Compositions and methods comprising Anti-NRP2 antibodies

Abstract

To provide compositions and methods comprising anti-NRP2 antibodies.SOLUTION: There are provided antibodies and antigen-binding fragments thereof that specifically bind to human neuropilin-2 (NRP2) polypeptides, including those which modulate binding interactions between human NRP2 and at least one NRP2 ligand, for example, human histidyl-tRNA synthetase (HRS), and which thereby modulate NRP2-mediated downstream signaling events, and including related therapeutic compositions and methods for modulating NRP2 activity and treating diseases such as NRP2-associated diseases.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 653,823, filed April 6, 2018, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Description The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named ATYR_134_01WO_ST25.txt. The text file is 228kB in size, was created on April 5, 2019, and has been submitted electronically via EFS-Web.

[0003] 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, such as human histidyl-tRNA synthetase (HRS), thereby modulating NRP2-mediated downstream signaling events, as well as related therapeutic compositions and methods for modulating NRP2 activity and treating diseases, such as NRP2-associated diseases. [Background technology]

[0004] Although tRNA synthetases are well-characterized in protein synthesis, recent findings suggest that they also play important roles in cellular responses. In particular, tRNA synthetases are increasingly recognized as having previously unidentified roles in cellular stress responses and tissue homeostasis in both the intracellular and extracellular milieu.

[0005] The resokine family of proteins (HRS polypeptides), derived from the histidyl-tRNA synthetase (HARS) gene via proteolysis or splicing, are important modulators of both intracellular and extracellular activities. Extracellular HARS can be readily detected in the circulation of healthy volunteers, and autoantibodies against HARS (Jo-1 antibodies) have been associated with inflammatory myopathies (IM) and in subjects with inflammatory pulmonary diseases (ILDs). Although the role of Jo-1 antibodies in disease progression is still not fully understood, subjects with Jo-1 antibodies tend to be less likely to develop cancer than subjects with inflammatory myopathies who do not have Jo-1 antibodies (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). (See

[0006] Significant progress has been made in elucidating the role of extracellular HARS-inducible proteins, including the identification of a putative cellular receptor, neuropilin-2 (NRP2 or NRP-2). HARS and NRP2 are thought to interact via the N-terminal region of HARS, and this interaction can result in important changes in the cellular function of NRP2.

[0007] Thus, new discoveries of the lyzokine / neuropilin-2 system have revealed previously unknown mechanisms by which it acts as a central regulator of cellular processes, including axon guidance, endocytosis, cell migration, proliferation, engraftment, apoptosis, lymphangiogenesis, cell differentiation, and cell adhesion, which are directly relevant to cancer development, growth, and metastasis, as well as muscle, vascular, neural, bone, and immune homeostasis. Deregulation of any of these processes may result in a variety of diseases, which may be clarified by developing anti-NRP2 antibodies that selectively target the lyzokine / neuropilin-2 system. The present disclosure provides such antibodies and related embodiments. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Lu et al., PLOS ONE 9(4) e94128, 2014 [Non-patent document 2] Modan et al., Clin. Exp. Dermatol. 34(5) 561-565, 2009 [Non-patent document 3] Shi et al., J. Rheum 44 (7) doi 10.3899 / jrheum.161480 Summary of the Invention [Means for solving the problem]

[0009] 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).

[0010] In certain embodiments, 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, and optionally binds to a human NRP2 polypeptide at a concentration of about 10 pM to about 500 pM, or about 10 pM to 50 nM, or at about, at least about, or at most about 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, or optionally with an affinity 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 500 pM, about 10 pM to about 600 pM, about 10 pM to about 700 pM, about 10 pM to about 800 pM, about 10 pM to about 900 pM, about 10 pM to about 100 pM, about 10 pM to about 250 pM, about 10 pM to about 300 pM, 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 500 pM, about 10 pM to about 500 pM, about 10 pM to about 600 pM, about 10 pM to about 250 pM, about 10 pM to about 500 pM, about 10 pM to about 250 pM, about 10 pM to about 300 pM, 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 500 pM, about 10 pM to about 500 pM, about 10 pM to about 25 ...300 pM, about 10 pM to about 500 pM, about 10 pM to about 5 M ~ about 200pM, about 10pM to about 100pM, about 10pM to about 50pM, or about 20pM to about 500pM, about 20pM to about 400pM, about 20pM to about 300pM, about 20pM to about 200pM, about 20p M ~ about 100pM, about 20pM - about 50pM, or about 30pM - about 500pM, about 30pM - about 400pM, about 30pM - about 300pM, about 30pM - about 200pM, about 30pM - about 100pM, about 30p In some examples, the at least one antibody or antigen-binding fragment thereof specifically binds to a human NRP2 polypeptide in its native form but does not substantially bind to a human NRP2 polypeptide in its denatured form.

[0011] In certain embodiments, the at least one antibody or antigen-binding fragment thereof is selected from the group consisting of a neuropilin A1 domain, a neuropilin A2 domain, a neuropilin B1 domain, a neuropilin B2 domain, a neuropilin C domain, a neuropilin A1 / A2 composite domain, a neuropilin B1 / B2 composite domain, a neuropilin A2 / B1 composite domain, a neuropilin B2 / C composite domain, a neuropilin A2 / B1 / B2 composite domain, a neuropilin A2 / B1 / B2 / C composite domain, a neuropilin A1 / A2 / B1 composite domain, a neuropilin specifically binds to at least one epitope within a neuropilin domain selected from one or more of a neuropilin A1 / A2 / B1 / B2 composite domain, a neuropilin A1 / A2 / B1 / B2 / C composite domain, and a neuropilin B1 / B2 / C composite domain, and optionally has a specific binding affinity of about 10 pM to about 500 pM or about 10 pM to about 50 nM, or about, at least about, or at most about 10 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, or optionally with an affinity of about 10 pM to about 500 pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM,It binds with an affinity in the range of about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

[0012] In certain embodiments, 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 complex domain, or within the adjacent linker region, e.g., near the following residue: (Neuropilin A1 domain) residues 20-148, 30-141, 40-141, 50-141, 60-141, 70-141, 80-141, 90-141, 100-141, 110-141, 120-141, 130-141, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 as defined in SEQ ID NO: 1 (FL human NRP2), or (Neuropilin A2 domain) residues 142-280, 150-265, 160-265, 170-265, 180-265, 190-265, 200-265, 210-265, 220-265, 230-265, 240-265, 250-265, 260-265, 141-270, 141-260, 141-250, 141-240, 141-230, 141-220, 141-210, 141-200, 141-190, 141-180, 141-170, 141-160, 141-150 ...260-265, 141-270, 141- 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; (Composite A1A2 domain) Residues 20-280, 30-280, 40-280, 50-280, 60-280, 70-280, 80-280, 90-280, 100-280, 110-280, 120-280, 130-280, 140-280, 150-280, 160-280, 170-280, 180-280, 190-280, 200-280, 210-280, 220-280, and 230-280 as defined in SEQ ID NO: 1 (FL human NRP2) , 240-280, 260-280, 270-280, 20-270, 20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-200, 20-190, 20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30.

[0013] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin B1 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), or within the adjacent linker region, e.g., near the following residues: (Neuropilin B1 domain) residues 266-426, 280-426, 290-426, 300-426, 310-426, 320-426, 330-426, 340-426, 350-426, 360-426, 370-426, 380-426, and 390-426 as defined in SEQ ID NO: 1 (FL human NRP2) , 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, (Neuropilin B2 domain) residues 438-591, 450-591, 460-591, 470-591, 480-591, 490-591, 500-591, 510-591, 520-591, 530-591, 540-591, 550-591, 560-591, 570-591, 580-591, 590-591, 600-601, 610-611, 620-621, 630-631, 640-641, 650-651, 660-661, 670-671, 680-681, 690-691, 700-701, 710-711, 720-721, 730-731, 740-731, 750-751, 760-761, 770-771, 780-781, 790-801, 800-801, 810-811, 820-811, 830-811, 840-811, 850-811, 860-811, 870-811, 880-811, 890-901, 900-901, 910-911, 920-921, 930-931, 940-941, 950-951, 960-961, 970-971, 980-981, 990- 70-591, 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, or (Neuropilin B1 / B2 complex domain) residues 266-591, 276-591, 286-591, 296-591, 306-591, 316-591, 326-591, 336-591, 346-591, 356-591, 366-591, 376-591, 386-591, 396-591, 406-591, 416-591, 426-591, 436-591, 446-591, 456-591, 466-591, 476-591, 486-591, 496-591, 506-591, 516-591, 526-591, 536-591, 546-591, 556-591, 566-591, 576-591, 586-591, 591-606-591, 606-606-591, 616-616-591, 626-626-591, 636-636-591, 646-646-591, 656-656-591, 666-666-591, 676-676-591, 686-686-591, 696-691, 706- ... 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 Combine with 6~431, 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.

[0014] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin A2 / B1 composite domain and / or the neuropilin B2C composite domain or the adjacent linker region, e.g., near the following residues: (Neuropilin A2B1 complex domain) Residues 149-437, 159-426, 169-426, 179-426, 189-426, 199-426, 209-426, 219-426, 229-426, 239-426, 249-426, 259-426, 269-426, 279-426, 289-426, 299-426, 309-426, 319-426, 329-426, 339-426, 349-426, 359-426, 369-426, 379-426, 389-426, 399-426, 409 as defined in SEQ ID NO: 1 (FL human NRP2) ~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, 1 49-296, 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, or (Neuropilin B2C complex domain) residues 438 to 794, 448 to 794, 458 to 794, 468 to 794, 478 to 794, 487 to 794, 497 to 794, 507 to 794, 517 to 794, 527 to 794, 537 to 794, 547 to 794, 557 to 794, and 567 to 794 as defined in SEQ ID NO: 1 (FL human NRP2); 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, 77 7~794, 787~794, 427~794, 438~784, 438~774, 438~764, 438~754, 438~744, 438~734, 438~728, 438~714, 438~704, 438~694, 438~684, 438~674, 438~664, 438~654, 438~644, 438~634, 438 ~624, 438~614, 438~604, 438~596, 438~586, 438~576, 438~566, 438~556, 438~546, 438~536, 438~526, 438~516, 438~506, 438~494, 438~484, 438~474, 438~464, 438~454, 438~444.

[0015] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within a neuropilin C domain or an adjacent linker region, e.g., near residues 591-794, 600-794, 610-794, 620-794, 630-794, 640-794, 650-794, 660-794, 670-794, 680-794, 690-794, 700-794, 710-794, 720-794, 730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 800-800, 810-810, 820-820, 830-830, 840-840, 850-850, 860-850, 870-850, 880-850, 890-900, 900-900, 910-910, 920-930, 930-940, 940-950, 950-960, 960-970, 970-980, 980-990, 990-1000, 1000-1000, 1010-1010, 1020-1020, 1030-1030, 1040-1040, 1050-1050, 1060-1060, 1070-1070, 1080- Combine with 794, 730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 591-790, 591-780, 591-770, 591-760, 591-750, 591-740, 591-730, 591-720, 591-710, 591-700, 591-690, 591-680, 591-670, 591-660, 591-650, 591-640, 591-630, 591-620, 591-610, or 591-600.

[0016] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin B1 / B2 / C complex domain or the adjacent linker region, e.g., near 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, 496-806, 506-794, 516-794, 526-794, 536-794, 546-794, 556-794, 566-794, 576-794, 587-806, 596-806, 606-794, 616-794, 626-794, 636-794, 646-794, 656-794, 666-794, 676-794, 687-794, 696-806, 706-794, 716-794, 726-794, 736-794, 746-794, 756-794, 766-794, 776-79 794, 396~794, 406~794, 416~794, 426~794, 436~794, 446~794, 456~794, 466~794, 476~794, 486~794, 496~794, 506~794, 516~794, 526~794, 536~794, 546~794, 556~794, 566~794, 576~794, 586~794, 596~794, 606~794, 616~794, 626~794, 636~794, 646~794, 656~794, 666~794, 676~794, 686~794, 696~794, 706~794, 716~794, 726~794, 736~794, 746~794, 756~794, 766~794, 776~794, 786~794, 266~794, 276~784, 276~774, 276~764, 276~754, 276~744, 276~734, 276~724, 276~714, 276~704, 276~694, 276~684, 276~674, 276~664, 276~ Combined with 654, 276-644, 276-634, 276-624, 276-614, 276-604, 276-594, 276-584, 276-574, 276-564, 276-554, 276-544, 276-534, 276-524, 276-514, 276-504, 276-594, 276-584, 276-574, 276-564, 276-554, 276-544, 276-534, 276-524, 276-514, 276-504, or 276-496.

[0017] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to a structural epitope comprised of two or more discontinuous epitope regions. (a) a first epitope region within the A1 domain and a second epitope region within the A2 domain of a human NPR2 polypeptide; (b) a first epitope region within the A1 domain and a second epitope region within the B1 domain of a human NPR2 polypeptide; (c) a first epitope region within the A1 domain and a second epitope region within the B2 domain of a human NPR2 polypeptide; (d) a first epitope region within the A1 domain and a second epitope region within the C domain of a human NPR2 polypeptide; (e) a first epitope region within the A2 domain and a second epitope region within the B1 domain of a human NPR2 polypeptide; (f) a first epitope region within the A2 domain and a second epitope region within the B2 domain of a human NPR2 polypeptide; (g) a first epitope region within the A2 domain and a second epitope region within the C domain of a human NPR2 polypeptide; (h) a first epitope region within the B1 domain and a second epitope region within the B2 domain of a human NPR2 polypeptide; (i) a first epitope region within the B1 domain and a second epitope region within the C domain of a human NPR2 polypeptide; or (j) specifically binds to a structural epitope comprising or consisting of a first epitope region in the B2 domain and a second epitope region in the C domain of human NPR2 polypeptide;

[0018] In some embodiments, at least one antibody or antigen-binding fragment thereof modulates (e.g., inhibits) binding of a 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 an HRS splice variant selected from Table H1, e.g., an 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)).

[0019] In certain embodiments, the at least one antibody or antigen-binding fragment thereof is a blocking antibody that inhibits about or at least about 80% to 100% of the theoretical maximum binding of a human NRP2 polypeptide to at least one NRP2 ligand after pre-incubation with the human NRP2 polypeptide in a stoichiometric equivalent amount, optionally about or at least about 80%, 85%, 90%, 95%, or 100% of the theoretical maximum binding.

[0020] In certain embodiments, at least one antibody or antigen-binding fragment thereof is a partial blocking antibody that inhibits about or at least about 20% to 80% of the theoretical maximum binding of a human NRP2 polypeptide to at least one NRP2 ligand after pre-incubation with the human NRP2 polypeptide in a stoichiometric equivalent amount, optionally about or at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the theoretical maximum binding.

[0021] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an HRS polypeptide-interacting region of an NRP2 polypeptide and mimics or stimulates one or more signaling activities of an HRS polypeptide bound to the NRP2 polypeptide.

[0022] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to the HRS polypeptide-interacting region of an NRP2 polypeptide and modulates the binding activity / signaling activity between the NRP2 polypeptide and at least one NRP2 ligand. In some embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between the NRP2 polypeptide and at least one NRP2 ligand. In some embodiments, at least one antibody or antigen-binding fragment thereof stimulates or enhances the binding activity / signaling activity between the NRP2 polypeptide and at least one NRP2 ligand.

[0023] In one embodiment, the at least one NRP2 ligand is a VEGF selected from one or more of VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, and PIGF-2; a VEGF receptor (VEGFR) selected from VEGFR2 and VEGFR3, a semaphorin selected from one or more of SEMA-3B, SEMA-3C, SEMA-3D, SEMA-3F, and SEMA-3G; a plexin selected from one or more of plexin A1, plexin A2, plexin A3, plexin A4, and plexin D1, growth factors selected from one or more of fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF); a growth factor receptor selected from one or more of fibroblast growth factor receptor (FGFR), hepatocyte growth factor receptor (HGFR), and platelet-derived growth factor receptor (PDGF); - galectins or galectin receptors, - a transcription factor selected from FAC1 and bromoprotein PHD finger transcription factors, an adaptor protein selected from one or more of GIPC1, GIPC2, and GIPC3, selected from Table N3, optionally α V β1, α V β3, α V β5, α V β6, α V an integrin selected from one or more of β8, α6β1, and α6β4; a transforming growth factor beta selected from one or more of TGFβ1, TGFβ2, TGFβ3, and their corresponding TGFβ receptors, and - an HRS polypeptide selected from Table H1, optionally HisRS N1 , HisRS N2 , HisRS N3 , HisRS N4 (SV9), HisRS N5 , HisRS C1 , HisRS C2 , HisRS C3 , HisRS C4 , HisRS C5 , HisRS C6 , HisRS C7 , HisRS C8 (SV11), and HisRS C9 (SV14), HRS splice variants selected from one or more of:

[0024] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor and / or a semaphorin without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and VEGFR3 or VEGF-C.

[0025] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor and / or a semaphorin without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and an HRS polypeptide.

[0026] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor and / or a semaphorin without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and an HRS polypeptide, and without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and VEGFR3 or VEGF-C.

[0027] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and VEGR3 without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor and / or a semaphorin.

[0028] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and VEGR3 or VEGF-C without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and an HRS polypeptide.

[0029] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor without substantially modulating ligand binding between semaphorin 3 and NRP2.

[0030] In some embodiments, the plexin receptor is selected from plexin A1, plexin A2, plexin A3, plexin A4, and plexin D1. In some embodiments, the semaphorin is selected from semaphorin 3B, semaphorin 3C, semaphorin 3D, semaphorin 3F, and semaphorin 3G.

[0031] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the A2 domain of human NRP2 comprising at least five consecutive 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 certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within amino acids 232 to 242 of human NRP2 set forth in SEQ ID NO: 1.

[0032] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the B1 domain of human NRP2 comprising at least five consecutive 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.

[0033] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the B2 domain of human NRP2 comprising at least five consecutive 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 also inhibits dimerization between NRP2 and Plexin A1.

[0034] In one embodiment, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the C domain of human NRP2 comprising at least five consecutive 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).

[0035] In certain embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ), and the affinity for (i) and (ii) is within 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.

[0036] In certain embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ), and the affinity for (i) and (ii) is within 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.

[0037] In certain embodiments, at least one antibody or antigen-binding fragment thereof is V of a complementarity-determining region selected from Table A1 that specifically binds to a human NRP2 polypeptide H CDR1 sequence, V H CDR2 sequence, and V H Heavy chain variable region (V) including CDR3 sequences and their variants H ) array, and V of a complementarity-determining region selected from Table A1 that specifically binds to a human NRP2 polypeptide L CDR1 sequence, V L CDR2 sequence, and V L The light chain variable region (V) including CDR3 sequences and their variants L) array, as well as affinity matured variants of the foregoing sequences that specifically bind to human NRP2 polypeptides.

[0038] In a specific embodiment, V H The CDR1 sequence is SEQ ID NO: 23 and its variants, V H The CDR2 sequence is SEQ ID NO: 24 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 25 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 26 and its variants, V L The CDR2 sequence is SEQ ID NO: 27 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 28 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and which specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 29 and its variants, V H The CDR2 sequences are SEQ ID NO: 30 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 31 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 32 and its variants, V L The CDR2 sequence is SEQ ID NO: 33 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 34 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 35 and its variants, V H The CDR2 sequence is SEQ ID NO: 36 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 37 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 38 and its variants, V L The CDR2 sequence is SEQ ID NO: 39 and variants thereof, and V Lthe CDR3 sequence comprises SEQ ID NO: 40 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 41 and its variants, V H The CDR2 sequence is SEQ ID NO: 42 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 43 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 44 and its variants, V L The CDR2 sequence is SEQ ID NO: 45 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 46 and variants thereof, which variants have one, two, three, four, or five alterations in the CDRs and specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 47 and its variants, V H The CDR2 sequence is SEQ ID NO: 48 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 49 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 50 and its variants, V L The CDR2 sequences are SEQ ID NO: 51 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 52 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 53 and its variants, V H The CDR2 sequence is SEQ ID NO: 54 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 55 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 56 and its variants, V L The CDR2 sequence is SEQ ID NO: 57 and variants thereof, and V Lthe CDR3 sequence comprises SEQ ID NO: 58 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and which specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 59 and its variants, V H The CDR2 sequence is SEQ ID NO: 60 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 61 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 62 and its variants, V L The CDR2 sequence is SEQ ID NO: 63 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 64 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 65 and its variants, V H The CDR2 sequence is SEQ ID NO: 66 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 67 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 68 and its variants, V L The CDR2 sequence is SEQ ID NO: 69 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 70 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and which specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 71 and its variants, V H The CDR2 sequence is SEQ ID NO: 72 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 73 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 74 and its variants, V L The CDR2 sequences are SEQ ID NO: 75 and variants thereof, and V Lthe CDR3 sequence comprises SEQ ID NO: 76 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and which specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 77 and its variants, V H The CDR2 sequence is SEQ ID NO: 78 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 79 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 80 and its variants, V L The CDR2 sequence is SEQ ID NO: 81 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 82 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and specifically bind to a human NRP2 polypeptide; V H The CDR1 sequence is SEQ ID NO: 83 and its variants, V H The CDR2 sequence is SEQ ID NO: 84 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 85 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 86 and its variants, V L The CDR2 sequence is SEQ ID NO: 87 and variants thereof, and V L The CDR3 sequence comprises SEQ ID NO: 88 and variants thereof, which have one, two, three, four, or five alterations in the CDRs and which specifically bind to a human NRP2 polypeptide.

[0039] In certain embodiments, at least one antibody or antigen-binding fragment thereof comprises an Fc domain of IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), or IgM, optionally a human Fc domain, or a hybrid and / or variant thereof. In certain embodiments, at least one antibody or antigen-binding fragment thereof comprises an Fc domain of an IgG with high effector function in humans, optionally comprising an IgG1 or IgG3 Fc domain. In certain embodiments, at least one antibody or antigen-binding fragment thereof comprises an Fc domain of an IgG with low effector function in humans, optionally comprising an IgG2 or IgG4 Fc domain. In certain embodiments, at least one antibody or antigen-binding fragment thereof comprises an IgG1 or IgG4 Fc domain, optionally selected from Table F1.

[0040] 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, an anticalin, an aptamer, an avimer, a camelid antibody, an engineered ankyrin repeat protein, or a fusion protein. proteins (DARPins), minibodies, nanobodies, or unibodies.

[0041] In certain embodiments, compositions of the invention are at least about 80%, 85%, 90%, 95%, 98%, or 99% pure on a protein basis for at least one antibody or antigen-binding fragment thereof of the invention, and are substantially free of aggregation. In certain embodiments, therapeutic compositions of the invention are substantially endotoxin-free.

[0042] In certain embodiments, the therapeutic compositions are sterile injectable solutions, optionally suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration.

[0043] 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 therapy agent, and a kinase inhibitor. In certain embodiments, the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint modulator, a cancer vaccine, an oncolytic virus, a cytokine, and a cellular immunotherapeutic agent. In certain embodiments, the immune checkpoint modulator is a polypeptide, optionally an antibody or antigen-binding fragment thereof, or a ligand, or a small molecule. In certain embodiments, the immune checkpoint modulator is (a) an antagonist of an inhibitory immune checkpoint molecule, or (b) agonists of stimulatory immune checkpoint molecules; For example, the immune checkpoint modulator specifically binds to an immune checkpoint molecule.

[0044] In one embodiment, the inhibitory immune checkpoint molecule is programmed death-ligand 1 (PD-L1), programmed death 1 (PD-1), programmed death-ligand 2 (PD-L2), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), V-domain Ig suppressor of T-cell activation (VISTA), B and T lymphocyte attenuator (BTLA), CD16 0, herpesvirus entry mediator (HVEM), and T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0045] In some embodiments, the antagonist is a PD-L1 antagonist and / or a PD-L2 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L1 and / or PD-L2, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736); the antagonist is a PD-1 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514, PDR001, and pidilizumab; the antagonist is a CTLA-4 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, and tremelimumab; the antagonist is an IDO antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to IDO, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman, 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat; the antagonist is a TDO antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91, and LM10; the antagonist is a TIM-3 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TIM-3; the antagonist is a LAG-3 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3, and BMS-986016; the antagonist is a VISTA antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to VISTA; the antagonist is an antagonist of BTLA, CD160, and / or HVEM, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to BTLA, CD160, and / or HVEM; and / or The antagonist is a TIGIT antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TIGIT.

[0046] In certain embodiments, the stimulatory immune checkpoint molecule is selected from one or more of OX40, CD40, glucocorticoid-induced TNFR family related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0047] In some embodiments, the agonist is an OX40 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40, OX86, Fc-OX40L, and GSK3174998; the agonist is a CD40 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40, CP-870893, dacetuzumab, Chi Lob7 / 4, ADC-1013, and rhCD40L; the agonist is a GITR agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1, and MEDI1873; the agonist is a CD137 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD137, utomilumab, and 4-1BB ligand; the agonist is a CD27 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27, varlilumab, and CDX-1127 (1F5); The agonist is a CD28 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28, and TAB08; and / or The agonist is an HVEM agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to HVEM.

[0048] In one embodiment, the cancer vaccine is Oncophage, a human papillomavirus HPV vaccine (optionally Gardasil or Cervarix), a hepatitis B vaccine (optionally Engerix B, Heptavax HB, or Twi One or more of the following: Twinrix (Twinrix), and Sipuleucel-T (Provenge) or human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor (VEGF) (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74 , CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, membrane Transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer (pancarcinoma) The cancer antigens include cancer antigens selected from one or more of the following: (Illegible) leukemia antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

[0049] In one embodiment, the oncolytic virus is talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H In some embodiments, the cytokine is selected from one or more of interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the cellular immunotherapeutic agent comprises 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 chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0050] In certain embodiments, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type I or type II), and a microtubule inhibitor.

[0051] In certain embodiments, the alkylating agent is selected from one or more of 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, and streptozotocin), tetrazines (optionally, dacarbazine, mitozolomide, and temozolomide), aziridines (optionally, thiotepa, mitomycin, and diaziquone (AZQ)), cisplatins and their derivatives (optionally, carboplatin and oxaliplatin), and non-classical alkylating agents (optionally, procarbazine and hexamethylmelamine); the antimetabolite is selected from one or more of antifolates (optionally methotrexate and pemetrexed), fluoropyrimidines (optionally 5-fluorouracil and capecitabine), deoxynucleoside analogues (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (optionally thioguanine and mercaptopurine); the cytotoxic antibiotic is selected from one or more of anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin; the topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin; and / or The microtubule inhibitor is selected from one or more of taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0052] 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 a progestogen (progestin), a corticosteroid (optionally prednisolone, methylprednisolone, or dexamethasone), an insulin-like growth factor, a VEGF-derived angiogenic and lymphangiogenic factor (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), a fibroblast growth factor (FGF), a galectin, a hepatocyte growth factor (HGF), a platelet-derived growth factor (PDGF), a transforming growth factor (TGF) beta, an androgen, an estrogen, and a somatostatin analog. In certain embodiments, the hormone antagonist is a combination of 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 against a hormone receptor, optionally including cizutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, loxatuzumab, thiazol-20 ... Robatumumab, alacizumab pegol, bevacizumab, icrucumab, ramucirumab, fresolimumab, meclomid, Metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, imgatsuma Imagatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitum Mab, tomuzotuximab, zalutumumab, april tumab ixadocin (aprutumab ixadotin), bemarituzumab, olaratumab, or tovetumab).

[0053] In certain embodiments, the kinase inhibitor is selected from the group consisting of adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, leuproreductase inhibitor ... and selected from one or more of patinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0054] Also included herein are methods for treating a disease or condition in a subject in need thereof, the methods 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, e.g., as a therapeutic composition described herein, wherein the at least one antibody or antigen-binding fragment thereof modulates (e.g., inhibits) binding of a 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).

[0055] In certain embodiments, the disease or condition is an NRP2-associated disease or condition, e.g., diseases and pathways associated with cancer, such as cancer and cancer cell proliferation, development, migration, adhesion, invasion, and / or metastasis; diseases associated with inflammation, autoimmune diseases, and related inflammatory diseases, e.g., diseases associated with inappropriate immune cell activation or migration, such as graft-versus-host disease (GVHD); diseases associated with lymphangiogenesis, lymphangiogenesis, and lymphatic damage, e.g., edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability; diseases associated with infectious diseases, e.g., latent infections; and diseases associated with chronic obstructive pulmonary disease (COPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, and systemic lupus erythematosus. diseases associated with allergic disorders / diseases and allergic reactions, such as rheumatoid arthritis, inflammasome-related diseases, and skin-related neutrophil-mediated diseases such as pyoderma gangrenosum; diseases associated with granulomatous inflammatory diseases, e.g., sarcoidosis and granulomas; diseases associated with fibrosis, e.g., fibrotic diseases, fibrosis, endothelial-mesenchymal transition (EMT), and wound healing; diseases associated with improper smooth muscle contractility and improper vascular smooth muscle cell migration and adhesion; diseases associated with improper autophagy, phagocytosis, and efferocytosis; diseases associated with improper migratory cell motility; diseases associated with neurological diseases, peripheral nervous system remodeling, and pain sensation; and diseases associated with bone development and bone remodeling.

[0056] In some embodiments, the disease is cancer, e.g., the cancer expresses or overexpresses NRP2. In some embodiments, the cancer exhibits NRP2-dependent proliferation, 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).

[0057] Also included herein are methods of reducing or preventing recurrence of cancer in a subject, wherein the administration of a therapeutic composition of the invention can create immune memory against the cancer. In some embodiments, the subject has diabetes or is at risk of developing diabetes.

[0058] Certain methods include administering to a subject at least one additional agent selected from one or more of a cancer immunotherapy agent, a chemotherapeutic agent, a hormonal therapy agent, and a kinase inhibitor, 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.

[0059] In some embodiments, the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint modulator, a cancer vaccine, an oncolytic virus, a cytokine, and a cellular immunotherapeutic agent. In some embodiments, the immune checkpoint modulator is a polypeptide, and optionally an antibody or antigen-binding fragment thereof, or a ligand, or a small molecule. In some embodiments, the immune checkpoint modulator is: (a) an antagonist of an inhibitory immune checkpoint molecule, or (b) an agonist of a stimulatory immune checkpoint molecule; For example, the immune checkpoint modulator specifically binds to, in one embodiment, an inhibitory immune checkpoint molecule is selected from the group consisting of programmed death-ligand 1 (PD-L1), programmed death 1 (PD-1), programmed death-ligand 2 (PD-L2), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), V-domain Ig suppressor of T-cell activation (VISTA), B and T lymphocyte attenuator (BTLA), CD160, herpes virus enzyme (HVE), and HIV-1. The immunoreceptor is selected from one or more of: human vein mediator (HVEM), and T cell immunoreceptor with Ig domain and ITIM domain (TIGIT).

[0060] In certain embodiments, the antagonist is a PD-L1 antagonist and / or a PD-L2 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L1 and / or PD-L2, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736); and optionally, the cancer is selected from one or more of colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer, and renal cell carcinoma; the antagonist is a PD-1 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514, PDR001, and pidilizumab, optionally the PD-1 antagonist is nivolumab, and the cancer is optionally selected from one or more of Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer; the PD-1 antagonist is pembrolizumab, and the cancer is optionally selected from one or more of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma; the antagonist is a CTLA-4 antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, and tremelimumab, and optionally the cancer is selected from one or more of melanoma, prostate cancer, lung cancer, and bladder cancer; the antagonist is an IDO antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds IDO, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman, 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, and the cancer is optionally selected from one or more of metastatic breast cancer and brain cancer (optionally glioblastoma multiforme, glioma, gliosarcoma, or malignant brain tumor); the antagonist is a TDO antagonist optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TDO, 680C91, and LM10; the antagonist is a TIM-3 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TIM-3; the antagonist is a LAG-3 antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to LAG-3, and BMS-986016; the antagonist is a VISTA antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to VISTA; the antagonist is an antagonist of BTLA, CD160, and / or HVEM, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to BTLA, CD160, and / or HVEM; The antagonist is a TIGIT antagonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to TIGIT.

[0061] In certain embodiments, the stimulatory immune checkpoint molecule is selected from one or more of OX40, CD40, glucocorticoid-induced TNFR family related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0062] In some embodiments, the agonist is an OX40 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to OX40, OX86, Fc-OX40L, and GSK3174998; the agonist is a CD40 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD40, CP-870893, dacetuzumab, Chi Lob7 / 4, ADC-1013, and rhCD40L, and the cancer is optionally selected from one or more of melanoma, pancreatic cancer, mesothelioma, and hematological cancer (optionally a lymphoma such as non-Hodgkin's lymphoma); the agonist is a GITR agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to GITR, INCAGN01876, DTA-1, and MEDI1873; the agonist is a CD137 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD137, utomilumab, and 4-1BB ligand; the agonist is a CD27 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD27, varlilumab, and CDX-1127 (1F5); The agonist is a CD28 agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to CD28, and TAB08; and / or The agonist is an HVEM agonist, optionally selected from one or more of an antibody or antigen-binding fragment or small molecule or ligand that specifically binds to HVEM.

[0063] In one embodiment, the cancer vaccine is Oncophage, a human papillomavirus HPV vaccine (optionally Gardasil or Cervarix), a hepatitis B vaccine (optionally Engerix B, Heptavax HB, or Twi One or more of the following: Twinrix (Twinrix), and Sipuleucel-T (Provenge) or human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor (VEGF) (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein Protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor and optionally, the subject has or is at risk of having a cancer comprising the corresponding cancer antigen, and the cancer antigen is selected from one or more of: glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

[0064] In one embodiment, the oncolytic virus is talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H In some embodiments, the cytokine is selected from one or more of interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the cellular immunotherapeutic agent comprises 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 chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0065] In certain embodiments, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type I or type II), and a microtubule inhibitor.

[0066] In certain embodiments, the alkylating agent is selected from one or more of 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, and streptozotocin), tetrazines (optionally, dacarbazine, mitozolomide, and temozolomide), aziridines (optionally, thiotepa, mitomycin, and diaziquone (AZQ)), cisplatins and their derivatives (optionally, carboplatin and oxaliplatin), and non-classical alkylating agents (optionally, procarbazine and hexamethylmelamine); the antimetabolite is selected from one or more of antifolates (optionally methotrexate and pemetrexed), fluoropyrimidines (optionally 5-fluorouracil and capecitabine), deoxynucleoside analogues (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (optionally thioguanine and mercaptopurine); the cytotoxic antibiotic is selected from one or more of anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin; the topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin; and / or The microtubule inhibitor is selected from one or more of taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0067] 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 a progestogen (progestin), a corticosteroid (optionally prednisolone, methylprednisolone, or dexamethasone), an insulin-like growth factor, a VEGF-derived angiogenic and lymphangiogenic factor (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), a fibroblast growth factor (FGF), a galectin, a hepatocyte growth factor (HGF), a platelet-derived growth factor (PDGF), a transforming growth factor (TGF) beta, an androgen, an estrogen, and a somatostatin analog. In certain embodiments, the hormone antagonist is a combination of 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 against a hormone receptor, optionally including cizutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, ), robatumumab, alacizumab pegol, bevacizumab, icrucumab, ramucirumab, fresolimumab ), metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, i Mugatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, paclitaxel Nitumumab, tomuzotuximab, zalutumumab, april 2019 The therapeutic agent is selected from one or more of: aprutumab ixadotin, bemarituzumab, olaratumab, or tovetumab.

[0068] In certain embodiments, the kinase inhibitor is selected from the group consisting of adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, leuproreductase inhibitor ... It is selected from one or more of: patinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib and vemurafenib.In some embodiments, the cancer is primary cancer.In some embodiments, the cancer is metastatic cancer, for example, metastatic cancer that expresses NRP2 and / or NRP2B. In certain embodiments, the cancer is selected from one or more of melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma (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), renal 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.

[0069] In one embodiment, the metastatic cancer is (a) bladder cancer that has metastasized to the bone, liver, and / or lungs; (b) breast cancer that has metastasized to the bone, brain, liver, and / or lungs; (c) colorectal cancer metastasized to the liver, lung, and / or peritoneum; (d) renal cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or lungs; (e) lung cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or other lung sites; (f) melanoma that has metastasized to bone, brain, liver, lung, and / or skin / muscle; (g) ovarian cancer that has metastasized to the liver, lung, and / or peritoneum; (h) pancreatic cancer metastasized to the liver, lung, and / or peritoneum; (i) prostate cancer that has metastasized to the adrenal glands, bone, liver, and / or lungs; (j) gastric cancer metastasized to the liver, lung, and / or peritoneum; (l) thyroid cancer that has metastasized to the bone, liver, and / or lungs, and (m) uterine cancer metastasized to bone, liver, lung, peritoneum, and / or vagina.

[0070] In one embodiment, the subject has and / or has elevated circulating or serum levels of at least one NRP2 ligand (e.g., an NRP2 ligand selected from Table N2 or Table N3 and / or an HRS polypeptide selected from Table H1), whether the NRP2 ligand is bound or free, compared to healthy or matched controls or subjects. and optionally, the level of the at least one NRP2 ligand (e.g., HRS polypeptide) is about or at least about 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 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, , 1600pM, 1700pM, 1800pM, 1900pM, 2000pM, 3000pM, 4000pM, or 5000pM, or the level of the at least one NRP2 ligand is about or at least about 30pM to 100pM, 40pM to 100pM, 50pM to 100pM, 30pM to 2000pM, 40pM to 2000pM, 50pM to 2000pM, 60pM to 2000pM, 70pM to 2000pM, 80pM to 8000pM, ~2000pM, 90pM~2000pM, 100pM~2000pM, 200pM~2000pM, 300pM~2000pM, 400pM~2000pM, 500pM~2000pM, 600pM~2000pM, 700pM~2000pM, 800pM~2000pM, 900pM~2000pM, 1000pM~2000pM, 2000pM~3000pM, 3000pM~4000pM, or 4000pM~5000pM.

[0071] In certain embodiments, the subject has, and / or is selected for treatment pursuant to having, a disease associated with elevated levels or expression of at least one NRP2 ligand (e.g., an NRP2 ligand selected from Table N2 or Table N3, and / or an HRS polypeptide selected from Table H1) and / or its encoding mRNA relative to a healthy control or matched control or subject group, optionally wherein the disease is a cancer associated with elevated levels or expression of at least one NRP2 ligand and / or its encoding mRNA relative to a non-cancerous control cell or tissue, optionally relative to a non-cancerous cell or tissue of the same type, and optionally wherein the HRS polypeptide is 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 A splice variant selected from:

[0072] In certain embodiments, the subject has, and / or is selected for treatment pursuant to, an elevated circulating or serum level of a soluble neuropilin-2 (NRP2) polypeptide (e.g., selected from Table N1), whether bound or free, relative to a healthy control or matched control or subject group level, optionally with a circulating or serum level of soluble NRP2 polypeptide 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, 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, 500 pM, 5100 pM, 00pM, 1400pM, 1500pM, 1600pM, 1700pM, 1800pM, 1900pM, 2000pM, 3000pM, 4000pM, or 5000pM, or optionally the circulating or serum level of soluble NRP2 polypeptide is about 30pM to 50pM, 50pM to 100pM, 100pM to 2000pM, 200pM to 3000pM, 3000pM, 4000pM, or 5000pM. ~2000pM, 300pM~2000pM, 400pM~2000pM, 500pM~2000pM, 600pM~2000pM, 700pM~2000pM, 800pM~2000pM, 900pM~2000pM, 1000pM~2000pM, 2000pM~3000pM, 3000pM~4000pM, or 4000pM~5000pM.

[0073] In one embodiment, the subject has, and / or is selected for treatment pursuant to having, a disease associated with elevated levels or expression of an NRP2 polypeptide (e.g., one selected from Table N1) and / or its encoding mRNA compared to healthy or matched controls or a group of subjects, and optionally the disease is a cancer associated with elevated levels or expression of an NRP2 polypeptide (e.g., one selected from Table N1) and / or its encoding mRNA compared to non-cancerous cells or tissues of the same type.

[0074] In some embodiments, the subject has, and / or is selected for treatment pursuant to having, a disorder associated with elevated levels or expression of NRP2A and / or NRP2B, or an altered NRP2A to NRP2B expression ratio, relative to a healthy or matched control, or subject group. In some embodiments, the subject has significantly elevated NRP2B expression or levels relative to a healthy or matched control, or subject group. In some embodiments, the level of NRP2B is about or at least about 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% elevated relative to a healthy or matched control, or subject group.

[0075] In one embodiment, the subject has elevated circulating levels of the HRS and NRP2 complex compared to healthy or matched controls or subjects, and / or is selected for treatment based on said elevated levels.

[0076] In one embodiment, the healthy or matched control or subject group comprises an average range of age-matched samples of the same type of cancerous or non-cancerous cells or tissue, including specific characteristics such as drug resistance, metastatic potential, aggressiveness, genetic signature (e.g., p53 mutation, PTEN deletion, IGFR expression), and / or expression pattern.

[0077] Certain embodiments include administering at least one anti-NRP2 antibody in an amount and frequency sufficient to achieve a sustained average serum or circulating level of soluble NRP2 polypeptide of about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM, or less than about 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM.

[0078] Certain embodiments include administering at least one anti-NRP2 antibody in an amount and frequency sufficient to reduce circulating levels of complexes between HRS and NRP2, 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%.

[0079] In certain embodiments, the at least one anti-NRP2 antibody enhances the immune response against cancer by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more compared to a control.

[0080] In certain embodiments, at least one anti-NRP2 antibody reduces the in vitro growth rate of a 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 compared to an untreated control.

[0081] In certain embodiments, at least one anti-NRP2 antibody reduces in vitro adhesion of cancer to a substrate by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more compared to an untreated control. In some examples, the substrate comprises laminin.

[0082] In certain embodiments, at least one anti-NRP2 antibody reduces cancer invasion 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 compared to untreated controls.

[0083] In certain embodiments, at least one anti-NRP2 antibody inhibits the rate of cancer migration or motility 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 compared to an untreated control.

[0084] In certain embodiments, at least one anti-NRP2 antibody inhibits the rate of autophagy or endosomal maturation (e.g., endosomal acidification) of 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 compared to untreated controls.

[0085] In certain embodiments, at least one anti-NRP2 antibody enhances the sensitivity of a cancer to a chemotherapeutic agent, hormone therapy agent, 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 compared to the chemotherapeutic agent alone.

[0086] In certain embodiments, at least one anti-NRP2 antibody enhances the anti-tumor activity and / or immunostimulatory activity of a cancer immunotherapeutic agent by about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000% or more compared to the cancer immunotherapeutic agent alone.

[0087] Certain embodiments include administering at least one anti-NRP2 antibody in an amount and frequency sufficient to achieve a steady-state or mean circulating concentration of about 1 nM to about 1 μM, about 1 nM to about 100 nM, about 1 nM to about 10 nM, or about 1 nM to about 3 μM.

[0088] A patient care kit is also included, the care kit including: (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, (b) at least one additional agent selected from one or more of a cancer immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapy agent, and a kinase inhibitor.

[0089] In some embodiments, (a) and (b) are contained in separate therapeutic compositions. In some embodiments, (a) and (b) are contained in the same therapeutic composition, as described herein.

[0090] In some patient care kits, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type I or type II), and a microtubule inhibitor.

[0091] In some patient care kits, the alkylating agent is selected from one or more of 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, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mitomycin, and diaziquone (AZQ)), cisplatins and their derivatives (optionally carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine); the antimetabolite is selected from one or more of antifolates (optionally methotrexate and pemetrexed), fluoropyrimidines (optionally 5-fluorouracil and capecitabine), deoxynucleoside analogues (optionally ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (optionally thioguanine and mercaptopurine); the cytotoxic antibiotic is selected from one or more of anthracyclines (optionally doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin; the topoisomerase inhibitor is selected from one or more of camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin; and / or The microtubule inhibitor is selected from one or more of taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0092] In some patient care kits, the at least one hormone therapy agent is a hormone agonist or a hormone antagonist. In some patient care kits, the hormone agonist is selected from one or more of a progestogen (progestin), a corticosteroid (optionally prednisolone, methylprednisolone, or dexamethasone), an insulin-like growth factor, a VEGF-derived angiogenic and lymphangiogenic factor (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), a fibroblast growth factor (FGF), a galectin, a hepatocyte growth factor (HGF), a platelet-derived growth factor (PDGF), a transforming growth factor (TGF) beta, an androgen, an estrogen, and a somatostatin analog.

[0093] In one patient care kit, the hormone antagonist comprises 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 against a hormone receptor, optionally cizutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, robatumumab, alacizumab pegol, pegol), bevacizumab, icrucumab, ramucirumab, fresolimumab, metelimumab, naxitamab, cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomuzotuximab, zalutumumab, aprutumab ixadotin, bemarituzumab, olaratumab, or tovetumab can be.

[0094] In one patient care kit, kinase inhibitors include adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, and imatinib. and vemurafenib.

[0095] Also included is a bioassay system, optionally comprising a substantially pure anti-NRP2 antibody or antigen-binding fragment thereof as described herein, and a host cell line expressing a human NRP2 polypeptide on the cell surface.

[0096] In some embodiments, the NRP2 polypeptide is labeled with a detectable label. In some embodiments, the anti-NRP2 antibody is labeled with a detectable label. In some embodiments, the NRP2 polypeptide is operably linked to an indicator or label, such as a fluorescent or luminescent label, that indicates 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 selected from Table N2 or Table N3 and / or a human histidyl-tRNA synthetase (HRS) polypeptide selected from Table H1), e.g., the host cell described above expresses the at least one NRP2 ligand. In some embodiments, the HRS polypeptide is selected from Table H1, e.g., the HRS polypeptide is 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 C9In certain embodiments, the at least one NRP2 ligand is selected from Table N2 or Table N3.

[0097] One embodiment includes a detection system comprising cells expressing a human neuropilin-2 (NRP2) polypeptide, 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 described herein, which modulates the interaction between the NRP2 polypeptide and the at least one NRP2 ligand. In one embodiment, the anti-NRP2 antibody is labeled with a detectable label. In one embodiment, the NRP2 polypeptide is selected from Table N1. In one embodiment, the HRS polypeptide is selected from Table H1, e.g., the HRS polypeptide is HisRS. N1 , HisRS N2 , HisRS N3 , HisRS N4 , HisRS N5 , HisRS C1 , HisRS C2 , HisRS C3 , HisRS C4 , HisRS C5 , HisRS C6 , HisRS C7 , HisRS C8 , and HisRS C9 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 at least one NRP2 ligand (e.g., an NRP2 ligand selected from Table N2 or Table N3, and / or an HRS polypeptide selected from Table H1) is operably linked to an indicator or label, such as a fluorescent or luminescent label, that indicates the biological activity of the NRP2 polypeptide or at least one NRP2 ligand.

[0098] Also included are diagnostic systems comprising cells containing 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 cells contain a labeled molecule that indicates an altered level or activity of the NRP2 polypeptide in response to interaction with the at least one NRP2 ligand.

[0099] Also included are cell compositions, which comprise an altered cell population, wherein at least one cell comprises one or more polynucleotides encoding a human or humanized anti-NRP2 antibody, or antigen-binding fragment thereof, as described herein, and wherein the cell is capable of growth in serum-free medium.

[0100] Also included is a cell growth device comprising a human or humanized anti-NRP2 antibody or antigen-binding fragment thereof described herein, a modified 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 explanation of the drawings]

[0101] [Figure 1A] 1A-1B show the general domain structure of neuropilin (FIG. 1A) and an example of the function of a neuropilin co-receptor (FIG. 1B). [Figure 1B] Same as above. [Figure 2] FIG. 1 shows the domain structure of specific NRP2 isoforms and examples of NRP2 ligand-binding domains. [Figure 3]Figure 1 shows Western blot results of cell lines blotted with a commercial antibody against human NRP2 (BAF2215, Boster, CA, USA) using a total of 6 μg of cell lysate per lane. The outer lanes show molecular weight markers. [Figure 4-1] Figures 4A-4D show flow cytometry cell surface binding plots for the C-domain-specific anti-NRP2 antibody 3F2 (aNRP2-2) on selected cell lines, detected with an AF647-conjugated goat anti-mouse IgG secondary antibody (GaM AF647). Binding of the secondary antibody alone is shown in light gray (curve on the left side of each figure). Figure 4A shows antibody binding to U251 cells, Figure 4B shows antibody binding to HUVEC cells, Figure 4C shows antibody binding to THP-1M1 cells, and Figure 4D shows antibody binding to HLEC cells. [Figure 4-2] Same as above. [Figure 5-1] Figures 5A-5D show flow cytometry surface binding plots of specific anti-NRP2 antibodies against various cell lines. Antibody binding was detected using an AF647-conjugated goat anti-mouse IgG secondary antibody (GaM AF647). Figure 5A shows antibody binding to HUVEC cells over an antibody concentration range of 0.06 nM to 1000 nM. Figure 5B shows binding to U251 cells over an antibody concentration range of 0.01 nM to 1000 nM. Figure 5C shows binding to A549 cells over an antibody concentration range of 0.01 nM to 1000 nM. Figure 5D shows binding to THP-1M1 cells over an antibody concentration range of 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 the cells at the concentrations described in the Examples. [Figure 5-2] Same as above. [Figure 6]Figures 6A and 6B show binding curves of anti-NRP2 antibodies to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 6A shows antibody concentrations from 0.05 nM to 30 nM, and Figure 6B shows results at antibody concentrations tested from 0.02 nM to 10 nM, both using 3-fold dilutions of antibody. Antibody binding was detected using an AF647-conjugated goat anti-mouse IgG secondary antibody (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) (data not shown) were incubated with cells at the concentrations described in the Examples. [Figure 7] Figures 7A and 7B show VEGF-C binding to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 7A shows the FACS binding curve for VEGF-C (R&D Systems) using a rabbit detection antibody (Abcam) and a goat antibody AF647-labeled secondary antibody. Figure 7B shows a flow cytometry scatter plot. The curves shown in Figure 7B, from left to right, are (aR AF647), (Rb Iso_Ctl + aR AF647), (a-VEGFc + aR AF647), and (VEGFc 100 nM + a-VEGFc + aR AF647). [Figure 8-1]Figures 8A-8M show blocking of anti-NRP2 antibodies, depicting the displacement curves of selected antibodies against VEGF-C binding to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 8A shows the results using an isotype control antibody, Figure 8B shows the results using antibody 8v2 (aNRP2-8) #1328, Figure 8C shows the results using antibody 9v2 (aNRP2-9) #1329, Figure 8D shows the results using antibody 11v2 (aNRP2-11) #1331, Figure 8E shows the results using antibody 14v2 (aNRP2-14) #1344, Figure 8F shows the results using antibody 2v1 (aNRP2-1) #1326, and Figure 8G shows the results using antibody 11v2 (aNRP2-11) #1331. Figure 8H shows the results using antibody 2v2 (aNRP2-2) #1327, Figure 8H shows the results using antibody 2v10 (aNRP2-10) #1330, Figure 8I shows the results using antibody #1333, Figure 8J shows the results using antibody #1334, Figure 8K shows the results using antibody 2v7 (aNRP2-7) #1335, Figure 8L shows the results using antibody 2v12 (aNRP2-12) #1336, and Figure 8M shows the results using antibody #1337. [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 9] Figure 9 shows the binding of SEMA3F-p95 and SEMA3F-p65 (0.05 nM to 100 nM) to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 9A shows the FACS binding curves for SEMA3F-p95 or p65 (manufactured in-house), using the anti-myc detection antibody described in the Examples. [Figure 10-1]10A-10H show blocking anti-NRP2 antibodies, depicting the displacement curves of selected antibodies for SEMA3F-p95 binding to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 10A shows the results using an isotype control antibody, Figure 10B shows the results using antibody 14v2 (aNRP2-14) #1344, Figure 10C shows the results using antibody 8v2 (aNRP2-8) #1328, Figure 10D shows the results using antibody 9v2 (aNRP2-9) #1329, Figure 10E shows the results using antibody 2v10 (aNRP2-10) #1330, Figure 10F shows the results using antibody 15v2 (aNRP2-15) #1347, Figure 10G shows the results using antibody 11v2 (aNRP2-11) #1331, and Figure 10H shows the results using antibody 2v2 (aNRP2-2) #1327. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 11-1] Figures 11A-11F show blocking of anti-NRP2 antibodies, showing the displacement curves of selected antibodies against SEMA3F-p95 binding to Expi293-hNRP2 clone cells overexpressing human NRP2. Figure 11A shows the results using antibody 14v2 (aNRP2-14) #1344, Figure 11B shows the results using antibody 8v2 (aNRP2-8) #1328, Figure 11C shows the results using antibody 9v2 (aNRP2-9) #1329, Figure 11D shows the results using antibody 2v10 (aNRP2-10) #1330, Figure 11E shows the results using antibody 15v2 (aNRP2-15) #1347, and Figure 11F shows the results using antibody 11v2 (aNRP2-11) #1331. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 12] FIG. 1 shows the effect of anti-NRP2 antibody binding on NRP2-Plexin A1-induced receptor heterodimerization described in the Examples when antibodies of the present disclosure are used. [Figure 13]FIG. 1 shows the effect of anti-NRP2 antibody binding on NRP2-FLT4 (VEGFR3)-induced receptor heterodimerization as described in the Examples when antibodies of the present disclosure are used. [Figure 14] FIG. 1 shows the crystal structure of human NRP2, depicting the a2 and b1 / b2 domains in complex with the variable heavy (VH) and variable light (VL) chains of aNRP2-14Fab. [Figure 15] FIG. 1 shows the interaction surface between human NRP2 and the heavy (VH) and light (VL) chains of aNRP2-14 Fab, with the a2 and b1 domains labeled, and the interacting amino acids. [Figure 16] Depicting the interaction interface between the a2 domain of human NRP2 (bottom) and the heavy (top left) and light (top right) chains of aNRP2-14Fab, the interaction between residue E237 of human NRP2 and residue S57 of the Fab heavy chain is circled. DETAILED DESCRIPTION OF THE INVENTION

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods, materials, compositions, reagents, or cells similar or equivalent to those described herein can be used in the practice or testing of the subject matter of the present disclosure, preferred methods and materials are described. All publications and references cited herein, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety, as if each individual publication or reference was specifically and individually indicated to be incorporated herein by reference as if fully set forth. In addition, any patent application to which this application claims priority is incorporated herein by reference in its entirety, as described above for publications and references.

[0103] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture, and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification procedures can be performed according to manufacturer's instructions, or as commonly practiced in the art, or as described herein. These and related techniques and procedures can generally be performed according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout the specification. Unless specifically defined otherwise, the terms used in connection with molecular biology, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are those well known and commonly used in the art. Standard techniques can be used for recombinant technology, molecular biological synthesis, microbiological synthesis, chemical synthesis, chemical analysis, drug preparation, formulation, and delivery, and patient treatment.

[0104] For purposes of this disclosure, the following terms are defined as follows:

[0105] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" includes "one element," "one or more elements," and / or "at least one element."

[0106] "About" means that the amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length varies by 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the referenced amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length.

[0107] The term "antigen" refers to a molecule or portion of a molecule that can bind to a specific binding agent, such as an antibody, and can be used in an animal to produce an antibody capable of binding to that antigen's epitope. An antigen may have one or more epitopes. As used herein, the term "antigen" includes any substance that, under the proper conditions, can induce an immune response and react with the products of that immune response. For example, antigens can be recognized by antibodies (humoral immune response) or sensitized T lymphocytes (T helper or T cell-mediated immune response), or both. Antigens can be soluble substances, such as toxins and foreign proteins, or particulate substances, such as bacteria and tissue cells; however, only portions of protein or polysaccharide molecules, known as antigenic determinants (epitopes), bind to specific receptors on antibodies or lymphocytes. In a broader sense, the term "antigen" includes any substance that binds to or has affinity for an antibody, regardless of whether it is immunogenic. Antibodies to such antigens can be identified using recombinant methods, independent of an immune response.

[0108] "Antagonist" refers to a biological structure or chemical agent that inhibits or reduces the physiological action of another drug or molecule. In some instances, antagonists specifically bind to other drugs or molecules. Full antagonists and partial antagonists are also included.

[0109] "Agonist" refers to a biological structure or chemical agent that increases or potentiates the physiological effect of another drug or molecule. In some instances, agonists specifically bind to other drugs or molecules. Full agonists and partial agonists are also included.

[0110] The term "anergy" refers to functional inactivation of T-cell or B-cell responses to antigen restimulation.

[0111] The term "amino acid," as used herein, is intended to refer to natural and unnatural amino acids, as well as amino acid analogs and amino acid mimetics. Natural amino acids include the 20 L-amino acids used during protein biosynthesis and other amino acids, such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Unnatural amino acids include, for example, D-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, which are known to those skilled in the art. Amino acid analogs include modified forms of natural and unnatural amino acids. Such modifications can include, for example, substitutions or replacements of chemical groups and moieties of amino acids or derivatization of amino acids. Amino acid mimetics include organic structures that have functionally similar properties, such as charge and charge-spatial configuration properties, of the reference amino acids. For example, organic structures that mimic arginine (Arg or R) include those that have a similar molecular spatial arrangement and a positively charged moiety with a similar degree of mobility as the e-amino group of the side chain of the natural Arg amino acid. Mimetics also include structures that are constrained to maintain optimal spatial arrangement and charge interactions of the amino acid 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.

[0112] The term "antibody," as used herein, encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAbs, Fab, Fab', F(ab')2, Fv, etc.), single-chain antibodies (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of predetermined specificity, humanized antibodies, and chimeric antibodies, as well as any other modified form of an immunoglobulin molecule that contains an antigen-binding site or fragment (epitope-recognizing site) with predetermined specificity. Specific properties and characteristics of antibodies (and antigen-binding fragments thereof) are described in more detail herein.

[0113] 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 can specifically bind to a target, such as an immune checkpoint molecule, via at least one epitope recognition site in the variable region of the immunoglobulin molecule.

[0114] The term "antigen-binding fragment," as used herein, refers to a polypeptide fragment comprising 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 includes a V CDR from the antibody that binds to a target molecule. H Sequence and V L It may comprise one, two, three, four, five or all six CDRs of the sequence.

[0115] The binding properties of antibodies and antigen-binding fragments thereof can be determined using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to a target molecule, e.g., an NRP2 polypeptide or an epitope thereof or a complex thereof, with an equilibrium dissociation constant of about 10 -7 M or less ~ about 10 -8 In one embodiment, the equilibrium dissociation constant is about 10 -9 M or less ~ about 10 -10 In certain exemplary embodiments, the affinity (Kd or EC 50) is about or at least 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, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 4 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, or less than about 0.01 nM, less than 0.05 nM, less than 0.1 nM, or less than 0.2 nM , less than 0.3 nM, less than 0.4 nM, less than 0.5 nM, less than 0.6 nM, less than 0.7 nM, less than 0.8 nM, less than 0.9 nM, less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM, less than 11 nM, less than 12 nM, less than 13 nM, less than 14 nM, less than 15 nM, less than 16 nM, less than 17 nM, less than 18 nM, less than 19 nM, less than 20 nM, less than 21 nM, less than 22 nM, less than 23 nM, less than 24 nM, less than 25 nM, less than 26 nM, less than 27 nM, less than 28 nM, less than 29 nM, less than 30 nM, less than 40 nM, or less than 50 nM.

[0116] A molecule, such as a polypeptide or antibody, is said to exhibit "specific binding" or "selective binding" if it reacts with or binds to a particular cell, substrate, or particular epitope more frequently, faster, for longer, and / or with higher affinity than it binds to other cells, substrates, or epitopes. An antibody "specifically binds" or "selectively binds" to a target molecule or epitope if it binds with higher affinity, avidity, ease, and / or duration than it binds to other substrates or epitopes, e.g., by a statistically significant amount. Typically, one moiety of a pair of molecules exhibiting specific binding has a surface region or depression that specifically binds to and is complementary to a unique spatial and / or polar configuration of the other moiety of its pair. Thus, the moieties of the pair possess the property of specifically binding to each other. For example, an antibody that specifically or selectively binds to a particular epitope is an antibody that binds that particular epitope with higher affinity, higher avidity, more readily, and / or for longer than it binds to other epitopes. It is also understood by reading this definition that, for example, an antibody (portion or epitope) that specifically or selectively binds to a first target may or may not specifically or selectively bind to a second target. This term also applies when, for example, an antibody is specific for a particular epitope possessed by multiple antigens, i.e., when a specific binding portion having an antigen-binding fragment or domain can bind to various antigens that share that epitope, and in this case, for example, the antibody may be cross-reactive with various forms of a target antigen from multiple species that share a common epitope.

[0117] Immunological binding typically refers to the type of noncovalent interaction that occurs between an immunoglobulin molecule and an antigen for which that immunoglobulin is specific, resulting from, by way of example only and not limitation, electrostatic, ionic, hydrophilic and / or hydrophobic affinities or repulsions, steric forces, hydrogen bonding, van der Waals forces, and other interactions. The strength or affinity of an immunological binding interaction can be expressed as the dissociation constant (Kd) of the interaction, with a smaller Kd indicating a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method measures the rates of formation and dissociation of antigen-binding site / antigen complexes, which vary with the concentrations of both components of the complex, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "on-rate constant" (Kon) and "off-rate constant" (Koff) can be calculated by calculating the concentrations and the actual rates of association and dissociation. The ratio Koff / Kon can be calculated to cancel out all parameters unrelated to affinity and is therefore equivalent to the dissociation constant Kd. The term "affinity" as used herein includes the equilibrium constant for the reversible binding of two drugs, and is not necessarily a constant that can be expressed as Kd or EC 50 The affinity of a binding protein for a ligand, e.g., the affinity of an antibody for an epitope, can range, for example, from about 0.1 nanomolar (nM) to about 100 nM, from about 1 picomolar (pM) to about 100 nM, or from about 1 femtomolar (fM) to about 100 nM. The term "avidity," as used herein, refers to the resistance of a complex of two or more agents to dissociation after dilution. In certain embodiments, affinity is measured using the 50% effective concentration (EC 50 EC ) refers to the concentration of an agent, such as an antibody or anti-NRP2 antibody disclosed herein, that induces a 50% maximum response from baseline after a specific exposure time. 50 is commonly used as a measure of antibody potency.

[0118] Antibodies can be prepared by any of a variety of techniques known to those skilled in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific to a polypeptide of interest can be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, or modifications thereof. Methods for expressing human antibodies using transgenic animals such as mice are also included. See, for example, Neuberger et al. See, Lonberg et al., Nature Biotechnology 14:826, 1996, Lonberg et al., Handbook of Experimental Pharmacology 113:49-101, 1994, and Lonberg et al., Internal Review of Immunology 13:65-93, 1995. A specific example is the VELOCIMMUNE® platform of REGENEREX® (e.g., U.S. Patent No. 6,596,541).

[0119] Antibodies can also be generated or identified using phage display or yeast display libraries (see, e.g., U.S. Pat. No. 7,244,592; Chao et al., Nature Protocols. 1:755-768, 2006). Non-limiting examples of libraries that can be used include cloned or synthetic libraries, such as HuCAL (Human Combinatorial Antibody Library), which are structural representations of the human antibody repertoire. The diversity is represented by seven heavy chain and seven light chain variable region genes. The combination of these genes constitutes 49 frameworks of the master library. By introducing highly variable gene cassettes (CDRs: complementarity determining regions) into these frameworks, a vast human antibody repertoire can be reproduced. Also included is a human library designed using fragments derived from human donors encoding the light chain variable region and heavy chain CDR-3, synthetic DNA encoding the diversity of heavy chain CDR-1, and synthetic DNA encoding the diversity of heavy chain CDR-2. Other libraries suitable for use will be apparent to those skilled in the art.

[0120] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy and light chain CDRs. The set of CDRs is inserted between a set of heavy and light chain framework regions (FRs), respectively, which support the CDRs and define their spatial relationship. The term "set of CDRs" as used herein refers to the three hypervariable regions of a heavy or light chain V region. These regions are referred to as "CDR1," "CDR2," and "CDR3," respectively, starting from the N-terminus of the heavy or light chain. Thus, an antigen-binding site contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analyses of multiple antigen-antibody complexes have revealed that amino acid residues in the CDRs make extensive contact with the bound antigen, with the antigen making the most extensive contact with the heavy chain CDR3. Thus, the molecular recognition unit primarily determines the specificity of the antigen-binding site.

[0121] As used herein, the term "FR set" refers to four adjacent amino acid sequences that define the CDRs of a CDR set in a heavy or light chain V region. While some FR residues may contact the bound antigen, the FRs, particularly those immediately adjacent to the CDRs, define the folding of the V region into the antigen-binding site. Within the FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form the antigen-binding surface. It is generally recognized that, regardless of the amino acid sequence of the CDR itself, there are conserved structural regions in the FRs that direct the folding shape of the CDR loops into certain "canonical" structures. Furthermore, certain FR residues are known to participate in noncovalent interdomain contacts that stabilize the interaction between the heavy and light chains of an antibody.

[0122] The structure and location of immunoglobulin variable domains are described in Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition, US Department of Health and Human Services. 1987 and its latest updates can be found by reference. do.

[0123] Also included are "monoclonal" antibodies, which refer to a homogeneous population of antibodies. Monoclonal antibodies are composed of amino acids (natural and non-natural) that are involved in selective binding to an epitope. Monoclonal antibodies are highly specific and act against a single epitope. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (scFv), variants thereof, fusion proteins containing the antibody-binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified form of an immunoglobulin molecule containing an antigen-binding fragment (epitope recognition site) with a given specificity and binding ability to the epitope. It is not intended to be limited as to the source of the antibody or the method by which it is produced (e.g., hybridoma, phage selection, recombinant expression, transgenic animals). The term encompasses whole immunoglobulins and the fragments described above that comply with the definition of "antibody."

[0124] The proteolytic enzyme papain preferentially cleaves IgG molecules to form multiple fragments, two of which (F(ab) fragments) comprise covalently linked heterodimers each containing an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to form multiple fragments, such as F(ab')2 fragments containing two antigen-binding sites. Fv fragments for use in certain embodiments can be generated by selective proteolytic cleavage of immunoglobulin molecules, such as IgM, and more rarely IgG or IgA. However, Fv fragments are more commonly obtained using recombinant techniques known in the art. Fv fragments include noncovalent VH-VL heterodimers containing an antigen-binding site, which retain much of the antigen recognition and binding capabilities of natural antibody molecules. See Inbar et al., PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980.

[0125] In certain embodiments, single-chain Fv (scFv) antibodies are of interest, such as kappabodies (Ill et al., Prot. Eng. 10:949-57, 1997), minibodies (Martin et al., EMBO J 13:5305-9, 1994), diabodies (Holliger et al., PNAS 90: 6444-8, 1993), or Janusins (Traunecker 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 following the teachings of the present application regarding the selection of antibodies with the desired specificity.

[0126] Single-chain Fv (scFv) polypeptides are covalent VH-VL heterodimers that are expressed from gene fusions containing VH- and VL-encoding genes linked by a peptide-encoding linker. See Huston et al., PNAS USA. 85(16):5879-5883, 1988. Spontaneously aggregated but chemically dissociated antibody VFvs are also known. Several methods have been reported to identify chemical structures that convert the light and heavy polypeptide chains of a domain into scFv molecules that fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, e.g., U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al. and U.S. Patent No. 4,946,778 to Ladner et al.

[0127] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are in the form of "diabodies." Diabodies are multimers of polypeptides, each of which contains a first domain comprising an immunoglobulin light chain binding region and a second domain comprising an immunoglobulin heavy chain binding region, and the two domains are linked (e.g., by a peptide linker) but cannot associate with each other to form an antigen-binding site. An antigen-binding site is formed by the association of a first domain of a particular polypeptide within the multimer with a second domain of another polypeptide within the multimer (WO 94 / 13804). A dAb fragment of an antibody consists of a VH domain (Ward et al., Nature 341:544-546, 1989). Diabodies and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO 94 / 13804 and Holliger et al., PNAS USA. 90:6444-6448, 1993).

[0128] Minibodies containing scFv linked to a CH3 domain are also included (Hu et al., Cancer Res. 56:3055-3061, 1996). Also see Ward et al., Nature. 341:544-546, 1989, Bird et al., Science. 242:423-426, 1988, Huston et al., PNAS USA. 85:5879-5883, 1988), International Application No. PCT / US92 / 09965, International Publication No. 94 See also Reiter et al., Nature Biotech. 14:1239-1245, 1996. I want to be illuminated.

[0129] When bispecific antibodies are used, conventional bispecific antibodies may be used. Bispecific antibodies can be produced in a variety of ways (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), for example, even if it is chemically prepared, The antibody fragment may be prepared from a hybrid hybridoma or any of the bispecific antibody fragments described above. Diabodies and scFvs can be constructed using only variable domains, without the Fc region, potentially reducing the effects of anti-idiotypic reaction.

[0130] Bispecific diabodies can also be particularly useful because, in contrast to bispecific whole antibodies, they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) with the correct binding specificity can be easily selected from libraries using phage display technology (WO 94 / 13804). If one arm of the diabody is kept stable, e.g., with specificity for antigen X, the other arm can be varied to generate a library from which antibodies with the correct specificity can be selected. Bispecific whole antibodies can also be generated by knobs-into-holes engineering (Ridgeway et al., Protein Eng., 9:616-621, 1996).

[0131] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are in the form of a UniBody®. A UniBody® is a polypeptide in which the hinge region has been removed. Unibody® is an IgG4 antibody (see GenMab, Utrecht, The Netherlands and U.S. Patent Application Publication No. 20090226421). This antibody technology provides a stable, smaller antibody format that is expected to have a broader therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and do not interact with the immune system. Fully human IgG4 antibodies can be engineered by removing the antibody hinge region to yield half-fragments that are more stable than the corresponding intact IgG4 (GenMab, Utrecht, The Netherlands). By bisecting the IgG4 molecule, Unibody® leaves only one region available for binding the cognate antigen (e.g., disease target), and thus the Unibody® binds monovalently to only one site on the target cell. With respect to a specific cancer cell surface antigen, this monovalent binding does not potentially stimulate cancer cells to proliferate, as occurs with bivalent antibodies with the same antigen specificity. Thus, Unibody® technology may offer a treatment option for cancer types that may be resistant to treatment with conventional antibodies. The small size of Unibodies® may be extremely beneficial in treating some forms of cancer, potentially improving the distribution of the molecule to larger solid tumor sizes and increasing efficacy.

[0132] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein are in the form of nanobodies. Minibodies are encoded by a single gene and are efficiently produced in almost all prokaryotic and eukaryotic hosts, such as Escherichia coli (see U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyveromyces, Hansenula, or Pichia) (see U.S. Pat. No. 6,838,254). The production process is amenable to large-scale production, and nanobodies have been produced in multi-kilogram quantities. Nanobodies can be formulated as ready-to-use solutions with long shelf lives. The Nanoclone method (see WO 06 / 079372) is a patented method that utilizes automated high-throughput sorting of B cells to generate nanobodies against desired targets.

[0133] In some embodiments, the antibodies or antigen-binding fragments thereof 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 are generally synthesized using the SELEX method (systematic evolution of ligands by exponential enrichment). "Selection" refers to nucleic acid species that have been engineered to bind to various molecular targets (e.g., small molecules, proteins, nucleic acids, as well as cells, tissues, and organisms) through repeated in vitro selection, such as in vitro selection (selection methods), or equivalent methods. See, e.g., U.S. Patent Nos. 6,376,190 and 6,387,620, which are incorporated by reference.

[0134] Peptide aptamers typically contain a variable peptide loop attached at both ends to a protein scaffold. This dual structural constraint increases the binding affinity of peptide aptamers to levels comparable to that of antibodies (nanomolar range). In certain embodiments, the variable loop may be approximately 10 to 20 amino acids in length (including all integers within that range), and the scaffold may comprise any protein with good solubility and compactness. In certain exemplary embodiments, the bacterial protein thioredoxin-A is used as the scaffold protein, with the variable loop inserted into the reductive active site (a -Cys-Gly-Pro-Cys-loop in the wild-type protein) and two cysteine side chains capable of forming a disulfide bridge. Methods for identifying peptide aptamers are described, for example, in U.S. Patent Application Publication No. 2003 / 0108532, which is incorporated by reference. Peptide aptamers can be selected using various systems known in the art, such as the yeast two-hybrid system.

[0135] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are in the form of avimers. Avimers are multimeric binding proteins or peptides engineered using in vitro exon shuffling and phage display techniques. Linking multiple binding domains can provide higher affinity and specificity than single epitope immunoglobulin domains. See, e.g., Silverman et al., Nature Biotechnology. 23:1556-1561, 2005, U.S. Patent No. 7,166,697, U.S. Patent Application Publication No. 200 See, e.g., Patent Applications Nos. 2005 / 04 / 0175756, 2005 / 0048512, 2005 / 0053973, 2005 / 0089932, and 2005 / 0221384, which are incorporated by reference.

[0136] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are in the form of Adnectins. Adnectins are a type of targeted biologic derived from human fibronectin, a large extracellular protein that naturally binds to other proteins. See, e.g., U.S. Patent Application Publication Nos. 2007 / 0082365, 2008 / 0139791, and 2008 / 0220049, which are incorporated by reference. Adnectins typically consist of a natural fibronectin scaffold and multiple targeting domains containing specific portions of human fibronectin. The targeting domains can be engineered to enable Adnectins to specifically recognize NRP2 polypeptides or epitopes thereof.

[0137] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are in the form of anticalins. Anticalins are a type of antibody mimics typically synthesized from human lipocalins, a family of binding proteins with hypervariable loop regions supported by a structurally rigid framework. See, e.g., U.S. Patent Application Publication No. 2006 / 0058510. Anticalins are typically approximately 20 kDa in size. Anticalins can be characterized by a barrel structure formed by eight antiparallel β-strands (a stable β-barrel backbone) connected in pairs by four peptide loops followed by α-helices. In certain embodiments, the conformation is altered in the hyperloop regions to achieve specific binding. See, e.g., Skerra, FEBS J. 275:2677-83, 2008. No. 6,299,133, which is incorporated by reference.

[0138] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are in the form of designed ankyrin repeat proteins (DARPins). DARPins are a class of non-immunoglobulin proteins that may offer advantages over antibodies in terms of target binding in drug discovery and development. DARPins possess favorable molecular properties, such as small size and high stability, making them highly suitable for in vivo imaging or delivery of toxins or payloads. DARPin-based methods are useful for drug discovery because they can be produced in bacteria at low cost and allow for the rapid generation of many target-specific DARPins. Furthermore, DARPins can be easily produced in a multispecific format, potentially enabling the targeting of effector DARPins to specific organs or the use of a single molecule composed of multiple DARPins to target multiple receptors. See, e.g., Stumpp et al., Curr Opin Drug Discov Devel. 10:153-159, 2007; U.S. Pat. See published application No. 2009 / 0082274 and international application PCT / EP2001 / 10454, which are incorporated by reference.

[0139] Heavy chain dimers, such as antibodies from camelids and sharks, are also included. Camelid and shark antibodies contain a two-chain homodimer pair of a V-like domain and a C-like domain (neither of which has a light chain). The VH region of heavy chain dimeric IgG in camelids does not have hydrophobic interactions with light chains, so the region of the heavy chain that normally contacts the light chain is made up of hydrophilic amino acid residues in camelids. The VH domain of heavy chain dimeric IgG is called a VHH domain. Shark Ig-NAR contains a homodimer of one variable domain (called the V-NAR domain) and five C-like constant domains (C-NAR domains).

[0140] In camelids, the diversity of the antibody repertoire is determined by complementarity-determining regions (CDRs) 1, 2, and 3 within the VH or VHH regions. CDR3 within the VHH regions of camelids is characterized by its relatively long length, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9): 1129). This contrasts with the CDR3 regions of antibodies from many other species. For example, the CDR3 of mouse VH consists of an average of nine amino acids. Libraries of antibody variable regions from Camelidae maintain the in vivo diversity of Camelidae variable regions and can be generated, for example, by the method disclosed in U.S. Patent Application Publication No. 20050037421, published February 17, 2005.

[0141] In certain embodiments, the antibody or antigen-binding fragment thereof is humanized. These embodiments refer to chimeric molecules, typically prepared using recombinant techniques, that have an antigen-binding site derived from an immunoglobulin of a non-human species and the immunoglobulin structure of the molecule based on the remainder of the human immunoglobulin structure and / or sequence. The antigen-binding site may comprise a complete variable domain fused to a constant domain, or it may comprise only the CDRs grafted into the appropriate framework regions of the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but leaves open the possibility of an immune response against the heterologous variable region (LoBuglio et al., PNAS USA 86:4220-4224, 1989). (Queen et al., PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327, 1988). Examples of methods for humanizing antibodies include those described in U.S. Patent No. 7,462,697.

[0142] Other methods focus on not only providing constant regions of human origin but also modifying the variable regions so that they can be reconstructed in a form as close to the human form as possible. Both the heavy and light chain variable regions contain three complementarity-determining regions (CDRs) that vary depending on the epitope of interest and determine the binding ability. These CDRs are known to be adjacent to four framework regions (FRs) that are relatively conserved among species and are said to provide a scaffold for the CDRs. When preparing a non-human antibody against a specific epitope, the variable region can be "reconstructed" or "humanized" by grafting CDRs from a non-human antibody into the FRs of the human antibody to be modified. The application of this method to various antibodies is described in Sato et al., Cancer Res. 53:851-856, 1993; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991; and Gorman et al., PNAS USA. 88:4181-4185, 1991, Tempest et al., Bio / Technology 9:266-271, 1991, Co et al., PNAS USA. 88:2869-2873, 1991, Carter et al., PNAS USA. 89:4285-4289, 1992, and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, a humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibodies). In other embodiments, a humanized antibody has one or more (one, two, three, four, five, or six) CDRs that are altered relative to the original antibody, which are also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody.

[0143] In certain embodiments, the antibody is a "chimeric" antibody. A chimeric antibody is composed of an antigen-binding fragment of an antibody operably linked or fused to a heterologous Fc portion of another antibody. In certain embodiments, the Fc domain or heterologous Fc domain is of human origin. In certain embodiments, the Fc domain or heterologous Fc domain is of murine origin. In other embodiments, the heterologous Fc domain may be derived from an Ig class other than that of the parent antibody, including, for example, IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be composed of CH2 and CH3 domains derived from one or more of the various Ig classes. As described above for humanized antibodies, an antigen-binding fragment of a chimeric antibody may include only one or more of the CDRs of an antibody described herein (e.g., one, two, three, four, five, or six of the CDRs of an antibody described herein), or may include the entire variable domain (VL, VH, or both).

[0144] As used herein, a subject "at risk" of developing a disease or adverse reaction may or may not have detectable disease or symptoms of a disease, and may or may not have detectable disease or symptoms of a disease before using the treatment methods described herein. "At risk" means that a subject has one or more risk factors, which are measurable parameters that correlate with the development of a disease, as described herein and known in the art. A subject with one or more risk factors is more likely to develop a disease or adverse reaction than a subject without one or more risk factors.

[0145] "Biocompatible" means a material or compound that does not generally impair the biological function of a cell or subject and does not cause any degree of unacceptable toxicity, such as an allergic or disease state.

[0146] The term "bond" refers to interactions such as covalent, electrostatic, hydrophobic, and ionic interactions, and / or hydrogen bonding interactions, e.g., salt bridges and water bridges. It refers to a direct association between two molecules by action.

[0147] A "coding sequence" refers to any nucleic acid sequence that provides a code for the polypeptide product of a gene, whereas a "non-coding sequence" refers to any nucleic acid sequence that does not directly provide a code for the polypeptide product of a gene.

[0148] In this disclosure, unless expressly stated otherwise, the terms "comprise," "comprises," and The words "comprising" and "comprising" are understood to mean the inclusion of the stated step or element or steps or elements, but not to the exclusion of any other step or element or steps or elements.

[0149] "Consisting of" means inclusive of and limited to everything that follows the phrase. Thus, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means that any of the listed elements that follow the phrase may be present. "Consisting essentially of" means that the recited elements are limited to elements that include, but are not limited to, other elements that do not inhibit or provide the activity or function detailed in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" does not mean that the recited elements are necessary or essential. However, other elements are optional and may or may not be present depending on whether they substantially affect the activity or action of the listed elements.

[0150] The term "effector function" or "ADCC effector function" in the context of antibodies refers to the ability of an antibody to interact with other arms of the immune system, including, for example, activation of the classical complement pathway or through Fc receptor engagement. The complement-dependent pathway is primarily driven by the interaction of C1q with the C1 complex, which contains the Fc domains of clustered antibodies. 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) with the Fc region of IgG bound to target cells. Fc receptors (FcRs) are key immunoregulatory receptors involved in antibody-mediated (humoral) immune responses through cellular effector functions. Receptors for all immunoglobulin classes have been identified, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM), and FcδR (IgD). At least three classes of receptors for human IgG found on leukocytes include CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb, and FcγRIIc), and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classified as a high-affinity receptor (Kd in the nanomolar range), whereas FcγRII and FcγRIII have low- to intermediate-affinity receptors (Kd in the micromolar range). Upon Fc binding, signaling is triggered, resulting in the secretion of various substances, such as lytic enzymes, perforin, granzymes, and tumor necrosis factor, which promote target cell destruction. The level of ADCC effector function varies depending on the human IgG subtype. Simply put, ADCC effector function is "high" for human IgG1 and human IgG3, and "low" for IgG2 and IgG4, although it also depends on the allotype and the specific FcvR.

[0151] The terms "endotoxin-free" or "substantially endotoxin-free" typically refer to compositions, solvents, and / or containers containing at most trace amounts of endotoxin (e.g., amounts that do not cause clinically adverse physiological effects in a subject), preferably amounts below the limit of detection. Endotoxins are toxins derived from certain microorganisms, e.g., bacteria, typically gram-negative bacteria, although endotoxins can also 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 are the primary pathogenic components of the disease-causing properties of gram-negative bacteria. In humans, small amounts of endotoxin can cause fever, decreased blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[0152] Therefore, in drug production, it is often desirable to remove most or all detectable endotoxins from the drug and / or drug container, since even small amounts can have harmful effects in humans. Temperatures above 300°C are typically required to destroy most endotoxins, and depyrogenation ovens can be used for this purpose. For example, when using primary packaging materials such as syringes or vials, a glass temperature of 250°C for 30 minutes is often sufficient to reduce endotoxin levels by three orders of magnitude. Other methods of endotoxin removal are also contemplated, including, for example, chromatographic and filtration methods described herein and known in the art.

[0153] Endotoxin can be detected using conventional techniques known in the art. For example, the Limulus amebocyte lysate assay, which utilizes horseshoe crab blood, is a highly sensitive assay for detecting the presence of endotoxin. The detection test can detect the coagulation of horseshoe crab cell lysates even at very low levels of LPS due to a powerful enzyme cascade that enhances the reaction. Endotoxin can also be quantified using enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, the endotoxin level may be less than about 0.001 EU / mg, less than 0.005 EU / mg, less than 0.01 EU / mg, less than 0.02 EU / mg, less than 0.03 EU / mg, less than 0.04 EU / mg, less than 0.05 EU / mg, less than 0.06 EU / mg, less than 0.08 EU / mg, less than 0.09 EU / mg, less than 0.1 EU / mg, less than 0.5 EU / mg, less than 1.0 EU / mg, less than 1.5 EU / mg, less than 2 EU / mg, less than 2.5 EU / mg, less than 3 EU / mg, less than 4 EU / mg, less than 5 EU / mg, less than 6 EU / mg, less than 7 EU / mg, less than 8 EU / mg, less than 9 EU / mg, or less than 10 EU / mg of active compound. Generally, 1 ng of lipopolysaccharide (LPS) corresponds to about 1 EU to about 10 EU.

[0154] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope includes a specific region of an antigen that binds to an antibody. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups, and in certain embodiments may have unique three-dimensional structural characteristics and / or unique charge characteristics. An epitope may be continuous or discontinuous, corresponding to the primary structure of the antigen, e.g., an NRP2 polypeptide. In certain embodiments, an epitope comprises, consists of, or consists essentially of about, at least about, or at most about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids (i.e., a linear epitope) or non-contiguous amino acids (i.e., a conformational epitope) of a reference sequence (see, e.g., Table N1) or a target molecule described herein.

[0155] An "epitope" includes a portion of an antigen or other macromolecule capable of forming a binding interactor with the variable region binding pocket of a binding protein. Such a binding interactor can be manifested as making intermolecular contacts 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., a "continuous" sequence) or comprised of discontinuous amino acid sequences (i.e., a "conformational" or "discontinuous" sequence). Because a binding protein can recognize one or more amino acid sequences, an epitope can be characterized by two or more unique amino acid sequences. Epitopes recognized by binding proteins can be identified using 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 recognizable by a binding protein of a denatured or proteolytic polypeptide. In the structure of unmodified polypeptide, the amino acid sequence that is not exposed or only partially exposed may be a cryptic epitope.If an epitope is not exposed or only partially exposed, it is considered to be buried inside the polypeptide.Candidates for cryptic epitopes can be identified, for example, by examining the three-dimensional structure of unmodified polypeptide.

[0156] "50% effective concentration" or "EC 50 The term "EC" refers to the concentration of an agent (e.g., an antibody) described herein that induces a response of 50% of the maximum from baseline after a specified exposure time. Thus, the EC of a graded dose-response curve 50 EC represents the compound concentration at which 50% of the maximum effect is obtained. 50 The EC also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. 90」 "EC" means the concentration of a drug or composition that provides 90% of its maximum effect. 90" is "EC 50 and the Hill slope, or can be calculated directly from the data using routine knowledge in the art. In one embodiment, the EC 50 is less than about 0.01 nM, less than 0.05 nM, less than 0.1 nM, less than 0.2 nM, less than 0.3 nM, less than 0.4 nM, less than 0.5 nM, less than 0.6 nM, less than 0.7 nM, less than 0.8 nM, less than 0.9 nM, less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM , less than 11 nM, less than 12 nM, less than 13 nM, less than 14 nM, less than 15 nM, less than 16 nM, less than 17 nM, less than 18 nM, less than 19 nM, less than 20 nM, less than 25 nM, less than 30 nM, less than 40 nM, less than 50 nM, less than 60 nM, less than 70 nM, less than 80 nM, less than 90 nM, less than 100 nM, 200, or less than 500 nM. 50 It has a value.

[0157] "Immune response" refers to an immunological response derived from the immune system, including responses from the cellular immune system, humoral immune system, innate immune system, and adaptive immune system. Examples of cellular immune cells include 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 function, cytokine release, phagocytosis, efferocytosis, translocation, trafficking, proliferation, differentiation, activation, suppression, cell-cell interaction, apoptosis, and the like. Humoral responses include, for example, IgG responses, IgM responses, IgA responses, IgE responses, and their corresponding effector functions.

[0158] The "half-life" of a drug, such as an antibody, can refer to the time required for the pharmacological, physiological, or other activity of the drug to decrease by half relative to the activity at the time of administration to the serum or tissues of an organism, or relative to the activity at any other defined time. "Half-life" can also refer to the time required for the amount or concentration of the drug to decrease by half relative to the initial amount administered to the serum or tissues of an organism, i.e., the amount or concentration at the time of administration to the serum or tissues of an organism, or relative to any other defined time. Half-life can be measured in serum and / or in any one or more selected tissues.

[0159] The terms "modulate" and "alter" include "increase," "enhance," or "stimulate," and "decrease" or "reduce," generally by a statistically significant or physiologically significant amount or degree, relative to a control. An "increase," "stimulate," or "enhance" amount is typically a "statistically significant" amount and may include a 1.1-fold, 1.2-fold, 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 (e.g., 500-fold, 1000-fold) increase (including all integers and ranges therebetween, e.g., 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, etc.) over the amount produced in the absence of the composition (e.g., in the absence of an agent) or over the amount produced by a control composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount and may include 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., without the drug) or the amount produced by a control composition. Examples of comparative values and "statistically significant" amounts are described herein.

[0160] The term "migratory cell" refers to a cell that can move from one location to another in response to a stimulus. Examples of migratory cells include monocytes, natural killer (NK) cells, dendritic cells (immature or mature), myeloid cells, plasmacytoid (also called lymphoid) cells, and subsets of dendritic cells including Langerhans cells, macrophages such as histiocytes, tissue-resident macrophages such as Kupffer cells, microglia of the central nervous system, alveolar macrophages, and peritoneal macrophages, and M0 macrophages, M1 macrophages, Mox macrophages, M2a macrophages, M2b macrophages, and the like. Examples of migratory cells include macrophage subtypes such as M2c macrophages, neutrophils, eosinophils, mast cells, basophils, B cells such as plasma B cells, memory B cells, B-1 cells, and B-2 cells, immune cells such as CD45RO cells (naive T cells), CD45RA cells (memory T cells), CD4 helper T cells such as Th1 cells, Th2 cells, and Tr1 / Th3 cells, CD8 cytotoxic T cells, regulatory T cells, γδ T cells, and thymocytes. Further 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 "modulating cell migration" refers to modulating the movement of any such migratory cells.

[0161] The terms "polypeptide," "protein," and "peptide" are used interchangeably and refer to a polymer of amino acids, not limited to any particular length. The term "enzyme" includes catalytic polypeptides or catalytic proteins. These terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The terms "polypeptide" or "protein" refer to one or more amino acid chains, each chain comprising amino acids covalently linked by peptide bonds. The polypeptide or protein may comprise multiple chains noncovalently and / or covalently linked to each other by peptide bonds, but these chains have the sequence of a native protein, i.e., a protein produced in nature, particularly in non-recombinant cells, or by genetically engineered or recombinant cells. The polypeptide or protein may also include molecules having the amino acid sequence of a native protein or molecules with one or more amino acid deletions, additions, and / or substitutions of the native sequence. In certain embodiments, a polypeptide is a "recombinant" polypeptide produced by a recombinant cell that contains one or more recombinant DNA molecules, which are generally composed of heterologous polynucleotide sequences or combinations of polynucleotide sequences not found in the cell.

[0162] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA. These terms refer to polymerized forms of nucleotides at least 10 bases in length, i.e., ribonucleotides, deoxyribonucleotides, or modified forms of either species of nucleotide. The terms include single- and double-stranded forms of DNA. The terms "isolated DNA," "isolated polynucleotide," and "isolated nucleic acid" refer to molecules isolated from total genomic DNA of a particular species. Thus, an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences but has been substantially isolated or purified free from total genomic DNA of the species from which the DNA segment was obtained. Non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode a polypeptide are also included. Recombinant vectors are also included, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, and the like.

[0163] Although not required, additional coding or non-coding sequences may be included in the polynucleotides described herein, and the polynucleotides may be linked to other molecules and / or support materials. Thus, a polynucleotide or expressible polynucleotide, regardless of the length of the coding sequence, may be associated with other sequences, for example, expression control sequences.

[0164] "Expression control sequences" include nucleic acid or corresponding 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, and subcellular localization signals. These can affect the transcription or translation, or subcellular or intracellular distribution of coding sequences in host cells. Examples of expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0165] A "promoter" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. A promoter sequence, as used herein, is one that is adjacent at its 3' end to the transcription initiation site and extends upstream (5' direction) to contain the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. A transcription initiation site (conveniently defined by mapping with nuclease S1) can be found within the promoter sequence and within protein binding domains (consensus sequences) that mediate RNA polymerase binding. Eukaryotic promoters often, but not always, contain "TATA" and "CAT" boxes. Prokaryotic promoters contain a Shine-Dalgarno sequence in addition to the consensus sequences at base pairs -10 and -35.

[0166] A large number of promoters are known in the art, including constitutive, inducible, and repressible promoters from a variety of sources. Representative sources include, for example, viral, mammalian, insect, plant, yeast, and bacterial cell types. Suitable promoters from these sources are readily available or can be synthesized based on sequences published online or obtained from depositories such as the ATCC and other commercial or private sources. Promoters can be unidirectional (i.e., transcription can be initiated in one direction) or bidirectional (i.e., transcription can be initiated in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No. et al., Proc. Natl. Acad. Sci. (1996) 93 (8): 3346-3351), the T-REx™ system (Invitrogen, Carlsbad, CA), LacSwitch® (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-induced These include promoters known in the art, such as transducible recombinase systems (Indra et al. Nuc. Acid. Res. (1999) 27(22):4324-4327, Nuc. Acid. Res. (2000) 28(23):e99, U.S. Patent No. 7,112,715, and Kramer & Fussenegger, Methods Mol. Biol. (2005) 308:123-144), or any promoter suitable for expression in the desired cells.

[0167] An "expressible polynucleotide" includes a cDNA, RNA, mRNA, or other polynucleotide that contains at least one coding sequence and, optionally, at least one expression control sequence (e.g., a transcriptional and / or translational regulatory element) and that is capable of expressing an encoded polypeptide when introduced into a cell, e.g., a cell of a subject.

[0168] Various viral vectors that can be used to deliver expressible polynucleotides include adenovirus vectors, herpesvirus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retrovirus vectors. In some instances, retrovirus vectors are derivatives of murine or avian retroviruses or lentivirus vectors. Examples of retroviral vectors that can insert a single foreign gene include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). Other retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate genes for selectable markers, allowing transduced cells to be identified and generated. Inserting a polypeptide sequence of interest into a viral vector in conjunction with another gene encoding a ligand for a specific target cell receptor can, for example, make the vector target-specific. For example, retroviral vectors can be made target-specific by inserting a polynucleotide encoding a protein.Specific targeting can be achieved by using an antibody that targets retroviral vectors.Those skilled in the art can easily identify or identify the specific polynucleotide sequence that is inserted into retroviral genome to enable target-specific delivery of retroviral vectors without undue experimentation.

[0169] In certain embodiments, the expressible polynucleotide is a modified RNA polynucleotide or a modified mRNA polynucleotide, e.g., a non-natural RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide contains one or more modified or non-natural bases, e.g., a nucleotide base other than adenine (A), guanine (G), cytosine (C), thymine (T), and / or uracil (U). In some embodiments, the modified mRNA contains one or more modified or non-natural internucleotide linkages. Expressible RNA polynucleotides that deliver encoded polypeptide formulations are described, for example, in Kormann et al., Nat Biotechnol. 29:154-7, 2011, and U.S. Patent Application Publication Nos. 2015 / 0111248, 2014 / 0243399, 2014 / 0147454, and 2013 / 0245104, which are incorporated by reference in their entireties.

[0170] As used herein, the term "isolated" polypeptide or protein means that the subject protein (1) is free from at least some other proteins that typically naturally coexist with the protein, (2) is essentially free from other proteins from the same source, e.g., from the same species, (3) is expressed by cells from a different species, (4) is separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials that naturally coexist with the protein, (5) is not associated (by covalent or noncovalent interactions) with portions of proteins that naturally associate with the "isolated protein," (6) is operably associated (by covalent or noncovalent interactions) with polypeptides that are not naturally associated with the protein, or (7) is not naturally occurring. Such isolated proteins may be encoded using genomic DNA, cDNA, mRNA, or other RNA, or may be synthetic, 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, etc.).

[0171] In certain embodiments, a range of "purity" of a given agent (e.g., a polypeptide such as an antibody) in a composition can be defined. For example, a particular composition may contain an agent, such as a polypeptide agent, that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure on a protein basis or weight / weight basis, or any percentage therebetween, including all decimal points and ranges therebetween. Methods for this measurement include, but are not limited to, 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.

[0172] "Lipid nanoparticle" or "solid lipid nanoparticle" refers to one or more spherical nanoparticles having an average diameter of about 10 to 1000 nanometers and comprising a solid lipid core matrix capable of dissolving lipophilic molecules. The lipid core is stabilized by a surfactant (e.g., an emulsifier) and may comprise one or more of triglycerides (e.g., tristearin), diglycerides (e.g., glycerol behenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate), and combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014, U.S. Patent Nos. 6,217,912, 6,881,421, 7,402,573, 7,404,969, 7,550,441, 7,727,969, 8,003,621, 8,691,750, 8,871,509, 9,017,726, 9,173,853, 9,220,779, 9,227,917, and 9,278,130, which are incorporated by reference in their entireties. Certain compositions described herein are formulated with one or more lipid nanoparticles.

[0173] The term "neuropilin-2-related disease" or "NRP2-related disease" refers to diseases and conditions in which the activity, expression, and / or spatial distribution of NRP2 influences the pathophysiology of the disease or condition. In some examples, an NRP2-related disease is alleviated by using an anti-NRP2 antibody of the present disclosure to alter the interaction of NRP2 with at least one NRP2 ligand, thereby affecting NRP2 activity, signaling, expression, and / or spatial distribution. Examples of NRP2-related diseases and conditions include, but are not limited to, cancer and cancer-related diseases or conditions, including cancer cell proliferation, cancer development, cancer migration, cancer cell adhesion, invasion, and metastasis. Diseases associated with inflammation and autoimmunity and related inflammatory diseases are also included, including diseases associated with inappropriate immune cell activation or migration, such as graft-versus-host disease (GVHD). Further examples include diseases related to lymphangiogenesis, lymphangiogenesis, and lymphatic damage, such as edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability. Diseases related to infectious diseases, such as latent infection, and diseases related to allergic diseases / disorders and allergic responses, such as chronic obstructive pulmonary disease (COPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, systemic lupus erythematosus, rheumatoid arthritis, inflammasome-related diseases, and skin-related neutrophil-mediated diseases such as pyoderma gangrenosum. Further examples include diseases related to granulomatous inflammatory diseases, such as sarcoidosis and granulomas, and diseases related to fibrosis, such as fibrotic diseases, fibrosis, endothelial-mesenchymal transition (EMT), and wound healing. Also included are diseases associated with inappropriate smooth muscle contractility and vascular smooth muscle cell migration and / or adhesion, and diseases associated with inappropriate autophagy, phagocytosis, and efferocytosis. Diseases associated with inappropriate migratory cell motility, as described herein, are also included. Further examples include neurological diseases, such as diseases associated with peripheral nervous system remodeling and pain sensation. Diseases associated with bone development and / or bone remodeling are also included. Generally, the term "inappropriate" refers to an activity or property that is associated with or causes a pathological or disease state.

[0174] The term "reference sequence" typically refers to a nucleic acid coding sequence or amino acid sequence that is compared to another sequence. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those sequences indicated by name and those sequences indicated in tables and sequence listings.

[0175] Certain embodiments include biologically active "variants" and "fragments" of the polypeptides (e.g., antibodies) and encoding polynucleotides described herein. "Variant" refers to a variant that contains one or more substitutions, additions, deletions, and / or insertions relative to a reference polypeptide or polynucleotide (see, e.g., Tables and Sequence Listing). A variant polypeptide or polynucleotide comprises an amino acid or nucleotide sequence that has 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, similarity, or homology to a reference sequence described herein, and substantially maintains the activity of the reference sequence. Also included are sequences that consist of a reference sequence or that differ from the reference sequence by the addition, deletion, insertion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more amino acids or nucleotides, while substantially maintaining the activity of the reference sequence. In certain embodiments, the additions or deletions include C- and / or N-terminal additions and / or deletions.

[0176] The term "sequence identity," as used herein, including examples such as "50% identical sequence," refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison region. Thus, "percent sequence identity" can be calculated by comparing two optimally aligned sequences over a comparison region, calculating the number of positions with identical nucleic acid groups (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) in both sequences to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison region (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison region can be performed using a computerized algorithm (Genetics Computer Group, Madison, Wisconsin, USA). This can be done by using the Wisconsin Genetics software package (GAP, BESTFIT, FASTA, and TFASTA in release 7.0) from Wisconsin Genetics (Wisconsin Genetics, Inc., 575 Science Drive), or by verification and optimal alignment (i.e., the one that gives the highest percentage of homology in the region of comparison) using any of the various methods selected. Also, see the BLAST family of programs, e.g., as disclosed in Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0177] The term "solubility" refers to the ability of a drug (e.g., an antibody) provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed in terms of concentration, such as the mass of solute per unit volume of solvent (e.g., grams of solute per kg of solvent, grams per dL (100 mL), mg / mL, etc.), molar concentration, molality, mole fraction, or other similar concentration descriptions. The maximum equilibrium amount of solute that can be dissolved per unit volume of solvent is the solubility of the solute in the solvent under specific conditions of temperature, pressure, pH, and solvent properties. In certain embodiments, solubility is measured at physiological pH or other pHs, such as pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In certain embodiments, solubility is measured in water or a physiological buffer solution (with or without NaPO4), such as PBS or NaCl. In certain embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and a relatively high salt concentration (e.g., 500 mM NaCl and 10 mM NaPO). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature may 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 drug 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, or 10 mg / ml at room temperature or 37°C. g / ml, 10mg / ml, 11mg / ml, 12mg / ml, 13mg / ml, 14mg / ml, 15mg / ml, 16mg / ml, 17mg / ml, 18mg / ml, 19mg / ml, 2 0mg / ml, 25mg / ml, 30mg / ml, 40mg / ml, 50mg / ml, 60mg / ml, 70mg / ml, 80mg / ml, 90mg / ml, or 100mg / ml.

[0178] A "subject" or "subject in need thereof" or a "patient" or "patient in need thereof" includes mammalian subjects, such as human subjects.

[0179] "Substantial" or "essentially" means nearly wholly or nearly completely, for example, 95%, 96%, 97%, 98%, 99% or more of a specified amount.

[0180] "Statistically significant" means that the results are unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. A commonly used measure of significance is the p-value, which is the frequency or probability that the observed event will occur if the null hypothesis is true. If the p-value obtained is less than the significance level, the null hypothesis is rejected. In a simple example, the significance level is defined as a p-value of 0.05 or less.

[0181] "Therapeutic response" means an improvement in symptoms (whether sustained or not) following administration of one or more therapeutic agents.

[0182] The terms "therapeutically effective amount," "therapeutic dose," "prophylactically effective amount," or "diagnostically effective amount," as used herein, refer to the amount of an agent (e.g., an anti-NRP2 antibody, an immunotherapeutic agent) required to elicit a desired biological response following administration.

[0183] "Treatment" of a subject (e.g., a mammal such as a human) or cell, as used herein, refers to any type of intervention used to alter the natural course of the individual or cell. Treatment includes, but is not limited to, the administration of pharmaceutical compositions, and can be administered prophylactically or after a pathological event or exposure to a pathogen. Also included are "prophylactic" treatments, which can reduce the rate of progression of the disease or condition being treated, delay the onset of the disease or condition, or induce a reduction in the severity of onset. "Treatment" or "prevention" does not necessarily imply complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

[0184] The term "wild-type" refers to the gene or gene product (e.g., polypeptide) that is most commonly observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form of the gene.

[0185] Each embodiment herein applies mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0186] Anti-NRP2 antibody 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., inhibits) binding of a human NRP2 polypeptide to at least one NRP2 ligand, such as a human histidyl-tRNA synthetase (HRS) polypeptide or another NRP2 ligand.

[0187] Neuropilin-2 is a cell surface receptor protein that regulates a wide range of cellular functions through its role as an important cell surface receptor and co-receptor for various ligands (see, e.g., Guo and Vander Kooi, J. Cell. Biol. 290 No. 49: 29120-29126. 2015). For example, neuropilin-2 mediates the regulation of various cellular functions during epithelial-mesenchymal transition (EMT), e.g., It functions 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 endothelial-mesenchymal transition (endo-EMT) in fibroblasts, myofibroblasts, and endothelial cells, promoting fibrogenesis (see, e.g., Pardali et al., Int. J. Mol. Sci. 18:2157, 2017).

[0188] Neuropilin-2 expression promotes lymphangiogenesis (see, e.g., Doci et al., Cancer Res. 75:2937-2948, 2015), and single nucleotide polymorphisms (SNPs) in NRP2 promote lymphangiogenesis. NRP2 has been associated with edema (see, e.g., Miaskowski et al., PLoS ONE 8(4) e60164, 2013). NRP2 also regulates smooth muscle contractility (see, e.g., Bielenberg et al., Amer. J. Path. 181:548-559, 2012), and, for example, in cancer, regulates autophagy (see, e.g., Stanton et al., Cancer Res. 73:160-171, 2013), contributing to tumor initiation, engraftment, 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). ), regulating immune cell activation and migration (see, e.g., Mendes-da-Cruz et al., PLoS ONE 9(7) e103405, 2014). Neuropilins also play a role in tumor initiation, growth, and metastasis. , and is a multifunctional coreceptor involved in immunity (see, e.g., Prud'homme et al., Oncotarget 3:921-939, 2012).

[0189] Neuropilin-2 is expressed in various cells of the immune system, including lymphoid cells such as B cells and T cells, and myeloid cells such as basophils, eosinophils, monocytes, dendritic cells, neutrophils, and macrophages (including tissue-specific macrophages, e.g., alveolar macrophages). Neuropilin-2 is expressed in endothelial and epithelial cells of the lung and other tissues, as well as in muscle cells (see, e.g., Bielenberg et al., Amer. J. Path. 181:548-559, 2012; Aung, et al., PLoS ONE 11(2) e0147358, 2016; Schellenburg et al., Mol. Imm 90:239-244, 2017; and Wild et al., Int. J. Exp. Path. 93:81-103, 2012).

[0190] Neuropilin-2 also plays a key role in endosomal maturation, for example, by regulating the maturation of late endosomes, and is an important aspect of phagocytosis, which contributes to the clearance of infectious agents, and efferocytosis, which contributes to the clearance of apoptotic cells (e.g., Diaz-Vera et al., J. Cell. Sci. 130:697-711, 2017 and Dutta et al., J. Cell. Sci. 130:697-711, 2017). et al., Cancer Res. 76:418-428, 2016).

[0191] Neuropilin-2 is known to be an important factor in the pathophysiology of many diseases (e.g., "NRP2-associated diseases") and interacts with a wide range of soluble ligands. Examples of soluble ligands include semaphorin 3F, VEGF-C, VEGF-D, and TGF-beta (see, e.g., Tables N2 and N3), as well as a series of cellular receptors and cofactors (see, e.g., Figures 1A-1B and 2). Furthermore, NRP2 is polysialylated in dendritic cells and strongly interacts with the chemokine CCL21, mediating immune cell migration. In this regard, single nucleotide polymorphisms associated with ILD and RA have been reported (see, e.g., Rey-Gallardo et al., 2014). (See, e.g., Parker et al., Structure and Biology 20:1139-1146, 2010; Stahl et al., Nat. Genet. 42:508-514, 2013; and Miller et al., Arthritis Rheum. 65:3239-3247). Also, a soluble circulating form of NRP-2 is known (see, e.g., Parker et al., Structure and Biology 20:1139-1146, 2010; Stahl et al., Nat. Genet. 42:508-514, 2013; and Miller et al., Arthritis Rheum. 65:3239-3247). 23(4) 677-687, 2015), and our research has shown that HRS polypeptides are secreted in the circulatory system. It has been confirmed that circulating complexes of NRP2 polypeptides with NRP-2 polypeptides exist. Thus, given the major role that NRP2 plays in the pathophysiology of a wide range of diseases, it is clear that the interaction between NRP2 and an NRP2 ligand (e.g., an NRP2 ligand selected from Tables N2 and N3) and modulation of this interaction with antibodies to NRP2 to selectively alter the corresponding biological activity offer broad possibilities for the treatment of diseases, including NRP2-related diseases.

[0192] NRP2 is a single transmembrane receptor with a prominent extracellular region containing two CUB domains (A1 / A2 complex domains), two factor V / factor VIII homology domains (B1 / B2 complex domains), and a MAM domain (C domain) (see Figures 1A-1B). The A1A2 complex domain interacts with the Sema domain of semaphorins, and the B1 domain interacts with the PSI and Ig-like domains of semaphorins. NRP2 has a relatively high affinity for SEMA3F and SEMA3G, whereas SEMA3A, SEMA3B, and SEMA3E preferentially interact with NRP1. Both NRP1 and NRP2 have similar affinity for SEMA3C. The B1B2 complex domain interacts with multiple growth factors, including heparin-binding domains (e.g., VEGF-C and VEGF-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) beta (see, e.g., Prud'homme et al., Oncotarget. 3:921-939, 2012). NRP2 also interacts with various growth factor-specific receptors, and these interactions occur regardless of whether they are bound to SEMA. In this context, integrins and growth factor receptors, such as VEGF receptor, TGF beta receptor, c-Met, EGFR, FGFR, and PDGFR, have been shown to interact with NRPs, collectively increasing the affinity of their respective ligands for the receptor and modulating downstream signaling. The C domain (Mam) is thought to be dispensable for ligand binding but essential for signal transduction.

[0193] Therefore, anti-NRP2 antibodies that bind to the A1 and / or A2 domains of NRP2 may be able to selectively modulate semaphorin binding. Similarly, anti-NRP2 antibodies that bind to the B1 domain may be able to modulate semaphorin and VEGF and growth factor binding, and anti-NRP2 antibodies that bind to the B2 domain may be able to selectively modulate VEGF and growth factor binding. Antibodies that bind to the C domain are not expected to directly affect NRP2 ligand binding but may be able to modulate NRP2 downstream signaling. Specific anti-NRP2 antibodies may have even more diverse functional effects based on their binding mode and as a result of steric effects that may indirectly affect ligand binding.

[0194] NRP2 can form homodimers and heterodimers and is heavily glycosylated. NRP2 exists in various splice variants ranging in length from approximately 551 to 926 amino acids. The two major NRP2 variants are classified as NRP2a and NRP2b. These differ in their intracellular C-terminal regions (Figures 1A-1B). NRP2a contains a 42-amino acid C-terminal domain with a C-terminal SEA amino acid sequence and a PDZ-binding domain. In contrast, NRP2b contains a 46-amino acid C-terminal domain that shares approximately 11% of the intracellular and transmembrane sequences of NRP2a. Additional splicing can occur between the MAM and transmembrane domains, adding an additional five amino acids (GENFK) to either the NRP2a or NRP2b forms. These variants are named according to the number of amino acids added by alternative splicing. Thus, two additional variants of NRP2 are designated NRP2a(17) and NRP2a(22), and two additional transmembrane variants of NRP2b are designated NRP2b(0) and NRP2b(5). In addition, a soluble form, designated sNRP2b, can also be formed. Examples of NRP2 polypeptide sequences are provided in Table N1 below. [Table N1-1]

Table N1-2

Table N1-3

Table N1-4

Table N1-5

Table N1-6

Table N1-7

[0195] In certain embodiments, at least one antibody or antigen-binding fragment thereof of the present invention specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1. In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to a human NRP2 polypeptide at a concentration of about 10 pM to about 500 pM or about 10 pM to 50 nM, or at about, at least about, or at most 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, 210 pM, 220 pM, 230 pM, 240 pM, 250 pM, 260 pM, 270 pM, 280 pM, 290 pM, 300 pM, 310 pM, 320 pM, 330 pM, 340 pM, 350 pM, 360 pM, 370 pM, 380 pM, 390 pM, 400 pM, 410 pM, 420 pM, 430 pM, 440 pM, 450 pM, 460 pM, 470 pM, 480 pM, 490 pM, 500 pM, 510 pM, 520 pM, 530 pM, 540 pM, 550 pM, 560 pM, 570 pM, 580 pM, 590 pM, 600 or optionally binds with an affinity 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 10 nM, or about 10 pM to about 25 nM. pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or It binds with an affinity 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 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.

[0196] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within at least one neuropilin domain. Examples of neuropilin domains include the neuropilin A1 domain (SEQ ID NO: 10), the neuropilin A2 domain (SEQ ID NO: 11), the neuropilin B1 domain (SEQ ID NO: 12), the neuropilin B2 domain (SEQ ID NO: 13), the neuropilin C domain (SEQ ID NO: 14), the neuropilin A1 / A2 combined domain (SEQ ID NO: 15), the neuropilin B1 / B2 combined domain (SEQ ID NO: 20), the neuropilin A2 / B1 combined domain (SEQ ID NO: 16), the neuropilin B2 / C combined domain (SEQ ID NO: 17), the neuropilin A1 / A2 combined domain (SEQ ID NO: 18), the neuropilin B1 / B2 combined domain (SEQ ID NO: 19), the neuropilin A2 / B1 combined domain (SEQ ID NO: 20), the neuropilin B2 / C combined domain (SEQ ID NO: 21), the neuropilin B2 / C combined domain (SEQ ID NO: 22), the neuropilin A1 / A2 combined domain (SEQ ID NO: 23), the neuropilin B1 / B2 combined domain (SEQ ID NO: 24), the neuropilin A2 / B1 combined domain (SEQ ID NO: 25), the neuropilin B2 / C combined domain (SEQ ID NO: 26), the neuropilin B2 / C combined domain (SEQ ID NO: 27), the neuropilin A1 / A2 combined domain (SEQ ID NO: 28), the neuropilin A2 / B1 combined domain (SEQ ID NO: 29), the neuropilin B2 / C combined domain (SEQ ID NO: 30), the neuropilin B1 / B2 combined domain (SEQ ID NO: 31), the neuropilin B2 / C combined domain (SEQ ID Neuropilin domains include one or more of the neuropilin A1 / A2 / B1 / B2 composite domain (SEQ ID NO: 19), neuropilin A2 / B1 / B2 / C composite domain (SEQ ID NO: 121), neuropilin A1 / A2 / B1 composite domain (SEQ ID NO: 17), neuropilin A1 / A2 / B1 / B2 composite domain (SEQ ID NO: 18), neuropilin A1 / A2 / B1 / B2 / C composite domain (SEQ ID NO: 122), and neuropilin B1 / B2 / C composite domain (SEQ ID NO: 90). In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin B1 domain, neuropilin B2 domain, and / or neuropilin B1 / B2 composite domain (see Table N1).In certain embodiments, the antibody or antigen-binding fragment thereof has a cytotoxicity of about 10 pM to about 500 pM or about 10 pM to 50 nM to at least one domain (or at least one epitope thereof), or about, at least about, or at most 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, 210 pM, 220 pM, 230 pM, 240 pM, 250 pM, 260 pM, 270 pM, 280 pM, 290 pM, 300 pM, 310 pM, 320 pM, 330 pM, 340 pM, 350 pM, 360 pM, 370 pM, 380 pM, 390 pM, 400 pM, 410 pM, 420 pM, 430 pM, 440 pM, 450 pM, 460 pM, 470 pM, 480 pM, 490 pM, 500 pM, 510 pM, 520 pM, 530 pM, 540 pM, 550 pM, 560 pM, 570 pM, 580 pM, 590 pM, 600 pM, 610 pM, 620 pM, 630 pM, 640 pM 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, 1 nM, 10 nM, 25 nM, or 50 nM, or optionally binds with an affinity 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 About 100 pM, about 10 pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM to about 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM or about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

[0197] In some embodiments, 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 complex domain, or an adjacent linker region, e.g., near the following residues (neuropilin A1 domain): residues 20-148, 30-141, 40-141, 50-141, 60-141, 70-141, 80-141, 90-141, 100-141, 110-141, 120-141, 130-141, 140-141, 150-141, 160-141, 170-141, 180-141, 190-141, 200-141, 210-141, 220-141, 230-141, 240-141, 250-141, 260-141, 270-141, 280-141, 290-141, 300-141, 310-141, 320-141, 330-141, 340-141, 350-141, 360-141, 370-141, 380-141, 390-141, 400-141, 410-141, 420-141, 430-141, 440-141, 450-141, 460-141, 470-141, 480-141, 490-141, 500 and residues 142-280, 150-265, 160-265, 170-265, 180-265, 190-265, 200-265, 210-265, 220-265, 230-265, 240-265, 250-265, 260-265, 270-265, 280-285, 290-295, 300-300, 310-310, 320-325, 330-330, 340-345, 350-350, 360-365, 370-375, 380-385, 390-400, 410-410, 420-425, 430-430, 440-445, 450-450, 460-465, 470-475, 480-485, 490-500, 510-510, 520-525, 530-530, 540-545, 550-550, 560-565, 570-575, 580-585, 590-600, 600-600, 610-610, 620-625, 630-630, 640-645, 650-650, 660-665, 670-675, 680-685, 690-700, 710-710, 720-725, 730 65, 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, 2 20-250, 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, 236-24 7, 227-246, 227-245, 227-244, 227-243, 227-242, 227-241, 227-240, 227-239, 227-238, 235-240, 236-239, 236-238, or residue 237, or residues 20-280, 30-280, 40-280, 50-280, 60-280, 70-280, 80-280, 90-280, 100-280, 110-280, 120-280, 130-280 as defined in SEQ ID NO: 1 (FL human NRP2), for example, around the following residues (composite A1A2 domain):140~280, 150~280, 160~280, 170~280, 180~280, 190~280, 200~280, 210~280, 220~280, 230~280, 240~280, 260~280, 270~280, 20~270, 20~260, 20~250, 20~240, 20~230, 2 Combine between 0~220, 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.

[0198] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin B1 domain (SEQ ID NO: 12), the neuropilin B2 domain (SEQ ID NO: 13), and / or the neuropilin B1 / B2 complex domain (SEQ ID NO: 20), or within the adjacent linker region, e.g., near the following residues (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, 430-436, 440-446, 450-450, 460-460, 470-470, 480-480, 490-490, 500-510, 510-510, 520-520, 530-530, 540-540, 550-550, 560-560, 570-570, 580-580, 590-600, 600-600, 610-610, 620-620, 630-630, 640-640, 650-650, 660-660, 670-670, 680-680, 690-700, 710-710, 720- 26, 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, or about the following residues (neuropilin B2 domain) as defined by SEQ ID NO: 1 (FL human NRP2). Residues 438-591, 450-591, 460-591, 470-591, 480-591, 490-591, 500-591, 510-591, 520-591, 530-591, 540-591, 550-591, 560-591, 570-591, 580-591, 438-590, 438-580, 438-570, 438-560, 438-550, 438-540, 438-530, 438-520, 438-510, 438-500, 438-490, 438-480, 438-470, 438-460, 438-450, or the following residues: residues 266-591, 276-591, 286-591, 296-591, 306-591, 316-591, 326-591, 336-591, 346-591, 356-591, 366-591, 376-591, 386-591, 396-591, 406-591, 416-591, 426-591, 436-591, 446-591, 456-591, 466-591, 476-591, 486-591, 498-591, 508-591 as defined in SEQ ID NO: 1 (FL human NRP2), which are near the base (neuropilin B1 / B2 complex domain);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~ Combine with 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.

[0199] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin A2 / B1 complex domain and / or the neuropilin B2C complex domain, or within the adjacent linker region, e.g., near the following residues (neuropilin A2B1 complex domain): residues 149-437, 159-426, 169-426, 179-426, 189-426, 199-426, 209-426, 219-426, 229-426, 239-437, 240-437, 250-437, 260-437, 270-437, 280-437, 290-437, 300-437, 310-437, 320-437, 330-437, 340-437, 350-437, 360-437, 370-437, 380-437, 390-437, 400-437, 410-437, 420-437, 437-437, 440-437, 450-437, 460-437, 470-437, 480-437, 490-437, 500-500, 510-510, 520-520, 530-537, 540-540, 550-550, 560-560, 570-570, 580-580, 590-590, 600-600, 610-61 26, 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, 3 99~426, 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, 149~31 6, 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, or residues 438-794, 448-794, 458-794, 468-794, 478-794, 487-794, 497-794 as defined in SEQ ID NO: 1 (FL human NRP2), e.g., near the following residues (neuropilin B2C complex domain): , 507~794, 517~794, 527~794, 537~794, 547~794, 557~794, 567~794, 587~794, 597~794, 607~794, 617~794, 627~794, 637~794, 647~794, 657~794, 667 ~794, 677~794, 687~794, 697~794, 707~794, 717~794, 727~794, 737~794, 747~794, 757~794, 767~794, 777~794, 787~794, 427~794, 438~784, 438~774,438~764, 438~754, 438~744, 438~734, 438~728, 438~714, 438~704, 438~694, 438~684, 438~674, 438~664, 438~654, 438~644, 438~634, 438~624, 438~614, 438~60 4, 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.

[0200] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within a neuropilin C domain or an adjacent linker region, e.g., near residues 591-794, 600-794, 610-794, 620-794, 630-794, 640-794, 650-794, 660-794, 670-794, 680-794, 690-794, 700-794, 710-794, 720-794, 730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 800-800, 810-810, 820-820, 830-830, 840-840, 850-850, 860-850, 870-850, 880-850, 890-900, 900-900, 910-910, 920-930, 930-940, 940-950, 950-960, 960-970, 970-980, 980-990, 990-1000, 1000-1000, 1010-1010, 1020-1020, 1030-1030, 1040-1040, 1050-1050, 1060-1060, 1070-1070, 1080- Combine with 794, 730-794, 740-794, 750-794, 760-794, 770-794, 780-794, 790-794, 591-790, 591-780, 591-770, 591-760, 591-750, 591-740, 591-730, 591-720, 591-710, 591-700, 591-690, 591-680, 591-670, 591-660, 591-650, 591-640, 591-630, 591-620, 591-610, or 591-600.

[0201] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope within the neuropilin B1 / B2 / C complex domain or the adjacent linker region, e.g., near 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, 496-806, 506-794, 516-794, 526-794, 536-794, 546-794, 556-794, 566-794, 576-794, 587-806, 596-806, 606-794, 616-794, 626-794, 636-794, 646-794, 656-794, 666-794, 676-794, 687-794, 696-806, 706-794, 716-794, 726-794, 736-794, 746-794, 756-794, 766-794, 776-79 794, 396~794, 406~794, 416~794, 426~794, 436~794, 446~794, 456~794, 466~794, 476~794, 486~794, 496~794, 506~794, 516~794, 526~794, 536~794, 546~794, 556~794, 566~794, 576~794, 586~794, 596~794, 606~794, 616~794, 626~794, 636~794, 646~794, 656~794, 666~794, 676~794, 686~794, 696~794, 706~794, 716~794, 726~794, 736~794, 746~794, 756~794, 766~794, 776~794, 786~794, 266~794, 276~784, 276~774, 276~764, 276~754, 276~744, 276~734, 276~724, 276~714, 276~704, 276~694, 276~684, 276~674, 276~664, 276~ Combined with 654, 276-644, 276-634, 276-624, 276-614, 276-604, 276-594, 276-584, 276-574, 276-564, 276-554, 276-544, 276-534, 276-524, 276-514, 276-504, 276-594, 276-584, 276-574, 276-564, 276-554, 276-544, 276-534, 276-524, 276-514, 276-504, or 276-496.

[0202] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to a structural epitope comprised of two or more discontinuous epitope regions. (a) a first epitope region within the A1 domain and a second epitope region within the A2 domain of a human NPR2 polypeptide; (b) a first epitope region within the A1 domain and a second epitope region within the B1 domain of a human NPR2 polypeptide; (c) a first epitope region within the A1 domain and a second epitope region within the B2 domain of a human NPR2 polypeptide; (d) a first epitope region within the A1 domain and a second epitope region within the C domain of a human NPR2 polypeptide; (e) a first epitope region within the A2 domain and a second epitope region within the B1 domain of a human NPR2 polypeptide; (f) a first epitope region within the A2 domain and a second epitope region within the B2 domain of a human NPR2 polypeptide; (g) a first epitope region within the A2 domain and a second epitope region within the C domain of a human NPR2 polypeptide; (h) a first epitope region within the B1 domain and a second epitope region within the B2 domain of a human NPR2 polypeptide; (i) a first epitope region within the B1 domain and a second epitope region within the C domain of a human NPR2 polypeptide; or (j) specifically binds to a structural epitope comprising or consisting of a first epitope region in the B2 domain and a second epitope region in the C domain of human NPR2 polypeptide;

[0203] In certain embodiments, an antibody or antigen-binding fragment thereof of the present invention specifically binds to at least one epitope within a region of a human NRP2 polypeptide, which binds to or interacts with at least one "NRP2 ligand," including, for example, any molecule that interacts with or reversibly binds to human NRP2. Typical examples of "NRP2 ligands" include polypeptides such as HRS polypeptides, soluble ligands, and receptors (e.g., cell surface receptors), including growth factors and growth factor receptors; specific examples of NRP2 ligands are described herein. In certain embodiments, at least one antibody or antigen-binding fragment thereof modulates (e.g., weakens, inhibits, stimulates, or enhances) binding between a human NRP2 polypeptide and at least one "NRP2 ligand."

[0204] As mentioned above, in certain embodiments, at least one NRP2 ligand is HRS polypeptide.Therefore, in certain embodiments, antibody or its antigen-binding fragment specifically binds to at least one epitope in the region of human NRP2 polypeptide that binds to or interacts with at least one human HRS polypeptide, thereby regulating the binding of human NRP2 polypeptide to human HRS polypeptide.Examples of HRS polypeptide sequences are provided in the following Table H1. [Table H1-1] [Table H1-2] [Table H1-3] [Table H1-4]

[0205] Thus, in certain embodiments, at least one NRP2 ligand is selected from Table H1, and the anti-NRP2 antibody or antigen-binding fragment thereof modulates (e.g., inhibits) binding between a human NRP2 ligand (e.g., a human NRP2 polypeptide selected from Table H1) and a human HRS polypeptide selected from Table H1. In certain embodiments, the anti-NRP2 antibody or antigen-binding fragment thereof specifically binds to an HRS polypeptide-interacting region of an NRP2 polypeptide and, in certain instances, mimics one or more signaling activities of an HRS polypeptide bound to the NRP2 polypeptide, e.g., as an agonist antibody. An "HRS polypeptide-interacting region" includes a region or domain of a human NRP2 polypeptide that interacts with a region or domain of a human HRS polypeptide. For example, such sites include ligand-binding sites for other NRP2 ligands (examples of which are described herein), dimerization domains, protein-protein interaction domains, or sites within an NRP2 polypeptide that are allosterically influenced to modulate the activity of the NRP2 polypeptide.

[0206] In certain embodiments, the antibody or antigen-binding fragment thereof is a "blocking antibody" that completely or substantially inhibits binding between a human NRP2 polypeptide (e.g., one selected from Table N1) and an NRP2 ligand, such as a human HRS polypeptide (e.g., one selected from Table H1) or another NRP2 ligand (e.g., one selected from Table N2 or Table N3). In certain embodiments, a "blocking antibody" inhibits about or at least about 80% to 100% (e.g., 80%, 85%, 90%, 95%, or 100%) of the theoretical maximal binding between an NRP2 polypeptide and an NRP2 ligand (e.g., an HRS polypeptide) after pre-incubation of the NRP2 polypeptide with the "blocking antibody" in substantially stoichiometric amounts. "Stoichiometric equivalent," as used herein, means that the number of moles of one substance (e.g., an anti-NRP2 antibody) is equal to or substantially equal to the number of moles of at least one other substance (e.g., an NRP2 polypeptide) in a given reaction equation or reaction.

[0207] In certain embodiments, the antibody or antigen-binding fragment thereof is a "partially blocking antibody" that at least partially, but not completely, inhibits binding between a human NRP2 polypeptide (e.g., one selected from Table N1) and an NRP2 ligand, such as a human HRS polypeptide (e.g., one selected from Table H1) or another NRP2 ligand (e.g., one selected from Table N2 or Table N3). In certain embodiments, a "partially blocking antibody" inhibits about or at least about 20% to 80% (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%) of the theoretical maximal binding between the NRP2 polypeptide and an NRP2 ligand (e.g., an HRS polypeptide) after pre-incubation of the NRP2 polypeptide with a stoichiometric equivalent of the "partially blocking antibody."

[0208] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically inhibits or attenuates binding between a human NRP2 polypeptide and an HRS polypeptide splice variant selected from Table H1. For example, the HRS splice variant is selected from the HisRS splice variant. 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).

[0209] As noted above, NRP2 also interacts with several NRP2 ligands other than HRS, which influence downstream signaling events. Further examples of NRP2 ligands are listed in Tables N2 and N3 below. [Table N2] [Table N3]

[0210] Thus, in certain embodiments, at least one NRP2 ligand is selected from Table N2 and / or Table N3.

[0211] For example, in some embodiments, 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 interaction is associated with promoting cell migration, cell proliferation, cell survival, and cell adhesion, as well as lymphangiogenesis, increasing vascular permeability, activating integrin signaling, promoting vesicular trafficking and internalization, and delaying cell differentiation. Thus, anti-NRP2 antibodies that modulate VEGF-associated NRP2 ligands may be useful for modulating one or more of these pathways.

[0212] In certain embodiments, the at least one NRP2 ligand is a semaphorin selected from one or more of SEMA-3B, SEMA-3C, SEMA-3D, SEMA-3F, and SEMA-3B, or a plexin receptor selected from one or more of Plexin A1, Plexin A2, Plexin A3, Plexin A4, and Plexin D1. There is a close dynamic interaction between the VEGF and SEMA signaling pathways, and SEMA counteracts the effects of VEGF-C. SEMA generally functions in the immune system to regulate cell motility, cell migration, cell-cell communication, and cell activation. The interaction of SEMA plexins with NRP2 is associated with inhibiting cell migration, inhibiting cell proliferation, promoting apoptosis, inhibiting cell adhesion, inhibiting integrin signaling, promoting cell differentiation, inhibiting lymphangiogenesis, reducing vascular permeability, promoting microtubule destabilization, mediating actin cytoskeleton collapse and cell contraction (e.g., growth cone collapse and actomyosin contraction), preventing neuronal spreading, and inhibiting axon outgrowth. Thus, anti-NRP2 antibodies that modulate SEMA-associated NRP2 ligands may be useful for modulating one or more of these pathways.

[0213] In some embodiments, the at least one NRP2 ligand is α V β1, α V β3, α V β5, α V β6, α V The integrin is selected from one or more of β8, α6β1, and α6β4. Interaction between integrins and NRP2 is associated with increased cell adhesion, cell proliferation, cancer growth, and invasion. Therefore, anti-NRP2 antibodies that modulate integrin-associated NRP2 ligands are believed to be useful for modulating one or more of these pathways.

[0214] In one embodiment, the at least one NRP2 ligand is selected from TGFβ1, TGFβ2, TGFβ3, and their corresponding TGFβ receptors. TGF-β signaling is strongly involved in the regulation of EMT in cancer and is also involved in the development of fibrosis (see, e.g., Gemmill et al., Sci. Signal. 10 eaag0528, 2017). Expression is preferentially upregulated in abnormal lungs by TGF-β signaling, with little or no expression in normal lungs. NRP2B expression enhances migration, invasion, metastasis, and tumor-like mass formation, and also enhances EMT-associated acquired EGFR inhibitor resistance in cancer cells. Therefore, anti-NRP2 antibodies that modulate TGF-β-associated NRP2 ligands are believed to be useful for modulating one or more of these pathways. Thus, in certain embodiments, the anti-NRP2 antibody or antigen-binding fragment thereof modulates the binding activity / signaling activity between an NRP2 polypeptide and at least one NRP2 ligand selected from Table N2 and / or Table N3, e.g., by specifically binding to the NRP2 ligand-interacting region of the NRP2 polypeptide.

[0215] In some examples, at least one antibody or antigen-binding fragment thereof attenuates binding / signaling activity between an NRP2 polypeptide and at least one NRP2 ligand. For example, in some embodiments, the anti-NRP2 antibody attenuates or reduces about or at least about 20% to 100% (e.g., about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 100%) of the theoretical maximum binding / signaling between an NRP2 polypeptide and an NRP2 ligand after pre-incubation of the anti-NRP2 antibody with the NRP2 polypeptide in a substantially stoichiometric equivalent amount.

[0216] In some examples, at least one antibody or antigen-binding fragment thereof stimulates or enhances binding / signaling activity between an NRP2 polypeptide and at least one NRP2 ligand. For example, in some embodiments, the anti-NRP2 antibody stimulates or enhances about or at least about 20% to 500% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, or 500%) of the theoretical maximum binding / signaling activity between an NRP2 polypeptide and at least one NRP2 ligand after pre-incubation of the anti-NRP2 antibody with the NRP2 polypeptide in a substantially stoichiometric amount.

[0217] In certain embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates binding and / or signaling between a semaphorin and an NRP2 polypeptide, or binding and / or signaling via a semaphorin and an NRP2 polypeptide. In certain aspects, such antibodies do not substantially block the interaction of VEGF-C or related NRP2 ligands. In certain aspects, such antibodies are agonistic antibodies against semaphorin signaling. In certain aspects, such antibodies are antagonistic antibodies against semaphorin signaling.

[0218] In certain embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates binding and / or signaling between VEGF-C or related NRP2 ligands and NRP2 polypeptides, or binding and / or signaling between VEGF-C or related NRP2 ligands and NRP2 polypeptides. In certain aspects, such antibodies do not substantially block semaphorin interactions. In certain embodiments, such antibodies selectively modulate binding between VEGF-C or related NRP2 ligands and NRP2 polypeptides, and binding between semaphorins and NRP2 polypeptides. In certain embodiments, such antibodies are agonistic antibodies against VEGF-C signaling. In certain aspects, such antibodies are antagonistic antibodies against VEGF-C signaling.

[0219] In certain embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates binding and / or signaling between an integrin or related NRP2 ligand and an NRP2 polypeptide.

[0220] In certain embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates binding and / or signaling between TGFβ1, TGFβ2, TGFβ3, or their corresponding TGFβ receptors, and an NRP2 polypeptide.

[0221] In certain embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates binding and / or signaling of fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor, and / or their corresponding receptors to an NRP2 polypeptide.

[0222] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor and / or a semaphorin without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and VEGFR3 or VEGF-C.

[0223] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor and / or a semaphorin without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and an HRS polypeptide.

[0224] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor and / or a semaphorin without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and an HRS polypeptide, and without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and VEGFR3 or VEGF-C.

[0225] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and VEGR3 without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor and / or a semaphorin.

[0226] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and VEGR3 or VEGF-C without substantially modulating the binding activity / signaling activity between an NRP2 polypeptide and an HRS polypeptide.

[0227] In certain embodiments, at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and a plexin receptor without substantially modulating ligand binding between semaphorin 3 and NRP2.

[0228] In some embodiments, the plexin receptor is selected from plexin A1, plexin A2, plexin A3, plexin A4, and plexin D1. In some embodiments, the semaphorin is selected from semaphorin 3B, semaphorin 3C, semaphorin 3D, semaphorin 3F, and semaphorin 3G.

[0229] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the A2 domain of human NRP2 comprising at least five consecutive 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 certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within amino acids 232 to 242 of human NRP2 set forth in SEQ ID NO: 1.

[0230] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the B1 domain of human NRP2 comprising at least five consecutive 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.

[0231] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the B2 domain of human NRP2 comprising at least five consecutive 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 also inhibits dimerization between NRP2 and Plexin A1.

[0232] In one embodiment, at least one antibody or antigen-binding fragment thereof specifically binds to an epitope within the C domain of human NRP2 comprising at least five consecutive 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).

[0233] In certain embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ), and the affinity for (i) and (ii) is within 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.

[0234] In certain embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ), and the affinity for (i) and (ii) is within 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.

[0235] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a complementarity-determining region V H CDR1 sequence, V H CDR2 sequence, and V H The heavy chain variable region (V H ) sequence and complementarity-determining region V L CDR1 sequence, V L CDR2 sequence, and V L The light chain variable region (V L ) sequence or comprising the sequence. H Array, V H CDR1 sequence, V H CDR2 sequence, V H CDR3 sequence, V L Array, VL CDR1 sequence, V L CDR2 sequence, and V L Examples of CDR3 sequences are shown in Table A1 below. [Table A1-1] [Table A1-2] [Table A1-3]

[0236] Thus, in certain embodiments, the antibody or antigen-binding fragment thereof comprises: A V complementarity determining region selected from Table A1 that specifically binds to a human NRP2 polypeptide (e.g., one selected from Table N1). H CDR1 sequence, V H CDR2 sequence, and V H Heavy chain variable region (V) including CDR3 sequences and their variants H ) array, and A V complementarity determining region selected from Table A1 that specifically binds to a human NRP2 polypeptide (e.g., one selected from Table N1). L CDR1 sequence, V L CDR2 sequence, and V L The light chain variable region (V) including CDR3 sequences and their variants L ) array, and In a particular embodiment, the CDR sequences are as follows: V H The CDR1 sequence is SEQ ID NO: 23 and its variants, V H The CDR2 sequence is SEQ ID NO: 24 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 25 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 26 and its variants, V L CDR2 is SEQ ID NO: 27 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 28 and variants thereof; VH The CDR1 sequence is SEQ ID NO: 29 and its variants, V H The CDR2 sequences are SEQ ID NO: 30 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 31 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 32 and its variants, V L CDR2 is SEQ ID NO: 33 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 34 and variants thereof; V H The CDR1 sequence is SEQ ID NO: 35 and its variants, V H The CDR2 sequence is SEQ ID NO: 36 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 37 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 38 and its variants, V L CDR2 is SEQ ID NO: 39 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 40 and variants thereof; V H The CDR1 sequence is SEQ ID NO: 41 and its variants, V H The CDR2 sequence is SEQ ID NO: 42 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 43 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 44 and its variants, V L CDR2 is SEQ ID NO: 45 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 46 and variants thereof; V H The CDR1 sequence is SEQ ID NO: 47 and its variants, V H The CDR2 sequence is SEQ ID NO: 48 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 49 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 50 and its variants, V L CDR2 is SEQ ID NO: 51 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 52 and variants thereof; V HThe CDR1 sequence is SEQ ID NO: 53 and its variants, V H The CDR2 sequence is SEQ ID NO: 54 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 55 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 56 and its variants, V L CDR2 is SEQ ID NO: 57 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 58 and variants thereof; V H The CDR1 sequence is SEQ ID NO: 59 and its variants, V H The CDR2 sequence is SEQ ID NO: 60 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 61 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 62 and its variants, V L CDR2 is SEQ ID NO: 63 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 64 and variants thereof; V H The CDR1 sequence is SEQ ID NO: 65 and its variants, V H The CDR2 sequence is SEQ ID NO: 66 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 67 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 68 and its variants, V L CDR2 is SEQ ID NO: 69 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 70 and variants thereof; V H The CDR1 sequence is SEQ ID NO: 71 and its variants, V H The CDR2 sequence is SEQ ID NO: 72 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 73 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 74 and its variants, V L CDR2 is SEQ ID NO: 75 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 76 and variants thereof; V HThe CDR1 sequence is SEQ ID NO: 77 and its variants, V H The CDR2 sequence is SEQ ID NO: 78 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 79 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 80 and its variants, V L CDR2 is SEQ ID NO: 81 and variants thereof, and V L the CDR3 sequence comprises SEQ ID NO: 82 and variants thereof; V H The CDR1 sequence is SEQ ID NO: 83 and its variants, V H The CDR2 sequence is SEQ ID NO: 84 and variants thereof, and V H CDR3 sequences include SEQ ID NO: 85 and variants thereof, V L The CDR1 sequence is SEQ ID NO: 86 and its variants, V L CDR2 is SEQ ID NO: 87 and variants thereof, and V L The CDR3 sequence includes SEQ ID NO: 88 and variants thereof.

[0237] Variants thereof, such as affinity matured variants that bind to human NRP2, are also included, which may comprise one or more CDR regions, e.g., one or more VCRs described herein. H CDR1 sequence, V H CDR2 sequence, V H CDR3 sequence, V L CDR1 sequence, V L CDR2 sequence, and / or V L The CDR3 sequence has 1, 2, 3, 4, 5, or 6 modifications. Examples of "modifications" include amino acid substitutions, additions, and deletions.

[0238] By way of example only, the binding interaction between human NRP2 polypeptide and an NRP2 ligand can be detected and quantified using a variety of conventional methods, including Biacore assays (e.g., using appropriately labeled soluble reagents bound to a sensor chip), FACS analysis using cells (native or recombinant) expressing NRP2 polypeptide on their cell surface, immunoassays, fluorescent staining assays, ELISA assays, and microcalorimetry methods such as ITC (isothermal titration calorimetry).

[0239] In certain embodiments, antibodies or antigen-binding fragments thereof include variant or modified Fc regions, e.g., those with different properties or biological activities than the wild-type Fc region. Examples of modified Fc regions include those with sequences mutated, e.g., by substituting, inserting, deleting, or truncating one or more amino acids relative to the wild-type sequence, i.e., hybrid Fc polypeptides comprising domains from different immunoglobulin classes / subclasses, Fc polypeptides with altered glycosylation / sialylation patterns, Fc polypeptides modified or derivatized, e.g., by biotinylation (e.g., U.S. Patent Application Publication No. 2010 / 0209424), phosphorylation, sulfation, or a combination thereof. These modified forms can be used to improve or modify the binding characteristics of the Fc region to one or more specific FcRs (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, FcγRIIIb, FcRn), 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, among other properties described herein. max , t max , C min、 The Fc region may have altered (e.g., increased or decreased) activity, immunogenicity, complement fixation or activation, and / or CDC / ADCC / ADCP-related activity relative to the corresponding wild-type Fc sequence of the antibody or antigen-binding fragment thereof. Modified Fc regions of human and / or murine origin are also included.

[0240] Also included are antibodies or antigen-binding fragments thereof comprising hybrid Fc regions, examples of which include those comprising a combination of Fc domains (e.g., hinge, CH2, CH3, CH4) derived from immunoglobulins of various species (e.g., human, mouse), various Ig classes, and / or various Ig subclasses.Common examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following CH2 / CH3 domain combinations: 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 / IgG 3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgE / IgA1, IgE / IgA2, IgE / IgD, IgE / IgE, IgE / IgG1, IgE / IgG2, IgE / IgG3, IgE / IgG4, IgE / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG 3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG 3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM, IgM / IgA1, IgM / IgA2, IgM / IgD, IgM / IgE, IgM / IgG1, IgM / IgG2, IgM / IgG3, IgM / IgG4, IgM / IgM (or a fragment or variant thereof), optionally comprising a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4, and / or a CH4 domain derived from IgE and / or IgM.In certain embodiments, the hinge domain, CH2 domain, CH3 domain, and CH4 domain are derived from a human Ig.

[0241] Further examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following CH2 / CH4 domain combinations, wherein the CH2 / CH4 domain combinations are 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 / IgE, IgD / IgM, IgE / IgM, IgG1 / IgE, IgD / IgM, IgD / IgM, IgD / IgM, IgD / IgM, IgD / IgM, IgD / IgM, IgD / IgM, IgD / IgM, IgD / IgE ... IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof), optionally including a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4, and / or a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In certain embodiments, the hinge domain, CH2 domain, CH3 domain, CH4 domain are derived from a human Ig.

[0242] Specific examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following CH3 / CH4 domain combinations, where the CH3 / CH4 domain combinations are IgA1 / IgE, IgA2 / IgE, IgD / IgE, IgE / IgE, IgG1 / IgE, IgG2 / IgE, IgG3 / IgE, IgG4 / IgE, IgM / IgE, IgA1 / IgM, IgA2 / IgM, IgD / IgM, IgE / IgM, IgG1 / IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or a fragment or variant thereof), optionally including a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4, and / or a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In certain embodiments, the hinge domain, CH2 domain, CH3 domain, CH4 domain are derived from a human Ig.

[0243] Specific examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following hinge / CH2 domain combinations: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 ...1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA gA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, I gG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA 2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), optionally including a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH4 domain derived from IgE and / or IgM. In certain embodiments, the hinge domain, CH2 domain, CH3 domain, CH4 domain are derived from a human Ig.

[0244] Specific examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following hinge / CH3 domain combinations: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 ...1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA gA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, I gG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA 2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), optionally including a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH4 domain derived from IgE and / or IgM. In certain embodiments, the hinge domain, CH2 domain, CH3 domain, CH4 domain are derived from a human Ig.

[0245] Some examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following hinge / CH4 domain combinations: IgA1 / IgE, IgA1 / IgM, IgA2 / IgE, IgA2 / IgM, IgD / IgE, IgD / IgM, IgG1 / IgE, IgG1 / IgM, IgG2 / IgE, IgG2 / IgM, IgG3 / IgE, IgG3 / IgM, Ig IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM (or fragments or variants thereof), and optionally comprising a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM.

[0246] Specific examples of hybrid Fc regions are shown, for example, in WO 2008 / 147143, in which the Fc regions are derived from a combination of IgG subclasses or a combination of human IgD and IgG.

[0247] Antibodies or antigen-binding fragments thereof with derivatized or altered Fc regions are also included. In certain aspects, the Fc region may be altered, e.g., by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, or amidation relative to a wild-type or native Fc region. In certain embodiments, the Fc region may comprise a wild-type or native glycosylation pattern, or may be more or less glycosylated than the native form, or may be entirely deglycosylated. As an example of an altered Fc glycoform, reduced glycosylation of the Fc region reduces binding to the C1q region of the first complement component C1, reduces ADCC-related activity, and / or reduces CDC-related activity. Accordingly, certain embodiments utilize deglycosylated or aglycosylated Fc regions. See, e.g., WO 2005 / 047337 for the production of exemplary aglycosylated Fc regions. Another example of an Fc region glycoform is the numbering scheme of Kabat et al. Depending on the system, an Fc region may be generated by substituting Q295 with a cysteine residue (see, e.g., U.S. Patent Application Publication No. 2010 / 0080794). Certain embodiments may include an Fc region in which approximately 80%-100% of the glycoproteins in the Fc region have a mature core glycan structure lacking fructose (see, e.g., U.S. Patent Application Publication No. 2010 / 0255013). Certain embodiments may include an Fc region that has been optimized by substitution or deletion to reduce fucosylation levels, e.g., increase affinity for FcγRI, FcγRIa, or FcγRIIIa, and / or enhance phagocytosis by FcγRIIa-expressing cells (see, e.g., U.S. Patent Application Publication Nos. 2010 / 0249382 and 2007 / 0148170).

[0248] As another example of an engineered Fc glycoform, the Fc region of an antibody or antigen-binding fragment thereof may comprise oligomannose-type N-glycans, and may optionally have one or more of the following characteristics relative to a corresponding Fc region containing complex-type N-glycans: increased ADCC effector activity, increased binding affinity for FcγRIIIA (and certain other FcRs), equivalent or increased binding specificity for the target of an NRP2 polypeptide, equivalent or increased binding affinity for the target of an NRP2 polypeptide, and / or equivalent or decreased binding affinity for the mannose receptor (see, e.g., U.S. Patent Application Publication No. 2007 / 0092521 and U.S. Patent No. 7,700,321). In another example, the affinity of the Fc region for FcγR has been increased by engineered glycoforms produced by expressing the antibody in engineered or variant cell lines (see, e.g., Umana et al., Nat Biotechnol. 17:176-180, 1999; Davies et al., Biotechnol Bioeng. 74:288-294, 2001; Shields et al., J Biol Chem. 277:26733-26740, 2002; Shinkawa et al., 2003, J Biol Chem. 278:3466-3473, 2003; and U.S. Patent Application Publication No. 2007 / 0111281). Certain Fc region glycoforms have a high proportion of N-glycosidically linked complex glycans that lack the 1-position of fucose linked to the 6-position of N-acetylglucosamine at the reducing end of the glycan (see, e.g., U.S. Patent Application Publication No. 2010 / 0092997). Certain embodiments may include an IgG Fc region glycosylated with at least one galactose moiety linked to a terminal sialic acid moiety via an α-2,6 linkage, optionally exhibiting enhanced anti-inflammatory activity relative to the corresponding wild-type Fc region (see, e.g., U.S. Patent Application Publication No. 2008 / 0206246).These and related engineered glycosylation techniques substantially improve the performance of the Fc region to selectively bind FcRs, such as FcγRIII, mediate ADCC, and modify other properties of the Fc region, as described herein.

[0249] Particular variant, fragment, hybrid, or modified Fc regions of antibodies or antigen-binding fragments thereof may have altered binding to one or more FcRs and / or altered associated effector functions compared to the corresponding wild-type Fc sequence (e.g., same species, same Ig class, same Ig subclass). For example, such Fc regions may have increased binding to one or more of Fcγ receptors, Fcα receptors, Fcε receptors, and / or fetal Fc receptors compared to the corresponding wild-type Fc sequence. In other embodiments, variant, fragment, hybrid, or modified Fc regions may have decreased binding to one or more of Fcγ receptors, Fcα receptors, Fcε receptors, and / or fetal Fc receptors compared to the corresponding wild-type Fc sequence. Specific FcRs are described elsewhere herein.

[0250] In certain embodiments, the antibody comprises an Fc domain with one or more mutations that increase binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or fetal Fc receptors compared to the corresponding wild-type Fc sequence. In certain embodiments, the antibody comprises an IgG1 or IgG3 Fc domain with one or more mutations that increase binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or fetal Fc receptors compared to the corresponding wild-type Fc sequence. In certain embodiments, the antibody comprises an Fc domain with one or more mutations that enhance effector function. In certain embodiments, at least one antibody comprises an Fc domain selected from human IgG1 and human IgG3 with one or more mutations that enhance effector function.

[0251] In some embodiments, the antibody is a blocking antibody comprising an Fc domain with enhanced effector activity. In some embodiments, the blocking antibody comprises an Fc domain selected from human IgG1 and human IgG3 with one or more mutations that enhance effector function. In some embodiments, the antibody is a partially blocking antibody comprising an Fc domain with enhanced effector activity. In some embodiments, the partially blocking antibody comprises an Fc domain selected from human IgG1 and human IgG3 with one or more mutations that enhance effector function. In some embodiments, the antibody is a non-blocking antibody comprising an Fc domain with enhanced effector activity. In some embodiments, the non-blocking antibody comprises an Fc domain selected from human IgG1 or human IgG3 with one or more mutations that enhance effector function.

[0252] In certain embodiments, the antibody comprises an Fc domain with one or more mutations that reduce binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or fetal Fc receptors compared to the corresponding wild-type Fc sequence. In certain embodiments, the antibody comprises an IgG1 or IgG3 Fc domain with one or more mutations that reduce binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or fetal Fc receptors compared to the corresponding wild-type Fc sequence. In certain embodiments, the antibody comprises an Fc domain with one or more mutations that reduce effector function. In certain embodiments, the antibody comprises an Fc domain selected from human IgG2 and human IgG4 with one or more mutations that reduce effector function.

[0253] In some embodiments, the antibody is a blocking antibody comprising an Fc domain with reduced effector activity. In some embodiments, the blocking antibody comprises an Fc domain selected from human IgG2 and human IgG4 with one or more mutations that reduce effector function. In some embodiments, the antibody is a partially blocking antibody comprising an Fc domain with reduced effector activity. In some embodiments, the partially blocking antibody comprises an Fc domain selected from human IgG2 and human IgG4 with one or more mutations that reduce effector function. In some embodiments, the antibody is a non-blocking antibody comprising an Fc domain with reduced effector activity. In some embodiments, the non-blocking antibody comprises an Fc domain selected from human IgG2 and human IgG4 with one or more mutations that reduce effector function.

[0254] Specific examples of Fc variants with altered (e.g., increased, decreased) effector function / FcR binding are provided, for example, in U.S. Patent Nos. 5,624,821 and 7,425,619, and U.S. Patent Application Publication Nos. 2009 / 0017023, 2009 / 0010921, and 2010 / 0203046, and WO 2000 / 42072 and WO 2004 / 016750. Specific examples include human Fc variants with one or more substitutions at positions 298, 333, and / or 334 (e.g., S298A, E333A, and / or K334A) (according to the EU index numbering of Kabat et al.). These include modified Fc regions, which have been shown to increase binding to the activating receptor FcγRIIIa and decrease binding to the inhibitory receptor FcγRIIb. These mutations can be combined to produce double and triple mutant variants that further improve binding to FcRs. A specific embodiment includes the triple mutant S298A / E333A / K334A, which increases binding to FcγRIIIa and decreases binding to FcγRIIb, increasing ADCC (see, e.g., Shields et al., J. Immunol. 2014, 14:191–192). Biol Chem. 276:6591-6604, 2001 and Presta et al., Biochem Soc Trans. 30:487-490, 2002). See also Umana et al., supra, and U.S. Pat. See also modified Fc glycoforms with increased binding to FcRs disclosed in Kabat et al., 2002, 2925. In one embodiment, the modified Fc glycoforms include those disclosed in Kabat et al., 434S, 2 Included are Fc regions containing one or more substitutions selected from 52Y / 428L, 252Y / 434S, and 428L / 434S (see, e.g., U.S. Patent Application Publication Nos. 2009 / 0163699 and 20060173170).

[0255] Certain variant, fragment, hybrid, or modified Fc regions may have altered effector function compared to the corresponding wild-type Fc sequence. For example, such Fc regions may have increased complement fixation or activation, increased Clq binding affinity, increased CDC-related activity, increased ADCC-related activity, and / or increased ADCP-related activity compared to the corresponding wild-type Fc sequence. In other embodiments, such Fc regions may have decreased complement fixation or activation, decreased Clq binding affinity, decreased CDC-related activity, decreased ADCC-related activity, and / or decreased ADCP-related activity compared to the corresponding wild-type Fc sequence. By way of example only, the Fc region may contain 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 using techniques routine in the art (see, e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Effector cells useful for such assays include, but are not limited to, natural killer (NK) cells, macrophages, and other peripheral blood mononuclear cells (PBMCs). Alternatively or additionally, specific Fc effector functions can be assayed using techniques such as those described, e.g., in Clynes et al. It can be evaluated in vivo using the animal model described in PNAS. 95:652-656, 1998.

[0256] The Fc region or modified Fc region of a particular variant complex may have altered stability or half-life compared to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have increased half-life compared to the corresponding wild-type Fc sequence. In other embodiments, the Fc region or modified Fc region of a variant complex may have decreased half-life compared to the corresponding wild-type Fc sequence. Half-life can be measured in vitro (e.g., under physiological conditions) or in vivo using methods routine in the art, such as radiolabeling, ELISA, or other methods. Stability or half-life can be measured in one or more body fluids, such as blood, serum, plasma, urine, or cerebrospinal fluid, or in specific tissues, such as liver, kidney, muscle, central nervous system tissue, or bone, in vivo. As an example, modifying the Fc region to alter its ability to bind to FcRn may alter in vivo half-life. Non-limiting examples of assays measuring in vivo pharmacokinetic properties (e.g., in vivo mean elimination half-life) and Fc modifications that alter binding to FcRn are described, for example, in U.S. Pat. Nos. 7,217,797 and 7,732,570, and U.S. Patent Application Publication Nos. 2010 / 0143254 and 2010 / 0143254.

[0257] Further non-limiting examples of modifications that alter stability or half-life include the numbering scheme of Kabat et al. The stem may be substituted / deleted at one or more amino acid residues selected from residues 251-256, 285-290, and 308-314 in the CH2 domain, and residues 385-389 and 428-436 in the CH3 domain. See U.S. Patent Application Publication No. 2003 / 0190311. Specific examples include substitution with leucine at position 251, substitution with tyrosine, tryptophan, or phenylalanine at position 252, substitution with threonine or serine at position 254, substitution with arginine at position 255, substitution with glutamine, arginine, serine, threonine, or glutamic acid at position 256, substitution with threonine at position 308, substitution with proline at position 309, substitution with serine at position 311, substitution with aspartic acid at position 312, substitution with leucine at position 314, substitution with arginine, aspartic acid at position 385, or serine, substitution with threonine or proline at position 386, substitution with arginine or proline at position 387, substitution with proline, asparagine or serine at position 389, substitution with methionine or threonine at position 428, substitution with tyrosine or phenylalanine at position 434, substitution with histidine, arginine, lysine or serine at position 433, and / or substitution with histidine, tyrosine, arginine or threonine at position 436, and any combination thereof. Such modifications optionally increase the affinity of the Fc region for FcRn, resulting in increased half-life, as compared to the corresponding wild-type Fc region.

[0258] The Fc region or modified Fc region of a particular variant complex may have altered solubility compared to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have increased solubility compared to the corresponding wild-type Fc sequence. In other embodiments, the Fc region or modified Fc region of a variant complex may have decreased solubility compared to the corresponding wild-type Fc sequence. Solubility can be measured, for example, in vitro (e.g., under physiological conditions) using techniques routine in the art. Exemplary methods for measuring solubility are described elsewhere herein.

[0259] Further exemplary variants include IgG Fc regions having conservative or non-conservative substitutions (as described anywhere herein) at one or more of heavy chain positions 250, 314, or 428, or any combination thereof, e.g., substitutions at positions 250 and 428, or 250 and 314, or 314 and 428, or 250, 314, and 428 (see, e.g., U.S. Patent Application Publication No. 2011 / 0183412). In specific embodiments, the residue at position 250 is substituted with glutamic acid or glutamine, and / or the residue at position 428 is substituted with leucine or phenylalanine. In another illustrative example of an IgG Fc variant, any one or more of the amino acid residues at positions 214-238, 297-299, 318-322, and / or 327-331 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., Ser228Pro and Leu235Ala mutations and human IgG4) to attenuate the effector function of the Fc region (see U.S. Pat. No. 7,030,226). Here, the numbering of residues in the heavy chain is that of the EU index (Kabat et al., "Sequences of Proteins of Immunological Interest," 5 th Ed., National Institutes of Health, Bethesda, Md. (1991). In some of these and related embodiments, FcRn binding and / or serum half-life are altered (e.g., increased, decreased), optionally without diminishing effector function, such as ADCC-related activity or CDC-related activity.

[0260] Further examples include variant Fc regions comprising one or more amino acid substitutions at positions 279, 341, 343, or 373 of the wild-type Fc region, or any combination thereof (see, e.g., U.S. Patent Application Publication No. 2007 / 0224188). The wild-type amino acid residues at these positions in human IgG are valine (279), glycine (341), proline (343), and tyrosine (373). The substitutions may be conservative or non-conservative, as described herein, and may include unnatural amino acids or mimetics. In certain embodiments, the following substitutions are used alone or in combination with these substitutions: 235G, 235R, 236F, 236R, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 244L, 245 R, 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W, 247Y, 248F, 248P, 248Q, 248W, 249L, 24 9M, 249N, 249P, 249Y, 251H, 251I, 251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P, 2 54Q, 254R, 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V, 256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260S, 262L, 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272 R, 279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279Y, 280T, 28 3F, 283G, 283H, 283I, 283K, 283L, 283M, 283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 2 92G, 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, 3 39E, 339F, 339G, 339H, 339I, 339K, 339L, 339M, 339N, 339Q, 339R, 339S, 33 9W, 339Y, 341D, 341E, 341F, 341H, 341I, 341K, 341L, 341M, 341N, 341P, 341 Q, 341R, 341S, 341T, 341V, 341W, 341Y, 343A, 343D, 343E, 343F, 343G, 343H , 343I, 343K, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 343V, 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373Q, 373R, 3 73S, 373T, 373V, 373W, 375R, 376E, 376F, 376G, 376H, 376I, 376L, 376M, 37 6N, 376P, 376Q, 376R, 376S, 376T, 376V, 376W, 376Y, 377G, 377K, 377P, 378N , 379N, 379Q, 379S, 379T, 380D, 380N, 380S, 380T, 382D, 382F, 382H, 382I, 382K, 382L, 382M, 382N, 382P, 382Q, 382R, 382S, 382T, 382V, 382W, 382Y, 3 85E, 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L, 427N, 429A, 429F, 42 9M, 430A, 430D, 430F, 430G, 430H, 430I, 430K, 430L, 430M, 430N, 430P, 430 Variant Fc regions may also be used that contain at least one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions selected from Q, 430R, 430S, 430T, 430V, 430W, 430Y, 431H, 431K, 431P, 432R, 432S, 438G, 438K, 438L, 438T, 438W, 439E, 439H, 439Q, 440D, 440E, 440F, 440G, 440H, 440I, 440K, 440L, 440M, 440Q, 440T, 440V, or 442K. As noted above, the numbering of residues in the heavy chain is as follows:The numbering is based on the EU index (see Kabat et al., supra). Such variant Fc regions typically alter the effector function or alter the serum half-life of the antibody to which they operably bind. Preferably, the altered effector function includes 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, relative to a corresponding Fc region lacking such amino acid substitutions.

[0261] Further examples include: 221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285 , 286, 288, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and / or 428 (see, e.g., U.S. Pat. No. 7,662,925). In certain embodiments, the variant Fc region is 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 a specific embodiment, the variant Fc region is 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 / I332E, V264I / A330L / I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D / I332E, L328V / I332E, L328T / I332E, L328I / I332E, S239E / V264I / I332E, S239Q / V264I / I332E, S239E / V264I / A330Y / I332E, S239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I332E, S239N / A330L / I332E, V264I / S298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, S239D / V264I / A330L / I332E, S239D / I332E / A330I, P230A, P230A / E233D / I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, and at least one amino acid substitution selected from the group consisting of S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L234I, S239D / A330Y / I332E / L235D, S239D / A330Y / I332E / V240I, S239D / A330Y / I332E / V264T, S239D / A330Y / I332E / K326E, and S239D / A330Y / I332E / K326T. In a more specific embodiment, the variant Fc region is N297D / I332E, F241Y / F243Y / V262T / V264T / N297D / I332E,and N297D / S298A / A330Y / I332E. In a specific embodiment, the variant Fc region comprises an amino acid substitution at position 332 (using the numbering of the EU index of 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. Residues in the Fc region are numbered using the EU index numbering system of Kabat et al. The properties described herein Among other things, these variant Fc regions may have increased affinity for FcγR, improved stability, and / or increased solubility compared to the corresponding wild-type Fc region.

[0262] Further examples include the following amino acid substitutions: 224N / Y, 225A, 228L, 230S, 239P, 240A, 241L, 243S / L / G / H / I, 244L, 246E, 247L / A, 252T, 254T / P, 258K, 261Y, 265V, 266A, 267G / N, 268N, 269K / G, 273A, 276D, 278H, 279M, 280N, 283G, 285R, 288R, 289M, 290M, 291M, 292M, 293M, 294M, 295M, 296M, 297M, 298M, 299M, 300M, 301M, 302M, 303M, 304M, 305M, 306M, 307M, 308M, 309M, 310M, 311M, 312M, 313M, 314M, 315M, 316M, 317M, 318M, 319M, 320M, 321M, 322M, 323M, 324M, 325M, 326M, 327M, 328M, 329M, 330M, 331M, 332M, 333M, 334M, 335M, 336M, 337M, 338M, 339M, 340M, 34 9A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A, 306I / F, 311R, 312N, 315D / K / S, 320R, 3 22E, 323A, 324T, 325S, 326E / R, 332T, 333D / G, 335I, 338R, 339T, 340Q, 341E, 342R, 344Q, 347R, 35 1S, 352A, 354A, 355W, 356G, 358T, 361D / Y, 362L, 364C, 365Q / P, 370R, 372L, 377V, 378T, 383N, 389 S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R, 411I, 412A, 414M, 421S, 422I, 426F / P, 428T, 4 Variant Fc regions include one or more of the following residues: 30K, 431S, 432P, 433P, 438L, 439E / R, 440G, 441F, 442T, 445R, 446A, 447E, optionally having altered Fc ligand recognition and / or altered effector function when compared to the parent Fc polypeptide. Residue numbering may be based on the EU numbering scheme of Kabat et al. Illustrative examples of these and related embodiments include the following series of substitutions: (1) N276D, R292Q, V305A, I377V, T394A, V412A, and K439E; (2) P244L, K246E, D399G, and K409R; (3) S304G, K320R, S324T, K326E, and M358T; (4) F243S, P247L, D265V, V266A, S383N, and T411I; (5) H224N, F243L, T393A, and H433P; (6) V240A, S267G, G341E, and E356G; (7) M252T, P291L, P35 2A, R355W, N390D, S408G, S426F, and A431S, (8) P228L, T289A, L365Q, N389S, and 5440G, (9) F241L, V273A, K340Q, and L441F, (10) F241L, T299A, I332T, and M428T, (11) E269K, Y300H, Q342R, V422I, and G446A, (12) T225A, R301c, S304G, D312N, N315D, L351S, and N421S, (13) S254T, L306I , K326R, and Q362L, (14) H224Y, P230S, V323A, E333D, K338R, and S364C, (15) T335I, K414M, and P445R, (16) T335I and K414M, (17) P247A, E258K, D280N, K288R, N297D, T299A, K322E, Q342R, S354A, and L365P, (18) H268N, V279M, A339T, N361D, and S426P, (19) C261Y, K290E, L306F, Q311R , E333G, and Q438L, (20) E283G, N315K, E333G, R344Q, L365P, and S442T, (21) Q347R, N361Y, and K439R, (22) S239P, S254P, S267N, H285R, N315S, F372L, A378T, N390D, Y391C, F404S, E430K, L432P, and K447E, and (23) variant Fc regions comprising or consisting of E269G, Y278H, N325S, and K370R.Residue numbering follows the EU index numbering system of Kabat et al. (See U.S. Patent Application Publication No. 2010 / 0184959).

[0263] A variant Fc region may also have one or more mutated hinge regions, e.g., as described in U.S. Patent Application Publication No. 2003 / 0118592. For example, one or more cysteines in the hinge region may be deleted or substituted with another amino acid. The mutated hinge region may have no cysteine residues, or may have one, two, or three fewer cysteine residues than the corresponding wild-type hinge region. In certain embodiments, an Fc region having such a mutated hinge region is less likely to dimerize than a wild-type Ig hinge region.

[0264] In certain embodiments, the Fc region is an Fc region derived from human IgG1 or human IgG4 (see, e.g., Allberse and Schuurman, Immunology. 105:9-19, 2002) or a fragment thereof. or variants thereof. Table F1 shows example sequences derived from human IgG1 and human IgG4 (CH1 region, hinge region (underlined), CH2 region, and CH3 region). Examples of variant IgG4 sequences that can be used are described, for example, in Peters et al., JBC. 287:24525-24533, 2012, and have substitutions at C227, C230, C127 (e.g., C127S), and C131 (e.g., C131S). Other variants that can be used have a substitution of L445P in IgG4 (denoted IgG4-2) and a substitution of D356E and L358M in IgG1 (denoted IgG1m(zf)). [Table F1]

[0265] As described above, antibodies with altered Fc regions have altered (improved, improved, or decreased) pharmacokinetic properties compared to the corresponding wild-type Fc region. Examples of pharmacokinetic properties include stability or half-life, bioavailability (the proportion of drug absorbed), tissue distribution, volume of distribution (the apparent volume into which a drug is distributed immediately after intravenous injection and equilibrates 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 balance elimination), area under the curve (AUC or exposure, which is the integral of the concentration-time curve after a single dose or at steady state), clearance (the plasma volume from which the drug is eliminated per unit time), and C max (maximum plasma concentration of the drug after oral administration), t max (C max time to reach C min (Minimum concentration reached before next dose) 、 Fluctuations (fluctuations in peak and trough values within each dosing interval at steady state) are included.

[0266] In certain embodiments, the antibody or antigen-binding fragment thereof is maintained at about pH 7.4, i.e., approximately physiological pH, at about 25°C or room temperature, and / or at about 37°C or human body temperature (e.g., in In vivo, in serum, in specific tissues, or in specific species such as rat, mouse, monkey, or human), a biological half-life of about or at least about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours, or about 144 hours or 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 half-life therebetween, including all ranges therebetween.

[0267] In one embodiment, the T mis 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 one embodiment, the T m is above 60°C.

[0268] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxic or chemotherapeutic agents, including, but not limited to, alkylating agents, antimetabolites, anthracyclines, anticancer antibiotics, platinums, 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, camptothecins, irinotecan, topotecan, amsacrine, etoposide, ethylenediamine phosphate Toposide, 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 , rasagiline, naltrexone, lubiprostone, safinamide, istradefylline, pimavanserin, pitolisant, isradipine, pridopidine (ACR16), tetrabenazine, bexarotene, glatiramer acetate, Further examples of cytotoxic or chemotherapeutic agents include alkylating agents such as thiotepa, cyclophosphamide (CYTOXAN™), busulfan, improsulfan, and mitoxantrone, as well as pharmaceutically acceptable salts and acids thereof. alkyl sulfonates such as benzodopa, kaolin, and piposulfan; Aziridines such as rubocone, meturedopa, and uredopa ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine, and chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, fenestrone, Nitrogen mustards such as phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycins, actinomycin, autramycin, azaserine, bleomycin, and kalimumab; Antibiotics such as cutinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, and methotrexate and 5-fluorouracil. folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; florinic acid Folic acid supplements such as aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, and bestrabucil , bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamt, pirarubicin, podophyllinic acid, 2- Ethyl hydrazide, procarbazine, PSK, razoxane, sizofiran, spirogermanium, tenuazonic acid, triazicon, 2,2',2''-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, and taxoids such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc, Antony, France). Rorer), 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, difluoromethylomithine (DMFO), retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin), ONTAK™ (trademark), Examples of suitable anti-inflammatory drugs include denileukin diftitox (denileukin diftitox), esperamicins, capecitabine, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0269] The antibody or antigen-binding fragment thereof can be used in any of the compositions, methods, and / or kits described herein and can be combined with one or more of the immunotherapeutic agents described herein.

[0270] Additional Therapeutic Agents and Compositions Immunotherapeutic Agents In certain embodiments, one or more cancer immunotherapeutic agents are used. In certain instances, the immunotherapeutic agent modulates the subject's immune response, e.g., increasing or maintaining a cancer-associated or cancer-specific immune response, thereby enhancing immune cell inhibition or reduction of cancer cells. Examples of immunotherapeutic agents include polypeptides, e.g., antibodies and antigen-binding fragments thereof, ligands, and small peptides, as well as combinations thereof. Immunotherapeutic agents also include small molecules, cells (e.g., immune cells such as T cells), various cancer vaccines, gene therapy agents or other polynucleotide-based agents, and viral agents such as oncolytic viruses, as well as other agents known in the art. Thus, in certain embodiments, the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint modulator, a cancer vaccine, an oncolytic virus, a cytokine, and a cellular immunotherapeutic agent.

[0271] In certain embodiments, the cancer immunotherapeutic agent is an immune checkpoint modulator. Specific examples include "antagonists" of one or more inhibitory immune checkpoint molecules and "agonists" of one or more stimulatory immune checkpoint molecules. Generally, immune checkpoint molecules are components of the immune system that either enhance (co-stimulatory molecules) or attenuate (decreasingly attenuated) signals. Targeting immune checkpoint molecules has potential for cancer therapy because cancer cells can disrupt the natural function of these molecules (see, e.g., Sharma and Allison, Science. 348:56-61, 2015; Topalian et al., Cancer Cell. 27:450-461, 2015; Pardoll, Nature Reviews Cancer. 12:252-264, 2012). In certain embodiments, immune checkpoint modulators (e.g., antagonists , agonists) "bind" or "specifically bind" to one or more immune checkpoint molecules, as described herein.

[0272] In certain embodiments, the immune checkpoint modulator is a polypeptide or peptide. The terms "peptide" and "polypeptide" are used interchangeably herein; however, in certain instances, the term "peptide" can refer to a short polypeptide, e.g., a polypeptide consisting of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids, or all integers and ranges therebetween (e.g., 5-10, 8-12, 10-15). Polypeptides and peptides can be composed of naturally occurring and / or unnatural amino acids, as described herein.

[0273] Also included as polypeptides are antibodies. Thus, in certain embodiments, the immune checkpoint modulating polypeptide agent is an "antibody" or "antigen-binding fragment thereof" as described anywhere herein.

[0274] In certain embodiments, the agent is or includes a "ligand," e.g., a natural ligand of an immune checkpoint molecule. "Ligand" generally refers to a substance or molecule that forms a complex with a target molecule (e.g., a biomolecule) in a biologically operative manner, and includes "protein ligands," which generally generate a signal by binding to a site on a target molecule or target protein. Thus, certain agents naturally bind to immune checkpoint molecules and generate a signal. Also included are "modified ligands," e.g., protein ligands fused to pharmacokinetic modifiers such as Fc regions from immunoglobulins.

[0275] The binding properties of a polypeptide can be determined using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In this embodiment, the polypeptide specifically binds to a target molecule, such as an immune checkpoint molecule or an epitope thereof, with an equilibrium dissociation constant of about 10 -7 M or less ~ about 10 -8 In one embodiment, the equilibrium dissociation constant is about 10 -9 M or less ~ about 10 -10 In certain exemplary embodiments, the affinity (Kd or EC 50) is about or at least 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, 31 nM, 32 nM, 33 nM, 34 nM, 35 nM, 36 nM, 37 nM, 38 nM, 39 nM, 40 nM, 41 nM, 42 nM, 43 nM, 44 nM, 45 nM, 46 nM, 47 nM, 48 nM, 49 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 4 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, or less than about 0.01 nM, less than 0.05 nM, less than 0.1 nM, or less than 0.2 nM , less than 0.3 nM, less than 0.4 nM, less than 0.5 nM, less than 0.6 nM, less than 0.7 nM, less than 0.8 nM, less than 0.9 nM, less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM, less than 11 nM, less than 12 nM, less than 13 nM, less than 14 nM, less than 15 nM, less than 16 nM, less than 17 nM, less than 18 nM, less than 19 nM, less than 20 nM, less than 21 nM, less than 22 nM, less than 23 nM, less than 24 nM, less than 25 nM, less than 26 nM, less than 27 nM, less than 28 nM, less than 29 nM, less than 30 nM, less than 40 nM, or less than 50 nM.

[0276] In some embodiments, the agent is a "small molecule," which refers to an organic compound of synthetic or biological origin (biomolecule), but which is generally not a polymer. Organic compounds refer to a broad class of chemical compounds whose molecules contain carbon and generally exclude those containing only carbonates, simple oxides of carbon, or cyanides. "Biomolecules" generally refer to organic molecules produced by living organisms, including large polymeric molecules (biopolymers) such as peptides, polysaccharides, and nucleic acids, as well as small molecules such as primary and secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins, and hormones. "Polymer" generally refers to a large molecule or macromolecule composed of repeating structural units linked by covalent chemical bonds.

[0277] In certain embodiments, the molecular weight of a small molecule is between about 1000 and 2000 daltons or less than about 1000 and 2000 daltons, typically between about 300 and 700 daltons, e.g., about 50 daltons, 100 daltons, 150 daltons, 200 daltons, 250 daltons, 300 daltons, 350 daltons, 400 daltons, 450 daltons, 500 daltons, 550 daltons, 500 daltons, 650 daltons, 600 daltons, 750 daltons, 700 daltons, 850 daltons, 800 daltons, 950 daltons, 1000 daltons, 1500 daltons, 200 daltons, 250 daltons, 300 daltons, 350 daltons, 400 daltons, 450 daltons, 500 daltons, 550 daltons, 500 daltons, 650 daltons, 600 daltons, 750 daltons, 700 daltons, 850 daltons, 800 daltons, 950 daltons, 1000 daltons, 1000 daltons, 1500 daltons, 200 daltons, 250 daltons, 300 daltons, 350 daltons, 400 daltons, 450 daltons, 500 daltons, 550 daltons, 60 000 or 2000 daltons, or is less than about 50 daltons, 100 daltons, 150 daltons, 200 daltons, 250 daltons, 300 daltons, 350 daltons, 400 daltons, 450 daltons, 500 daltons, 550 daltons, 500 daltons, 650 daltons, 600 daltons, 750 daltons, 700 daltons, 850 daltons, 800 daltons, 950 daltons, 1000 daltons, or 2000 daltons.

[0278] Certain small molecules may have "specific binding" properties as described for polypeptides, such as antibodies, herein. For example, in certain embodiments, a small molecule specifically binds to a target, e.g., an immune checkpoint molecule, and its binding affinity (Kd or EC 50) is about or at least 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, 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, 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, or less than about 0.01 nM, less than 0.05 nM, less than 0.1 nM, or less than 0.2 nM, less than 0.3 nM, less than 0.4 nM, less than 0.5 nM, less than 0.6 nM, less than 0.7 nM, less than 0.8 nM, less than 0.9 nM, less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM, less than 11 nM, less than 12 nM, less than 13 nM, less than 14 nM, less than 15 nM, less than 16 nM, less than 17 nM, less than 18 nM, less than 19 nM, less than 20 nM, less than 21 nM, less than 22 nM, less than 23 nM, less than 24 nM, less than 25 nM, less than 26 nM, less than 27 nM, less than 28 nM, less than 29 nM, less than 30 nM, less than 40 nM, or less than 50 nM.

[0279] In some embodiments, the immune checkpoint modulator is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules, such as programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), programmed death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), V-domain Ig suppressor of T-cell activation (VISTA), B- and T-lymphocyte attenuator (BTLA), CD160, and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0280] In certain embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, the targeting of which has been reported to 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 pro-B cells. PD-1 interacts with two ligands, PD-L1 and PD-L2. PD-1 functions as an inhibitory immune checkpoint molecule, for example, by reducing or preventing T cell activation, thereby reducing autoimmunity and promoting self-tolerance. The inhibitory effect of PD-1 occurs, at least in part, through two mechanisms: promoting apoptosis of antigen-specific T cells in lymph nodes while reducing apoptosis in regulatory T cells (suppressor T cells). Examples of PD-1 antagonists or inhibitors include antibodies, or antigen-binding fragments, or small molecules that specifically bind to PD-1 and impair one or more immunosuppressive activities of PD-1, such as downstream signaling of PD-1 or 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. Patent Nos. 8,008,449, 8,993,731, and 9,073,994). Nos. 9,084,776, 9,102,727, 9,102,728, 9,181,342, 9,217,034, 9,387,247, 9,492,539, 9,492,540, and U.S. Patent Application Publication Nos. 2012 / 0039906 and 2015 / 0203579.

[0281] 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. Typical 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 of PD-L1, such as the binding of PD-L1 to the PD-1 receptor. Specific examples of PD-L1 antagonists include the 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, and 9,439,962).

[0282] In one embodiment, the agent is a PD-L2 antagonist or inhibitor. As discussed above, PD-L2 is one of the natural ligands for the PD-1 receptor. Exemplary 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 immunosuppressive activities of PD-L2, such as the binding of PD-L2 to the PD-1 receptor.

[0283] In one embodiment, the agent is a CTLA-4 antagonist or inhibitor. CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an inhibitory immune checkpoint molecule by transmitting an inhibitory signal to T cells when CTLA-4 binds to CD80 or CD86 on the surface of antigen-presenting cells, for example. Exemplary CTLA-4 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to CTLA-4. Specific examples include the antibodies ipilimumab and tremelimumab, and their antigen-binding fragments. At least some of the activity of ipilimumab is believed to be mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing of CTLA-4-expressing suppressor Tregs.

[0284] In some embodiments, the agent is an antagonist or inhibitor of IDO or an antagonist or inhibitor of TDO. IDO and TDO are tryptophan-degrading enzymes with immunosuppressive properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. Exemplary antagonists or inhibitors of IDO and TDO include antibodies, antigen-binding fragments, or small molecules that specifically bind to IDO or TDO (see, e.g., Platten et al., Front Immunol. 5: 673, 2014) and reduce or inhibit one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman, 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).

[0285] In one embodiment, the agent is an antagonist or inhibitor of TIM-3. 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 inducing cell death through interaction with its ligand, galectin-9. TIM-3 confers a suppressive tumor microenvironment, and its overexpression is associated with poor prognosis in various cancers (e.g., Li et al., Acta Oncol. 54:1706-13, 2015). TIM-3 antagonists or inhibitors Exemplary agents include antibodies, or antigen-binding fragments, or small molecules that specifically bind to TIM-3 and reduce or inhibit one or more immunosuppressive activities of TIM-3.

[0286] In one embodiment, the agent is an antagonist or inhibitor of LAG-3. Lymphocyte activation gene-3 (LAG-3) is expressed in activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3, like CTLA-4 and PD-1, negatively regulates T cell proliferation, activation, and homeostasis (see, e.g., Workman and Vignali, European Journal of Immun. 33: 970-9, 2003, and Workman et al., Journal of Immun. 172: 5450-5, 2004), and has a Treg suppressor function. LAG-3 has been reported to be involved in the immune system's immune system function (see, e.g., Huang et al., Immunity. 21: 503-13, 2004). LAG-3 also maintains CD8+ T cells in a tolerogenic state and, together with PD-1, maintains the exhausted state of CD8 T cells. Exemplary LAG-3 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to LAG-3 and inhibit one or more immunosuppressive activities of LAG-3. Specific examples include the antibody BMS-986016 and its antigen-binding fragments.

[0287] In one embodiment, the agent is a VISTA antagonist or inhibitor. V-domain Ig suppressor of T-cell activation (VISTA) is an inhibitory immune checkpoint regulator that is primarily expressed in hematopoietic cells and is highly expressed in the tumor microenvironment, where it suppresses T-cell activation, induces Foxp3 expression, and suppresses anti-tumor T-cell responses (see, e.g., Lines et al., Cancer Res. 74:1924-32, 2014). Exemplary VISTA antagonists or inhibitors include antibodies, or antigen-binding fragments, or small molecules that specifically bind to VISTA and reduce one or more of VISTA's immunosuppressive activities.

[0288] In one embodiment, the agent is a BTLA antagonist or inhibitor. During T cell activation, expression of B and T lymphocyte attenuator (BTLA, CD272) is induced, which inhibits T cells through interaction with tumor necrosis family receptors (TNF-R) and the B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily member 14 (TNFRSF14), also known as herpesvirus 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, e.g., Derre et al., J Clin Invest 120:157-67, 2009). A typical example of a BTLA antagonist or inhibitor is BTL. The present invention also includes antibodies, or antigen-binding fragments, or small molecules that specifically bind to BTLA-4 and reduce one or more immunosuppressive activities of BTLA-4.

[0289] In one embodiment, the agent is an antagonist or inhibitor of HVEM, e.g., an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD 160. Exemplary antagonists or inhibitors of HVEM include antibodies, or antigen-binding fragments, or small molecules that specifically bind to HVEM and optionally reduce the HVEM / BTLA and / or HVEM / CD 160 interaction, thereby reducing one or more immunosuppressive activities of HVEM.

[0290] In certain 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. Exemplary CD160 antagonists or inhibitors include antibodies, or antigen-binding fragments, or small molecules that specifically bind to CD160 and optionally reduce CD160 / HVEM interaction, thereby reducing or inhibiting one or more immunosuppressive activities of CD160.

[0291] In some embodiments, the agent is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor found on the surface of various lymphoid cells and suppresses anti-tumor immunity, for example, via Tregs (Kurtulus et al., J Clin Invest. 125:4053-4062, 2015). Exemplary TIGIT antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to TIGIT and reduce one or more immunosuppressive activities of TIGIT (see, e.g., Johnston et al., Cancer Cell. 26:923-37, 2014).

[0292] In certain embodiments, the immune checkpoint modulator is an agonist of one or more stimulatory immune checkpoint molecules, such as OX40, CD40, glucocorticoid-induced TNFR family related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0293] In one embodiment, the agent is an agonist of OX40. OX40 (CD134) promotes the proliferation of effector and memory T cells and suppresses the differentiation and activity of T regulatory cells (see, e.g., Croft et al., Immunol Rev. 229:173-91, 2009). OX40's ligand is OX40L (CD252). OX40 signaling influences both T cell activation and survival and plays an important role in initiating antitumor immune responses in lymph nodes and maintaining them in the tumor microenvironment. Typical examples of OX40 agonists include antibodies, antigen-binding fragments, 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), as well as antigen-binding fragments thereof.

[0294] In one embodiment, the agent is a CD40 agonist. CD40 is expressed on antigen-presenting cells (APCs) and some malignant tumors. The ligand for CD40 is CD40L (CD154). In APCs, ligation can upregulate costimulatory molecules, potentially eliminating the need for T cell help in antitumor immune responses. CD40 agonist therapy plays an important role in APC maturation and migration from tumors to lymph nodes, thereby accelerating antigen presentation and T cell activation. Anti-CD40 agonist antibodies have induced substantial responses and durable anticancer immunity in animal models, an effect mediated, at least in part, by cytotoxic T cells (see, e.g., Johnson et al., Clin Cancer Res. 21: 1321-1328, 2015, and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). Typical examples of CD40 agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to CD40 and increase one or more immunostimulatory activities of CD40. Specific examples include CP-870893, dacetuzumab, Chi Lob 7 / 4, ADC-1013, CD40L, rhCD40L, and antigen-binding fragments thereof.

[0295] In one embodiment, the agent is a GITR agonist. Glucocorticoid-induced TNFR family-related gene (GITR) increases T cell proliferation, inhibits Treg suppressive activity, and extends the survival of T effector cells. GITR agonists have been shown to promote anti-tumor responses by destabilizing the Treg lineage (see, e.g., Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These diverse mechanisms suggest that GITR plays an important role in initiating immune responses in lymph nodes and maintaining immune responses in tumor tissues. The ligand for GITR is GITRL. Exemplary GITR agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to GITR and increase one or more immunostimulatory activities of GITR. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and antigen-binding fragments thereof.

[0296] In one embodiment, the agent is a CD137 agonist. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and cross-linking of CD137 increases T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells, such as CD8+ T cells, from activation-induced cell death. Exemplary CD137 agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to CD137 and increase one or more immunostimulatory activities of CD137. Specific examples include CD137 (or 4-1BB) ligands (see, e.g., Shao and Schwarz, J. Leukoc. Biol. 89:21-9, 2011). (see reference), and the antibody utomilumab and its antigen-binding fragments.

[0297] In one embodiment, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific proliferation of naive T cells and influences T cell memory and long-term persistence of T cell immunity. The ligand for CD27 is CD70. Combined targeting of human CD27 with agonistic antibodies stimulates T cell activation and anti-tumor immunity (see, e.g., Thomas et al., Oncoimmunology. 2014;3:e27255. doi:10.4161 / onci.27255, and He et al., J Immunol. 191:4174-83, 2013). Typical examples of antibodies include antibodies, or antigen-binding fragments, or small molecules, or ligands that specifically bind to CD27 and increase one or more immunostimulatory activities of CD27. Specific examples include CD70 and the antibodies varlilumab and CDX-1127 (1F5) and antigen-binding fragments thereof.

[0298] In one embodiment, the agent is a CD28 agonist. CD28 is constitutively expressed on CD4+ T cells and a subset of CD8+ cells. Ligands for CD28 include CD80 and CD86, and stimulation of CD28 increases T cell proliferation. Typical examples of CD28 agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to CD28 and increase one or more immunostimulatory activities of CD28. Specific examples include CD80, CD86, the antibody TAB08, and antigen-binding fragments thereof.

[0299] In some embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares a ligand with TIGIT. Unlike TIGIT, binding of CD226 enhances T cell activation (see, for example, Kurtulus et al., J Clin Invest. 125:4053-4062, 2015; Bottino et al., J Exp Med. 1984:557-567, 2003; and Tahara-Hanaoka et al., Int Immunol. 16:533-538, 2004). Typical 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 one or more immunostimulatory activities of CD226.

[0300] In one embodiment, the agent is an agonist of HVEM. Herpesvirus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF-receptor superfamily. HVEM is found on a variety of cells, including T cells, APCs, and other immune cells. Unlike other receptors, HVEM is expressed at high levels on resting T cells and is downregulated upon activation. HVEM signaling has been shown to play an important role in the early stages of T cell activation and during the proliferation of tumor-specific lymphocyte populations in lymph nodes. Typical examples of HVEM agonists include antibodies, antigen-binding fragments, small molecules, or ligands that specifically bind to HVEM and increase one or more of its immunostimulatory activities.

[0301] In certain embodiments, the cancer immunotherapeutic agent is a cancer vaccine. Examples of cancer vaccines include Oncophage, human papillomavirus HPV vaccines (such as Gardasil or Cervarix), hepatitis B vaccines (such as Engerix B, Heptavax HB, or Twinrix), and sipuleucel-T (Provenge). In some embodiments, the cancer vaccine comprises or utilizes one or more cancer antigens or cancer-associated antigens.Examples of 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, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, Integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PSMA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAM) MF7), EGP40 pancarcinoma antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

[0302] In certain embodiments, the cancer immunotherapeutic agent is an oncolytic virus. Oncolytic viruses are viruses that preferentially infect and kill cancer cells. These viruses include natural and artificial oncolytic viruses, or recombinant oncolytic viruses. Many oncolytic viruses have been modified for tumor selectivity, but examples of natural viruses include reovirus and SVV-001 Seneca Valley virus. Common examples of oncolytic viruses include VSV, poliovirus, reovirus, Seneca virus, and RIGVIR, as well as modified versions thereof. Non-limiting examples of oncolytic viruses include herpes simplex virus (HSV) and modified versions thereof, talimogene laherparepvec (T-VEC), coxsackievirus A21 (CAVATAK™), Oncorine (H101), pelareorep (REOLYSIN®), Seneca Valley virus, among others. These include 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.

[0303] In certain embodiments, the cancer immunotherapeutic agent is a cytokine. Examples of cytokines include interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0304] In certain embodiments, the cancer immunotherapeutic agent is a cellular immunotherapeutic agent, e.g., a T cell-based adoptive immunotherapeutic agent. In some embodiments, the cellular 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 chimeric antigen receptor (CAR)-modified T cells and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells. In a specific embodiment, the CAR-modified T cells target CD-19 (see, e.g., Maude et al., Blood. 125:4017-4023, 2015).

[0305] In certain examples, the cancer to be treated is associated with a cancer antigen, and the cancer antigen-specific T cells target or are enriched for at least one antigen known to be associated with the cancer to be treated. In certain embodiments, the cancer antigen is selected from the group consisting of CD19, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor (VEGF) (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD 74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor Body 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer (p ancarcinoma antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

[0306] Additional cancer antigens include 5T4, 707-AP, 9D7, AFP, and AlbZIP HPG1, α5β1 integrin, α5β6 integrin, α-actinin-4 / m, α-methylacyl-coenzyme A racemase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, β-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA15-3 / CA27-29, 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, coactosin-like protein, collagen XXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1, cyclin B2, cyclin B3, cyclin B4, cyclin B5, cyclin B6, cyclin B7, cyclin B8, cyclin B9, cyclin B10, cyclin B11, cyclin B12, cyclin B13, cyclin B14, cyclin B15, cyclin B16, cyclin B17, cyclin B18, cyclin B19, cyclin B29, cyclin B19, cyclin B29, cyclin B15, cyclin B29, cyclin B19, cyclin B29, cyclin B15, cyclin B16, cyclin B19, cyclin B29 ... LinD1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, HEPSIN, Her2 / neu, HERV-K-MEL, HL A-A*0201-R17I, 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, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, matrix protein 22, MCI R, M-CSF, ME1 / m, mesothelin, MG50 / PXDN, MMP11, MN / CAIX antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class l / m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-B, NY-ESO-1, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS-9 / m, osteoca Lucin, osteopontin, pi5, p190 minor, bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 kinase, Pin-1, Pml / PARalpha, 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-beta, TGF-beta RII, 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. Specific 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.

[0307] In certain embodiments, the antigen is MAGE-A1 (e.g., MAGE-A1 of accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g., MAGE-A6 of accession number NM_005363), MAGE-C1, MAGE-C2, Melan-A (e.g., Melan-A of accession number NM_00551 1), GP100 (e.g., GP100 of accession number M77348), tyrosinase (e.g., tyrosinase of accession number NM_000372), survivin (e.g., survivin of accession number AF077350), CEA (e.g., CEA of accession number NM_004363), Her-2 / neu (e.g., MAGE-C1 730), Her-2 / neu), WT1 (e.g., WT1 under accession number NM_000378), PRAME (e.g., PRAME under accession number NM_0061 15), EGFRI (epidermal growth factor receptor 1) (e.g., EGFRI under accession number AF288738), MUC1, mucin-1 (e.g., mucin-1 under accession number NM_002456), SEC61G (e.g., SEC61G under accession number NM_014302), hTERT (e.g., hTERT under accession number NM_198253), 5T4 (e.g., 5T4 under accession number NM_006670), TRP-2 (e.g., TRP-2 under accession number NM_001 922), STEAP1 (prostate six-transmembrane epithelial antigen 1), PSCA, PSA, PSMA, and the like.

[0308] In some embodiments, the cancer antigen is selected from PCA, PSA, PSMA, STEAP, and optionally MUC-1, and fragments, variants, and derivatives thereof. In some embodiments, the cancer antigen is selected from NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and optionally MUC-1, and fragments, variants, and derivatives thereof.

[0309] In some examples, the cancer antigen comprises an idiotypic antigen associated with a cancer or tumor disease, particularly a lymphoma or lymphoma-related disease, for example, the idiotypic antigen is an immunoglobulin idiotype of a lymphoid blood cell or a T cell receptor idiotype of a lymphoid blood cell.

[0310] In some examples, the cancer antigen-specific T cells are selected from one or more of chimeric antigen receptor (CAR)-modified T cells (e.g., targeting a cancer antigen) and T cell receptor (TCR)-modified T cells, tumor-infiltrating lymphocytes (TILs), and peptide-induced T cells.

[0311] One of skill in the art will understand that the various cancer immunotherapeutics described herein can be used in combination with one or more of the various anti-NRP2 antibodies (including antigen-binding fragments thereof) described herein in accordance with any one or more of the methods or compositions described herein.

[0312] In certain embodiments, one or more chemotherapeutic agents are used, such as small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or type II), and microtubule inhibitors, among others.

[0313] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, and diaziquone (AZQ)), cisplatins and their derivatives (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally, procarbazine and hexamethylmelamine).

[0314] 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); Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin.

[0315] Examples of microtubule inhibitors include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

[0316] Those skilled in the art will understand that the various chemotherapeutic agents described herein can be used in combination with one or more of the various anti-NRP2 antibodies (including antigen-binding fragments thereof) described herein in accordance with any one or more of the methods or compositions described herein.

[0317] Hormonal Therapy Agents In certain embodiments, at least one hormonal therapy agent is used. Common examples of hormonal therapy agents include hormone agonists and hormone antagonists. Specific examples of hormone agonists include progestogens (progestins), 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 factors (FGF), galectins, hepatocyte growth factors (HGF), platelet-derived growth factors (PDGF), transforming growth factors (TGF) beta, 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) and their analogs. 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).

[0318] Also included are hormone pathway inhibitors, such as antibodies acting against hormone receptors. Examples include inhibitors of IGF receptors (e.g., IGF-IR1), such as cizutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, and robatumumab, and inhibitors of IGF receptors (e.g., IGF-IR1), such as alacizumab pegol, bevacizumab, and ixuclovir. Inhibitors of vascular endothelial growth factor 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as icrucumab and ramucirumab; inhibitors of TGF-beta receptor R1, R2, or R3, such as fresolimumab and methimumab; inhibitors of c-Met, such as naxitamab; cetuximab and depatuxizumab Depatuxizumab mafodotin, futuximab, imgatuzumab imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab EGF receptor inhibitors such as tomuzotuximab and zalutumumab, FGF receptor inhibitors such as aprutumab ixadotin and bemarituzumab, and P-anticoagulants such as olaratumab and tovetumab. and inhibitors of the DGF receptor.

[0319] Those skilled in the art will understand that the various hormone therapies described herein can be used in combination with one or more of the various anti-NRP2 antibodies (including antigen-binding fragments thereof) described herein in accordance with any one or more of the methods or compositions described herein.

[0320] Kinase Inhibitors In certain embodiments, at least one kinase inhibitor is used, including a tyrosine kinase inhibitor. Examples of kinase inhibitors include, but are not limited to, adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, and fostamatinib. tetanib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0321] One of skill in the art will understand that the various kinase inhibitors described herein can be used in combination with one or more of the various anti-NRP2 antibodies (including antigen-binding fragments thereof) described herein in accordance with any one or more of the methods or compositions described herein.

[0322] Methods of Use and Therapeutic Compositions Embodiments of the present disclosure involve the discovery that human histidyl-tRNA synthetase (HRS) polypeptides have unexpected biological properties suitable for treating a wide range of diseases and conditions, some of which are related to the interaction of HRS with human neuropilin-2 (NRP2). Accordingly, antibodies against human NRP2 inhibit the binding of NRP2 to an NRP2 ligand (e.g., human HRS) and can be used as the sole therapeutic agent or in combination with other therapeutic agents described herein to treat diseases, e.g., NRP2-related diseases.

[0323] Certain embodiments include methods for treating a disease or condition, ameliorating symptoms of a disease or condition, and / or inhibiting progression of a disease or condition in a subject, 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 examples, the at least one antibody or antigen-binding fragment thereof attenuates the binding activity / signaling activity between an NRP2 polypeptide and at least one NRP2 ligand. In some examples, the at least one antibody or antigen-binding fragment thereof mimics or enhances one or more signaling activities of the NRP2 / NRP2 ligand interaction, for example, by acting as an agonist antibody.

[0324] In some examples, at least one antibody or antigen-binding fragment thereof inhibits binding between a human NRP2 polypeptide and a human HRS polypeptide. In some examples, at least one antibody or antigen-binding fragment thereof mimics or enhances one or more signaling activities of an HRS polypeptide that binds to an NRP2 polypeptide, for example, by acting as an agonist antibody. Examples of anti-NRP2 antibodies and therapeutic compositions comprising the same are described elsewhere herein.

[0325] In certain embodiments, the disease or condition is an NRP2-associated disease or condition. In certain embodiments, the NRP2-associated disease or condition is a disease or pathway associated with cancer, e.g., cancer and cancer cell proliferation, development, migration, adhesion, invasion, and / or metastasis; a disease associated with inflammation, autoimmune diseases, and related inflammatory diseases, e.g., diseases associated with inappropriate immune cell activation or migration, e.g., graft-versus-host disease (GVHD); a disease associated with lymphangiogenesis, lymphangiogenesis, and lymphatic damage, e.g., edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability; a disease associated with infectious diseases, e.g., latent infections; and a disease associated with chronic obstructive pulmonary disease (COPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, systemic vasculitis, and other conditions. diseases associated with allergic disorders / diseases and allergic reactions, such as allergic lupus erythematosus, rheumatoid arthritis, inflammasome-related diseases, and skin-related neutrophil-mediated diseases such as pyoderma gangrenosum; diseases associated with granulomatous inflammatory diseases, e.g., sarcoidosis and granulomas; diseases associated with fibrosis, e.g., fibrotic diseases, fibrosis, endothelial-mesenchymal transition (EMT), and wound healing; diseases associated with improper smooth muscle contractility and improper vascular smooth muscle cell migration and adhesion; diseases associated with improper autophagy, phagocytosis, and efferocytosis; diseases associated with neurological diseases, peripheral nervous system remodeling, and pa...

Claims

[Claim 1] The invention as described in the drawings.

Images