Compositions and methods comprising Anti-NRP2 antibodies

Abstract

To provide compositions and methods comprising anti-NRP2 antibodies.SOLUTION: Provided are affinity-matured and humanized 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 and thereby modulate subsequent NRP2-mediated downstream signaling events, including related therapeutic compositions and methods for modulating NRP2 activity and treating diseases such as NRP2-associated diseases.SELECTED DRAWING: None

Description

[Technical Field]

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

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

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

[0004] Related field description Recent developments suggest that tRNA synthetases play important roles in cellular responses beyond their well-characterized role in protein synthesis. In particular, there is growing recognition that tRNA synthetases are involved in a range of as yet unrecognized roles in response to cellular stress and tissue homeostasis, both in the intracellular and extracellular milieu.

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

[0006] Thus, the recent discovery of this novel regulatory pathway represents a previously unknown mechanism that serves as a central regulator of cellular processes including, for example, axon guidance, endocytosis, cell migration, proliferation, survival, apoptosis, lymphangiogenesis, cell differentiation and cell adhesion directly related to cancer initiation, growth, metastasis, and chemoresistance, as well as muscle, vascular, neural, bone, and immune homeostasis. Deregulation of any of these processes can result in a wide variety of diseases, which may be addressed by the development of anti-NRP2 antibodies that selectively target the neuropilin-2 axis. The present disclosure provides such antibodies and related embodiments. Summary of the Invention [Means for solving the problem]

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

[0008] Certain embodiments are directed to 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), wherein the at least one antibody or antigen-binding fragment thereof specifically binds to a complementarity-determining region V selected from Table A1 or Table A3. H CDR1, V H CDR2 and V H Heavy chain variable region (V) including CDR3 sequences and their variants H ) sequence; and a complementarity determining region V selected from Table A1 or Table A3 that specifically binds to a human NRP2 polypeptide L CDR1, V L CDR2 and V L The light chain variable region (V) including CDR3 sequences and their variants L ) sequences.

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

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

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

[0012] In some embodiments, at least one antibody or antigen-binding fragment thereof binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1, as appropriate, at a concentration of between about 10 pM and about 500 pM, or between about 10 pM and about 50 nM, or at about, at least about, or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, with an affinity of 200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or, as appropriate, 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, or about 20 pM 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 8 specifically binds with an affinity in the range of 0 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM, and optionally, at least one antibody or antigen-binding fragment thereof specifically binds to human NRP2 polypeptide in its native form but does not substantially bind to human NRP2 polypeptide in its denatured form.

[0013] In some embodiments, at least one antibody or antigen-binding fragment thereof is selected from the group consisting of a neuropilin b1 domain, a neuropilin a1 domain, a neuropilin a2 domain, a neuropilin b2 domain, a neuropilin c domain, a neuropilin a1 / a2 composite domain, a neuropilin b1 / b2 composite domain, a neuropilin a2 / b1 composite domain, a neuropilin b2 / c composite domain, a neuropilin a2 / b1 / b2 composite domain, a neuropilin a2 / b1 / b2 / c composite domain, a neuropilin a1 / a2 / b1 composite domain, and optionally, about 10 pM to about 500 pM or about 10 pM to about 50 nM, or about, at least about, or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20 ... with an affinity of 0, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or, as appropriate, from about 10 pM to about 500 pM, from about 10 pM to about 400 pM, from about 10 pM to about 300 pM, from about 10 pM to about 200 pM, from about 10 pM to about 100 pM, from about 10 pM to about 50 pM, or from about 20 pM to about 500 pM, from about 20 pM to about 400 pM, from about 20 pM to about 300 pM, from about 20 pM to about 200 pM, from about 20 pM to about 100 pM, from about 20 pM to about 50 pM, or from about 30 pM to about 500 pM, It specifically binds with an affinity in the range of about 100 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20 pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

[0014] In some embodiments, at least one antibody or antigen-binding fragment thereof is directed to at least one epitope in the neuropilin a1 domain, the neuropilin a2 domain and / or the neuropilin a1a2 complex domain, and the adjacent linker region, as appropriate. (Neuropilin a1 domain) residues 20-148, 30-141, 40-141, 50-141, 60-141, 70-141, 80-141, 90-141, 100-141, 110-141, 120-141, 130-141; 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 as defined by the human NRP2 precursor sequence (see Table N1); (Neuropilin a2 domain) Residues 142-280, 150-265, 160-265, 170-265, 180-265, 190-265, 200-265, 210-265, 220-265, 230-265, 240-265, 250-265, 260-265, 141-270, 141-260, 141-250, 141-240, 141-230, 141-220, 141-210, 141-200, 141-190, 141-180, 141-170, 141-160, 141-171, 141-182, 141-183, 141-184, 141-185, 141-186, 141-187, 141-188, 141-189, 141-190, 141-201, 141-202, 141-213, 141-214, 141-225, 141-236, 141-246, 141-257, 141-260, 141-261, 141-262, 141-263, 141-264, 141-265, 141-276, 141-265, 141-266, 141-267, 141-268, 141-269, 14 50, 200-250, 210-250, 220-250, 230-250, 200-240, 210-240, 220-240, 230-240, 227-247, 228-247, 229-247, 230-247, 231-247, 232-247, 233-247, 234-2 47, 235-247, 236-247; 227-246, 227-245, 227-244, 227-243, 227-242, 227-241, 227-240, 227-239, 227-238; 235-240, 236-239, 236-238, or residue 237; or (Composite a1a2 domain) Residues 20-280, 30-280, 40-280, 50-280, 60-280, 70-280, 80-280, 90-280, 100-280, 110-280, 120-280, 130-280, 140-280, 150-280, 160-280, 170-280, 180-280, 190-280, 200-280, 210-280, 220-280, and 230-280 are defined by the human NRP2 precursor sequence (see Table N1). 0, 230-280, 240-280, 260-280, 270-280, 20-270, 20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-200, 20-190, 20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30 It specifically binds in the vicinity of

[0015] In some embodiments, at least one antibody or antigen-binding fragment thereof is directed to at least one epitope in the neuropilin b1 domain, the neuropilin b2 domain and / or the neuropilin b1 / b2 complex domain, and the adjacent linker region, as appropriate: (Neuropilin b1 domain) residues 299-420, 266-426, 280-426, 290-426, 300-426, 310-426, 320-426, 330-426, 340-426, 350-426, 360-426, 370-426, 380-426, 390-426, 400-426, 410-426, 420 ... 0, 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, optionally wherein the epitope is a discontinuous epitope comprising one, two, or three of residues 299Y, 354N, and / or 416S, as defined by the human NRP2 precursor sequence; (Neuropilin b2 domain) residues 438-591, 450-591, 460-591, 470-591, 480-591, 490-591, 500-591, 510-591, 520-591, 530-591, 540-591, 550-591, 560-591, 570-591, 580-591, 590-591, 600-600, 610-610, 620-620, 630-630, 640-640, 650-650, 660-660, 670-670, 680-680, 690-691, 700-700, 710-710, 720-720, 730-730, 740-740, 750-750, 760-760, 770-770, 780-780, 790-800, 800-800, 810-810, 820-820, 830-830, 840-840, 850-850, 860-860, 870-870, 880-880, 890-900, 900-900, 910-910, 920-920, 930-930, 940-940, 950-950, 960-960, 970-970, 980-980, 990- 0-591, 570-591, 580-591, 438-590, 438-580, 438-570, 438-560, 438-550, 438-540, 438-530, 438-520, 438-510, 438-500, 438-490, 438-480, 438-470, 438-460, or 438-450; 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, 596-591, 606-591, 616-591, 626-591, 636-591, 646-591, 656-591, 666-591, 676-591, 686-591, 696-691, 706-706, 716-716, 726-726, 736-736, 746-746, 756-756, 766-766, 776-776, 786-786, 796-806, 806-806, 76~591, 386~591, 396~591, 406~591, 416~591, 426~591, 436~591, 446~591, 456~591, 466~591, 476~591, 486~591, 498~591, 508~591, 518~591, 528~591, 538~591, 548~591, 55 8~591, 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, 26 6~441, 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 It specifically binds in the vicinity of

[0016] In some embodiments, at least one antibody or antigen-binding fragment thereof is directed to at least one epitope in the neuropilin a2 / b1 composite domain and / or the neuropilin b2c composite domain, and the adjacent linker region, as appropriate. (Neuropilin a2b1 complex domain) residues 149-437, 159-426, 169-426, 179-426, 189-426, 199-426, 209-426, 219-426, 229-426, 239-426, 249-426, 259-426, 269-426, 279-426, 289-426, 299-426, 309-426, 319-426, 329-426, 339-426, 349-426, 359-426, 369-426, 379-426, 389-426, 399-426, 409-426, 419-426, 429-426, 439-426, 449-426, 459-426, 469-426, 479-426, 489-426, 499-426, 509-426, 519-426, 529-426, 539-426, 549-426, 559-426, 569-426, 579-426, 589-426, 599-426, 609-426, 619-426, 629-426, 639-426, 649-426, 659-426, 669-426, 679-426, 689-426, 6 426, 409-426, 419-426, 149-436, 149-426, 149-416, 149-406, 149-396, 149-386, 149-376, 149-366, 149-356, 149-346, 149-336, 149-326, 149-316, 149-3 06, 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 (Neuropilin b2c complex domain) residues 438-794, 448-794, 458-794, 468-794, 478-794, 487-794, 497-794, 507-794, 517-794, 527-794, 537-794, 547-794, 557-794, as defined by the human NRP2 precursor sequence (see Table N1). 4, 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, 7 67~794, 777~794, 787~794, 427~794, 438~784, 438~774, 438~764, 438~754, 438~744, 438~734, 438~728, 438~714, 438~704, 438~694, 438~684, 438~674, 438~664, 438~654, 438~644, 438~ 634, 438~624, 438~614, 438~604, 438~596, 438~586, 438~576, 438~566, 438~556, 438~546, 438~536, 438~526, 438~516, 438~506, 438~494, 438~484, 438~474, 438~464, 438~454, 438~444 It specifically binds in the vicinity of

[0017] In some embodiments, at least one antibody or antigen-binding fragment thereof is directed to at least one epitope in the neuropilin c domain and adjacent linker region, optionally including 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, 1080-1080, 1 It specifically binds in the vicinity of 0 to 794, 740 to 794, 750 to 794, 760 to 794, 770 to 794, 780 to 794, 790 to 794, 591 to 790, 591 to 780, 591 to 770, 591 to 760, 591 to 750, 591 to 740, 591 to 730, 591 to 720, 591 to 710, 591 to 700, 591 to 690, 591 to 680, 591 to 670, 591 to 660, 591 to 650, 591 to 640, 591 to 630, 591 to 620, 591 to 610, or 591 to 600.

[0018] In some embodiments, at least one antibody or antigen-binding fragment thereof is directed to at least one epitope in the neuropilin b1 / b2 / c complex domain and adjacent linker region, optionally to 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, 506-506, 516-506, 526-506, 536-506, 546-506, 556-506, 566-506, 576-506, 587-506, 596-606, 606-606, 616-616, 626-626, 636-636, 646-646, 656-656, 666-656, 676-676, 687-687, 696-794, 706-794, 716-794, 726-794, 736-794, 746-794, 756-794, 766-794, 776-794, 787- 96~794, 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, 666~79 4, 676~794, 686~794, 696~794, 706~794, 716~794, 726~794, 736~794, 746~794, 756~794, 766~794, 776~794, 786~794, 266~794, 276~784, 276~774, 276~764, 276~754, 276~744, 276~734, 276~724, 276~714, 276~704, 276~694, 276~684, 276~674, 276~664, 276~654, 276 Specifically binds to the vicinity of ~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.

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

[0020] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to a conformational epitope comprised of two or more discontinuous epitope regions, optionally the conformational epitope being (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 a1 domain and a second epitope region within the juxtamembrane domain of a human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; (f) a first epitope region within the a2 domain and a second epitope region within the b1 domain of a human NPR2 polypeptide; (g) a first epitope region within the a2 domain and a second epitope region within the b2 domain of a human NPR2 polypeptide; (h) a first epitope region within the a2 domain and a second epitope region within the c domain of a human NPR2 polypeptide; (i) a first epitope region within the a2 domain and a second epitope region within the juxtamembrane domain of a human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; (j) a first epitope region within the b1 domain and a second epitope region within the b2 domain of a human NPR2 polypeptide; (k) a first epitope region within the b1 domain and a second epitope region within the c domain of a human NPR2 polypeptide; (l) a first epitope region within the b1 domain and a second epitope region within the juxtamembrane domain of a human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; (m) a first epitope region within the b2 domain and a second epitope region within the c domain of a human NPR2 polypeptide; (n) a first epitope region within the b2 domain and a second epitope region within the juxtamembrane domain of a human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; or (o) a first epitope region within the c domain and a second epitope region within the juxtamembrane domain of a human NPR2 polypeptide selected from variants 1, 2, 3, 4, and 5; It comprises or consists of:

[0021] In some embodiments, at least one antibody or antigen-binding fragment thereof modulates binding of a human NRP2 polypeptide to at least one NRP2 ligand (optionally an NRP2 ligand selected from Table N2 or Table N3, and / or a human histidyl-tRNA synthetase (HRS) polypeptide selected from Table H1, optionally 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))).

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

[0023] In some embodiments, the at least one antibody or antigen-binding fragment thereof agonizes or enhances the binding / signaling activity between an NRP2 polypeptide and at least one NRP2 ligand. In some embodiments, 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 SEMA3-A, SEMA-3B, SEMA-3C, SEMA-3D, SEMA-3F and SEMA-3G; - a plexin selected from one or more of plexins A1, A2, A3, A4 and D1; - a growth factor selected from one or more of fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF); - a growth factor receptor selected from one or more of fibroblast growth factor receptor (FGFR), hepatocyte growth factor receptor (HGFR) and platelet-derived growth factor receptor (PDGF); - 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; - Table N3, if necessary, 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 a HisRS N1 , HisRS N2 , HisRS N3 , HisRS N4 (SV9), HisRS N5 , HisRS C1 , HisRS C2 , HisRS C3 , HisRS C4 , HisRS C5 , HisRS C6 , HisRSC7 , HisRS C8 (SV11) and HisRS C9 HRS splice variants selected from one or more of (SV14) is selected from.

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

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

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

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

[0028] In some embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ) 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 to about 1.2 nM, about 0.9 to about 5.5 nM, about 0.9 to about 5 nM, or about 1 nM to about 10 nM. In some embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC) for each of the corresponding regions of (i) the human NRP2 polypeptide and (ii) the mouse NRP2 polypeptide. 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.

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

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

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

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

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

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

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

[0036] In some embodiments, the antagonist is a PD-L1 and / or 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 an antagonist of PD-1 optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to PD-1, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514, PDR001, and pidilizumab; the antagonist is an antagonist of CTLA-4 optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab and tremelimumab; the antagonist is an antagonist of IDO 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 an antagonist of TDO 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 an antagonist of TIM-3 optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds to TIM-3; the antagonist is an antagonist of LAG-3 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 an antagonist of VISTA optionally selected from one or more of an antibody or antigen-binding fragment or a 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 an antagonist of TIGIT optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds to TIGIT.

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

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

[0039] In some embodiments, the cancer vaccine is selected from one or more of Oncophage, human papillomavirus HPV vaccine, optionally Gardasil or Cervarix, hepatitis B vaccine, optionally Engerix-B, Recombivax HB or Twinrix, and sipuleucel-T (Provenge), or a vaccine selected from human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD 33, 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, insulin Tegrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAM) MF7), EGP40 pan-cancer antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

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

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

[0042] In some embodiments, the cell-based immunotherapeutic comprises cancer antigen-specific T cells, optionally ex vivo-derived T cells, hi 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.

[0043] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type I or type II), and an anti-microtubule agent.

[0044] In some embodiments, the alkylating agents are selected from one or more of nitrogen mustards (mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan, as appropriate), nitrosoureas (N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin, as appropriate), tetrazines (dacarbazine, mitozolomide, and temozolomide, as appropriate), aziridines (thiotepa, mitomycin, and diaziquone (AZQ), as appropriate), cisplatin and its derivatives (carboplatin and oxaliplatin, as appropriate), and non-classical alkylating agents (procarbazine and hexamethylmelamine, as appropriate); the antimetabolite is selected from one or more of antifolates (optionally, methotrexate and pemetrexed), fluoropyrimidines (optionally, 5-fluorouracil and capecitabine), deoxynucleoside analogs (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), bleomycin, 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 anti-microtubule agent is selected from one or more of taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0045] In some embodiments, at least one hormonal therapeutic agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of: progestogen (progestin), corticosteroid (optionally prednisolone, methylprednisolone or dexamethasone), insulin-like growth factor, VEGF-derived angiogenic factor and lymphangiogenic factor (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgen, estrogen and somatostatin analog. In some embodiments, the hormone antagonist is a hormone synthesis inhibitor, optionally an aromatase inhibitor or gonadotropin-releasing hormone (GnRH) or an analog thereof, and a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an antiandrogen, or an antibody to a hormone receptor, optionally cizutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, lobatum. and / or cyclosporine, selected from one or more of: cyclosporine, ...

[0046] In some embodiments, the kinase inhibitor is adavosertib, afatinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, 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.

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

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

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

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

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

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

[0053] In some embodiments, the antagonist is an antibody or antigen-binding fragment or small molecule that specifically binds to PD-L1 and / or PD-L2, an antagonist of PD-L1 and / or PD-L2 optionally selected from one or more of atezolizumab (MPDL3280A), avelumab (MSB0010718C) and durvalumab (MEDI4736), and optionally the cancer is selected from one or more of colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer and renal cell carcinoma; the antagonist is an antagonist of PD-1 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, where optionally the PD-1 antagonist is nivolumab, and the cancer is optionally selected from one or more of Hodgkin lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer; the PD-1 antagonist is pembrolizumab and the cancer is optionally selected from one or more of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial cancer; the antagonist is an antagonist of CTLA-4 optionally selected from one or more of an antibody or antigen-binding fragment or small molecule that specifically binds to CTLA-4, ipilimumab, tremelimumab, and optionally the cancer is selected from one or more of melanoma, prostate cancer, lung cancer, and bladder cancer; the antagonist is an 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, 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 an antagonist of TDO 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 an antagonist of TIM-3 optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds to TIM-3; the antagonist is an antagonist of LAG-3 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 an antagonist of VISTA optionally selected from one or more of an antibody or antigen-binding fragment or a 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 an antagonist of TIGIT optionally selected from one or more of an antibody or antigen-binding fragment or a small molecule that specifically binds to TIGIT.

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

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

[0056] In some embodiments, the cancer vaccine is Oncophage, a human papillomavirus HPV vaccine, optionally Gardasil or Cervarix, a hepatitis B vaccine, optionally Engerix-B, RecombivaxHB or 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, CD4 0, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate Receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-cancer antigen, B-cell activating factor (BAFF), platelets 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: mitochondrial 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.

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

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

[0059] In some embodiments, the cell-based immunotherapeutic comprises cancer antigen-specific T cells, optionally ex vivo-derived T cells, hi 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.

[0060] In some embodiments, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type I or type II), and an anti-microtubule agent.

[0061] In some embodiments, the alkylating agents are selected from one or more of nitrogen mustards (mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan, as appropriate), nitrosoureas (N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin, as appropriate), tetrazines (dacarbazine, mitozolomide, and temozolomide, as appropriate), aziridines (thiotepa, mitomycin, and diaziquone (AZQ), as appropriate), cisplatin and its derivatives (carboplatin and oxaliplatin, as appropriate), and non-classical alkylating agents (procarbazine and hexamethylmelamine, as appropriate); the antimetabolite is selected from one or more of antifolates (optionally, methotrexate and pemetrexed), fluoropyrimidines (optionally, 5-fluorouracil and capecitabine), deoxynucleoside analogs (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), bleomycin, 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 anti-microtubule agent is selected from one or more of taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0062] In some embodiments, at least one hormonal therapeutic agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of: progestogen (progestin), corticosteroid (optionally prednisolone, methylprednisolone or dexamethasone), insulin-like growth factor, VEGF-derived angiogenic factor and lymphangiogenic factor (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgen, estrogen and somatostatin analog. In some embodiments, the hormone antagonist is a hormone synthesis inhibitor, optionally an aromatase inhibitor or gonadotropin-releasing hormone (GnRH) or an analog thereof, and a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an antiandrogen, or an antibody to a hormone receptor, optionally cizutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, lobatum. and / or cisplatin, ...

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

[0064] In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer, optionally a metastatic cancer that expresses NRP2a and / or NRP2b. In some embodiments, the cancer is selected from one or more of melanoma (e.g., metastatic melanoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatocellular carcinoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), kidney cancer (e.g., renal cell carcinoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer. (a) bladder cancer that has metastasized to bone, liver, and / or lung; (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) kidney 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 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 that has metastasized to the bone, liver, lung, peritoneum, and / or vagina is selected from one or more of:

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

[0066] In some embodiments, the subject has and / or is selected for treatment based on having a disease associated with an increased level or expression of at least one NRP2 ligand (optionally an NRP2 ligand from Table N2 or Table N3, and / or an HRS polypeptide from Table H1) and / or its encoding mRNA compared to a population of healthy or matched control standards or subjects, and optionally the cancer has an increased level or expression of at least one NRP2 ligand and / or its encoding mRNA compared to cells or tissue of a non-cancerous control, optionally compared to non-cancerous cells or tissue of the same type as the cancer, and optionally the HRS polypeptide is selected for treatment based on having 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:

[0067] In some embodiments, the subject has an increased circulating or serum level of a soluble neuropilin 2 (NRP2) polypeptide (optionally selected from Table N1), either bound or free, compared to levels in a population of healthy or matched controls or subjects, optionally of about or at least about 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, or 4000, 5000, or, as appropriate, about 30-50, 50-100, 100-2000, 200-2000, 300-2000, 400-2000, 500-2000, 600-2000, 700-2000, 800-2000, 900-2000, 1000-2000, 2000-3000, 3000-4000, 4000-5000 pM of circulating or serum levels of soluble NRP2 polypeptide.

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

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

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

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

[0072] In some embodiments, at least one anti-NRP2 antibody reduces the aggressiveness of the cancer by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to an untreated control. In some embodiments, at least one anti-NRP2 antibody inhibits the rate of cancer migration or movement 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 embodiments, at least one anti-NRP2 antibody inhibits the rate of autophagy or endosomal maturation (optionally, endosomal acidification) of cancer or related immune cells by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to untreated controls. In some embodiments, at least one anti-NRP2 antibody enhances the sensitivity of the cancer to an additional agent selected from one or more of a chemotherapeutic agent, a hormonal therapy agent, and a 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 additional agent alone. In some embodiments, at least one anti-NRP2 antibody enhances the anti-tumor 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.

[0073] Certain embodiments include administering at least one anti-NRP2 antibody in an amount and frequency sufficient to achieve a steady-state or average circulating concentration of at least one anti-NRP2 antibody 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.

[0074] 1. A patient care kit comprising: (a) at least one antibody or antigen-binding fragment thereof that specifically binds to a human neuropilin-2 (NRP2) polypeptide; and optionally (b) at least one additional drug selected from a cancer immunotherapy agent, a chemotherapy agent, a hormone therapy agent, and a kinase inhibitor; Also included is a patient care kit including:

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

[0076] In some embodiments, the alkylating agents are selected from one or more of nitrogen mustards (mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan, as appropriate), nitrosoureas (N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin, as appropriate), tetrazines (dacarbazine, mitozolomide, and temozolomide, as appropriate), aziridines (thiotepa, mitomycin, and diaziquone (AZQ), as appropriate), cisplatin and its derivatives (carboplatin and oxaliplatin, as appropriate), and non-classical alkylating agents (procarbazine and hexamethylmelamine, as appropriate); the antimetabolite is selected from one or more of antifolates (optionally, methotrexate and pemetrexed), fluoropyrimidines (optionally, 5-fluorouracil and capecitabine), deoxynucleoside analogs (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), bleomycin, 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 anti-microtubule agent is selected from one or more of taxanes (optionally paclitaxel and docetaxel) and vinca alkaloids (optionally vinblastine, vincristine, vindesine, vinorelbine).

[0077] In some embodiments, at least one hormonal therapeutic agent is a hormone agonist or a hormone antagonist. In some embodiments, the hormone agonist is selected from one or more of: progestogen (progestin), corticosteroid (optionally prednisolone, methylprednisolone or dexamethasone), insulin-like growth factor, VEGF-derived angiogenic factor and lymphangiogenic factor (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgen, estrogen and somatostatin analog. In some embodiments, the hormone antagonist is a hormone synthesis inhibitor, optionally an aromatase inhibitor or gonadotropin-releasing hormone (GnRH) or an analog thereof, and a hormone receptor antagonist, optionally a selective estrogen receptor modulator (SERM) or an antiandrogen, or an antibody to a hormone receptor, optionally cizutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, lobatum. and / or cisplatin, ...

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

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

[0080] Also included is a detection system comprising cells expressing a human neuropilin 2 (NRP2) polypeptide, at least one NRP2 ligand (optionally a recombinant NRP2 ligand selected from Table N2 or Table N3, and / or a human histidyl-tRNA synthetase (HRS) polypeptide selected from Table H1), and optionally a human or humanized anti-NRP2 antibody or antigen-binding fragment thereof as defined herein, which modulates the interaction between the NRP2 polypeptide and at least one NRP2 ligand. In some embodiments, the anti-NRP2 antibody is labeled with a detectable label. In some embodiments, the NRP2 polypeptide is selected from Table N1. In some embodiments, the HRS polypeptide is an HRS splice variant selected from Table H1, optionally HisRS. N1 , HisRS N2 , HisRS N3 , HisRS N4 , HisRS N5 , HisRS C1 , HisRS C2 , HisRS C3 , HisRS C4 , HisRS C5 , HisRS C6 , HisRS C7 , HisRS C8 and HisRS C9In 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 is operably linked to a readout or indicator, such as a fluorescent or luminescent indicator, of the biological activity of the NRP2 polypeptide or at least one NRP2 ligand.

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

[0082] Also included are cell compositions comprising a population of engineered cells, wherein at least one cell comprises one or more polynucleotides encoding a human or humanized anti-NRP2 antibody or antigen-binding fragment thereof as defined herein, and wherein the cells are capable of growing in serum-free medium.

[0083] Also included is a cell growth device comprising a human or humanized anti-NRP2 antibody or antigen-binding fragment thereof as defined herein, a population of engineered cells wherein at least one cell comprises one or more polynucleotides encoding said 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]

[0084] [Figure 1] 1A-1B illustrate the general domain structure of neuropilins (1A) and the function of exemplary neuropilin co-receptors (1B).

[0085] [Figure 2-1] Figures 2A-2B illustrate the domain structure of NRP2 isoforms and an exemplary NRP2 ligand-binding domain. All NRP2 isoforms are identical throughout the MAM domain, followed by a short, potentially retained intron encoding GENFK. Next, there are two possible splicing frames (forms a and b), which encode different juxtamembrane domains, transmembrane helices, and cytoplasmic domains. Within the first is an alternative splicing acceptor (variant 3 or form c) that removes 17 amino acids. Each form has different spacing from the membrane, which may affect coreceptor specificity. The A / C and B forms also have different transmembrane domains, with the A / C form containing a dimerization motif (GXXXG). [Figure 2-2] Figures 2A-2B illustrate the domain structure of NRP2 isoforms and an exemplary NRP2 ligand-binding domain. All NRP2 isoforms are identical throughout the MAM domain, followed by a short, potentially retained intron encoding GENFK. Next, there are two possible splicing frames (forms a and b), which encode different juxtamembrane domains, transmembrane helices, and cytoplasmic domains. Within the first is an alternative splicing acceptor (variant 3 or form c) that removes 17 amino acids. Each form has different spacing from the membrane, which may affect coreceptor specificity. The A / C and B forms also have different transmembrane domains, with the A / C form containing a dimerization motif (GXXXG).

[0086] [Figure 3] Figure 3 shows binding of anti-NRP2 antibodies to clonal Expi293 cells expressing human NRP2. Four anti-NRP2 antibodies and an isotype control antibody (human IgG4) were added to human NRP2-expressing cells at the concentrations indicated, and cell binding was determined by FACS analysis as described in the Examples.

[0087] [Figure 4] Figure 4 shows binding of anti-NRP2 antibodies to clonal Expi293 cells expressing cynomolgus monkey NRP2. Four anti-NRP2 antibodies and an isotype control antibody (human IgG4) were added to cynomolgus monkey NRP2-expressing cells at the concentrations indicated, and cell binding was determined by FACS analysis as described in the Examples.

[0088] [Figure 5] Figures 5A-5B show the effect of anti-NRP2 antibodies on NRP2 receptor dimerization with KDR in the presence and absence of VEGF-A. The indicated antibodies were evaluated in the receptor dimerization assay as described in the Examples. 5A shows the normalized response for each individual well, which was calculated relative to the time point before addition of the ligand and then normalized to the no antibody / no ligand (baseline signal). Repeats were then run to obtain the mean response and standard deviation. 5B shows the net effect at 80 minutes.

[0089] [Figure 6] Figures 6A-6B show the effect of anti-NRP2 antibodies on NRP2 receptor dimerization with FLT4 in the presence and absence of VEGF-C. The indicated antibodies were evaluated in a receptor dimerization assay as described in the Examples. 6A shows the normalized response for each individual well, calculated relative to the time point before addition of the ligand and then normalized to the no antibody / no ligand (baseline signal). Repeated replicates were then performed to obtain the mean response and standard deviation. 6B shows the net effect at 80 minutes.

[0090] [Figure 7]Figures 7A-7B show the effect of anti-NRP2 antibodies on NRP2 receptor dimerization with PLXNA1 in the presence and absence of SEMA 3F. The indicated antibodies were evaluated in the receptor dimerization assay as described in the Examples. 7A shows the normalized response for each individual well, calculated relative to the time point before addition of the ligand and then normalized to the no antibody / no ligand (baseline signal). Repeats were then run to obtain the mean response and standard deviation. 7B shows the net effect at 80 minutes.

[0091] [Figure 8] Figures 8A-8B show the effect of anti-NRP2 antibodies on NRP2 receptor dimerization with KDR in the presence and absence of VEGF-A. The indicated antibodies were evaluated in the receptor dimerization assay as described in the Examples. 8A shows the normalized response for each individual well, which was calculated relative to the time point before addition of the ligand and then normalized to the no antibody / no ligand (baseline signal). Repeated measurements were then performed to obtain the mean response and standard deviation. 8B shows the net effect at 80 minutes.

[0092] [Figure 9] Figures 9A-9B show the effect of anti-NRP2 antibodies on NRP2 receptor dimerization with FLT4 in the presence and absence of VEGF-C. The indicated antibodies were evaluated in a receptor dimerization assay as described in the Examples. Figure 9A shows the normalized response for each individual well, calculated relative to the time point before addition of the ligand and then normalized to the no antibody / no ligand (baseline signal). Repeated measurements were then performed to obtain the mean response and standard deviation. Figure 9B shows the net effect at 80 minutes.

[0093] [Figure 10]Figures 10A-10B show the effect of anti-NRP2 antibodies on NRP2 receptor dimerization with PLXN A1 in the presence and absence of SEMA 3F. The indicated antibodies were evaluated in the receptor dimerization assay as described in the Examples. Figure 10A shows the normalized response for each individual well, calculated relative to the time point before addition of the ligand and then normalized to the no antibody / no ligand (baseline signal). Replicates were then processed to obtain the mean response and standard deviation. Figure 10B shows the net effect at 80 minutes.

[0094] [Figure 11] Figures 11A-11B show measurements of antibody binding to Expi293 cells overexpressing NRP2. 11A shows an example of an MFI profile stained with a mouse / human reactive control antibody, where the histogram of untransfected cells is shown in light gray, while cells overexpressing the mouse NRP2 I383V variant are shown in dark gray. Gating for NRP2-overexpressing cells is shown. 11B shows a chart of MFI staining of aNRP2-10v10 at 10 nM human and mouse wild-type NRP2, as well as the mouse NRP2 receptor containing individual mutations representing human residues.

[0095] [Figure 12] Figure 12 shows the structure of the b1 domain of human NRP2 from PBD file 2QQK. A ribbon diagram showing amino acids 280-426 is displayed. The side chains of residues 299Y, 354N, 416S, and 319T are highlighted in white. The distances between the alpha carbons of 299Y, 354N, and 416S in angstroms are shown along with the dotted lines.

[0096] [Figure 13]Figures 13A-13B show the inhibitory effect of anti-NRP2 antibodies on anchorage-independent growth and sensitivity to chemotherapy in triple-negative breast cancer (TNBC) cells in a 3D soft agar colony formation assay. Fluorescence readouts from the colony formation assay are shown as dot plots and mean ± SEM for MDA-MB-231 (13A) or BT549 (13B) TNBC cells treated with a-NRP2-10v10 versus isotype control hIgG4 at 100 nM in combination with the chemotherapy drugs cisplatin or 5-FU. Statistical significance is indicated by asterisks (*<0.05, **<0.01, ****<0.0001 by Student's t-test). In both cases, the antitumor effect of anti-NRP2 antibodies is significantly more pronounced in combination with cisplatin or 5-FU.

[0097] [Figure 14] Figure 14 shows the inhibitory effect of anti-NRP2 antibodies in combination with chemotherapeutic agents and a VEGF-A antibody drug (bevacizumab) on the anchorage-independent growth of TNBC MDA-MB-231 cells in a 3D methylcellulose colony formation assay. Luminescence readouts of the colony formation assay are shown as dot plots and mean ± SEM for TNBC MDA-MB-231 cells treated with aNRP2-10v10 at 100 nM versus isotype control hIgG4 in combination with the chemotherapeutic agents cisplatin (2 doses) or bevacizumab (100 nM). Statistical significance is indicated by asterisks (**p<0.01, ***p<0.001, ****p<0.0001 by Student's t-test).

[0098] [Figure 15] Figure 15 shows the inhibition of human lymphatic endothelial cell (HLEC) migration to vascular endothelial growth factor C (VEGF-C) by antibodies aNRP2-10v10 and aNRP2-11v7. HLEC were inhibited by both NRP2 antibodies to levels consistent with positive control antibodies (αKDR or 3C5) against VEGFR2 or VEGFR3, respectively.

[0099] [Figure 16] Figure 16 shows that treatment of cells with antibody aNRP2-14v10 (NRP2_14) or aNRP2-11v7 (NRP2_11) blocks the reduction in phospho-Akt levels upon Semaphorin 3F treatment in U251 glioblastoma cells. Treatment of U251 cells with Sema3F results in an approximately 50% reduction in intracellular phospho-Akt levels that can be blocked by both NRP2_11 and NRP2_14, but not by the VEGF-C-blocking antibody aNRP2-10v10 or by mIgG1 and hIgG4 control antibodies.

[0100] [Figure 17] Figure 17 shows a schematic of Sema3F signaling through NRP2. Treatment with Sema3F results in blocking PI3K activity and thereby reducing phospho-Akt levels.

[0101] [Figure 18] Figure 18 shows that antibodies aNRP2-14v10 and aNRP22-11v7, but not antibody aNRP2-10v10, block Semaphorin 3F-mediated inhibition of AKT phosphorylation. Treatment of U251 cells with Sema3F resulted in an approximately 50% reduction in intracellular phospho-Akt levels, which can be blocked by both semaphorin blocking antibodies aNRP2-11v7 and aNRP2-14v10, but not by the VEGF-C-blocking antibody aNRP2-10v10 or by the mIgG1 and hIgG4 control antibodies.

[0102] [Figure 19]Figures 19A-19B show that the murine surrogate VEGF-blocking antibody aNRP2-28 inhibits tumor growth in a murine melanoma model (B16.F10). Animals bearing B16-F10 tumors and treated with three doses of the murine surrogate antibody aNRP2-28 recognize murine NRP2 (and are a functional surrogate for the anti-human NRP2 antibody aNRP2-10v10). Figure 19A shows that treatment with the murine surrogate antibody aNRP2-28 (gray triangles) demonstrated tumor growth inhibition compared to the IgG control group (black squares) and bevacizumab (black triangles), reaching statistical significance (p≦0.05) on days 14, 16, and 19. Figure 19B shows the final tumor weights for each treatment group.

[0103] [Figure 20] Figures 20A-20B show the anti-metastatic effect of a VEGF blocker (aNRP2-28) in a model of spontaneous metastasis (4T1). Figure 20A shows that animals bearing 4T1 tumors and treated with the mouse surrogate antibody aNRP2-28 ultimately showed a reduction in metastatic nodules in the lungs compared with IgG controls (black bars), reaching statistical significance (p≦0.05). Figure 20B shows serum antibody concentrations in the lungs of pulmonary metastases, demonstrating a linear correlation between final antibody concentration and the number of metastatic nodules.

[0104] [Figure 21] Figure 21 shows the synergistic effect of VEGF blockers (aNRP2-10v10 / aNRP2-28) in combination with cisplatin in a TNBC xenograft model (MDA-MB-231). The addition of pooled anti-NRP2 antibodies (black symbols) to the cisplatin treatment regimen increased the tumor inhibitory effect of cisplatin starting at day 40 post-inoculation compared to control animals (gray symbols), reaching statistical significance at day 60.

[0105] [Figure 22]Figure 22 shows that a VEGF blocker (aNRP2-10v10) enhances the activity of the chemotherapy drug 5-FU in an NSCLC xenograft model (A549). The combination of 5-FU and aNRP2-10 (black symbols) performed better than 5-FU with control IgG (gray symbols), demonstrating that targeting NRP2 increases the efficacy of the chemotherapy drug 5-FU in an art-accepted model of NSCLC. Statistical significance was reached 51 days after cell inoculation.

[0106] [Figure 23] Figures 23A-23B show that a VEGF blocker (aNRP2-28) inhibits neolymphangiogenesis in an animal model of corneal injury. Figure 23A shows representative image scans of Lyve-1 (a lymphangiogenesis marker) stained corneas from the IgG control and aNRP2-28 groups. The mean % Lyve-1+ area is plotted for each treatment group. Figure 23B shows a clear trend toward reduced sprouting of lymphatic vessels in the aNRP2-10 treatment group compared to the IgG control group. DETAILED DESCRIPTION OF THE INVENTION

[0107] Detailed Description Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this disclosure, 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. Any patent application to which this application claims priority is also incorporated herein by reference in its entirety, in the manner described above for publications and references.

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

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

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

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

[0112] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent, such as an antibody, and can be used in an animal to generate antibodies capable of binding to that antigen's epitope. An antigen may have one or more epitopes. As used herein, the term "antigen" includes a substance that, under appropriate conditions, can induce an immune response to the substance and react with the products of the immune response. For example, an antigen can be recognized by antibodies (humoral immune response) or sensitized T lymphocytes (helper T 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 is desired by an antibody, regardless of whether the substance is immunogenic. For such antigens, antibodies can be identified by recombinant methods, independent of any immune response.

[0113] "Antagonist" refers to a biological structure or chemical agent that prevents or otherwise reduces the physiological action of another drug or molecule. In some instances, antagonists specifically bind to other drugs or molecules. Includes full antagonists and partial antagonists.

[0114] "Agonist" refers to a biological structure or chemical agent that increases or enhances 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 included.

[0115] The term "anergy" refers to the functional inactivation of T cell or B cell responses to restimulation with an antigen.

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

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

[0118] 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 located in the variable region of the immunoglobulin molecule.

[0119] The term "antigen-binding fragment," as used herein, refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy chain and / or light chain that binds to an antigen of interest. In this regard, an antigen-binding fragment of an antibody described herein is a V CDR from an antibody that binds to a target molecule. H Array and V L It may comprise one, two, three, four, five or all six CDRs of the sequence.

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

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

[0122] Immunological binding generally refers to the type of noncovalent interaction that occurs between an immunoglobulin molecule and an antigen specific for that immunoglobulin as a result of, for example, but not limited to, electrostatic, ionic, hydrophilic and / or hydrophobic attractions 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 in terms of the dissociation constant (Kd) of the interaction, with a smaller Kd representing a higher affinity. The immunological binding of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, where these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, both the "on" rate constant (Kon) and the "off" rate constant (Koff) can be determined by calculating the concentrations and the actual rates of association and dissociation. The ratio Koff / Kon allows for the cancellation of all parameters not related to affinity and is therefore equal to the dissociation constant Kd. As used herein, the term "affinity" includes the equilibrium constant for the reversible binding of two drugs, and is not necessarily related to Kd or EC 50 The affinity of a binding protein for a ligand, e.g., the affinity of an antibody for an epitope, can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM). As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. In some embodiments, affinity is measured using the 50% effective concentration (EC 50 ) which refers to the concentration of an agent, such as an antibody or anti-NRP2 antibody disclosed herein, that induces a response halfway between baseline and maximum after a specific exposure time. 50 is commonly used as a measure of antibody potency.

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

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

[0125] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy and light chain CDRs inserted between a set of heavy and light chain framework regions (FRs), respectively, which provide support for the CDRs and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3," respectively. Thus, an antigen-binding site comprises six CDRs, comprising the CDR sets 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 analysis of several antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact occurring with the heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0126] As used herein, the term "FR set" refers to the four adjacent amino acid sequences that constitute the CDRs of a CDR set in a heavy or light chain V region. While some FR residues may contact a bound antigen, the FRs, particularly those directly adjacent to the CDRs, are primarily responsible for folding 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. Regardless of the exact amino acid sequence of the CDRs, it is generally recognized that there are conserved structural regions of the FRs that affect the folded 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.

[0127] The structure and location of immunoglobulin variable domains are described in Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. USDapartment of Health and Human Services. 1987 and its revisions.

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

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

[0130] In certain embodiments, single-chain Fv (scFv) antibodies are contemplated. For example, kappabodies (Ill et al., Prot. Eng. 10:949-57, 1997); minibodies (Martin et al., EMBO J 13:5305-9, 1994); diabodies (Holliger et al., PNAS90:6444-8, 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 used to select antibodies with desired specificity. These can be prepared using standard molecular biology techniques, following the teachings of the present application regarding selection.

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

[0132] In certain embodiments, the antibodies or antigen-binding fragments described herein are in the form of "diabodies." Diabodies are multimers of polypeptides, each of which comprises a first domain comprising an immunoglobulin light chain binding region and a second domain comprising an immunoglobulin heavy chain binding region, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site. An antigen-binding site is formed by the association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer (WO 94 / 13804). 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 synthesized, for example, by genetically modifying the VH domain. It can be constructed by gene fusion (see WO 94 / 13804; and Holliger et al., PNAS USA. 90:6444-6448, 1993).

[0133] Also included are minibodies containing scFvs linked to CH3 domains (Hu et al., Cancer Res. 56:3055-3061, 1996). 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. WO 94 / 13804; and Reiteret et al., Nature Biotech. 14:1239-1245, 1996.

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

[0135] 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) of appropriate binding specificity can be easily selected from libraries using phage display (WO 94 / 13804). If one arm of the diabody is held constant, for example, with specificity for antigen X, the other arm can then be varied to create a library from which antibodies of appropriate specificity can be selected. Bispecific whole antibodies can also be generated by knob-into-hole engineering (Ridgeway et al., Protein Eng., 9:616-621, 1996).

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

[0137] In certain embodiments, the antibodies and antigen-binding fragments 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 E. coli (see U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see U.S. Pat. No. 6,838,254)). The production process is scalable, and multi-kilogram quantities of nanobodies have been produced. Nanobodies may also be formulated as ready-to-use solutions with long shelf lives. The Nanoclone method (see WO 06 / 079372) is a proprietary method for generating nanobodies against desired targets based on automated high-throughput selection of B cells.

[0138] In some embodiments, the antibody or antigen-binding fragment described herein is in the form of an aptamer (see, for example, Ellington et al., Nature. 346, 818-22, 1990; and Tuerk et al., Science. 249, 505-10, 1990, which are incorporated herein by reference). Examples of aptamers include nucleic acid aptamers (e.g., DNA aptamers, RNA aptamers) and peptide aptamers. Nucleic acid aptamers generally refer to nucleic acid species that have been engineered by repeated rounds of in vitro selection, such as SELEX (systematic evolution of ligands by exponential enrichment) or equivalent methods, to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. See, for example, U.S. Patent Nos. 6,376,190 and 6,387,620, which are incorporated herein by reference.

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

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

[0141] In some embodiments, the antibody or antigen-binding fragment described herein is in the form of Adnectin. Adnectin refers to a class of targeting biologics derived from human fibronectin, an abundant extracellular protein that naturally binds to other proteins. For example, see US Patent Application Publication Nos. 2007 / 0082365; 2008 / 0139791; and 2008 / 0220049, which are incorporated herein by reference. Adnectins typically consist of a natural fibronectin backbone and multiple targeting domains of specific parts of human fibronectin. The targeting domain can be engineered to enable Adnectin to specifically recognize NRP2 polypeptide or its epitope.

[0142] In some embodiments, the antibody or antigen-binding fragment described herein is in the form of anticalin. Anticalin refers to a class of antibody mimetics that are typically synthesized from human lipocalin, a family of binding proteins with hypervariable loop regions supported by a structurally rigid framework. See, for example, US Patent Application Publication No. 2006 / 0058510. Anticalins typically have a size of about 20 kDa. Anticalins can be characterized by a barrel structure formed by eight antiparallel β-strands (stable β-barrel backbone) connected in pairs by four peptide loops and linked α-helices. In certain aspects, conformational abnormalities are created in the hypervariable loop regions to achieve specific binding. See, for example, Skerra, FEBS J. 275:2677-83, 2008, which is incorporated herein by reference.

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

[0144] Heavy chain dimers, such as antibodies from camelids and sharks, are also included. Camelid and shark antibodies contain two homodimeric pairs of V-like and C-like domains (neither of which has a light chain). Because the VH region of heavy chain dimeric IgG in camelids does not interact hydrophobically with light chains, the region in the heavy chain that normally contacts the light chain is modified to 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 (referred to as the V-NAR domain) and five C-like constant domains (C-NAR domains).

[0145] In camelids, the diversity of the antibody repertoire is determined by complementarity-determining regions (CDRs) 1, 2, and 3 in the VH or VHH regions. CDR3 in camelid VHH regions is characterized by its relatively long length, averaging 16 amino acids (Muylderman 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 has an average of 9 amino acids. Libraries of antibody variable regions from camelids maintain the in vivo diversity of camelid variable regions and can be generated, for example, by the method disclosed in U.S. Patent Application Publication No. 20050037421, published February 17, 2005.

[0146] In certain embodiments, the antibody or antigen-binding fragment thereof is humanized. These embodiments refer to chimeric molecules, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either a complete variable domain fused to a constant domain or only the CDRs grafted into appropriate framework regions in the variable domain. The epitope-binding site may be wild-type or may be modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but leaves open the possibility of an immune response to the foreign variable region (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al., J. Immunol. 2004, 103:1047-1052, 1989). al., PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327, 1988). Illustrative methods for antibody humanization include those described in U.S. Patent No. 7,462,697.

[0147] Another approach focuses not only on providing constant regions of human origin, but also on modifying the variable regions so as to reshape them as closely as possible to human types. It is known that the variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) that vary depending on the epitope in question and determine the binding ability, flanked by four framework regions (FRs) that are relatively conserved in a given species and that presumably provide a scaffold for the CDRs. When a non-human antibody is prepared for a specific epitope, the variable region can be "reshaped" or "humanized" by grafting CDRs from the non-human antibody into the FRs present in the modified human antibody. The application of this technique to various antibodies has been reviewed by Sato et al., Cancer Res. 53:851-856, 1993; Riechmann et al., Nature 332:323-327, 1988; Verhoeye 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; Gorma 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; Carteret al., PNAS USA.89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154,1992 reported by . In some embodiments, a humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from the mouse antibody). In other embodiments, a humanized antibody has one or more (1, 2, 3, 4, 5, 6) CDRs that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

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

[0149] As used herein, a subject who is "at risk" of developing a disease or adverse reaction may or may not have detectable disease or disease symptoms, and may or may not show detectable disease or disease symptoms before the treatment method described herein. "At risk" refers to a subject who has one or more risk factors, which are measurable parameters that correlate with the occurrence of disease, as described herein and known in the art. A subject who has one or more of these risk factors has a higher probability of developing a disease or adverse reaction than a subject who does not have one or more of these risk factors.

[0150] "Biocompatible" refers to a material or compound that generally does not impair the biological function of a cell or subject and does not cause any degree of unacceptable toxicity, including allergies and disease states.

[0151] The term "bond" refers to a direct association between two molecules due to covalent, electrostatic, hydrophobic and ionic and / or hydrogen-bonding interactions, including, for example, interactions such as salt bridges and water bridges.

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

[0153] By "coding sequence" is meant any nucleic acid sequence that contributes to the coding of the polypeptide product of a gene, whereas the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the coding of the polypeptide product of a gene.

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

[0155] "Consisting of" means including and limited to everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that no other elements are present. "Consisting essentially of" means including any elements listed after this phrase, limited to other elements that do not interfere with or contribute to the activity or action described in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that 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.

[0156] The term "effector function" or "ADCC effector function" in the context of an antibody refers to the antibody's ability to participate in other arms of the immune system, including, for example, activation of the classical complement pathway or engagement of Fc receptors. 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, which itself binds to target cells. Fc receptors (FcRs) are key immunoregulatory receptors involved in antibody-mediated (humoral) immune responses to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM), and FcδR (IgD). There are at least three classes of receptors for human IgG found on leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb, and FcγRIIc), and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classified as a high-affinity receptor (KD in the nanomolar range), whereas FcγRII and FcγRIII are low- to intermediate-affinity receptors (KD in the micromolar range). Upon Fc binding, signaling pathways are initiated that result in the secretion of various substances, such as lytic enzymes, perforin, granzymes, and tumor necrosis factor, which mediate target cell destruction. The level of ADCC effector function varies for human IgG subtypes. This depends on the allotype and the specific FcvR; briefly, ADCC effector function is "high" for human IgG1 and IgG3 and "low" for IgG2 and IgG4.

[0157] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents, and / or containers that contain at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effect on a subject), preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically gram-negative bacteria, although endotoxins are not found in Listeria. Endotoxins can be found in Gram-positive bacteria such as S. monocytogenes. The most widespread endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), which are found in the outer membrane of various Gram-negative bacteria and represent a central pathogenic property in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans can produce fever, a drop in blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

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

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

[0160] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope includes the 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 specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope can be continuous or discontinuous with respect to the primary structure of an antigen, e.g., an NRP2 polypeptide. In certain embodiments, an epitope comprises, consists of, or consists essentially of about, at least about, or at most about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids (i.e., 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.

[0161] An "epitope" includes a portion of an antigen or other macromolecule that can form a binding interaction with the variable region binding pocket of a binding protein. Such a binding interaction can be referred to as an intermolecular contact with one or more amino acid residues of a CDR. Antigen binding can involve a CDR3 or CDR3 pair. An epitope can be a linear peptide sequence (i.e., "continuous") or can be composed of discontinuous amino acid sequences (i.e., "conformational" or "discontinuous"). A binding protein can recognize one or more amino acid sequences; therefore, an epitope can define two or more distinct amino acid sequences. Epitopes recognized by binding proteins can be determined by peptide mapping and sequence analysis techniques well known to those skilled in the art. A "cryptic epitope" or "cryptic binding site" is an epitope or binding site of a protein sequence that is not exposed or substantially protected from recognition in an unmodified polypeptide but can be recognized by a binding protein in a denatured or proteolytically digested polypeptide. In the unmodified polypeptide structure, 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 may be buried inside the polypeptide.Candidates for cryptic epitopes can be identified, for example, by examining the three-dimensional structure of unmodified polypeptide.

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

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

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

[0165] The terms "modulating" and "altering" include "increasing," "enhancing," or "stimulating," and "decreasing," or "reducing," typically by a statistically significant or physiologically significant amount or degree compared to a control. An "increased," "stimulated," or "enhanced" amount is typically a "statistically significant" amount and can include a 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold or higher (e.g., 500, 1000-fold) (including all integers and ranges therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) increase over the amount produced by no composition (e.g., absence of agent) or a control composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount and can 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% reduction (including all integers and ranges therebetween) from the amount produced by no composition (e.g., absence of agent) or a control composition. Examples of comparisons and "statistically significant" amounts are described herein.

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

[0167] The terms "polypeptide," "protein," and "peptide" are used interchangeably and refer to a polymer of amino acids, not limited to any particular length. The term "enzyme" includes polypeptide or protein catalysts. These terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The term "polypeptide" or "protein" refers to one or more chains of amino acids, each chain comprising amino acids covalently linked by peptide bonds. The polypeptide or protein can comprise multiple chains noncovalently and / or covalently linked together by peptide bonds, and includes native proteins, i.e., proteins produced by specific non-recombinant cells occurring in nature, or by genetically engineered or recombinant cells, including molecules having the amino acid sequence of the native protein or molecules with deletions, additions, and / or substitutions of one or more amino acids of the native sequence. In certain embodiments, a polypeptide is a "recombinant" polypeptide produced by a recombinant cell containing one or more recombinant DNA molecules, and is typically composed of a heterologous polynucleotide sequence or combination of polynucleotide sequences not otherwise found in the cell.

[0168] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA. The terms refer to polymeric forms of nucleotides, typically at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of any type 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 that have been isolated free of total genomic DNA of a particular species. Thus, an isolated DNA segment that encodes a polypeptide refers to a DNA segment that contains one or more coding sequences and that has been substantially isolated from or purified free of total genomic DNA of the species from which the DNA segment is obtained. Non-coding polynucleotides that do not encode a polypeptide (e.g., primers, probes, oligonucleotides) are also included. Recombinant vectors include, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, and the like.

[0169] Additional coding or non-coding sequences may, but need not, be present within the polynucleotides described herein, and polynucleotides may, but need not, be linked to other molecules and / or supporting materials. Thus, a polynucleotide or expressible polynucleotide, regardless of the length of the coding sequence itself, may be associated with other sequences, for example, expression control sequences.

[0170] "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, subcellular localization signals, and the like, which are capable of influencing the transcription or translation of a coding sequence in a host cell, or its intracellular or cellular location. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

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

[0172] Numerous promoters, including constitutive promoters, inducible promoters, and repressible promoters from various different sources, are well known in the art. Representative sources include, for example, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available or can be synthesized based on publicly available sequences online, or sequences from depositories such as ATCC and other commercial or personal sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, 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-inducible recombinase system (Indra et al., Nuc. Acid. Res. (1999) 27 (22): 4324-4327; Nuc. Acid. Res. (2000) 28(23): e99; U.S. Pat. No. 6,464,758). US Patent No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol. (2005) 308: 123-144), or any other method in the art suitable for expression in the desired cells. Any known promoter can be used.

[0173] An "expressible polynucleotide" includes a cDNA, RNA, mRNA, or other polynucleotide comprising at least one coding sequence and, optionally, at least one expression control sequence, e.g., a transcriptional and / or translational regulatory element, which, upon introduction into a cell, e.g., a cell in a subject, is capable of expressing the encoded polypeptide.

[0174] 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 examples, retrovirus vectors are derivatives of murine or avian retroviruses, or lentivirus vectors. Examples of retrovirus vectors that can insert a single foreign gene include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). Some additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate genes for selectable markers, allowing transduced cells to be identified and generated. For example, a polypeptide sequence of interest can be inserted into the viral vector along with another gene encoding a ligand for a receptor on a specific target cell, making the vector target-specific. Retroviral vectors can be made target-specific, for example, by inserting a polynucleotide encoding a protein. Illustrative targeting may be achieved by using antibodies to target the retroviral vector. Those skilled in the art will know, or can readily ascertain, without undue experimentation, specific polynucleotide sequences that can be inserted into a retroviral genome to enable target-specific delivery of the retroviral vector.

[0175] In certain embodiments, the expressible polynucleotide is a modified RNA polynucleotide or a modified mRNA polynucleotide, for example, a non-naturally occurring RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide comprises one or more modified or unnatural bases, such as nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and / or uracil (U). In some embodiments, the modified mRNA comprises one or more modified or unnatural internucleotide bonds. Expressible RNA polynucleotides for delivering encoded therapeutic polypeptides are described, for example, in Kormannet 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 herein by reference in their entirety.

[0176] The term "isolated" polypeptide or protein, as referred to herein, means that the protein of interest (1) is free from at least some other proteins with which it would typically be found in nature; (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 with which it is naturally associated; (5) is not associated (by covalent or non-covalent interactions) with portions of proteins with which the "isolated protein" is naturally associated; (6) is operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated; or (7) is not naturally occurring. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, may be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free from proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, preventative, research, or otherwise).

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

[0178] "Lipid nanoparticles" or "solid lipid nanoparticles" refer to one or more spherical nanoparticles having an average diameter of about 10 to about 1000 nanometers and comprising a solid lipid core matrix capable of solubilizing lipid-soluble molecules. The lipid core is stabilized by a surfactant (e.g., an emulsifier) ​​and can 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), as well as combinations thereof. Lipid nanoparticles are described, for example, in Petrillie et al., Curr Pharm Biotechnol. 15:847-55, 2014; and U.S. Patent Nos. 6,217,912 and 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 herein by reference in their entirety.Certain compositions described herein are formulated with one or more lipid nanoparticles.

[0179] 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 plays a role in the pathophysiology of the disease or condition. In some examples, NRP2-related diseases are modulated by the anti-NRP2 antibodies of the present disclosure by altering the interaction of NRP2 with at least one NRP2 ligand, thereby affecting the activity, signaling, expression, and / or spatial distribution of NRP2. Exemplary NRP2-related diseases and conditions include, but are not limited to, cancer and cancer-related diseases or conditions, including cancer cell growth, cancer initiation, cancer migration, cancer cell adhesion, invasion, chemoresistance, and metastasis. Diseases associated with inflammation, 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). Additional examples include diseases related to lymphangiogenesis, lymphangiogenesis, and lymphatic vessel damage, including edema, lymphedema, secondary lymphedema, inappropriate fat absorption and deposition, excessive fat deposition, and vascular permeability. Diseases related to infection, including latent infection, and diseases related to allergic disorders / diseases and allergic reactions, including chronic obstructive pulmonary disorder (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. Additional examples include, among others, granulomatous inflammatory diseases, including sarcoidosis and granulomas, and diseases related to fibrotic diseases, including endometriosis, fibrosis, endothelial-mesenchymal transition (EMT), and wound healing. Also included are diseases related to inappropriate smooth muscle contractility, smooth muscle compensation and decompensation, vascular smooth muscle cell migration and / or adhesion, and diseases related to inappropriate autophagy, phagocytosis and efferocytosis.Also included are diseases related to inappropriate migratory cell migration, as described herein.Additional examples include neurological diseases, including diseases related to peripheral nervous system remodeling and pain sensation.Also included are diseases related to bone development and / or bone remodeling.Typically, the term "inappropriate" refers to an activity or characteristic that is associated with or causes a medical condition or disease state.

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

[0181] Certain embodiments include biologically active "variants" and "fragments" of the polypeptides (e.g., antibodies) and encoding polynucleotides described herein. "Variant" contains one or more substitutions, additions, deletions, and / or insertions compared to a reference polypeptide or polynucleotide (see, e.g., Tables and Sequence Listing). Variant polypeptides or polynucleotides comprise 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 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 acid or nucleotide additions, deletions, insertions, or substitutions, while substantially maintaining the activity of the reference sequence. In certain embodiments, the additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[0182] The term "sequence identity" or, for example, "a sequence 50% identical to" as used herein refers to the degree to which sequences are identical nucleotide by nucleotide or amino acid by amino acid across a comparison window.Therefore, "sequence identity percentage" can be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur, to obtain the number of identical positions, dividing the number of identical positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity.The optimal alignment of sequences for aligning a comparison window can be calculated by computer implementation of an algorithm (Wisconsin Genetics Software This may be performed by using a sequence matching algorithm (GAP, BESTFIT, FASTA, and TFASTA in the Genetics Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA), or by verifying and finding the best alignment (i.e., the one that gives the highest percentage of homology over the comparison window) produced by any of a variety of methods selected. For example, the method described by Altschul et al., Nucl. Acids Res. 25:3389, 1997. For purposes of illustration, reference may be made to the BLAST family of programs, as disclosed herein.

[0183] 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 as a concentration, either by mass of solute per unit volume of solvent (e.g., g of solute per kg of solvent, g per dL (100 mL), mg / mL, etc.), molar concentration, molality, mole fraction, or other similar concentration description. The maximum equilibrium amount of solute that can be dissolved per volume of solvent is the solubility of the solute in the solvent under specified conditions, including 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, such as PBS or NaCl (with or without NaPO). In specific embodiments, solubility is measured at a relatively low pH (e.g., pH 6.0) and a relatively high salt (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 can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain embodiments, the agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml at room temperature or 37°C.

[0184] A "subject" or "subject in need thereof" or a "patient" or "patient in need thereof" includes a mammalian subject, such as a human subject.

[0185] "Substantial" or "essentially" means nearly entirely or nearly completely, e.g., 95%, 96%, 97%, 98%, 99% or more of a given amount of some part.

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

[0187] "Therapeutic response" refers to an improvement in symptoms (whether sustained or not) upon administration of one or more therapeutic agents.

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

[0189] As used herein, "treatment" of a subject (e.g., a mammal such as a human) or cell is any type of intervention used to attempt to alter the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and can be performed either prophylactically or after the onset of a pathological event or contact with a pathogenic agent. "Prophylactic" treatment is also included, which may be aimed at reducing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily refer to the complete eradication, cure, or prevention of the disease or condition or its associated symptoms.

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

[0191] Each embodiment in this specification shall apply to all other embodiments unless expressly stated otherwise. Anti-NRP2 antibody

[0192] 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., prevents) 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.

[0193] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a complementarity-determining region V H CDR1, V H CDR2 and V H The heavy chain variable region (V H ) sequence, and complementarity-determining region V L CDR1, V L CDR2 and V L The light chain variable region (V L ) sequences. H , V H CDR1, V H CDR2, V H CDR3, V L , V L CDR1, V L CDR2 and V L The CDR3 sequences are provided in Tables A1, A2 and A3 below. [Table A1-1] [Table A1-2] [Table A2-1] [Table A2-2] [Table A2-3]

[0194] Thus, in certain embodiments, the antibody or antigen-binding fragment thereof A complementarity determining region V selected from Table A1 that specifically binds to a human NRP2 polypeptide (e.g., selected from Table N1). H CDR1, V H CDR2 and V H Heavy chain variable region (V) including CDR3 sequences and their variants H ) array; and A complementarity determining region V selected from Table A1 that specifically binds to a human NRP2 polypeptide (e.g., selected from Table N1). L CDR1, V L CDR2 and V L The light chain variable region (V) including CDR3 sequences and their variants L ) array.

[0195] In certain embodiments, the CDR sequences are as follows: V H CDR1, V H CDR2 and V H CDR3 sequences include SEQ ID NOs: 1 to 3 and variants thereof, respectively, and V L CDR1, V L CDR2 and V L the CDR3 sequences comprise SEQ ID NOs: 4 to 6 and variants thereof, respectively; V H CDR1, V H CDR2 and V H CDR3 sequences include SEQ ID NOs: 7 to 9 and variants thereof, respectively, and V L CDR1, V L CDR2 and V L the CDR3 sequences comprise SEQ ID NOs: 10 to 12 and variants thereof, respectively; V H CDR1, VH CDR2 and V H CDR3 sequences include SEQ ID NOs: 13 to 15 and variants thereof, respectively, and V L CDR1, V L CDR2 and V L the CDR3 sequences comprise SEQ ID NOs: 16 to 18 and variants thereof, respectively; V H CDR1, V H CDR2 and V H CDR3 sequences include SEQ ID NOs: 19 to 21 and variants thereof, respectively, and V L CDR1, V L CDR2 and V L the CDR3 sequences comprise SEQ ID NOs: 22 to 24 and variants thereof, respectively; V H CDR1, V H CDR2 and V H CDR3 sequences include SEQ ID NOs: 25 to 27 and variants thereof, respectively, and V L CDR1, V L CDR2 and V L the CDR3 sequences comprise SEQ ID NOs: 28 to 30 and variants thereof, respectively; V H CDR1, V H CDR2 and V H CDR3 sequences include SEQ ID NOs: 31 to 33 and variants thereof, respectively, and V L CDR1, V L CDR2 and V L the CDR3 sequences comprise SEQ ID NOs: 34 to 36 and variants thereof, respectively; V H CDR1, V H CDR2 and V H CDR3 sequences include SEQ ID NOs: 34 to 39 and variants thereof, respectively, and V L CDR1, V L CDR2 and V L the CDR3 sequences comprise SEQ ID NOs: 40 to 42 and variants thereof, respectively; V H CDR1, V HCDR2 and V H CDR3 sequences include SEQ ID NOs: 57 to 59 and variants thereof, respectively, and V L CDR1, V L CDR2 and V L the CDR3 sequence comprises SEQ ID NOs: 60-62 and variants thereof, respectively; or V H CDR1, V H CDR2 and V H CDR3 sequences include SEQ ID NOs: 63 to 65 and variants thereof, respectively, and V L CDR1, V L CDR2 and V L The CDR3 sequences include SEQ ID NOs: 66 to 68 and variants thereof, respectively.

[0196] Also included are variants thereof, including affinity matured variants that bind to human NRP2, e.g., variants that bind to one or more CDR regions, e.g., one or more VCRs described herein. H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and / or V L It has 1, 2, 3, 4, 5 or 6 alterations in the CDR3 sequence. Exemplary "alterations" include amino acid substitutions, additions and deletions.

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

[0198] In some embodiments, V L The sequence is at least 80, 85, 90, 95, 97, 98, 99 or 100% identical to a sequence selected from Table A2, e.g., V LSequences include those with 1, 2, 3, 4 or 5 changes in one or more framework regions.

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

[0200] Also included are variants thereof, for example, variants having 1, 2, 3, 4 or 5 alterations in one or more framework regions. Exemplary "alterations" include amino acid substitutions, additions and deletions.

[0201] Table A3 below summarizes the CDRs of exemplary redundant antibodies, including exemplary consensus CDR sequences, and provides the amino acid codes therefor. [Table A3-1] [Table A3-2]

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

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

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

[0205] 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 tissue-specific macrophages, including alveolar macrophages. It is also expressed in endothelial and epithelial cells in the lung and other tissues, as well as in muscle cells (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). See the reference.

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

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

[0208] NRP2 is a single transmembrane receptor with a predominant extracellular region containing two CUB domains (a1 / a2 complex domains), two factor V / factor VIII homology domains (b1 / b2 complex domains), a MAM domain (c domain) (see Figures 1A-1B), and a short juxtamembrane region (spanning the plasma membrane) connecting the c domain to the transmembrane domain. 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 higher affinity for SEMA3F and 3G; in contrast, SEMA3A, 3B, and 3E interact preferentially with NRP1. Both NRP1 and NRP2 have similar affinity for SEMA3C. The b1b2 complex domain interacts with several growth factors containing heparin-binding domains, including VEGF C and D, placental growth factor (PIGF)-2, fibroblast growth factor (FGF), galectins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF)-beta (see, e.g., Prud'homme et al., Oncotarget. 3:921-939, 2012). NRP2 also interacts with various growth factor-specific receptors, and interactions with these receptors occur independently of binding to SEMA. In this context, it has been shown that integrins, as well as growth factor receptors such as VEGF receptors, TGF-beta receptors, c-Met, EGFR, FGFR, and PDGFR, interact with NRPs, which generally appears to increase the affinity of their respective ligands for their receptors and modulate downstream signaling. The c domain (Mam) is not thought to be directly required for ligand binding but may influence ligand specificity, receptor signaling, and NRP2 dimerization. The juxtamembrane region differs significantly between the NRP2a and NRP2b isoforms and may also influence ligand specificity, dimerization, and signaling.

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

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

[0211] NRP2 can form homodimers and heterodimers and is highly glycosylated. NRP2 has different splice variants ranging from approximately 551 to 926 amino acids in length. The two main variants of NRP2 are classified as NRP2a and NRP2b. They differ in their intracellular C-terminal portions (Figures 1A-1B), where, for NRP2a, the C-terminal domain contains 42 amino acids with a C-terminal SEA amino acid sequence and a PDZ-binding domain. In contrast, NRP2b contains a 46-amino acid C-terminal domain that shares approximately 11% sequence identity with the intracellular, juxtamembrane, and transmembrane sequences of NRP2a. Additional splicing can occur between the MAM and transmembrane domains, adding an additional 5 amino acids (GENFK) to either the NRP2a or NRP2b forms; these variants are named based on the number of additional amino acids added by alternative splicing. Thus, the two variants of NRP2a are designated NRP2a(17) (or variant 1) and NRP2a(22) (or variant 2), and the two transmembrane variants for NRP2b are designated NRP2b(0) (or variant 4) and NRP2b(5) (or variant 5). In addition, a soluble form called sNRP2b (or variant 6) can be produced. Exemplary NRP2 polypeptide sequences, including the mature (after cleavage of the N-terminal signal peptide) and precursor forms of the various isoforms of NRP2, are provided in Table N1 below. [Table N1-1] [Table N1-2] [Table N1-3] [Table N1-4] [Table N1-5]

Table N1-6

Table N1-7

Table N1-8

Table N1-9

[0212] In certain embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to a full-length human NRP2 polypeptide or a human NRP2 polypeptide selected from Table N1. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a human NRP2 polypeptide at about 10 pM to about 500 pM or about 10 pM to about 50 nM, or at about, at least about, or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20 ...40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 18 with an affinity of 0, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or, as appropriate, from about 10 pM to about 500 pM, from about 10 pM to about 400 pM, from about 10 pM to about 300 pM, from about 10 pM to about 200 pM, from about 10 pM to about 100 pM, from about 10 pM to about 50 pM, or from about 20 pM to about 500 pM, 0 pM to about 400 pM, about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20 pM to about 200 pM, about 30 The antibody binds with an affinity in the range of 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 about 25 nM, or about 25 nM to about 50 nM.

[0213] In some embodiments, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in at least one neuropilin domain. Exemplary neuropilin domains include one or more 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 a1 / a2 / b1 / b2 composite domain, a neuropilin a1 / a2 / b1 / b2 / c composite domain, a1 / a2 / b1 / b2 / c / juxtamembrane composite domain, and a neuropilin b1 / b2 / c composite domain (see Table N1 for domain residues). In a specific embodiment, at least one antibody or antigen-binding fragment thereof specifically binds to at least one epitope in the neuropilin b1 domain, the neuropilin b2 domain, and / or the neuropilin b1 / b2 complex 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 about 50 nM in at least one domain (or at least one epitope therein), or about, at least about, or at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, with an affinity of 70, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM, or, as appropriate, from about 10 pM to about 500 pM, from about 10 pM to about 400 pM, from about 10 pM to about 300 pM, from about 10 pM to about 200 pM, from about 10 pM to about 100 pM, from about 10 pM to about 50 pM, or about 20 pM M ~ about 500pM, about 20pM - about 400pM, about 20pM - about 300pM, about 20pM - about 200pM, about 20pM - about 100pM, about 20pM - about 50pM, or about 30pM - about 500pM, about 30pM to about 400pM, about 30pM to about 300pM, about 30pM to about 200pM, about 30pM to about 100pM, about 30pM to about 50pM, or about 20pM to about 200 pM, about 30 pM to about 300 pM, about 40 pM to about 400 pM, about 50 pM to about 500 pM, about 60 pM to about 600 pM, about 70 pM to about 700 pM, about 80 pM to about 800 pM, about 90 pM to about 900 pM, about 100 pM to about 1 nM, about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to 25 nM, or about 25 nM to about 50 nM.

[0214] In some embodiments, at least one antibody or antigen-binding fragment thereof is directed to at least one epitope in the neuropilin a1 domain, the neuropilin a2 domain and / or the neuropilin a1a2 complex domain, and the adjacent linker region, e.g., residues 20-148, 30-141, 40-141, 50-141, 60-141, 70-141, 80-148, 90-148, 100-148, 110-148, 120-148, 130-141, 140-141, 150-141, 160-141, 170-141, 180-148, 190-148, 200-200, 210-210, 220-220, 230-230, 240-240, 250-250, 260-260, 270-270, 280-280, 290-300, 300-300, 310-310, 320-320, 330-330, 340-340, 350-350, 360-360, 370-370, 380-380, 390-400, 410-410, 420-420, 430-430, 440-440, 450-450, 460-460, 470-470, 480-480, 490-500, 500-500, 510-510, 520-520, 5 or at about residues 142-280, 150-265, 160-265, 170-265, 180, 190-285, 200-285, 210-285, 211-212, 213-214, 215-216, 217-218, 219-220, 221-222, 222-223, 223-224, 224-225, 225-226, 226-227, 227-228, 228-229, 229-230, 230-231, 231-232, 232-233, 233-234, 234-235, 235-236, 236-237, 237-238, 238-239, 239-240, 241-242, 241-243, 241-244, 241-245, 242-246, 243-247, 244-248, 245-249, 246-249, 247-250, 248-251, 249-252, 253-254, 254-255, 255-256, 256-257, 257-258, 258-259, 260-260, 261-262, 261-262, 262-263, 263-264, 264-265, 265-2 ~265, 190~265, 200~265, 210~265, 220~265, 230~265, 240~265, 250~265, 260~265, 141~270, 141~260, 141~250, 141~240, 141~230, 141~220, 141~210, 141~200, 141~190, 141~180, 141~170, 141~160, 141~150, 200~250, 210~250, 220~250, 230~250, 200~240, 210-240, 220-240, 230-240, 227-247, 228-247, 229-247, 230-247, 231-247, 232-247, 233-247, 234-247, 235-247, 236-247; 227-246, 227-245, 227-244, 227-243, 227-242, 227-241, 227-240, 227-239, 227-238; 235-240, 236-239, 236-238, or near residue 237;or, for example, residues 20-280, 30-280, 40-280, 50-280, 60-280, 70-280, 80-280, 90-280, 100-280, 110-280, 120-280, 130-280, 140-280, 150-280, 160-280, 170-280, 180-280, 190-280, 200-280, 210-280, 220-280, 230-280, 240-280, 250-280, 260-280, 270-280, 280-280, 290-280, 300-280, 310-280, 320-280, 330-280, 340-280, 350-280, 360-280, 370-280, 380-280, 390-280, 400-280, 410-280, 420-280, 430-280, 440-280, 450-280, 460-280, 470-280, 480-490, 490-500, 500-510, 510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 580-590 specifically binds at or near 30-280, 240-280, 260-280, 270-280, 20-270, 20-260, 20-250, 20-240, 20-230, 20-220, 20-210, 20-200, 20-190, 20-180, 20-170, 20-160, 20-150, 20-140, 20-130, 20-120, 20-110, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, or 20-30;

[0215] In certain embodiments, at least one antibody or antigen-binding fragment thereof is directed to at least one epitope in the neuropilin b1 domain, neuropilin b2 domain, and / or neuropilin b1 / b2 complex domain, and the adjacent linker region, e.g., (neuropilin b1 domain) residues 266-426, 280-426, 290-426, 291-292, 292-293, 293-294, 294-295, 295-296, 296-300, 297-301, 298-302, 299-403, 304-305, 306-307, 308-309, 309-408, 310-311, 311-312, 312-313, 313-314, 314-315, 315-316, 316-317, 317-318, 318-319, 320-321, 321-322, 322-323, 323-324, 324-325, 325-326, 326-327, 327-328, 328-329, 330-331, 331-332, 332-333, 334-335, 336-337, 338-339, 340-341, 342-343, 344-345, 346-347, 348-349, 349-350, 349-351, 349-352, 34 99~420, 300~426, 310~426, 320~426, 330~426, 340~426, 350~426, 360~426, 370~426, 380~426, 390~426, 400~426, 410~426, 420~426, 280~420, 280~410, 280~400, 280~390, 280~380, 280~370, 280~360, 280~350, 280~340, 280~330, 280~3 a discontinuous epitope comprising residues 20, 280-310, 280-300, or 280-290, and one, two, or three of residues Y299, N354, and / or S416 as defined by the human NRP2 precursor sequence; (neuropilin b2 domain) residues 438-591, 450-591, 460-591, 470-591, 480-591, 490-591 as defined by the human NRP2 precursor sequence (see Table N1); 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 (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, 596-591, 606-591, 616-591, 626-591, 636-591, 646-591, 656-591, 666-591, 676-591, 686-591, 696-691, 706-706, 716-716, 726-726, 736-736, 746-746, 756-756, 766-766, 776-776, 786-786, 796-806, 806-806, 6~591, 386~591, 396~591, 406~591, 416~591, 426~591, 436~591, 446~591, 456~591, 466~591, 476~591, 486~591, 498~591, 508~591, 518~591, 528~591, 538~591, 548~591, 558~591 , 568~591, 578~591, 588~591, 266~581, 266~571, 266~561, 266~551, 266~541, 266~531, 266~521, 266~511, 266~501, 266~491, 266~481, 266~471, 266~461, 266~451, 266~441, 266~ specifically binds near 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;

[0216] In some embodiments, at least one antibody or antigen-binding fragment thereof targets at least one epitope in the neuropilin a2 / b1 composite domain and / or neuropilin b2c composite domain, and the adjacent linker region, e.g., residues 149-437, 159-426, 169-426, 179-426, 189-426, 199-426, 209-426, 219-426, 229-426, 239-426, 249-426, 259-426, 269-426, 279-426, 289-426, 299-426, 309-426, 319-426, 329-426, 339-426, 349-426, 359-426, 369-426, 379-426, 389-426, 399-426, 409-426, 419-426, 429-426, 439-426, 449-426, 459-426, 469-426, 479-426, 489-426, 499-426, 509-426, 519-426, 529-426, 539-426, 549-426, 559-426, 569-426, 579-426, 589-426, 599-426, 609-426, 619-426, 629-426, 639-426, 6 6, 339-426, 349-426, 359-426, 369-426, 379-426, 389-426, 399-426, 409-426, 419-426, 149-436, 149-426, 149-416, 149-406, 149-396, 149-386, 149-376, 149-366, 149-356, 149-346, 149 ~336, 149~326, 149~316, 149~306, 149~296, 149~286, 149~276, 149~266, 149~256, 149~246, 149~236, 149~226, 149~216, 149~206, 149~196, 146~186, 146~176, 146~166, or near 146~155;or residues 438-794, 448-794, 458-794, 468-794, 478-794, 487-794, 497-794, 507-794, 517-794, 527-794, 537-794, 547-794, 557-794, 567-794, 570-794, 580-794, 590-800, 601-602, 611-612, 613-614, 615-615, 616-616, 617-617, 620-621, 622-623, 624-625, 626-627, 628-629, 630-631, 632-633, 634-635, 636-637, 638-640, 639-641, 642-643, 644-645, 646-647, 648-648, 650-651, 652-653, 654-655, 656-657, 658-659, 658-659, 660-661, 662-663, 664-665, 670-671, 672-673, 674-675, 676-677, 678-679, 680-681, 682-683, 684-685, 686-686, 688 794, 567~794, 587~794, 597~794, 607~794, 617~794, 627~794, 637~794, 647~794, 657~794, 667~794, 677~794, 687~794, 697~794, 707~794, 717~794, 727~794, 737~794, 747~794, 757~794, 767~ 794, 777~794, 787~794, 427~794, 438~784, 438~774, 438~764, 438~754, 438~744, 438~734, 438~728, 438~714, 438~704, 438~694, 438~684, 438~674, 438~664, 438~654, 438~644, 438~634, 438~ 624, 438-614, 438-604, 438-596, 438-586, 438-576, 438-566, 438-556, 438-546, 438-536, 438-526, 438-516, 438-506, 438-494, 438-484, 438-474, 438-464, 438-454, 438-444;

[0217] In some embodiments, at least one antibody or antigen-binding fragment thereof is directed to at least one epitope in the neuropilin c domain and adjacent linker region, e.g., 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, 860-860, 870-860, 880-880, 890-894, 900-900, 910-910, 920-920, 930-940, 940-940, 950-950, 960-960, 970-970, 980-980, 990-1000, 1010-1010, 1020-1020, 1030-1030, 1040-1040, 1050-1050, 1060-1060, 1070-1070, 1080-1080, 1090-1090 It specifically binds in the vicinity of 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.

[0218] In some embodiments, at least one antibody or antigen-binding fragment thereof is directed to at least one epitope in the neuropilin b1 / b2 / c complex domain and adjacent linker region, e.g., residues 276-794, 286-794, 296-794, 306-794, 316-794, 326-794, 336-794, 346-794, 356-794, 366-794, 376-794, 387-794, 396-794, 406-794, 416-794, 426-794, 436-794, 446-794, 456-794, 466-794, 476-794, 487-794, 496-794, 506-794, 516-794, 526-794, 536-794, 546-794, 556-794, 566-794, 576-794, 587-794, 596-794, 606-794, 616-794, 626-794, 636-794, 646-794, 656-794, 666-794, 676-794, 687-794, 696-794, 706-794, 716-794, 726-794, 736-794, 746-794, 756-794, 766-794, 776-794, 7 ~794, 406~794, 416~794, 426~794, 436~794, 446~794, 456~794, 466~794, 476~794, 486~794, 496~794, 506~794, 516~794, 526~794, 536~794, 546~794, 556~794, 566~794, 576~794, 586~794, 596~794, 606~794, 616~794, 626~794, 636~794, 646~794, 656~794, 666~794, 676~794, 686~794, 696~794, 706~794, 716~794, 726~794, 736~794, 746~794, 756~794, 766~794, 776~794, 786~794, 266~794, 276~784, 276~774, 276~764, 276~754, 276~744, 276~734, 276~724, 276~714, 276~704, 276~694, 276~684, 276~674, 276~664, 276~654, 276~ It specifically binds to the vicinity of 644, 276 to 634, 276 to 624, 276 to 614, 276 to 604, 276 to 594, 276 to 584, 276 to 574, 276 to 564, 276 to 554, 276 to 544, 276 to 534, 276 to 524, 276 to 514, 276 to 504, 276 to 594, 276 to 584, 276 to 574, 276 to 564, 276 to 554, 276 to 544, 276 to 534, 276 to 524, 276 to 514, 276 to 504, or 276 to 496.

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

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

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

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

[0223] 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, which binds to or interacts with at least one human HRS polypeptide, thereby modulating the binding of human NRP2 polypeptide to human HRS polypeptide.Exemplary HRS polypeptide is provided in the following table H1. [Table H1-1] [Table H1-2] [Table H1-3] [Table H1-4]

[0224] Thus, in certain embodiments, at least one NRP2 ligand is selected from Table H1, and the anti-NRP2 antibody or antigen-binding fragment thereof modulates (e.g., prevents) the binding of a human NRP2 polypeptide (e.g., a human NRP2 polypeptide selected from Table H1) to a human HRS polypeptide selected from Table H1. In some embodiments, the anti-NRP2 antibody or antigen-binding fragment specifically binds to an HRS polypeptide-interacting region of an NRP2 polypeptide and, in some 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, e.g., at a ligand-binding site for a different NRP2 ligand (examples of which are provided herein), a dimerization domain, a protein-protein interaction domain, or an allosterically sensitive site within the NRP2 polypeptide to modulate the activity of the NRP2 polypeptide.

[0225] 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., selected from Table N1) and an NRP2 ligand, such as a human HRS polypeptide (e.g., selected from Table H1) or another NRP2 ligand (e.g., selected from Table N2 or Table N3). In some embodiments, a "blocking antibody" inhibits about or at least about 80-100% (e.g., 80, 85, 90, 95, or 100%) of the theoretical maximum binding between an NRP2 polypeptide and an NRP2 ligand (e.g., an HRS polypeptide) after pre-incubation of the "blocking antibody" with the NRP2 polypeptide in a substantially stoichiometric equivalent amount. As used herein, "stoichiometric equivalent" refers to a situation in which 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 equation or reaction.

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

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

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

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

[0230] For example, in some embodiments, 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 growth, cell survival and cell adhesion, and is also associated with lymphangiogenesis, increased vascular permeability, activation of integrin signaling, promotion of vesicle transport and internalization, and delayed cell differentiation. Therefore, it is expected that anti-NRP2 antibodies that modulate VEGF-related NRP2 ligands will find utility in modulating one or more of these pathways.

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

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

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

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

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

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

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

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

[0239] In some embodiments, at least one antibody or its antigen-binding fragment selectively modulates the binding and / or signal transduction of semaphorin to or through an NRP2 polypeptide. In some aspects, such an antibody does not substantially block the interaction of VEGF-C or related NRP2 ligands. In some aspects, such an antibody is an agonist antibody for semaphorin signal transduction. In some aspects, such an antibody is an antagonist antibody for semaphorin signal transduction.

[0240] In some embodiments, at least one antibody or antigen-binding fragment thereof selectively modulates the binding and / or signal transduction of VEGF-C or related NRP2 ligands to or via NRP2 polypeptides. In some aspects, such antibodies do not substantially block semaphorin interactions. In some embodiments, such antibodies selectively modulate both the binding of VEGF-C or related NRP2 ligands and semaphorins to NRP2 polypeptides. In some embodiments, such antibodies are agonistic antibodies for VEGF-C signal transduction. In some aspects, such antibodies are antagonistic antibodies for VEGF-C signal transduction.

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

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

[0243] In some 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.

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

[0245] In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes the binding activity / signaling activity between 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.

[0246] In some embodiments, at least one antibody or antigen-binding fragment thereof antagonizes 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 a VEGFR2, VEGFR3 and / or VEGF-C.

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

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

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

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

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

[0252] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope of at least five amino acids within the b1 domain of human NRP2, and 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.

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

[0254] In some embodiments, the at least one antibody or antigen-binding fragment thereof specifically binds to an epitope of at least five amino acids within the c-domain of human NRP2, and 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).

[0255] In some 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 to about 1.2 nM, about 0.9 to about 5.5 nM, about 0.9 to about 5 nM, or about 1 nM to about 10 nM.

[0256] In some embodiments, at least one antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ), 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.

[0257] In certain embodiments, at least one antibody or antigen-binding fragment thereof selectively binds to a human NRP2 polypeptide (see Table N1) compared to a mouse NRP2 polypeptide, e.g., its affinity for a human NRP2 polypeptide is significantly stronger, e.g., about or at least about 2, 5, 10, 20, 30, 40, 50, 100, 500, or 1000 times higher, than its affinity for a mouse NRP2 polypeptide. In certain embodiments, at least one antibody or antigen-binding fragment thereof selectively binds to a human NRP2 polypeptide and does not substantially bind to a mouse NRP2 polypeptide. Certain exemplary mouse NRP2 polypeptides include Mus musculus NRP2 polypeptides (see, e.g., UniProt O35375).

[0258] By way of illustration only, binding interactions between human NRP2 polypeptides and NRP2 ligands can be detected and quantified using a variety of routine methods, including biacore assays (e.g., using appropriately tagged soluble reagents bound to a sensor chip), FACS analysis using cells (either native or recombinant) expressing NRP2 polypeptides on their cell surface, immunoassays, fluorescent staining assays, ELISA assays, and microcalorimetry techniques such as ITC (isothermal titration calorimetry).

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

[0260] Also included are antibodies or antigen-binding fragments thereof that contain hybrid Fc regions, e.g., Fc regions that contain a combination of Fc domains (e.g., hinge, CH2, CH3, CH4) from immunoglobulins of different species (e.g., human, mouse), different Ig classes, and / or different Ig subclasses.Common examples include 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 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / Ig E, 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, Ig E / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG 2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM, Included are hybrid Fc regions comprising, consisting of, or consisting essentially of IgM / IgA1, IgM / IgA2, IgM / IgD, IgM / IgE, IgM / IgG1, IgM / IgG2, IgM / IgG3, IgM / IgG4, IgM / IgM (or fragments or variants 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 a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from human Ig.

[0261] Additional examples include the following CH2 / CH3 domain combinations: 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 any combination thereof). and hybrid Fc regions comprising, consisting of, or consisting essentially of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4 (or a fragment or variant thereof), optionally comprising a hinge derived from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from a human Ig.

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

[0263] Specific examples include 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 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM gM, 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, IgG 2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / I gG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2 , IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), optionally comprising a CH3 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH4 domain derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from a human Ig.

[0264] Certain examples include 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 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA 2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, Ig G2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / I gG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2 , IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or fragments or variants thereof), optionally comprising a CH2 domain derived from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH4 domain derived from IgE and / or IgM. In a specific embodiment, the hinge, CH2, CH3, and CH4 domains are derived from a human Ig.

[0265] 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, IgG4 / IgE, IgG4 / IgM (or fragments or variants thereof), optionally comprising a CH2 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM.

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

[0267] Antibodies or antigen-binding fragments thereof having derivatized or otherwise modified Fc regions are also included. In certain aspects, the Fc region may be modified, e.g., by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc., relative to a wild-type or naturally occurring Fc region. In certain embodiments, the Fc region may comprise a wild-type or native glycosylation pattern, or alternatively, may comprise increased glycosylation relative to the native form, decreased glycosylation relative to the native form, or may be entirely deglycosylated. As an example of a modified Fc glycoform, decreased glycosylation of the Fc region reduces binding to the C1q region of the first complement component C1, reduced ADCC-related activity, and / or reduced CDC-related activity. Accordingly, certain embodiments utilize deglycosylated or aglycosylated Fc regions. See, e.g., WO 2005 / 047337 for the generation of exemplary aglycosylated Fc regions. Another example of an Fc region glycoform can be generated by substituting a cysteine ​​residue at position Q295 according to the numbering system of Kabat et al. (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 comprise a mature core carbohydrate structure lacking fructose (See, e.g., U.S. Patent Application Publication No. 2010 / 0255013). Some embodiments may include an Fc region optimized by substitution or deletion to reduce the level of fucosylation, e.g., to increase affinity for FcγRI, FcγRIa, or FcγRIIIa, and / or to improve phagocytosis by FcγRIIa-expressing cells (See, U.S. Patent Application Publication Nos. 2010 / 0249382 and 2007 / 0148170).

[0268] 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, optionally, have one or more of the following compared to a corresponding Fc region containing complex-type N-glycans: increased ADCC effector activity, increased binding affinity to FcγRIIIA (and certain other FcRs), similar or increased binding specificity to a target of an NRP2 polypeptide, similar or higher binding affinity to a target of an NRP2 polypeptide, and / or similar or lower binding affinity to the mannose receptor (see, e.g., U.S. Patent Application Publication No. 2007 / 0092521 and U.S. Patent No. 7,700,321). As another example, enhanced affinity of the Fc region for FcγRs has been achieved using engineered glycoforms generated by expression of 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 contain an increased proportion of N-glycosidically linked complex glycans that do not have 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 connected by an α-2,6 linkage to each terminal sialic acid moiety, optionally with an Fc region having enhanced anti-inflammatory activity relative to the corresponding wild-type Fc region (see, e.g., U.S. Patent Application Publication No. 2008 / 0206246). Certain of these and related altered glycosylation approaches, as described herein, result in a substantial enhancement of the ability of the Fc region to selectively bind FcRs, such as FcγRIII, mediate ADCC, and alter other properties of the Fc region.

[0269] Certain variant, fragment, hybrid, or otherwise modified Fc regions of antibodies or antigen-binding fragments thereof may have altered binding to one or more FcRs and / or correspondingly altered effector function 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 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 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.

[0270] In some embodiments, the antibody comprises an Fc domain comprising 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 some embodiments, the antibody comprises an IgG1 or IgG3 Fc domain comprising 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 some embodiments, the antibody comprises an Fc domain comprising one or more mutations that increase effector function. In some embodiments, at least one antibody comprises an Fc domain selected from human IgG1 and human IgG3 comprising one or more mutations that increase effector function.

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

[0272] In some embodiments, the antibody comprises an Fc domain comprising 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 some embodiments, the antibody comprises an IgG1 or IgG3 Fc domain comprising 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 some embodiments, the antibody comprises an Fc domain comprising one or more mutations that reduce effector function. In some embodiments, the antibody comprises an Fc domain selected from human IgG2 and IgG4 comprising one or more mutations that reduce effector function.

[0273] 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 IgG4 comprising 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 IgG4 comprising 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 IgG4 comprising one or more mutations that reduce effector function.

[0274] Specific examples of Fc variants with altered (e.g., increased, decreased) effector function / FcR binding can be found, for example, in U.S. Patent Nos. 5,624,821 and 7,425,619; U.S. Patent Application Publication Nos. 2009 / 0017023, 2009 / 0010921, and 2010 / 0203046; and WO 2000 / 42072 and WO 2004 / 016750. Certain examples include human Fc regions with one or more substitutions at positions 298, 333, and / or 334, e.g., S298A, E333A, and / or K334A (based on the EU index numbering of Kabat et al.), which have been shown to increase binding to the activating receptor FcγRIIIa and reduce binding to the inhibitory receptor FcγRIIb. These mutations can be combined to obtain double and triple mutant variants with further improved FcR binding. A specific embodiment includes the triple mutant S298A / E333A / K334A, which increases binding to FcγRIIIa, decreases binding to FcγRIIb, and increases ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604, 2001; and Presta et al., Biochem Soc Trans. 30:487-490, 2002). Umana et al. and others, supra, See also engineered Fc glycoforms with increased binding to FcRs as disclosed in U.S. Patent No. 7,662,925. Some embodiments include an Fc region containing one or more substitutions selected from 434S, 252Y / 428L, 252Y / 434S, and 428L / 434S based on the EU index of Kabat et al. (see U.S. Patent Application Publication Nos. 2009 / 0163699 and 20060173170).

[0275] 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 illustrative example only, the Fc region may contain deletions or substitutions in complement binding sites, such as the Clq binding site, and / or may contain deletions or substitutions in ADCC sites. Examples of such deletions / substitutions are described, for example, in U.S. Patent No. 7,030,226. Many Fc effector functions, such as ADCC, can be assayed according to routine techniques 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 in addition, certain Fc effector functions can be assayed using animal models, such as those described in, e.g., Clynesetal. PNAS. 95:652-656, 1998. The assay may be performed in vivo.

[0276] Certain variant hybrids or modified Fc regions 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, variant hybrids or modified Fc regions 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 according to routine techniques in the art, such as radiolabeling, ELISA, or other methods. In vivo measurements of stability or half-life can be measured in one or more body fluids, including blood, serum, plasma, urine, or cerebrospinal fluid, or in a given tissue such as liver, kidney, muscle, central nervous system tissue, bone, etc.

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

[0278] Additional non-limiting examples of stability- or half-life-altering modifications include substitution / deletion of one or more amino acid residues selected from 251-256, 285-290, and 308-314 in the CH2 domain, and 385-389 and 428-436 in the CH3 domain, according to the numbering system of Kabat et al. 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, and substitution with arginine or 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, thereby increasing half-life, as compared to the corresponding wild-type Fc region.

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

[0280] Additional examples of variants include IgG Fc regions with conservative or non-conservative substitutions (as described elsewhere herein) at one or more of positions 250, 314, or 428 of the heavy chain, or any combination thereof, e.g., positions 250 and 428, or positions 250 and 314, or positions 314 and 428, or positions 250, 314, and 428 (see, e.g., U.S. Patent Application Publication No. 2011 / 0183412). In specific embodiments, the residue at position 250 is substituted with glutamic acid or glutamine, and / or the residue at position 428 is substituted with leucine or phenylalanine. As another example of an IgG Fc variant, any one or more of the amino acid residues at positions 214-238, 297-299, 318-322, and / or 327-331 may be used as suitable targets for modification (e.g., conservative or non-conservative substitutions, deletions). In certain embodiments, the Fc variant CH2 domain of an IgG contains amino acid substitutions at positions 228, 234, 235, and / or 331 (e.g., human IgG4 with Ser228Pro and Leu235Ala mutations) to attenuate the effector function of the Fc region (see U.S. Pat. No. 7,030,226), where 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(See, e.g., Ed., National Institutes of Health, Bethesda, Md. (1991)). Certain of these and related embodiments The FcRn binding and / or serum half-life of the antibody may be altered (e.g., increased, decreased), as needed, without reducing effector function, such as ADCC or CDC-related activity.

[0281] Additional examples include variant Fc regions containing 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 for human IgG are valine (279), glycine (341), proline (343), and tyrosine (373). Substitutions may be conservative or non-conservative, as described herein, or may include non-naturally occurring amino acids or mimetics. Alone or in combination with these substitutions, certain embodiments may also employ variant Fc regions comprising at least one, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions selected from the following: 235G, 235R, 236F, 236R, 236Y, 237K, 237N, 237R, 238E, 238G, 238H, 238I, 238L, 238V, 238W, 238Y, 24 4L, 245R, 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W, 247Y, 248F, 248P, 248Q, 248W, 249L , 249M, 249N, 249P, 249Y, 251H, 251I, 251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P, 2 54Q, 254R, 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V, 256W, 256Y, 257A, 257I, 257M, 257N, 257 S, 258D, 260S, 262L, 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272R, 279A, 279D, 279F, 279G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279Y, 280T, 283F, 283G, 28 3H, 283I, 283K, 283L, 283M, 283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 292G, 292I, 292L,293S, 293V, 301W, 304E, 307E, 307M, 312P, 315F, 315K, 315L, 315P, 315R, 316F, 316K, 317P, 317T, 318N, 318P, 318T, 332F, 332G, 332L, 332M, 332S, 3 32V, 332W, 339D, 339E, 339F, 339G, 339H, 339I, 339K, 339L, 339M, 339N, 33 9Q, 339R, 339S, 339W, 339Y, 341D, 341E, 341F, 341H, 341I, 341K, 341L, 341 M, 341N, 341P, 341Q, 341R, 341S, 341T, 341V, 341W, 341Y, 343A, 343D, 343 E, 343F, 343G, 343H, 343I, 343K, 343L, 343M, 343N, 343Q, 343R, 343S, 343T , 343V, 343W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373Q, 373R, 373S, 373T, 373V, 373W, 375R, 376E, 376F, 376G, 376H, 3 76I, 376L, 376M, 376N, 376P, 376Q, 376R, 376S, 376T, 376V, 376W, 376Y, 3 77G, 377K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D, 380N, 380S, 380T, 38 2D, 382F, 382H, 382I, 382K, 382L, 382M, 382N, 382P, 382Q, 382R, 382S, 382 T, 382V, 382W, 382Y, 385E, 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L , 427N, 429A, 429F, 429M, 430A, 430D, 430F, 430G, 430H, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430S, 430T, 430V, 430W, 430Y, 431H, 431K, 431P, 432R, 432S, 438G, 438K, 438L, 438T, 438W, 439E, 439H, 439Q, 440D, 440E, 440F, 440G, 440H, 440I, 440K, 440L, 440M, 440Q, 440T, 440V or 442K. As noted above, the numbering of residues in the heavy chain is:The numbering is EU index (see Kabat et al., supra). Such variant Fc regions typically confer altered effector function or altered serum half-life on antibodies to which they operably bind. Preferably, the altered effector function is increased ADCC, decreased ADCC, increased CDC, decreased CDC, increased Clq binding affinity, decreased Clq binding affinity, increased FcR (preferably FcRn) binding affinity, or decreased FcR (preferably FcRn) binding affinity, compared to a corresponding Fc region lacking such amino acid substitutions.

[0282] Additional 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, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 35 and variant Fc regions comprising one or more amino acid substitutions at positions 8, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 302, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336 and / or 428 (see, e.g., U.S. Patent No. 7,662,925). In a specific embodiment, the variant Fc region 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, F275 and at least one amino acid substitution selected from the group consisting of W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y. In other specific embodiments, the variant Fc region is V264I, F243L / V264I, L328M, I332E, L328M / I332E, V264I / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, A330Y, I332D, L328I / I332E, L328Q / I332E, V264T, V240I, V266I, S239D, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q,S239E / I332D, S239E / I332N, S239E / I332Q, S239N / I332D, S239N / I332E, S239Q / I332D, A330Y / I332E, V264I / A330Y / I332E, A33 0L / I332E, V264I / A330L / I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D / I 332E, 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, S23 9N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I332E, S239D / V264I / A330L / I332E, S239D / I332E / A330I, P230A, P 230A / E233D / I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I , K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L234I, S239D / A330Y / I332E / L235D, S239D / A330Y / I332E / V240I, S239D / A330Y / I332E / V264T, S239D / A330Y / I332E / K326E and S239D / A330Y / I332E / K326T. In more specific embodiments, the variant Fc region is selected from the group consisting of N297D / I332E, F241Y / F243Y / V262T / V264T / N297D / I332E, S239D / N297D / I332E, S239E / N297D / I332E, S239D / D265Y / N297D / I332E,In a specific embodiment, the variant Fc region comprises an amino acid substitution at position 332 (using the numbering of the EU index, Kabat et al., supra). Exemplary substitutions include 332A, 332D, 332E, 332F, 332G, 332H, 332K, 332L, 332M, 332N, 332P, 332Q, 332R, 332S, 332T, 332V, 332W, and 332Y. The numbering of residues in the Fc region is that of the EU index of Kabat et al. Among other properties described herein, such variant Fc regions may have increased affinity for FcγRs, increased stability, and / or increased solubility compared to the corresponding wild-type Fc region.

[0283] 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, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A, 306I / F, 311R, 312N, 315D / K / S, 320R, 322E, 323A, 324T, 325S, 326E / R, 332T, 333D / G, 335I, 338R, 339T, 340Q, 341E, 342R, 344Q, 34 7R, 351S, 352A, 354A, 355W, 356G, 358T, 361D / Y, 362L, 364C, 365Q / P, 370R, 372L, 377V, 378T, 383N, 3 89S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R, 411I, 412A, 414M, 421S, 422I, 426F / P, 428T, 430 and variant Fc regions comprising one or more of the following residues: 431K, 431S, 432P, 433P, 438L, 439E / R, 440G, 441F, 442T, 445R, 446A, 447E, optionally wherein the variants have altered recognition of Fc ligands and / or altered effector function compared to the parent Fc polypeptide, and the numbering of residues is that of the EU index as in Kabat et al.Illustrative examples of these and related embodiments include the following sets of substitutions: (1) N276D, R292Q, V305A, I377V, T394A, V412A, and K439E; (2) P244L, K246E, D399G, and K409R; (3) S304G, K320R, S324T, K326E, and M358T; (4) F243S, P247L, D265V, V266A, S383N, and T411I; (5) H224N, F243L, T393A, and H433P; (6) V240A, S267G, G341E, and E356G; (7) M252T, P291L, P352A, R355W, N390D, S408G, S426F, and A431S; (8) P228L, T289A, L365Q, N389S, and 5440G; (9) F241L, V273A, K340Q, and L441F; (10) F241L, T299A, I332T, and M428T; (11) E269K, Y300H, Q342R, V422I, and G446A; (12) T225A, R301c, S304G, D312N, N315D, L351S, and N421S; (13) S254T, L306I, K326 R 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, (21) Q347R, N361Y, and K439R; (22) S239P, S254P, S267N, H285R, N315S, F372L, A378T, N390D, Y391C, F404S, E430K, L432P, and K447E; and (23) E269G, Y278H, N325S, and K370R, wherein residue numbering is that of the EU index as in Kabatetal. (e.g., See, for example, U.S. Patent Application Publication No. 2010 / 0184959.

[0284] The variant Fc region can also have one or more mutated hinge regions, for example, as described in U.S. Patent Application Publication No. 2003 / 0118592. For example, one or more cysteines in the hinge region can be deleted or substituted with different amino acids. The mutated hinge region can contain no cysteine ​​residues, or it can contain one, two, or three fewer cysteine ​​residues than the corresponding wild-type hinge region. In some embodiments, Fc regions with this type of mutated hinge region exhibit reduced dimerization ability compared to wild-type Ig hinge regions.

[0285] In certain embodiments, the Fc region is an Fc region derived from human IgG1 or IgG4 (see, e.g., Allberse and Schuurman, Immunology. 105:9-19, 2002), or The antibody may comprise, consist of, or consist essentially of a fragment or variant of any of the following: (i) a fragment of a human IgG1 or IgG4; (ii) a fragment of a human IgG1 or IgG4; (iii) a fragment of a human IgG1 or IgG4; (iv) a fragment of a human IgG1 or IgG4; (v) a fragment of a human IgG1 or IgG4; (vi ... [Table F1]

[0286] As noted above, antibodies with altered Fc regions typically have altered (e.g., improved, increased, or decreased) pharmacokinetic properties compared to the corresponding wild-type Fc region. Examples of pharmacokinetic properties include stability or half-life, bioavailability (fraction of drug absorbed), tissue distribution, volume of distribution (apparent volume into which a drug distributes immediately after intravenous injection and equilibrates between plasma and surrounding tissues), concentration (initial or steady-state concentration of drug in plasma), elimination rate constant (rate at which a drug is eliminated from the body), elimination rate (rate of infusion required to balance elimination), area under the curve (AUC or exposure; the integral of the concentration-time curve after a single dose or at steady state), clearance (plasma volume excreted of drug per unit time), C max (peak plasma concentration of drug after oral administration), t max (C max time to reach C min (the minimum concentration reached by the drug before the next dose is administered) and fluctuation (peak-trough fluctuation within one dosing interval at steady state).

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

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

[0289] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxic or chemotherapeutic agents. Common examples of cytotoxic or chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, anthracyclines, antitumor antibiotics, platinum, type I topoisomerase inhibitors, type II topoisomerase inhibitors, vinca alkaloids, and taxanes. Specific examples of cytotoxic or chemotherapeutic agents include, but are not limited to, cyclophosphamide, cilengitide, lomustine (CCNU), melphalan, procarbazine, carmustine (BCNU), enzastaurin, busulfan, daunorubicin, doxorubicin, gefitinib, erlotinib, idarubicin, temozolomide, epirubicin, mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin, camptothecin, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, temsirolimus, everolimus, vincristine, vinblastine, vinorelbine, vindesine, CT52923, paclitaxel, imatinib, dasatinib, sorafenib, paclitaxel, These include zopanib, sunitinib, vatalanib, geftinib, erlotinib, AEE-788, dichloroacetate, tamoxifen, fasudil, SB-681323, semaxanib, donepezil, galantamine, memantine, rivastigmine, tacrine, rasagiline, naltrexone, lubiprostone, safinamide, istradefylline, pimavanserin, pitolisant, isradipine, pridopidine (ACR16), tetrabenazine, bexarotene, glatiramer acetate, fingolimod, and mitoxantrone, and pharmaceutically acceptable salts and acids thereof.Further examples of cytotoxic or chemotherapeutic agents include alkylating agents such as thiotepa, cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; ethylenimines and methylamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolmelamine. Melamine; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novoenbiquine, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; and aclacinomycin. , actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, queramycin, rodorubicin, streptomycin, Antibiotics such as leptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine. Pyrimidine analogs such as 5-FU; androgens such as calucelone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; Bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; Lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;Taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®; hne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; C PT-11; the topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene) and Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0290] 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. Additional Therapeutic Agents and Compositions

[0291] Immunotherapeutic Agents. Certain embodiments employ one or more cancer immunotherapeutic agents. In certain instances, the immunotherapeutic agent modulates a subject's immune response, e.g., increasing or maintaining a cancer-associated or cancer-specific immune response, thereby inhibiting or reducing increased immune cells in cancer cells. Exemplary immunotherapeutic agents include polypeptides, e.g., antibodies and antigen-binding fragments thereof, ligands, and small peptides, as well as mixtures 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 and others known in the art. Thus, in certain embodiments, the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint modulating agent, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapy.

[0292] In certain embodiments, the cancer immunotherapeutic agent is an immune checkpoint modulating agent. 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 strengthen (costimulatory molecules) or weaken (depress) signals, and targeting them has the potential to treat cancer because cancer cells can disrupt the natural function of immune checkpoint 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 some embodiments, an immune checkpoint modulating agent (e.g., antagonist, agonist) "binds" or "specifically binds" to one or more immune checkpoint molecules, as described herein.

[0293] In certain embodiments, the immune checkpoint modulating agent is a polypeptide or peptide. The terms "peptide" and "polypeptide" are used interchangeably herein; however, in certain instances, the term "peptide" can refer to shorter polypeptides, e.g., polypeptides 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, and all integers and ranges therebetween (e.g., 5-10, 8-12, 10-15). Polypeptides and peptides can be composed of naturally occurring and / or non-naturally occurring amino acids, as described herein.

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

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

[0296] The binding affinity of a polypeptide can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, a polypeptide specifically binds to a target molecule, e.g., an immune checkpoint molecule or epitope thereof, with an equilibrium dissociation constant of, or in a range of, about ≦10 M to about 10 M. In some embodiments, the equilibrium dissociation constant is, or in a range of, about ≦10 M to about ≦10 M. In certain illustrative embodiments, the polypeptide has a concentration of about or at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM, or about 0 an affinity (Kd or EC 50 )

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

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

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

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

[0301] In certain embodiments, the agent is a PD-1 (receptor) antagonist or inhibitor, the targeting of which has been shown 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, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is achieved, at least in part, through a dual mechanism: promoting apoptosis of antigen-specific T cells in lymph nodes while also reducing apoptosis in regulatory T cells (suppressor T cells). Some examples of PD-1 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to PD-1 and reduce one or more of its immunosuppressive activities, such as its downstream signaling or its interaction with PD-L1. Specific examples of PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP-224, AMP-514, and pidilizumab, and antigen-binding fragments thereof (see, e.g., U.S. Patent Nos. 8,008,449; 8,993,731; 9,073,994; See U.S. Patent 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; 2015 / 0203579).

[0302] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As mentioned above, PD-L1 is one of the natural ligands for the PD-1 receptor. Common 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 of its immunosuppressive activities, such as its binding 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, for example, U.S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; and 9,439,962).

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

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

[0305] In some embodiments, the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor. IDO and TDO are tryptophan degrading enzymes with immunosuppressive properties. For example, IDO is known to suppress T cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis. General examples of IDO and TDO antagonists or inhibitors include those that specifically bind to IDO or TDO (see, for example, Platte et al., Front Immunol. 5: 673, 2014) and reduce one or more immunosuppressive activities. Specific examples of IDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that inhibit or inhibit IDO. 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, for example, Sheridan, NatureBiotechnology. 33:321-322, 2015). Specific examples of agonists or inhibitors include 680C91 and LM10 (see, for example, Pilotte et al., PNAS USA. 109:2497-2502, 2012).

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

[0307] In some embodiments, the agent is an antagonist or inhibitor of LAG-3. Lymphocyte activation gene-3 (LAG-3) is expressed in activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It negatively regulates T cell proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1 (see, 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 plays a role in Treg suppressive function. LAG3 has also been reported to play a role in maintaining CD8+ T cells in a tolerogenic state and in PD. In combination with LAG-3-1, it maintains the exhaustion of CD8 T cells. General examples of LAG-3 antagonists or inhibitors include antibodies, antigen-binding fragments, or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016 and its antigen-binding fragments.

[0308] In some embodiments, 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, suppresses T-cell activation, induces Foxp3 expression, and is highly expressed in the tumor microenvironment, suppressing anti-tumor T-cell responses (see, for example, Lineset et al., Cancer Res. 74:1924-32, 2014). General examples of VISTA antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to VISTA and reduce one or more of its immunosuppressive activities.

[0309] In some embodiments, the agent is a BTLA antagonist or inhibitor. Expression of B and T lymphocyte attenuator (BTLA; CD272) is induced during T cell activation, inhibiting 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) and is 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). General examples of BTLA antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to BTLA-4 and reduce one or more of its immunosuppressive activities.

[0310] In some embodiments, the agent is an antagonist or inhibitor of HVEM, e.g., an antagonist or inhibitor that specifically binds to HVEM and prevents its interaction with BTLA or CD 160. Common examples of antagonists or inhibitors of HVEM include antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM and optionally reduce the HVEM / BTLA and / or HVEM / CD 160 interaction, thereby reducing one or more of the immunosuppressive activities of HVEM.

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

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

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

[0314] In some embodiments, the agent is an agonist of OX40. OX40 (CD134) promotes the expansion 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). Its ligand is OX40L (CD252). OX40 signaling affects both T cell activation and survival, so it plays an important role in eliciting antitumor immune responses in lymph nodes and maintaining antitumor immune responses in the tumor microenvironment. Common examples of OX40 agonists include antibodies or 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 (mouse OX4 agonist), and MEDI6383 (OX40 agonist), as well as antigen-binding fragments thereof.

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

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

[0317] In some embodiments, 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 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. CD137-mediated signaling also protects T cells, such as CD8+ T cells, from activation-induced cell death. General examples of CD137 agonists include antibodies or antigen-binding fragments, small molecules, or ligands that specifically bind to CD137 and increase one or more of its immunostimulatory activities. Specific examples include CD137 (or 4-1BB) ligands (see, for example, Shao and Schwarz, J. Leukoc. Biol. 89:21-9, 2011), and the antibody utomilumab and its antigen-binding fragments.

[0318] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases the antigen-specific expansion of naive T cells and contributes to the long-term maintenance of T cell memory and T cell immunity. Its ligand is CD70. Targeting human CD27 with 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., JImmunol. 191:4174-83, 2013). Common examples of CD27 agonists include CD27-specific agonists, CD70 ... These include antibodies or antigen-binding fragments, or small molecules or ligands that specifically bind to and increase one or more of its immunostimulatory activities. Specific examples include CD70, and the antibodies varlilumab and CDX-1127 (1F5), and antigen-binding fragments thereof.

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

[0320] In some embodiments, the agent is a CD226 agonist. CD226 is a stimulatory receptor that shares the same ligand with TIGIT, and unlike TIGIT, CD226 engagement 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). Common 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 of its immunostimulatory activities.

[0321] In some embodiments, 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 in various cells, including T cells, APCs, and other immune cells. Unlike other receptors, HVEM is expressed at high levels in resting T cells and is downregulated upon activation. HVEM signaling has been shown to play an important role in the early stages of T cell activation and during the expansion and proliferation of tumor-specific lymphocyte populations in lymph nodes. General examples of HVEM agonists include antibodies or antigen-binding fragments, small molecules, or ligands that specifically bind to HVEM and increase one or more of its immunostimulatory activities.

[0322] In certain embodiments, the cancer immunotherapeutic agent is a cancer vaccine.Exemplary cancer vaccines include human papillomavirus HPV vaccines such as Oncophage, Gardasil or Cervarix, hepatitis B vaccines such as Engerix-B, Recombivax HB or Twinrix, and sipuleucel-T (Provenge).In some embodiments, the cancer vaccine comprises or utilizes one or more cancer antigens or cancer-associated antigens.Exemplary cancer antigens include, but are not limited to, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, VEGFR-3, NRP2, CD30, CD33, CD37, CD40, CD 44, 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 These include bar 7 (SLAMF7), EGP40 pan-cancer antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

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

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

[0325] In certain embodiments, the cancer immunotherapeutic agent is a cell-based immunotherapy, for example, a T cell-based adoptive immunotherapy. In some embodiments, the cell-based immunotherapy comprises 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 (TIL), and peptide-induced T cells. In a specific embodiment, the CAR-modified T cells are targeted against CD-19 (see, for example, Maude et al., Blood. 125:4017-4023, 2015).

[0326] In certain instances, the cancer to be treated is associated with a cancer antigen, i.e., cancer antigen-specific T cells are targeted to or enriched for at least one antigen known to be associated with the cancer to be treated. In some 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, CD5 6, 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 α5β 1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PMSA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), Selected from one or more of EGP40 pan-cancer antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death 1, protein disulfide isomerase (PDI), regenerating liver phosphatase 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (a disialoganglioside expressed in tumors of neuroectodermal origin), glypican 3 (GPC3), and mesothelin.

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

[0328] In some embodiments, the antigen is MAGE-A1 (e.g., MAGE-A1 with accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g., MAGE-A6 with accession number NM_005363), MAGE-C1, MAGE-C2, Melan-A (e.g., Melan-A with accession number NM_00551 1), GP100 (e.g., GP100 with accession number M77348), tyrosinase (e.g., tyrosinase with accession number NM_000372), survivin (e.g., survivin with accession number AF077350), CEA (e.g., CEA with accession number NM_004363), Her-2 / neu (e.g., MAGE-C1 with accession number M1 1), or a combination thereof. 730), WT1 (e.g., WT1 according to accession number NM_000378), PRAME (e.g., PRAME according to accession number NM_0061 15), EGFRI (epidermal growth factor receptor 1) (e.g., EGFRI (epidermal growth factor receptor 1) according to accession number AF288738), MUC1, mucin-1 (e.g., mucin-1 according to accession number NM_002456), SEC61 G (e.g., SEC61 G according to accession number NM_014302), hTERT (e.g., hTERT according to accession number NM_198253), 5T4 (e.g., 5T4 according to accession number NM_006670), TRP-2 (e.g., TRP-2 according to accession number NM_001 922), STEAP1 (prostate six-transmembrane epithelial antigen 1), PSCA, PSA, PSMA, and the like.

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

[0330] In some cases, cancer antigens include idiotypic antigens associated with cancer or tumor diseases, particularly lymphoma or lymphoma-related diseases, 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.

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

[0332] One of skill in the art will understand that the various cancer immunotherapeutics described herein can be combined with any one or more of the various anti-NRP2 antibodies (including antigen-binding fragments thereof) described herein and used in accordance with any one or more of the methods or compositions described herein.

[0333] Chemotherapeutic Agents. Certain embodiments employ one or more chemotherapeutic agents, e.g., small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or type II), microtubule inhibitors, among others.

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

[0335] 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), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin.

[0336] Examples of anti-microtubule agents include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

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

[0338] Hormonal Therapy Agents. Certain embodiments employ at least one hormonal therapy agent. General 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 factors and lymphangiogenic factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D,...

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a human neuropilin-2 (NRP2) polypeptide at an epitope in the neuropilin a2 domain of NRP2, wherein the antibody or antigen-binding fragment thereof is: a heavy chain variable region (VH) sequence, including complementarity determining regions VH CDR1, VH CDR2, and VH CDR3 sequences, and a light chain variable region (VL) sequence, including complementarity determining regions VL CDR1, VL CDR2, and VL CDR3 sequences; Including, the VH CDR1, VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 31-33, respectively, and the VL CDR1, VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 34-36, respectively, and variants thereof which differ by a single amino acid substitution in VH CDR3, VLCDR1 and / or VLCDR3; or the VH CDR1, VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 37-39, respectively, and the VL CDR1, VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 40-42, respectively, including variants thereof which differ by one amino acid substitution in VH CDR3, VLCDR1 and / or VLCDR3; 1. An isolated antibody or antigen-binding fragment thereof, wherein the VHCDR3 sequence comprises SEQ ID NO: 81, the VLCDR1 sequence comprises SEQ ID NO: 82, and the VLCDR3 sequence comprises SEQ ID NO: 83, and X38 of SEQ ID NO: 83 is F, G, I, K, L, N, R, T, V, or Y.

2. The VH CDR1, VH CDR2 and VH CDR3 sequences comprise SEQ ID NOs: 31 to 33 and variants thereof, respectively, and the VL CDR1, VL CDR2 and VL CDR3 sequences comprise SEQ ID NOs: 34 to 36 and variants thereof, respectively; or 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the VH CDR1, VH CDR2, and VH CDR3 sequences comprise SEQ ID NOs: 37-39 and variants thereof, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences comprise SEQ ID NOs: 40-42 and variants thereof, respectively. Claim 3: The V H sequence comprises a sequence at least 95% identical to SEQ ID NO: 53 and the V L sequence comprises a sequence at least 95% identical to SEQ ID NO: 54; or 3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the VH sequence comprises a sequence at least 95% identical to SEQ ID NO:55 and the VL sequence comprises a sequence at least 95% identical to SEQ ID NO:

56. Claim 4: The V H sequence comprises SEQ ID NO: 53 and the V L sequence comprises SEQ ID NO: 54; or 4. The isolated antibody or antigen-binding fragment thereof of claim 3, wherein the VH sequence comprises SEQ ID NO:55 and the VL sequence comprises SEQ ID NO:

56.

5. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising an Fc domain or a variant thereof of IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3 and IgG4) or IgM, and optionally the Fc domain is a human Fc domain.

6. An isolated antibody or antigen-binding fragment thereof described in claim 5, comprising an Fc domain of IgG1 or IgG3.

7. An isolated antibody or antigen-binding fragment thereof described in claim 5, comprising an IgG2 or IgG4 Fc domain.

8. An isolated antibody or antigen-binding fragment thereof described in claim 7, comprising an IgG1 or IgG4 Fc domain, and optionally comprising an IgG1 or IgG4 Fc domain selected from SEQ ID NOs: 144 to 148.

9. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising a modified IgG1 or IgG4 Fc domain having altered binding to FcRn, optionally wherein the modified IgG1 or IgG4 Fc domain comprises one or more of the following mutations (EU numbering): YD (M252Y / T256D), DQ (T256D / T307Q), DW (T256D / T307W), YTE (M252Y / S254T / T256E), AAA (T307A / E380A / N434A), LS (M428L / N434S), M252Y, T256D / E, K288D / N, T307Q / W, E380C, N434F / Y and / or Y436H / N / W.

10. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 4, which is a monoclonal antibody and / or a humanized antibody.

11. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 4, which is an Fv fragment, a single-chain Fv (scFv) polypeptide, or a minibody.

12. An isolated antibody or antigen-binding fragment thereof described in any one of claims 1 to 4, which is a bispecific antibody.

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