiments, the HRS polypeptide is an HRS splice variant selected from Table H1, optionally HisRS N1 , HisRS N2 , HisRS N3 , HisRS N4 , HisRS N5 , HisRS C1 , HisRS C2 , HisRS C3 , HisRS C4 , HisRS C5 , HisRS C6 , HisRS C7 , HisRS C8 , HisRS C9 . N1 , HisRS N2 , HisRS N3 , HisRS N4 , HisRS N5 , HisRS C1 , HisRS C2 , HisRS C3 , HisRS C4 , HisRS C5 , HisRS C6 , HisRS C7 , HisRS C8 and HisRS C9It includes an HRS splice variant selected from. In some embodiments, at least one NRP2 ligand is selected from Table N2 or Table N3. In some embodiments, the NRP2 polypeptide and / or at least one NRP2 ligand is functionally linked to a readout or indicator, such as a fluorescent or luminescent indicator of the biological activity of the NRP2 polypeptide or at least one NRP2 ligand.

[0081] A diagnostic system, comprising a cell containing a neuropilin 2 (NRP2) polypeptide, and at least one NRP2 ligand that specifically binds to the NRP2 polypeptide (optionally, an NRP2 ligand selected from Table N2 or Table N3, and / or a human histidyl tRNA synthetase (HRS) polypeptide selected from Table H1), wherein the cell contains an indicator molecule that exhibits a change in the level or activity of the NRP2 polypeptide in response to interaction with at least one NRP2 ligand, is also included.

[0082] A cell composition, comprising a population of engineered cells in which at least one cell contains one or more polynucleotides encoding a human or humanized anti-NRP2 antibody or an antigen-binding fragment thereof as defined herein, and the cells are capable of growing in serum-free medium, is also included.

[0083] A cell growth device, comprising a human or humanized anti-NRP2 antibody or an antigen-binding fragment thereof as defined herein, a population of engineered cells in which at least one cell contains one or more polynucleotides encoding the anti-NRP2 antibody or an antigen-binding fragment thereof, at least about 10 liters of serum-free growth medium, and a sterile container, is also included.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0107] Detailed Description 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 disclosure belongs. Any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this disclosure, but preferred methods and materials are described. All publications and references, including but not limited to patents and patent applications, cited herein are hereby incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Any patent application for which this application claims priority is also incorporated by reference in its entirety in the manner described above for publications and references.

[0108] Standard techniques for recombinant DNA, oligonucleotide synthesis, and tissue culture, and transformation (e.g., electroporation, lipofection) can be used. Enzyme reactions and purification techniques can be performed according to the manufacturer's specifications, or as commonly accomplished in the art, or as described herein. These and related techniques and procedures can generally be performed according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. Unless specific definitions are provided, the nomenclature, laboratory procedures, and techniques utilized in connection with molecular biology, analytical chemistry, synthetic organic chemistry, and pharmaceuticals and pharmaceutical chemistry described herein are well known and generally used in the art. Standard techniques for recombinant technology, molecular biological synthesis, microbiological synthesis, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients can be used.

[0109] For the purposes of the present disclosure, the following terms are defined below.

[0110] The articles "a" and "an" are used herein to refer to the object of the article being one or more (i.e., at least one). By way of example, "an element" includes "one element", "one or more elements", and / or "at least one element".

[0111] "About" means that a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% with respect to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0112] The term "antigen" refers to a molecule or a part of a molecule that can be bound by a selective binding agent such as an antibody and can further be used in an animal to produce an antibody that can bind to an epitope of the antigen. An antigen may have one or more epitopes. As used herein, the term "antigen" includes a substance that can induce an immune response with a substance under appropriate conditions and a substance that can react with the product of an immune response. For example, an antigen can be recognized by an antibody (humoral immune response) or a sensitized T lymphocyte (helper T or T cell-mediated immune response) or both. An antigen can be a soluble substance such as a toxin and a foreign protein, or a particulate substance such as bacteria and tissue cells. However, only a part of a protein or polysaccharide molecule known as an antigenic determinant (epitope) binds to a specific receptor on an antibody or lymphocyte. In a broader sense, the term "antigen" includes any substance that binds to an antibody or to which an antibody is desired to bind, whether or not the substance is immunogenic. For such an antigen, an antibody can be identified by recombinant methods independent of any immune response.

[0113] "Antagonist" refers to a biological structure or chemical agent that impedes or otherwise reduces the physiological action of another agent or molecule. In some examples, an antagonist specifically binds to another agent or molecule. Full antagonists and partial antagonists are included.

[0114] An "agonist" refers to a biological structure or chemical agent that increases or enhances the physiological action of another agent or molecule. In some examples, an agonist specifically binds to another agent or molecule. Full agonists and partial agonists are included.

[0115] The term "anergy" refers to the functional inactivation of the response of T cells or B cells to restimulation by an antigen.

[0116] As used herein, the term "amino acid" is intended to mean both naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis, as well as other amino acids such as, for example, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, etc., which are known to those of skill in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties in the amino acid, or derivatization of the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties such as the charge and charge-spacing characteristics of a reference amino acid. For example, an organic structure that mimics arginine (Arg or R) will have a positively charged moiety located in a molecular space similar to the e-amino group of the side chain of the naturally occurring Arg amino acid and will have a similar degree of mobility. Mimetics also include restricted structures that maintain the optimal spacing and charge interactions of the amino acid or amino acid functional group. Those of skill in the art know or can determine which structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0117] As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab’, F(ab’)2, Fv, etc.), single-chain antibodies (scFv), their synthetic variants, naturally occurring variants, fusion proteins containing antibody moieties having antigen-binding fragments of the required specificity, humanized antibodies, chimeric antibodies, and any other modified conformation of an immunoglobulin molecule containing an antigen-binding site or fragment (epitope recognition site) of the required specificity. Certain properties and characteristics of antibodies (and their antigen-binding fragments) are described in more detail herein.

[0118] The antibody or antigen-binding fragment can be essentially of any type. As is well known in the art, an antibody is an immunoglobulin molecule that can specifically bind to a target, such as an immune checkpoint molecule, by at least one epitope recognition site located in the variable region of the immunoglobulin molecule.

[0119] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of the heavy and / or light chain of an immunoglobulin that binds to the antigen of interest. In this regard, the antigen-binding fragments of the antibodies described herein may contain 1, 2, 3, 4, 5, or all 6 CDRs of the V H sequence and the V L sequence.

[0120] The binding of antibodies and their antigen-binding fragments can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, the antibody or its antigen-binding fragment specifically binds to a target molecule, such as an NRP2 polypeptide or an epitope or complex thereof, and the equilibrium dissociation constant is about ≦10 -7 M to about 10 -8is M or in that range. In some embodiments, the equilibrium dissociation constant is about ≦10 -9 M to about ≦10 -10 M or in that range. In certain exemplary embodiments, the antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ) for a target molecule (which it specifically binds) of about or at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50 nM, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50 nM.

[0121] A molecule such as a polypeptide or an antibody is said to exhibit "specific binding" or "preferential binding" when it reacts or associates with a particular cell, substance, or specific epitope more frequently, more rapidly, for a longer period, and / or with a higher affinity than it does with an alternative cell or substance, or epitope. An antibody "specifically binds" or "preferentially binds" to a target molecule or epitope when it binds to that target molecule or epitope in a statistically significant amount, with a higher affinity, avidity, more readily, and / or for a longer period than it binds to other substances or epitopes. Typically, one member of a pair of molecules that exhibit specific binding binds specifically to a particular spatial and / or polar configuration of the other member of the pair of molecules and thus has a region or cavity on its surface that is complementary. Thus, the members of the pair have the property of binding specifically to each other. For example, an antibody that specifically or preferentially binds to a particular epitope is an antibody that binds to that specific epitope with a higher affinity, avidity, more readily, and / or for a longer period than it binds to other epitopes. For example, it is understood from this definition that an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. This term is applicable, for example, when an antibody is specific for a particular epitope that some antigens possess, or when a specific binding member having an antigen-binding fragment or domain is capable of binding to various antigens having that epitope, for example, this may be cross-reactive with several different forms of a target antigen from multiple species that share a common epitope.

[0122] Immunological binding generally refers to non-covalent interactions that occur between immunoglobulin molecules and antigens specific to those immunoglobulins, such as, by way of example and not limitation, electrostatic, ionic, hydrophilic and / or hydrophobic attractive or repulsive forces, steric forces, hydrogen bonds, van der Waals forces, and other types of interactions resulting from such interactions. The strength or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, with a smaller Kd representing a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. Such a method involves measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, where those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, both the "association rate constant" (Kon) and the "dissociation rate constant" (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of Koff / Kon allows for the cancellation of all parameters not related to affinity and thus is equal to the dissociation constant Kd. As used herein, the term "affinity" includes the equilibrium constant for the reversible binding of two agents and is represented as Kd or EC 50 and is expressed as such. The affinity of a binding protein for its ligand, for example, the affinity of an antibody for an epitope, can be, for example, from about 100 nanomolar concentration (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar concentration (pM), or from about 100 nM to about 1 femtomolar concentration (fM). As used herein, the term "avidity" refers to the resistance to dissociation of a complex of two or more agents after dilution. In some embodiments, affinity is expressed in terms of the 50% effective concentration (EC 50 ), which refers to the concentration of an agent, such as an antibody or an anti-NRP2 antibody disclosed herein, that induces a response halfway between baseline and maximum after a specific exposure time. EC 50 is generally used as a measure of the potency of an antibody.

[0123] Antibodies can be prepared by any of a variety of techniques known to those skilled in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific for the polypeptide of interest can be prepared, for example, using the techniques of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976 and improvements thereto. Also included are methods of expressing human antibodies using transgenic animals such as mice. See, for example, Neuberger et al., Nature Biotechnology 14:826, 1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101, 1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995. A specific example is the VELOCIMMUNE® platform by REGENEREX® (see, for example, U.S. Patent No. 6,596,541).

[0124] Antibodies can also be made or identified by use of phage display or yeast display libraries (see, e.g., U.S. Patent No. 7,244,592; Chao et al., Nature Protocols. 1:755-768, 2006). Non-limiting examples of available libraries include cloned or synthetic libraries, such as the Human Combinatorial Antibody Library (HuCAL), which represents the structural diversity of the human antibody repertoire by seven heavy chain and seven light chain variable region genes. Combinations of these genes result in 49 frameworks in the master library. By overlaying highly variable gene cassettes (CDR = complementarity determining regions) in these frameworks, an enormous human antibody repertoire can be reproduced. Also included are human libraries designed using fragments of human donor origin encoding the light chain variable region, the heavy chain CDR-3, synthetic DNA encoding the diversity of the heavy chain CDR-1, and synthetic DNA encoding the diversity of the heavy chain CDR-2. Other libraries suitable for use will be apparent to those of skill in the art.

[0125] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise sets of heavy and light chain complementarity determining regions (CDRs) inserted between sets of heavy and light chain framework regions (FRs) that provide support for the CDRs and define the spatial relationship of the CDRs relative to one another. As used herein, the term "set of CDRs" refers to the three hypervariable regions of the V region of a heavy or light chain. In order from the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Thus, the antigen-binding site comprises six CDRs, a set of CDRs from each of the V regions of the heavy and light chains. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit". Crystal structures of several antigen-antibody complexes have demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, where the most extensive antigen contacts are with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of the antigen-binding site.

[0126] As used herein, the term "set of FRs" refers to four contiguous amino acid sequences that constitute the CDRs of a set of CDRs of the V region of a heavy or light chain. Some FR residues may contact the bound antigen; however, the FRs, particularly the FR residues immediately adjacent to the CDRs, are primarily responsible for the folding of the V region into the antigen-binding site. Within the FRs, certain amino residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form the antigen-binding surface. It is generally recognized that there are conserved structural regions of the FRs that influence the folded shape of the CDR loops into a particular "canonical" structure, regardless of the exact amino acid sequence of the CDRs. In addition, certain FR residues are known to be involved in non-covalent interdomain contacts that stabilize the interaction between the heavy and light chains of the antibody.

[0127] The structure and location of immunoglobulin variable domains can be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, 1987 and its revisions.

[0128] Also included are "monoclonal" antibodies, which refer to a population of homogeneous antibodies, where monoclonal antibodies are composed of amino acids (naturally occurring and non-naturally occurring) involved in the selective binding of an epitope. Monoclonal antibodies are highly specific and are made against a single epitope. The term "monoclonal antibody" includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (Fab, Fab’, F(ab’)2, Fv, etc.), single-chain antibodies (ScFv), their variants, fusion proteins containing antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified conformation of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) with the required specificity and ability to bind an epitope. It is not intended to be limiting with respect to the source of the antibody or the method by which the antibody is made (e.g., hybridoma, phage selection, recombinant expression, transgenic animals). The term includes whole immunoglobulins and fragments such as those described above under the definition of "antibody".

[0129] The protease papain preferentially cleaves IgG molecules, generating several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer containing an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including an F(ab’)2 fragment that contains both antigen-binding sites. Fv fragments, used according to certain embodiments, can be generated by the preferential proteolytic cleavage of immunoglobulin molecules of IgM, and rarely IgG or IgA. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments include non-covalently linked VH::VL heterodimers that contain the antigen-binding site and retain much of the antigen recognition and antigen-binding capabilities of native antibody molecules. See Inbar et al., PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980.

[0130] In certain embodiments, single-chain Fv (scFv) antibodies are contemplated. For example, camelids (Ill et al., Prot. Eng. 10:949-57, 1997); minibodies (Martin et al., EMBO J 13:5305-9,1994); diabodies (Holliger et al., PNAS 90: 6444-8, 1993); or Janusins (Traunecker etal., EMBO J 10: 3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl.7:51-52, 1992) can be prepared using standard molecular biology techniques according to the teachings of this application regarding the selection of antibodies having the desired specificity.

[0131] A single-chain Fv (scFv) polypeptide is a covalently linked VH::VL heterodimer expressed from a gene fusion that includes VH and VL coding genes linked by a peptide-coding linker. Huston et al. (PNAS USA. 85(16):5879-5883, 1988). Several methods have been described for identifying chemical structures that convert the light and heavy polypeptide chains derived from the antibody V regions, which are naturally aggregated but chemically separated, into scFv molecules that fold into a three-dimensional structure substantially similar to that of the antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Patent No. 4,946,778 to Ladner et al.

[0132] In certain embodiments, the antibodies or antigen-binding fragments described herein are in the form of a "diabody." A diabody is a multimer of polypeptides, each polypeptide comprising a first domain that includes a binding region of an immunoglobulin light chain and a second domain that includes a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site. The antigen-binding site is formed by the association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer (WO 94 / 13804). The dAb fragment of an antibody consists of a VH domain (Ward et al., Nature 341:544-546, 1989). Diabodies and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO 94 / 13804; and Holliger et al., PNAS USA. 90:6444-6448, 1993).

[0133] Also included are minibodies that contain an scFv linked to a CH3 domain (see Hu et al., Cancer Res. 56:3055-3061, 1996). See also Ward et al., Nature. 341:544-546, 1989; Bird et al., Science. 242:423-426, 1988; Huston et al., PNAS USA. 85:5879-5883, 1988); International Application No. PCT / US92 / 09965; International Publication No. 94 / 13804; and Reiter et al., Nature Biotech. 14:1239-1245, 1996.

[0134] When bispecific antibodies are used, these may be conventional bispecific antibodies, which can be produced in a variety of ways (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), for example, chemically or prepared from hybrid hybridomas, or may be any of the bispecific antibody fragments described above. Diabodies and scFvs can be constructed using only variable domains without an Fc region, potentially reducing the effect of anti-idiotypic reactions.

[0135] Bispecific diabodies may also be particularly useful because, in contrast to bispecific whole antibodies, they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) with appropriate binding specificities can be readily selected from libraries using phage display (International Publication No. 94 / 13804). If one arm of a diabody is kept constant, for example, with specificity for antigen X, it is possible to create a library in which the other arm is varied and antibodies with appropriate specificities are selected. Bispecific whole antibodies may be produced by a knob-into-hole operation (Ridgeway et al., Protein Eng., 9:616-621, 1996).

[0136] In certain embodiments, the antibodies or antigen-binding fragments described herein are in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, the Netherlands; see also, e.g., US Patent Application Publication No. 20090226421). This antibody technology creates a stable and smaller antibody format with a longer therapeutic range than current small antibody formats. IgG4 antibodies are thought to be inert and thus do not interact with the immune system. A fully human IgG4 antibody may be modified by removing the hinge region of the antibody to obtain a half-molecule fragment with distinct stability compared to the corresponding intact IgG4 (GenMab, Utrecht). By splitting the IgG4 molecule in half, only one region remains on the UniBody® that can bind to a homologous antigen (e.g., a disease target), and thus the UniBody® binds monovalently to only one site on the target cell. For certain cancer cell surface antigens, this monovalent binding does not stimulate and grow cancer cells as can be seen using a bivalent antibody with the same antigen specificity, and thus UniBody® technology may provide an option for treating some types of cancer that may be resistant to treatment with conventional antibodies. The small size of the UniBody® can be highly beneficial when treating some forms of cancer, allowing for better distribution of the molecule throughout larger solid tumors and potentially increasing efficacy.

[0137] In certain embodiments, the antibodies and antigen-binding fragments described herein are in the form of nanobodies. Nanobodies are encoded by a single gene and can be efficiently produced in almost all prokaryotic and eukaryotic hosts, such as E. coli (see U.S. Patent No. 6,765,087), fungi (e.g., Aspergillus or Trichoderma), and yeast (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see U.S. Patent No. 6,838,254)). The production process is scalable, and nanobodies have been produced in kilogram quantities. Nanobodies may be formulated as ready-to-use solutions with a long shelf life. The nanocloning method (see International Publication No. 06 / 079372) is an exclusive method for generating nanobodies against a desired target based on the automated high-throughput selection of B cells.

[0138] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of aptamers (see, e.g., Ellington et al., Nature. 346, 818-22, 1990; and Tuerk et al., Science. 249, 505-10, 1990, which are incorporated herein by reference). Examples of aptamers include nucleic acid aptamers (e.g., DNA aptamers, RNA aptamers) and peptide aptamers. Nucleic acid aptamers generally refer to nucleic acid species that are engineered by repeated rounds of in vitro selection, such as SELEX (systematic evolution of ligands by exponential enrichment), or equivalent methods, to bind to a variety of molecular targets, including small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. See, e.g., U.S. Patent No. 6,376,190; and U.S. Patent No. 6,387,620, which are incorporated herein by reference.

[0139] Peptide aptamers typically contain a variable peptide loop attached at both ends to a protein backbone, and the dual structural constraints typically increase the binding affinity of the peptide aptamer to a level comparable to that of an antibody (e.g., in the nanomolar range). In certain embodiments, the length of the variable loop may consist of about 10 to 20 amino acids (including all integers therebetween), and the backbone may include any protein having good solubility and compactness. Certain exemplary embodiments utilize the bacterial protein thioredoxin-A as the backbone protein, and the variable loop is inserted into the reducing active site (the -Cys-Gly-Pro-Cys- loop in the wild-type protein), and the two cysteine side chains are capable of forming a disulfide bridge. Methods for identifying peptide aptamers are described, for example, in U.S. Patent Application Publication No. 2003 / 0108532, which is incorporated herein by reference. Peptide aptamer selection can be performed using different systems known in the art, including yeast two-hybrid systems.

[0140] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of an avimer. An avimer refers to a multimeric binding protein or peptide engineered using in vitro exon shuffling and phage display. Multiple binding domains are linked, resulting in higher affinity and higher specificity compared to a single epitope immunoglobulin domain. See, for example, Silverman et al., Nature Biotechnology. 23:1556-1561, 2005; U.S. Patent No. 7,166,697; and U.S. Patent Application Publications No. 2004 / 0175756, 2005 / 0048512, 2005 / 0053973, 2005 / 0089932, and 2005 / 0221384, which are incorporated herein by reference.

[0141] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of anadnectins. Anadnectins refer to a class of targeted biologics derived from human fibronectin, an abundant extracellular protein that binds naturally to other proteins. See, for example, U.S. Patent Application Publication Nos. 2007 / 0082365; 2008 / 0139791; and 2008 / 0220049, which are incorporated herein by reference. Anadnectins typically consist of a natural fibronectin backbone and multiple targeting domains of specific portions of human fibronectin. The targeting domains can be engineered to enable the anadnectin to specifically recognize the NRP2 polypeptide or an epitope thereof.

[0142] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of anticalins. Anticalins refer to a class of antibody mimetics typically synthesized from human lipocalins, a family of binding proteins having hypervariable loop regions supported by a structurally robust framework. See, for example, U.S. Patent Application Publication No. 2006 / 0058510. Anticalins typically have a size of about 20 kDa. Anticalins can be characterized by a barrel structure formed by eight antiparallel β-strands (stable β-barrel backbone) connected in pairs by four peptide loops and a bound α-helix. In certain embodiments, steric aberrations for achieving specific binding are created in the hypervariable loop regions. See, for example, Skerra, FEBS J. 275:2677-83, 2008, which is incorporated herein by reference.

[0143] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of designed ankyrin repeat proteins (DARPin). DARPin includes a class of non-immunoglobulin proteins that can provide advantages over antibodies for target binding in drug discovery and development. Among other uses, DARPin is ideally suited for in vivo imaging or delivery of toxins or other therapeutic payloads due to their favorable molecular properties, including small size and high stability. Low-cost production in bacteria and rapid generation of many target-specific DARPin make DARPin a useful approach for drug discovery. In addition, DARPin can be easily generated in multispecific formats, offering the possibility of targeting effector DARPin to specific organs or targeting multiple receptors using one molecule composed of several DARPin. See, for example, Stumpp et al., Curr Opin Drug Discov Devel. 10:153-159, 2007; US Patent Application Publication No. 2009 / 0082274; and International Application No. PCT / EP2001 / 10454, which are incorporated herein by reference.

[0144] Also included are heavy-chain dimers such as antibodies from camels and sharks. Camelid and shark antibodies contain a homodimer pair of two chains of V-like and C-like domains (both lacking a light chain). Since the VH region of the heavy-chain dimer IgG in camelids does not make hydrophobic interactions with a light chain, the region in the heavy chain that normally contacts the light chain has been changed to hydrophilic amino acid residues in camelids. The VH domain of the heavy-chain dimer IgG is called the VHH domain. Shark Ig-NAR contains a homodimer of one variable domain (referred to as the V-NAR domain) and five C-like constant domains (C-NAR domains).

[0145] In camels, the diversity of the antibody repertoire is determined by complementarity-determining regions (CDRs) 1, 2, and 3 in the VH or VHH regions. CDR3 in the VHH region of camels is characterized by its relatively long length of an average of 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9): 1129). This is in contrast to the CDR3 regions of antibodies of many other species. For example, the CDR3 of mouse VH has an average of 9 amino acids. A library of antibody variable regions derived from camels maintains the in vivo diversity of camel variable regions and can be prepared by the methods disclosed in, for example, US Patent Application Publication No. 20050037421, published on February 17, 2005.

[0146] In certain embodiments, the antibody or its antigen-binding fragment is humanized. These embodiments refer to chimeric molecules generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species, as well as the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site can include either a complete variable domain fused to a constant domain or only the CDRs grafted into an appropriate framework region in the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals but leaves the possibility of an immune response to the foreign variable region (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al., PNAS USA.86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327, 1988). An example of a method for humanizing an antibody is the method described in US Patent No. 7,462,697.

[0147] Another approach focuses not only on providing human-derived constant regions so as to reform them as close as possible to the human form, but also on modifying the variable regions. The variable regions of both the heavy and light chains vary according to the epitope in question, adjacent to four framework regions (FRs) that are relatively conserved in a given species and presumably provide a scaffold for the CDRs, and are known to contain three complementarity-determining regions (CDRs) that determine binding ability. When a non-human antibody is prepared with respect to a particular epitope, the variable region can be "refashioned" or "humanized" by grafting the CDRs derived from the non-human antibody onto the FRs present in the human antibody to be modified. The application of this approach to various antibodies has been reported by Sato et al., Cancer Res. 53:851-856, 1993; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNAS USA. 88:4181-4185, 1991; Tempest et al., Bio / Technology 9:266-271, 1991; Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA. 89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, the humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more (1, 2, 3, 4, 5, 6) CDRs that have been changed relative to the original antibody, and these are also referred to as one or more CDRs that "derive" from one or more CDRs from the original antibody.

[0148] In certain embodiments, the antibody is a "chimeric" antibody. In this regard, a chimeric antibody is composed of an antigen-binding fragment of an antibody that is operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the Fc domain or heterologous Fc domain is of human origin. In certain embodiments, the Fc domain or heterologous Fc domain is of murine origin. In other embodiments, the heterologous Fc domain may be derived from an Ig class different from the parental antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be composed of CH2 and CH3 domains derived from one or more of different Ig classes. As described above with regard to humanized antibodies, the antigen-binding fragment of a chimeric antibody may contain only one or more of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or may contain the entire variable domain (VL, VH, or both).

[0149] As used herein, a subject "at risk" of developing a disease or adverse reaction may or may not have a detectable disease or symptom of a disease, and may or may not exhibit a detectable disease or symptom of a disease prior to the treatment methods described herein. "At risk" means that the subject has one or more risk factors that are measurable parameters correlated with the development of a disease described herein and known in the art. A subject having one or more of these risk factors has a higher likelihood of developing a disease or adverse reaction than a subject without one or more of these risk factors.

[0150] "Biocompatible" refers to a material or compound that generally does not impair the biological functions of cells or a subject and does not result in any unacceptable toxicity, including allergic and disease states.

[0151] The term "binding" refers to the direct association between two molecules resulting from covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges.

[0152] The term "chemoresistance" refers to the change in the therapeutic sensitivity of a cancer cell population over time after exposure to chemotherapy, including resistance to at least one of a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent and / or a kinase inhibitor. Ultimately, chemoresistance leads to cancer recurrence and / or metastasis, challenging the improvement of the clinical outcome for cancer patients. This remains a major obstacle to long-term successful cancer treatment. For example, approximately 30% of women diagnosed with early-stage breast cancer will ultimately develop resistance and progress to metastatic breast cancer. Molecular mechanisms of chemoresistance include induction of transporter pumps, cancer genes, tumor suppressor genes, mitochondrial changes, DNA repair, autophagy, epithelial-mesenchymal transition (EMT), cancer stem cellness and exosome production. These processes can operate alone or in combination with each other through distinct mechanisms, but ultimately act in concert to prevent cell death in response to specifically targeted chemotherapeutic agents. For example, such processes provide alternative pro-growth signals and / or eliminate or otherwise reduce the apoptotic pathway. Therefore, agents that reduce chemoresistance may find utility in the treatment or reduction of chemoresistant cancers.

[0153] "Coding sequence" means any nucleic acid sequence that contributes to the coding of the polypeptide product of a gene. On the other hand, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the coding of the polypeptide product of a gene.

[0154] Throughout this disclosure, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are understood to mean including the stated step or element or group of steps or elements but not excluding any other step or element or group of steps or elements.

[0155] "Consisting of" means including and limited to all following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or essential and that no other elements are present. "Consisting essentially of" means including any element limited to those that do not interfere with or contribute to the activity or action described in this disclosure for the recited elements following this phrase. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or essential, but that other elements may or may not be present, depending on whether or not they substantially affect the activity or action of the recited elements.

[0156] The terms "effector function" or "ADCC effector function" in the context of an antibody refer to the ability of that antibody to engage other arms of the immune system, including, for example, activation of the classical complement pathway or association with Fc receptors. The complement-dependent pathway is primarily driven by the interaction of C1q with the C1 complex that has the Fc domain of the clustered antibody. Antibody-dependent cell-mediated cytotoxicity (ADCC) is primarily driven by the interaction of Fc receptors (FcRs) on the surface of effector cells (natural killer cells, macrophages, monocytes, and eosinophils) that bind to the Fc region of IgG, which itself binds to the target cell. Fc receptors (FcRs) are important immunoregulatory receptors involved in the antibody-mediated (humoral) immune response to cell effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγR (for IgG), FcεRI (for IgE), FcαRI (for IgA), FcμR (for IgM), and FcδR (for IgD). There are at least three classes of receptors for human IgG found on leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb, and FcγRIIc), and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classified as a high-affinity receptor (KD in the nanomolar concentration range), while FcγRII and FcγRIII are low- to medium-affinity (KD in the micromolar concentration range). Upon Fc binding, signaling pathways are initiated that result in the secretion of various substances such as lysozyme, perforin, granzyme, and tumor necrosis factor, which mediate the destruction of the target cell. The level of ADCC effector function varies for the subtypes of human IgG. This depends on the allotype and specific FcvR, but briefly, the ADCC effector function is "high" for human IgG1 and IgG3 and "low" for IgG2 and IgG4.

[0157] The terms "endotoxin-free" or "substantially endotoxin-free" generally relate to a composition, solvent and / or container that contains, at most, trace amounts (e.g., an amount that has no clinically harmful physiological effect on a subject), preferably undetectable amounts, of endotoxin. Endotoxins are toxins associated with certain microorganisms, typically bacteria, such as Gram-negative bacteria, although endotoxins may also be found in Gram-positive bacteria such as Listeria monocytogenes. The most widespread endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found in the outer membranes of various Gram-negative bacteria, which represent central pathogenic characteristics in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can cause, among other harmful physiological effects, heat, a drop in blood pressure, and activation of inflammation and coagulation.

[0158] Therefore, in pharmaceutical manufacturing, since even small amounts can cause harmful effects in humans, it is often desirable to remove most or all traces of endotoxin from pharmaceutical products and / or drug containers. Since temperatures above 300 °C are typically required to destroy most endotoxins, depyrogenation ovens may be used for this purpose. For example, depending on the main packaging material, such as a syringe or vial, a combination of a glass temperature of 250 °C and a holding time of 30 minutes is often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods of removing endotoxin are contemplated, including, for example, chromatographic and filtration methods described herein and known in the art.

[0159] Endotoxins can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay using horseshoe crab blood is a very sensitive assay for detecting the presence of endotoxins. In this test, very low levels of LPS can cause detectable coagulation of the horseshoe crab lysate due to a powerful enzyme cascade that amplifies this reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). Since it is substantially endotoxin-free, the endotoxin level can be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10 EU / mg of the active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1 - 10 EU.

[0160] The term "epitope" includes any determinant, preferably a polypeptide determinant, that can specifically bind to an immunoglobulin or T cell receptor. An epitope includes the region of an antigen that binds to an antibody. In certain embodiments, an epitope determinant includes a chemically active surface population of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope can be continuous or discontinuous with respect to the primary structure of an antigen, e.g., an NRP2 polypeptide. In certain embodiments, an epitope comprises, consists of, or consists essentially of about, at least about, or at most about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids (i.e., linear epitope) or discontinuous amino acids (i.e., conformational epitope) of a reference sequence (see, e.g., Table N1) or a target molecule described herein.

[0161] An "epitope" includes an antigen or a portion of another macromolecule that can form a binding interaction that interacts with the variable region binding pocket of a binding protein. Such a binding interaction can be referred to as an intermolecular contact with one or more amino acid residues of a CDR. Antigen binding can include a CDR3 or CDR3 pair. An epitope can be a linear peptide sequence (i.e., "continuous") or can be composed of a discontinuous amino acid sequence (i.e., "conformational" or "discontinuous"). A binding protein can recognize one or more amino acid sequences, and thus, an epitope can define two or more distinct amino acid sequences. The epitope recognized by a binding protein can be determined by techniques of peptide mapping and sequence analysis well known to those of skill in the art. A "cryptic epitope" or "cryptic binding site" is an epitope or binding site of a protein sequence that is not exposed or is substantially protected from recognition in an unmodified polypeptide but can be recognized by a binding protein of a denatured or proteolyzed polypeptide. In an unmodified polypeptide structure, an amino acid sequence that is not exposed or is only partially exposed can be a potential cryptic epitope. If an epitope is not exposed or is only partially exposed, as a result, this can be buried within the polypeptide. Candidates for cryptic epitopes can be identified, for example, by examining the three-dimensional structure of an unmodified polypeptide.

[0162] The term "50% effective concentration" or "EC 50 " refers to the concentration of an agent (e.g., an antibody) described herein that induces a response halfway between baseline and maximum after some specified exposure time, and thus, the EC 50 of a stepwise dose-response curve represents the concentration of a compound at which 50% of the maximal effect is observed. EC50 also represents the plasma concentration required to obtain 50% of the maximal effect in vivo. Similarly, "EC 90 " refers to the concentration of an agent or composition at which 90% of the maximal effect is observed. "EC 90" can be calculated from the "EC50" and the Hill slope, or it can be determined directly from the data using common knowledge in the art. In some embodiments, the EC50 of an agent (e.g., an antibody) is less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200 or 500 nM. In some embodiments, the agent has an EC 50 value of about 1 nM or lower.

[0163] "Immune response" means any immunological response where the immune system is the origin, including responses from the cellular and humoral, innate and adaptive immune systems. Exemplary cellular immune cells include, for example, lymphocytes, macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, dendritic cells, mast cells, monocytes and all subsets thereof. Cellular responses include, for example, effector functions, cytokine release, phagocytosis, efferocytosis, translocation, transport, proliferation, differentiation, activation, inhibition, cell-cell interaction, apoptosis, etc. Humoral responses include, for example, responses of IgG, IgM, IgA, IgE, and their corresponding effector functions.

[0164] The "half-life" of an agent such as an antibody can refer to the time it takes for the agent to lose half of its pharmacological, physiological or other activity, compared to such activity at the time of administration to the serum or tissue of an organism, or compared to any other defined time point. "Half-life" can also refer to the time it takes for the amount or concentration of the agent to be reduced to half of the starting amount administered to the serum or tissue of the organism, compared to such amount or concentration at the time of administration to the serum or tissue of an organism, or compared to any other defined time point. The half-life can be measured in serum and / or any one or more selected tissues.

[0165] The terms "modulate" and "alter" typically include, relative to a control, "increase", "enhance" or "stimulate", and "decrease" or "reduce", in a statistically or physiologically significant amount or degree. An "increased", "stimulated" or "enhanced" amount is typically a "statistically significant" amount, and can include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold or higher (e.g., 500, 1000-fold) (including all integers and ranges therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amount produced without the composition (e.g., in the absence of the agent) or by the control composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount, and can include a decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (including all integers and ranges therebetween) of the amount produced without the composition (e.g., in the absence of the agent) or by the control composition. Examples of comparisons and "statistically significant" amounts are described herein.

[0166] The term "migratory cell" refers to a cell that can move from one location to another in response to a stimulus. Exemplary migratory cells include monocytes, natural killer (NK) cells, dendritic cells (immature or mature), myeloid cells, plasmacytoid (also called lymphoid) cells and subsets of dendritic cells including Langerhans cells, macrophages such as histiocytes, Kupffer cells, microglial cells of the CNS, tissue resident macrophages such as alveolar macrophages and peritoneal macrophages, macrophage subtypes such as M0, M1, Mox, M2a, M2b and M2c macrophages, neutrophils, eosinophils, mast cells, basophils, plasma B cells, memory B cells, B cells including B-1 cells and B-2 cells, CD45RO (naive T) cells, CD45RA (memory T) cells, CD4 helper T cells including Th1, Th2 and Tr1 / Th3 cells, CD8 cytotoxic T cells, regulatory T cells, gamma delta T cells, and immune cells such as thymocytes. Additional examples of migratory cells include fibroblasts, fibrocytes, tumor cells and stem cells. The term "cell migration" refers to the movement of migratory cells, and the term "modulation of cell migration" refers to the modulation of the movement of any such migratory cells.

[0167] The terms "polypeptide", "protein" and "peptide" are used interchangeably and mean polymers of amino acids not limited to any particular length. The term "enzyme" includes polypeptide or protein catalysts. These terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation and the addition or deletion of signal sequences. The term "polypeptide" or "protein" means a chain of one or more amino acids, each chain containing amino acids covalently linked by peptide bonds, and said polypeptide or protein can include multiple chains non-covalently and / or covalently linked together by peptide bonds, which is a native protein, i.e., having the sequence of a protein produced by a particular non-recombinant cell that occurs in nature, or a genetically engineered or recombinant cell, and includes a molecule having the amino acid sequence of a native protein or a molecule having one or more amino acid deletions, additions and / or substitutions of the native sequence. In certain embodiments, the polypeptide is a "recombinant" polypeptide produced by a recombinant cell containing one or more recombinant DNA molecules and typically consists of a heterologous polynucleotide sequence or combination of polynucleotide sequences that would not otherwise be found in a cell.

[0168] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA and DNA. This term typically refers to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. This term includes single-stranded and double-stranded forms of DNA. The terms "isolated DNA" and "isolated polynucleotide" and "isolated nucleic acid" refer to a molecule that is isolated without the genomic DNA of a particular species. Thus, an isolated DNA segment encoding a polypeptide contains one or more coding sequences and, further, is a DNA segment that has been substantially isolated from or purified away from the genomic DNA of the species from which the DNA segment is obtained. Non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode polypeptides are also included. Recombinant vectors are also included, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, etc.

[0169] Additional coding or non-coding sequences may be present within the polynucleotides described herein, although they are not required, and the polynucleotides may, although not required, be linked to other molecules and / or support materials. Thus, a polynucleotide or an expressible polynucleotide may be associated with other sequences, such as expression control sequences, regardless of the length of the coding sequence itself.

[0170] "Expression control sequence" includes regulatory sequences of nucleic acids or corresponding amino acids, such as promoters, leaders, enhancers, introns, recognition motifs for RNA or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES) within the sequence, secretion signals, intracellular localization signals, etc., which have the ability to affect transcription or translation of the coding sequence in the host cell, or the intracellular or cellular location. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0171] "Promoter" is a DNA regulatory region that can bind to RNA polymerase in the cell and initiate transcription of the downstream (3' direction) coding sequence. As used herein, the promoter sequence binds to the transcription start site at its 3' end and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level above background. The transcription start site (conveniently defined by mapping with nuclease S1) can be found within the promoter sequence and within the protein binding domain (consensus sequence) responsible for binding of RNA polymerase. Eukaryotic promoters can often, but not always, contain a "TATA" box and a "CAT" box. Prokaryotic promoters contain a Shine-Dalgarno sequence in addition to the -10 and -35 consensus sequences.

[0172] A number of promoters, including constitutive, inducible, and repressible promoters from a variety of different sources, are well known in the art. Representative sources include, for example, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available or can be synthesized based on publicly available sequences online or sequences from depository institutions such as the ATCC and other commercial or personal sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter. Inducible promoters include the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci. (1996) 93 (8): 3346-3351), the T-RExTM system (Invitrogen, Carlsbad, CA), LacSwitch® (Stratagene (San Diego, CA)), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al. Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc. Acid. Res. (2000) 28(23): e99; U.S. Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol. (2005) 308: 123-144), or any promoter known in the art suitable for expression in the desired cell.

[0173] "Expressible polynucleotide" includes cDNA, RNA, mRNA or at least one coding sequence, and optionally at least one expression control sequence, such as other polynucleotides including transcriptional and / or translational regulatory elements, which can express the encoded polypeptide when introduced into cells, such as cells in a subject.

[0174] Various viral vectors that can be used to deliver expressible polynucleotides include adenoviral vectors, herpes viral vectors, vaccinia viral vectors, adeno-associated virus (AAV) vectors and retroviral vectors. In some examples, retroviral vectors are derivatives of murine or avian retroviruses or are lentiviral vectors. Examples of retroviral vectors that can insert a single foreign gene include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous sarcoma virus (RSV). Some additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker, and as a result, transduced cells can be identified and generated. The vector may be made target-specific, for example, by inserting the polypeptide sequence of interest together with another gene encoding a ligand for the receptor of a specific target cell into the viral vector. Retroviral vectors can be made target-specific, for example, by inserting a polynucleotide encoding a protein. Targeting can be achieved, for example, by using an antibody that targets the retroviral vector. Those skilled in the art know or can readily elucidate specific polynucleotide sequences that can be inserted into the retroviral genome to enable target-specific delivery of the retroviral vector without undue experimentation.

[0175] In certain embodiments, the expressible polynucleotide is a modified RNA polynucleotide or a modified mRNA polynucleotide, e.g., an RNA analog that does not occur naturally. In certain embodiments, the modified RNA or mRNA polypeptide comprises one or more modified or non-natural bases, e.g., nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and / or uracil (U). In some embodiments, the modified mRNA comprises one or more modified or non-natural internucleotide linkages. Expressible RNA polynucleotides for delivering the encoded therapeutic polypeptide are described, e.g., in Kormann et al., Nat Biotechnol. 29:154-7, 2011; and U.S. Patent Application Publication Nos. 2015 / 0111248; 2014 / 0243399; 2014 / 0147454; and 2013 / 0245104, which are hereby incorporated by reference in their entirety.

[0176] The term "isolated" polypeptide or protein as referred to in this specification means that the protein of interest (1) does not contain at least some of the other proteins that would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by cells from different species, (4) is separated from at least about 50% of the polynucleotides, lipids, carbohydrates or other substances that naturally associate with it, (5) does not associate (by covalent or non-covalent interactions) with a portion of the protein that naturally associates with the "isolated protein", (6) operably associates (by covalent or non-covalent interactions) with a polypeptide that does not naturally associate, or (7) does not exist in nature. Such isolated proteins can be encoded by genomic DNA, cDNA, mRNA, or other RNA, or can be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins, polypeptides or other contaminating substances found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0177] In certain embodiments, the "purity" of any given agent (e.g., a polypeptide such as an antibody) in a composition can be defined. For example, a particular composition may contain an agent such as a polypeptide agent that is pure at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, and all decimals and ranges therebetween, as measured, for example, but not by way of limitation, by column chromatography in a well-known form frequently used in high performance liquid chromatography (HPLC), biochemistry and analytical chemistry to separate, identify and quantify compounds, on a protein basis or a weight-weight basis.

[0178] "Lipid nanoparticles" or "solid lipid nanoparticles" refer to one or more spherical nanoparticles having an average diameter of about 10 to about 1000 nanometers and containing a solid lipid core matrix capable of solubilizing lipophilic molecules. The lipid core is stabilized by a surfactant (e.g., an emulsifier) and can contain one or more of triglycerides (e.g., tristearin), diglycerides (e.g., glyceryl behenate), monoglycerides (e.g., glyceryl monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate), and combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014; and U.S. Patent Nos. 6,217,912; 6,881,421; 7,402,573; 7,404,969; 7,550,441; 7,727,969; 8,003,621; 8,691,750; 8,871,509; 9,017,726; 9,173,853; 9,220,779; 9,227,917; and 9,278,130, which are hereby incorporated by reference in their entirety. Certain compositions described herein are formulated using one or more lipid nanoparticles.

[0179] The terms "neuropilin 2-related disease" or "NRP2-related disease" refer to diseases and conditions in which the activity, expression, and / or spatial distribution of NRP2 play a role in the pathophysiology of that disease or condition. In some examples, NRP2-related diseases are modulated by the anti-NRP2 antibodies of the present disclosure by altering the interaction between NRP2 and at least one NRP2 ligand, thereby affecting the activity, signaling, expression, and / or spatial distribution of NRP2. Exemplary NRP2-related diseases and conditions include, but are not limited to, cancer, and diseases or conditions associated with cancer, including cancer cell growth, cancer induction, cancer migration, cancer cell adhesion, invasion, chemoresistance, and metastasis. Also included are diseases associated with inflammation, autoimmunity, and related inflammatory diseases, including diseases associated with inappropriate activation or migration of immune cells such as graft-versus-host disease (GVHD). Additional examples include diseases associated with lymphangiogenesis, lymphovenous angiogenesis, and lymphatic vessel injury, including edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability. Also included are diseases associated with allergies / allergic disorders and allergic reactions, including diseases associated with infection, including latent infection, and skin-related neutrophil-mediated diseases such as chronic obstructive pulmonary disease (COPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, systemic lupus erythematosus, rheumatoid arthritis, inflammasome-related diseases, and pyoderma gangrenosum. Additional examples include diseases associated with granulomatous inflammatory diseases, including sarcoidosis and granulomas, and fibrotic diseases, including endometriosis, fibrosis, epithelial-mesenchymal transition (EMT), and wound healing. Also included are diseases associated with inappropriate smooth muscle contractility, smooth muscle compensation and decompensation, vascular smooth muscle cell migration and / or adhesion, and diseases associated with inappropriate autophagy, phagocytosis, and efferocytosis. Also included are diseases associated with inappropriate migratory cell movement as described herein. Additional examples include neurological diseases, including diseases associated with peripheral nervous system remodeling and pain sensation. Also included are diseases associated with bone development and / or bone remodeling.Typically, the term "inappropriate" refers to an activity or characteristic that is associated with or causes a medical condition or disease state.

[0180] The term "reference sequence" generally refers to a nucleic acid coding sequence or amino acid sequence that is being compared to another sequence. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those described by name as well as those described in the tables and sequence listings.

[0181] Certain embodiments include biologically active "variants" and "fragments" of the polypeptides (e.g., antibodies) and polynucleotides described herein. A "variant" contains one or more substitutions, additions, deletions and / or insertions as compared to a reference polypeptide or polynucleotide (see, e.g., the tables and sequence listings). Variant polypeptides or polynucleotides include amino acid or nucleotide sequences having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, similarity or homology to the reference sequences described herein and that substantially maintain the activity of the reference sequences. Also included are sequences that differ from the reference sequences by the addition, deletion, insertion or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides and that substantially maintain the activity of the reference sequences. In certain embodiments, the addition or deletion includes C-terminal and / or N-terminal additions and / or deletions.

[0182] As used herein, the term "sequence identity" or terms that include, for example, "a sequence that is 50% identical" refer to the degree to which sequences are identical nucleotide by nucleotide or amino acid by amino acid over a window of comparison. Thus, "percent sequence identity" can be calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., window size), and multiplying the result by 100 to obtain the percent sequence identity. Optimal alignment of sequences for aligning the comparison window may be performed by computerized execution of an algorithm (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA), or by verification and best alignment created by any of a variety of selected methods (i.e., resulting in the highest percentage of homology over the comparison window). For example, reference may be made to the programs of the BLAST family, as disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0183] The term "solubility" refers to the property of dissolving a drug (e.g., an antibody) provided herein in a liquid solvent to form a homogeneous solution. Solubility is typically expressed as a concentration by any of the descriptions of the mass of solute per unit volume of solvent (g of solute per kg of solvent, g per 1 dL (100 mL), mg / ml, etc.), molar concentration, molality, mole fraction, or other similar concentrations. The maximum equilibrium amount of solute that can be dissolved per amount of solvent is the solubility of the solute in the solvent under specific conditions including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH or other pH values, e.g., pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5 - 8). In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaPO4). In specific embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO4). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25 °C) or about body temperature (37 °C). In certain embodiments, the drug has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml at room temperature or 37 °C.

[0184] "Subject" or "subject in need thereof" or "patient" or "patient in need thereof" includes mammalian subjects such as human subjects.

[0185] "Substantial" or "substantially" means almost entirely or almost completely, e.g., 95%, 96%, 97%, 98%, 99%, or higher of a given amount.

[0186] "Statistically significant" means that the result is unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. A commonly used measure of significance is the p-value, which is the frequency or probability with which the observed event occurs if the null hypothesis is true. If the obtained p-value is less than the significance level, the null hypothesis is rejected as a result. In a simple example, the significance level is defined as a p-value of 0.05 or lower.

[0187] "Treatment response" refers to an improvement in symptoms (whether persistent or not) based on the administration of one or more therapeutic agents.

[0188] As used herein, the terms "therapeutically effective amount", "therapeutic dose", "prophylactically effective amount" or "diagnostically effective amount" refer to the amount of an agent (e.g., an anti-NRP2 antibody, an immunotherapeutic agent) required to induce a desired biological response after administration.

[0189] As used herein, "treatment" of a subject (e.g., a mammal such as a human) or cell is any type of intervention used to attempt to alter the natural course of an individual or cell. Treatments include, but are not limited to, the administration of a pharmaceutical composition, which can be done prophylactically or either after the onset of a pathological event or after contact with a pathogen. "Preventive" treatments are also included, which can be directed at reducing the rate of progression of a disease or condition being treated, delaying the onset of a disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily imply complete eradication, cure or prevention of a disease or condition or their associated symptoms.

[0190] The term "wild type" refers to a gene or gene product (e.g., a polypeptide) that is most frequently observed in a population and, thus, to which the "normal" or "wild type" form of the gene is arbitrarily designed.

[0191] Each embodiment of this specification should be applicable to all other embodiments unless otherwise explicitly stated. Anti-NRP2 antibody

[0192] Certain embodiments include antibodies and antigen-binding fragments thereof that specifically bind to human neuropilin 2 (NRP2) polypeptides. In some embodiments, at least one antibody or antigen-binding fragment thereof modulates (e.g., inhibits) binding to at least one NRP2 ligand, such as the human histidyl tRNA synthetase (HRS) polypeptide of the human NRP2 polypeptide or other NRP2 ligands.

[0193] In certain embodiments, the antibody or antigen-binding fragment thereof has a heavy chain variable region (V H CDR1, V H CDR2, and V H CDR3 sequences, and a light chain variable region (V H ) sequence that includes complementarity-determining regions V L CDR1, V L CDR2, and V L CDR3 sequences, or is characterized by or includes them. Exemplary V L , V H , V H CDR1, V H CDR2, V H CDR3, V L , V L CDR1, V L CDR2, and V L CDR3 sequences are provided in Tables A1, A2, and A3 below.

Table A1-1

Table A1-2

Table A2-1

Table A2-2

Table A2-3

[0194] Thus, in certain embodiments, an antibody or antigen-binding fragment thereof comprises complementary determining regions V selected from Table A1 that specifically bind to a human NRP2 polypeptide (e.g., selected from Table N1), H CDR1, V H CDR2, and V H CDR3 sequences, and variants thereof, a heavy chain variable region (V H H); and complementary determining regions V selected from Table A1 that specifically bind to a human NRP2 polypeptide (e.g., selected from Table N1), L CDR1, V L CDR2, and V L CDR3 sequences, and variants thereof, a light chain variable region (V L L) sequence.

[0195] In certain embodiments, the CDR sequences are as follows: V H CDR1, V H CDR2, and V H CDR3 sequences each comprise SEQ ID NOs: 1-3 and variants thereof, and V L CDR1, V L CDR2, and V L CDR3 sequences each comprise SEQ ID NOs: 4-6 and variants thereof; or V H CDR1, V H CDR2, and V H CDR3 sequences each comprise SEQ ID NOs: 7-9 and variants thereof, and V L CDR1, V L CDR2, and V L CDR3 sequences each comprise SEQ ID NOs: 10-12 and variants thereof; V H CDR1, V H CDR2 and V H The CDR3 sequences each include SEQ ID NOs: 13-15 and their variants, V L CDR1, V L CDR2 and V L Do the CDR3 sequences each include SEQ ID NOs: 16-18 and their variants? V H CDR1, V H CDR2 and V H The CDR3 sequences each include SEQ ID NOs: 19-21 and their variants, V L CDR1, V L CDR2 and V L Do the CDR3 sequences each include SEQ ID NOs: 22-24 and their variants? V H CDR1, V H CDR2 and V H The CDR3 sequences each include SEQ ID NOs: 25-27 and their variants, V L CDR1, V L CDR2 and V L Do the CDR3 sequences each include SEQ ID NOs: 28-30 and their variants? V H CDR1, V H CDR2 and V H The CDR3 sequences each include SEQ ID NOs: 31-33 and their variants, V L CDR1, V L CDR2 and V L Do the CDR3 sequences each include SEQ ID NOs: 34-36 and their variants? V H CDR1, V H CDR2 and V H The CDR3 sequences each include SEQ ID NOs: 34-39 and their variants, V L CDR1, V L CDR2 and V L Do the CDR3 sequences each include SEQ ID NOs: 40-42 and their variants? V H CDR1, V H CDR2 and V H The CDR3 sequences each include SEQ ID NOs: 57-59 and their variants, V L CDR1, V L CDR2 and V L The CDR3 sequences each include SEQ ID NOs: 60-62 and their variants; or V H CDR1, V H CDR2 and V H The CDR3 sequences each include SEQ ID NOs: 63-65 and their variants, V L CDR1, V L CDR2 and V L The CDR3 sequences each include SEQ ID NOs: 66-68 and their variants.

[0196] Those variants also include affinity matured variants that bind to human NRP2. For example, the variant has one or more CDR regions, such as one or more V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and / or V L has 1, 2, 3, 4, 5 or 6 changes in the CDR3 sequence. Exemplary "changes" include amino acid substitutions, additions and deletions.

[0197] In certain embodiments, V H the sequence is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to a sequence selected from Table A2. For example, V H sequences include those having 1, 2, 3, 4 or 5 changes in one or more framework regions.

[0198] In some embodiments, V L the sequence is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to a sequence selected from Table A2. For example, VL The array includes those having 1, 2, 3, 4 or 5 modifications in one or more framework regions.

[0199] In some embodiments, the V of the antibody or antigen-binding fragment H and V L arrays are as follows: V H The array includes SEQ ID NO: 43 and the V L Does the array include SEQ ID NO: 44; V H The array includes SEQ ID NO: 45 and the V L Does the array include SEQ ID NO: 46; V H The array includes SEQ ID NO: 47 and the V L Does the array include SEQ ID NO: 48; V H The array includes SEQ ID NO: 49 and the V L Does the array include SEQ ID NO: 50; V H The array includes SEQ ID NO: 51 and the V L Does the array include SEQ ID NO: 52; V H The array includes SEQ ID NO: 53 and the V L Does the array include SEQ ID NO: 54; V H The array includes SEQ ID NO: 55 and the V L Does the array include SEQ ID NO: 56; V H The array includes SEQ ID NO: 69 and the V L Does the array include SEQ ID NO: 70; V H The array includes SEQ ID NO: 71 and the V L Does the array include SEQ ID NO: 72; or V H The array includes SEQ ID NO: 73 and the V L The array includes SEQ ID NO: 74.

[0200] Those variants, e.g., variants having 1, 2, 3, 4 or 5 changes in one or more framework regions are also included. Exemplary "changes" include amino acid substitutions, additions and deletions.

[0201] Table A3 below summarizes the CDRs of exemplary redundant antibodies containing exemplary consensus CDR sequences and provides the amino acid codes therefor.

Table A3-1

Table A3-2

[0202] Thus, in certain embodiments, at least one anti-NRP2 antibody or antigen-binding fragment thereof comprises a CDR sequence, e.g., a CDR1 consensus sequence from Table A3.

[0203] Neuropilin-2 is a cell surface receptor protein that modulates a wide range of cellular functions by virtue of its role as an essential cell surface receptor and co-receptor for various ligands (see, e.g., Guo and Vander Kooi, J. Cell. Biol. 290 No 49: 29120-29126, 2015). For example, it functions during epithelial-mesenchymal transition (EMT) by promoting, e.g., TGF-β1-mediated EMT in colorectal cancer cells and other cancer cells (see, e.g., Grandclement et al., PLoS ONE 6(7) e20444, 2011), and by promoting fibrosis by mediating EMT or endo-EMT in fibroblasts, myofibroblasts and endothelial cells (see, e.g., Pardali et al., Int. J. Mol. Sci. 18:2157, 2017).

[0204] Neuropilin-2 expression promotes lymphangiogenesis (see, e.g., Doci et al., Cancer Res. 75:2937-2948, 2015), and single nucleotide polymorphisms (SNPs) in NRP2 are associated with lymphedema (see, e.g., Miaskowski et al., PLoS ONE 8(4) e60164, 2013). NRP2 also regulates smooth muscle contractility (see, e.g., Bielenberg et al., Amer. J. Path. 181:548-559, 2012), and, for example, regulates autophagy in cancer (see, e.g., Stanton et al., Cancer Res. 73:160-171, 2013), contributes to tumor initiation, survival and metastasis (see, e.g., Goel et al., EMBO Mol. Med. 5:488-508, 2013; and Samuel et al., PLoS ONE 6(10) e23208, 2011), and regulates activation and migration of immune cells (see, e.g., Mendes-da-Cruz et al., PLoS ONE 9(7) e103405, 2014). Neuropilin is also a multifunctional coreceptor involved in tumor initiation, growth, metastasis and immunity (see, e.g., Prud'homme et al., Oncotarget 3:921-939, 2012).

[0205] Neuropilin-2 is expressed in a variety of cells of the immune system, including lymphoid cells such as B and T cells, and myeloid cells such as basophils, eosinophils, monocytes, dendritic cells, neutrophils and tissue-specific macrophages, such as alveolar macrophages. It is also expressed in endothelial and epithelial cells in the lung and other tissues, as well as in muscle cells (see, for example, Bielenberg et al., Amer. J. Path. 181:548-559, 2012; Aung, et al., PLoS ONE 11(2) e0147358, 2016; Schellenburg et al., Mol. Imm 90:239-244, 2017; and Wild et al., Int. J. Exp. Path. 93:81-103, 2012).

[0206] Neuropilin-2 also plays an important role in endosome development, for example, by regulating late endosome maturation, and important aspects of phagocytosis and efferocytosis contribute to the elimination of the infected area and apoptotic cells, respectively (see, for example, Diaz-Vera et al., J. Cell. Sci. 130:697-711, 2017; Dutta et al., Cancer Res. 76:418-428, 2016).

[0207] Neuropilin-2 is known to be an important factor in the pathophysiology of many diseases (e.g., "NRP2-related diseases") and interacts with a wide range of soluble ligands, including semaphorin 3F, VEGF-C and D, and TGF-beta (see, e.g., Tables N2 and N3), as well as a series of cell receptors and cofactors (see, e.g., FIGS. 1A-1B and 2). NRP2 is also polysialylated in dendritic cells and actively interacts with chemokine CCL21 to mediate the migration of immune cells, for which single nucleotide polymorphisms associated with ILD and RA have been described (see, e.g., Rey-Gallardo et al., Glycobiology 20:1139-1146, 2010; Stahl et al., Nat. Genet. 42:508-514, 2013; and Miller et al., Arthritis Rheum. 65:3239-3247). In addition, a soluble circulating form of NRP-2 is known (see, e.g., Parker et al., Structure 23(4) 677-687, 2015), and in-house studies have confirmed the presence of a circulating complex of HRS polypeptide and NRP-2 polypeptide in circulation. Thus, considering the central role played by NRP2 in the pathophysiology of a wide range of diseases, this is evidence that modulation of the interaction between NRP2 and NRP2 ligands (e.g., NRP2 ligands from Tables N2 and N3), as well as their interaction with antibodies to NRP2 that selectively alter the corresponding biological activity, offers broad potential for the treatment of diseases, including NRP2-related diseases.

[0208] NRP2 is a single transmembrane receptor with a dominant extracellular region containing two CUB domains (a1 / a2 composite domain), two factor V / factor VIII homology domains (b1 / b2 composite domain), a MAM domain (c domain) (see FIGS. 1A-1B), and a short juxtamembrane region that connects the c domain to the transmembrane domain (transversing the plasma membrane). The a1a2 composite domain interacts with the sema region of semaphorins, and the b1 domain interacts with the PSI domain and Ig-like domain of semaphorins. NRP2 has a higher affinity for SEMA3F and 3G. In contrast, SEMA3A, 3B, and 3E preferentially interact with NRP1. Both NRP1 and NRP2 have a similar affinity for SEMA3C. The b1b2 composite domain interacts with several growth factors including VEGF C and D, heparin-binding domains, placental growth factor (PIGF)-2, fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF)-beta (see, e.g., Prud'homme et al., Oncotarget. 3:921-939, 2012). NRP2 also interacts with various growth factor-specific receptors, and the interaction with these receptors occurs independently of binding to SEMAs. In this context, integrins, as well as growth factor receptors such as VEGF receptor, TGF-beta receptor, c-Met, EGFR, FGFR, PDGFR, are shown to interact with NRP and generally increase the affinity of their respective ligands for the receptor and modulate downstream signaling. The domain of the c domain (Mam) is not thought to be directly required for ligand binding but can affect ligand specificity, receptor signaling, and NRP2 dimerization. The juxtamembrane region varies significantly between the NRP2a and NRP2b isoforms and can also affect ligand specificity, dimerization, and signaling.

[0209] Therefore, anti-NRP2 antibodies and antigen-binding fragments thereof that bind to the a1 and / or a2 domains of NRP2 have the potential to selectively modulate semaphorin binding. Similarly, anti-NRP2 antibodies and antigen-binding fragments thereof that bind to the b1 domain have the potential to modulate both semaphorin and VEGF and growth factor binding, and anti-NRP2 antibodies that bind to the b2 domain have the potential to selectively modulate VEGF and growth factor binding. Antibodies and antigen-binding fragments thereof that bind to the c domain do not have the potential to directly affect NRP2 ligand binding, but have the potential to modulate NRP2 downstream signaling, for example, by modulating (e.g., promoting or enhancing) NRP2 receptor dimerization.

[0210] Anti-NRP2 antibodies and antigen-binding fragments thereof that promote the homodimerization of the NRP2 receptor can modulate NRP2 receptor activity and provide agonist or antagonist antibodies depending on the nature of the binding site. Such antibodies and antigen-binding fragments thereof can modulate (e.g., enhance) the activity of NRP2 ligands even if they do not directly modulate ligand binding. Additional diversity in the functional effects of specific anti-NRP2 antibodies can be predicted based on their binding modes and as a result of steric effects, and they can indirectly affect ligand binding.

[0211] NRP2 can form homodimers and heterodimers and is highly glycosylated. NRP2 has different splice variants that are approximately 551 - 926 amino acids in length. Two major variants of NRP2 are classified as NRP2a and NRP2b. They differ in their intracellular C - terminal portions (Figures 1A - 1B), where, for NRP2a, the C - terminal domain contains 42 amino acids with a C - terminal SEA amino acid sequence and a PDZ - binding domain. In contrast, NRP2b contains a 46 - amino - acid C - terminal domain that shares approximately 11% sequence homology with the intracellular, juxtamembrane, and transmembrane sequences of NRP2a. Additional splicing can occur between the MAM domain and the transmembrane domain, and an additional 5 amino acids (GENFK) can be added to either the NRP2a or NRP2b form. These variants are named based on the number of additional amino acids added by alternative splicing. Thus, two variants of NRP2a are named NRP2a(17) (or variant 1) and NRP2a(22) (or variant 2), and two transmembrane variants of NRP2b are named NRP2b(0) (or variant 4) and NRP2b(5) (or variant 5). In addition, a soluble form called sNRP2b (or variant 6) can be created. Exemplary NRP2 polypeptide sequences, including the mature (after cleavage of the N - terminal signal peptide) and precursor forms of various isoforms of NRP2, are provided in Table N1 below.

Table N1 - 1

Table N1 - 2

Table N1 - 3

Table N1 - 4

Table N1-5

Table N1-6

Table N1-7

Table N1-8

Table N1-9

[0212] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide selected from Table N1 or to a human NRP2 polypeptide. In some embodiments, the antibody or antigen-binding fragment thereof binds to the human NRP2 polypeptide with an affinity of about 10 pM to about 500 pM or about 10 pM to about 50 nM, or about, at least about, or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or, optionally, in the range of about 10 pM to about 500 pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

[0213] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in at least one neuropilin domain. Exemplary neuropilin domains include the neuropilin a1 domain, neuropilin a2 domain, neuropilin b1 domain, neuropilin b2 domain, neuropilin c domain, neuropilin a1 / a2 composite domain, neuropilin b1 / b2 composite domain, neuropilin a2 / b1 composite domain, neuropilin b2 / c composite domain, neuropilin a2 / b1 / b2 composite domain, neuropilin a2 / b1 / b2 / c composite domain, neuropilin a1 / a2 / b1 composite domain, neuropilin a1 / a2 / b1 / b2 composite domain, neuropilin a1 / a2 / b1 / b2 / c composite domain, a1 / a2 / b1 / b2 / c / near-membrane composite domain, and one or more of the neuropilin b1 / b2 / c composite domain (see Table N1 for domain residues). In a specific embodiment, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin b1 domain, neuropilin b2 domain, and / or neuropilin b1 / b2 composite domain (see Table N1).In certain embodiments, the antibody or antigen-binding fragment thereof binds to at least one domain (or at least one epitope therein) with an affinity in the range of about 10 pM to about 500 pM or about 10 pM to about 50 nM, or about, at least about, or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or, optionally, in the range of about 10 pM to about 500 pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

[0214] In some embodiments, at least one antibody or antigen-binding fragment thereof binds to at least one epitope in the neuropilin a1 domain, neuropilin a2 domain and / or neuropilin a1a2 composite domain, and the adjacent linker region, for example, residues 20 - 148, 30 - 141, 40 - 141, 50 - 141, 60 - 141, 70 - 141, 80 - 141, 90 - 141, 100 - 141, 110 - 141, 120 - 141, 130 - 141; 20 - 130, 20 - 120, 20 - 110, 20 - 100, 20 - 90, 20 - 80, 20 - 70, 20 - 60, 20 - 50, 20 - 40, or 20 - 30 as defined by, for example, (neuropilin a1 domain) the human NRP2 precursor sequence (see Table N1); or near residues 142 - 280, 150 - 265, 160 - 265, 170 - 265, 180 - 265, 190 - 265, 200 - 265, 210 - 265, 220 - 265, 230 - 265, 240 - 265, 250 - 265, 260 - 265, 141 - 270, 141 - 260, 141 - 250, 141 - 240, 141 - 230, 141 - 220, 141 - 210, 141 - 200, 141 - 190, 141 - 180, 141 - 170, 141 - 160, 141 - 150, 200 - 250, 210 - 250, 220 - 250, 230 - 250, 200 - 240, 210 - 240, 220 - 240, 230 - 240, 227 - 247, 228 - 247, 229 - 247, 230 - 247, 231 - 247, 232 - 247, 233 - 247, 234 - 247, 235 - 247, 236 - 247; 227 - 246, 227 - 245, 227 - 244, 227 - 243, 227 - 242, 227 - 241, 227 - 240, 227 - 239, 227 - 238; 235 - 240, 236 - 239, 236 - 238, or near residue 237 as defined by, for example, (neuropilin a2 domain) the human NRP2 precursor sequence (see Table N1).Alternatively, for example, specifically binds in the vicinity of residues 20 - 280, 30 - 280, 40 - 280, 50 - 280, 60 - 280, 70 - 280, 80 - 280, 90 - 280, 100 - 280, 110 - 280, 120 - 280, 130 - 280, 140 - 280, 150 - 280, 160 - 280, 170 - 280, 180 - 280, 190 - 280, 200 - 280, 210 - 280, 220 - 280, 230 - 280, 240 - 280, 260 - 280, 270 - 280, 20 - 270, 20 - 260, 20 - 250, 20 - 240, 20 - 230, 20 - 220, 20 - 210, 20 - 200, 20 - 190, 20 - 180, 20 - 170, 20 - 160, 20 - 150, 20 - 140, 20 - 130, 20 - 120, 20 - 110, 20 - 100, 20 - 90, 20 - 80, 20 - 70, 20 - 60, 20 - 50, 20 - 40, or 20 - 30, as defined by, for example, the (composite a1a2 domain) human NRP2 precursor sequence (see Table N1).;

[0215] In certain embodiments, at least one antibody or antigen-binding fragment thereof binds to at least one epitope in the neuropilin b1 domain, neuropilin b2 domain, and / or neuropilin b1 / b2 composite domain, and the adjacent linker region, for example, residues 266-426, 280-426, 290-426, 299-420, 300-426, 310-426, 320-426, 330-426, 340-426, 350-426, 360-426, 370-426, 380-426, 390-426, 400-426, 410-426, 420-426, 280-420, 280-410, 280-400, 280-390, 280-380, 280-370, 280-360, 280-350, 280-340, 280-330, 280-320, 280-310, 280-300, or 280-290, as defined by the (neuropilin b1 domain) human NRP2 precursor sequence (see Table N1), and one, two, or three of residues Y299, N354, and / or S416, which are discontinuous epitopes; residues 438-591, 450-591, 460-591, 470-591, 480-591, 490-591, 500-591, 510-591, 520-591, 530-591, 540-591, 550-591, 560-591, 570-591, 580-591, 438-590, 438-580, 438-570, 438-560, 438-550, 438-540, 438-530, 438-520, 438-510, 438-500, 438-490, 438-480, 438-470, 438-460, 438-450, as defined by the (neuropilin b2 domain) human NRP2 precursor sequence (see Table N1);Or specifically binds in the vicinity of residues 266 - 591, 276 - 591, 286 - 591, 296 - 591, 306 - 591, 316 - 591, 326 - 591, 336 - 591, 346 - 591, 356 - 591, 366 - 591, 376 - 591, 386 - 591, 396 - 591, 406 - 591, 416 - 591, 426 - 591, 436 - 591, 446 - 591, 456 - 591, 466 - 591, 476 - 591, 486 - 591, 498 - 591, 508 - 591, 518 - 591, 528 - 591, 538 - 591, 548 - 591, 558 - 591, 568 - 591, 578 - 591, 588 - 591, 266 - 581, 266 - 571, 266 - 561, 266 - 551, 266 - 541, 266 - 531, 266 - 521, 266 - 511, 266 - 501, 266 - 491, 266 - 481, 266 - 471, 266 - 461, 266 - 451, 266 - 441, 266 - 431, 266 - 421, 266 - 411, 266 - 401, 266 - 391, 266 - 381, 266 - 371, 266 - 361, 266 - 351, 266 - 341, 266 - 331, 266 - 321, 266 - 311, 266 - 301, 266 - 291, 266 - 281, or 266 - 271, as defined by the (neuropilin b1 / b2 composite domain) human NRP2 precursor sequence (see Table N1).;

[0216] In some embodiments, at least one antibody or antigen-binding fragment thereof binds to at least one epitope in the neuropilin a2 / b1 complex domain and / or neuropilin b2c complex domain, and the adjacent linker region, for example, residues 149 - 437, 159 - 426, 169 - 426, 179 - 426, 189 - 426, 199 - 426, 209 - 426, 219 - 426, 229 - 426, 239 - 426, 249 - 426, 259 - 426, 269 - 426, 279 - 426, 289 - 426, 299 - 426, 309 - 426, 319 - 426, 329 - 426, 339 - 426, 349 - 426, 359 - 426, 369 - 426, 379 - 426, 389 - 426, 399 - 426, 409 - 426, 419 - 426, 149 - 436, 149 - 426, 149 - 416, 149 - 406, 149 - 396, 149 - 386, 149 - 376, 149 - 366, 149 - 356, 149 - 346, 149 - 336, 149 - 326, 149 - 316, 149 - 306, 149 - 296, 149 - 286, 149 - 276, 149 - 266, 149 - 256, 149 - 246, 149 - 236, 149 - 226, 149 - 216, 149 - 206, 149 - 196, 146 - 186, 146 - 176, 146 - 166, or in the vicinity of 146 - 155, as defined by, for example, the (neuropilin a2b1 complex domain) human NRP2 precursor sequence (see Table N1).Alternatively, for example, specifically binds in the vicinity of residues 438 - 794, 448 - 794, 458 - 794, 468 - 794, 478 - 794, 487 - 794, 497 - 794, 507 - 794, 517 - 794, 527 - 794, 537 - 794, 547 - 794, 557 - 794, 567 - 794, 587 - 794, 597 - 794, 607 - 794, 617 - 794, 627 - 794, 637 - 794, 647 - 794, 657 - 794, 667 - 794, 677 - 794, 687 - 794, 697 - 794, 707 - 794, 717 - 794, 727 - 794, 737 - 794, 747 - 794, 757 - 794, 767 - 794, 777 - 794, 787 - 794, 427 - 794, 438 - 784, 438 - 774, 438 - 764, 438 - 754, 438 - 744, 438 - 734, 438 - 728, 438 - 714, 438 - 704, 438 - 694, 438 - 684, 438 - 674, 438 - 664, 438 - 654, 438 - 644, 438 - 634, 438 - 624, 438 - 614, 438 - 604, 438 - 596, 438 - 586, 438 - 576, 438 - 566, 438 - 556, 438 - 546, 438 - 536, 438 - 526, 438 - 516, 438 - 506, 438 - 494, 438 - 484, 438 - 474, 438 - 464, 438 - 454, 438 - 444, as defined by, for example, the (neuropilin b2c complex domain) human NRP2 precursor sequence (see Table N1).;

[0217] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin c domain and adjacent linker region, for example, residues 591-794, 600-794, 610-794, 620-794, 630-794, 640-794, 650-794, 660-794, 670-794, 680-794, 690-794, 700-794, 710-794, 720-794, 730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 591-790, 591-780, 591-770, 591-760, 591-750, 591-740, 591-730, 591-720, 591-710, 591-700, 591-690, 591-680, 591-670, 591-660, 591-650, 591-640, 591-630, 591-620, 591-610, or 591-600, as defined by the human NRP2 precursor sequence (see Table N1), in the vicinity of.

[0218] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin b1 / b2 / c complex domain and adjacent linker region, e.g., residues 276 - 794, 286 - 794, 296 - 794, 306 - 794, 316 - 794, 326 - 794, 336 - 794, 346 - 794, 356 - 794, 366 - 794, 376 - 794, 387 - 794, 396 - 794, 406 - 794, 416 - 794, 426 - 794, 436 - 794, 446 - 794, 456 - 794, 466 - 794, 476 - 794, 486 - 794, 496 - 794, 506 - 794, 516 - 794, 526 - 794, 536 - 794, 546 - 794, 556 - 794, 566 - 794, 576 - 794, 586 - 794, 596 - 794, 606 - 794, 616 - 794, 626 - 794, 636 - 794, 646 - 794, 656 - 794, 666 - 794, 676 - 794, 686 - 794, 696 - 794, 706 - 794, 716 - 794, 726 - 794, 736 - 794, 746 - 794, 756 - 794, 766 - 794, 776 - 794, 786 - 794, 266 - 794, 276 - 784, 276 - 774, 276 - 764, 276 - 754, 276 - 744, 276 - 734, 276 - 724, 276 - 714, 276 - 704, 276 - 694, 276 - 684, 276 - 674, 276 - 664, 276 - 654, 276 - 644, 276 - 634, 276 - 624, 276 - 614, 276 - 604, 276 - 594, 276 - 584, 276 - 574, 276 - 564, 276 - 554, 276 - 544, 276 - 534, 276 - 524, 276 - 514, 276 - 504, 276 - 496, as defined by the human NRP2 precursor sequence (see Table N1).

[0219] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin C / membrane-proximal complex domain.

[0220] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin membrane-proximal domain (variant 1) of NRP2a. In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin membrane-proximal domain (variant 2) of NRP2a. In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin membrane-proximal domain (variant 3) of NRP2a. In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin membrane-proximal domain (variant 4) of NRP2b. In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin membrane-proximal domain (variant 5) of NRP2b.

[0221] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to a conformational epitope composed of two or more discontinuous epitope regions. In some embodiments, at least one antibody or antigen-binding fragment thereof is (a) a first epitope region within the a1 domain and a second epitope region within the a2 domain of the human NPR2 polypeptide; (b) a first epitope region within the a1 domain and a second epitope region within the b1 domain of the human NPR2 polypeptide; (c) a first epitope region within the a1 domain and a second epitope region within the b2 domain of the human NPR2 polypeptide; (d) a first epitope region within the a1 domain and a second epitope region within the c domain of the human NPR2 polypeptide; (e) a first epitope region within the a1 domain and a second epitope region within the membrane-proximal domain of the human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; (f) The first epitope region within the a2 domain and the second epitope region within the b1 domain of the human NPR2 polypeptide; (g) The first epitope region within the a2 domain and the second epitope region within the b2 domain of the human NPR2 polypeptide; (h) The first epitope region within the a2 domain and the second epitope region within the c domain of the human NPR2 polypeptide; (i) The first epitope region within the a2 domain and the second epitope region within the membrane-proximal domain of the human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; (j) The first epitope region within the b1 domain and the second epitope region within the b2 domain of the human NPR2 polypeptide; (k) The first epitope region within the b1 domain and the second epitope region within the c domain of the human NPR2 polypeptide; (l) The first epitope region within the b1 domain and the second epitope region within the membrane-proximal domain of the human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; (m) The first epitope region within the b2 domain and the second epitope region within the c domain of the human NPR2 polypeptide; (n) The first epitope region within the b2 domain and the second epitope region within the membrane-proximal domain of the human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; or (o) The first epitope region within the c domain and the second epitope region within the membrane-proximal domain of the human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5 specifically binds to a conformational epitope comprising or consisting of these.

[0222] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to at least one epitope within a region of any molecule that binds to or interacts with at least one "NRP2 ligand", or interacts with or binds reversibly to human NRP2, including a human NRP2 polypeptide and any one or more variants of human NRP2. General examples of "NRP2 ligands" include polypeptides such as the HRS polypeptide, soluble ligands, receptors (e.g., cell surface receptors), and growth factors, growth factor receptors, etc., and specific examples of NRP2 ligands are detailed herein. In some embodiments, at least one antibody or antigen-binding fragment thereof modulates (e.g., antagonizes, inhibits, agonizes, enhances) the binding of a human NRP2 polypeptide to at least one "NRP2 ligand".

[0223] As described above, in certain embodiments, at least one NRP2 ligand is the HRS polypeptide. Thus, in certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to at least one epitope within a region of a human NRP2 polypeptide that binds to or interacts with at least one human HRS polypeptide, thereby modulating the binding of the human NRP2 polypeptide to the human HRS polypeptide. Exemplary HRS polypeptides are provided in Table H1 below.

Table H1-1

Table H1-2

Table H1-3

Table H1-4

[0224] Thus, in certain embodiments, at least one NRP2 ligand is selected from Table H1, and the anti-NRP2 antibody or antigen-binding fragment thereof modulates (e.g., inhibits) the binding of a human NRP2 polypeptide (e.g., a human NRP2 polypeptide selected from Table N1) to a human HRS polypeptide selected from Table H1. In some embodiments, the anti-NRP2 antibody or antigen-binding fragment specifically binds to the HRS polypeptide interaction region of the NRP2 polypeptide, and in some instances, mimics, e.g., as an agonist antibody, one or more signaling activities of an HRS polypeptide that binds to the NRP2 polypeptide. The "HRS polypeptide interaction region" includes, for example, a ligand binding site for different NRP2 ligands (examples of which are provided herein), a dimerization domain, a protein-protein interaction domain, or an allosterically sensitive site within the NRP2 polypeptide for modulating the activity of the NRP2 polypeptide, which interacts with a region or domain of a human HRS polypeptide.

[0225] In certain embodiments, the antibody or antigen-binding fragment thereof is a "blocking antibody", which completely or substantially inhibits the binding between a human NRP2 polypeptide (e.g., selected from Table N1) and an NRP2 ligand such as a human HRS polypeptide (e.g., selected from Table H1) or another NRP2 ligand (e.g., selected from Table N2 or Table N3). In some embodiments, the "blocking antibody" inhibits about or at least about 80-100% (e.g., 80, 85, 90, 95 or 100%) of the theoretical maximum binding between the NRP2 polypeptide and the NRP2 ligand (e.g., HRS polypeptide) after pre-incubation of the "blocking antibody" with the NRP2 polypeptide at substantially stoichiometric equivalents. As used herein, "stoichiometric equivalents" refers to a situation in which, in a given formula or reaction, the number of moles of one substance (e.g., an anti-NRP2 antibody) is equal to or substantially equal to the number of moles of at least one other substance (e.g., an NRP2 polypeptide).

[0226] In certain embodiments, the antibody or antigen-binding fragment thereof is a "partial blocking antibody" which at least partially inhibits, but not completely, the binding between a human NRP2 polypeptide (e.g., selected from Table N1), and an NRP2 ligand such as a human HRS polypeptide (e.g., selected from Table H1) or other NRP2 ligands (e.g., selected from Table N2 or Table N3). In some embodiments, a "partial blocking antibody" inhibits about or at least about 20-80% (e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80%) of the theoretical maximum binding between an NRP2 polypeptide and an NRP2 ligand (e.g., HRS polypeptide) after pre-incubation of the "partial blocking antibody" with the NRP2 polypeptide in stoichiometric amounts.

[0227] In a specific embodiment, at least one antibody or antigen-binding fragment thereof specifically inhibits or otherwise reduces the binding between a human NRP2 polypeptide and an HRS polypeptide splice variant selected from Table H1, e.g., HisRS N1 、HisRS N2 、HisRS N3 、HisRS N4 (SV9), HisRS N5 、HisRS C1 、HisRS C2 、HisRS C3 、HisRS C4 、HisRS C5 、HisRS C6 、HisRS C7 、HisRS C8 (SV11) and HisRS C9 (SV14).

[0228] As described above, NRP2 interacts with multiple NRP2 ligands other than HRS, which mediate downstream signaling events. Additional examples of NRP2 ligands are provided in Tables N2 and N3 below.

Table N2

Table N3

[0229] Thus, in certain embodiments, at least one NRP2 ligand is selected from Table N2 and / or Table N3.

[0230] For example, in some aspects, at least one NRP2 ligand is a VEGF (vascular endothelial growth factor) ligand selected from VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, and PIGF-2. The VEGF-VEGFR2 / 3-NRP2 interaction is associated with promoting cell migration, cell growth, cell survival, and cell adhesion, as well as lymphangiogenesis, increased vascular permeability, activation of integrin signaling, promotion of vesicular transport and internalization, and delay of cell differentiation. Thus, anti-NRP2 antibodies that modulate VEGF-related NRP2 ligands would be expected to find utility in modulating one or more of these pathways.

[0231] In certain embodiments, at least one NRP2 ligand is a semaphorin selected from one or more of SEMA-3B, SEMA-3C, SEMA-3D, SEMA-3F and SEMA-3B, or a plexin receptor selected from one or more of plexin A1, A2, A3, A4 and D1. SEMA typically antagonizes the effects of VEGF-C, but there is a close dynamic interaction between the VEGF and Sema signaling pathways. SEMA typically functions in the immune system to control cell migration, cell trafficking, cell-cell communication and cell activation. The SEMA-plexin-NRP2 interaction is associated with inhibition of cell migration, inhibition of cell growth, promotion of apoptosis, inhibition of cell adhesion, inhibition of integrin signaling, promotion of cell differentiation, inhibition of lymphangiogenesis, reduction of vascular permeability, promotion of microtubule destabilization, disruption of the actin cytoskeleton and mediation of cell contraction including disruption and actomyosin contraction of the growth cone, as well as prevention of neuronal cell spreading and inhibition of axonal elongation. Accordingly, anti-NRP2 antibodies that modulate SEMA-related NRP2 ligands would be expected to find utility in modulating one or more of these pathways.

[0232] In some embodiments, at least one NRP2 ligand is an integrin selected from one or more of αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α6β1 and α6β4. The integrin-NRP2 interaction is generally associated with increased cell adhesion, cell growth, cancer growth and invasiveness. Accordingly, anti-NRP2 antibodies that modulate NRP2 ligands associated with integrins would be expected to find utility in modulating one or more of these pathways.

[0233] In some embodiments, at least one NRP2 ligand is selected from TGFβ1, TGFβ2, TGFβ3 and their corresponding TGFβ receptors. TGF-β signaling is strongly involved in the regulation of EMT in cancer and the development of fibrosis (see, e.g., Gemmill et al., Sci. Signal. 10 eaag0528, 2017). NRP2b expression is preferentially upregulated by TGF-β signaling in abnormal lungs and shows little or no expression in normal lungs. NRP2b expression enhances migration, invasion, metastasis, chemoresistance and tumor-like mass formation, and also enhances the acquired EGFR inhibitor resistance associated with EMT in cancer cells. Thus, anti-NRP2 antibodies that modulate NRP2 ligands associated with TGF-β are expected to be found useful in modulating one or more of these pathways and in the treatment of cancer chemoresistance. In certain embodiments, the anti-NRP2 antibody or antigen-binding fragment thereof modulates the binding activity / signaling activity between the NRP2 polypeptide and at least one of the NRP2 ligands from Table N2 and / or Table N3, for example, by specifically binding to the NRP2 ligand interaction region of the NRP2 polypeptide.

[0234] In some embodiments, at least one antibody or antigen-binding fragment thereof selectively binds to the NRP2a isoform of NRP2 (e.g., variants 1, 2 and / or 3 of Table N1) and does not substantially bind to the NRP2b isoform of NRP2 (e.g., variants 4 and / or 5 of Table N1). In some embodiments, at least one antibody or antigen-binding fragment thereof selectively binds to the NRP2b isoform of NRP2 (e.g., variants 4 and / or 5 of Table N1) and does not substantially bind to the NRP2a isoform of NRP2 (e.g., variants 1, 2 and / or 3 of Table N1).

[0235] In some examples, at least one antibody or an antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between an NRP2 polypeptide and at least one NRP2 ligand. For example, in some embodiments, an anti-NRP2 antibody antagonizes or reduces the theoretical maximum binding / signal transduction between an NRP2 polypeptide and an NRP2 ligand by about or at least about 20-100% (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or 100%) after pre-incubation of the anti-NRP2 antibody with the NRP2 polypeptide / ligand in substantially stoichiometric equivalents.

[0236] In some examples, at least one antibody or an antigen-binding fragment thereof reduces or inhibits dimerization between two NRP2 polypeptides. For example, in some embodiments, an anti-NRP2 antibody antagonizes or reduces the theoretical maximum dimerization between two NRP2 polypeptides by about or at least about 20-100% (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or 100%) after pre-incubation of the anti-NRP2 antibody with the NRP2 polypeptide in substantially stoichiometric equivalents.

[0237] In some examples, at least one antibody or an antigen-binding fragment thereof agonizes or enhances dimerization between two NRP2 polypeptides. For example, in some embodiments, an anti-NRP2 antibody agonizes or enhances the basal dimerization state of two NRP2 polypeptides by about or at least about 20%-500% (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500%) after pre-incubation of the anti-NRP2 antibody with the NRP2 polypeptide in substantially stoichiometric equivalents.

[0238] In some examples, at least one antibody or antigen-binding fragment thereof agonizes or enhances the binding activity / signaling activity between the NRP2 polypeptide and at least one NRP2 ligand. For example, in some embodiments, the anti-NRP2 antibody agonizes or enhances the theoretical maximum binding activity / signaling activity between the NRP2 polypeptide and at least one NRP2 ligand by about or at least about 20% to 500% (e.g., about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500%) after pre-incubation of the anti-NRP2 antibody with the NRP2 polypeptide in substantially stoichiometric equivalents.

[0239] In some embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates the binding of semaphorin to or through the NRP2 polypeptide and / or signaling. In some aspects, such antibodies do not substantially block the interaction of VEGF-C or related NRP2 ligands. In some aspects, such antibodies are agonist antibodies with respect to semaphorin signaling. In some aspects, such antibodies are antagonist antibodies with respect to semaphorin signaling.

[0240] In some embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates the binding of VEGF-C or related NRP2 ligands to or through the NRP2 polypeptide and / or signaling. In some aspects, such antibodies do not substantially block the interaction of semaphorin. In some embodiments, such antibodies selectively modulate both the binding of VEGF-C or related NRP2 ligands and semaphorin to the NRP2 polypeptide. In some embodiments, such antibodies are agonist antibodies with respect to VEGF-C signaling. In some aspects, such antibodies are antagonist antibodies with respect to VEGF-C signaling.

[0241] In some embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates the binding and / or signaling of an integrin or related NRP2 ligand to the NRP2 polypeptide.

[0242] In some embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates the binding and / or signaling of TGFβ1, TGFβ2, TGFβ3 or their corresponding TGFβ receptors to the NRP2 polypeptide.

[0243] In some embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates the binding and / or signaling of fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor and / or their corresponding receptors to the NRP2 polypeptide.

[0244] In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signaling activity between the NRP2 polypeptide and the plexin receptor and / or semaphorin without substantially modulating the binding activity / signaling activity between the NRP2 polypeptide and VEGFR2, VEGFR3 and / or VEGF-C.

[0245] In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signaling activity between the NRP2 polypeptide and the plexin receptor and / or semaphorin without substantially modulating the binding activity / signaling activity between the NRP2 polypeptide and the HRS polypeptide.

[0246] In some embodiments, at least one antibody or antigen-binding fragment thereof substantially antagonizes the binding activity / signal transduction activity between an NRP2 polypeptide and a plexin receptor and / or a semaphorin without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and an HRS polypeptide, without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and VEGFR2, VEGFR3, and / or VEGF-C.

[0247] In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between an NRP2 polypeptide and VEGFR2 and / or VEGFR3 without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and a plexin receptor and / or a semaphorin.

[0248] In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between an NRP2 polypeptide and VEGFR2, VEGFR3, and / or VEGF-C without substantially modulating the binding activity / signal transduction activity between the NRP2 polypeptide and an HRS polypeptide.

[0249] In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signal transduction activity between an NRP2 polypeptide and a plexin receptor without substantially modulating the ligand binding of semaphorin 3 to NRP2.

[0250] In some embodiments, the plexin receptor is selected from plexin A1, A2, A3, A4, and D1. In some embodiments, the semaphorin is selected from semaphorin 3B, 3C, 3D, 3F, and 3G.

[0251] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope of at least 5 amino acids within the a2 domain of human NRP2, and at least one antibody or antigen-binding fragment thereof selectively inhibits receptor dimerization between NRP2 and plexin A1 without substantially inhibiting dimerization between NRP2 and FLT4 (VEGFR3). In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within amino acids 232-242 of the human NRP2 precursor (see Table N1).

[0252] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope of at least 5 amino acids within the b1 domain of human NRP2, and at least one antibody or antigen-binding fragment thereof selectively inhibits receptor dimerization between NRP2 and plexin A1 without substantially inhibiting dimerization between NRP2 and FLT4 (VEGFR3).

[0253] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope of at least 5 amino acids within the b2 domain of human NRP2, and at least one antibody or antigen-binding fragment thereof inhibits receptor dimerization between NRP2 and FLT4 (VEGFR3) and inhibits dimerization between NRP2 and plexin A1.

[0254] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope of at least 5 amino acids within the c domain of human NRP2, and at least one antibody or antigen-binding fragment thereof inhibits receptor dimerization between NRP2 and plexin A1 and partially inhibits dimerization between NRP2 and FLT4 (VEGFR3).

[0255] In some embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ) for each of the corresponding regions of (i) a human NRP2 polypeptide and (ii) a cynomolgus NRP2 polypeptide (see, e.g., UniProt G7PL91), and the affinity for (i) and (ii) is in the range of about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 0.4 to about 1.2 nM, about 0.9 to about 5.5 nM, about 0.9 to about 5 nM, or about 1 nM to about 10 nM.

[0256] In some embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ) for each of the corresponding regions of (i) a human NRP2 polypeptide and (ii) a mouse NRP2 polypeptide, and the affinity for (i) and (ii) is in the range of about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, or about 1 nM to about 10 nM.

[0257] In certain embodiments, at least one antibody or antigen-binding fragment thereof selectively binds to a human NRP2 polypeptide (see Table N1) as compared to a mouse NRP2 polypeptide, e.g., its affinity for the human NRP2 polypeptide is, for example, about or at least about 2, 5, 10, 20, 30, 40, 50, 100, 500 or 1000-fold or more higher than its affinity for the mouse NRP2 polypeptide, and is significantly stronger. In certain embodiments, at least one antibody or antigen-binding fragment thereof selectively binds to the human NRP2 polypeptide and does not substantially bind to the mouse NRP2 polypeptide. As a particular exemplary mouse NRP2 polypeptide, there may be mentioned the NRP2 polypeptide of Mus musculus (see, e.g., UniProt O35375).

[0258] For illustrative purposes only, the binding interaction between a human NRP2 polypeptide and an NRP2 ligand can be detected and quantified using a variety of routine methods, including biacore assays (e.g., using appropriately tagged soluble reagents bound to a sensor chip), FACS analysis using cells expressing the NRP2 polypeptide on the cell surface (either native or recombinant), immunoassays, fluorescence staining assays, ELISA assays, and microcalorimetry techniques such as ITC (isothermal titration calorimetry).

[0259] In certain embodiments, the antibody or antigen-binding fragment thereof includes a variant or otherwise modified Fc region, and those having altered properties or biological activities compared to the wild-type Fc region. Examples of modified Fc regions include, for example, those having a sequence mutated by substitution, insertion, deletion, or truncation of one or more amino acids compared to the wild-type sequence, hybrid Fc polypeptides composed of domains from different immunoglobulin classes / subclasses, Fc polypeptides having an altered glycosylation / sialylation pattern, and Fc polypeptides modified or derivatized, for example, by biotinylation (see, e.g., U.S. Patent Application Publication No. 2010 / 0209424), phosphorylation, sulfation, etc. or any combination of the foregoing. Using such modifications, compared to the corresponding wild-type Fc sequence of the antibody or antigen-binding fragment thereof, among other properties described herein, the binding to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn) of the Fc region, its pharmacokinetic properties (e.g., stability or half-life, bioavailability, tissue distribution, volume of distribution, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, C max , t max , C min , variability), its immunogenicity, its complement binding or activation, and / or the CDC / ADCC / ADCP-related activities of the Fc region can be altered (e.g., increased, decreased). Modified Fc regions of human and / or mouse origin are included.

[0260] Also included are antibodies or antigen-binding fragments thereof that include a hybrid Fc region, e.g., an Fc region that includes a combination of Fc domains (e.g., hinge, CH2, CH3, CH4) from immunoglobulins of different species (e.g., human, mouse), different Ig classes and / or different Ig subclasses.As general examples, there may be mentioned hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of CH2 / CH3 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgE / IgA1, IgE / IgA2, IgE / IgD, IgE / IgE, IgE / IgG1, IgE / IgG2, IgE / IgG3, IgE / IgG4, IgE / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM, IgM / IgA1, IgM / IgA2, IgM / IgD, IgM / IgE, IgM / IgG1, IgM / IgG2, IgM / IgG3, IgM / IgG4, IgM / IgM (or fragments or variants thereof), and optionally comprising one or more hinges derived from IgA1, IgA2, IgD, IgG1, IgG2, IgG3 or IgG4, and / or CH4 domains derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3 and CH4 domains are derived from human Ig.

[0261] As an additional example, there are hybrid Fc regions that include, consist of, or consist essentially of the following combinations of CH2 / CH3 domains: IgA1 / IgE, IgA2 / IgE, IgD / IgE, IgE / IgE, IgG1 / IgE, IgG2 / IgE, IgG3 / IgE, IgG4 / IgE, IgM / IgE, IgA1 / IgM, IgA2 / IgM, IgD / IgM, IgE / IgM, IgG1 / IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof), and optionally include a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0262] As a specific example, there are hybrid Fc regions that include, consist of, or consist essentially of the following combinations of CH3 / CH4 domains: IgA1 / IgE, IgA2 / IgE, IgD / IgE, IgE / IgE, IgG1 / IgE, IgG2 / IgE, IgG3 / IgE, IgG4 / IgE, IgM / IgE, IgA1 / IgM, IgA2 / IgM, IgD / IgM, IgE / IgM, IgG1 / IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof), and optionally include a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0263] As specific examples, there are the following combinations of hinge / CH2 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), including, consisting of, or essentially consisting of a hybrid Fc region, and optionally including a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM, and / or a CH4 domain derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3 and CH4 domains are of human Ig origin.

[0264] As a specific example, there are the following combinations of hinge / CH3 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), including, consisting of, or essentially consisting of a hybrid Fc region, and optionally including a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM, and / or a CH4 domain derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3 and CH4 domains are of human Ig origin.

[0265] Examples include hybrid Fc regions that include, consist of, or consist essentially of the following hinge / CH4 domain combinations: IgA1 / IgE, IgA1 / IgM, IgA2 / IgE, IgA2 / IgM, IgD / IgE, IgD / IgM, IgG1 / IgE, IgG1 / IgM, IgG2 / IgE, IgG2 / IgM, IgG3 / IgE, IgG3 / IgM, IgG4 / IgE, IgG4 / IgM (or fragments or variants thereof), and optionally include CH2 domains from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM, and / or CH3 domains from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM.

[0266] Specific examples of hybrid Fc regions can be found, for example, in WO 2008 / 147143, which are derived from combinations of IgG subclasses or combinations of human IgD and IgG.

[0267] Also included are antibodies or antigen-binding fragments thereof having a derivatized Fc region or otherwise modified Fc region. In certain embodiments, the Fc region may be modified, for example, by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc., relative to the wild-type or naturally occurring Fc region. In certain embodiments, the Fc region may comprise the wild-type or native glycosylation pattern, or alternatively, may comprise increased glycosylation compared to the native form, decreased glycosylation compared to the native form, or may be overall deglycosylated. As an example of a modified Fc glycoform, decreased glycosylation of the Fc region reduces binding to the C1q region of the first complement component C1, decreases ADCC-related activity, and / or decreases CDC-related activity. Accordingly, certain embodiments utilize a deglycosylated or aglycosylated Fc region. See, for example, International Publication No. WO 2005 / 047337 for an exemplary method of generating an aglycosylated Fc region. Another example of an Fc region glycoform can be created by substituting the cysteine residue at position Q295 according to the Kabat et al. numbering system (see, for example, U.S. Patent Application Publication No. 2010 / 0080794). Certain embodiments may comprise an Fc region in which about 80-100% of the glycoprotein in the Fc region comprises a mature core carbohydrate structure lacking fructose (see, for example, U.S. Patent Application Publication No. 2010 / 0255013). Some embodiments may comprise an Fc region optimized by substitution or deletion to reduce the level of fucosylation, for example, to increase affinity for FcγRI, FcγRIa or FcγRIIIa, and / or to improve phagocytosis by FcγRIIa-expressing cells (see U.S. Patent Application Publication Nos. 2010 / 0249382 and 2007 / 0148170).

[0268] As another example of modified Fc glycoforms, the Fc region of an antibody or antigen-binding fragment thereof may contain oligomannose-type N-glycans and, optionally, may have one or more of the following compared to the corresponding Fc region containing complex-type N-glycans: increased ADCC effector activity, increased binding affinity for FcγRIIIA (and certain other FcRs), similar or increased binding specificity for the target of the NRP2 polypeptide, similar or higher binding affinity for the target of the NRP2 polypeptide, and / or similar or lower binding affinity for the mannose receptor (see, e.g., U.S. Patent Application Publication No. 2007 / 0092521 and U.S. Patent No. 7,700,321). As another example, enhanced affinity of the Fc region for FcγR has been achieved using engineered glycoforms generated by expression of the antibody in engineered cell lines or variant cell lines (see, e.g., Umana et al., Nat Biotechnol. 17:176-180, 1999; Davies et al., Biotechnol Bioeng. 74:288-294, 2001; Shields et al., J Biol Chem. 277:26733-26740, 2002; Shinkawa et al., 2003, J Biol Chem. 278:3466-3473, 2003; and U.S. Patent Application Publication No. 2007 / 0111281). Certain Fc region glycoforms contain an increased proportion of N-glycosidically linked complex glycans that do not have a fucose at the 1-position attached to the 6-position of N-acetylglucosamine at the reducing end of the glycan (see, e.g., U.S. Patent Application Publication No. 2010 / 0092997). Certain embodiments may include an Fc region of IgG that is glycosylated with at least one galactose moiety connected by an α-2,6 linkage to each terminal sialic acid moiety, and optionally, the Fc region may have higher anti-inflammatory activity compared to the corresponding wild-type Fc region (see U.S. Patent Application Publication No. 2008 / 0206246).Certain of these and related modified glycosylation techniques result in a substantial enhancement of the capabilities of the Fc region, selectively binding to FcRs such as FcγRIII, mediating ADCC, and altering other properties of the Fc region, as described herein.

[0269] Certain variants, fragments, hybrids, or otherwise modified Fc regions of an antibody or antigen-binding fragment thereof may alter binding to one or more FcRs and / or correspondingly alter effector function, compared to the corresponding wild-type Fc sequence (e.g., of the same species, same Ig class, same Ig subclass). For example, such Fc regions may increase binding to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor compared to the corresponding wild-type Fc sequence. In other embodiments, the variant, fragment, hybrid, or modified Fc region may decrease binding to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor compared to the corresponding wild-type Fc sequence. Specific FcRs are described elsewhere herein.

[0270] In some embodiments, the antibody comprises an Fc domain comprising one or more mutations that increase binding to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor compared to the corresponding wild-type Fc sequence. In some embodiments, the antibody comprises an Fc domain of IgG1 or IgG3 comprising one or more mutations that increase binding to one or more of the Fcγ receptor, Fcα receptor, Fcε receptor, and / or neonatal Fc receptor compared to the corresponding wild-type Fc sequence. In some embodiments, the antibody comprises an Fc domain comprising one or more mutations that increase effector function. In some embodiments, at least one antibody comprises an Fc domain selected from human IgG1 and human IgG3 comprising one or more mutations that increase effector function.

[0271] In some embodiments, the antibody is a blocking antibody that includes an Fc domain having high effector activity. In some embodiments, the blocking antibody includes an Fc domain selected from human IgG1 and human IgG3 that includes one or more mutations that increase effector function. In some embodiments, the antibody is a partial blocking antibody that includes an Fc domain having high effector activity. In some embodiments, the partial blocking antibody includes an Fc domain selected from human IgG1 and human IgG3 that includes one or more mutations that increase effector function. In some embodiments, the antibody is a non-blocking antibody that includes an Fc domain having high effector activity. In some embodiments, the non-blocking antibody includes an Fc domain selected from human IgG1 or IgG3 that includes one or more mutations that increase effector function.

[0272] In some embodiments, the antibody includes an Fc domain that includes one or more mutations that decrease binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and / or a neonatal Fc receptor as compared to the corresponding wild-type Fc sequence. In some embodiments, the antibody includes an Fc domain of IgG1 or IgG3 that includes one or more mutations that decrease binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and / or a neonatal Fc receptor as compared to the corresponding wild-type Fc sequence. In some embodiments, the antibody includes an Fc domain that includes one or more mutations that decrease effector function. In some embodiments, the antibody includes an Fc domain selected from human IgG2 and IgG4 that includes one or more mutations that decrease effector function.

[0273] In some embodiments, the antibody is a blocking antibody that includes an Fc domain having low effector activity. In some embodiments, the blocking antibody includes an Fc domain selected from human IgG2 and IgG4 that includes one or more mutations that reduce effector function. In some embodiments, the antibody is a partial blocking antibody that includes an Fc domain having low effector activity. In some embodiments, the partial blocking antibody includes an Fc domain selected from human IgG2 and IgG4 that includes one or more mutations that reduce effector function. In some embodiments, the antibody is a non-blocking antibody that includes an Fc domain having low effector activity. In some embodiments, the non-blocking antibody includes an Fc domain selected from human IgG2 and IgG4 that includes one or more mutations that reduce effector function.

[0274] Specific examples of Fc variants having an altered (e.g., increased, decreased) effector function / FcR binding can be found, for example, in U.S. Pat. Nos. 5,624,821 and 7,425,619; U.S. Patent Application Publication Nos. 2009 / 0017023, 2009 / 0010921, and 2010 / 0203046; and International Publication Nos. 2000 / 42072 and 2004 / 016750. As certain examples, human Fc regions having one or more substitutions at positions 298, 333, and / or 334, e.g., S298A, E333A, and / or K334A (based on the Kabat et al. EU index numbering), which have been shown to increase binding to the activating receptor FcγRIIIa and decrease binding to the inhibitory receptor FcγRIIb, are included. By combining these mutations, double and triple mutant variants with further improved binding to FcR can be obtained. As certain embodiments, the triple mutant S298A / E333A / K334A is included, which has been shown to increase binding to FcγRIIIa, decrease binding to FcγRIIb, and increase ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604, 2001; and Presta et al., Biochem Soc Trans. 30:487-490, 2002). See also engineered Fc glycoforms with increased binding to FcR as disclosed in Umana et al. and U.S. Pat. No. 7,662,925 above. As some embodiments, Fc regions are included that contain one or more substitutions selected from 434S, 252Y / 428L, 252Y / 434S, and 428L / 434S based on the Kabat et al. EU index (see U.S. Patent Application Publication Nos. 2009 / 0163699 and 20060173170).

[0275] Certain variants, fragments, hybrids or modified Fc regions may have altered effector functions compared to the corresponding wild-type Fc sequence. For example, such Fc regions may have increased complement binding or complement activation, increased Clq binding affinity, increased CDC-related activity, increased ADCC-related activity, and / or increased ADCP-related activity compared to the corresponding wild-type Fc sequence. In other embodiments, such Fc regions may have decreased complement binding or complement activation, decreased Clq binding affinity, decreased CDC-related activity, decreased ADCC-related activity, and / or decreased ADCP-related activity compared to the corresponding wild-type Fc sequence. By way of mere example, the Fc region may contain deletions or substitutions in complement binding sites such as the C1q binding site, and / or may contain deletions or substitutions in the ADCC site. Examples of such deletions / substitutions are described, for example, in U.S. Patent No. 7,030,226. Many Fc effector functions, such as ADCC, can be assayed according to routine techniques in the art (see, for example, Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Effector cells useful in such assays include, but are not limited to, natural killer (NK) cells, macrophages and other peripheral blood mononuclear cells (PBMC). Alternatively, or in addition, certain Fc effector functions may be evaluated in vivo, for example, by using the animal models described in Clynes et al. PNAS. 95:652-656, 1998.

[0276] Certain variant hybrids or modified Fc regions may have an altered stability or half-life compared to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have an increased half-life compared to the corresponding wild-type Fc sequence. In other embodiments, the variant hybrid or modified Fc region may have a decreased half-life compared to the corresponding wild-type Fc sequence. The half-life can be measured in vitro (e.g., under physiological conditions) or in vivo according to routine techniques in the art, such as radiolabeling, ELISA, or other methods. In vivo measurements of stability or half-life can be made in one or more body fluids including blood, serum, plasma, urine, or cerebrospinal fluid, or in a given tissue such as liver, kidney, muscle, central nervous system tissue, bone, etc.

[0277] As an example, modifications to the Fc region that alter its ability to bind to FcRn can change its in vivo half-life or other properties. In some embodiments, the modified Fc domain, e.g., the Fc domain of modified IgG1 or IgG4, contains at least one mutation that alters FcRn binding, as described, for example, by Zalevsky et al. (Nature Biotechnology. 28(2): 157-159, 2010) or Mackness et al. (mAbs. 11(7): 1276-1288, 2019). In a specific embodiment, the Fc domain of modified IgG1 or IgG3 contains any one or more of the YD (M252Y / T256D), DQ (T256D / T307Q), DW (T256D / T307W), YTE (M252Y / S254T / T256E), AAA (T307A / E380A / N434A), LS (M428L / N434S), M252Y, T256D / E, K288D / N, T307Q / W, E380C, N434FY or Y436H / N / W mutations (EU numbering) and combinations thereof. In some embodiments, the Fc domain of modified IgG1 or IgG3 contains any one or more of the M252Y, T256D / E, T307Q / W and / or N434F / Y mutations (EU numbering), and combinations thereof. Assays for measuring in vivo pharmacokinetic properties (e.g., in vivo mean elimination half-life), and non-limiting examples of Fc modifications that alter its binding to FcRn are described, for example, in U.S. Pat. Nos. 7,217,797 and 7,732,570; and U.S. Patent Application Publication Nos. 2010 / 0143254 and 2010 / 0143254.

[0278] Non-limiting examples of additional modifications that alter stability or half-life include substitutions / deletions at one or more of the amino acid residues selected from positions 251-256, 285-290, and 308-314 in the CH2 domain, and positions 385-389 and 428-436 in the CH3 domain, according to the Kabat et al. numbering system. See U.S. Patent Application Publication No. 2003 / 0190311. Specific examples include substitution with leucine at position 251, substitution with tyrosine, tryptophan, or phenylalanine at position 252, substitution with threonine or serine at position 254, substitution with arginine at position 255, substitution with glutamine, arginine, serine, threonine, or glutamate at position 256, substitution with threonine at position 308, substitution with proline at position 309, substitution with serine at position 311, substitution with aspartic acid at position 312, substitution with leucine at position 314, substitution with arginine, aspartic acid, or serine at position 385, substitution with threonine or proline at position 386, substitution with arginine or proline at position 387, substitution with proline, asparagine, or serine at position 389, substitution with methionine or threonine at position 428, substitution with tyrosine or phenylalanine at position 434, substitution with histidine, arginine, lysine, or serine at position 433, and / or substitution with histidine, tyrosine, arginine, or threonine at position 436, and any combination thereof. Such modifications, if desired, increase the affinity of the Fc region for FcRn compared to the corresponding wild-type Fc region, thereby increasing the half-life.

[0279] Certain variant hybrids or modified Fc regions may have altered solubility compared to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have increased solubility compared to the corresponding wild-type Fc sequence. In other embodiments, the variant hybrid or modified Fc region may have decreased solubility compared to the corresponding wild-type Fc sequence. Solubility can be measured, for example, in vitro (e.g., under physiological conditions) according to routine techniques in the art. Exemplary solubility measurements are described elsewhere in this specification.

[0280] Additional examples of variants include the Fc region of IgG having conservative or non-conservative substitutions (described elsewhere herein) at one or more of positions 250, 314, or 428 of the heavy chain, or any combination thereof, e.g., positions 250 and 428, or positions 250 and 314, or positions 314 and 428, or positions 250, 314, and 428 (see, e.g., U.S. Patent Application Publication No. 2011 / 0183412). In specific embodiments, the residue at position 250 is substituted with glutamic acid or glutamine, and / or the residue at position 428 is substituted with leucine or phenylalanine. As another example of an IgG Fc variant, any one or more of the amino acid residues at positions 214-238, 297-299, 318-322, and / or 327-331 may be used as suitable targets for modification (e.g., conservative or non-conservative substitution, deletion). In certain embodiments, the IgG Fc variant CH2 domain contains amino acid substitutions at positions 228, 234, 235, and / or 331 (e.g., human IgG4 having Ser228Pro and Leu235Ala mutations), which attenuate the effector function of the Fc region (see U.S. Patent No. 7,030,226). Here, the residue numbering in the heavy chain is according to the EU index numbering (Kabat et al., "Sequences of Proteins of Immunological Interest," 5 th(See, e.g., Ed., National Institutes of Health, Bethesda, Md. (1991)). Certain of these and related embodiments have altered (e.g., increased, decreased) FcRn binding and / or serum half-life without, if desired, reducing effector functions such as ADCC or CDC-related activities.

[0281] Examples of additions include variant Fc regions that contain one or more amino acid substitutions at positions 279, 341, 343, or 373 of the wild-type Fc region, or any combination thereof (see, e.g., US Patent Application Publication No. 2007 / 0224188). The wild-type amino acid residues at these positions for human IgG are valine (279), glycine (341), proline (343), and tyrosine (373). The substituents can be conservative or non-conservative, as described herein, or can include non-naturally occurring amino acids or mimetics. Alone or in combination with these substitutions, certain embodiments can also use variant Fc regions that contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions selected from: 235G, 235R, 236F, 236R, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L, 245R, 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W, 247Y, 248F, 248P, 248Q, 248W, 249L, 249M, 249N, 249P, 249Y, 251H, 251I, 251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P, 254Q, 254R, 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V, 256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272R, 279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279Y, 280T, 283F, 283G, 283H, 283I, 283K, 283L, 283M, 283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 292G, 292I, 292L,293S, 293V, 301W, 304E, 307E, 307M, 312P, 315F, 315K, 315L, 315P, 315R, 316F, 316K, 317P, 317T, 318N, 318P, 318T, 332F, 332G, 332L, 332M, 332S, 332V, 332W, 339D, 339E, 339F, 339G, 339H, 339I, 339K, 339L, 339M, 339N, 339Q, 339R, 339S, 339W, 339Y, 341D, 341E, 341F, 341H, 341I, 341K, 341L, 341M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y, 343A, 343D, 343E, 343F, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 343V, 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 376E, 376F, 376G, 376H, 376I, 376L, 376M, 376N, 376P, 376Q, 376R, 376S, 376T, 376V, 376W, 376Y, 377G, 377K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D, 380N, 380S, 380T, 382D, 382F, 382H, 382I, 382K, 382L, 382M, 382N, 382P, 382Q, 382R, 382S, 382T, 382V, 382W, 382Y, 385E, 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L, 427N, 429A, 429F, 429M, 430A, 430D, 430F, 430G, 430H, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V, 430W, 430Y, 431H, 431K, 431P, 432R, 432S, 438G, 438K, 438L, 438T, 438W, 439E, 439H, 439Q, 440D, 440E, 440F, 440G, 440H, 440I, 440K, 440L, 440M, 440Q, 440T, 440V or 442K. As described above, the numbering of residues in the heavy chain isNumbering of the EU index (see Kabat et al. supra). Such variant Fc regions typically confer altered effector function or altered serum half-life in the antibody to which the variant Fc region is operably linked. Preferably, the altered effector function is an increase in ADCC, a decrease in ADCC, an increase in CDC, a decrease in CDC, an increase in Clq binding affinity, a decrease in Clq binding affinity, an increase in FcR (preferably, FcRn) binding affinity, or a decrease in FcR (preferably, FcRn) binding affinity as compared to the corresponding Fc region lacking such amino acid substitutions.,

[0282] Examples of additions include variants of the Fc region that contain one or more amino acid substitutions at positions 221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 and / or 428 (see, for example, U.S. Patent No. 7,662,925). In a specific embodiment, the variant Fc region is P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R and T335Y, and contains at least one amino acid substitution selected from the group consisting of. In other specific embodiments, the variant Fc region is V264I, F243L / V264I, L328M, I332E, L328M / I332E, V264I / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, A330Y, I332D, L328I / I332E, L328Q / I332E, V264T, V240I, V266I, S239D, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q,At least one amino acid substitution selected from the group consisting of S239E / I332D, S239E / I332N, S239E / I332Q, S239N / I332D, S239N / I332E, S239Q / I332D, A330Y / I332E, V264I / A330Y / I332E, A330L / I332E, V264I / A330L / I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D / I332E, L328V / I332E, L328T / I332E, L328I / I332E, S239E / V264I / I332E, S239Q / V264I / I332E, S239E / V264I / A330Y / I332E, S239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E, V264I / S298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, S239D / V264I / A330L / I332E, S239D / I332E / A330I, P230A, P230A / E233D / I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L234I, S239D / A330Y / I332E / L235D, S239D / A330Y / I332E / V240I, S239D / A330Y / I332E / V264T, S239D / A330Y / I332E / K326E and S239D / A330Y / I332E / K326T. In a more specific embodiment, the variant Fc region is N297D / I332E, F241Y / F243Y / V262T / V264T / N297D / I332E, S239D / N297D / I332E, S239E / N297D / I332E, S239D / D265Y / N297D / I332E,It comprises a series of substitutions selected from the group consisting of S239D / D265H / N297D / I332E, V264E / N297D / I332E, Y296N / N297D / I332E, N297D / A330Y / I332E, S239D / D265V / N297D / I332E, S239D / D265I / N297D / I332E and N297D / S298A / A330Y / I332E. In a specific embodiment, the variant Fc region comprises an amino acid substitution at position 332 (using the EU index, Kabat et al., the numbering as above). Examples of substitutions include 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W and 332Y. The numbering of residues in the Fc region is the EU index numbering of Kabat et al. Among other properties described herein, such variant Fc regions may have increased affinity for FcγR, increased stability, and / or increased solubility compared to the corresponding wild-type Fc region.,

[0283] As a further example, there are provided variant Fc regions comprising one or more of the following amino acid substitutions: 224N / Y, 225A, 228L, 230S, 239P, 240A, 241L, 243S / L / G / H / I, 244L, 246E, 247L / A, 252T, 254T / P, 258K, 261Y, 265V, 266A, 267G / N, 268N, 269K / G, 273A, 276D, 278H, 279M, 280N, 283G, 285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A, 306I / F, 311R, 312N, 315D / K / S, 320R, 322E, 323A, 324T, 325S, 326E / R, 332T, 333D / G, 335I, 338R, 339T, 340Q, 341E, 342R, 344Q, 347R, 351S, 352A, 354A, 355W, 356G, 358T, 361D / Y, 362L, 364C, 365Q / P, 370R, 372L, 377V, 378T, 383N, 389S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R, 411I, 412A, 414M, 421S, 422I, 426F / P, 428T, 430K, 431S, 432P, 433P, 438L, 439E / R, 440G, 441F, 442T, 445R, 446A, 447E, wherein, optionally, the variant has an altered Fc ligand recognition and / or an altered effector function as compared to the parental Fc polypeptide, and the residue numbering is according to Kabat et al.is the numbering of the EU index as in. Specific examples of these and related embodiments include the following sets of substitutions: (1) N276D, R292Q, V305A, I377V, T394A, V412A and K439E; (2) P244L, K246E, D399G and K409R; (3) S304G, K320R, S324T, K326E and M358T; (4) F243S, P247L, D265V, V266A, S383N and T411I; (5) H224N, F243L, T393A and H433P; (6) V240A, S267G, G341E and E356G; (7) M252T, P291L, P352A, R355W, N390D, S408G, S426F and A431S; (8) P228L, T289A, L365Q, N389S and 5440G; (9) F241L, V273A, K340Q and L441F; (10) F241L, T299A, I332T and M428T; (11) E269K, Y300H, Q342R, V422I and G446A; (12) T225A, R301c, S304G, D312N, N315D, L351S and N421S; (13) S254T, L306I, K326R and Q362L; (14) H224Y, P230S, V323A, E333D, K338R and S364C; (15) T335I, K414M and P445R; (16) T335I and K414M; (17) P247A, E258K, D280N, K288R, N297D, T299A, K322E, Q342R, S354A and L365P; (18) H268N, V279M, A339T, N361D and S426P; (19) C261Y, K290E, L306F, Q311R, E333G and Q438L; (20) E283G, N315K, E333G, R344Q, L365P and S442T; (21) Q347R, N361Y and K439R; (22) S239P, S254P, S267N, H285R, N315S, F372L, A378T, N390D, Y391C, F404S, E430K, L432P and K447E; and (23) E269G, Y278H, N325S and K370R, or variant Fc regions comprising or consisting of them, and the residue numbering is according to Kabat et al.numbering of EU indexes as in (see, e.g., U.S. Patent Application Publication No. 2010 / 0184959).

[0284] The variant Fc region can also have one or more mutated hinge regions, as described, for example, in U.S. Patent Application Publication No. 2003 / 0118592. For example, one or more cysteines in the hinge region can be deleted or substituted with a different amino acid. The mutated hinge region may not contain cysteine residues, or it may contain 1, 2, or 3 fewer cysteine residues than the corresponding wild-type hinge region. In some embodiments, an Fc region having this type of mutated hinge region exhibits a reduced ability to dimerize compared to the wild-type Ig hinge region.

[0285] In certain embodiments, the Fc region comprises, consists of, or consists essentially of an Fc derived from human IgG1 or IgG4 (see, e.g., Allberse and Schuurman, Immunology. 105:9-19, 2002) or a fragment or variant thereof. Table F1 below provides exemplary sequences (CH1, hinge (underlined), CH2, and CH3 regions) derived from human IgG1 and IgG4. Examples of variant IgG4 sequences that can be used are described, for example, in Peters et al., JBC. 287:24525-24533, 2012, and include substitutions at C227, C230, C127 (e.g., C127S), and C131 (e.g., C131S). Other variants that can be used include the L445P substitution in IgG4 (designated IgG4-2) or the D356E and L358M substitutions in IgG1 (designated IgG1m(zf)).

Table F1

[0286] As described above, antibodies having an altered Fc region typically have altered (e.g., improved, increased, decreased) pharmacokinetic properties compared to the corresponding wild-type Fc region. Examples of pharmacokinetic properties include stability or half-life, bioavailability (the fraction of drug absorbed), tissue distribution, volume of distribution (the apparent volume when the drug is distributed immediately after intravenous injection and equilibrates between plasma and surrounding tissues), concentration (the initial or steady-state concentration of drug in plasma), elimination rate constant (the rate at which the drug is removed from the body), elimination rate (the rate of infusion required to maintain equilibrium with elimination), area under the curve (AUC or exposure; the integrated value of the concentration-time curve after a single dose or at steady state), clearance (the plasma volume from which drug is cleared per unit time), C max (the maximum plasma concentration of the drug after oral administration), t max (C max (the time to reach), C min (the lowest concentration the drug reaches before the next dose is administered) and variability (peak-to-trough variability within a dosing interval at steady state).

[0287] In certain embodiments, the antibody or antigen-binding fragment thereof has a biological half-life of about or at least about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours or about 144 hours, or longer, or about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks, or longer, or any half-life in between including all ranges in between, at an approximate physiological pH of about pH 7.4, at about 25°C or room temperature and / or at about 37°C or human body temperature (e.g., in vivo, in serum, in a given tissue, in a given species such as rat, mouse, monkey or human).

[0288] In some embodiments, the antibody or antigen-binding fragment thereof has a T of about or at least about 60, 62, 64, 66, 68, 70, 72, 74 or 75 °C m In some embodiments, the antibody or antigen-binding fragment thereof has a T m of about 60 °C or higher

[0289] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxic or chemotherapeutic agents. General examples of cytotoxic or chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, anthracyclines, antitumor antibiotics, platinum, type I topoisomerase inhibitors, type II topoisomerase inhibitors, vinca alkaloids, and taxanes. Specific examples of cytotoxic or chemotherapeutic agents include, but are not limited to, cyclophosphamide, thalidomide, lomustine (CCNU), melphalan, procarbazine, carmustine (BCNU), enzastaurin, busulfan, daunorubicin, doxorubicin, gefitinib, erlotinib idarubicin, temozolomide, epirubicin, mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin, camptothecin, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, temsirolimus, everolimus, vincristine, vinblastine, vinorelbine, vindesine, CT52923, paclitaxel, imatinib, dasatinib, sorafenib, pazopanib, sunitinib, batrafenib, gefitinib, erlotinib, AEE-788, dichloroacetate, tamoxifen, fasudil, SB-681323, semaxanib, donepezil, galantamine, memantine, rivastigmine, tacrine, rasigiline, naltrexone, lubiprostone, safinamide, istradefylline, pimavanserin, pitolisant, isradipine, pridopidine (ACR16), tetrabenazine, bexarotene, glatiramer acetate, fingolimod, and mitoxantrone, and their pharmaceutically acceptable salts and acids. Further examples of cytotoxic or chemotherapeutic agents include alkylating agents such as thiotepa, cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;Ethylenimine and methylmelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobomycin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chloroozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, aclarubicin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, calminomycin, cardinophilin, chromomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodomycin, streptozocin, streptonigrin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine;Dexamethasone; diacquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gasitocin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, for example, paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; zeloda; ibandronate; CPT-11; RFS2000, a topoisomerase inhibitor; difluoromethylomithine (DMFO); retinoid acid derivatives such as Targretin® (bexarotene), Panretin® (alitretinoin); ONTAK® (denileukin diftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.;

[0290] An antibody or antigen-binding fragment thereof can be used in any of the compositions, methods and / or kits described herein and can be combined with one or more of the immunotherapeutic agents described herein. Additional Therapeutic Agents and Compositions

[0291] Immunotherapy agent. In certain embodiments, one or more cancer immunotherapy agents are used. In certain examples, the immunotherapy agent modulates the immune response of a subject to increase or maintain, for example, a cancer-related immune response or a cancer-specific immune response, thereby resulting in inhibition or reduction of increased immune cells against cancer cells. Exemplary immunotherapy agents include polypeptides, such as antibodies and antigen-binding fragments thereof, ligands, as well as small peptides, and mixtures thereof. Immunotherapy agents also include small molecules, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapy agents or other polynucleotide-based agents, as well as viral agents such as oncolytic viruses and others known in the art. Thus, in certain embodiments, the cancer immunotherapy agent is selected from one or more of immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapies.

[0292] In certain embodiments, the cancer immunotherapy agent is an immune checkpoint modulator. Specific examples include "antagonists" of one or more inhibitory immune checkpoint molecules and "agonists" of one or more stimulatory immune checkpoint molecules. Generally, immune checkpoint molecules are components of the immune system that enhance (co-stimulatory molecules) or weaken signals, and targeting them has the potential for cancer treatment because cancer cells can disrupt the original function of immune checkpoint molecules (see, for example, Sharma and Allison, Science. 348:56-61, 2015; Topalian et al., Cancer Cell. 27:450-461, 2015; Pardoll, Nature Reviews Cancer. 12:252-264, 2012). In some embodiments, the immune checkpoint modulator (e.g., antagonist, agonist) "binds" or "specifically binds" to one or more immune checkpoint molecules as described herein.

[0293] In certain embodiments, the immune checkpoint modulating agent is a polypeptide or a peptide. The terms "peptide" and "polypeptide" are used interchangeably herein; however, in certain instances, the term "peptide" can refer to a shorter polypeptide, e.g., a polypeptide consisting of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 amino acids, as well as all integers and ranges therebetween (e.g., 5 - 10, 8 - 12, 10 - 15) of amino acids. Polypeptides and peptides can be composed of naturally occurring amino acids and / or non-naturally occurring amino acids, as described herein.

[0294] Antibodies are also included as polypeptides. Thus, in some embodiments, the immune checkpoint modulating polypeptide agent is an antibody or an "antigen-binding fragment thereof" as described elsewhere herein.

[0295] In some embodiments, the agent is a "ligand", e.g., the natural ligand of an immune checkpoint molecule or includes the same. A "ligand" generally refers to a substance or molecule that forms a complex with a target molecule (e.g., a biomolecule) to serve a biological purpose, and includes "protein ligands", which generally generate a signal by binding to a site on the target molecule or target protein. Thus, certain agents are, in fact, protein ligands that bind to an immune checkpoint molecule and generate a signal. Also included are "modified ligands", e.g., pharmacokinetic modifiers, such as a protein ligand fused to an Fc region derived from an immunoglobulin.

[0296] The binding properties of the polypeptide can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, the polypeptide specifically binds to a target molecule, such as an immune checkpoint molecule or an epitope thereof, and the equilibrium dissociation constant is about ≤10-7 to about 10-8 M, or in that range. In some embodiments, the equilibrium dissociation constant is about ≤10-9 M to about ≤10-10 M, or in that range. In certain exemplary embodiments, the polypeptide has an affinity (Kd or EC 50 ) for a target as described herein of about or at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50 nM, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50 nM (to which it specifically binds).

[0297] In some embodiments, the agent is a "small molecule", which refers to an organic compound that is of synthetic or biological origin (biomolecule), but is typically not a polymer. Organic compounds refer to a large class of compounds whose molecules contain carbon, typically excluding those that contain only carbonates, simple oxides of carbon or cyanides. "Biomolecule" generally refers to organic molecules produced by a living organism, including large polymeric molecules (biopolymers) such as peptides, polysaccharides and nucleic acids, as well as small molecules such as primary and secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins and hormones. "Polymer" generally refers to a large molecule or macromolecule typically composed of repeating structural units connected by covalent chemical bonds.

[0298] In certain embodiments, the small molecule has a molecular weight of about 1000 to 2000 Daltons or less than about 1000 to 2000 Daltons, typically about 300 to 700 Daltons, as well as about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, 1000 or 2000 Daltons, or less than about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, 1000 or 2000 Daltons.

[0299] Certain small molecules can have the "specific binding" characteristics described herein for polypeptides such as antibodies. For example, in some embodiments, the small molecule has a binding affinity (Kd or EC 50 ) of about or at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50 nM, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50 nM and specifically binds to a target, such as an immune checkpoint molecule.

[0300] In some embodiments, the immune checkpoint modulating agent is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules. Exemplary inhibitory immune checkpoint molecules include programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), programmed death 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), V domain Ig suppressor of T cell activation (VISTA), B and T lymphocyte attenuator (BTLA), CD160, and T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0301] In certain embodiments, the agent is an antagonist or inhibitor of PD-1 (receptor), and its targeting has been shown to restore immune function in the tumor microenvironment (see, e.g., Phillips et al., Int Immunol. 27:39-46, 2015). PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and is expressed in T cells and pro-B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 functions as an inhibitory immune checkpoint molecule, for example, by reducing or preventing T cell activation, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is achieved, at least in part, by a dual mechanism that promotes apoptosis of antigen-specific T cells in the lymph nodes while also reducing apoptosis in regulatory T cells (suppressor T cells). Some examples of antagonists or inhibitors of PD-1 include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1 and reduce one or more of its immunosuppressive activities, such as its downstream signaling or its interaction with PD-L1. Specific examples of antagonists or inhibitors of PD-1 include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pidilizumab, and their antigen-binding fragments (see, e.g., U.S. Patent Nos. 8,008,449; 8,993,731; 9,073,994; 9,084,776; 9,102,727; 9,102,728; 9,181,342; 9,217,034; 9,387,247; 9,492,539; 9,492,540; and U.S. Patent Application Publication Nos. 2012 / 0039906; 2015 / 0203579).

[0302] In some embodiments, the agent is an antagonist or inhibitor of PD-L1. As described above, PD-L1 is one of the natural ligands for the PD-1 receptor. General examples of antagonists or inhibitors of PD-L1 include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L1 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor. Specific examples of antagonists of PD-L1 include the antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), and their antigen-binding fragments (see, e.g., U.S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; 9,439,962).

[0303] In some embodiments, the agent is an antagonist or inhibitor of PD-L2. As described above, PD-L2 is one of the natural ligands for the PD-1 receptor. General examples of antagonists or inhibitors of PD-L2 include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L2 and reduce one or more of its immunosuppressive activities, such as its binding to the PD-1 receptor.

[0304] In some embodiments, the agent is an antagonist or inhibitor of CTLA-4. CTLA4 or CTLA-4 (cytotoxic T lymphocyte-associated protein 4), also known as CD152 (surface antigen classification 152), is a protein receptor that functions as an inhibitory immune checkpoint molecule, for example, by transmitting an inhibitory signal to T cells when it binds to CD80 or CD86 on the surface of antigen-presenting cells. General examples of antagonists or inhibitors of CTLA-4 include antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4. Specific examples include the antibodies ipilimumab and tremelimumab, and their antigen-binding fragments. At least some of the activity of ipilimumab is thought to be mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing of suppressor Tregs that express CTLA-4.

[0305] In some embodiments, the agent is an antagonist or inhibitor of IDO or an antagonist or inhibitor of TDO. IDO and TDO are tryptophan-degrading enzymes with immunosuppressive properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. General examples of antagonists or inhibitors of IDO and TDO include antibodies or antigen-binding fragments or small molecules that specifically bind to IDO or TDO (see, e.g., Platten et al., Front Immunol. 5: 673, 2014) and reduce or inhibit one or more immunosuppressive activities. Specific examples of antagonists or inhibitors of IDO include indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples of antagonists or inhibitors of TDO include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).

[0306] In some embodiments, the agent is an antagonist or inhibitor of TIM-3. T cell immunoglobulin domain and mucin domain 3 (TIM-3) is expressed in activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by causing cell death upon interaction with its ligand, galectin-9. TIM-3 contributes to an immunosuppressive tumor microenvironment, and its overexpression is associated with poor prognosis in various cancers (see, for example, Li et al., Acta Oncol. 54:1706-13, 2015). General examples of antagonists or inhibitors of TIM-3 include antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3 and reduce or inhibit one or more of its immunosuppressive activities.

[0307] In some embodiments, the agent is an antagonist or inhibitor of LAG-3. Lymphocyte activation gene-3 (LAG-3) is expressed in activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It negatively regulates T cell proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1 (see, for example, Workman and Vignali, European Journal of Immun. 33: 970-9, 2003; and Workman et al., Journal of Immun. 172: 5450-5, 2004), and has been reported to play a role in Treg suppressive function (see, for example, Huang et al., Immunity. 21: 503-13, 2004). LAG3 also maintains CD8+ T cells in a tolerant state and, in combination with PD-1, maintains the exhaustion of CD8 T cells. General examples of antagonists or inhibitors of LAG-3 include antibodies or antigen-binding fragments or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016 and its antigen-binding fragments.

[0308] In some embodiments, the agent is an antagonist or inhibitor of VISTA. V-domain Ig suppressor of T cell activation (VISTA) is an inhibitory immune checkpoint regulator that is mainly expressed in hematopoietic cells, suppresses T cell activation, induces Foxp3 expression, and is highly expressed in the tumor microenvironment that suppresses anti-tumor T cell responses (see, e.g., Lines et al., Cancer Res. 74:1924-32, 2014). General examples of antagonists or inhibitors of VISTA include antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA and reduce one or more of its immunosuppressive activities.

[0309] In some embodiments, the agent is an antagonist or inhibitor of BTLA. Expression of B and T lymphocyte attenuator (BTLA; CD272) is induced during T cell activation, which inhibits T cells through interaction with the tumor necrosis factor receptor (TNF-R) and the B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14) and is also known as herpesvirus entry mediator (HVEM). The BTLA-HVEM complex negatively regulates the immune response of T cells, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, e.g., Derre et al., J Clin Invest 120:157-67, 2009). General examples of antagonists or inhibitors of BTLA include antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA-4 and reduce one or more of its immunosuppressive activities.

[0310] In some embodiments, the agent is an antagonist or inhibitor of HVEM, for example, an antagonist or inhibitor that specifically binds to HVEM and prevents its interaction with BTLA or CD160. General examples of antagonists or inhibitors of HVEM include antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM and, optionally, reduce the interaction of HVEM / BTLA and / or HVEM / CD160, thereby reducing one or more of the immunosuppressive activities of HVEM.

[0311] In some embodiments, the agent is an antagonist or inhibitor of CD160, for example, an antagonist or inhibitor that specifically binds to CD160 and prevents its interaction with HVEM. General examples of antagonists or inhibitors of CD160 include antibodies or antigen-binding fragments or small molecules that specifically bind to CD160 and, optionally, reduce the CD160 / HVEM interaction, thereby reducing or inhibiting one or more of its immunosuppressive activities.

[0312] In some embodiments, the agent is an antagonist or inhibitor of TIGIT. T cell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor found on the surface of various lymphoid cells that suppresses antitumor immunity, for example, via Tregs (Kurtulus et al., J Clin Invest. 125:4053-4062, 2015). General examples of antagonists or inhibitors of TIGIT include antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT and reduce one or more of its immunosuppressive activities (see, for example, Johnstonet al., Cancer Cell. 26:923-37, 2014).

[0313] In certain embodiments, the immune checkpoint modulating agent is an agonist of one or more stimulatory immune checkpoint molecules. Exemplary stimulatory immune checkpoint molecules include OX40, CD40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0314] In some embodiments, the agent is an agonist of OX40. OX40 (CD134) promotes the expansion and proliferation of effector and memory T cells and inhibits the differentiation and activity of regulatory T cells (see, for example, Croft et al., Immunol Rev. 229:173-91, 2009). Its ligand is OX40L (CD252). Since OX40 signaling affects both T cell activation and survival, it plays an important role in the induction of antitumor immune responses in lymph nodes and the maintenance of antitumor immune responses in the tumor microenvironment. General examples of OX40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40 and increase one or more of its immune-stimulatory activities. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (mouse OX4 agonist), and MEDI6383 (OX40 agonist), and antigen-binding fragments thereof.

[0315] In some embodiments, the agent is an agonist of CD40. CD40 is expressed on antigen-presenting cells (APCs) and some malignancies. Its ligand is CD40L (CD154). In APCs, ligation results in upregulation of costimulatory molecules and potentially circumvents the need for T cell help in the anti-tumor immune response. CD40 agonist therapy plays an important role in APC maturation and their migration from tumors to lymph nodes, resulting in increased antigen presentation and T cell activation. Anti-CD40 agonist antibodies result in substantial responses and durable anti-cancer immunity in animal models, which is at least in part an effect mediated by cytotoxic T cells (see, for example, Johnson et al. Clin Cancer Res. 21: 1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). General examples of CD40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD40 and increase one or more of its immunostimulatory activities. Specific examples include CP-870,893, dacetuzumab, Chi Lob 7 / 4, ADC-1013, CD40L, rhCD40L, and antigen-binding fragments thereof.

[0316] In some embodiments, the agent is an agonist of GITR. Glucocorticoid-induced TNFR family-related gene (GITR) increases the expansion and proliferation of T cells, inhibits the suppressive activity of Tregs, and prolongs the survival of T effector cells. Agonists of GITR have been shown to promote an antitumor response through loss of Treg lineage stability (see, e.g., Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These diverse mechanisms indicate that GITR plays an important role in the induction of immune responses in lymph nodes and the maintenance of immune responses in tumor tissues. Its ligand is GITRL. General examples of GITR agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITR and increase one or more of its immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and antigen-binding fragments thereof.

[0317] In some embodiments, the agent is an agonist of CD137. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells, such as CD8+ T cells, from activation-induced cell death. General examples of CD137 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137 and increase one or more of its immunostimulatory activities. Specific examples include CD137 (or 4-1BB) ligand (see, e.g., Shao and Schwarz, J Leukoc Biol. 89:21-9, 2011), as well as the antibody utomilumab and antigen-binding fragments thereof.

[0318] In some embodiments, the agent is an agonist of CD27. Stimulation of CD27 increases antigen-specific expansion and proliferation of naive T cells and contributes to the long-term maintenance of T cell memory and T cell immunity. Its ligand is CD70. Targeting with an agonist antibody of human CD27 stimulates T cell activation and anti-tumor immunity (see, e.g., Thomas et al., Oncoimmunology. 2014;3:e27255. doi:10.4161 / onci.27255; and He et al., J Immunol. 191:4174-83, 2013). General examples of agonists of CD27 include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27 and increase one or more of its immunostimulatory activities. Specific examples include CD70, as well as the antibodies balstilimab and CDX-1127 (1F5), and their antigen-binding fragments.

[0319] In some embodiments, the agent is an agonist of CD28. CD28 is constitutively expressed in CD4+ T cells and some CD8+ cells. Its ligands include CD80 and CD86, and its stimulation increases the expansion and proliferation of T cells. General examples of agonists of CD28 include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD28 and increase one or more of its immunostimulatory activities. Specific examples include CD80, CD86, the antibody TAB08, and their antigen-binding fragments.

[0320] In some embodiments, the agent is an agonist of CD226. CD226 is a stimulatory receptor that shares a ligand with TIGIT, and unlike TIGIT, the engagement of CD226 enhances T cell activation (see, e.g., Kurtulus et al., J Clin Invest. 125:4053-4062, 2015; Bottino et al., J Exp Med. 1984:557-567, 2003; and Tahara-Hanaoka et al., Int Immunol. 16:533-538, 2004). General examples of agonists of CD226 include antibodies or antigen-binding fragments or small molecules or ligands (e.g., CD112, CD155) that specifically bind to CD226 and increase one or more of its immunostimulatory activities.

[0321] In some embodiments, the agent is an agonist of HVEM. Herpes virus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF-receptor superfamily. HVEM is found on a variety of cells, including T cells, APCs and other immune cells. Unlike other receptors, HVEM is expressed at high levels on resting T cells and is downregulated upon activation. HVEM signaling has been shown to play an important role in the early stages of T cell activation and during the expansion of tumor-specific lymphocyte populations in lymph nodes. General examples of agonists of HVEM include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM and increase one or more of its immunostimulatory activities.

[0322] In certain embodiments, the cancer immunotherapeutic agent is a cancer vaccine. Exemplary cancer vaccines include oncolytic phages, human papillomavirus (HPV) vaccines such as Gardasil or Cervarix, hepatitis B vaccines such as Engerix-B, Recombivax HB or Twinrix, and sipuleucel-T (Provenge). In some embodiments, the cancer vaccine comprises or utilizes one or more cancer antigens or cancer-related antigens.Exemplary cancer antigens include, but are not limited to, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, VEGFR-3, NRP2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin alpha v beta 3, integrin alpha 5 beta 1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PSMA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostate acid phosphatase, Lewis-Y antigen, GD2 (diasialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

[0323] In certain embodiments, the cancer immunotherapeutic agent is an oncolytic virus. Oncolytic viruses are viruses that preferentially infect and kill cancer cells. Natural and artificial or engineered oncolytic viruses are included. Most oncolytic viruses are engineered for tumor selectivity, but there are natural examples such as reovirus and the Seneca Valley virus SVV-001. General examples of oncolytic viruses include VSV, poliovirus, reovirus, Seneca virus, and RIGVIR, as well as engineered versions thereof. Non-limiting examples of oncolytic viruses include, inter alia, herpes simplex virus (HSV) and engineered versions thereof, talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus (NTX-010), Seneca virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS, and DNX-2401.

[0324] In certain embodiments, the cancer immunotherapeutic agent is a cytokine. Exemplary cytokines include interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte macrophage colony-stimulating factor (GM-CSF).

[0325] In certain embodiments, the cancer immunotherapy agent is a cell-based immunotherapy, such as adoptive immunotherapy based on T cells. In some embodiments, the cell-based immunotherapy includes cancer antigen-specific T cells, optionally including ex vivo-derived T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TIL), and peptide-induced T cells. In a specific embodiment, the CAR-modified T cells are targeted against CD-19 (see, e.g., Maude et al., Blood. 125:4017-4023, 2015).

[0326] In certain examples, the cancer to be treated is targeted or enriched for at least one antigen known to associate with a cancer antigen, i.e., cancer antigen-specific T cells associate with the cancer to be treated. In some embodiments, the cancer antigen is selected from one or more of CD19, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin alpha v beta 3, integrin alpha 5 beta 1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), phosphatase of regenerating liver 3 (PRL-3), prostate acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

[0327] Additional exemplary cancer antigens include 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4 / m, alpha-methylacyl-Coenzyme A racemase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, beta-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA 15-3 / CA 27-29, CA 19-9, CA72-4, CA125, calretinin, CAMEL, CASP-8 / m, cathepsin B, cathepsin L, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, coactosin-like protein, collagen XXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, hepsin, Her2 / neu, HERV-K-MEL, HLA-A *0201-R1 7I, HLA-A1 1 / m, HLA-A2 / m, HNE, homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1 R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205 / m, KK-LC-1, K-Ras / m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, matrix protein 22, MCI R, M-CSF, ME1 / m, mesothelin, MG50 / PXDN, MMP1 1, MN / CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class l / m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-B, NY-ESO-1, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS-9 / m, osteocalcin, osteopontin, pi 5, p190 minor bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 kinase, Pin-1, Pml / PAR alpha, POTE, PRAME, PRDX5 / m, prostain, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp1 7, SSX-1, SSX-2 / HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF-beta, TGF beta RII, TGM-4, TPI / m, TRAG-3, TRG, TRP-1, TRP-2 / 6b, TRP / INT2, TRP-p8, tyrosine kinase, UPA, VEGFR1, VEGFR-2 / FLK-1 and WT1. Certain preferred antigens include p53, CA125, EGFR, Her2 / neu, hTERT, PAP, MAGE-A1, MAGE-A3, mesothelin, MUC-1, GP100, MART-1, tyrosine kinase, PSA, PSCA, PSMA, STEAP-1, Ras, CEA and WT1, more preferably PAP, MAGE-A3, WT1 and MUC-1.

[0328] In some embodiments, the antigen is selected from MAGE-A1 (e.g., MAGE-A1 according to accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g., MAGE-A6 according to accession number NM_005363), MAGE-C1, MAGE-C2, Melan-A (e.g., Melan-A according to accession number NM_005511), GP100 (e.g., GP100 according to accession number M77348), Tyrosinase (e.g., Tyrosinase according to accession number NM_000372), Survivin (e.g., Survivin according to accession number AF077350), CEA (e.g., CEA according to accession number NM_004363), Her-2 / neu (e.g., Her-2 / neu according to accession number M11730), WT1 (e.g., WT1 according to accession number NM_000378), PRAME (e.g., PRAME of accession number NM_006115), EGFRI (Epidermal Growth Factor Receptor 1) (e.g., EGFRI (Epidermal Growth Factor Receptor 1) according to accession number AF288738), MUC1, Mucin-1 (e.g., Mucin-1 according to accession number NM_002456), SEC61G (e.g., SEC61G according to accession number NM_014302), hTERT (e.g., hTERT of accession number NM_198253), 5T4 (e.g., 5T4 according to accession number NM_006670), TRP-2 (e.g., TRP-2 according to accession number NM_001922), STEAP1 (Prostate Six-Transmembrane Epithelial Antigen 1), PSCA, PSA, PSMA, etc.

[0329] In some embodiments, the cancer antigen is selected from PCA, PSA, PSMA, STEAP, and optionally MUC-1, and fragments, variants and derivatives thereof. In some embodiments, the cancer antigen is selected from NY-ESO-1, MAGE-C1, MAGE-C2, Survivin, 5T4, and optionally, MUC-1, and fragments, variants and derivatives thereof.

[0330] In some examples, the cancer antigen includes idiotypic antigens associated with cancer or tumor diseases, particularly lymphoma or lymphoma-related diseases. For example, the idiotypic antigen is an immunoglobulin idiotypic of lymphoid blood cells or a T cell receptor idiotypic of lymphoid blood cells.

[0331] In some examples, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells (e.g., targeted to the cancer antigen), T cell receptor (TCR)-modified T cells, tumor infiltrating lymphocytes (TIL), and peptide-induced T cells.

[0332] Those skilled in the art will understand that the various cancer immunotherapeutic agents described herein can be combined with any one or more of the various anti-NRP2 antibodies (including their antigen-binding fragments) described herein and used according to any one or more of the methods or compositions described herein.

[0333] Chemotherapeutic agent. Certain embodiments use one or more chemotherapeutic agents, e.g., small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include, inter alia, alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or II), and microtubule inhibitors.

[0334] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (e.g., procarbazine and hexamethylmelamine, as required).

[0335] Examples of antimetabolites include folic acid antagonists (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine). Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, melvalonate, and aclarubicin.

[0336] Examples of antimicrotubule agents include taxanes (e.g., paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine).

[0337] One of ordinary skill in the art will understand that the various chemotherapeutic agents described herein can be combined with any one or more of the various anti-NRP2 antibodies (including their antigen-binding fragments) described herein and used according to any one or more of the methods or compositions described herein.

[0338] Hormonal therapeutic agents. In certain embodiments, at least one hormonal therapeutic agent is used. General examples of hormonal therapeutic agents include hormonal agonists and hormonal antagonists. Specific examples of hormonal agonists include progestogens (progestins), corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone...

Claims

1. A therapeutic composition comprising at least one antibody or antigen-binding fragment thereof (anti-NRP2 antibody) that specifically binds to a human neuropilin-2 (NRP2) polypeptide, wherein the at least one antibody or antigen-binding fragment thereof Complementary determining region V Ｈ CDR1, V Ｈ CDR2 and V Ｈ Heavy chain variable region (V Ｈ ) sequence containing the CDR3 sequence, as well as complementary determining region V Ｌ CDR1, V Ｌ CDR2 and V Ｌ Light chain variable region (V Ｌ ) sequence containing the CDR3 sequence comprises said V Ｈ CDR1, V Ｈ CDR2 and V Ｈ The CDR3 sequences each contain SEQ ID NOs: 19 to 21, and said V Ｌ CDR1, V Ｌ CDR2 and V Ｌ The CDR3 sequences each contain SEQ ID NOs: 22 to 24, a therapeutic composition.

2. Said V Ｈ The array contains an array that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO: 49, and said V Ｌ The therapeutic composition according to claim 1, wherein the array contains an array that is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to SEQ ID NO:

50.

3. Said V Ｈ array contains SEQ ID NO: 49, and said V Ｌ array contains SEQ ID NO: 50, the therapeutic composition according to claim 2.

4. The therapeutic composition according to claim 1 or 2, wherein the at least one antibody or antigen-binding fragment thereof comprises an Fc domain of IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4) or IgM, optionally a human Fc domain or hybrids and / or variants thereof.

5. The therapeutic composition according to claim 4, wherein the at least one antibody or antigen-binding fragment thereof comprises an Fc domain of IgG having high effector function in humans, optionally an Fc domain of IgG1 or IgG3.

6. The therapeutic composition according to claim 4, wherein the at least one antibody or antigen-binding fragment thereof comprises an Fc domain of IgG having low effector function in humans, optionally an Fc domain of IgG2 or IgG4.

7. The therapeutic composition according to claim 6, wherein the at least one antibody or antigen-binding fragment thereof comprises an Fc domain of IgG1 or IgG4 selected from Table F1, optionally.

8. The therapeutic composition according to any one of claims 1 to 7, wherein the at least one antibody or antigen-binding fragment thereof comprises a modified Fc domain of IgG1 or IgG4 with altered binding to FcRn, and optionally, the modified Fc domain of IgG1 or IgG4 comprises any one or more of the mutations of YD (M252Y / T256D), DQ (T256D / T307Q), DW (T256D / T307W), YTE (M252Y / S254T / T256E), AAA (T307A / E380A / N434A), LS (M428L / N434S), M252Y, T256D / E, K288D / N, T307Q / W, E380C, N434FY and / or Y436H / N / W (EU numbering), and combinations thereof.

9. The therapeutic composition according to any one of claims 1 to 8, wherein the at least one antibody or antigen-binding fragment thereof is a monoclonal antibody and / or a humanized antibody.

10. The therapeutic composition according to any one of claims 1 to 9, wherein the at least one antibody or antigen-binding fragment thereof is an Fv fragment, a single-chain Fv (scFv) polypeptide, an adnectin, an anticalin, an aptamer, an avimer, a camel antibody, a designed ankyrin repeat protein (DARPin), a minibody, a nanobody or a unibody.

11. The therapeutic composition according to any one of claims 1 to 10, wherein the at least one antibody or antigen-binding fragment has a purity of at least about 80%, 85%, 90%, 95%, 98% or 99% on a protein basis and is substantially non-aggregated.

12. The therapeutic composition according to any one of claims 1 to 11, which is substantially endotoxin-free.

13. The therapeutic composition according to any one of claims 1 to 12, which is a sterile injectable solution, preferably a sterile injectable solution suitable for intravenous, intramuscular, subcutaneous or intraperitoneal administration, as required.

14. The therapeutic composition according to any one of claims 1 to 13 for treating a disease or condition in a subject in need of treatment of the disease or condition.

15. The therapeutic composition according to claim 14, wherein the disease or condition is an NRP2-related disease or condition.

16. The disease or condition is selected from one or more of cancer and diseases and pathways associated with cancer, including cancer cell growth, induction, migration, adhesion, invasion, chemoresistance, and / or metastasis; diseases associated with inappropriate activation or migration of immune cells such as graft-versus-host disease (GVHD), including inflammatory, autoimmune, and related inflammatory diseases; diseases associated with lymphangiogenesis, lymphangioma, lymphovenous angiogenesis, and lymphatic vessel injury, including edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability; diseases associated with infection, including latent infection; allergic disorders / diseases, diseases associated with allergic reactions, including, for example, allergic disorders / diseases, diseases associated with allergic reactions, including, for example, chronic obstructive pulmonary disease (COPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, systemic lupus erythematosus, rheumatoid arthritis, inflammasome-related diseases, and skin-related neutrophil-mediated diseases such as pyoderma gangrenosum; diseases associated with granulomatous inflammatory diseases, including sarcoidosis and granuloma; diseases associated with fibrosis, including fibrosis, endothelial-mesenchymal transition (EMT), and wound healing; diseases associated with inappropriate smooth muscle contractility, smooth muscle compensation and decompensation, and inappropriate migration and adhesion of vascular smooth muscle cells; diseases associated with inappropriate autophagy, phagocytosis, and efferocytosis; diseases associated with inappropriate migratory cell movement; neurological diseases, peripheral nervous system remodeling, and diseases associated with pain sensation; and diseases associated with bone development and bone remodeling, the therapeutic composition according to claim 14 or 15.

17. The disease is cancer, and optionally, the cancer expresses or overexpresses NRP2, and optionally, the cancer exhibits NRP2-dependent growth, NRP2-dependent adhesion, NRP2-dependent migration, and / or NRP2-dependent invasion, the therapeutic composition according to claim 16.

18. The cancer expresses or overexpresses NRP2 but does not substantially express neuropilin-1 (NRP1), the therapeutic composition according to claim 17.

19. The subject has lymphedema, the therapeutic composition according to any one of claims 16 to 18.

20. The therapeutic composition according to any one of claims 14 to 19, wherein the therapeutic composition is administered to the subject in combination with at least one additional agent selected from one or more of a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent, and a kinase inhibitor.

21. A cell growth device comprising a human or humanized anti-NRP2 antibody or an antigen-binding fragment thereof defined according to any one of claims 1 to 13, a population of engineered cells in which at least one cell contains one or more polynucleotides encoding the anti-NRP2 antibody or an antigen-binding fragment thereof, at least about 10 liters of serum-free growth medium, and a sterile container.

22. The therapeutic composition according to claim 17, wherein the cancer is prostate cancer or glioblastoma multiforme.

23. The therapeutic composition according to claim 20, wherein the cancer immunotherapeutic agent is selected from an immune checkpoint modulating agent, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapy.

24. The therapeutic composition according to claim 23, wherein the immune checkpoint modulating agent is a PD-1 or PD-L1 antagonist, or the cell-based immunotherapy comprises chimeric antigen receptor (CAR)-modified T cells.

25. The therapeutic composition according to claim 24, wherein the disease or condition is prostate cancer or glioblastoma multiforme.

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