Compositions and methods comprising anti-NRP2 antibodies

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Patent Application No. 62 / 653,823, filed April 6, 2018, which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The text file containing the sequence listing is named ATYR_134_01WO_ST25.txt. The text file is 228KB, was created on April 5, 2019, and was submitted electronically via EFS-Web. Technical Field

[0005] Embodiments of the present disclosure relate to antibodies and antigen-binding fragments thereof that specifically bind to human neuropilin-2 (NRP2) polypeptides, including antibodies and antigen-binding fragments thereof that modulate the binding interaction between human NRP2 and at least one NRP2 ligand (e.g., human histidyl-tRNA synthetase (HRS)) and thereby modulate subsequent NRP2-mediated downstream signaling events, including related therapeutic compositions and methods for modulating NRP2 activity and treating diseases such as NRP2-related diseases. Background Art

[0006] Recent advances have revealed that tRNA synthetases, beyond their well-characterized roles in protein synthesis, also play important roles in cellular responses. Specifically, it is increasingly recognized that tRNA synthetases play a range of previously unknown roles in responses to cellular stress and tissue homeostasis, both within and outside the cell.

[0007] The resokine protein family (HRS polypeptides) are derived from the histidyl-tRNA synthetase gene (HARS) by proteolysis or alternative splicing and are important regulators of intracellular and extracellular activity. Extracellular HARS can be easily detected in the blood circulation of normal healthy volunteers, and autoantibodies to HARS (Jo-1 antibodies) have been characterized in subjects with inflammatory myopathy (IM) and inflammatory lung disease (ILD). Although the role of Jo-1 antibodies in disease progression is unclear, subjects with inflammatory myopathies who have Jo-1 antibodies tend to be less likely to develop cancer than subjects without Jo-1 antibodies (see, e.g., Lu et al., PLOS ONE 9(4)e94128, 2014; Modan et al., Clin. Exp. Dermatol. 34(5)561-565, 2009; and Shi et al., J. Rheum 44(7)doi 10.3899 / jrheum.161480).

[0008] Significant progress has been made in elucidating the roles of extracellular HARS-derived proteins, including the identification of a putative cellular receptor, neuropilin-2 (NRP2 ​​or NRP-2). The interaction of HARS with NRP2 appears to be mediated by the N-terminal region of HARS and may lead to important changes in the cellular function of NRP2.

[0009] Thus, the current discovery of the Reoskine / neuropilin-2 axis represents a previously unknown mechanism that acts as a core regulator of cellular processes including, for example, axon guidance, endocytosis, cell migration, proliferation, survival, apoptosis, lymphangiogenesis, cell differentiation, and cell attachment, which are directly related to the initiation, growth, and metastasis of cancer, as well as muscle, vascular, neuronal, bone, and immune homeostasis. Abnormalities in any of these processes may lead to a range of diseases that can be addressed by developing anti-NRP2 antibodies that selectively target the Reoskine / neuropilin-2 axis. The present disclosure provides such antibodies and related embodiments. Summary of the Invention

[0010]

[0014] Embodiments of the present disclosure include therapeutic compositions comprising at least one antibody or antigen-binding fragment thereof that specifically binds to a human neuropilin-2 (NRP2) polypeptide (anti-NRP2 antibody).

[0011] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1, optionally at an expression level of about 10 pM to about 500 pM or to about 50 nM, or about, at least about, or no more than about 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 110 pM, 120 pM, 130 pM, 140 pM, 150 pM, pM, 160 pM, 170 pM, 180 pM, 190 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 10 nM, 25 nM or 50 nM, or optionally binds with an affinity ranging from 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 In some cases, the at least one antibody or antigen-binding fragment thereof specifically binds to the human NRP2 polypeptide in its native form but does not substantially bind to the human NRP2 polypeptide in its denatured form.

[0012] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in a neuropilin domain selected from one or more of the following: neuropilin A1 domain, neuropilin A2 domain, neuropilin B1 domain, neuropilin B2 domain, neuropilin C domain, neuropilin A1 / A2 combined domain, neuropilin B1 / B2 combined domain, neuropilin A2 / B1 combined domain, neuropilin B2 / C combined domain, neuropilin A2 / B1 / B2 combined domain, neuropilin A2 / B1 / B2 / C combined domain, neuropilin The A1 / A2 / B1 combination domain, the neuropilin A1 / A2 / B1 / B2 combination domain, the neuropilin A1 / A2 / B1 / B2 / C combination domain, and the neuropilin B1 / B2 / C combination domain, are optionally present in an amount of about 10 pM to about 500 pM or to about 50 nM, or about, at least about, or no more than about 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 110 pM, 120 pM, 130 pM, 140 pM, 150 pM, 160 pM, 170 pM, 180 pM, 190 pM, 200 pM, 300 pM, 400 pM, 00 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or optionally binds with an affinity ranging from 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. 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.

[0013] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin A1 domain, the neuropilin A2 domain, and / or the neuropilin A1A2 combined domain, including the adjacent linker region, e.g., at approximately the following residues:

[0014] (neuropilin A1 domain) 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 in SEQ ID NO: 1 (FL human NRP2); or

[0015] (Neuropilin A2 domain) 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 0, 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, as in SEQ defined by ID NO: 1 (FL human NRP2); or

[0016] (Combined A1A2 domains) 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, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 20-140, 20-150, 20-160, 20-170, 20-180, 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 SEQ ID NO: 1 (FL human NRP2).

[0017] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin Bl domain (SEQ ID NO: 12), the neuropilin B2 domain (SEQ ID NO: 13), and / or the neuropilin B1 / B2 combined domain (SEQ ID NO: 20), including the adjacent linker region, e.g., at approximately the following residues:

[0018] (Neuropilin B1 domain) 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, as defined by SEQ ID NO: 1 (FL human NRP2);

[0019] (Neuropilin B2 domain) 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, as defined in SEQ ID NO: 1 (FL human NRP2); or

[0020] (Neuropilin B1 / B2 combined domain) 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 91, 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, 578-5 91, 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 SEQ ID NO: 1 (FL human NRP2).

[0021] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin A2 / B1 combined domain and / or the neuropilin B2C combined domain, including the adjacent linker region, e.g., at approximately the following residues:

[0022] (Neuropilin A2B1 combined domain) 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 26, 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 SEQ defined by ID NO: 1 (FL human NRP2); or

[0023] (Neuropilin B2C assembly domain) 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, 438-634 8-614, 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 SEQ ID NO: 1 (FL human NRP2).

[0024] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin C domain, including the adjacent linker region, e.g., at approximately the following 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, 94, 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 SEQ ID NO: 1 (FL human NRP2).

[0025] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin B1 / B2 / C combined domain, including the adjacent linker region, e.g., at approximately the following 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, 487-794, 496-794, 506-794, 516-794, 526-794, 536-794, 546-794, 556-794, 566-794, 576-794, 587-794, 596-794, 5 794, 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,

[0026] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to a conformational epitope comprised of two or more discontinuous epitope regions. In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to a conformational epitope comprising or consisting of:

[0027] (a) a first epitope region within the A1 domain and a second epitope region within the A2 domain of the human NPR2 polypeptide;

[0028] (b) a first epitope region within the A1 domain and a second epitope region within the B1 domain of the human NPR2 polypeptide;

[0029] (c) a first epitope region within the A1 domain and a second epitope region within the B2 domain of the human NPR2 polypeptide;

[0030] (d) a first epitope region within the A1 domain and a second epitope region within the C domain of the human NPR2 polypeptide;

[0031] (e) a first epitope region within the A2 domain and a second epitope region within the B1 domain of the human NPR2 polypeptide;

[0032] (f) a first epitope region within the A2 domain and a second epitope region within the B2 domain of the human NPR2 polypeptide;

[0033] (g) a first epitope region within the A2 domain and a second epitope region within the C domain of the human NPR2 polypeptide;

[0034] (h) a first epitope region within the B1 domain and a second epitope region within the B2 domain of the human NPR2 polypeptide;

[0035] (i) a first epitope region within the B1 domain and a second epitope region within the C domain of the human NPR2 polypeptide; or

[0036] (j) a first epitope region within the B2 domain and a second epitope region within the C domain of the human NPR2 polypeptide.

[0037] In some embodiments, the at least one antibody or antigen-binding fragment thereof modulates (e.g., interferes with) the binding of the human NRP2 polypeptide to at least one NRP2 ligand (e.g., an NRP2 ligand selected from Table N2 or Table N3 and / or a human histidyl-tRNA synthetase (HRS) polypeptide selected from Table H1). In some embodiments, the at least one NRP2 ligand is a HRS splice variant selected from Table H1, e.g., a HRS splice variant selected from one or more of SV9 (HRS (1-60)), SV11 (HRS (1-60) + (399-509)), and SV14 (HRS (1-100) + (399-509)).

[0038] In some embodiments, the at least one antibody or antigen-binding fragment thereof is a blocking antibody that, when pre-incubated with the human NRP2 polypeptide in stoichiometric equivalents, inhibits about or at least about 80-100% of the theoretical maximal binding between the human NRP2 polypeptide and the at least one NRP2 ligand, optionally about or at least about 80%, 85%, 90%, 95% or 100% of the theoretical maximal binding.

[0039] In some embodiments, the at least one antibody or antigen-binding fragment thereof is a partially blocking antibody that, upon preincubation with the human NRP2 polypeptide in stoichiometric equivalents, inhibits about or at least about 20-80% of the theoretical maximal binding between the human NRP2 polypeptide and the at least one NRP2 ligand, optionally about or at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% of the theoretical maximal binding.

[0040] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to the HRS polypeptide interacting region of the NRP2 polypeptide and mimics or agonizes one or more signaling activities of the HRS polypeptide to which the NRP2 polypeptide is bound.

[0041] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to the HRS polypeptide-interacting region of the NRP2 polypeptide and modulates the binding / signaling activity between the NRP2 polypeptide and at least one NRP2 ligand. In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the at least one NRP2 ligand. In some embodiments, the at least one antibody or antigen-binding fragment thereof agonizes or enhances the binding / signaling activity between the NRP2 polypeptide and the at least one NRP2 ligand.

[0042] In some embodiments, the at least one NRP2 ligand is selected from one or more of the following:

[0043] -VEGF selected from one or more of VEGF-A145, VEGF-A165, VEGF-C, VEGF-D and PIGF-2;

[0044] - a VEGF receptor (VEGFR) selected from the group consisting of VEGFR2 and VEGFR3;

[0045] - a semaphorin selected from one or more of SEMA-3B, SEMA-3C, SEMA-3D, SEMA-3F and SEMA-3G;

[0046] - a plexin selected from one or more of plexins A1, A2, A3, A4 and D1;

[0047] - a growth factor selected from one or more of fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF);

[0048] - 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);

[0049] -Galectins or galectin receptors;

[0050] - a transcription factor selected from the group consisting of FAC1 and bromoprotein PHD finger transcription factors;

[0051] - an adaptor protein selected from one or more of GIPC1, GIPC2 and GIPC3;

[0052] - an integrin selected from Table N3, optionally α V β1, α V β3, α V β5, α V β6, α V One or more of β8, α6β1, and α6β4;

[0053] - Transforming growth factor β, which is selected from one or more of TGFβ1, TGFβ2, TGFβ3 and their corresponding TGFβ receptors; and

[0054] - a HRS polypeptide selected from Table H1, optionally a HRS splice variant selected from one or more of the following: 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).

[0055] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the plexin receptor and / or scutellum protein, without substantially modulating the binding / signaling activity between the NRP2 polypeptide and VEGFR3 or VEGF-C.

[0056] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the plexin receptor and / or scutellum protein, while not substantially modulating the binding / signaling activity between the NRP2 polypeptide and the HRS polypeptide.

[0057] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the plexin receptor and / or scutellum protein, but does not substantially modulate the binding / signaling activity between the NRP2 polypeptide and the HRS polypeptide, and does not substantially modulate the binding / signaling activity between the NRP2 polypeptide and VEGFR3 or VEGF-C.

[0058] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and VEGR3 without substantially modulating the binding / signaling activity between the NRP2 polypeptide and the plexin receptor and / or schizolin.

[0059] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and VEGR3 or VEGF-C, but does not substantially modulate the binding / signaling activity between the NRP2 polypeptide and the HRS polypeptide.

[0060] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the plexin receptor without substantially modulating ligand binding of schirmlin 3 to NRP2.

[0061] In some embodiments, the plexin receptor is selected from plexin A1, A2, A3, A4, and D1. In some embodiments, the sagittal protein is selected from sagittal protein 3B, 3C, 3D, 3F, and 3G.

[0062] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the human NRP2 A2 domain, the epitope comprising at least 5 contiguous amino acids of SEQ ID NO: 11, wherein the 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, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within amino acids 232-242 of human NRP2 SEQ ID NO: 1.

[0063] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the human NRP2 B1 domain, the epitope comprising at least 5 contiguous amino acids of SEQ ID NO: 12, wherein the at least one antibody or antigen-binding fragment thereof selectively inhibits receptor dimerization between NRP2 and FLT4 (VEGFR3) without substantially inhibiting dimerization between NRP2 and Plexin A1.

[0064] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the human NRP2 B2 domain, said epitope comprising at least 5 contiguous amino acids of SEQ ID NO: 13, wherein the 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.

[0065] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the human NRP2 C domain, the epitope comprising at least 5 contiguous amino acids of SEQ ID NO: 14, wherein the 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).

[0066] In some embodiments, the at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC) for each of (i) a human NRP2 polypeptide and (ii) a corresponding region of a cynomolgus monkey NRP2 polypeptide. 50), wherein 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 nM to about 1.2 nM, about 0.9 nM to about 5.5 nM, about 0.9 nM to about 5 nM, or about 1 nM to about 10 nM.

[0067] In some embodiments, the at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC) for each of (i) a human NRP2 polypeptide and (ii) a corresponding region of a murine NRP2 polypeptide. 50 ), wherein 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.

[0068] In some embodiments, the at least one antibody or antigen-binding fragment thereof comprises:

[0069] Heavy chain variable region (V H ) sequence, the V H The sequence includes a complementary determining region V selected from Table A1 that specifically binds to the human NRP2 polypeptide H CDR1, V H CDR2 and V H CDR3 sequences and variants thereof; and

[0070] Light chain variable region (V L ) sequence, the V L The sequence includes a complementary determining region V selected from Table A1 that specifically binds to the human NRP2 polypeptide L CDR1, V L CDR2 and V L CDR3 sequences and variants thereof,

[0071] The invention also provides an affinity matured variant comprising the aforementioned items that specifically binds to the human NRP2 polypeptide.

[0072] In a specific embodiment:

[0073] The V H CDR1, V H CDR2 and V HThe CDR3 sequences include SEQ ID NOs: 23-25, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 26-28, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0074] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 29-31, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 32-34, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in the one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0075] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 35-37, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 38-40, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0076] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 41-43, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 44-46, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in the one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0077] The V H CDR1, V H CDR2 and V HThe CDR3 sequences include SEQ ID NOs: 47-49, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 50-52, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0078] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 53-55, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 56-58, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0079] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 59-61, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 62-64, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0080] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 65-67, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 68-70, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0081] The V H CDR1, V H CDR2 and V HThe CDR3 sequences include SEQ ID NOs: 71-73, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 74-76, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0082] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 77-79, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 80-82, respectively, including variants thereof, wherein the variants have 1, 2, 3, 4, or 5 alterations in one or more CDRs and specifically bind to the human NRP2 polypeptide;

[0083] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 83-85, respectively, and the V L CDR1, V L CDR2 and V L The CDR3 sequences include SEQ ID NOs: 86-88, respectively, including variants thereof having 1, 2, 3, 4 or 5 alterations in one or more CDRs and specifically binding to the human NRP2 polypeptide.

[0084] In some embodiments, the at least one antibody or antigen-binding fragment thereof comprises an IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), or IgM Fc domain, optionally a human Fc domain or a hybrid and / or variant thereof. In some embodiments, the at least one antibody or antigen-binding fragment thereof comprises an IgG Fc domain with high effector function in humans, optionally an IgG1 or IgG3 Fc domain. In some embodiments, the at least one antibody or antigen-binding fragment thereof comprises an IgG Fc domain with low effector function in humans, optionally an IgG2 or IgG4 Fc domain. In some embodiments, the at least one antibody or antigen-binding fragment thereof comprises an IgG Fc domain, optionally an IgG2 or IgG4 Fc domain, optionally selected from Table F1.

[0085] In some embodiments, the at least one antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the at least one antibody or antigen-binding fragment thereof is a humanized antibody. In some embodiments, the at least one antibody or antigen-binding fragment thereof is an Fv fragment, a single-chain Fv (scFv) polypeptide, an adnectin, anticalin, aptamer, avimer, camelid antibody, designed ankyrin repeat protein (DARPin), minibody, nanobody, or unibody.

[0086] In some embodiments, the composition is at least about 80%, 85%, 90%, 95%, 98% or 99% pure with respect to the at least one antibody or antigen-binding fragment, calculated as protein, and is substantially free of aggregates. In some embodiments, the therapeutic composition is substantially free of endotoxins.

[0087] In some embodiments, the therapeutic composition is a sterile injectable solution, optionally suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration.

[0088] In certain 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 regulator, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapy. In some embodiments, the immune checkpoint regulator is a polypeptide, optionally an antibody or an antigen-binding fragment thereof, or a ligand or a small molecule. In some embodiments, the immune checkpoint regulator comprises:

[0089] (a) antagonists of inhibitory immune checkpoint molecules; or

[0090] (b) agonists that stimulate immune checkpoint molecules,

[0091] For example, optionally wherein the immune checkpoint modulator specifically binds to the immune checkpoint molecule.

[0092] In some embodiments, the inhibitory immune checkpoint molecule is selected from one or more of the following: programmed death-ligand 1 (PD-L1), programmed death receptor 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), T cell activation V domain Ig inhibitor (VISTA), B and T lymphocyte attenuation factor (BTLA), CD160, herpes virus entry mediator (HVEM) and T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0093] In some embodiments, the antagonist is a PD-L1 and / or PD-L2 antagonist optionally selected from one or more of the following: 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);

[0094] The antagonist is a PD-1 antagonist optionally selected from one or more of the following: 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;

[0095] The antagonist is a CTLA-4 antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, and tremelimumab;

[0096] The antagonist is an IDO antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to IDO, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat;

[0097] The antagonist is a TDO antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91, and LM10;

[0098] The antagonist is a TIM-3 antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to TIM-3;

[0099] The antagonist is a LAG-3 antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3 and BMS-986016;

[0100] The antagonist is a VISTA antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to VISTA;

[0101] The antagonist is a BTLA, CD160 and / or HVEM antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to BTLA, CD160 and / or HVEM; and / or

[0102] The antagonist is a TIGIT antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or a small molecule that specifically binds to TIGIT.

[0103] In some embodiments, the stimulatory immune checkpoint molecule is selected from one or more of the following: OX40, CD40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226 and herpes virus entry mediator (HVEM).

[0104] In some embodiments, the agonist is an OX40 agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40, OX86, Fc-OX40L, and GSK3174998;

[0105] The agonist is a CD40 agonist optionally selected from one or more of the following: 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;

[0106] The agonist is a GITR agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1 and MEDI1873;

[0107] The agonist is a CD137 agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD137, utomilumab, and a 4-1BB ligand;

[0108] The agonist is a CD27 agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27, varlilumab, and CDX-1127 (1F5);

[0109] The agonist is a CD28 agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28 and TAB08; and / or

[0110] The agonist is an HVEM agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to HVEM.

[0111] In some embodiments, the cancer vaccine is selected from one or more of the following: Oncophage; human papillomavirus (HPV) vaccine, optionally Gardasil or Cervarix; hepatitis B vaccine, optionally Engerix-B, Recombivax HB or Twinrix; and sipuleucel-T (Provenge), or the cancer vaccine comprises one or more cancer antigens selected from the group consisting 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 (VE) GF (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), guanylate cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein α (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 TRA The antibodies included IL-17 receptor 2 (IL-17R2), 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 receptor-1, protein disulfide isomerase (PDI), phosphatase for liver regeneration 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin.

[0112] In some embodiments, the oncolytic virus is selected from one or more of the following: Talilakh (talimogenelaherparepvec, T-VEC), Coxsackievirus A21 (coxsackievirus A21, CAVATAKTM ), Oncorine (H101), pelareorep ( ), Seneca Valley virus (NTX-010), Seneca virus SVV-001 (Senecavirus SVV-001), ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS and DNX-2401. In some embodiments, the cytokine is selected from one or more of the following: interferon (IFN) -α, IL-2, IL-12, IL-7, IL-21 and granulocyte-macrophage colony stimulating factor (GM-CSF). In some embodiments, the cell-based immunotherapeutic agent includes cancer antigen-specific T cells, optionally ex vivo derived T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of the following: chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor infiltrating lymphocytes (TIL) and peptide-induced T cells.

[0113] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of the following: an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent.

[0114] In some embodiments, the alkylating agent is selected from one or more of the following: nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine (fo Tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mytomycin, and diaziquone (AZQ)), cisplatin and its derivatives (optionally carboplatin and oxaliplatin), and atypical alkylating agents (optionally procarbazine and hexamethylmelamine);

[0115] The antimetabolite is selected from one or more of the following: an antifolate (optionally methotrexate and pemetrexed), a fluoropyrimidine (optionally 5-fluorouracil and capecitabine), a deoxynucleoside analog (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine and pentostatin) and a thiopurine (optionally thioguanine and mercaptopurine);

[0116] The cytotoxic antibiotic is selected from one or more of the following: anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin;

[0117] The topoisomerase inhibitor is selected from one or more of the following: camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, aclarubicin; and / or

[0118] The anti-microtubule agent is selected from one or more of the following: a taxane (optionally paclitaxel and docetaxel) and a vinca alkaloid (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0119] In some embodiments, the at least one hormone therapy agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of the following: progestogen (progesterone), corticosteroids (optionally prednisolone, methylprednisolone or dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis 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)-β, androgen, estrogen and somatostatin analogs. In some embodiments, the hormone antagonist is selected from one or more of the following: a hormone synthesis inhibitor, optionally an aromatase inhibitor or gonadotropin-releasing hormone (GnRH) or an analog thereof; and a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an anti-androgen; or an antibody to a hormone receptor, optionally cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, robatumumab, alacizumab, or a combination thereof. pegol), bevacizumab, icrucumab, ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomozuotuximab, zalutumumab, aprutumabixadotin, bemarituzumab, olaratumab, or tovetumab.

[0120] In some embodiments, the kinase inhibitor is selected from one or more of the following: adavosertib, afanitib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemuafenib.

[0121] Also included are methods 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, wherein the at least one antibody or antigen-binding fragment thereof, e.g., as a therapeutic composition described herein, modulates (e.g., interferes with) binding of the human NRP2 polypeptide to at least one NRP2 ligand (e.g., an NRP2 ligand from Table N2 or Table N3 and / or a human histidyl-tRNA synthetase (HRS) polypeptide from Table H1).

[0122] In some embodiments, the disease or condition is an NRP2-related disease or condition. In some embodiments, the NRP2-related disease or condition is selected from one or more of the following: cancer and cancer-related diseases and pathways, including cancer cell growth, initiation, migration, adhesion, invasion and / or metastasis; diseases associated with inflammation, autoimmunity and related inflammatory diseases, including diseases associated with inappropriate immune cell activation or migration, such as graft-versus-host disease (GVHD); diseases associated with lymphatic development, lymphangiogenesis and lymphatic damage, 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; diseases associated with allergic conditions / diseases, allergic reactions, including, for example, chronic obstructive pulmonary disease (COPD), neutrophilic asthma, 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 related to granulomatous inflammatory diseases, including sarcoidosis and granulomas; diseases related to fibrosis, including fibrosis, endothelial-mesenchymal transition (EMT), and wound healing; diseases related to inappropriate smooth muscle contractility and inappropriate vascular smooth muscle cell migration and adhesion; diseases related to inappropriate autophagy, phagocytosis, and efferocytosis; diseases related to inappropriate migratory cell migration; diseases related to neuronal diseases, peripheral nervous system remodeling, and pain perception; and diseases related to skeletal development and skeletal remodeling.

[0123] In some embodiments, the disease is cancer, e.g., wherein the cancer expresses or overexpresses NRP2. In some embodiments, 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).

[0124] Also included are methods for reducing or preventing recurrence of cancer in a subject in need thereof, wherein administration of the therapeutic composition generates immune memory against the cancer. In some embodiments, the subject has or is at risk of developing diabetes.

[0125] Certain methods include administering to the subject 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, e.g., as described herein. 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, e.g., as a therapeutic composition described herein.

[0126] In some embodiments, the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint regulator, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapy. In some embodiments, the immune checkpoint regulator is a polypeptide, optionally an antibody or an antigen-binding fragment thereof, a ligand, or a small molecule. In some embodiments, the immune checkpoint regulator includes:

[0127] (a) antagonists of inhibitory immune checkpoint molecules; or

[0128] (b) Agonists that stimulate immune checkpoint molecules.

[0129] For example, wherein the immune checkpoint regulator specifically binds. In some embodiments, the inhibitory immune checkpoint molecule is selected from one or more of the following: programmed death-ligand 1 (PD-L1), programmed death receptor 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), T cell activation V domain Ig inhibitor (VISTA), B and T lymphocyte attenuation factor (BTLA), CD160, herpes virus entry mediator (HVEM) and T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0130] In some embodiments, the antagonist is a PD-L1 and / or PD-L2 antagonist optionally selected from one or more of the following: 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), optionally wherein the cancer is selected from one or more of the following: colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer, and renal cell carcinoma;

[0131] The antagonist is a PD-1 antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514PDR001, and pidilizumab, optionally wherein the PD-1 antagonist is nivolumab, and the cancer is optionally selected from one or more of the following: Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer;

[0132] The PD-1 antagonist is pembrolizumab, and the cancer is optionally selected from one or more of the following: melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma;

[0133] The antagonist is a CTLA-4 antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, and tremelimumab, optionally wherein the cancer is selected from one or more of the following: melanoma, prostate cancer, lung cancer, and bladder cancer;

[0134] The antagonist is an IDO antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to IDO, indomod (NLG-8189), 1-methyl-tryptophan (1MT), a β-carboline (norharman; 9H-pyrido[3,4-b]indole), and icardostat, and wherein the cancer is optionally selected from one or more of the following: metastatic breast cancer and brain cancer, optionally glioblastoma multiforme, glioma, gliosarcoma, or a malignant brain tumor;

[0135] The antagonist is a TDO antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91, and LM10;

[0136] The antagonist is a TIM-3 antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to TIM-3;

[0137] The antagonist is a LAG-3 antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3 and BMS-986016;

[0138] The antagonist is a VISTA antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to VISTA;

[0139] The antagonist is a BTLA, CD160 and / or HVEM antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule that specifically binds to BTLA, CD160 and / or HVEM;

[0140] The antagonist is a TIGIT antagonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or a small molecule that specifically binds to TIGIT.

[0141] In some embodiments, the stimulatory immune checkpoint molecule is selected from one or more of the following: OX40, CD40, glucocorticoid-induced TNFR family-related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226 and herpes virus entry mediator (HVEM).

[0142] In some embodiments, the agonist is an OX40 agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40, OX86, Fc-OX40L, and GSK3174998;

[0143] The agonist is a CD40 agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40, CP-870,893, daclizumab, Chi Lob 7 / 4, ADC-1013, and rhCD40L, and wherein the cancer is optionally selected from one or more of the following: melanoma, pancreatic cancer, mesothelioma, and a hematological cancer, optionally a lymphoma, such as Non-Hodgkin's lymphoma;

[0144] The agonist is a GITR agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1 and MEDI1873;

[0145] The agonist is a CD137 agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD137, utamilumab and 4-1BB ligand;

[0146] The agonist is a CD27 agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27, valirumab, and CDX-1127 (1F5);

[0147] The agonist is a CD28 agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28 and TAB08; and / or

[0148] The agonist is an HVEM agonist optionally selected from one or more of the following: an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to HVEM.

[0149] In some embodiments, the cancer vaccine is selected from one or more of the following: Oncophage; human papillomavirus HPV vaccine, optionally Gardasil or Cervarix; hepatitis B vaccine, optionally Anzaci-B, Recombivax HB or Twinrix; and Sipulusel-T (Provenge), or the cancer vaccine comprises one or more cancer antigens selected from the group consisting 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, VEGF R-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), α-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylate cyclase C, NY-ESO-1, p 53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein α (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 receptor-1, protein disulfide isomerase (PDI), liver regeneration phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3) and mesothelin, optionally wherein the subject has or is at risk of having cancer, and the cancer comprises the corresponding cancer antigen.

[0150] In some embodiments, the oncolytic virus is selected from one or more of the following: Talillah (T-VEC), Coxsackie virus A21 (CAVATAK TM ), Ankori (H101), Perareo Repu Senegal Valley virus (NTX-010), Senegal virus SVV-001, ColoAd1, SEPREHVIR (HSV-1716), CGTG-102 (Ad5 / 3-D24-GMCSF), GL-ONC1, MV-NIS and DNX-2401. In some embodiments, the cytokine is selected from one or more of the following: interferon (IFN) -α, IL-2, IL-12, IL-7, IL-21 and granulocyte-macrophage colony stimulating factor (GM-CSF). In some embodiments, the cell-based immunotherapeutic agent includes cancer antigen-specific T cells, optionally ex vivo derived T cells. In some embodiments, the cancer antigen-specific T cells are selected from one or more of the following: chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor infiltrating lymphocytes (TIL) and peptide-induced T cells.

[0151] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of the following: an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent.

[0152] In some embodiments, the alkylating agent is selected from one or more of the following: nitrogen mustards (optionally dichloromethane, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mitomycin, and diazocone (AZQ)), cisplatin and its derivatives (optionally carboplatin and oxaliplatin), and atypical alkylating agents (optionally procarbazine and altretamine);

[0153] The antimetabolite is selected from one or more of the following: an antifolate (optionally methotrexate and pemetrexed), a fluoropyrimidine (optionally 5-fluorouracil and capecitabine), a deoxynucleoside analog (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine and pentostatin) and a thiopurine (optionally thioguanine and mercaptopurine);

[0154] The cytotoxic antibiotic is selected from one or more of the following: anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin;

[0155] The topoisomerase inhibitor is selected from one or more of the following: camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mepalong, aclarubicin; and / or

[0156] The anti-microtubule agent is selected from one or more of the following: taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0157] In some embodiments, the at least one hormone therapy agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of the following: progestogen (progesterone), corticosteroids (optionally prednisolone, methylprednisolone or dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis 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)-β, androgen, estrogen and somatostatin analogs. In some embodiments, the hormone antagonist is selected from one or more of the following: a hormone synthesis inhibitor, optionally an aromatase inhibitor or gonadotropin-releasing hormone (GnRH) or an analog thereof; and a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an antiandrogen; or an antibody to a hormone receptor, optionally citrullumab, doxetuzumab, fentocillin, ganituzumab, isotuzumab, rotuzumab, peglucituzumab, bevacizumab, irucurumab, ramucirumab, fusumumab, metitumumab, nasituximab, cetuximab, moftuximab depatuximab, fusutuximab, imaclizumab, encinlatuximab, matuzumab, motuximab, nexitumomab, nimotuzumab, panitumumab, tomatuximab, zalutumumab, aprutumab ixadotin, bemarituzumab, olaratumumab, or toviromab.

[0158] In some embodiments, the kinase inhibitor is selected from one or more of the following: adasotinib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostantinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mulitinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib and vemurafenib. In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer, for example, a metastatic cancer that expresses NRP2 and / or NRP2B. In some embodiments, the cancer is selected from one or more of the following: 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 myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancies, 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.

[0159] In some embodiments, the metastatic cancer is selected from one or more of the following:

[0160] (a) bladder cancer that has metastasized to the bones, liver, and / or lungs;

[0161] (b) breast cancer that has metastasized to the bones, brain, liver, and / or lungs;

[0162] (c) colorectal cancer that has metastasized to the liver, lungs, and / or peritoneum;

[0163] (d) renal cancer that has metastasized to the adrenal glands, bones, brain, liver, and / or lungs;

[0164] (e) Lung cancer that has metastasized to the adrenal glands, bones, brain, liver and / or other lung sites;

[0165] (f) melanoma that has metastasized to the bone, brain, liver, lung, and / or skin / muscle;

[0166] (g) ovarian cancer that has metastasized to the liver, lungs, and / or peritoneum;

[0167] (h) pancreatic cancer that has metastasized to the liver, lungs, and / or peritoneum;

[0168] (i) Prostate cancer that has metastasized to the adrenal gland, bone, liver, and / or lung;

[0169] (j) gastric cancer that has metastasized to the liver, lung, and / or peritoneum;

[0170] (l) thyroid cancer that has metastasized to the bones, liver, and / or lungs; and

[0171] (m) Uterine cancer that has metastasized to the bones, liver, lungs, peritoneum, and / or vagina.

[0172] In some embodiments, the subject has an increased level and / or is selected for treatment based on an increased circulating or serum level of at least one bound or free NRP2 ligand (e.g., an NRP2 ligand from Table N2 or Table N3 and / or a HRS polypeptide from Table H1) relative to a healthy control or matched control standard or a subject population consisting of one or more subjects, optionally at a level of about or at least about 30 pM. , 40pM, 50pM, 60pM, 70pM, 80pM, 90pM, 100pM, 200pM, 300pM, 400pM, 500pM, 600pM, 700pM, 800pM ,900pM, 1000pM, 1100pM, 1200pM, 1300pM, 1400pM, 1500pM, 1600pM, 1700pM, 1800pM, 1900pM, 2 000 pM, 3000 pM, 4000 pM or 5000 pM of the at least one NRP2 ligand (e.g., HRS polypeptide) or about or at least about 30-100 pM, 40-100 pM, 50-100 pM, 30-2000 pM, 40-2000 pM, 50-2000 pM, 60-2000 pM, 70-2000 pM, 80-2000 pM, 90-2000 pM, 100 -2000 pM, 200-2000 pM, 300-2000 pM, 400-2000 pM, 500-2000 pM, 600-2000 pM, 700-2000 pM, 800-2000 pM, 900-2000 pM, 1000-2000 pM, 2000-3000 pM, 3000-4000 pM or 4000-5000 pM of the at least one NRP2 ligand.

[0173] In some embodiments, the subject has a disease associated with increased levels or expression of at least one NRP2 ligand (e.g., an NRP2 ligand from Table N2 or Table N3 and / or a HRS polypeptide from Table H1) and / or its encoding mRNA relative to a healthy control or matched control standard or a subject population consisting of one or more subjects and / or is selected for treatment based on having the disease, optionally a cancer in which the levels or expression of at least one NRP2 ligand and / or its encoding mRNA are increased relative to non-cancerous control cells or tissues, optionally relative to non-cancerous cells or tissues of the same type as the cancer, optionally wherein the HRS polypeptide is a splice variant selected from the group consisting of: 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 .

[0174] In some embodiments, the subject has an increased level and / or is selected for treatment based on an increased level of circulating or serum bound or free soluble neuropilin 2 (NRP2) polypeptide (e.g., selected from Table N1) relative to a healthy control or matched control standard or a subject population consisting of one or more subjects, optionally a circulating or serum level of about or at least about 10 pM, 20 pM, 30 pM, 50 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1000 pM, 1100 pM, 1200 pM, 1300 pM, 1400 pM, 1500 pM, 1600 pM, 1700 pM, 1800 pM, 1900 pM, 2000 pM, 2100 pM, 2200 pM, 2300 pM, 2400 pM, 2500 pM, 2600 pM, 2700 pM, 2800 pM, 2900 pM, 3000 pM, 3100 pM, 3200 pM, 3300 pM, 3400 pM, 3500 pM, 3600 pM, 3700 pM, 3800 pM, 3900 pM, 4000 pM, 4100 pM, 4200 pM, 4300 pM, 4400 pM, 4500 pM, 4600 pM, 4700 pM, 4800 pM, 4900 pM, 5000 pM, 5100 pM, 5 600 pM, 1700 pM, 1800 pM, 1900 pM, 2000 pM, 3000 pM, 4000 pM, 5000 pM of said soluble NRP2 polypeptide, or optionally a circulating or serum level of about 30-50 pM, 50-100 pM, 100-2000 pM, 200-2000 pM, 300-2000 pM The soluble NRP2 polypeptide is preferably present in an amount of 0.1-1.0 M, 0.2-1.0 M, 0.3-1.0 M, 0.4-1.0 M, 0.5-1.0 M, 0.6-1.0 M, 0.7-1.0 M, 0.8-1.0 M, 0.9-1.0 M, 0.9-1.0 M, 0.1-1.0 M, 0.2-1.0 M, 0.3-1.0 M, 0.4-1.0 M, 0.5-1.0 M, 0.6-1.0 M, 0.7-1.0 M, 0.8-1.0 M, 0.9-1.0 M, 0.8-1.0 M, 0.9-1.0 M,

[0175] In some embodiments, the subject has a disease associated with increased levels or expression of an NRP2 polypeptide (e.g., selected from Table N1) and / or its encoding mRNA relative to a healthy control or matched control standard or a subject population consisting of one or more subjects and / or is selected for treatment based on having the disease, optionally a cancer in which the levels or expression of an NRP2 polypeptide (e.g., selected from Table N1) and / or its encoding mRNA are increased relative to non-cancerous control cells or tissues, optionally relative to cells or tissues of the same type as the cancer.

[0176] In some embodiments, the subject is suffering from an increased level or expression of NRP2A and / or NRP2B or an altered ratio of NRP2A:NRP2B expression relative to a healthy control or matched control standard or a subject population consisting of one or more subjects and / or is selected for treatment based on suffering from the disease. In some embodiments, the subject's NRP2B expression or level is significantly higher relative to a healthy control or matched control standard or a subject population consisting of one or more subjects. 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 matched control standard or a subject population consisting of one or more subjects.

[0177] In some embodiments, the subject has increased circulating levels of the HRS:NRP2 complex relative to a healthy or matched control standard or a population of one or more subjects and / or is selected for treatment based on increased circulating levels of the HRS:NRP2 complex.

[0178] In some embodiments, the healthy control or matched control standard or subject population consisting of one or more subjects includes an average range of age-matched samples 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, genetic characteristics (e.g., p53 mutation, PTEN loss, IGFR expression) and / or expression pattern.

[0179] Certain embodiments comprise administering the at least one anti-NRP2 antibody in an amount and at a frequency sufficient to achieve an average sustained serum or circulating level of about 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 of soluble NRP2 polypeptide.

[0180] Certain embodiments comprise administering the at least one anti-NRP2 antibody in an amount and at a frequency sufficient to reduce said circulating levels 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%.

[0181] In some embodiments, the at least one anti-NRP2 antibody enhances the immune response to the cancer by 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 relative to a control.

[0182] In some embodiments, the at least one anti-NRP2 reduces the in vitro growth rate of the cancer by 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 relative to an untreated control.

[0183] In some embodiments, the at least one anti-NRP2 antibody reduces the in vitro adhesion of the cancer to a matrix 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 relative to an untreated control. In some cases, the matrix comprises laminin.

[0184] In some embodiments, the at least one anti-NRP2 antibody reduces the aggressiveness of the 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 relative to an untreated control.

[0185] In some embodiments, the at least one anti-NRP2 antibody inhibits the migration or motility rate of the 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 relative to an untreated control.

[0186] In some embodiments, the at least one anti-NRP2 antibody inhibits the rate of autophagy or endosomal maturation (e.g., endosomal acidification) of the cancer or associated 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 relative to an untreated control.

[0187] In some embodiments, the at least one anti-NRP2 antibody increases the susceptibility of the cancer to the chemotherapeutic agent, hormonal therapy, or kinase inhibitor 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 relative to the chemotherapeutic agent alone.

[0188] In some embodiments, the at least one anti-NRP2 antibody enhances the anti-tumor and / or immunostimulatory activity of the 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 relative to the cancer immunotherapeutic agent alone.

[0189] Certain embodiments comprise administering the at least one anti-NRP2 antibody in an amount and at a frequency sufficient to achieve a steady-state concentration or mean circulating concentration of the at least one anti-NRP2 antibody of between about 1 nM and about 1 μM, between about 1 nM and about 100 nM, between about 1 nM and about 10 nM, or between about 1 nM and about 3 μM.

[0190] Also included is a patient care kit comprising:

[0191] (a) at least one antibody or antigen-binding fragment thereof that specifically binds to a human neuropilin-2 (NRP2) polypeptide as described herein; and optionally

[0192] (b) 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.

[0193] In some embodiments, (a) and (b) are in separate therapeutic compositions. In some embodiments, (a) and (b) are in the same therapeutic composition as described herein.

[0194] In some patient care kits, in some embodiments, the at least one chemotherapeutic agent is selected from one or more of the following: an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an antimicrotubule agent.

[0195] In some patient care kits, the alkylating agent is selected from one or more of the following: nitrogen mustards (optionally dichloromethane, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mitomycin, and diazocone (AZQ)), cisplatin and its derivatives (optionally carboplatin and oxaliplatin), and atypical alkylating agents (optionally procarbazine and altretamine);

[0196] The antimetabolite is selected from one or more of the following: an antifolate (optionally methotrexate and pemetrexed), a fluoropyrimidine (optionally 5-fluorouracil and capecitabine), a deoxynucleoside analog (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine and pentostatin) and a thiopurine (optionally thioguanine and mercaptopurine);

[0197] The cytotoxic antibiotic is selected from one or more of the following: anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone), bleomycin, mitomycin C, mitoxantrone and actinomycin;

[0198] The topoisomerase inhibitor is selected from one or more of the following: camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mepalong, aclarubicin; and / or

[0199] The anti-microtubule agent is selected from one or more of the following: taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0200] In some patient care kits, in some embodiments, the at least one hormone therapy agent is a hormone agonist or a hormone antagonist. In some patient care kits, in some embodiments, the hormone agonist is selected from one or more of the following: progestogen (progesterone), corticosteroids (optionally prednisolone, methylprednisolone or dexamethasone), insulin-like growth factor, VEGF-derived angiogenic and lymphangiogenic factors (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)-β, androgen, estrogen and somatostatin analogs.

[0201] In some patient care kits, the hormone antagonist is selected from one or more of the following: a hormone synthesis inhibitor, optionally an aromatase inhibitor or gonadotropin-releasing hormone (GnRH) or an analog thereof; and a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an antiandrogen; or an antibody to a hormone receptor, optionally citrullumab, doxetuzumab, fentocillin, ganituzumab, isotuzumab, rotuzumab, peglucituzumab, bevacizumab, irucurumab, ramucirumab, fusumumab, metitumumab, nasituximab, cetuximab, moftuximab, depatuximab, fusutuximab, imaclizumab, encinlatuximab, matuzumab, motuximab, nexitumomab, nimotuzumab, panitumumab, tomatuximab, zalutumumab, aprutumab ixadotin, bemarituzumab, olaratumumab, or toviromab.

[0202] In some patient care kits, in some embodiments, the kinase inhibitor is selected from one or more of the following: adasotinib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostaminib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mulitinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib and vemurafenib.

[0203] Also encompassed are bioassay systems comprising a substantially pure anti-NRP2 antibody or antigen-binding fragment thereof, optionally as defined herein, and a host cell line that expresses a human NRP2 polypeptide on the cell surface.

[0204] In some embodiments, the NRP2 polypeptide is labeled with a detectable marker. In some embodiments, the anti-NRP2 antibody is labeled with a detectable marker. In some embodiments, the NRP2 polypeptide is functionally coupled 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. Some bioassay systems include at least one NRP2 ligand (e.g., an NRP2 ligand from Table N2 or Table N3 and / or a human histidyl-tRNA synthetase (HRS) polypeptide from Table H1), for example, wherein the host cell expresses the at least one NRP2 ligand. In some embodiments, the HRS polypeptide is selected from Table H1, for example, wherein the HRS polypeptide includes optionally selected from 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 In some embodiments, the at least one NRP2 ligand is selected from Table N2 or Table N3.

[0205] Some embodiments include a detection system comprising a cell expressing a human neuropilin 2 (NRP2) polypeptide and at least one NRP2 ligand (e.g., 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 a human or humanized anti-NRP2 antibody or antigen-binding fragment thereof that modulates the interaction between the NRP2 polypeptide and the at least one NRP2 ligand as described herein. 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 selected from Table H1, for example, wherein the HRS polypeptide comprises optionally selected from 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 In some embodiments, the at least one NRP2 ligand is selected from Table N2 or Table N3. In some embodiments, the NRP2 polypeptide and / or the at least one NRP2 ligand (e.g., an NRP2 ligand from Table N2 or Table N3 and / or a HRS polypeptide from Table H1) is functionally coupled to a readout or indicator, such as a fluorescent or luminescent indicator of the biological activity of the NRP2 polypeptide or the at least one NRP2 ligand.

[0206] Also included are diagnostic systems comprising a cell comprising a neuropilin 2 (NRP2) polypeptide and at least one NRP2 ligand that specifically binds to the NRP2 polypeptide (e.g., 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 comprises an indicator molecule that indicates a change in the level or activity of the NRP2 polypeptide in response to interaction with the at least one NRP2 ligand.

[0207] Also included are cell compositions comprising an engineered cell population, wherein at least one cell in the engineered cell population comprises one or more polynucleotides encoding a human or humanized anti-NRP2 antibody or antigen-binding fragment thereof as described herein, wherein the cells are capable of growing in a serum-free medium.

[0208] Also included is a cell growth device comprising a human or humanized anti-NRP2 antibody or antigen-binding fragment thereof as described herein, an engineered cell population wherein at least one cell comprises one or more polynucleotides encoding the anti-NRP2 antibody or antigen-binding fragment thereof, at least about 10 liters of serum-free growth medium, and a sterile container. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] Figures 1A-1B The general domain structure of neuropilin (1A) and exemplary neuropilin co-receptor functions (1B) are shown.

[0210] Figure 2 The domain structures of certain NRP2 isoforms and exemplary NRP2 ligand-binding domains are shown.

[0211] Figure 3Shown are Western blots of the indicated cell lines blotted with a commercially available antibody against human NRP2 (BAF2215, Boster, CA) using a total of 6 μg of cell lysate per lane. Molecular weight markers are shown in the outer lanes.

[0212] Figures 4A-4D Figure 2 shows flow cytometry cell surface binding profiles of the c-domain-specific anti-NRP2 antibody 3F2 (aNRP2-2) on the indicated cell lines, detected using a goat anti-mouse IgG secondary antibody conjugated to AF647 (GaMAF647). Binding of the secondary antibody alone is shown in light grey (left curve in each figure). Figure 4A shows the binding of the antibody to U251 cells, Figure 4B shows the binding of antibodies to HUVEC cells, Figure 4C Binding of the antibody to THP-1M1 cells is shown, and Figure 4D Binding of antibodies to HLEC cells is shown.

[0213] Figures 5A-5D Flow cytometric surface binding profiles to various cell lines using the indicated anti-NRP2 antibodies are shown. Antibody binding was detected using AF647-conjugated goat anti-mouse IgG secondary antibody (GaM AF647). Figure 5A Binding of antibodies to HUVEC cells is shown at antibody concentrations ranging from 0.06 nM to 1000 nM. Figure 5B Binding to U251 cells is shown at antibody concentrations ranging from 0.01 nM to 1000 nM. Figure 5C Binding to A549 cells is shown at antibody concentrations ranging from 0.01 nM to 1000 nM. Figure 5D Binding to THP-1M1 cells is shown at antibody concentrations ranging from 0.01 nM to 1000 nM. Antibodies 14v2 (aNRP2-14), 10v2 (aNRP2-10), 11v2 (aNRP2-11), 2v2 (aNRP2-2), and an isotype control (cMOPC21) were incubated with cells at the indicated concentrations as described in the Examples.

[0214] Figures 6A-6B Shown are the binding curves of anti-NRP2 antibodies to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 6A Antibody concentrations ranging from 0.05 to 30 nM are shown, and Figure 6BResults are shown for antibody concentrations tested in the range of 0.02-10 nM, with both antibody dilutions 3-fold. Antibody binding was detected using a goat anti-mouse IgG secondary antibody conjugated to AF647 (GaM AF647). Antibodies 8v2 (aNRP2-8) #1328, 9v2 (aNRP2-9) #1329, 10v2 (aNRP2-10) #1330, 14v2, 11v2 (aNRP2-11) #1331, 14v2 (aNRP2-14) #1344, 15v2 (aNRP2-15) #1347, and an isotype control (cMOPC21) were incubated with cells at the indicated concentrations described in the Examples.

[0215] Figures 7A-7B Shown is the binding of VEGF-C to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 7A Shown are FACS binding curves using VEGF-C (R&D systems), a secondary antibody labeled with a rabbit detection antibody (Abcam) and goat antibody AF647. Figure 7B Flow cytometry scatter plots are shown. From left to right, Figure 7B The curves in FIG show (aR AF647); (Rb Iso_Ctl + aR AF647); (a-VEGFc + a-RAF647); and (VEGFc 100 nM + a-VEGFc + aR AF647).

[0216] Figures 8A-8M Anti-NRP2 antibody blocking displacement curves are shown for the indicated antibodies binding to VEGF-C and Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 8A Results obtained using an isotype control antibody are shown. Figure 8B The results obtained using antibody 8v2 (aNRP2-8) #1328 are shown. Figure 8C The results obtained using antibody 9v2 (aNRP2-9) #1329 are shown. Figure 8D The results obtained using antibody 11v2 (aNRP2-11) #1331 are shown. Figure 8E The results obtained using antibody 14v2 (aNRP2-14) #1344 are shown. Figure 8F The results obtained using antibody 2v1 (aNRP2-1) #1326 are shown. Figure 8G The results obtained using antibody 2v2 (aNRP2-2) #1327 are shown. Figure 8H The results obtained using antibody 2v10 (aNRP2-10) #1330 are shown. Figure 8IResults obtained using antibody #1333 are shown. Figure 8J Results obtained using antibody #1334 are shown. Figure 8K The results obtained using antibody 2v7 (aNRP2-7) #1335 are shown. Figure 8L Results obtained using antibody 2v12 (aNRP2-12) #1336 are shown, and Figure 8M Results obtained using antibody #1337 are shown.

[0217] Figure 9 Shown is the binding of Sema3F-p95 and Sema 3F-p65 (0.05-100 nM) to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 9 Shown are FACS binding curves using Sema 3F-p95 or p65 (inside), using anti-myc detection antibodies as described in the Examples.

[0218] Figures 10A-10H Anti-NRP2 antibody blocking displacement curves are shown for the indicated antibodies for Sema 3F-p95 binding to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 10A The results obtained using the same type of control antibody are shown. Figure 10B The results obtained using antibody 14v2 (aNRP2-14) #1344 are shown. Figure 10C The results obtained using antibody 8v2 (aNRP2-8) #1328 are shown. Figure 10D The results obtained using antibody 9v2 (aNRP2-9) #1329 are shown. Figure 10E The results obtained using antibody 2v10 (aNRP2-10) #1330 are shown. Figure 10F The results obtained using antibody 15v2 (aNRP2-15) #1347 are shown. Figure 10G Results obtained using antibody 11v2 (aNRP2-11) #1331 are shown, and Figure 10H Results obtained using antibody 2v2 (aNRP2-2) #1327 are shown.

[0219] Figures 11A-11F Anti-NRP2 antibody blocking displacement curves are shown for the indicated antibodies for Sema 3F-p95 binding to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 11A The results obtained using antibody 14v2 (aNRP2-14) #1344 are shown. Figure 11B The results obtained using antibody 8v2 (aNRP2-8) #1328 are shown. Figure 11CThe results obtained using antibody 9v2 (aNRP2-9) #1329 are shown. Figure 11D The results obtained using antibody 2v10 (aNRP2-10) #1330 are shown. Figure 11E Results obtained using antibody 15v2 (aNRP2-15) #1347 are shown, and Figure 11F Results obtained using antibody 11v2 (aNRP2-11) #1331 are shown.

[0220] Figure 12 Shown are the effects of anti-NRP2 antibody binding achieved using the disclosed antibodies on NRP2-Plexin A1 induced receptor heterodimerization as described in the Examples.

[0221] Figure 13 Shown are the effects of anti-NRP2 antibody binding achieved using the disclosed antibodies on NRP2-FLT4 (VEGFR3)-induced receptor heterodimerization as described in the Examples.

[0222] Figure 14 The crystal structure of human NRP2 is shown, showing the a2 domain and the b1 / b2 domains together with the variable heavy chain (V H ) and variable light chain (V L )complex.

[0223] Figure 15 The heavy chains of human NRP2 and aNRP2-14 Fab showing the a2 and b1 domains are shown (V H ) and light chain (V L ) and the interaction surfaces between the interacting amino acids.

[0224] Figure 16 Shown is a view of the interaction interface between the human NRP2 a2 domain (bottom) and the heavy (upper left) and light (upper right) chains of aNRP2-14 Fab. Interacting residues E237 of human NRP2 and S57 of the Fab heavy chain are circled. DETAILED DESCRIPTION

[0225] Unless otherwise defined, the meaning of all technical terms and scientific terms used herein is the same as that generally understood by those of ordinary skill in the art to which the present disclosure pertains. Although methods, materials, compositions, reagents, cells similar to or equivalent to any method, material, composition, reagent, cell described herein can be used when practicing or testing the subject matter of the present disclosure, preferred methods and materials are described. All publications and references cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety, as if clearly and individually indicating that each individual publication or reference is incorporated herein by reference as if fully elaborated. Any patent application for which this application claims priority is also incorporated herein by reference in its entirety in the manner described above for publications and references.

[0226] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and conversion (for example, electroporation, liposome transfection).Can be according to manufacturer's specification sheets or as this area is usually realized or as described herein, perform enzymatic reaction and purification technique.These techniques and procedures and related techniques and procedures can usually be according to conventional methods well known in the art and as described in the various general and more specific references of quoting and discussing in whole this specification sheets and perform.Unless specific definition is provided, otherwise the naming used in combination with molecular biology described herein, analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry and the laboratory procedures and techniques of described molecular biology, analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry are well known in the art and commonly used naming and laboratory procedures and techniques.Standard techniques can be used for recombinant technology, molecular biosynthesis, microbial synthesis, chemosynthesis, chemical analysis, pharmaceutical preparation, deployment and delivery and patient treatment.

[0227] For the purposes of this disclosure, the following terms are defined below.

[0228] The articles “a” and “an” are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, “an element” includes “an element,” “one or more elements,” and / or “at least one element.”

[0229] "About" means that an amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight or length varies by as much as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% relative to a reference amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight or length.

[0230] 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 additionally be used in an animal to produce an antibody that can bind to the epitope of the antigen. An antigen can have one or more epitopes. As used herein, the term "antigen" includes substances that can induce an immune response to a substance under appropriate conditions and can react with the product of the immune response. For example, an antigen can be recognized by an antibody (humoral immune response) or a sensitized T lymphocyte (helper T cell or cell-mediated immune response) or both. An antigen can be a soluble substance (such as toxins and foreign proteins) or a particle (such as bacteria and tissue cells); however, only the part of a protein or polysaccharide molecule that is called an antigenic determinant (epitope) is combined with a specific receptor on an antibody or lymphocyte. More broadly speaking, the term "antigen" includes any substance to which an antibody binds or requires an antibody, regardless of whether the substance is immunogenic. For such antigens, antibodies can be identified by recombinant methods, which has nothing to do with any immune response.

[0231] "Antagonist" refers to a biological structure or chemical agent that interferes with or otherwise reduces the physiological effect of another agent or molecule. In some cases, an antagonist specifically binds to another agent or molecule. This includes full antagonists and partial antagonists.

[0232] An "agonist" refers to a biological structure or chemical agent that increases or enhances the physiological effects of another agent or molecule. In some cases, an agonist specifically binds to another agent or molecule. This includes full agonists and partial agonists.

[0233] The term "anergy" refers to the functional inactivation of T cell or B cell responses to restimulation with antigen.

[0234] As used herein, the term "amino acid" is intended to refer to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. For example, naturally occurring amino acids include the 20 (L)-amino acids used during protein biosynthesis, as well as other amino acids such as 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, and the like, known to those skilled 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 on the amino acids, or derivatization of the amino acids. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties to those of the reference amino acids, such as charge and charge spacing characteristics. For example, an organic structure that mimics arginine (Arg or R) will have a positively charged moiety positioned in a similar molecular space and with the same mobility as the ε-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures to maintain optimal spacing and charge interactions between amino acids or amino acid functional groups. Those skilled in the art know or can determine which structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0235] As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chains (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion having an antigen-binding fragment with the desired specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding site or fragment (epitope recognition site) with the desired specificity. Certain features and properties of antibodies (and antigen-binding fragments thereof) are described in more detail herein.

[0236] The antibody or antigen-binding fragment can be of essentially any type. As is well known in the art, an antibody is an immunoglobulin molecule that is capable of specifically binding to a target, such as an immune checkpoint molecule, through at least one epitope recognition site located in the variable region of the immunoglobulin molecule.

[0237] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy chain and / or light chain that binds to an antigen of interest. In this regard, an antigen-binding fragment of an antibody described herein may include a V or V region from an antibody that binds to a target molecule. H and V L 1, 2, 3, 4, 5 or all 6 CDRs of a sequence.

[0238] The binding properties of antibodies and antigen-binding fragments thereof 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 antigen-binding fragment thereof is expressed as about or in the range of about ≤10 -7 M to about 10 -8 The equilibrium dissociation constant of M specifically binds to a target molecule (e.g., an NRP2 polypeptide or an epitope or complex thereof). In some embodiments, the equilibrium dissociation constant is about or ranges from about ≤10 -9 M to approximately ≤10 -10 In certain illustrative embodiments, the affinity (Kd or EC) of an antibody or antigen-binding fragment thereof for a target molecule to which it specifically binds is 50 ) is about, at least about, or less than about 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 40 nM, or 50 nM.

[0239] A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a specific cell, substance, or specific epitope more frequently, more rapidly, for a longer duration, and / or with a higher affinity than a molecule, such as a polypeptide or antibody, to react or associate with a specific cell, substance, or epitope to an alternative cell, substance, or epitope. An antibody "specifically binds" or "preferentially binds" to a target molecule or epitope if it binds with higher affinity, higher avidity, more readily, and / or for a longer duration than it binds to other substances or epitopes, e.g., if the superiority reaches a statistically significant amount. Typically, one member of a pair of molecules exhibiting specific binding has an area on its surface or has a cavity that specifically binds to and is therefore complementary to a specific spatial and / or polar organization of the other member of the pair. Thus, the members of a pair of molecules have the property of specifically binding to each other. For example, an antibody that specifically or preferentially binds to a particular epitope is an antibody that binds to a particular epitope with greater affinity, with greater avidity, more readily, and / or for a longer duration than it binds to other epitopes. It should also be understood from reading this definition that, for example, 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. The term also applies to situations where, for example, an antibody is specific for a particular epitope carried by multiple antigens, in which case a particular binding member carrying an antigen binding fragment or domain will be able to bind to the various antigens carrying the epitope; for example, it may be cross-reactive to multiple different forms of a target antigen from multiple species that share a common epitope.

[0240] Immunobinding generally refers to the non-covalent interaction of the type occurring between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example but not limited to, due to electrostatic, ionic, hydrophilic and / or hydrophobic attraction or repulsion, steric drag, hydrogen bonding, van der Waals forces, and other interactions. The intensity or affinity of the interaction can be represented by the dissociation constant (Kd) of the immunobinding interaction, where the smaller the Kd, the greater the affinity. The immunobinding properties of the selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where those rates depend on the concentration of the complex partner, the affinity of the interaction, and on geometric parameters that affect the rate equally in both directions. Therefore, the "association rate constant" (Kon) and the "dissociation rate constant" (Koff) can be determined by calculating the concentration and actual rate of association and dissociation. The ratio of Koff / Kon can achieve the elimination of all parameters unrelated to affinity, and is therefore equal to the dissociation constant Kd. As used herein, the term "affinity" encompasses the equilibrium constant for the reversible binding of two agents and is expressed as Kd or EC50 The affinity of a binding protein for a ligand, such as the affinity of an antibody for an epitope, can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM). Alternatively, as used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. In some embodiments, affinity is expressed as a half-maximal effective concentration (EC 50 ), the half-maximal effect concentration refers to the concentration of an agent (such as an antibody or anti-NRP2 antibody) as disclosed herein that induces a response between baseline and maximum after a specific exposure time. 50 Often used as a measure of antibody potency.

[0241] Antibodies can be prepared by any of a variety of techniques known to those of ordinary skill 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 modifications thereof. Methods for expressing human antibodies using transgenic animals such as mice are also included. 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. Specific examples include of Platform (see, eg, US Patent No. 6,596,541).

[0242] Antibodies can also be produced or identified by using phage display libraries or yeast display libraries (see, for example, 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), in which the structural diversity of the human antibody repertoire is represented by seven heavy chain variable region genes and seven light chain variable region genes. The combination of these genes produces 49 frameworks in the master library. By superimposing highly variable gene cassettes (CDR=complementarity determining regions) on these frameworks, a large human antibody repertoire can be replicated. Human libraries designed with human donor-derived fragments encoding light chain variable regions, heavy chain CDR-3, synthetic DNA encoding the diversity in heavy chain CDR-1, and synthetic DNA encoding the diversity in heavy chain CDR-2 are also included. Other applicable libraries will be obvious to those skilled in the art.

[0243] In certain embodiments, antibodies and Fabs thereof as described herein comprise heavy chain and light chain CDR groups inserted respectively between heavy chain framework region (FR) group and light chain FR group, and the heavy chain and light chain FR groups provide support to CDR and limit the spatial relationship of CDR relative to each other. As used herein, the term "CDR group" refers to three hypervariable regions in heavy chain or light chain V district. Starting from the N-terminal of heavy chain or light chain, these districts are respectively expressed as "CDR1", "CDR2" and "CDR3". Therefore, the antigen binding site comprises six CDRs, which include CDR groups from each of the heavy chain and light chain V districts. The polypeptide comprising a single CDR (e.g., CDR1, CDR2 or CDR3) is referred to as a "molecular recognition unit" in this article. Crystallographic analysis of multiple antigen-antibody complexes has shown that the amino acid residues of CDR form extensive contacts with the bound antigen, wherein the most extensive antigen contact is the antigen contact with the heavy chain CDR3. Therefore, the molecular recognition unit is primarily responsible for the specificity of the antigen binding site.

[0244] As used herein, the term "FR group" refers to the four flanking amino acid sequences of the CDRs in the CDR group that frame the heavy or light chain V region. Some FR residues may contact the bound antigen; however, the FR is primarily responsible for folding the V region into an antigen binding site, particularly the FR residues directly adjacent to the CDRs. Within the FR, certain amino acid residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop consisting of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are displayed as protruding loop motifs that form the antigen binding surface. It is generally believed that there are conserved structural regions of FR that influence the folding shape of the CDR loops into certain "typical" structures - regardless of the precise CDR amino acid sequence. Further, it is known that certain FR residues are involved in non-covalent domain contacts that stabilize the interaction between the antibody heavy and light chains.

[0245] The structure and location of immunoglobulin variable domains can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4th ed. US Department of Health and Human Services 1987, and updates thereto.

[0246] Also included are "monoclonal" antibodies, which refer to homogeneous antibody populations, wherein monoclonal antibodies include amino acids (naturally occurring and non-naturally occurring) that participate in the selective binding of epitopes. Monoclonal antibodies are highly specific, directed against a single epitope. The term "monoclonal antibody" encompasses not only complete monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chains (ScFv), variants thereof, fusion proteins including antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and immunoglobulin molecules that include antigen-binding fragments (epitope recognition sites) with desired specificity and that can be combined with epitopes. It is not intended to limit the source of the antibody or its formation mode (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals). The term encompasses all immunoglobulins and the fragments described above under the definition of "antibody," etc.

[0247] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each comprise a covalent heterodimer containing an intact antigen-binding site. The enzyme pepsin is capable of cleaving IgG molecules to provide several fragments, including a F(ab')2 fragment comprising two antigen-binding sites. The Fv fragments for use according to certain embodiments can be produced by preferential proteolytic cleavage of IgM, and in rare cases by preferential proteolytic cleavage of IgG or IgA immunoglobulin molecules. However, more commonly, Fv fragments are obtained using recombinant techniques known in the art. Fv fragments comprise non-covalent VH::VL heterodimers comprising an antigen-binding site that retains many of the antigen recognition and binding capabilities of a native antibody molecule. 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.

[0248] In certain embodiments, single-chain Fv (scFV) antibodies are contemplated. For example, kappa bodies (111 et al., Prot. Eng. 10:949-57, 1997); minibodies (Martin et al., EMBO J. 13:5305-9, 1994); bifunctional antibodies (Holliger et al., PNAS 90:6444-8, 1993); or Janusins ​​(Traunecker et al., 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 selection of antibodies with desired specificity.

[0249] Single-chain Fv (scFv) polypeptides are covalently linked VH::VL heterodimers expressed from a gene fusion comprising VH and VL encoding genes connected by a peptide-encoding linker. Huston et al., Proc. Natl. Acad. Sci. USA 85(16):5879-5883, 1988). Various methods have been described for identifying chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to that of the antigen binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Pat. No. 4,946,778 to Ladner et al.

[0250] In certain embodiments, the antibodies or antigen-binding fragments described herein take the form of "bifunctional antibodies". Bifunctional antibodies are multimers formed by polypeptides, each polypeptide comprising a first domain and a second domain, wherein the first domain comprises a binding domain for an immunoglobulin light chain, and the second domain comprises a binding domain for an immunoglobulin heavy chain, wherein the two domains are connected (e.g., by a peptide linker) but cannot associate with each other to form an antigen binding site: the antigen binding site is formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94 / 13804). The dAb fragment of the antibody consists of a VH domain (Ward et al., Nature 341:544-546, 1989). Bifunctional antibodies and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO94 / 13804; and Holliger et al., Proceedings of the National Academy of Sciences of the United States of America 90:6444-6448, 1993).

[0251] Also included are minibodies comprising 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., Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988); PCT / US92 / 09965; WO 94 / 13804; and Reiter et al., Nature Biotech., 14:1239-1245, 1996).

[0252] When bispecific antibodies are used, the antibodies can be conventional bispecific antibodies that can be produced in various ways (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), for example, chemically prepared or produced by hybridomas, or any of the bispecific antibody fragments mentioned above. Bifunctional antibodies and scFvs can be constructed without an Fc region using only the variable domains, thereby potentially reducing the impact of anti-idiotypic reactions.

[0253] In contrast to bispecific complete antibodies, bispecific bifunctional antibodies may also be particularly useful because they can be easily constructed and expressed in E. coli. Phage display (WO94 / 13804) can be used to easily select bifunctional antibodies (and many other polypeptides, such as antibody fragments) with suitable binding specificities from libraries. If one arm of a bifunctional antibody is to be kept constant, for example, to have specificity for antigen X, a library in which the other arm is altered can be produced and antibodies with suitable specificity can be selected. Bispecific complete antibodies can be produced by knobs-into-holes engineering (Ridgeway et al., Protein Engineering, 9:616-621, 1996).

[0254] In certain embodiments, the antibodies or antigen-binding fragments described herein are form. is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, for example, US20090226421). This antibody technology produces stable smaller antibody formats with a therapeutic window expected to be longer than that of current small antibody formats. IgG4 antibodies are considered inert and therefore do not interact with the immune system. Fully human IgG4 antibodies can be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments with different stability properties relative to the corresponding intact IgG4 (GenMab Utrecht). Splitting the IgG4 molecule in half allows for the Only the region that can bind to the cognate antigen (e.g., disease target) is left on the Binds monovalently to only one site on the target cell. For some cancer cell surface antigens, this monovalent binding may not stimulate cancer cell growth as can be seen using bivalent antibodies with the same antigen specificity, and therefore, Technology could provide treatment options for certain types of cancer that are refractory to treatment with conventional antibodies. The small size of the molecule can bring great benefits, allowing for better distribution of the molecule on larger solid tumors and potentially improving efficacy.

[0255] In certain embodiments, antibody or antigen-binding fragment described herein adopt the form of nanobody.Minibody is encoded by a single gene and is efficiently produced in almost all prokaryotic and eukaryotic hosts, such as Escherichia coli (see U.S. Patent No. 6,765,087), mold (such as Aspergillus (Aspergillus) or Trichoderma (Trichoderma)) and yeast (such as Saccharomyces (Saccharomyces), Kluyvermyces (Kluyvermyces), Hansenula (Hansenula) or Pichia (Pichia) (see U.S. Patent No. 6,838,254).The production process is scalable, and several kilograms of nanobody have been produced.Nanobody can be formulated as a ready-to-use solution with a longer shelf life.Nanoclone method (see WO 06 / 079372) is a proprietary method for producing nanobody for desired target based on the automation high throughput selection of B cells.

[0256] 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 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 have been engineered to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms by repeated rounds of in vitro selection or equivalent methods such as SELEX (systematic evolution of ligands by exponential enhancement). See, e.g., U.S. Patent Nos. 6,376,190 and 6,387,620, which are incorporated by reference.

[0257] Peptide aptamers are generally included in a variable peptide loop connected to a protein scaffold at both ends, and the protein scaffold is a dual structural constraint that generally increases the binding affinity of the peptide aptamer to a level comparable to that of the antibody (e.g., in the nanomolar range). In certain embodiments, the variable loop length can be composed of about 10-20 amino acids (including all integers therebetween), and the scaffold can include any protein with good solubility and compatibility. Certain exemplary embodiments use bacterial protein - Thioredoxin-A as a scaffold protein, and the variable loop is inserted into the reduction active site (-Cys-Gly-Pro-Cys-loop in the wild-type protein), wherein two cysteine ​​side chains can form a disulfide bridge. For example, a method for identifying peptide aptamers is described in U.S. Application No. 2003 / 0108532, which is incorporated by reference. Peptide aptamer selection can be performed using different systems known in the art (including yeast two-hybrid system).

[0258] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of affinity multimers. Affinity multimers refer to multimeric binding proteins or peptides engineered using in vitro exon shuffling and phage display. Multiple binding domains are connected, thereby producing greater affinity and specificity than single-epitope immunoglobulin domains. See, for example, Silverman et al., Nature Biotechnology. 23:1556-1561, 2005; U.S. Patent No. 7,166,697; and U.S. Application Nos. 2004 / 0175756, 2005 / 0048512, 2005 / 0053973, 2005 / 0089932, and 2005 / 0221384, which are incorporated by reference.

[0259] In some embodiments, the antibodies or antigen-binding fragments described herein take the form of adnectins. Adnectins refer to a class of targeted biologics derived from human fibronectin, an abundant extracellular protein that naturally binds to other proteins. See, for example, U.S. Application Nos. 2007 / 0082365; 2008 / 0139791; and 2008 / 0220049, which are incorporated by reference. Adnectins typically consist of a native fibronectin backbone and multiple targeting domains derived from specific portions of human fibronectin. Targeting domains can be engineered to enable the adnectin to specifically recognize an NRP2 polypeptide or an epitope thereof.

[0260] In some embodiments, the antibodies or antigen-binding fragments described herein take the form of anticalins. Anticalins refer to a class of antibody mimics typically synthesized from human lipocalin, a family of binding proteins with hypervariable loop regions supported by a structurally rigid framework. See, for example, U.S. Application No. 2006 / 0058510. Anticalins are typically about 20 kDa in size. Anticalins can be characterized by a barrel structure formed by eight antiparallel β-strands (a stable β-barrel scaffold) connected in pairs by four peptide loops and a connected α-helix. In certain aspects, conformational deviations that enable specific binding are generated in one or more hypervariable loop regions. See, for example, Skerra, FEBS J., 275:2677-83, 2008, which is incorporated by reference.

[0261] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of pre-designed ankyrin repeat proteins (DARPins). DARPins comprise a class of non-immunoglobulin proteins that can offer advantages over antibodies for target binding in drug discovery and development. Among other uses, DARPins are well suited for in vivo imaging or delivery of toxins or other therapeutic payloads because they have favorable molecular properties, including small size and high stability. The low-cost bacterial production and rapid production of many target-specific DARPins make the DARPin approach useful for drug discovery. In addition, DARPins can be easily produced in a multispecific format, making it possible to target effector DARPins to specific organs or to target multiple receptors with a single molecule composed of several DARPins. See, for example, Stumpp et al., Curr Opin Drug Discov Devel., 10:153-159, 2007; U.S. Application No. 2009 / 0082274; and PCT / EP2001 / 10454, which are incorporated by reference.

[0262] Also included are heavy chain dimers, such as antibodies from camelids and sharks. Camelid and shark antibodies include homodimer pairs composed of two chains (neither of which has a light chain) with V-like and C-like domains. Because the VH region of the heavy chain dimer IgG in camelids does not need to interact hydrophobically with the light chain, the region in the heavy chain that normally contacts the light chain in camelids is converted to hydrophilic amino acid residues. The VH domain of the heavy chain dimer IgG is called a VHH domain. Shark Ig-NARs include homodimers composed of one variable domain (called a V-NAR domain) and five C-like constant domains (C-NAR domains).

[0263] In camelids, the diversity of the antibody repertoire is determined by the complementarity determining regions (CDRs) 1, 2, and 3 in the VH or VHH regions. The CDR3 in the camelid VHH region is characterized by its relatively long length, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering, 7(9):1129). This is in contrast to the CDR3 regions of antibodies from many other species. For example, the average length of the mouse VH CDR3 is 9 amino acids. Libraries composed of camelid-derived antibody variable regions that maintain the in vivo diversity of camelid variable regions can be generated, for example, by the methods disclosed in U.S. patent application serial number 20050037421, published on February 17, 2005.

[0264] In certain embodiments, the antibody or its antigen-binding fragment is humanized. These embodiments relate to chimeric molecules prepared using recombinant technology, wherein the chimeric molecule has an antigen binding site derived from an immunoglobulin from a non-human species, and the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of human immunoglobulin. The antigen binding site may include a complete variable domain fused to a constant domain or a CDR only transplanted to an appropriate framework region in the variable domain. The epitope binding site may be wild type or may be modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but there is still the possibility of an immune response to exogenous variable regions (LoBuglio et al., Proceedings of the National Academy of Sciences of the United States of America 86:4220-4224, 1989; Queen et al., Proceedings of the National Academy of Sciences of the United States of America 86:10029-10033, 1988; Riechmann et al., Nature 332:323-327, 1988). Illustrative methods for humanizing antibodies include those described in US Patent No. 7,462,697.

[0265] Another approach focuses not only on providing humanized constant regions, but also on modifying the variable regions to reshape them into human form as much as possible. It is known that the variable regions of both heavy and light chains contain three complementary determining regions (CDRs) flanked by four framework regions (FRs), which vary in response to the epitope in question and determine the binding capacity. The four FR regions are relatively conserved in a given species and presumably provide a scaffold for the CDRs. When preparing non-human antibodies for a specific epitope, the variable regions can be "reshaped" or "humanized" by transplanting CDRs derived from non-human antibodies onto FRs present in the human antibody to be modified. The application of this method to various antibodies has been reported in the following literature: Sato et al., Cancer Research 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 and Hybridomas Hybridoma 2:124-134, 1991; Gorman et al., PNAS 88:4181-4185, 1991; Tempest et al., Bio / Technology 9:266-271, 1991; Co et al., PNAS 88:2869-2873, 1991; Carter et al., PNAS 89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, a humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" the CDR(s) from the original antibody.

[0266] In certain embodiments, antibody is " chimeric " antibody. In this respect, chimeric antibody is made up of the antigen binding fragment that is operably connected or otherwise fused to the heterologous Fc portion of antibody with different antibodies. In certain embodiments, Fc domain or heterologous Fc domain are derived from people. In certain embodiments, Fc domain or heterologous Fc domain are derived from mice. In other embodiments, heterologous Fc domain can be from Ig class different from parent antibody, comprise IgA (comprising subclass IgA1 and IgA2), IgD, IgE, IgG (comprising subclass IgG1, IgG2, IgG3 and IgG4) and IgM. In another embodiment, heterologous Fc domain can be by CH2 and CH3 domain from one or more classes in different Ig classes. As described above for humanized antibodies, the antigen-binding fragment of a chimeric antibody can include only one or more of the CDRs of an antibody described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of an antibody described herein), or can include the entire variable domain (VL, VH, or both).

[0267] As used herein, a subject who is "at risk" of developing a disease or producing an adverse reaction may or may not have detectable disease or disease symptoms and may or may not have exhibited detectable disease or disease symptoms prior to the treatment methods described herein. "At risk" means that the subject has one or more risk factors, which are measurable parameters associated with the development of a disease as described herein and known in the art. A subject with one or more of these risk factors has a higher likelihood of developing a disease or producing an adverse reaction than a subject without one or more of these risk factors.

[0268] "Biocompatible" refers to materials or compounds that generally do not cause damage to the biological functions of cells or subjects and do not cause any degree of unacceptable toxicity, including allergic and disease states.

[0269] The term "binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions (including interactions such as salt bridges and water bridges).

[0270] "Coding sequence" refers to any nucleic acid sequence that contributes to the coding of a polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the coding of a polypeptide product of a gene.

[0271] Throughout this disclosure, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of stated steps or elements or groups of steps or elements but not the exclusion of any other steps or elements or groups of steps or elements.

[0272] "Consisting of is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present. "Consisting essentially of" is meant to include any elements listed after the phrase, and is limited to other elements that do not interfere with or facilitate the activity or action specified for the listed elements in this disclosure. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they materially affect the activity or action of the listed elements.

[0273] In the context of antibodies, the term "effector function" or "ADCC effector function" refers to the ability of the antibody to connect to other arms of the immune system (including, for example, activation of the classical complement pathway, or connection through Fc receptors). The complement-dependent pathway is primarily driven by the interaction of C1q with the C1 complex of aggregated antibody Fc domains. Antibody-dependent cellular 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), which bind to the Fc region of IgG that is bound to the target cell. Fc receptors (FcRs) are key immunomodulatory receptors that associate antibody-mediated (humoral) immune responses with cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγRs (IgG), FcεRI (IgE), FcαRI (IgA), FcμRs (IgM), and FcδRs (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 (nanomolar range KD), while FcγRII and FcγRIII have low to moderate affinities (micromolar range KD). When Fc binding occurs, signaling pathways are triggered, which lead to the secretion of various substances such as lytic enzymes, perforins, granzymes, and tumor necrosis factor that mediate the destruction of target cells. The level of ADCC effector function varies among human IgG subtypes. Although this depends on the allotype and specific FcvR, in simple terms, ADCC effector function is "higher" for human IgG1 and IgG3, and "lower" for IgG2 and IgG4.

[0274] The term "endotoxin-free" or "substantially endotoxin-free" generally refers to a composition, solvent, and / or blood vessel that contains at most trace amounts (e.g., an amount that has no clinically adverse physiological effect on a subject), and preferably no detectable amounts, of endotoxins. Endotoxins are toxins associated with certain microorganisms, such as bacteria (usually Gram-negative bacteria), but can be found in Gram-positive bacteria such as Listeria monocytogenes. The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found in the outer membrane of various Gram-negative bacteria, which represent central pathogenic features in the ability of these bacteria to cause disease. Small amounts of endotoxins in humans may cause fever, decreased blood pressure, and inflammation and coagulation activation, among other adverse physiological effects.

[0275] Therefore, in pharmaceutical production, it is usually desirable to remove most or all traces of endotoxin from pharmaceuticals and / or pharmaceutical containers, because even a small amount may also produce adverse effects on people. A pyrogen oven can be used for this purpose, because decomposing most endotoxins usually requires a temperature exceeding 300°C. For example, based on primary packaging materials such as syringes or bottles, a combination of a glass temperature of 250°C and a hold time of 30 minutes is usually enough to achieve a 3-log reduction in endotoxin levels. Other methods of removing endotoxins are envisioned, including chromatography and filtration as described herein and as known in the art.

[0276] Endotoxins can be detected using conventional 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 LPS levels may cause detectable coagulation of the horseshoe crab lysate, as a powerful enzymatic cascade amplifies this reaction. Endotoxins can also be quantitatively measured by enzyme-linked immunosorbent assay (ELISA). In order to be substantially free of endotoxins, endotoxin levels 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 active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1-10 EU.

[0277] The term "epitope" includes any determinant, preferably a polypeptide determinant, that is capable of specific binding to an immunoglobulin or T cell receptor. An epitope comprises a region of an antigen that binds to an antibody. In certain embodiments, an epitope determinant comprises a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments may have specific three-dimensional structural properties and / or specific charge properties. With respect to the primary structure of an antigen (e.g., an NRP2 polypeptide), an epitope may be continuous or non-continuous. In specific embodiments, an epitope comprises about, at least about, or no more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids (i.e., linear epitopes) or non-continuous amino acids (i.e., conformational epitopes) of a reference sequence described herein (see, e.g., Table N1) or a target molecule, consisting of, or consisting essentially of.

[0278] An "epitope" comprises a portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of a binding protein. This binding interaction can be manifested as intermolecular contact with one or more amino acid residues of a CDR. Antigen binding can involve a CDR3 or a CDR3 pair. An epitope can be a linear peptide sequence (i.e., "continuous") or can be composed of discontinuous amino acid sequences (i.e., "conformational" or "discontinuous"). A binding protein can recognize one or more amino acid sequences; thus, an epitope can be defined by more than one distinct amino acid sequence. The epitope recognized by a binding protein can be determined by peptide mapping and sequence analysis techniques well known to those skilled 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 substantially not recognized in an unmodified polypeptide, but is recognized by a binding protein of a denatured or proteolytically hydrolyzed polypeptide. Amino acid sequences that are not exposed or only partially exposed in the unmodified polypeptide structure are potential cryptic epitopes. If the epitope is not exposed or only partially exposed, it is likely buried in the interior of the polypeptide.Candidate cryptic epitopes can be identified, for example, by examining the three-dimensional structure of the unmodified polypeptide.

[0279] The term "half-maximal effect concentration" or "EC 50 "" refers to the concentration of an agent (e.g., an antibody) described herein at which the agent induces a response between baseline and maximum after a specified exposure time; thus, the EC of a graded dose response curve is 50 It represents the concentration of a compound at which 50% of its maximum effect is observed. EC50 also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. Similarly, “EC 90 ” refers to the concentration of an agent or composition at which 90% of the maximum effect is observed. “EC 90" can be calculated based on "EC50" and Hill slope, or can be determined directly from the data using routine knowledge in the art. In some embodiments, the EC50 of an agent (e.g., an antibody) is less than about 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 11nM, 12nM, 13nM, 14nM, 15nM, 16nM, 17nM, 18nM, 19nM, 20nM, 25nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 200nM or 500nM. In some embodiments, the EC of the agent is 50 The value was about 1 nM or less.

[0280] "Immune response" means any immunological response originating from the immune system, including responses from cells and organs, innate and acquired 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 interactions, apoptosis, and the like. Organismic responses include, for example, IgG, IgM, IgA, IgE responses, and their corresponding effector functions.

[0281] 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 when administered to the serum or tissue of an organism, or relative to any other defined time point. "Half-life" can also refer to the time it takes for the amount or concentration of an agent to decrease to half of the initial amount administered to the serum or tissue of an organism, relative to such amount or concentration when administered to the serum or tissue of an organism, or relative to any other defined time point. Half-life can be measured in serum and / or any one or more selected tissues.

[0282] The terms "modulate" and "alter" include "increasing," "enhancing," or "stimulating," as well as "reducing" or "decreasing" relative to a control, typically by a statistically significant or physiologically significant amount or degree. An "increased," "stimulated," or "enhanced" amount is typically a "statistically significant" amount and may 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 or more times (e.g., 500 times, 1000 times) relative to the amount produced by the absence of the composition (e.g., in the absence of the agent) or in the presence of a control composition (e.g., all integers and ranges therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.). A "reduced" or "decreased" amount is typically a "statistically significant" amount and can comprise a 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% decrease (including all integers and ranges therebetween) relative to the amount produced in the absence of the composition (e.g., in the absence of the agent) or in the control composition. Examples of comparisons and "statistically significant" amounts are described herein.

[0283] The term "migratory cell" refers to a cell that is able to move from one place to another in response to a stimulus. Exemplary migratory cells include immune cells, such as monocytes; natural killer (NK) cells; dendritic cells (immature or mature); dendritic cell subsets, including myeloid cells, plasmacytoid cells (also known as lymphoid cells) and Langerhans cells; macrophages, such as histiocytes, such as Kupffer's cells; Cells) and other tissue-resident macrophages, microglia in the CNS, alveolar macrophages and peritoneal macrophages; macrophage subtypes such as M0, M1, Mox, M2a, M2b and M2c macrophages; neutrophils; eosinophils; mast cells; basophils; B cells, including plasma B cells, memory B cells, B-1 cells and B-2 cells; CD45RO (natural T) cells; CD45RA (memory T) cells; CD4 helper T cells, including Th1, Th2 and Tr1 / Th3 cells; CD8 cytotoxic T cells; regulatory T cells; γδ T cells; and 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 "regulation of cell migration" refers to the regulation of the movement of any such migratory cells.

[0284] The terms "polypeptide," "protein," and "peptide" are used interchangeably and are intended to refer to polymers of amino acids that are not limited to any particular length. The term "enzyme" encompasses polypeptide or protein catalysts. The term encompasses modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The term "polypeptide" or "protein" means one or more amino acid chains, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein the polypeptide or protein may comprise multiple chains having a sequence consisting of a natural protein (i.e., a protein produced by a naturally occurring cell, particularly a non-recombinant cell, or a genetically engineered cell or recombinant cell) linked together non-covalently and / or covalently by peptide bonds, and includes molecules having the amino acid sequence of a natural protein or molecules having deletions, additions, and / or substitutions of one or more amino acids of the natural sequence. In certain embodiments, a polypeptide is a "recombinant" polypeptide produced by a recombinant cell that includes one or more recombinant DNA molecules that are typically made from a heterologous polynucleotide sequence or combination of polynucleotide sequences that cannot otherwise be found in the cell.

[0285] The terms "polynucleotide" and "nucleic acid" encompass mRNA, RNA, cRNA, cDNA, and DNA. The terms generally refer to a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide. The terms encompass both single-stranded and double-stranded forms of DNA. The terms "isolated DNA," "isolated polynucleotide," and "isolated nucleic acid" refer to molecules that have been separated from the total genomic DNA of a particular species. Thus, an isolated DNA fragment encoding a polypeptide refers to a DNA fragment that contains one or more coding sequences but is substantially separated from or purified from the total genomic DNA of the species from which the DNA fragment was obtained. Non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode a polypeptide are also encompassed. Recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, bacteriophages, viruses, and the like, are also encompassed.

[0286] Additional coding or non-coding sequences may, but need not, be present within the polynucleotides described herein, and the polynucleotides may, but need not, be linked to other molecules and / or support materials. Thus, a polynucleotide or expressible polynucleotide, regardless of the length of the coding sequence itself, may be combined with other sequences, such as expression control sequences.

[0287] "Expression control sequences" include nucleic acid or amino acid regulatory sequences, such as promoters, leaders, enhancers, introns, recognition motifs for RNA or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, subcellular localization signals, and the like, which have the ability to affect the transcription or translation or subcellular or cellular localization of a coding sequence in a host cell. Exemplary expression control sequences are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0288] " Promoter " is a DNA regulatory region that can bind to the RNA polymerase in the cell and start the transcription of the downstream (3 ' direction) coding sequence. As used herein, the promoter sequence is defined by the transcription start site at its 3 ' end and extends upstream (5 ' direction) to include the minimum number of bases or elements necessary for starting transcription under a detectable level higher than background. The transcription start site can be found in the promoter sequence and the protein binding domain (consensus sequence) responsible for the combination of RNA polymerase (conveniently defined by mapping with nuclease S1). Eukaryotic promoters can usually, but not always, contain " TATA " boxes and " CAT " boxes. Prokaryotic promoters also contain Shine-Dalgarno sequences (Shine-Dalgarno sequence) in addition to -10 and -35 consensus sequences.

[0289] A large amount of promoters (comprising constitutive, inducible and repressible promoters) from various sources are well known in the art. Representative sources include, for example, viruses, mammals, insects, plants, yeast and bacterial cell types, and suitable promoters from these sources are easily available, or can be prepared synthetically based on online publicly available or, for example, from depository institutions such as ATCC and other commercial or personal sources. Promoter can be unidirectional (that is, start transcription in one direction) or bidirectional (that is, start transcription in 3' or 5' direction). Non-limiting examples of promoter include, for example, T7 bacterial expression system, pBAD (araA) bacterial expression system, cytomegalovirus (CMV) promoter, SV40 promoter, 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. USA (1996) 93(8):3346-3351); the T-REx™ system (Invitrogen, Carlsbad, CA); (Stratagene, San Diego, CA) and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nuc. Acid. Res. (1999) 27(22):4324-4327; Nuc. Acids Res. (2000) 28(23):e99; U.S. Pat. No. 7,112,715; and Kramer and Fussenegger, Methods Mol Biol (2005) 308:123-144), or any promoter known in the art suitable for expression in the desired cells.

[0290] An "expressible polynucleotide" comprises a cDNA, RNA, mRNA or other polynucleotide that includes at least one coding sequence and, optionally, at least one expression control sequence (e.g., transcriptional and / or translational regulatory elements) and that can express the encoded polypeptide when introduced into a cell (e.g., a cell of a subject).

[0291] Various viral vectors that can be used to deliver expressible polynucleotides include adenoviral vectors, herpesvirus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors and retroviral vectors. In some cases, retroviral vectors are derivatives of mouse or avian retroviruses, or are lentiviral vectors. Examples of retroviral vectors into which a single exogenous gene can be inserted include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous sarcoma virus (Rous Sarcoma Virus, RSV). Many other retroviral vectors can be combined with multiple genes. All of these vectors can transfer or combine the gene of a selectable marker so that transduced cells can be identified and produced. By, for example, inserting the polypeptide sequence of interest into a viral vector together with another gene encoding a ligand for a receptor on a specific target cell, the vector can be made target-specific. Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a protein. Illustrative targeting can be achieved by targeting the retroviral vector using antibodies. One skilled in the art will know or can readily determine, without undue experimentation, specific polynucleotide sequences that can be inserted into the retroviral genome to allow target-specific delivery of the retroviral vector.

[0292] In certain embodiments, the expressible polynucleotide is a modified RNA or modified mRNA polynucleotide, for example, a non-natural RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide includes one or more modified or non-natural bases, for example, nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and / or uracil (U). In certain embodiments, the modified mRNA includes one or more modified or non-natural internucleotide connections. For example, expressible RNA polynucleotides for delivering encoded therapeutic polypeptides are described in the following documents: Kormann et al., Nature Biotechnology 29: 154-7, 2011; and U.S. Application No. 2015 / 0111248; No. 2014 / 0243399; No. 2014 / 0147454; and No. 2013 / 0245104, which are incorporated by reference in their entirety.

[0293] As used herein, the term "isolated" polypeptide or protein means that the subject protein: (1) is free of at least some other proteins with which the subject protein is normally found in nature; (2) is substantially free of other proteins from the same source, e.g., from the same species; (3) is expressed by cells from a different species; (4) has been separated by at least about 50% from polynucleotides, lipids, carbohydrates, or other materials with which the subject protein is naturally associated; (5) is not associated (by covalent or non-covalent interactions) with protein moieties with which the "isolated protein" is associated in nature; (6) is operably associated (by covalent or non-covalent interactions) with polypeptides with which the subject protein is not naturally associated; or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA, or other RNA, or can be produced synthetically, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0294] 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, certain compositions can include an agent, such as a polypeptide agent, that has a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, including all decimals and ranges therebetween, on a protein or weight-by-weight basis, as measured, for example, but in no way limited to, by high performance liquid chromatography (HPLC), a well-known form of column chromatography commonly used in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0295] "Lipid nanoparticles" or "solid lipid nanoparticles" refer to one or more spherical nanoparticles having an average diameter between about 10 nanometers and about 1000 nanometers and comprising a solid lipid core matrix that can solubilize lipophilic molecules. The lipid core is stabilized by a surfactant (e.g., an emulsifier) ​​and can include one or more of a triglyceride (e.g., tristearin), a diglyceride (e.g., behenyl glyceryl), a monoglyceride (e.g., monostearin), a fatty acid (e.g., stearic acid), a steroid (e.g., cholesterol), and a wax (e.g., cetyl palmitate), including combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014; and U.S. Pat. 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 incorporated by reference in their entirety. Certain compositions described herein are formulated with one or more lipid nanoparticles.

[0296] The term or "neuropilin 2-related disease" or "NRP2-related disease" refers to diseases and conditions in which NRP2 activity, expression and / or spatial distribution play a role in the pathophysiology of the disease or condition. In some cases, the anti-NRP2 antibodies of the present disclosure modulate NRP2-related diseases by altering the interaction of NRP2 with at least one NRP2 ligand to affect NRP2 activity, signaling, expression and / or spatial distribution. Exemplary NRP2-related diseases and conditions include, but are not limited to, cancer and cancer-related diseases or lesions, including cancer cell growth, cancer initiation, cancer migration, cancer cell adhesion, invasion and metastasis. Also included are diseases associated with inflammation and autoimmunity and related inflammatory diseases, including diseases associated with inappropriate immune cell activation or migration, such as graft-versus-host disease (GVHD); additional examples include diseases associated with lymphatic development, lymphangiogenesis and lymphatic damage, including edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition and vascular permeability. Also comprising the disease relevant to infection (comprising latent infection) and the disease relevant to allergic condition / disease and anaphylaxis, comprising chronic obstructive pulmonary disease (COPD), neutrophilic asthma, the systemic vasculitis relevant to antineutrophil cytoplasmic antibodies (ANCA), systemic lupus erythematosus, rheumatoid arthritis, one or more inflammasome related diseases and one or more skin-related neutrophil-mediated diseases, such as pyoderma gangrenosum.Other examples include the disease relevant to granulomatous inflammatory diseases, comprising sarcoidosis and granuloma, and fibrotic diseases, comprising endometriosis, fibrosis, endothelial-mesenchymal transition (EMT) and wound healing etc. Also comprising the disease relevant to inappropriate smooth muscle contractility and vascular smooth muscle cell migration and / or adhesion, and the disease relevant to inappropriate autophagy, phagocytosis and burial. Also comprising the disease relevant to inappropriate wandering cell movement, as described herein.Other examples include neuronal diseases, comprising the disease relevant to peripheral nervous system remodeling and pain perception. Also comprising the disease relevant to skeletal development and / or skeletal remodeling. Generally, the term "inappropriate" refers to an activity or characteristic that is associated with or causes a pathology or disease state.

[0297] The term "reference sequence" generally refers to a nucleic acid coding sequence or amino acid sequence to which another sequence is compared. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including sequences described by name and sequences described in the tables and sequence listing.

[0298] Certain embodiments comprise biologically active "variants" and "fragments" of the polypeptides (e.g., antibodies) described herein, and polynucleotides encoding the same. A "variant" contains one or more substitutions, additions, deletions, and / or insertions relative to a reference polypeptide or polynucleotide (see, e.g., Tables and Sequence Listings). A variant polypeptide or polynucleotide comprises an amino acid or nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity or homology to a reference sequence described herein, and substantially retains the activity of the reference sequence. Also included are sequences consisting 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 or sequences that differ from a reference sequence by additions, deletions, insertions or substitutions of said amino acids or nucleotides and that substantially retain the activity of said reference sequence. In certain embodiments, additions or deletions comprise C-terminal and / or N-terminal additions and / or deletions.

[0299] As used herein, the term "sequence identity" or, for example, "a sequence that is 50% identical to ..." refers to the degree to which sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within a comparison window. Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within the comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occurs in the two sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity. The optimal alignment for comparing the sequence of the comparison window can be by the computerized implementation scheme of the algorithm (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package 7.0 version of No. 575, Madison Science Avenue, Wisconsin, USA, Genetics Computer Group (Genetics Computer Group, 575 Science Drive Madison, Wis., USA), or by testing and by the optimal alignment (that is, producing the highest percentage homology in the comparison window) produced by any method in a variety of selected methods. It can also be carried out with reference to the BLAST program family disclosed in, for example, Altschul et al., " Nucleic Acids Research " 25:3389,1997.

[0300] The term "solubility" refers to that the agent (e.g., antibody) provided herein is dissolved in a liquid solvent and forms a homogeneous solution. Solubility is typically expressed as concentration, or solute mass / unit volume solvent (grams of solute / kilogram of solvent, g / dL (100mL), mg / ml, etc.), molar concentration, mass molarity, molar fraction or other similar concentration descriptions. The maximum equilibrium amount of the solute that a unit amount of solvent can dissolve is the solubility of the solute in the solvent under the specified conditions comprising temperature, pressure, pH and solvent properties. In certain embodiments, solubility is measured at physiological pH or other pH, for example, at 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 physiological buffers such as PBS or NaCl (with or without NaPO 4 ). In certain embodiments, solubility is measured at 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° C., 21° C., 22° C., 23° C., 24° C., 25° C.) or about body temperature (37° C.). In certain embodiments, the solubility of the agent at room temperature or 37°C is at least about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 or 100 mg / ml.

[0301] "Subject" or "subject in need thereof" or "patient" or "patient in need thereof" includes mammalian subjects, such as human subjects.

[0302] "Substantially" or "essentially" means almost completely or completely, for example, 95%, 96%, 97%, 98%, 99%, or more of a given amount.

[0303] "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. Common measures of significance include p-values, which are the frequency or probability that the observed event would occur if the null hypothesis were true. If the p-value obtained is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is limited to p-values ​​of 0.05 or less.

[0304] A "therapeutic response" refers to an improvement in symptoms (whether sustained or not) achieved upon administration of one or more therapeutic agents.

[0305] As used herein, the terms "therapeutically effective amount," "therapeutic dose," "prophylactically effective amount," or "diagnostically effective amount" are the amount of an agent (e.g., anti-NRP2 antibody, immunotherapeutic agent) required to elicit a desired biological response upon administration.

[0306] As used herein, "treatment" of a subject (e.g., a mammal, such as a human) or cell is any type of intervention intended to alter the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and may be performed prophylactically or after the initiation of a pathological event or after contact with a pathogen. Also included are "preventative" treatments, which may be intended to reduce the rate of progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. "Treatment" or "prevention" does not necessarily mean complete eradication, cure, or prevention of a disease or condition or its associated symptoms.

[0307] The term "wild-type" refers to a gene or gene product (eg, polypeptide) that is most commonly observed in a population, and is therefore arbitrarily designated as the "normal" or "wild-type" form of the gene.

[0308] Unless expressly stated otherwise, each embodiment in this specification is applicable to every other embodiment.

[0309] Anti-NRP2 antibodies

[0310] Certain embodiments include antibodies and antigen-binding fragments thereof that specifically bind to a human neuropilin 2 (NRP2) polypeptide. In some embodiments, at least one antibody or antigen-binding fragment thereof modulates (e.g., interferes with) the binding of the human NRP2 polypeptide to at least one NRP2 ligand, such as a human histidyl-tRNA synthetase (HRS) polypeptide or other NRP2 ligand.

[0311] Neuropilin-2 is a cell surface receptor protein that regulates a wide range of cellular functions through its role as an essential cell surface receptor and a co-receptor for various ligands (see, e.g., Guo and Vander Kooi, J. Cell. Biol. 290(49):29120-29126, 2015). For example, it plays a role during epithelial-mesenchymal transition (EMT), for example, by promoting 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 mediating EMT or endo-EMT in fibroblasts, myofibroblasts, and endothelial cells to promote fibrosis formation (see, e.g., Pardali et al., Int. J. Mol. Sci. 18:2157, 2017).

[0312] 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., Int J Mol Sci 8(4)e60164, 2013). NRP2 also regulates smooth muscle contractility (see, e.g., Bielenberg et al., Amer. J. Path. 181:548-559, 2012), regulates autophagy, for example, in cancer (see, e.g., Stanton et al., Cancer Res. 73:160-171, 2013), promotes 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 immune cell activation and migration (see, e.g., Mendes-da-Cruz et al., PLoS ONE 9(7)e103405, 2014). Neuropilin is also a multifunctional co-receptor involved in tumor initiation, growth, metastasis, and immunity (see, eg, Prud'homme et al., Oncotarget 3:921-939, 2012).

[0313] Neuropilin-2 is expressed in various cells of the immune system, including lymphocytes, such as B cells and T cells; and myeloid cells, such as basophils, eosinophils, monocytes, dendritic cells, neutrophils and macrophages, including tissue-specific macrophages, for example, 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., Am. J. Pathol. 181:548-559, 2012; Aung et al., PLoS ONE 11(2)e0147358, 2016; Schellenburg et al., Mol. Immunol., 90:239-244, 2017; and Wild et al., Int. J. Exp. Path. 93:81-103, 2012).

[0314] Neuropilin-2 also plays a key role in endosomal development, for example, by regulating late endosomal maturation, which are important aspects of phagocytosis and endocytosis that promote clearance of infected and apoptotic cells, respectively (see, e.g., Diaz-Vera et al., J. Cell. Sci. 130:697-711, 2017; Dutta et al., Cancer Res 76:418-428, 2016).

[0315] Neuropilin-2 is known to be a key factor in the pathophysiology of many diseases (e.g., "NRP2-related diseases") and interacts with various soluble ligands including scutellum 3F, VEGF-C and D, and TGF-β (see, e.g., Tables N2 and N3), as well as a large number of cellular receptors and cofactors (see, e.g., Tables N4 and N5). Figures 1A-1B and Figure 2) interaction. NRP2 is also polysialylated on dendritic cells and actively interacts with the chemokine CCL21 to mediate immune cell migration, and for this reason single nucleotide polymorphisms associated with ILD and RA have been described (see, for example, Rey-Gallardo et al., " Glycobiology (Glycobiology) " 20: 1139-1146, 2010; Stahl et al., " Nature - Genetics (Nat.Genet.) " 42: 508-514, 2013; and Miller et al., " Arthritis and Rheumatology (Arthritis Rheum.) " 65: 3239-3247). In addition, the soluble circulating form of NRP-2 is known (see, for example, Parker et al., " Structure (Structure) " 23 (4) 677-687, 2015), and internal studies have confirmed the presence of a circulating complex of HRS polypeptides and NRP-2 polypeptides in the circulation. Therefore, given the central role that NRP2 plays in the pathophysiology of a variety of diseases, it is clear that the interaction between NRP2 and one or more NRP2 ligands (e.g., NRP2 ligands from Table N2 and Table N3) and the modulation of those interactions with anti-NRP2 antibodies to selectively alter the corresponding biological activities offer broad potential for the treatment of diseases including NRP2-related diseases.

[0316] NRP2 is a single-pass transmembrane receptor whose main extracellular region contains two CUB domains (A1 / A2 combination domains), two factor V / VIII homology domains (B1 / B2 combination domains) and a MAM domain (C domain) (see Figures 1A-1B). The A1A2 combined domain interacts with the sema region of the arm plate protein, and the B1 domain interacts with the arm plate protein PSI and Ig-like domain. NRP2 has a higher affinity for SEMA3F and 3G; in contrast, SEMA 3A, 3B and 3E preferentially interact with NRP1. Both NRP1 and NRP2 have similar affinities for SEMA 3C. The B1B2 combined domain interacts with several growth factors containing heparin binding domains, including VEGF C and D, placental growth factor (PIGF)-2, fibroblast growth factor (FGF), galectins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β (see, for example, 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 SEMA. In this context, it has been shown that integrins and growth factor receptors (such as VEGF receptor, TGF-β receptor, c-Met, EGFR, FGFR, PDGFR) interact with NRPs and overall appear to increase the affinity of each ligand for its receptor and modulate downstream signaling. The C domain (Mam) domain does not appear to be required for ligand binding but appears to be required for signaling.

[0317] Therefore, anti-NRP2 antibodies that bind to the A1 and / or A2 domains of NRP2 have the potential to selectively regulate arm plate protein binding. Similarly, anti-NRP2 antibodies that bind to the B1 domain have the potential to regulate arm plate protein binding to VEGF and growth factors, and anti-NRP2 antibodies that bind to the B2 domain have the potential to selectively regulate VEGF and growth factor binding. Antibodies that bind to the C domain may not directly affect NRP2 ligand binding, but may regulate NRP2 downstream signaling. Based on the binding mode of specific anti-NRP2 antibodies and due to steric effects that may indirectly affect ligand binding, additional diversity in the functional effects of specific anti-NRP2 antibodies can be expected.

[0318] NRP2 can form homodimers as well as heterodimers and is highly glycosylated. NRP2 has different splice variants with lengths ranging from approximately 551 amino acids to 926 amino acids. The two main variants of NRP2 are classified as NRP2a and NRP2b. They differ in that their intracellular C-terminal portion ( Figures 1A-1B), where for NRP2a, the C-terminal domain includes 42 amino acids and a PDZ binding domain with a C-terminal SEA amino acid sequence. In contrast, NRP2b includes a C-terminal domain of 46 amino acids, which share approximately 11% of the intracellular and transmembrane sequences of NRP2a. Between the MAM domain and the transmembrane domain, additional splicing can occur, and 5 additional amino acids (GENFK) can be added to either the NRP2a or NRP2b forms - these variants are named according to the number of additional amino acids added by alternative splicing. Thus, two additional variants of NRP2 are named NRP2a(17) and NRP2a(22), and two different transmembrane variants of NRP2b are named NRP2b(0) and NRP2b(5). In addition, a soluble form known as sNRP2b can also be produced. Exemplary NRP2 polypeptide sequences are provided in Table N1 below.

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1. pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM affinity, or optionally with an affinity ranging from 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 50 0 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 The human NRP2 polypeptide is preferably bound to the human NRP2 polypeptide with an affinity of 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.

[0330] In some embodiments, the 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 one or more of the following: neuropilin A1 domain (SEQ ID NO: 10), neuropilin A2 domain (SEQ ID NO: 11), neuropilin B1 domain (SEQ ID NO: 12), neuropilin B2 domain (SEQ ID NO: 13), neuropilin C domain (SEQ ID NO: 14), neuropilin A1 / A2 combined domain (SEQ ID NO: 15), neuropilin B1 / B2 combined domain (SEQ ID NO: 20), neuropilin A2 / B1 combined domain (SEQ ID NO: 16), neuropilin B2 / C combined domain (SEQ ID NO: 89), neuropilin A2 / B1 / B2 combined domain (SEQ ID NO: 19), neuropilin A2 / B1 / B2 / C combined domain (SEQ ID NO: 121), neuropilin A1 / A2 / B1 combined domain (SEQ ID NO: 132). NO: 17), neuropilin A1 / A2 / B1 / B2 combined domain (SEQ ID NO: 18), neuropilin A1 / A2 / B1 / B2 / C combined domain (SEQ ID NO: 122), and neuropilin B1 / B2 / C combined domain (SEQ ID NO: 90). In specific embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin B1 domain, the neuropilin B2 domain, and / or the neuropilin B1 / B2 combined domain (see Table N1).In certain embodiments, the antibody or antigen-binding fragment thereof is present in an amount of about 10 pM to about 500 pM or to about 50 nM, or about, at least about, or no more than about 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 110 pM, 120 pM, 130 pM, 140 pM, 150 pM, 160 pM, 170 pM, 180 pM, 190 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1000 pM, 110 pM, 120 pM, 130 pM, 140 pM, 150 pM, 160 pM, 170 pM, 180 pM, 190 pM, 200 pM, 300 pM, 00 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 10 nM, 25 nM or 50 nM affinity, 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 25 nM or about 50 nM affinity. 0 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 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 The at least one domain (or at least one epitope therein) is bound to the at least one domain (or at least one epitope therein) with an affinity of about 10 nM to about 25 nM, about 10 nM to about 50 nM, about 25 nM to about 50 nM, 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.

[0331] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope of the neuropilin A1 domain, the neuropilin A2 domain, and / or the neuropilin A1A2 combined domain, including the adjacent linker region, e.g., at approximately the following residues: (neuropilin A1 domain) 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 set forth in SEQ ID NO: NO: 1 (FL human NRP2); or for example, binds at about the following residues: (neuropilin A2 domain) 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, 141-15 0, 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 as defined in SEQ ID NO: 1 (FL human NRP2);Or, for example, at about the following residues: (combined A1A2 domains) 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, 20-240, 20-280, 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 SEQ ID NO: 1 (FL human NRP2).

[0332] In certain embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope of the neuropilin Bl domain (SEQ ID NO: 12), the neuropilin B2 domain (SEQ ID NO: 13) and / or the neuropilin B1 / B2 combined domain (SEQ ID NO: 20), including the adjacent linker region, e.g., at approximately the following residues: (neuropilin Bl domain) 266-426, 280-426, 290-426, 300-426, 310-426, 320-426, 330-426, 340-426, 350-426, 360-426, 370-426, 380-426, 26, 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 SEQ ID NO:1 (FL human NRP2) defined; (neuropilin B2 domain) 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, 57 0-591, 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 SEQ ID NO: 1 (FL human NRP2);or (neuropilin B1 / B2 combined domain) 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 SEQ ID NO: 1 (FL human NRP2);

[0333] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope of the neuropilin A2 / B1 combined domain and / or the neuropilin B2C combined domain, including the adjacent linker region, e.g., at approximately the following residues: (neuropilin A2B1 combined domain) 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 SEQ ID NO: 1 (FL human NRP2);Or, for example, binds at approximately the following residues: (neuropilin B2C combining domain) 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 4, 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, 438-6 24, 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 SEQ ID NO: 1 (FL human NRP2);

[0334] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin C domain, including the adjacent linker region, e.g., at approximately the following 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, 94, 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 SEQ ID NO: 1 (FL human NRP2).

[0335] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin B1 / B2 / C combined domain, including the adjacent linker region, e.g., at approximately the following 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, 487-794, 496-794, 506-794, 516-794, 526-794, 536-794, 546-794, 556-794, 566-794, 576-794, 587-794, 596-794, 5 794, 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,

[0336] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to a conformational epitope comprised of two or more discontinuous epitope regions. In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to a conformational epitope comprising or consisting of:

[0337] (a) a first epitope region within the A1 domain and a second epitope region within the A2 domain of the human NPR2 polypeptide;

[0338] (b) a first epitope region within the A1 domain and a second epitope region within the B1 domain of the human NPR2 polypeptide;

[0339] (c) a first epitope region within the A1 domain and a second epitope region within the B2 domain of the human NPR2 polypeptide;

[0340] (d) a first epitope region within the A1 domain and a second epitope region within the C domain of the human NPR2 polypeptide;

[0341] (e) a first epitope region within the A2 domain and a second epitope region within the B1 domain of the human NPR2 polypeptide;

[0342] (f) a first epitope region within the A2 domain and a second epitope region within the B2 domain of the human NPR2 polypeptide;

[0343] (g) a first epitope region within the A2 domain and a second epitope region within the C domain of the human NPR2 polypeptide;

[0344] (h) a first epitope region within the B1 domain and a second epitope region within the B2 domain of the human NPR2 polypeptide;

[0345] (i) a first epitope region within the B1 domain and a second epitope region within the C domain of the human NPR2 polypeptide; or

[0346] (j) a first epitope region within the B2 domain and a second epitope region within the C domain of the human NPR2 polypeptide.

[0347] In some 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 "NRP2 ligand," including any molecule that interacts or reversibly binds to human NRP2. General examples of "NRP2 ligands" include polypeptides such as HRS polypeptides, soluble ligands, receptors (e.g., cell surface receptors), including growth factors, growth factor receptors, and the like, and specific examples of NRP2 ligands are described in detail herein. In some embodiments, the at least one antibody or antigen-binding fragment thereof modulates (e.g., antagonizes, interferes with, excites, enhances) the binding of the human NRP2 polypeptide to at least one "NRP2 ligand."

[0348] As described above, in certain embodiments, the at least one NRP2 ligand is a 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, and thereby modulates the binding of the human NRP2 polypeptide to the human HRS polypeptide. Table H1 below provides exemplary HRS polypeptides.

[0349]

[0350]

[0351]

[0352]

[0353] Thus, in certain embodiments, the at least one NRP2 ligand is selected from Table H1, and the anti-NRP2 antibody or antigen-binding fragment thereof modulates (e.g., interferes with) the binding of a human NRP2 polypeptide (e.g., a human NRP2 polypeptide selected from Table H1) 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 cases, mimics one or more signaling activities of the binding of the HRS polypeptide to the NRP2 polypeptide, for example, as an agonist antibody. A "HRS polypeptide interaction region" comprises a region or domain of a human NRP2 polypeptide that interacts with a region or domain of a human HRS polypeptide, for example, at a ligand binding site of a different NRP2 ligand (examples of which are provided herein), a dimerization domain, a protein-protein interaction domain, or at an allosterically sensitive site within the NRP2 polypeptide to modulate the activity of the NRP2 polypeptide.

[0354] In certain embodiments, the antibody or antigen-binding fragment thereof is a "blocking antibody" that 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 other NRP2 ligand (e.g., selected from Table N2 or Table N3). In some embodiments, after pre-incubation of the "blocking antibody" with the NRP2 polypeptide in substantially stoichiometric amounts, 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). As used herein, "stoichiometric equivalence" refers to a situation in which the number of moles of one substance (e.g., an anti-NRP2 antibody) in a given equation or reaction is equal to or substantially equal to the number of moles of at least one other substance (e.g., an NRP2 polypeptide).

[0355] In certain embodiments, the antibody or antigen-binding fragment thereof is a "partial blocking antibody" that at least partially, but not completely, 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 other NRP2 ligands (e.g., selected from Table N2 or Table N3). In some embodiments, after pre-incubation of the "partial blocking antibody" with the NRP2 polypeptide in a stoichiometric amount, the "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 the NRP2 polypeptide and the NRP2 ligand (e.g., HRS polypeptide).

[0356] In a specific embodiment, the at least one antibody or antigen-binding fragment thereof specifically inhibits or otherwise reduces the binding between a human NRP2 polypeptide and a HRS polypeptide splice variant selected from Table H1, such as a HRS splice variant selected from one or more of the following: 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).

[0357] As described above, NRP2 interacts with a variety of NRP2 ligands other than HRS that mediate downstream signaling events. Additional examples of NRP2 ligands are provided in Tables N2 and N3 below.

[0358]

[0359]

[0360]

[0361]

[0362] Thus, in certain embodiments, the at least one NRP2 ligand is selected from Table N2 and / or Table N3.

[0363] For example, in some aspects, the 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. VEGF-VEGFR2 / 3-NRP2 interactions are associated with promoting cell migration, cell growth, cell survival, and cell attachment, and are also associated with lymphangiogenesis, increasing vascular permeability, activating integrin signaling, promoting vesicle trafficking and internalization, and slowing cell differentiation. Therefore, it is expected that anti-NRP2 antibodies that modulate VEGF-related NRP2 ligands will be used to modulate one or more of these pathways.

[0364] In some aspects, the at least one NRP2 ligand is an arm plate protein 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 through a tight dynamic interaction between VEGF and the Sema signaling pathway. SEMA typically functions in the immune system to control cell movement, cell migration, intercellular communication and cell activation. The SEMA plexin-NRP2 interaction is associated with inhibiting cell migration, inhibiting cell growth, promoting apoptosis, inhibiting cell attachment, inhibiting integrin signaling, promoting cell differentiation, inhibiting lymphangiogenesis, reducing vascular permeability, promoting microtubule destabilization, mediating the collapse of the actin cytoskeleton and cell contraction (including growth cone collapse and actomyosin contraction), and preventing neuronal cell proliferation and inhibiting axonal proliferation. Therefore, it is expected that anti-NRP2 antibodies that modulate SEMA-related NRP2 ligands will be useful in modulating one or more of these pathways.

[0365] In some aspects, the at least one NRP2 ligand is an integrin selected from one or more of αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, α6β1, and α6β4. Integrin-NRP2 interactions are generally associated with cell adhesion, cell growth, cancer growth, and increased invasiveness. Therefore, it is expected that anti-NRP2 antibodies that modulate integrin-associated NRP2 ligands will be useful in modulating one or more of these pathways.

[0366] In some aspects, the at least one NRP2 ligand is selected from TGFβ1, TGFβ2, TGFβ3 and its corresponding TGFβ receptor. TGF-β signaling is largely involved in the regulation of EMT in cancer and is also involved in the development of fibrosis (see, for example, Gemmill et al., "Science Signaling (Sci.Signal.)" 10eaag0528, 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 and tumor sphere formation, and also enhances acquired EGFR inhibitor resistance associated with EMT in cancer cells. Therefore, it is expected that anti-NRP2 antibodies that regulate TGF-β related NRP2 ligands will be used to regulate one or more of these pathways. Thus, in certain embodiments, the anti-NRP2 antibody or antigen-binding fragment thereof modulates the binding / signaling activity between an 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.

[0367] In some cases, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the at least one NRP2 ligand. For example, in some embodiments, the anti-NRP2 antibody antagonizes or reduces 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%) of the theoretical maximal binding / signaling between the NRP2 polypeptide and the NRP2 ligand after preincubation of the anti-NRP2 antibody with the NRP2 polypeptide in substantially stoichiometric amounts.

[0368] In some cases, the at least one antibody or antigen-binding fragment thereof agonizes or enhances the binding / signaling activity between the NRP2 polypeptide and the at least one NRP2 ligand. For example, in some embodiments, the anti-NRP2 antibody agonizes or enhances the binding / signaling activity between the NRP2 polypeptide and the at least one NRP2 ligand 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%) of the theoretical maximal binding / signaling activity between the NRP2 polypeptide and the at least one NRP2 ligand after preincubation of the anti-NRP2 antibody and the NRP2 polypeptide in substantially stoichiometric amounts.

[0369] In some embodiments, the at least one antibody or antigen-binding fragment thereof selectively modulates the binding and / or signaling of scutellum to an NRP2 polypeptide or the binding and / or signaling of scutellum through an NRP2 polypeptide. In some aspects, the antibody does not substantially block the interaction of VEGF-C or related NRP2 ligands. In some aspects, the antibody is an agonist antibody with respect to scutellum signaling. In some aspects, the antibody is an antagonist antibody with respect to scutellum signaling.

[0370] In some embodiments, the at least one antibody or antigen-binding fragment thereof selectively modulates the binding and / or signaling of VEGF-C or a related NRP2 ligand to an NRP2 polypeptide or the binding and / or signaling of VEGF-C or a related NRP2 ligand through an NRP2 polypeptide. In some aspects, the antibody does not substantially block the interaction of arm plate protein. In some embodiments, the antibody selectively modulates the binding of VEGF-C or a related NRP2 ligand and arm plate protein to an NRP2 polypeptide. In some embodiments, the antibody is an agonist antibody with respect to VEGF-C signaling. In some aspects, the antibody is an antagonist antibody with respect to VEGF-C signaling.

[0371] In some embodiments, the at least one antibody or antigen-binding fragment thereof selectively modulates binding and / or signaling of an integrin or related NRP2 ligand to an NRP2 polypeptide.

[0372] In some embodiments, the at least one antibody or antigen-binding fragment thereof selectively modulates binding and / or signaling of TGFβ1, TGFβ2, TGFβ3, or their corresponding TGFβ receptors to an NRP2 polypeptide.

[0373] In some embodiments, the at least one antibody or antigen-binding fragment thereof selectively regulates 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.

[0374] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the plexin receptor and / or scutellum protein, without substantially modulating the binding / signaling activity between the NRP2 polypeptide and VEGFR3 or VEGF-C.

[0375] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the plexin receptor and / or scutellum protein, while not substantially modulating the binding / signaling activity between the NRP2 polypeptide and the HRS polypeptide.

[0376] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the plexin receptor and / or scutellum protein, but does not substantially modulate the binding / signaling activity between the NRP2 polypeptide and the HRS polypeptide, and does not substantially modulate the binding / signaling activity between the NRP2 polypeptide and VEGFR3 or VEGF-C.

[0377] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and VEGR3 without substantially modulating the binding / signaling activity between the NRP2 polypeptide and the plexin receptor and / or schizolin.

[0378] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and VEGR3 or VEGF-C, but does not substantially modulate the binding / signaling activity between the NRP2 polypeptide and the HRS polypeptide.

[0379] In some embodiments, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the plexin receptor without substantially modulating ligand binding of schirmlin 3 to NRP2.

[0380] In some embodiments, the plexin receptor is selected from plexin A1, A2, A3, A4, and D1. In some embodiments, the sagittal protein is selected from sagittal protein 3B, 3C, 3D, 3F, and 3G.

[0381] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the human NRP2 A2 domain, the epitope comprising at least 5 contiguous amino acids of SEQ ID NO: 11, wherein the 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, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within amino acids 232-242 of human NRP2 SEQ ID NO: 1.

[0382] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the human NRP2 B1 domain, the epitope comprising at least 5 contiguous amino acids of SEQ ID NO: 12, wherein the at least one antibody or antigen-binding fragment thereof selectively inhibits receptor dimerization between NRP2 and FLT4 (VEGFR3) without substantially inhibiting dimerization between NRP2 and Plexin A1.

[0383] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the human NRP2 B2 domain, said epitope comprising at least 5 contiguous amino acids of SEQ ID NO: 13, wherein the 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.

[0384] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the human NRP2 C domain, the epitope comprising at least 5 contiguous amino acids of SEQ ID NO: 14, wherein the 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).

[0385] In some embodiments, the at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC) for each of (i) a human NRP2 polypeptide and (ii) a corresponding region of a cynomolgus monkey NRP2 polypeptide. 50 ), wherein 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 nM to about 1.2 nM, about 0.9 nM to about 5.5 nM, about 0.9 nM to about 5 nM, or about 1 nM to about 10 nM.

[0386] In some embodiments, the at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC) for each of (i) a human NRP2 polypeptide and (ii) a corresponding region of a murine NRP2 polypeptide. 50), wherein 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.

[0387] In certain embodiments, the antibody or antigen-binding fragment thereof is characterized by a heavy chain variable region (V H ) sequence and light chain variable region (V L ) sequence is characterized by or includes the V H Sequence and V L Sequence, the V H The sequence includes the complementarity determining region V H CDR1, V H CDR2 and V H CDR3 sequence, the V L The sequence includes the complementarity determining region V L CDR1, V L CDR2 and V L CDR3 sequences are provided in Table A1 below. H 、V H CDR1, V H CDR2, V H CDR3, V L 、V L CDR1, V L CDR2 and V L CDR3 sequence.

[0388]

[0389]

[0390]

[0391]

[0392]

[0393] Thus, in certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0394] Heavy chain variable region (V H ) sequence, the V H The sequence includes a complementary determining region V selected from Table A1 that specifically binds to a human NRP2 polypeptide (e.g., selected from Table N1) H CDR1, V H CDR2 and VH CDR3 sequences and variants thereof; and

[0395] Light chain variable region (V L ) sequence, the V L The sequence includes a complementary determining region V selected from Table A1 that specifically binds to the human NRP2 polypeptide (e.g., selected from Table N1) L CDR1, V L CDR2 and V L CDR3 sequences and variants thereof.

[0396] In certain embodiments, the CDR sequences are as follows:

[0397] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 23-25, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 26-28, respectively, including variants thereof;

[0398] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 29-31, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 32-34, respectively, including variants thereof;

[0399] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 35-37, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 38-40, respectively, including variants thereof;

[0400] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 41-43, respectively, and the V L CDR1, V L CDR2 and V LCDR3 sequences include SEQ ID NOs: 44-46, respectively, including variants thereof;

[0401] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 47-49, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 50-52, respectively, including variants thereof;

[0402] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 53-55, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 56-58, respectively, including variants thereof;

[0403] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 59-61, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 62-64, respectively, including variants thereof;

[0404] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 65-67, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 68-70, respectively, including variants thereof;

[0405] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 71-73, respectively, and the V L CDR1, V L CDR2 and V LCDR3 sequences include SEQ ID NOs: 74-76, respectively, including variants thereof;

[0406] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 77-79, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 80-82, respectively, including variants thereof;

[0407] The V H CDR1, V H CDR2 and V H The CDR3 sequences include SEQ ID NOs: 83-85, respectively, and the V L CDR1, V L CDR2 and V L CDR3 sequences include SEQ ID NOs: 86-88, respectively, including variants thereof.

[0408] Also included are variants thereof that bind to human NRP2, including affinity matured variants, e.g., variants having 1, 2, 3, 4, 5, or 6 alterations in one or more of the CDR regions, e.g., V ... H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and / or V L One or more of the CDR3 sequences. Exemplary "alterations" include amino acid substitutions, additions, and deletions.

[0409] For illustrative purposes only, the binding interaction between human NRP2 polypeptide and NRP2 ligand can be detected and quantified using various conventional methods, including biacore assays (e.g., using appropriately labeled soluble reagents bound to a sensor chip), FACS analysis using cells expressing NRP2 polypeptide on the cell surface (natural or recombinant), immunoassays, fluorescent staining assays, ELISA assays, and microcalorimetry, such as ITC (isothermal titration calorimetry).

[0410] In certain embodiments, the antibodies or antigen-binding fragments thereof include one or more variant Fc regions or otherwise modified Fc regions comprising regions with altered properties or biological activities relative to one or more wild-type Fc regions. Examples of modified Fc regions include: Fc regions having mutant sequences, e.g., by substitution, insertion, deletion, or truncation of one or more amino acids relative to the wild-type sequence; hybrid Fc polypeptides composed of domains from different immunoglobulin classes / subclasses; Fc polypeptides with altered glycosylation / sialylation patterns; and Fc polypeptides modified or derivatized, e.g., by biotinylation (see, e.g., U.S. Application No. 2010 / 0209424), phosphorylation, sulfation, etc.; or any combination thereof. Such modifications can be employed to alter (e.g., increase, decrease) the binding properties of the Fc region to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn), 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 , fluctuation), its immunogenicity, its complement binding or activation and / or CDC / ADCC / ADCP related activities of the Fc region and other properties described herein. Contains a modified Fc region of human and / or mouse origin.

[0411] Also encompassed are antibodies or antigen-binding fragments thereof that comprise a hybrid Fc region, e.g., an Fc region comprising 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. General examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of the following 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, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgA2 gD / 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 / IgG 3. IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, Ig G3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / I IgM / IgM (or fragments or variants thereof), and optionally comprises a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3 or IgG4 and / or a CH4 domain from IgE and / or IgM.In specific embodiments, the hinge, CH2, CH3, and CH4 domains are from human Ig.

[0412] Additional examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of the following CH2 / 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, IgM IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof), and optionally comprising a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4 and / or a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM. In specific embodiments, the hinge, CH2, CH3 and CH4 domains are from human Ig.

[0413] Certain examples comprise hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of the following 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, IgM IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof), and optionally comprising a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4 and / or a CH2 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM. In specific embodiments, the hinge, CH2, CH3 and CH4 domains are from human Ig.

[0414] Specific examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of the following 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, Ig A2 / 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, I gG2 / 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), and optionally comprising a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM and / or a CH4 domain from IgE and / or IgM. In specific embodiments, the hinge, CH2, CH3 and CH4 domains are from human Ig.

[0415] Certain examples include hybrid Fc regions comprising, consisting of, or consisting essentially of 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, Ig A2 / 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, I gG2 / 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), and optionally comprising a CH2 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM and / or a CH4 domain from IgE and / or IgM. In specific embodiments, the hinge, CH2, CH3 and CH4 domains are from human Ig.

[0416] Some examples comprise hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of hinge / CH4 domains: 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 comprising a CH2 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM and / or a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM.

[0417] Specific examples of hybrid Fc regions derived from a combination of IgG subclasses or a combination of human IgD and IgG can be found, for example, in WO 2008 / 147143.

[0418] Also encompassed are antibodies or antigen-binding fragments thereof having a derivatized or otherwise modified Fc region. In certain aspects, the Fc region can be modified relative to a wild-type or naturally occurring Fc region by, for example, phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, or the like. In certain embodiments, the Fc region can comprise a wild-type or native glycosylation pattern, or alternatively, its glycosylation can be increased relative to the native form, decreased relative to the native form, or completely deglycosylated. As an example of a modified Fc glycoform, reduced glycosylation in the Fc region can reduce binding to the C1q region of the first complement component C1, reduce ADCC-associated activity, and / or reduce CDC-associated activity. Therefore, certain embodiments utilize a deglycosylated or aglycosylated Fc region. For the generation of exemplary aglycosylated Fc regions, see, for example, WO 2005 / 047337. Another example of an Fc region glycoform can be generated by replacing the Q295 position according to the numbering system of Kabat et al. with a cysteine ​​residue (see, e.g., U.S. Application No. 2010 / 0080794). Certain embodiments may comprise an Fc region in which about 80-100% of the glycoproteins in the Fc region comprise a mature core carbohydrate structure lacking fructose (see, e.g., U.S. Application No. 2010 / 0255013). Some embodiments may comprise an Fc region optimized by substitution or deletion to reduce the level of fucosylation, e.g., to increase affinity for FcγRI, FcγRIa, or FcγRIIIa and / or to improve phagocytosis of FcγRIIa-expressing cells (see, e.g., U.S. Application Nos. 2010 / 0249382 and 2007 / 0148170).

[0419] As another example of a modified Fc glycoform, the Fc region of an antibody or antigen-binding fragment thereof can include mannooligosaccharide-type N-glycans and optionally have one or more of the following relative to a 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 a target of an NRP2 polypeptide, similar or greater binding affinity for a target of an NRP2 polypeptide, and / or similar or less binding affinity for the mannose receptor (see, e.g., U.S. Appl. No. 2007 / 0092521 and U.S. Pat. No. 7,700,321). As another example, engineered glycoforms produced by expressing antibodies in engineered or variant cell lines have been used to enhance the affinity of the Fc region for FcγRs (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., J Biol Chem. 278:3466-3473, 2003; and U.S. Appl. No. 2007 / 0111281). Certain Fc region glycoforms include an increased proportion of N-glycosidically bonded complex carbohydrate chains that do not have a fucose-containing position 1 at the reducing end of the carbohydrate chain that is bound to the N-acetylglucosamine at the 6th position (see, e.g., U.S. Application No. 2010 / 0092997). Specific embodiments may comprise an IgG Fc region glycosylated with at least one galactose moiety linked to a corresponding terminal sialic acid moiety via an α-2,6 linkage, optionally wherein the Fc region has increased anti-inflammatory activity relative to a corresponding wild-type Fc region (see, e.g., U.S. Application No. 2008 / 0206246). Certain of these and related methods for altering glycosylation significantly enhance the ability of an Fc region to selectively bind to an FcR (e.g., FcγRIII) to mediate ADCC and alter other properties of the Fc region as described herein.

[0420] Relative to the corresponding wild-type Fc sequence (e.g., same species, same Ig class, same Ig subclass), certain variant Fc regions, fragment Fc regions, hybrid Fc regions, or otherwise modified Fc regions of an antibody or antigen-binding fragment thereof may have altered binding to one or more FcRs and / or corresponding changes in their effector functions. For example, relative to the corresponding wild-type Fc sequence, such Fc regions may have increased binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and / or a neonatal Fc receptor. In other embodiments, relative to the corresponding wild-type Fc sequence, the binding of a variant Fc region, a fragment Fc region, a hybrid Fc region, or a modified Fc region to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and / or a neonatal Fc receptor may be reduced. Specific FcRs are described elsewhere herein.

[0421] In some embodiments, the antibody comprises an Fc domain comprising one or more mutations that increase binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and / or a neonatal Fc receptor relative to a corresponding wild-type Fc sequence. In some embodiments, the antibody comprises an IgG1 or IgG3 Fc domain comprising one or more mutations that increase binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and / or a neonatal Fc receptor relative to a 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, the at least one antibody comprises an Fc domain selected from human IgG1 and IgG3 comprising one or more mutations that increase effector function.

[0422] In some embodiments, the antibody is a blocking antibody comprising an Fc domain with high effector activity. In some embodiments, the blocking antibody comprises an Fc domain selected from human IgG1 and IgG3, and the Fc domain comprises one or more mutations that increase effector function. In some embodiments, the antibody is a partially blocking antibody comprising an Fc domain with high effector activity. In some embodiments, the partially blocking antibody comprises an Fc domain selected from human IgG1 and IgG3, and the Fc domain comprises one or more mutations that increase effector function. In some embodiments, the antibody is a non-blocking antibody comprising an Fc domain with high effector activity. In some embodiments, the non-blocking antibody comprises an Fc domain selected from human IgG1 or IgG3, and the Fc domain comprises one or more mutations that increase effector function.

[0423] In some embodiments, the antibody comprises an Fc domain comprising one or more mutations that reduce binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and / or a neonatal Fc receptor relative to a corresponding wild-type Fc sequence. In some embodiments, the antibody comprises an IgG1 or IgG3 Fc domain comprising one or more mutations that reduce binding to one or more of an Fcγ receptor, an Fcα receptor, an Fcε receptor, and / or a neonatal Fc receptor relative to a corresponding wild-type Fc sequence. In some embodiments, the antibody comprises an Fc domain comprising one or more mutations that reduce effector function. In some embodiments, the antibody comprises an Fc domain selected from human IgG2 and IgG4 comprising one or more mutations that reduce effector function.

[0424] In some embodiments, the antibody is a blocking antibody comprising an Fc domain with low effector activity. In some embodiments, the blocking antibody comprises an Fc domain selected from human IgG2 and IgG4, and the Fc domain comprises one or more mutations that reduce effector function. In some embodiments, the antibody is a partially blocking antibody comprising an Fc domain with low effector activity. In some embodiments, the partially blocking antibody comprises an Fc domain selected from human IgG2 and IgG4, and the Fc domain comprises one or more mutations that reduce effector function. In some embodiments, the antibody is a non-blocking antibody comprising an Fc domain with low effector activity. In some embodiments, the non-blocking antibody comprises an Fc domain selected from human IgG2 and IgG4, and the Fc domain comprises one or more mutations that reduce effector function.

[0425] Specific examples of Fc variants with altered (e.g., increased, decreased) effector function / FcR binding can be found, for example, in U.S. Patent Nos. 5,624,821 and 7,425,619; U.S. Application Nos. 2009 / 0017023, 2009 / 0010921, and 2010 / 0203046; and WO 2000 / 42072 and WO 2004 / 016750. Certain examples include human Fc regions with one or more substitutions at positions 298, 333, and / or 334, such as S298A, E333A, and / or K334A (numbering based on the EU index of Kabat et al.), which have been shown to increase binding to the activating receptor FcγRIIIa and reduce binding to the inhibitory receptor FcγRIIb. These mutations can be combined to obtain double and triple mutation variants that further improve binding to FcRs. Certain embodiments include the S298A / E333A / K334A triple mutant, which has increased binding to FcγRIIIa, decreased binding to FcγRIIb, and increased 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 FcRs, such as those disclosed in Umana et al., supra; and U.S. Pat. No. 7,662,925. Some embodiments comprise an Fc region comprising one or more substitutions selected from 434S, 252Y / 428L, 252Y / 434S, and 428L / 434S based on the EU index of Kabat et al. (see US Application Nos. 2009 / 0163699 and 20060173170).

[0426] Certain variant Fc regions, fragment Fc regions, hybrid Fc regions, or modified Fc regions may exhibit altered effector functions relative to a corresponding wild-type Fc sequence. For example, such an Fc region may exhibit increased complement fixation or activation, increased Clq binding affinity, increased CDC-related activity, increased ADCC-related activity, and / or increased ADCP-related activity relative to a corresponding wild-type Fc sequence. In other embodiments, such an Fc region may exhibit decreased complement fixation or activation, decreased Clq binding affinity, decreased CDC-related activity, decreased ADCC-related activity, and / or decreased ADCP-related activity relative to a corresponding wild-type Fc sequence. As an illustrative example only, an Fc region may include deletions or substitutions in complement binding sites, such as the Clq binding site, and / or deletions or substitutions in ADCC sites. 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 conventional techniques in the art. (See, e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978.) Useful effector cells for such assays include, but are not limited to, natural killer (NK) cells, macrophages, and other peripheral blood mononuclear cells (PBMCs). Alternatively or additionally, certain Fc effector functions can be assessed in vivo, for example, by employing the animal model described in Clynes et al., Proc. Natl. Acad. Sci. USA, 95:652-656, 1998.

[0427] The stability or half-life of certain variant Fc regions, hybrid Fc regions, or modified Fc regions may be altered relative to the corresponding wild-type Fc sequence. In certain embodiments, the half-life of such Fc regions may be increased relative to the corresponding wild-type Fc sequence. In other embodiments, the half-life of variant Fc regions, hybrid Fc regions, or modified Fc regions may be reduced relative to the corresponding wild-type Fc sequence. Half-life can be measured in vitro (e.g., under physiological conditions) or in vivo according to conventional techniques in the art (e.g., radiolabeling, ELISA, or other methods). In vivo measurements of stability or half-life can be measured in one or more body fluids or given tissues, including blood, serum, plasma, urine, or cerebrospinal fluid, such as liver, kidney, muscle, central nervous system tissue, bone, etc. As an example, modifications that alter the binding ability of the Fc region to FcRn can alter its in vivo half-life. Non-limiting examples of assays for measuring in vivo pharmacokinetic properties (e.g., mean elimination half-life in vivo) and Fc modifications that alter Fc binding to FcRn are described, for example, in U.S. Patent Nos. 7,217,797 and 7,732,570; and U.S. Application Nos. US 2010 / 0143254 and 2010 / 0143254.

[0428] Additional non-limiting examples of modifications that alter stability or half-life include substitutions / deletions at one or more amino acid residues selected from amino acid residues 251-256, 285-290, and 308-314 in the CH2 domain and 385-389 and 428-436 in the CH3 domain, numbered according to the numbering system of Kabat et al., see U.S. Application No. 2003 / 0190311. Specific examples include substitution at position 251 with leucine, substitution at position 252 with tyrosine, tryptophan, or phenylalanine, substitution at position 254 with threonine or serine, substitution at position 255 with arginine, substitution at position 256 with glutamine, arginine, serine, threonine, or glutamic acid, substitution at position 308 with threonine, substitution at position 309 with proline, substitution at position 311 with serine, substitution at position 312 with aspartic acid, and substitution at position 314 with leucine. , substitution at position 385 with arginine, aspartic acid or serine, substitution at position 386 with threonine or proline, substitution at position 387 with arginine or proline, substitution at position 389 with proline, asparagine or serine, substitution at position 428 with methionine or threonine, substitution at position 434 with tyrosine or phenylalanine, substitution at position 433 with histidine, arginine, lysine or serine and / or substitution at position 436 with histidine, tyrosine, arginine or threonine, including any combination thereof. Such modifications optionally increase the affinity of the Fc region for FcRn and thereby increase half-life relative to a corresponding wild-type Fc region.

[0429] The solubility of certain variant Fc regions, hybrid Fc regions or modified Fc regions may change relative to the corresponding wild-type Fc sequence. In certain embodiments, the solubility of such Fc regions may increase relative to the corresponding wild-type Fc sequence. In other embodiments, the solubility of variant Fc regions, hybrid Fc regions or modified Fc regions may decrease relative to the corresponding wild-type Fc sequence. Solubility can be measured, for example, in vitro (e.g., under physiological conditions) according to conventional techniques in the art. Exemplary solubility measurements are described elsewhere herein.

[0430] Additional examples of variants include IgG Fc regions having conservative or non-conservative substitutions (as described elsewhere herein) at one or more of positions 250, 314, or 428 of the heavy chain, or any combination thereof (e.g., at positions 250 and 428, or at positions 250 and 314, or at positions 314 and 428, or at positions 250, 314, and 428). 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 illustrative example of an IgG Fc variant, any one or more of the amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, and / or 327 to 331 can be used as suitable targets for modification (e.g., conservative or non-conservative substitutions, deletions). 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 with Ser228Pro and Leu235Ala mutations) to reduce the effector function of the Fc region (see U.S. Patent No. 7,030,226). Here, the numbering of the residues in the heavy chain is that of the EU index (see Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., National Institutes of Health, Bethesda, Md. (1991)). Certain embodiments of these and related embodiments have altered (e.g., increased, decreased) FcRn binding and / or serum half-life, while optionally having no effector function such as ADCC or CDC-related activity.

[0431] Additional examples include variant Fc regions comprising one or more amino acid substitutions or any combination thereof at positions 279, 341, 343, or 373 of a wild-type Fc region (see, e.g., U.S. Application No. 2007 / 0224188). The wild-type amino acid residues at these positions of human IgG are valine (279), glycine (341), proline (343), and tyrosine (373). The one or more substitutions may be conservative or non-conservative, or may comprise non-naturally occurring amino acids or mimetics as described herein. Certain embodiments also employ variant Fc regions comprising at least one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions selected from the group consisting of 235G, 235R, 236F, 236R, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L, 245R, 247A, 247D, 247E, 247F, 247M, 247N, 247R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L, 245R, 247A, 247D, 247E, 247F, 247M, 247N, 247R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L, 245 , 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, 2 56V, 256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, 27 1T, 272H, 272K, 272L, 272R, 279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279 Y, 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、339 W、339Y、341D、341E、341F、341H、341I、341K、341L、341M、341N、341P、341Q、341R、341S、341T、341V、341W、341Y、343A、343D、343E、34 3F、343G、343H、343I、343K、343L、343M、343N、343Q、343R、343S、343T、343V、343W、343Y、373D、373E、373F、373G、373H、373I、373K、3 73L、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、382 L、382M、382N、382P、382Q、382R、382S、382T、382V、382W、382Y、38 5E、385P、386K、423N、424H、424M、424V、426D、426L、427N、429A、42 9F、429M、430A、430D、430F、430G、430H、430I、430K、430L、430M、430N、430P、430Q、430R、430S、430T、430V、430W、430Y、431H、431K、4 31P、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 the residues in the heavy chain is that of the EU index (see Kabat et al., supra). Such variant Fc regions typically alter the effector function or serum half-life of the antibody to which the variant Fc region is operably linked. Preferably, the altered effector function is increased ADCC, decreased ADCC, increased CDC, decreased CDC, increased Clq binding affinity, decreased Clq binding affinity, increased FcR (preferably FcRn) binding affinity, or decreased FcR (preferably FcRn) binding affinity, compared to a corresponding Fc region lacking such one or more such amino acid substitutions.

[0432] Additional examples include variant Fc regions comprising an amino acid substitution at one or more of the following 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 3, 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, e.g., U.S. Patent No. 7,662,925). In a specific embodiment, the variant Fc region comprises at least one amino acid substitution selected from the group consisting of: 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. In other specific embodiments, the variant Fc region comprises at least one amino acid substitution selected from the group consisting of 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, S239E / I332D,S239E / I332N, S239E / I332Q, S239N / I332D, S239N / I332E, S239Q / I332D, A330Y / I332E, V264I / A330Y / I332E, A330L / I332 E. V264I / A330L / I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D / I33 2E, L328V / I332E, L328T / I332E, L328I / I332E, S239E / V264I / I332E, S239Q / V264I / I332E, S239E / V264I / A330Y / I332E, S 239D / 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 / I332 E / A330I, P230A, P230A / E233D / I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S32 4D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L234I, S239D / A330Y / I332E / L 235D, 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 comprises a series of substitutions selected from the group consisting of: N297D / I332E, F241Y / F243Y / V262T / V264T / N297D / I332E, S239D / N297D / I332E, S239E / N297D / I332E, S239D / D265Y / N297D / I332E, 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 (numbering using the EU index, Kabat et al., supra). 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 that of the EU index of Kabat et al. Among other properties described herein, such variant Fc regions may have increased affinity for FcγRs, increased stability, and / or increased solubility relative to a corresponding wild-type Fc region.

[0433] Further examples include 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, 274L, 275V, 276A, 277G / N, 278N, 279K / G, 280A, 281L, 282S / L / G / H / I, 283S / L / G / H / I, 284L, 285E, 286L / A, 287T, 288 6D, 278H, 279M, 280N, 283G, 285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 30 5A, 306I / F, 311R, 312N, 315D / K / S, 320R, 322E, 323A, 324T, 325S, 326E / R, 332T, 333D / G, 335I, 338 R, 339T, 340Q, 341E, 342R, 344Q, 347R, 351S, 352A, 354A, 355W, 356G, 358T, 361D / Y, 362L, 364C, 3 65Q / 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, optionally wherein the variant has altered recognition of an Fc ligand and / or effector function thereof compared to a parent Fc polypeptide, and wherein the numbering of the residues is that of the EU index as in Kabat et al.Specific examples of these and related embodiments comprise variant Fc regions comprising or consisting of the following substitution groups: (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, G341 E 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) S 254T, 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, E333 G 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, where the numbering of the residues is that of the EU index as in Kabat et al. (see, e.g., U.S. application No. 2010 / 0184959).

[0434] Variant Fc regions may also have one or more mutant hinge regions, such as those described in U.S. Application No. 2003 / 0118592. For example, one or more cysteines in the hinge region may be deleted or substituted with different amino acids. The mutant hinge region may not include a cysteine ​​residue, or it may include one, two, or three fewer cysteine ​​residues than the corresponding wild-type hinge region. In some embodiments, Fc regions having such mutant hinge regions exhibit reduced dimerization capacity relative to wild-type Ig hinge regions.

[0435] In certain embodiments, the Fc region comprises, consists of, or consists essentially of an Fc 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 from human IgG1 and IgG4 (CH1, hinge (underlined), CH2, and CH3 regions). Examples of variant IgG4 sequences that can be employed are described, for example, in Peters et al., Journal of Biological Chemistry 287:24525-24533, 2012, and include substitutions at the following positions: C227, C230, C127 (e.g., C127S), and C131 (e.g., C131S). Other variants that can be used include substitutions at the L445P position in IgG4 (expressed as IgG4-2) or substitutions at the D356E and L358M position in IgG1 (expressed as IgG1m(zf)).

[0436]

[0437] As described above, antibodies with altered Fc regions generally have altered (e.g., improved, increased, decreased) pharmacokinetic properties relative to a corresponding wild-type Fc region. Examples of pharmacokinetic properties include stability or half-life, bioavailability (the fraction of the drug that is absorbed), tissue distribution, volume of distribution (the apparent volume of the drug distributed immediately after intravenous injection and after equilibrium is reached between plasma and surrounding tissues), concentration (the initial or steady-state concentration of the drug in plasma), elimination rate constant (the rate at which the drug is removed from the body), elimination rate (the infusion rate required to achieve equilibrium elimination), area under the curve (AUC or exposure; the integral of the concentration-time curve after a single dose or at steady state), clearance (the volume of plasma cleared of the drug per unit time), C max (peak plasma concentration of the drug after oral administration), t max (reach C max time), C min (the lowest concentration of a drug reached before the next dose) and fluctuation (peak-to-trough fluctuations within a dosing interval at steady state).

[0438] In specific embodiments, the biological half-life of the antibody or antigen-binding fragment thereof at about pH 7.4, about physiological pH, about 25°C or room temperature and / or 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) is 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 more, 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 more, or any intermediate half-life including all ranges therebetween.

[0439] In some embodiments, the T of the antibody or antigen-binding fragment thereof m is about or at least about 60° C., 62° C., 64° C., 66° C., 68° C., 70° C., 72° C., 74° C., or 75° C. In some embodiments, the T of the antibody or antigen-binding fragment thereof is about or at least about 60° C., 62° C., 64° C., 66° C., 68° C., m is about 60°C or higher.

[0440] 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, cilengitide, 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, and daptomycin. phosphate), teniposide, temsirolimus, everolimus, vincristine, vinblastine, vinorelbine, vindesine, CT52923, paclitaxel, imatinib, dasatinib, sorafenib, pazopanib, sunitinib, vatalanib, gefitinib, erlotinib, AEE-788, dichoroacetate, tamoxifen, fasudil, SB-681323, semaxanib, donepezil, galantamine , memantine, rivastigmine, tacrine, rasigiline, naltrexone, lubiprostone, safinamide, istradefylline, pimavanserin, pitolisant, isradipine, pridopidine (ACR16), tetrabenazine, bexarotene, glatirimer acetate, fingolimod and mitoxantrone, including pharmaceutically acceptable salts and acids thereof. Additional examples of cytotoxic or chemotherapeutic agents include: alkylating agents such as thiotepa, cyclophosphamide, TM); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide, and trimethylolomelamine; nitrogen mustards, such as chlornaphazine, cholophosphamide, estramustine, ifosfamide, dichloromethyldiethylamine, and mechlorethamine oxide; hydrochloride), melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Antibiotics, such as aclacinomycin, dactinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, icin), 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, tri-iron azotocin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate, rexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone;Antiadrenal agents, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diazocine; elformithine; elliptinium acetate; etoglucid; gallium nitrate nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (; Bristol-Myers Squibb Oncology, Princeton, NJ, and doxetaxel Rhône-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; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid derivatives, such as targretin TM (Bexarotene), Panretin TM (alitretinoin); ONTAK TM (denileukin diftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any one of the foregoing.

[0441] The antibodies or antigen-binding fragments 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 immunotherapeutics described herein.

[0442] Additional therapeutic agents and compositions

[0443] Immunotherapeutic agents Certain embodiments use one or more cancer immunotherapeutics. In some cases, immunotherapeutics regulate the immune response of the subject, for example to increase or maintain cancer-related or cancer-specific immune responses, and thereby increase immune cell suppression or reduce cancer cells. Exemplary immunotherapeutics include polypeptides, for example, antibodies and their antigen-binding fragments, ligands and small peptides and mixtures thereof. Immunotherapeutics also include small molecules, cells (for example, such as immune cells such as T cells), various cancer vaccines, gene therapy agents or other polynucleotide-based medicaments, including viral agents such as oncolytic viruses and other medicaments known in the art. Therefore, in certain embodiments, cancer immunotherapeutics are selected from one or more of immune checkpoint regulators, cancer vaccines, oncolytic viruses, cytokines and cell-based immunotherapies.

[0444] In certain embodiments, cancer immunotherapy agent is immune checkpoint regulator.Specific examples include " antagonists " of one or more inhibitory immune checkpoint molecules and " agonists " of one or more stimulating immune checkpoint molecules. Generally, immune checkpoint molecules are components that enhance signals (co-stimulatory molecules) or weaken signals of the immune system, and the targeting of the components has therapeutic potential for cancer, because cancer cells may disrupt the natural 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, " Natural Cancer Review (Nature Reviews Cancer) " 12:252-264,2012). In certain embodiments, immune checkpoint regulators (e.g., antagonists, agonists) are " combined " or " specifically bound " to one or more immune checkpoint molecules as described herein.

[0445] In a specific embodiment, immune checkpoint regulator is a polypeptide or peptide.The terms "peptide" and "polypeptide" are used interchangeably herein, however, in some cases, the term "peptide" can refer to a shorter polypeptide, for example, 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, including all integers and ranges therebetween (e.g., 5-10, 8-12, 10-15). Polypeptide and peptide can be made up of naturally occurring amino acids and / or non-naturally occurring amino acids as described herein.

[0446] Antibodies are also encompassed as polypeptides. Thus, in some embodiments, the immune checkpoint modulating polypeptide agent is an antibody or "antigen-binding fragment thereof" as described elsewhere herein.

[0447] In some embodiments, the agent is or includes a "ligand" of an immune checkpoint molecule, for example, a natural ligand. "Ligand" generally refers to a substance or molecule that forms a complex with a target molecule (e.g., a biomolecule) for biological purposes and includes a "protein ligand," which typically generates a signal by binding to a site on a target molecule or target protein. Therefore, some agents are protein ligands that naturally bind to immune checkpoint molecules and generate signals. Also included are "modified ligands," for example, protein ligands fused to a pharmacokinetic modifier, such as an Fc region derived from an immunoglobulin.

[0448] The binding properties of the polypeptide can be quantified using methods well known in the art (see Davies et al., Annual Review of Biochemistry 59: 439-473, 1990). In some embodiments, the polypeptide specifically binds to a target molecule (e.g., an immune checkpoint molecule or an epitope thereof) with an equilibrium dissociation constant of about or in the range of about ≤10-7M to about 10-8M. In some embodiments, the equilibrium dissociation constant is about or in the range of about ≤10-9M to about ≤10-10M. In certain illustrative embodiments, the affinity (Kd or EC) of the polypeptide for a target described herein (to which the polypeptide specifically binds) is 50 ) is about, at least about, or less than about 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, 40 nM, or 50 nM.

[0449] In some embodiments, the pharmaceutical agent is a "small molecule," which refers to an organic compound that is of synthetic or biological origin (biomolecules) but is generally not a polymer. An organic compound refers to a large class of compounds whose molecules contain carbon, and generally does not include compounds that contain only carbonates, simple carbon oxides, or cyanides. A "biomolecule" generally refers to an organic molecule produced by a living organism and includes large polymeric molecules (biopolymers) such as peptides, polysaccharides, and nucleic acids, as well as small molecules such as primary secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins, and hormones. A "polymer" generally refers to a macromolecule or polymer composed of repeating structural units, which are generally connected by covalent chemical bonds.

[0450] In certain embodiments, the molecular weight of a small molecule is about or less than about 1000-2000 Daltons, typically between about 300 and 700 Daltons, and includes about 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.

[0451] Certain small molecules may have "specific binding" properties as described for polypeptides herein, such as antibodies. For example, in some embodiments, a small molecule binds to a polypeptide with a binding affinity (Kd or EC) of about, at least about, or less than about 1 Å. 50 ) specifically binds to a target (e.g., an immune checkpoint molecule): 0.01 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 11 nM, 12nM, 13nM, 14nM, 15nM, 16nM, 17nM, 18nM, 19nM, 20nM, 21 nM, 22nM, 23nM, 24nM, 25nM, 26nM, 27nM, 28nM, 29nM, 30nM, 40nM or 50nM.

[0452] In some embodiments, the immune checkpoint modulator 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 receptor 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 inhibitor of T cell activation (VISTA), B and T lymphocyte attenuator (BTLA), CD160, and T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0453] In certain embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, which has been shown to target and restore immune function in the tumor environment (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 expressed on T cells and progenitor B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 acts as an inhibitory immune checkpoint molecule, for example, by reducing or preventing the activation of T cells, which reduces or prevents autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is achieved at least in part by promoting apoptosis of antigen-specific T cells in lymph nodes while also reducing apoptosis of regulatory T cells (suppressor T cells). Some examples of PD-1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1 and reduce one or more immunosuppressive activities thereof (e.g., its downstream signaling or its interaction with PD-L1). Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pidilizumab, and antigen-binding fragments thereof (see, e.g., U.S. Pat. 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. App. Nos. 2012 / 0039906, 2015 / 0203579).

[0454] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As described above, PD-L1 is one of the natural ligands of the PD-1 receptor. General examples of PD-L1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L1 and reduce one or more immunosuppressive activities thereof (e.g., its binding to the PD-1 receptor). Specific examples of PD-L1 antagonists include antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C) and durvalumab (MEDI4736) and antigen-binding fragments thereof (see, e.g., U.S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; 9,439,962).

[0455] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As described above, PD-L2 is one of the natural ligands of the PD-1 receptor. General examples of PD-L2 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-L2 and reduce one or more of its immunosuppressive activities (e.g., its binding to the PD-1 receptor).

[0456] In some embodiments, the agent is a CTLA-4 antagonist or inhibitor. CTLA4 or CTLA-4 (cytotoxic T lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that acts as an inhibitory immune checkpoint molecule by transmitting inhibitory signals to T cells when bound to CD80 or CD86 on the surface of antigen-presenting cells. General examples of CTLA-4 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4. Specific examples include antibodies ipilimumab and tremelimumab and their antigen-binding fragments. It is believed that at least some of the activity of ipilimumab is mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing of inhibitory factor Treg expressing CTLA-4.

[0457] In some embodiments, the agent is an IDO antagonist or inhibitor or a TDO antagonist or inhibitor. IDO and TDO are tryptophan catabolism enzymes with immunosuppressive properties. For example, IDO is known to inhibit T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. General examples of IDO and TDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IDO or TDO (see, for example, Platten et al., Front Immunol. 5:673, 2014) and reduce or inhibit one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include indomod (NLG-8189), 1-methyl-tryptophan (1MT), β-carbolines (norharman, 9H-pyrido[3,4-b]indole), rosmarinic acid, and icardostat (see, for example, Sheridan, Nature Biotechnology 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, eg, Pilotte et al., Proc. Natl. Acad. Sci. USA 109:2497-2502, 2012).

[0458] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T cell immunoglobulin domain and mucin domain 3 (TIM-3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by triggering cell death when interacting with its ligand, galectin-9. TIM-3 contributes to the suppressive 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 TIM-3 antagonists or inhibitors 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.

[0459] In some embodiments, the agent is a LAG-3 antagonist or inhibitor. Lymphocyte activation gene-3 (LAG-3) is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It negatively regulates cell proliferation, activation, and homeostasis of T cells in a manner similar to that of CTLA-4 and PD-1 (see, for example, Workman and Vignali, Eur. J. Immunol. 33:970-9, 2003; and Workman et al., Eur. J. Immunol. 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 keeps CD8+ T cells in a tolerogenic state and is combined with PD-1 to maintain CD8+ T cell exhaustion. General examples of LAG-3 antagonists or inhibitors include antibodies, 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.

[0460] In certain embodiments, medicament is VISTA antagonist or inhibitor.T cell activation V domain Ig inhibitor (VISTA) is mainly expressed on hematopoietic cells and is an inhibitory immune checkpoint regulator, which suppresses T cell activation, induces Foxp3 expression and is highly expressed in tumor microenvironment, in which it suppresses anti-tumor T cell response (see, for example, Lines et al., Cancer Research 74:1924-32,2014). General examples of VISTA antagonists or inhibitors include antibodies or antigen binding fragments or small molecules that specifically bind to VISTA and reduce its immunosuppressive activity.

[0461] In some embodiments, the agent is a BTLA antagonist or inhibitor. B and T lymphocyte attenuator (BTLA; CD272) expression is induced during T cell activation, and it inhibits T cells by interacting with tumor necrosis family receptors (TNF-R) and the B7 cell surface receptor family. BTLA is a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). The BTLA-HVEM complex negatively regulates T cell immune responses, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, for example, Derré et al., Journal of Clinical Research (J Clin Invest) 120: 157–67, 2009). General examples of BTLA antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA-4 and reduce one or more of its immunosuppressive activities.

[0462] In some embodiments, the agent is an HVEM antagonist or inhibitor, e.g., an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD 160. General examples of HVEM antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM, optionally reduce the HVEM / BTLA and / or HVEM / CD160 interaction, and thereby reduce one or more of the immunosuppressive activities of HVEM.

[0463] In some embodiments, the agent is a CD160 antagonist or inhibitor, e.g., an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. General examples of CD160 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CD160, optionally reduce the CD160 / HVEM interaction, and thereby reduce or inhibit one or more of its immunosuppressive activities.

[0464] In some embodiments, the agent is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domains (TIGIT) are co-inhibitory receptors found on the surface of various lymphocytes and, for example, inhibit anti-tumor immunity by Treg (Kurtulus et al., Journal of Clinical Investigation 125: 4053-4062, 2015). General examples of TIGIT antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIGIT and reduce its immunosuppressive activity (see, for example, Johnston et al., Cancer Cell 26: 923-37, 2014).

[0465] In certain embodiments, the immune checkpoint modulator is an agonist of one or more stimulatory immune checkpoint molecules. Exemplary stimulatory immune checkpoint molecules include OX40, CD40, glucocorticoid-induced TNFR family-related genes (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0466] In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the expansion of effector T cells and memory T cells and inhibits the differentiation and activity of T regulatory 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 initiating anti-tumor immune responses in lymph nodes and maintaining anti-tumor 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 immunostimulatory activities. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (murine OX4 agonist), and MEDI6383 (OX40 agonist), and antigen-binding fragments thereof.

[0467] In some embodiments, the agent is a CD40 agonist. CD40 is expressed on antigen presenting cells (APC) and some malignant tumors. Its ligand is CD40L (CD154). On APC, connection causes costimulatory molecules to be upregulated, thereby potentially bypassing the need for T cell assistance in the anti-tumor immune response. CD40 agonist therapy plays an important role in APC maturation and its migration from tumor to lymph node (thereby improving antigen presentation and T cell activation). Anti-CD40 agonist antibodies produce substantial responses and lasting anti-cancer immunity in animal models, which is at least partially mediated by cytotoxic T cells (see, for example, Johnson et al., Clinical Cancer Research (Clin Cancer Res.) 21: 1321-1328, 2015; and Vonderheide and Glennie, Clinical Cancer Research 19: 1035-43, 2013). General examples of CD40 agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to CD40 and increase one or more of its immunostimulatory activities. Specific examples include CP-870,893, daclizumab, Chi Lob 7 / 4, ADC-1013, CD40L, rhCD40L, and antigen-binding fragments thereof.

[0468] In certain embodiments, medicament is GITR agonist. Glucocorticoid-induced TNFR family related gene (GITR) increases T cell expansion, suppresses the inhibitory activity of Tregs and prolongs the survival of T effector cells. It has been shown that GITR agonist promotes anti-tumor response by the loss of Treg lineage stability (see, for example, Schaer et al., " Cancer Immunology Research (Cancer Immunol Res.) " 1:320-31,2013). These different mechanisms show that GITR plays an important role in starting the immune response in lymph nodes and maintaining the immune response in tumor tissue. Its ligand is GITRL. The general example of GITR agonist includes antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITR and increase one or more immunostimulatory activities in its immunostimulatory activity. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873 and its antigen-binding fragment.

[0469] In certain embodiments, the agent is a CD137 agonist.CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and the cross-linking of CD137 can enhance T cell proliferation, IL-2 secretion, survival and cytolytic activity. The signal transduction mediated by CD137 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 immunostimulatory activities in its immunostimulatory activity. Specific examples include CD137 (or 4-1BB) ligands (see, for example, Shao and Schwarz, " Journal of Leukocyte Biology (J Leukoc Biol.) " 89: 21-9, 2011) and antibody utamilumab, including its antigen-binding fragment.

[0470] In some embodiments, the agent is a CD27 agonist. Stimulating CD27 increases the antigen-specific expansion of naive T cells and contributes to the long-term maintenance of T cell memory and T cell immunity. Its ligand is CD70. Targeting human CD27 with agonist antibodies stimulates T cell activation and anti-tumor immunity (see, for example, Thomas et al., Oncoimmunology 2014; 3:e27255.doi:10.4161 / onci.27255; and He et al., Journal of Immunology 191:4174-83, 2013). General examples of CD27 agonists 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 and the antibodies wanlilumab and CDX-1127 (1F5), including antigen-binding fragments thereof.

[0471] In some embodiments, the agent is a CD28 agonist. CD28 is constitutively expressed on CD4+ T cells and some CD8+ T cells. Its ligands include CD80 and CD86, and its stimulation increases T cell expansion. General examples of CD28 agonists 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 its antigen-binding fragments.

[0472] In certain embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT, and in contrast to TIGIT, the engagement of CD226 enhances T cell activation (see, for example, Kurtulus et al., Journal of Clinical Investigation 125: 4053-4062, 2015; Bottino et al., Journal of Experimental Medicine (J Exp Med.) 1984: 557-567, 2003; and Tahara-Hanaoka et al., International Journal of Immunology 16: 533-538, 2004). General examples of CD226 agonists include antibodies or antigen-binding fragments or small molecules or ligands (e.g., CD112, CD155) that specifically bind to CD226 and increase its immunostimulatory activity.

[0473] In some embodiments, the agent is an HVEM agonist. 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 various 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. It has been shown that HVEM signaling plays 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 HVEM agonists 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.

[0474] In certain embodiments, the cancer immunotherapeutic agent is a cancer vaccine. Exemplary cancer vaccines include Oncophage, such as Gardasil or Cervarix, human papillomavirus HPV, hepatitis B vaccines such as Anzaci-B, Recombivax HB or Twinrix, and Siprussin-T (Provenge). In some embodiments, the cancer vaccine includes or utilizes one or more cancer antigens or cancer-associated 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, VEGR-3, NRP2, CD30, CD3 3. 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), α-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylate cyclase C, NY-ESO -1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein α (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 receptor-1, protein disulfide isomerase (PDI), phosphatase of liver regeneration 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin.

[0475] In certain embodiments, the cancer immunotherapeutic agent is an oncolytic virus. Oncolytic viruses are viruses that preferentially infect and kill cancer cells. Includes naturally occurring and artificial or engineered oncolytic viruses. Most oncolytic viruses are engineered for tumor selectivity, but there are naturally occurring examples such as reovirus and SVV-001 Seneca Valley virus. General examples of oncolytic viruses include VSV, poliovirus, reovirus, Senecavirus and RIGVIR and engineered versions thereof. Non-limiting examples of oncolytic viruses include herpes simplex virus (HSV) and engineered versions thereof, Talillah (T-VEC), Coxsackie virus A21 (CAVATAK TM ), Ankori (H101), Perareo Repu 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, etc.

[0476] 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).

[0477] In certain embodiments, cancer immunotherapeutic agents are cell-based immunotherapies, such as adoptive immunotherapy based on T cells. In certain embodiments, cell-based immunotherapies include cancer antigen-specific T cells, optionally ex vivo derived T cells. In certain embodiments, the cancer antigen-specific T cells are selected from one or more of the following: 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, CAR-modified T cells target CD-19 (see, for example, Maude et al., " Blood (Blood) " 125:4017-4023,2015).

[0478] In some cases, the cancer to be treated is associated with a cancer antigen, i.e., cancer antigen-specific T cells target at least one antigen known to be associated with the cancer to be treated or are enriched for said at least one antigen. In some embodiments, the cancer antigen is selected from one or more of the following: 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), α-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylate cyclase C, NY-ESO- 1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein α (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 receptor-1, protein disulfide isomerase (PDI), phosphatase of liver regeneration 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin.

[0479] Additional exemplary cancer antigens include: 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, α-5-β-1-integrin, α-5-β-6-integrin, α-actinin-4 / m, α-methylacyl-CoA racemase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, β-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA 15-3 / CA 27-29, CA19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8 / m, cathepsin B, cathepsin L, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, hairy 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, GAG E-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-A11 / 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-1R, 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, MAG E-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-C 2. 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, MMP11, MN / CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, class I myosin / 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 small bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 kinase, Pin-1, Pml / PARα, POTE, PRAME, PRDX5 / m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD1 68, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp17, 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-β, TGFβRII, TGM-4, TPI / m, TRAG-3, TRG, TRP-1, TRP-2 / 6b, TRP / INT2, TRP-p8, tyrosinase, 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, tyrosinase, PSA, PSCA, PSMA, STEAP-1, Ras, CEA and WT1, and more preferably PAP, MAGE-A3, WT1 and MUC-1.

[0480] In some embodiments, the antigen is selected from the group consisting of: MAGE-A1 (e.g., MAGE-A1 according to Accession No. M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g., MAGE-A6 according to Accession No. NM_005363), MAGE-C1, MAGE-C2, melan-A (e.g., melan-A according to Accession No. NM_005511), GP100 (e.g., GP100 according to Accession No. M77348), tyrosinase (e.g., tyrosinase according to Accession No. NM_000372), survivin (e.g., survivin according to Accession No. AF0773), tyrosinase (e.g., tyrosinase according to Accession No. NM_000372), survivin (e.g., survivin according to Accession No. AF0773), tyrosinase (e.g., tyrosinase according to Accession No. NM_000372), tyrosinase (e.g., tyrosinase according to Accession No. NM_000372), tyrosinase (e.g., tyrosinase according to Accession No. AF0773 ... 50), 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 according to 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), SEC61 G (e.g., SEC61 G according to accession number NM_014302), hTERT (e.g., hTERT 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.

[0481] In some embodiments, the cancer antigen is selected from PCA, PSA, PSMA, STEAP, and optionally MUC-1, including 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, including fragments, variants, and derivatives thereof.

[0482] In some cases, cancer antigens encompass idiotypic antigens associated with cancer or tumor diseases, particularly, for example, lymphoma or lymphoma-related diseases, wherein the idiotypic antigen is the immunoglobulin idiotype of a lymphocyte or the T cell receptor idiotype of a lymphocyte.

[0483] In some cases, the cancer antigen-specific T cells are selected from one or more of the following: chimeric antigen receptor (CAR)-modified T cells (e.g., targeting a cancer antigen) and T cell receptor (TCR)-modified T cells, tumor infiltrating lymphocytes (TIL), and peptide-induced T cells.

[0484] The skilled artisan will understand that the various cancer immunotherapeutics described herein can be combined with any one or more of the various anti-NRP2 antibodies (including antigen-binding fragments thereof) described herein and used in accordance with any one or more of the methods or compositions described herein.

[0485] Chemotherapeutic Agents Certain embodiments employ one or more chemotherapeutic agents, such as small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), antimicrotubule agents, and the like.

[0486] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mechlorethamine, 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 diazocone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and atypical alkylating agents (optionally procarbazine and altretamine).

[0487] Examples of antimetabolites include antifolates (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).

[0488] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and dactinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mepalong, and aclarubicin.

[0489] Examples of anti-microtubule agents include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

[0490] The skilled artisan will understand that the various chemotherapeutic agents described herein can be combined with any one or more of the various anti-NRP2 antibodies (including antigen-binding fragments thereof) described herein and used in accordance with any one or more of the methods or compositions described herein.

[0491] Hormonal Therapeutics Certain embodiments employ at least one hormonal therapeutic agent. General examples of hormonal therapeutic agents include hormone agonists and hormone antagonists. Specific examples of hormone agonists include progestogens (progesterone), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factors, VEGF-derived angiogenic and lymphangiogenic factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, androgens, estrogens, and somatostatin analogs. Examples of hormone antagonists include hormone synthesis inhibitors such as aromatase inhibitors and gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), including analogs thereof. Also included are hormone receptor antagonists such as selective estrogen receptor modulators (SERMs, e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0492] Also included are hormone pathway inhibitors, such as antibodies against hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1), such as citumumab, doxetuzumab, phentocinumab, ganituzumab, isotuzumab, and rotuinumab; inhibitors of vascular endothelial growth factor receptors 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as peglucuzumab, bevacizumab, irukumab, and ramucirumab; inhibitors of TGF-β receptors R1, R2, and R3, such as fusumumab and metitumumab; c- Met inhibitors, such as nasituximab; EGF receptor inhibitors, such as cetuximab, mofortin depatuximab, vortuximab, imatinib, encinlatuximab, matuzumab, mofortinib, necimumab, nimotuzumab, panitumumab, tomatuximab, and zalutumumab; FGF receptor inhibitors, such as aprutumabixadotin and bemarituzumab; PDGF receptor inhibitors, such as olaratumumab and tovelumab.

[0493] The skilled artisan will understand that the various hormonal therapeutics described herein can be combined with any one or more of the various anti-NRP2 antibodies (including antigen-binding fragments thereof) described herein and used in accordance with any one or more of the methods or compositions described herein.

[0494] Kinase inhibitors Certain embodiments utilize at least one kinase inhibitor, including a tyrosine kinase inhibitor. Examples of kinase inhibitors include: adasotinib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fotantinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mulitinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0495] The skilled artisan will understand that the various kinase inhibitors described herein can be combined with any one or more of the various anti-NRP2 antibodies (including antigen-binding fragments thereof) described herein and used in accordance with any one or more of the methods or compositions described herein.

[0496] Methods of use and therapeutic compositions

[0497] Embodiments of the present disclosure relate to the discovery that human histidyl-tRNA synthetase (HRS) polypeptides have unexpected biological properties relevant to the treatment of a wide range of diseases and conditions, and that certain of these properties are related to the interaction between HRS and human neuropilin 2 (NRP2). Thus, antibodies against human NRP2 that interfere with the binding between NRP2 and NRP2 ligands (including, for example, human HRS) can be used as stand-alone therapies for the treatment of diseases (including, NRP2-related diseases) or can be used in combination with other therapeutic agents described herein.

[0498] Thus, certain embodiments include methods of treating a disease or condition in a subject in need thereof, ameliorating symptoms thereof, and / or slowing progression thereof, comprising administering to the subject at least one antibody or antigen-binding fragment thereof that specifically binds to a human neuropilin-2 (NRP2) polypeptide. In some cases, the at least one antibody or antigen-binding fragment thereof antagonizes the binding / signaling activity between the NRP2 polypeptide and the at least one NRP2 ligand. In some cases, the at least one antibody or antigen-binding fragment thereof mimics or otherwise enhances one or more signaling activities of the NRP2 / NRP2 ligand interaction, for example, by acting as an agonist antibody.

[0499] In some cases, the at least one antibody or antigen-binding fragment thereof interferes with the binding of a human NRP2 polypeptide to a human HRS polypeptide. In some cases, the at least one antibody or antigen-binding fragment thereof mimics one or more signaling activities of the binding of a HRS polypeptide to an NRP2 polypeptide, for example, by acting as an agonist antibody. Exemplary anti-NRP2 antibodies and therapeutic compositions comprising the same are described elsewhere herein.

[0500] In certain embodiments, the disease or condition is an NRP2-related disease or condition. In some embodiments, the NRP2-related disease or condition is selected from one or more of the following: cancer and cancer-related diseases and pathways, including cancer cell growth, initiation, migration, adhesion, invasion and / or metastasis; diseases associated with inflammation, autoimmunity and related inflammatory diseases, including diseases associated with inappropriate immune cell activation or migration, such as graft-versus-host disease (GVHD); diseases associated with lymphatic development, lymphangiogenesis and lymphatic damage, 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; diseases associated with allergic conditions / diseases, 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-associated neutrophil-mediated diseases, such as pyoderma gangrenosum; diseases associated with granulomatous inflammatory diseases, including sarcoidosis and granulomas; diseases associated with fibrosis, including fibrotic diseases, fibrosis, endothelial-mesenchymal transition (EMT), and wound healing; diseases associated with inappropriate smooth muscle contractility and inappropriate vascular smooth muscle cell migration and adhesion; diseases associated with inappropriate autophagy, phagocytosis, and efferocytosis; diseases associated with neuronal diseases, peripheral nervous system remodeling, and pain perception; and diseases associated with skeletal development and skeletal remodeling.

[0501] In some embodiments, the disease is cancer. Here, the upregulation of NRP2 expression is associated with tumorigenesis, especially tumor metastasis, and is associated with more aggressive diseases in a variety of tumor types. In addition, the arm plate protein / plexin / neuropilin signaling axis affects many cancer hallmarks (see, for example, Franzolin and Tamagnone, International Journal of Molecular Sciences, 20, 377; doi: 10.3390 / ijms20020377, 2019; Nasarre et al., OncoTargets and Therapy, 2014: 7 1663-1687; Neufeld et al., Cold Spring Harb Perspect Med, 2: a006718, 2012). Consistent with these studies, increased NRP2 expression in prostate cancer cells is induced by phosphatase and tensin homolog (PTEN loss), and its expression is associated with Gleason grade (see, for example, Zhao et al., Thoracic Cancer 8: 203-213, 2017). In addition, p53 mutations upregulate NRP2 expression by inhibiting DLX2 transcription, thereby increasing cell motility. About 50% of human tumors and cancers contain mutations in the p53 gene, the vast majority of which occur in the DNA binding domain, and this is associated with poor prognosis (see, for example, Drabkin et al., Oncotarget 8 (No. 57) 96464-96465, 2017). Furthermore, TGF-β signaling is involved in the upregulation of NRP2B expression and EMT in cancer, which may explain why higher TGF-β production is positively correlated with tumor aggressiveness and poor prognosis in advanced tumors (see, e.g., Malfettone et al., Cancer Lett. 392:39-50, 2017).

[0502] Thus, certain embodiments include methods for treating cancer in a subject in need thereof, ameliorating symptoms thereof, or inhibiting progression thereof, comprising administering to the subject at least one antibody or antigen-binding fragment thereof that specifically binds to a human NRP2 polypeptide (anti-NRP2 antibody) and modulates (e.g., interferes with) the binding of a human NRP2 polypeptide to an NRP2 ligand (e.g., an NRP2 ligand from Table N2 or Table N3 and / or a human HRS polypeptide from Table H1). Certain embodiments include reducing or preventing cancer in a subject in need thereof, such as recurrence of metastatic cancer, wherein administration of the therapeutic composition is capable of generating immune memory against the cancer. In some embodiments, the subject has diabetes (e.g., type 1 diabetes or type 2 diabetes) or is at risk of developing diabetes.

[0503] In certain embodiments, relative to healthy controls, the subject suffers from a disease associated with an increase in the level or expression of at least one NRP2 part (e.g., an NRP2 part from Table N2 or Table N3 and / or an HRS polypeptide from Table H1) and / or its encoding mRNA and / or is selected for treatment based on suffering from the disease. For example, in certain embodiments, the level of the at least one NRP2 part in a disease subject, cell or tissue is about or at least about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times or more of the level of the at least one NRP2 part in a healthy control. In some embodiments, the subject suffers from a cancer in which the level or expression of at least one NRP2 ligand (e.g., an NRP2 ligand from Table N2 or Table N3 and / or an HRS polypeptide from Table H1) and / or its encoding mRNA is increased relative to a non-cancerous control cell or tissue and / or is selected for treatment based on suffering from the cancer. For example, in some embodiments, the level of the at least one NRP2 ligand in a cancer cell or tissue is about or at least about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times or more of the level of the at least one NRP2 ligand in a non-cancerous control or standard. Thus, certain embodiments include methods of selecting a subject for treatment, comprising (i) detecting an increase in the level of at least one NRP2 ligand (e.g., an NRP2 ligand from Table N2 or Table N3 and / or a HRS polypeptide from Table H1) and / or encoding mRNA in the subject relative to a control or reference, and (ii) administering to the subject a therapeutic composition comprising at least one anti-NRP2 antibody as described herein. In certain embodiments, the HRS polypeptide is a splice variant of full-length HRS. In some embodiments, the HRS splice variant is selected from one or more of the following: 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 .

[0504] In some embodiments, a subject has increased circulating or serum levels of a soluble neuropilin 2 (NRP2) polypeptide (e.g., selected from Table N1) - bound to a HRS polypeptide or free - relative to a healthy or matched control population of one or more subjects and / or is selected for treatment based on increased levels. For example, in certain embodiments, circulating or serum levels are about or at least about 10 pM, 20 pM, 30 pM, 50 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1000 pM, 1100 pM, 1200 pM, 1300 pM, 1400 pM, 1500 pM, 1600 pM, 1700 pM, 1800 pM, 1900 pM, 2000 pM, 3000 pM, 4000 pM, 5000 pM or a circulating or serum level of about 30-50 pM, 50-100 pM, 100-2000 pM, 200-2000 pM, 300-2000 pM, 400-2000 pM, 500-2000 pM, 600-2000 pM, 700-2000 pM, 800-2000 pM, 900-2000 pM, 1000-2000 pM, 2000-3000 pM, 3000-4000 pM, 4000-5000 pM of soluble NRP2 polypeptide.

[0505] In certain embodiments, the subject has a disease associated with increased levels or expression of an NRP2 polypeptide (optionally selected from Table N1) and / or its encoding mRNA relative to a healthy control (e.g., an NRP2-related disease) and / or is selected for treatment based on having the disease. For example, in certain embodiments, the level of an NRP2 polypeptide in the diseased subject, cell, or tissue is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more times the level of the NRP2 polypeptide in the healthy control. In some embodiments, the subject has a cancer in which the level or expression of an NRP2 polypeptide (e.g., selected from Table N1) and / or its encoding mRNA is increased relative to a control cell or tissue, optionally relative to a non-cancerous cell or tissue of the same type as the cancer, and / or is selected for treatment based on having the cancer. For example, in some embodiments, the level of NRP2 polypeptide in cancer cells or tissue is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more times greater than the level of NRP2 polypeptide in a non-cancerous control or standard. Accordingly, some embodiments include methods of selecting a subject for treatment comprising (i) detecting an increase in the expression level of an NRP2 polypeptide and / or its encoding mRNA in a subject relative to a control or reference, and (ii) administering to the subject a therapeutic composition comprising at least one anti-NRP2 antibody as described herein.

[0506] In some embodiments, the subject is suffering from an increased level or expression of NRP2A and / or NRP2B or an altered ratio of NRP2A:NRP2B expression relative to a healthy control or matched control standard or a subject population consisting of one or more subjects and / or is selected for treatment based on suffering from the disease. In some embodiments, the subject's NRP2B expression or level is significantly higher...

Claims

1. A therapeutic composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to a human neuropilin-2 (NRP2) polypeptide, wherein the antibody or the antigen-binding fragment thereof specifically binds to at least one epitope in the B1 domain of the human NRP2 polypeptide, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (V H ) sequence and light chain variable region (V L ) sequence, the V H The sequence contains the complementarity determining region V H CDR1, V H CDR2 and V H CDR3 sequence; and the V L The sequence contains the complementarity determining region V L CDR1, V L CDR2 and V L CDR3 sequence, wherein: The V H CDR1, V H CDR2 and V H The CDR3 sequences are shown in SEQ ID NOs: 59-61, respectively, and the V L CDR1, V L CDR2 and V L The CDR3 sequences are shown in SEQ ID NOs: 62-64, respectively.

2. The therapeutic composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a human IgA, IgD, IgE, IgG or IgM Fc domain.

3. The therapeutic composition of claim 2, wherein the IgA Fc domain is an IgA1 or IgA2 Fc domain, or wherein the IgG Fc domain is an IgG1, IgG2, IgG3 or IgG4 Fc domain.

4. The therapeutic composition of claim 2, wherein the antibody or antigen-binding fragment thereof comprises an IgG1 or IgG3 Fc domain.

5. The therapeutic composition of claim 2, wherein the antibody or antigen-binding fragment thereof comprises an IgG2 or IgG4 Fc domain.

6. The therapeutic composition of claim 2, wherein the antibody or antigen-binding fragment thereof comprises an IgG1 or IgG4 Fc domain selected from SEQ ID NOs: 116-118.

7. The therapeutic composition of claim 1, wherein the antibody or antigen-binding fragment thereof is a Fv fragment.

8. The therapeutic composition of claim 1, wherein the antibody or antigen-binding fragment thereof is a single-chain Fv (scFv) polypeptide or a unibody.

9. The therapeutic composition of claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal humanized antibody.

10. The therapeutic composition of claim 1 or 2, wherein the composition is at least 90% pure for the antibody or antigen-binding fragment, based on protein, and has less than 10% high molecular weight aggregated proteins.

11. The therapeutic composition of claim 10, wherein the composition is at least 95% pure, based on protein, with respect to the antibody or antigen-binding fragment.

12. The therapeutic composition of claim 10, wherein the composition is at least 98% pure on a protein basis with respect to the antibody or antigen-binding fragment.

13. The therapeutic composition of claim 10, wherein the composition is at least 99% pure on a protein basis with respect to the antibody or antigen-binding fragment.

14. The therapeutic composition of claim 10, wherein the therapeutic composition is at least 99% free of endotoxin.

15. The therapeutic composition of claim 1 or 2, wherein the therapeutic composition is a sterile injectable solution.

16. The therapeutic composition of claim 15, wherein the sterile injectable solution is suitable for intravenous, intramuscular, subcutaneous or intraperitoneal administration.

17. The therapeutic composition of claim 1 or 2, further comprising at least one additional agent selected from the group consisting of a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapeutic agent, and a kinase inhibitor.

18. The therapeutic composition of claim 17, wherein the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint regulator, an oncolytic virus, and a cytokine.

19. The therapeutic composition of claim 17, wherein the cancer immunotherapeutic agent is a cancer vaccine.

20. The therapeutic composition of claim 17, wherein the cancer immunotherapeutic is a cell-based immunotherapeutic.

21. A cell composition comprising an engineered cell population, at least one cell in the engineered cell population comprising one or more polynucleotides encoding an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 20, wherein the cells are capable of growing in a serum-free medium.

22. A cell growth device comprising an antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 20; an engineered cell population, at least one cell in the engineered cell population comprising one or more polynucleotides encoding the antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 20; at least 10 liters of serum-free growth medium and a sterile container.

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