Compositions and methods comprising anti-NRP2a antibodies

Abstract

Provided are antibodies and antigen-binding fragments thereof that preferentially or selectively bind human neuropilin-2a (NRP2a) variant 1 (v1) and / or variant 2 (v2) relative to other NRP2a or NRP2b isoforms and modulate the binding interaction between the NRP2a v1 / v2 ligand and downstream signaling events. Also included are related therapeutic compositions and methods for treating diseases, such as cancer and inflammatory and autoimmune diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Patent Application No. 63 / 272,374, filed October 27, 2021, which is incorporated by reference in its entirety.

[0002] Description of sequence listing The sequence listing XML associated with this application is provided in XML file format and is incorporated herein by reference. The name of the XML file containing the sequence listing XML is ATYR_137_01WO_ST26.xml. The XML file is approximately 222,086 bytes, was created on October 26, 2022, and has been submitted electronically via the USPTO Patent Center.

[0003] The present disclosure relates to antibodies and antigen-binding fragments thereof that preferentially or selectively bind human neuropilin-2a (NRP2a) variant 1 (v1) and / or variant 2 (v2) isoforms relative to the NRP2b isoform and selectively modulate the binding interactions between human NRP2a v1 / v2 ligands and downstream signaling events. Also included are related therapeutic compositions and methods for treating diseases, such as cancer and inflammatory and autoimmune diseases. [Background technology]

[0004] 2. Description of Related Art NRP2 is a single-pass transmembrane protein that forms heterodimeric complexes with a number of other plasma membrane receptors, including growth factor receptors such as FLT-4, KDR, and cMET (Nararre et al., (2014) OncoTargets and Therapy doi:10.2147 / OTT.S377744), as well as integrins and other signaling systems (Goel et al., J. Cell Sci 125 597-506, 2012). The extracellular domain of NRP2 binds to ligands of the VEGF and semaphorin families, while its short intracellular domain participates in a variety of protein-protein interactions. These interactions appear to enhance or regulate the intracellular trafficking and signaling output of growth factor receptors, integrins, and other co-receptors to promote cellular plasticity, among other processes that allow cells to rapidly respond and adapt to changing environments and cellular stresses (Favier et al., Blood doi:10.1182 / blood-2005-11-4447).

[0005] NRP2 is typically expressed in vivo as a mixture of various closely related splice variants, which are typically grouped together as NRP2a, which includes variants v1, v2, and v3, and NRP2b, which includes variants v4 and v5. Variant v6 is the soluble form of NRP2 found in circulation. While NRP2a and NRP2b share sequence identity over most (but not all) of their surface-exposed domains, the NRP2a and NRP2b variants differ significantly in their juxtamembrane, transmembrane, and intracellular C-terminal regions. In all three splice variants of NRP2a, the unique c-terminal domain contains 42 amino acids and an intracellular PDZ-binding domain with a C-terminal SEA amino acid sequence motif. In contrast, the two splice variants of NRP2b contain a 46 amino acid cytoplasmic domain region that lacks the C-terminal SEA. NRP2a and NRP2b share only about 11% sequence homology between their intracellular, juxtamembrane, and transmembrane sequences.

[0006] Increasing evidence suggests that NRP2a and NRP2b splice variants of NRP2 play distinct roles in regulating cellular functions, leading to differential patterns of subcellular localization and signaling. Emerging data suggest that the ratio of NRP2a and NRP2b expression in specific tissues varies over time, based on cellular stress and activation status, both in normal and pathophysiological conditions. Thus, the development of NRP2a and NRP2b selective antibodies provides an opportunity to develop a new generation of more selective and potent therapeutic and diagnostic agents. However, a major limitation to developing isoform-specific therapeutic and diagnostic reagents has been the lack of suitable isoform-selective, high affinity, and function-blocking antibodies, a limitation that has been overcome by the development and validation of NRP2a-specific antibodies described herein. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Nararre et al.,(2014)OncoTargets and Therapy doi:10.2147 / OTT.S377744 [Non-Patent Document 2] Goel et al.,J.Cell Sci 125 597-506,2012 [Non-Patent Document 3] Favier et al.,Blood doi:10.1182 / blood-2005-11-4447 Summary of the Invention [Means for solving the problem]

[0008]

[0023] Embodiments of the present disclosure include antibodies, or antigen-binding fragments thereof, that bind to a Neuropilin-2A (NRP2a) variant 1 (v1) or variant 2 (v2) polypeptide with an epitope comprising, consisting of, or consisting essentially of a sequence selected from Table N2, comprising about or at least about 8, 9, 10, 11, or 12 or more contiguous amino acids of a sequence selected from Table N2. In some embodiments, the epitope comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 96-104, comprising about or at least about 8, 9, 10, 11, or 12 contiguous amino acids of a sequence selected from SEQ ID NOs: 96-104. In certain embodiments, the epitope comprises, consists of, or consists essentially of SEQ ID NO: 100, or about or at least about 8, 9, 10, 11, or 12 contiguous amino acids of SEQ ID NO: 100.

[0009] In certain embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) sequence comprising complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 sequences, and a light chain variable region (VL) sequence comprising complementarity determining regions VLCDR1, VLCDR2, and VLCDR3 sequences, in which: The VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 13, 127, and GX1X2X3X4X5, respectively (wherein X1 is G, A, or S, X2 is Y, F, K, L, or R, X3 is T, A, G, I, L, Q, or V, X4 is D, A, G, K, N, Q, R, or S, and X5 is Y, A, D, E, F, G, H, I, K, L, N, Q, R, S, T, or V), and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 16, 17, and X6X7X8X9X, respectively. 10 X 11 X 12 X 13 (wherein X6 is S, A, G, I, L, P, T, or V; X7 is Q, A, G, R, or S; X8 is S, A, H, K, L, Q, or T; X9 is T, F, G, H, I, K, L, N, Q, R, S, V, or Y; and X 10is H, A, D, E, F, G, I, K, L, N, Q, R, S, T, or Y, and X 11 is V, A, E, F, G, H, I, K, L, N, P, Q, R, S, T, or Y; X 12 is L, A, E, H, I, N, P, Q, S, T, or V, and X 13 is T, A, D, E, F, G, I, K, L, N, Q, R, S, or V); VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 130-132, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 133-135, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 136-138, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 139-141, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 142-144, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 145-147, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 148-150, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 151-153, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 154-156, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 157-159, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 1-3, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 4-6, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 7-9, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 10-12, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 13-15, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 16-18, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 19-21, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 22-24, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs:25-27, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs:28-30, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 31-33, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 34-36, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 37-39, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 40-42, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 43-45, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 46-48, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 49-51, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 52-54, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; the VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 55-57, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 58-60, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; or The VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 61-63, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 64-66, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions.

[0010] In certain embodiments, the VH sequence comprises SEQ ID NO:170 and the VL sequence comprises SEQ ID NO:171; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 160 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 161; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 162 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 163; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 164 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 165; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 166 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 167; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 168 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 169; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:67 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:68; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:69 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:70; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:71 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:72; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:73 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:74; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:75 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:76; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:77 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:78; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:79 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:80; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:81 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:82; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:83 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:84; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:85 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:86; or The VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:87 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:88.

[0011] In some embodiments, the antibody, or antigen-binding fragment thereof, does not substantially bind to human neuropilin-2B (NRP2b) variant 4 (v4) polypeptide and / or human NRP2b variant 5 (v5) polypeptide. In some embodiments, the antibody, or antigen-binding fragment thereof, binds to an NRP2a v1 or v2 polypeptide or epitope at about 10 pM to about 500 pM or about 50 nM, or at about, at least about, or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 18 00, 900 pM, 1 nM, 10 nM, 25 nM, or 50 nM or less, or, optionally, 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 , about 20 pM to about 300 pM, about 20 pM to about 200 pM, about 20 pM to about 100 pM, about 20 pM to about 50 pM, or about 30 pM to about 500 pM, about 30 pM to about 400 pM, about 30 pM to about 300 pM, about 30 pM to about 200 pM, about 30 pM to about 100 pM, about 30 pM to about 50 pM, or about 20 pM to about 200 pM, about 30 pM to about 30 The antibody binds with an affinity in the range of about 0 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.

[0012] In certain embodiments, the binding affinity of the antibody, or antigen-binding fragment thereof, for an NRP2a v1 or v2 polypeptide is at least about 1.5 times, 2 times, 4 times, 6 times, 8 times, 10 times, 20 times, 40 times, 60 times, 80 times, 100 times, 200 times, 400 times, 600 times, 800 times, or 1000 times stronger than its binding affinity for an NRP2b v4 polypeptide and / or an NRP2b v5 polypeptide.

[0013] In some embodiments, the antibody, or antigen-binding fragment thereof, blocks or otherwise reduces binding between an NRP2a v1 or v2 polypeptide and its ligand, optionally where the ligand is selected from Table L1 or Table L2. In some embodiments, the antibody, or antigen-binding fragment thereof, blocks or otherwise reduces binding between an NRP2a v1 or v2 polypeptide and a chemokine (CC motif) ligand 21 (CCL21) polypeptide, optionally in an in vitro binding assay, an in vitro or ex vivo cell-based assay, or in vivo. In some embodiments, the antibody, or antigen-binding fragment thereof, blocks or otherwise reduces binding between an NRP2a v1 or v2 polypeptide and a CCL21 polypeptide by about or at least about 20-100% or more (optionally about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100% or more) relative to a control or reference. In certain embodiments, the antibody, or antigen-binding fragment thereof, blocks or otherwise reduces binding, including dimerization, between an NRP2a v1 or v2 polypeptide and a CC chemokine receptor type 7 (CCR7) polypeptide, optionally in an in vitro binding assay, in vitro or ex vivo cell-based assay, or in vivo. In certain embodiments, the antibody, or antigen-binding fragment thereof, blocks or otherwise reduces binding, including dimerization, between an NRP2a v1 or v2 polypeptide and a CCR7 polypeptide by about or at least about 20-100% or more (optionally about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100% or more) compared to a control or reference.

[0014] In some embodiments, the antibody, or antigen-binding fragment thereof, modulates (optionally antagonizes) signaling activity between an NRP2a v1 or v2 polypeptide and a CCL21 and / or CCR7 polypeptide, optionally by about or at least about 20-100% or more (optionally about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100% or more) relative to a control or reference. In certain embodiments, the signaling activity comprises induction of immune cell migration, optionally dendritic cells or mature T cells, in which the antibody, or antigen-binding fragment thereof, reduces the signaling activity; and / or in which the signaling activity comprises induction of tumor cell migration, and in which the antibody, or antigen-binding fragment thereof, reduces the signaling activity.

[0015] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises an IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), or IgM Fc domain, optionally a human Fc domain, or hybrids and / or variants thereof. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises an IgG Fc domain with high effector function in humans, optionally an IgG1 or IgG3 Fc domain; or it comprises an IgG Fc domain with low effector function in humans, optionally an IgG2 or IgG4 Fc domain. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises an IgG1 or IgG4 Fc domain, optionally selected from Table F1.

[0016] Particular embodiments include monoclonal antibodies, humanized antibodies, Fv fragments, single chain Fv (scFv) polypeptides, adnectins, anticalins, aptamers, avimers, camelid antibodies, designed ankyrin repeat proteins (DARPins), minibodies, nanobodies, and / or unibodies.

[0017] Also included are therapeutic compositions comprising a pharma- ceutically acceptable carrier and an antibody, or antigen-binding fragment thereof, as described herein. In some embodiments, the 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 thereof, and is substantially free of aggregates. In some embodiments, the therapeutic composition is substantially free of endotoxins. In some embodiments, the therapeutic composition is a sterile injectable solution, optionally suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration. Certain therapeutic compositions and methods further comprise 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.

[0018] Also included are methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutic composition described herein. In some embodiments, the disease or condition is a neuropilin 2 (NRP2)-associated disease or condition, optionally an NRP2a-associated disease or condition. In some embodiments, the disease or condition is selected from cancer, an inflammatory disease, an autoimmune disease, a lymphatic disease or associated condition, a fibrotic disease, and a disease associated with reduced smooth muscle contractility.

[0019] In certain 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 certain embodiments, the cancer expresses or overexpresses NRP2, but does not substantially express neuropilin-1 (NRP1). Certain methods are for reducing or preventing re-emergence of cancer in a subject in need thereof, where administration of a therapeutic composition allows for the generation of immune memory to the cancer. In some embodiments, the subject has lymphedema.

[0020] 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-NRPa2 antibody or antigen-binding fragment thereof and the at least one agent are administered separately as separate compositions. In some embodiments, the at least one anti-NRP2 antibody and the at least one agent are administered together as part of the same therapeutic composition, optionally as a therapeutic composition described herein.

[0021] 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 certain embodiments, the immune checkpoint modulating agent is a polypeptide, optionally an antibody or an antigen-binding fragment thereof, or a ligand, or a small molecule. In some embodiments, the immune checkpoint modulating agent is (a) an antagonist of an inhibitory immune checkpoint molecule; or (b) an agonist of a stimulatory immune checkpoint molecule; In some cases, the immune checkpoint modulating agent specifically binds to an immune checkpoint molecule.

[0022] 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), attenuation factor for B and T lymphocytes (BTLA), CD160, herpes virus entry mediator (HVEM), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT). In certain 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 a small molecule that specifically binds thereto, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), and optionally the cancer is selected from one or more of colorectal cancer, melanoma, breast cancer, non-small cell lung cancer, bladder cancer, and renal cell carcinoma; the antagonist is a PD-1 antagonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule that specifically binds thereto, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514PDR001, and pidilizumab, optionally where the PD-1 antagonist is nivolumab, and the cancer is optionally selected from one or more of Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer; the PD-1 antagonist is pembrolizumab and the cancer is optionally selected from one or more of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma; the antagonist is a CTLA-4 antagonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule that specifically binds thereto, 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 a small molecule that specifically binds thereto, indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat, and the cancer is optionally selected from one or more of metastatic breast cancer and brain cancer, optionally glioblastoma multiforme, glioma, gliosarcoma, or malignant brain tumor; the antagonist is a TDO antagonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule that specifically binds thereto, 680C91, and LM10; the antagonist is a TIM-3 antagonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule that specifically binds thereto; the antagonist is a LAG-3 antagonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule that specifically binds thereto, and BMS-986016; the antagonist is a VISTA antagonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule that specifically binds thereto; the antagonist is a BTLA, CD160, and / or HVEM antagonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule that specifically binds thereto; The antagonist is a TIGIT antagonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule that specifically binds thereto.

[0023] 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). the agonist is an OX40 agonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule or ligand that specifically binds thereto, OX86, Fc-OX40L, and GSK3174998; the agonist is a CD40 agonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule or ligand that specifically binds thereto, CP-870,893, dacetuzumab, Chi Lob 7 / 4, ADC-1013, and rhCD40L, and the cancer is optionally selected from one or more of melanoma, pancreatic carcinoma, mesothelioma, and hematological cancer, and optionally is a lymphoma, such as non-Hodgkin's lymphoma; the agonist is a GITR agonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule or ligand that specifically binds thereto, INCAGN01876, DTA-1, and MEDI1873; the agonist is a CD137 agonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule or ligand that specifically binds thereto, utomirumab, and 4-1BB ligand; the agonist is a CD27 agonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule or ligand that specifically binds thereto, varlilumab, and CDX-1127 (1F5); the agonist is a CD28 agonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule or ligand that specifically binds thereto, and TAB08; and / or The agonist is an HVEM agonist optionally selected from one or more of an antibody or antigen-binding fragment, or a small molecule or ligand that specifically binds thereto.

[0024] 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 human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3, C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, leukocyte Acid 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-carcinoma 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 on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin, and optionally the subject has or is at risk of having a cancer that comprises the corresponding cancer antigen.

[0025] In some embodiments, the oncolytic virus is selected from one or more of talimogene laherparepvec (T-VEC), Coxsackievirus A21 (CAVATAK™), Oncoline (H101), Peraleorep (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. In some embodiments, the cytokine is selected from one or more of interferon (IFN)-α, IL-2, IL-12, IL-7, IL-21, and granulocyte macrophage colony stimulating factor (GM-CSF). In some embodiments, the cell-based immunotherapeutic agent 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.

[0026] In certain embodiments, the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent. the alkylating agent is selected from one or more of nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (optionally N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and its derivatives (optionally carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine); the antimetabolite is selected from one or more of antifolates (optionally methotrexate and pemetrexed), fluoropyrimidines (optionally 5-fluorouracil and capecitabine), deoxynucleoside analogs (optionally acitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacytidine, 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).

[0027] In some embodiments, at least one hormonal therapy agent is a hormone agonist or a hormone antagonist. In certain embodiments, the hormone agonist is selected from one or more of a progestogen (progestin), a corticosteroid (optionally prednisolone, methylprednisolone, or dexamethasone), an insulin-like growth factor, an angiogenic and lymphangiogenic factor derived from VEGF (optionally VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), a fibroblast growth factor (FGF), a galectin, a hepatocyte growth factor (HGF), a platelet-derived growth factor (PDGF), a transforming growth factor (TGF)-beta, an androgen, an estrogen, and a somatostatin analog. In some embodiments, the hormone antagonist is a hormone synthesis inhibitor, optionally an aromatase inhibitor or a 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 directed against a hormone receptor, optionally cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, lobatumumab. , alacizumab pegol, bevacizumab, icrucumab, ramucirumab, fresolimumab, metelimuamab, naxitamab, cetuximab, depatuximab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomzotuximab, zalutumumab, apultumab ixadotin, bemarituzumab, olaratumab, or tobetumab.

[0028] In some embodiments, the kinase inhibitor is selected from one or more of adavosertib, afantinib, 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 bemafenib.

[0029] In some embodiments, the cancer is a primary cancer. In certain embodiments, the cancer is a metastatic cancer, optionally a metastatic cancer that expresses NRP2a and / or NRP2b.

[0030] 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.

[0031] In some embodiments, the metastatic cancer is selected from one or more of the following: (a) bladder cancer that has metastasized to the bone, liver, and / or lungs; (b) breast cancer that has metastasized to the bone, brain, liver, and / or lungs; (c) colorectal cancer metastasizing to the liver, lung, and / or peritoneum; (d) renal cancer that has metastasized to the adrenal glands, bones, 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 metastasizing to the liver, lung, and / or peritoneum; (h) pancreatic cancer that has metastasized to the liver, lung, and / or peritoneum; (i) prostate cancer that has metastasized to the adrenal glands, bone, liver, and / or lungs; (j) gastric cancer metastasizing 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.

[0032] Also included is a patient care kit containing: (a) an antibody, or antigen-binding fragment thereof, as described herein; and optionally, (b) at least one additional agent selected from a cancer immunotherapy agent, a chemotherapeutic agent, a hormonal therapy agent, and a kinase inhibitor.

[0033] 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 1 or type II), and an anti-microtubule agent.

[0034] Certain embodiments include a bioassay system comprising an antibody, or antigen-binding fragment thereof, described 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 antibody, or antigen-binding fragment thereof, is labeled with a detectable label. In some embodiments, the NRP2 polypeptide is functionally coupled to a readout or indicator, such as a fluorescent or luminescent indicator of the biological activity of the NRP2 polypeptide. In certain embodiments, the NRP2 polypeptide is selected from Table N1, optionally NRP2a v1 and / or v2 polypeptides. Certain bioassay systems include at least one NRP2a ligand, optionally selected from Table L1 or Table L2, and optionally in which the host cell expresses at least one NRP2a ligand.

[0035] Certain embodiments include a detection system comprising a human neuropilin 2a (NRP2a) polypeptide, at least one NRP2a ligand, and a cell expressing a human or humanized anti-NRP2a antibody, or antigen-binding fragment thereof, described herein, which modulates the interaction between the NRP2a polypeptide and at least one NRP2a ligand. In some embodiments, the anti-NRP2a antibody, or antigen-binding fragment thereof, is labeled with a detectable label. In some embodiments, the NRP2a polypeptide is an NRP2a variant 1 and / or variant 2 polypeptide selected from Table N1. In some embodiments, the at least one NRP2a ligand is selected from Table L1 or Table L2. In some embodiments, the NRP2a polypeptide and / or at least one NRP2a ligand are functionally coupled to a readout or indicator, such as a fluorescent or luminescent indicator of the biological activity of the NRP2a polypeptide or at least one NRP2a ligand.

[0036] Also included are cell compositions comprising an engineered cell population, where at least one cell comprises one or more polynucleotides encoding a human or humanized anti-NRP2a antibody, or antigen-binding fragment thereof, described herein, wherein the cells are capable of growing in serum-free medium.

[0037] Some embodiments include a cell growth device comprising a human or humanized anti-NRP2a antibody, or antigen-binding fragment thereof, described herein, an engineered cell population in which at least one cell comprises one or more polynucleotides encoding the anti-NRP2a antibody, or antigen-binding fragment thereof, at least about 10 liters of serum-free growth medium, and a sterile container. [Brief description of the drawings]

[0038] [Figure 1] Figure 1 shows a model of the interaction of NRP2a v1 / v2 with CCL21 and CCR7. NRP2a v3 and NRP2b v4 / v5 do not significantly interact with CCL21 or CCR7, in part because these NRP2 isoforms lack the CCL21-interacting sequence within the juxtamembrane domain. [Figure 2A] 2A-2E show the association between NRP2a v1 / v2 isoforms and CCR7 in the presence of CCL21 or CCL19. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 3A] 3A-3E show the effect of mutations in the CCL21 binding site of NRP2a on ligand-induced dimerization of NRP2a and CCR7. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 4A]Figures 4A-4C show the relative binding of antibodies to human and mouse NRP2A (4A), human NRP2a and NRP2b (4B), and human NRP2a v2 and NRP2a v3 (4C). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 5A] 5A-5D show the ability of antibodies to block NRP2a / CCR7-CCL21 induced receptor dimerization. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 6] FIG. 6 shows sequence analysis of an exemplary anti-NRP2a antibody epitope (SEQ ID NO: 95 (NRP2a) and the corresponding homologous murine sequence, SEQ ID NO: 124 (mNRP2a)). [Figure 7A] 7A-7B show the workflow (7A) and gating strategy (7B) of the in vivo dendritic cell migration assay. [Figure 7B] Same as above. [Figure 7C] FIG. 7C shows the results of FITC+ dendritic cells in lymph nodes of NRP2 KO compared to wild-type mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods, materials, compositions, reagents, 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, including but not limited to patents and patent applications cited herein, 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.

[0040] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). 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 molecular biology, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as the laboratory procedures and techniques thereof, are those well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0041] For purposes of this disclosure, the following terms are defined below.

[0042] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of an article. By way of example, "an element" includes "one element," "one or more elements," and / or "at least one element."

[0043] By "about" is meant an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0044] The term "antigen" refers to a molecule or a 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 an antibody capable of binding to an epitope of that antigen. An antigen can have one or more epitopes. As used herein, the term "antigen" includes a substance that, under suitable conditions, is capable of inducing an immune response against the substance and reacting with the products of the immune response. For example, an antigen can be recognized by an antibody (humoral immune response) or a sensitized T lymphocyte (T helper or cell-mediated immune response), or both. Antigens can be soluble substances, such as toxins and foreign proteins, or particles, such as bacteria and tissue cells; however, only parts of a protein or polysaccharide molecule, known as antigenic determinants (epitopes), combine with antibodies or specific receptors on lymphocytes. More broadly, the term "antigen" includes any substance to which an antibody binds or for which an antibody is desired, regardless of whether the substance is immunogenic or not. For such antigens, antibodies can be identified by recombinant methods, independent of any immune response.

[0045] "Antagonist" refers to a biological structure or chemical agent that interferes with or otherwise reduces the physiological action of another agent or molecule. In some instances, antagonists specifically bind to other agents or molecules. Included are full and partial antagonists.

[0046] "Agonist" refers to a biological structure or chemical agent that increases or enhances the physiological action of another agent or molecule. In some instances, agonists specifically bind to other agents or molecules. Included are full and partial agonists.

[0047] The term "anergy" refers to the functional inactivation of T cell or B cell responses to restimulation with an antigen.

[0048] As used herein, the term "amino acid" is intended to mean both natural and unnatural amino acids, as well as amino acid analogs and mimetics. Natural amino acids include the 20 (L) amino acids utilized during protein biosynthesis, as well as others, such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline, and ornithine. Unnatural amino acids include, for example, the (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, and the like, which are known to those of skill in the art. Amino acid analogs include modified forms of natural and unnatural amino acids. Such modifications can include, for example, the substitution or replacement of chemical groups and moieties on the amino acid, or can be by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures that exhibit functionally similar properties, such as the charge and charge spacing characteristics of the reference amino acid. For example, an organic structure that mimics arginine (Arg or R) will have a positively charged moiety that is located in a similar molecular space and has the same degree of flexibility as the e-amino group of the side chain of the natural Arg amino acid. Mimetics also include structures that are constrained to maintain optimal spacing and charge interactions of the amino acid or of the 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.

[0049] As used herein, the term "antibody" includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv, etc.), single chain (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configurations of immunoglobulin molecules that contain an antigen-binding site or fragment (epitope recognition site) of the required specificity. Specific features and characteristics of antibodies (and their antigen-binding fragments) are described in more detail herein.

[0050] 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 capable of specifically binding to a target, such as an immune checkpoint molecule, through at least one epitope recognition site located in the variable region of the immunoglobulin molecule.

[0051] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy 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-type polypeptide fragment from an antibody that binds to a target molecule. H and V L It may comprise one, two, three, four, five or all six CDRs of the sequence.

[0052] The binding properties of antibodies and their antigen-binding fragments can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, an antibody or antigen-binding fragment thereof binds to a target molecule, e.g., an NRP2a v1 and / or v2 polypeptide, or an epitope or complex thereof, with a binding affinity of about ≦10 -7 M~about 10 -8 M. In some embodiments, the equilibrium dissociation constant is about ≦10 -9 M~approx.≦10-10 In certain exemplary embodiments, the antibody or antigen-binding fragment thereof has an affinity (Kd or EC for the target molecule to which it specifically binds) of about, at least about, 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 ).

[0053] A molecule, such as a polypeptide or an 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, with greater duration, and / or with greater affinity than with alternative cells or substances or epitopes. An antibody "specifically binds" or "preferentially binds" to a target molecule or epitope if it binds, for example, by a statistically significant amount, with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances or epitopes. Typically, one member of a pair of molecules that exhibits specific binding has an area, or cavity, on its surface that specifically binds to, and is therefore complementary to, a particular spatial and / or polar structure of the other member of the pair of molecules. In this way, the members of the pair have 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 particular epitope with greater affinity, avidity, more readily, and / or with greater 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. The term is also applicable, for example, when an antibody is specific for a particular epitope carried by multiple antigens, in which case a specific binding member carrying an antigen-binding fragment or domain can bind to a variety of antigens carrying the epitope; for example, it may be cross-reactive to multiple different forms of a target antigen from multiple species that share a common epitope.

[0054] Immunological binding generally refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example, by way of illustration and not limitation, as a result of 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, where a smaller Kd represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentrations of the complex partners, geometric parameters that affect the affinity of the interaction, and equally on the rates in both directions. Thus, both the "on-rate constant" (Kon) and the "off-rate constant" (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio Koff / Kon allows cancellation of all parameters not related to affinity and is thus 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 expressed as Kd or EC 50 The affinity of a binding protein to a ligand, such as the affinity of an antibody for an epitope, can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from 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 upon dilution. In some embodiments, affinity is measured using the half maximal effective concentration (EC 50 EC is expressed in terms of the concentration of an agent disclosed herein, such as an anti-NRP2a antibody, that induces a response halfway between the baseline and maximum after a specified exposure time. 50 is commonly used as a measure of antibody potency.

[0055] Antibodies may be prepared by any of a variety of techniques known to those of skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific for a polypeptide of interest may be prepared using, e.g., Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereon. Also included are methods of expressing human antibodies using transgenic animals, such as mice. See, e.g., Neuberger et al., Nature Biotechnology 14:826, 1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101, 1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995. A specific example includes the VELOCIMMUNE® platform by REGENEREX® (see, eg, US Pat. No. 6,596,541).

[0056] Antibodies can also be generated or identified by the use of phage display libraries or yeast display libraries (see, e.g., U.S. Pat. No. 7,244,592; Chao et al., Nature Protocols. 1:755-768, 2006). Non-limiting examples of available libraries include cloned or synthetic libraries, such as the Human Combinatorial Antibody Library (HuCAL), in which the structural diversity of the human antibody repertoire is represented by seven heavy and seven light chain variable region genes. The combination of these genes gives rise to 49 frameworks in the master library. By stacking highly variable gene cassettes (CDRs=complementarity determining regions) on these frameworks, the vast human antibody repertoire can be recreated. Also included are human libraries designed with human donor-supplied fragments encoding the light chain variable region, synthetic DNA encoding diversity in heavy chain CDR-3, 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.

[0057] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein each comprise a set of heavy and light chain CDRs, interposed between a set of heavy and light chain framework regions (FRs) that provide support for the CDRs and define the spatial relationship of the CDRs to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2", and "CDR3", respectively. An antigen-binding site thus comprises six CDRs, comprising a set of CDRs from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit". Crystallographic analysis of numerous antigen-antibody complexes has demonstrated that amino acid residues of the CDRs form extensive contacts with bound antigen, with the most extensive antigen contacts being with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of the antigen-binding site.

[0058] As used herein, the term "FR set" refers to four adjacent amino acid sequences that frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact the bound antigen; however, FRs are primarily involved in folding the V region into an antigen binding site, particularly the FR residues directly adjacent to the CDRs. 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 about 90 amino acid residues. When the V region folds into the binding site, the CDRs are presented as protruding loop motifs that form an antigen binding surface. Regardless of the exact CDR amino acid sequence, 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. In addition, certain FR residues are known to be involved in non-covalent interdomain contacts that stabilize the interaction of antibody heavy and light chains.

[0059] The structure and location of immunoglobulin variable domains can be determined by reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof.

[0060] Also included are "monoclonal" antibodies, which refer to homogeneous antibody populations, in which the monoclonal antibody is composed of amino acids (natural and non-natural) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed against a single epitope. The term "monoclonal antibody" includes intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, etc.), single chain (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) of the required specificity and ability to bind to the epitope. It is not intended to be limited as to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals). The term includes whole immunoglobulins, as well as the fragments, etc., described above under the definition of "antibody".

[0061] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer containing an intact antigen binding site. The enzyme pepsin can also cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which contains both antigen binding sites. Fv fragments for use according to certain embodiments can be produced by preferential proteolytic cleavage of IgM, and in rare cases, IgG or IgA immunoglobulin molecules. Fv fragments, however, are more commonly derived using recombinant techniques known in the art. Fv fragments contain a non-covalent VH::VL heterodimer containing an antigen binding site that retains much of the antigen recognition and binding ability of the native antibody molecule. See Inbar et al., PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980.

[0062] In certain embodiments, single chain Fv (scFV) antibodies are contemplated. For example, kappa bodies (Ill et al., Prot. Eng. 10:949-57, 1997); minibodies (Martin et al., EMBO J 13:5305-9, 1994); diabodies (Holliger et al., PNAS 90:6444-8, 1993); or Janusins ​​(Traunecker et al., EMBO J 10:3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl. 7:51-52, 1992) may be prepared using standard molecular biology techniques following the teachings of the present application for the selection of antibodies with the desired specificity.

[0063] Single-chain Fv (scFv) polypeptides are covalently linked VH::VL heterodimers that are expressed from gene fusions containing VH and VL coding genes linked by a peptide-encoding linker. Huston et al. (PNAS USA.85(16):5879-5883,1988). Numerous methods have been described for identifying chemical structures for converting naturally aggregated, but chemically separated, light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to that of the antigen-binding site. See, for example, U.S. Pat. Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Pat. No. 4,946,778 to Ladner et al.

[0064] In certain embodiments, the antibodies or antigen-binding fragments described herein are in the form of a "diabody". Diabodies are multimers of polypeptides, each of which comprises a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site: the antigen-binding site is formed by association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer (WO94 / 13804). dAb fragments of antibodies consist of a VH domain (Ward et al., Nature 341:544-546, 1989). Diabodies and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO94 / 13804; and Holliger et al., PNAS USA. 90:6444-6448, 1993).

[0065] Also included are minibodies comprising an scFv linked to a CH3 domain (see Hu et al., Cancer Res. 56:3055-3061, 1996). See also Ward et al., Nature. 341:544-546, 1989; Bird et al., Science. 242:423-426, 1988; Huston et al., PNAS USA. 85:5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; and Reiter et al., Nature Biotech. 14:1239-1245, 1996.

[0066] If 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 mentioned above. Diabodies and scFvs can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reaction.

[0067] Bispecific diabodies, as opposed to bispecific whole antibodies, can also be particularly useful because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) of suitable binding specificity can be easily selected using phage display (WO94 / 13804) from libraries. If one arm of the diabody is kept constant, for example with specificity directed against antigen X, then a library can be created in which the other arm is varied and an antibody of suitable specificity is selected. Bispecific whole antibodies can be made by knob-into-hole engineering (Ridgeway et al., Protein Eng., 9:616-621, 1996).

[0068] In certain embodiments, the antibodies or antigen-binding fragments described herein are in the form of UniBody®. UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, for example, US20090226421). This antibody technology creates a stable, smaller antibody format with a predicted longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and thus do not interact with the immune system. Fully human IgG4 antibodies can be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments with different stability characteristics 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 the UniBody® therefore binds monovalently to only one site on the target cell. For certain cancer cell surface antigens, this monovalent binding will 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 refractory to treatment with conventional antibodies. The small size of UniBody® may be of great benefit in treating some forms of cancer, allowing for better distribution of the molecule over larger solid tumors, potentially increasing efficacy.

[0069] 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 can be formulated as a ready-to-use solution with a long shelf life. The Nanoclone method (see WO06 / 079372) is a proprietary method for generating nanobodies against desired targets based on automated high-throughput selection of B cells.

[0070] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of aptamers (see, e.g., Ellington et al., Nature. 346, 818-22, 1990; and Tuerk et al., Science. 249, 505-10, 1990, which are incorporated by reference). Examples of aptamers include nucleic acid aptamers (e.g., DNA aptamers, RNA aptamers) and peptide aptamers. Nucleic acid aptamers generally refer to nucleic acid species that are engineered through repeated rounds of in vitro selection, or equivalent methods, such as SELEX (systematic evolution of ligands by exponential enrichment), to bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. See, e.g., U.S. Patent Nos. 6,376,190; and 6,387,620, which are incorporated by reference.

[0071] Peptide aptamers typically comprise a variable peptide loop attached at both ends to a protein scaffold, typically a dual structural constraint that increases the binding affinity of the peptide aptamer to levels comparable to that of antibodies (e.g., nanomolar range). In certain embodiments, the variable loop length may be comprised of about 10-20 amino acids (including all integers in between), and the scaffold may comprise any protein with good solubility and capacity properties. Certain exemplary embodiments utilize the bacterial protein thioredoxin A as the scaffold protein, in which the variable loop is inserted into the reducing active site (-Cys-Gly-Pro-Cys-loop in the wild-type protein), and the two cysteine ​​side chains can form a disulfide bridge. Methods for identifying peptide aptamers are described, for example, in U.S. Patent Application Publication No. 2003 / 0108532, which is incorporated by reference. Peptide aptamer selection can be performed using different systems known in the art, including the yeast two-hybrid system.

[0072] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of avimers. Avimers refer to multimeric binding proteins or peptides engineered using in vitro exon shuffling and phage display. Multiple binding domains are linked, resulting in greater affinity and specificity compared to single epitope immunoglobulin domains. See, for example, Silverman et al., Nature Biotechnology. 23:1556-1561, 2005; U.S. Patent No. 7,166,697; and U.S. Patent Application Nos. 2004 / 0175756, 2005 / 0048512, 2005 / 0053973, 2005 / 0089932, and 2005 / 0221384, all of which are incorporated by reference.

[0073] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of adnectins. Adnectins refer to a class of targeted biologics derived from human fibronectin, an abundant extracellular protein that naturally binds to other proteins. See, for example, U.S. Patent Application Nos. 2007 / 0082365; 2008 / 0139791; and 2008 / 0220049, which are incorporated by reference. Adnectins typically consist of a natural fibronectin backbone and multiple targeting domains of specific portions of human fibronectin. The targeting domains can be engineered to enable the adnectin to specifically recognize the NRP2 polypeptide or an epitope thereof.

[0074] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of anticalins. Anticalins refer to a class of antibody mimics that are typically synthesized from human lipocalins, a family of binding proteins with hypervariable loop regions supported by a structurally rigid framework. See, for example, US Patent Application 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 (a stable β-barrel scaffold) connected in pairs by four peptide loops and an attached α-helix. In certain aspects, conformational deviations are made in the hypervariable loop regions to achieve specific binding. See, for example, Skerra, FEBS J. 275:2677-83, 2008, incorporated by reference.

[0075] 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 for 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 the DARPin approach useful for drug discovery. In addition, DARPins can be easily produced in a multispecific format, offering the potential to target effector DARPins to specific organs or to target multiple receptors using one molecule composed of several DARPins. See, e.g., Stumpp et al., Curr Opin Drug Discov Devel. 10:153-159, 2007; U.S. Patent Application No. 2009 / 0082274; and PCT / EP2001 / 10454, which are incorporated by reference.

[0076] Also included are heavy chain dimers, such as antibodies from camelids and sharks. Camelid and shark antibodies contain a homodimeric pair of two chains (neither of which has a light chain) of V-like and C-like domains. The VH region of heavy chain dimeric IgG in camelids does not have to make hydrophobic interactions with the light chains, so the regions in the heavy chains that normally contact the light chains are altered to hydrophilic amino acid residues in camelids. The VH domain of heavy chain dimeric IgG is called the VHH domain. Shark Ig-NAR contains a homodimer of one variable domain (termed the V-NAR domain) and five C-like constant domains (C-NAR domains).

[0077] 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 (Muyldermans et al., 1994, Protein Engineering 7(9):1129). This is in contrast to the CDR3 regions of antibodies of many other species. For example, the CDR3 of mouse VH has an average of 9 amino acids. Libraries of camelid-derived antibody variable regions maintain the in vivo diversity of camelid variable regions and can be generated, for example, by the methods disclosed in U.S. Patent Application No. 20050037421, published Feb. 17, 2005.

[0078] In certain embodiments, the antibodies or antigen-binding fragments thereof are humanized. These embodiments refer to chimeric molecules, generally prepared using recombinant techniques, that have 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 onto a constant domain, or only the CDRs grafted onto suitable framework regions in the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, but still leaves the possibility of an immune response against the foreign variable region (LoBuglio et al., PNAS USA 86:4220-4224, 1989; Queen et al., PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327, 1988). Exemplary methods for antibody humanization include those described in US Pat. No. 7,462,697.

[0079] Another approach focuses on not only providing constant regions of human origin, but also modifying the variable regions to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity determining regions (CDRs) that vary in response to the epitope in question and determine the binding ability, and are relatively conserved in a given species, flanked by four framework regions (FRs) that presumptively 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 the CDRs derived from the non-human antibody onto the FRs present in the modified human antibody. Application of this approach to a variety of antibodies has been reported in Sato et al., Cancer Res. 53:851-856, 1993; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988; Kettleborough et al., Protein Engineering. 4:773-3783, 1991; Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991; Gorman et al., PNAS USA. 88:4181-4185, 1991; Tempest et al., Bio / Technology 9:266-271, 1991; Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA. 89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, a humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibodies). In other embodiments, a humanized antibody has one or more CDRs (one, two, three, four, five, six) that are altered with respect to the original antibody, which are also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0080] 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 operatively 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 from a different Ig class than 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 from one or more of the different Ig classes. As described above for humanized antibodies, an antigen-binding fragment of a chimeric antibody may include only one or more of the CDRs of an antibody described herein (e.g., one, two, three, four, five, or six CDRs of an antibody described herein) or may include the entire variable domain (VL, VH, or both).

[0081] As used herein, a subject "at risk" of developing a disease or adverse reaction may or may not have detectable disease or symptoms of a disease, and may or may not exhibit detectable disease or symptoms of a disease prior to the treatment methods described herein. "At risk" means that the subject has one or more risk factors, which are measurable parameters that correlate with the development of a disease, as described herein and known in the art. A subject with one or more of these risk factors has a higher probability of developing a disease or adverse reaction than a subject without one or more of these risk factors.

[0082] "Biocompatible" refers to a material or compound that is not generally damaging to the biological functions of a cell or a subject and does not cause any degree of unacceptable toxicity, including allergenicity and disease conditions.

[0083] The term "bond" refers to a direct association between two molecules, for example, due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including, for example, salt bridges and water bridges.

[0084] The term "chemo-resistance" refers to changes in the therapeutic sensitivity of a cancer cell population over time after exposure to chemotherapy, including resistance to at least one of cancer immunotherapeutic agents, chemotherapeutic agents, hormonal therapeutic agents, and / or kinase inhibitors. Ultimately, chemotherapy resistance leads to cancer recurrence and / or metastasis, challenging improved clinical outcomes for cancer patients. It remains a major obstacle to long-term successful cancer treatment. For example, approximately 30 percent of women diagnosed with early-stage breast cancer ultimately develop resistance and eventually progress to metastatic breast cancer. Molecular mechanisms of chemotherapy resistance include 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 through different mechanisms, alone or in combination with each other, but are ultimately regulated to prevent cell death in response to specific targeted chemotherapeutic agents. For example, such processes provide alternative growth-promoting signals and / or eliminate or otherwise reduce apoptotic pathways. Thus, agents that reduce chemotherapy resistance may find utility in treating or reducing chemotherapy-resistant cancers.

[0085] By "coding sequence" is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the code for the polypeptide product of a gene.

[0086] Throughout this disclosure, unless the context requires otherwise, the terms "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0087] "Consisting of" means inclusive and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that no other elements will be present. By "consisting essentially of," it means including any elements recited after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" means that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they materially affect the activity or action of the recited elements.

[0088] The term "effector function" in the context of an antibody, or "ADCC effector function", refers to the ability of that antibody to associate with other arms of the immune system, including, for example, activation of the classical complement pathway or through the association of Fc receptors. The complement-dependent pathway is driven primarily by the interaction of C1q with the C1 complex with clustered antibody Fc domains. Antibody-dependent cellular cytotoxicity (ADCC) is driven primarily by the interaction of Fc receptors (FcRs) on the surface of effector cells (natural killer cells, macrophages, monocytes, and eosinophils) that bind to the Fc region of IgG, which itself is bound to the target cell. Fc receptors (FcRs) are key immunoregulatory receptors that connect antibody-mediated (humoral) immune responses to cellular effector functions. Receptors for all classes of immunoglobulins have been identified and include 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 (nanomolar range KD) while FcγRII and FcγRIII are low to intermediate affinity (micromolar range KD). Upon Fc binding, signaling pathways are triggered that result in the secretion of various substances such as lytic enzymes, perforin, granzymes and tumor necrosis factor, which mediate the destruction of the target cell. Levels of ADCC effector function vary for human IgG subtypes. This is dependent on the allotype and the specific FcvR, but simply put, ADCC effector function is "high" for human IgG1 and IgG3, and "low" for IgG2 and IgG4.

[0089] The terms "endotoxin-free" or "substantially free of endotoxin" generally refer to compositions, solvents, and / or containers that contain only trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects in a subject), and preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically gram-negative bacteria, although endotoxins can also be found in gram-positive bacteria, such as Listeria monocytogenes. The most prevalent endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), found in the outer membrane of various gram-negative bacteria, which represent a central pathogenic feature 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.

[0090] Thus, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from the drug product and / or drug container, since even small amounts can cause adverse effects in humans. Dehydrogenation ovens can be used for this purpose, since temperatures above 300°C are typically required to break down most endotoxins. For example, based on primary packaging materials, such as syringes or vials, a combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels. Other methods of removing endotoxin are contemplated, including, for example, chromatography and filtration methods described herein and known in the art.

[0091] Endotoxin can be detected using routine techniques known in the art. For example, the Limulus amebocyte lysis assay utilizes blood from horseshoe crabs and is a highly sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable clotting of the Limulus 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, endotoxin levels can be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to approximately 1-10 EU.

[0092] 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 is bound by an antibody. In certain embodiments, epitopic determinants include molecules, e.g., chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics. Epitopes may be contiguous or discontinuous with respect to the primary structure of the antigen, e.g., the NRP2 polypeptide. In certain embodiments, an epitope comprises, consists of, or consists essentially of about, at least about, or no more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 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, Table N2) or target molecule described herein.

[0093] An "epitope" includes that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of a binding protein. Such a binding interaction may manifest itself as an intermolecular contact with one or more amino acid residues of a CDR. Antigen binding may involve a CDR3 or a CDR3 pair. An epitope may be a linear peptide sequence (i.e., "continuous") or may be composed of a non-contiguous amino acid sequence (i.e., "conformational" or non-contiguous). A binding protein may recognize one or more amino acid sequences; thus, an epitope may define more than one distinct amino acid sequence. Epitopes recognized by a binding protein may 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 is capable of being recognized by a binding protein in a denatured or proteolytic polypeptide. An amino acid sequence that is not exposed or only partially exposed in the unmodified polypeptide structure is a potential cryptic epitope. If an epitope is not exposed or only partially exposed, it is likely to be buried within the interior of the polypeptide. Candidate cryptic epitopes can be identified, for example, by examining the three-dimensional structure of the unmodified polypeptide.

[0094] The term "half maximum effective concentration" or "EC 50 " refers to the concentration of an agent (e.g., an antibody) described herein that induces a response halfway between the baseline and maximum after some specified exposure time; EC 50 Thus, "EC50" represents the concentration of a compound at which 50% of its maximum effect is observed. EC50 also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. Similarly, "EC 90 "EC" refers to the concentration of a drug or composition at which 90% of its maximum effect is observed. 90" can be calculated from the "EC50" and Hill slope, or can be determined directly from the data using routine knowledge in the art. In some embodiments, the EC50 of an agent (e.g., an antibody) is less than about 0.01, 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 value of about 1 nM or less.

[0095] "Immune response" refers to any immunological response derived from the immune system, including responses from the cellular and cellular, innate and adaptive immune systems. Exemplary cellular immune cells include, for example, lymphocytes, macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, dendritic cells, mast cells, monocytes, and all subsets thereof. Cellular responses include, for example, effector functions, cytokine release, phagocytosis, efferocytosis, translocation, trafficking, proliferation, differentiation, activation, suppression, cell-cell interactions, apoptosis, and the like. cellular responses include, for example, IgG, IgM, IgA, IgE, responses and their corresponding effector functions.

[0096] The "half-life" of a drug, such as an antibody, can refer to the time at which the drug loses half of its pharmacological, physiological, or other activity, as compared to such activity upon administration into the serum or tissues of an organism, or as compared to any other defined time point. "Half-life" can also refer to the time at which the amount or concentration of the drug is reduced by half of the starting amount administered in the serum or tissues of an organism, as compared to such amount or concentration upon administration into the serum or tissues of an organism, or as compared to any other defined time point. Half-life can be measured in serum and / or in any one or more selected tissues.

[0097] The terms "modulating" and "modulating" include "increasing," "enhancing," or "stimulating," as well as "decreasing" or "reducing," typically in 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 an increase that is 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 more (e.g., 500, 1000-fold) (including all integers and ranges therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amount produced without the composition (e.g., absence of agent) or by 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% decrease (including all integers and ranges therebetween) in the amount produced without the composition (e.g., absence of agent) or with a control composition. Examples of comparisons and "statistically significant" amounts are described herein.

[0098] The term "migratory cell" refers to a cell that is capable of movement 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), subsets of dendritic cells, myeloid cells, plasmacytoid (also called lymphocytes), and Langerhans cells; macrophages, such as histiocytes, tissue resident macrophages, such as Kupffer cells, microglial cells in the CNS, alveolar macrophages, and peritoneal macrophages; macrophage subtypes, such as M0, M1, Mox, M2a, M2b, and M2c macrophages; neutrophils, eosinophils, mast cells, basophils, B cells, plasma B cells, memory B cells, B-1 cells, 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. Further examples of migratory cells include fibroblasts, fibrocytes, tumor cells, and stem cells. The term "cell migration" refers to the movement of migratory cells, and the term "modulation of cell migration" refers to the modulation of the movement of any such migratory cells.

[0099] 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. This term includes modifications, such as myristoylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of a signal sequence. The term "polypeptide" or "protein" refers to one or more chains of amino acids, where each chain includes amino acids covalently linked by peptide bonds, and where the polypeptide or protein can include multiple chains non-covalently and / or covalently linked together by peptide bonds, and includes molecules having the amino acid sequence of a native protein, i.e., a protein that occurs in nature and in particular is produced by a non-recombinant cell, or a genetically engineered or recombinant cell, or molecules having a deletion from, addition to, and / or substitution of one or more amino acids of the native sequence. In certain embodiments, a polypeptide is a "recombinant" polypeptide produced by a recombinant cell that contains one or more recombinant DNA molecules, which are typically made with heterologous polynucleotide sequences, or combinations of polynucleotide sequences that would not otherwise be found in the cell.

[0100] 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 either type of nucleotide. The terms include single-stranded and double-stranded forms of DNA. The terms "isolated DNA" and "isolated polynucleotide" and "isolated nucleic acid" refer to molecules that are 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 that has been substantially isolated away from or purified free of total genomic DNA of the species from which the DNA segment is obtained. Also included are non-coding polynucleotides (e.g., primers, probes, oligonucleotides) that do not encode a polypeptide. Also included are recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, and the like.

[0101] Additional coding or non-coding sequences may, but need not be, present within the polynucleotides described herein, and the polynucleotides may, but need not be linked to other molecules and / or supporting materials. Thus, a polynucleotide or expressible polynucleotide, regardless of the length of the coding sequence itself, may be combined with other sequences, for example, expression control sequences.

[0102] "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 coding sequences in a host cell, within a cell, or cellular location. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0103] 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 bounded at its 3' end by a transcription initiation site, extends upstream (5' direction), and includes 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, as well as protein binding domains (consensus sequences) responsible for binding RNA polymerase. 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.

[0104] Numerous promoters are well known in the art, including constitutive, inducible, and repressible promoters from a variety of different sources. Representative sources include, for example, viral, mammalian, insect, plant, yeast, and bacterial cell types), and suitable promoters from these sources are readily available online or based on publicly available sequences, for example, from repositories, such as ATCC, as well as other commercial or individual sources, or can be synthetically produced. 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, Calif.), LacSwitch® (Stratagene, San Diego, Calif.), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al. Nuc. Acid. Res. (1999) 27(22):4324-4327; Nuc. Acid. Res. (2000) 28(23):e99; U.S. Pat. No. 7,112,715; and Kramer & Fussenegger, Methods Mol. Biol. (2005) 308:123-144), or any promoter known in the art that is suitable for expression in the desired cell.

[0105] An "expressible polynucleotide" includes a cDNA, RNA, mRNA, or other polynucleotide that contains at least one coding sequence, and optionally at least one expression control sequence, e.g., a transcriptional and / or translational regulatory element, and is capable of expressing an encoded polypeptide upon introduction into a cell, e.g., a cell in a subject.

[0106] Various viral vectors that can be utilized to deliver expressible polynucleotides include adenoviral vectors, herpes virus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retroviral vectors. In some cases, the retroviral vector is a derivative of a murine or avian retrovirus, or is a lentiviral vector. Examples of retroviral vectors that can insert a single foreign gene include, but are not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), SIV, BIV, HIV, and Rous sarcoma virus (RSV). Many additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker, allowing transduced cells to be identified and generated. The vector can be made target specific, for example, by inserting a polypeptide sequence of interest into the viral vector along with another gene that codes for a ligand for a receptor on a specific target cell. Retroviral vectors can be made target specific, for example, by inserting a polynucleotide encoding a protein. An exemplary targeting can be achieved by targeting the retroviral vector using an antibody. Those skilled in the art will know or can easily ascertain, without undue experimentation, specific polynucleotide sequences that can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector.

[0107] In certain embodiments, the expressible polynucleotide is a modified RNA or modified mRNA polynucleotide, e.g., a non-natural RNA analog. In certain embodiments, the modified RNA or mRNA polypeptide comprises one or more modified or non-natural bases, e.g., nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and / or uracil (U). In some embodiments, the modified mRNA comprises one or more modified or non-natural internucleotide linkages. Expressible RNA polynucleotides for delivering encoded therapeutic polypeptides are described, for example, in Kormann et al., Nat Biotechnol. 29:154-7, 2011; and U.S. Patent Application Nos. 2015 / 0111248; 2014 / 0243399; 2014 / 0147454; and 2013 / 0245104, which are incorporated by reference in their entireties.

[0108] The term "isolated" polypeptide or protein as referred to herein means that the subject protein (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 percent of the polynucleotides, lipids, sugars, or other materials with which it is associated in nature, (5) is not associated (by covalent or non-covalent interactions) with portions of proteins with which it is associated in nature, (6) is operatively associated (by covalent or non-covalent interactions) with polypeptides with which it is not associated in nature, or (7) does not occur in nature. Such isolated proteins can be encoded by genomic DNA, cDNA, mRNA, or other RNA, or can be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides that would interfere with its use (therapeutic, diagnostic, preventative, research, or other methods) or other contaminants found in its natural environment.

[0109] In certain embodiments, the "purity" of any given agent (e.g., a polypeptide, such as an antibody) in a composition may be defined. For example, a particular composition may include 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 basis, including all decimal points and ranges therebetween, as measured, for example, but in no way limited to, by high performance liquid chromatography (HPLC), a well-known form of column chromatography frequently used in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0110] "Lipid nanoparticle" or "solid lipid nanoparticle" refers to one or more spherical nanoparticles with an average diameter between about 10 and about 1000 nanometers and comprising a solid lipid core matrix capable of solubilizing lipophilic molecules. The lipid core is stabilized by a surfactant (e.g., an emulsifier) ​​and can include one or more of triglycerides (e.g., tristearin), diglycerides (e.g., glycerol bahenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate), including combinations thereof. Lipid nanoparticles are described, for example, in Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014; and U.S. Patent Nos. 6,217,912; 6,881,421; 7,402,573; 7,404,969; 7,550,441; 7,727,969; 8,003,621; 8,691,750; 8,871,509; 9,017,726; 9,173,853; 9,220,779; 9,227,917; and 9,278,130, which are incorporated by reference in their entirety. Certain compositions described herein are formulated with one or more lipid nanoparticles.

[0111] The terms or "neuropilin 2-related disease" or "NRP2-related disease" refer to diseases and conditions in which NRP2 activity, expression, and / or spatial distribution play a role in the pathophysiology of the disease or condition. In some examples, the NRP2-related disease is modulated by the anti-NRP2 antibodies of the present disclosure by altering the interaction of NRP2 with at least one NRP2 ligand, thereby affecting NRP2 activity, signaling, expression, and / or spatial distribution. In certain embodiments, the NRP2 is NRP2a variant 1 or variant 2, and the NRP2 ligand is CCL21 and / or CCR7. Thus, in certain embodiments, the NRP2-related disease or condition is an "NRP2a-related disease or condition." Exemplary NRP2-related diseases and conditions include cancer and diseases or pathologies associated with cancer, including, but not limited to, cancer cell growth, cancer initiation, cancer migration, cancer cell adhesion, invasion, chemotherapy resistance, and metastasis. Also included are diseases associated with inflammation and autoimmunity, as well as related inflammatory diseases, including diseases associated with inappropriate immune cell activation or migration, such as graft-versus-host disease (GVHD). Particular embodiments include diseases associated with lymphatic vessel development, lymphangiogenesis, and lymphatic vessel damage, such as edema, lymphedema, secondary lymphedema, inappropriate fat absorption and accumulation, excessive fat accumulation, and vascular permeability; diseases associated with infection, including latent infection; allergic disorders / diseases, diseases associated with allergic responses, such as chronic obstructive pulmonary disease (COPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, systemic lupus erythematosus, rheumatoid arthritis, inflammasome-associated diseases, and skin-associated neutrophil-mediated diseases, such as rheumatoid arthritis, ... 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 neurological diseases, peripheral nervous system remodeling, and pain perception; diseases associated with bone development and bone remodeling.Typically, the term "inappropriate" refers to an activity or characteristic that is associated with or causes a pathology or disease state.

[0112] The term "reference sequence" generally refers to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is compared. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those described by name and those described in the Tables and Sequence Listing.

[0113] Certain embodiments include biologically active "variants" and "fragments" of the polypeptides (e.g., antibodies) described herein, as well as polynucleotides encoding the same. "Variants" include one or more substitutions, additions, deletions, and / or insertions compared to a reference polypeptide or polynucleotide (see, e.g., Tables and Sequence Listing). A variant polypeptide or polynucleotide comprises an amino acid or nucleotide sequence with at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity or homology to a reference sequence, as described herein, and substantially retains the activity of the reference sequence. Also included are sequences that consist of or differ from a 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, which substantially retain the activity of the reference sequence. In certain embodiments, the additions or deletions include C-terminal and / or N-terminal additions and / or deletions.

[0114] The term "sequence identity" as used herein also includes, for example, "a sequence 50% identical to," and refers to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a window of comparison. Thus, "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions where identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to yield a number of matching positions, dividing the number of matching positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield a percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window can be performed by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA) or by inspection and selection of the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by any of the various methods. Reference can also be made to the BLAST family of programs, for example, as disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0115] The term "solubility" refers to the ability of an agent (e.g., an antibody) provided herein to dissolve in a liquid solvent and form a homogenous solution. Solubility is typically expressed as a concentration by either mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg / ml, etc.), molality, molality, molar fraction, or other similar description of concentration. The maximum equilibrium amount of solute that can be dissolved per volume of solvent is the solubility of the solute in that solvent under specific conditions including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH or other 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, such as PBS or NaCl (with or without NaPO4). In certain embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO4). In certain embodiments, solubility is measured in a biological fluid (solvent), such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 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 at 37°C.

[0116] A "subject" or "subject in need thereof" or a "patient" or "patient in need thereof" includes mammalian subjects, such as human subjects.

[0117] "Substantially" or "essentially" means nearly entirely or completely, for example, 95%, 96%, 97%, 98%, 99%, or more of a given amount of a portion.

[0118] By "statistically significant", it means that the result is unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. A commonly used measure of significance includes p-value, which is the frequency or probability that the observed event may occur if the null hypothesis is true. If the p-value obtained is less than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0119] "Therapeutic response" refers to an improvement in symptoms (whether sustained or not) following administration of one or more therapeutic agents.

[0120] As used herein, the terms "therapeutically effective amount," "therapeutic dose," "prophylactically effective amount," or "diagnostically effective amount" are the amount of an agent (e.g., an anti-NRP2a antibody, an immunotherapeutic agent) required to elicit a desired biological response following administration.

[0121] As used herein, "treatment" of a subject (e.g., a mammal, such as a human) or cell is any type of intervention used in an attempt to alter the natural course of an individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition and can be performed either prophylactically or secondary to the initiation of a pathological event or contact with a pathogenic agent. Also included is "prophylactic" treatment, which can be directed to 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 indicate a complete eradication, cure, or prevention of the disease or condition, or its associated symptoms.

[0122] The term "wild-type" refers to a gene or gene product (eg, a polypeptide) that is most frequently observed in a population and thus arbitrarily designed as the "normal" or "wild-type" form of the gene.

[0123] Each embodiment in this specification applies to all other embodiments unless expressly stated otherwise.

[0124] Anti-NRP2a antibody Certain embodiments include antibodies, and antigen-binding fragments thereof, that bind to human neuropilin 2a (NRP2a) polypeptides, specifically human NRP2a variant 1 (v1) and / or variant 2 (v2) polypeptides. In certain embodiments, the antibodies, and antigen-binding fragments thereof, specifically bind to human NRP2a v1 and v2 polypeptides and also bind to human NRP2a variant 3 (v3) polypeptides. In some embodiments, the antibodies or antigen-binding fragments thereof selectively modulate (e.g., directly or indirectly interfere with, inhibit, reduce, stimulate, increase) binding of human NRP2a v1 and / or v2 polypeptides to at least one NRP2a ligand, such as a plasma membrane receptor, a growth factor, a signaling molecule, an integrin, a plexin, or other ligand. Specific examples of NRP2a ligands include chemokine (CC motif) ligand 21 (CCL21) polypeptide and / or CC chemokine receptor type 7 (CCR7) polypeptide.

[0125] NRP2 is a single transmembrane receptor with a predominant extracellular region that includes two CUB domains (a1 / a2 combinatorial domains), two factor V / VIII homology domains (b1 / b2 combinatorial domains), a MAM domain (c domain) (see Figures 1A-1B), and a short juxtamembrane region that connects the c domain to the transmembrane domain (spanning the cell membrane). NRP2 is typically expressed in vivo as a mixture of various closely related splice variants, which are often grouped together as NRP2a, which includes variants v1, v2, and v3, and NRP2b, which includes variants v4 and v5. Variant v6 is the soluble form of NRP2 found in the circulation.

[0126] The NRP2a and NRP2b splice variants have identical amino acid sequences across the a1, a2, b1, b2 and c domains, but differ in sequence across the juxtamembrane, transmembrane and cytoplasmic regions. NRP2a variants v1, v2 and v3 also differ in amino acid sequence across these regions based on their patterns of alternative splicing, with NRP2a v1 (931aa) and NRP2a v2 (926aa) having larger insertions compared to the relatively smaller NRP2a variant 3 (909aa). The different sizes of these alternatively spliced ​​forms of NRP2a reflect the loss of a 5 amino acid stretch at the N-terminus of the juxtamembrane sequence from v1 to v2, followed by an additional loss of 17 amino acids immediately C-terminal to the 5 amino acid deletion in the v3 variant. The juxtamembrane region, transmembrane helix and the C-terminal half of the cytoplasmic domain remain identical in all three NRP2a variants.

[0127] In both NRP2a and NRP2b, the a1a2 combining domain of NRP2 interacts with the sema region of semaphorins, and the b1 domain interacts with the semaphorin PSI and Ig-like domains. NRP2 has a higher affinity for SEMA3F and 3G; in contrast, SEMA3A, 3B, and 3E preferentially interact with NRP1. Both NRP1 and NRP2 have similar affinity for SEMA 3C. The b1b2 combining domain of NRP2 interacts with several growth factors that contain heparin-binding domains, including VEGF C&D, placental growth factor (PIGF)-2, fibroblast growth factor (FGF), galectins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF)-beta (see, e.g., Prud'homme et al., Oncotarget. 3:921-939, 2012). NRP2 also interacts with various growth factor specific receptors, and these interactions with receptors can occur independently of binding to SEMA. In this context, integrins and growth factor receptors such as VEGFR2 and VEGFR3, TGF-beta receptor, c-Met, EGFR, FGFR, PDGFR, etc., have been shown to interact with NRPs, generally increasing the affinity of each ligand for its receptor and modulating downstream signaling. The c domain (Mam) domain does not appear to be directly required for ligand binding, but may affect ligand specificity, receptor signaling, and NRP2 dimerization.

[0128] Neuropilin-2 regulates a wide range of cellular functions through its role as an essential cell surface receptor and co-receptor for various ligands (see, e.g., Guo and Vander Kooi, J. Cell. Biol. 290 No 49:29120-29126, 2015). In addition, recent data suggest that NRP2a and NRP2b are differentially expressed in normal tissues and in certain pathological conditions, suggesting that the relative expression of NRP2 splice variants plays an important role in driving receptor crosstalk in a context-dependent manner in multiple NRP2-related diseases. NRP2a and NRP2b also appear to play different, and sometimes opposing, roles in a wide range of cellular functions. Thus, the development of isoform-specific antibodies holds the promise of selectively modulating the activity of NRP2a variants, providing the opportunity to create a new generation of therapeutic agents with significantly enhanced cell type selectivity, isotype specificity, higher potency, and reduced toxicity compared to other anti-NRP2 antibodies that cannot distinguish between these isoforms.

[0129] Neuropilin-2 expression is associated with increased cell plasticity and epithelial to mesenchymal transition (EMT) in both normal and cancer cells, thereby increasing cell survival and the development of chemotherapy resistance during cancer treatment (see, e.g., Gandclement et al., PLoS ONE 6(7)e20444, 2011). In addition, NRP2 expression is increased by TGF-beta exposure leading to both EMT and the development of fibrosis in fibroblasts and endothelial cells (see, e.g., Pardali et al., Int. J. Mol. Sci. 18:2157, 2017). Thus, the development of NRP2a-specific antibodies offers the possibility to selectively modulate cell plasticity and survival, chemotherapy resistance, and the development of fibrosis.

[0130] 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). Thus, the development of NRP2a-specific antibodies offers the possibility of selectively modulating the function of these isoforms to regulate lymphangiogenesis and treat lymphedema. NRP2 also regulates smooth muscle contractility and tone (see, e.g., Bielenberg et al., Amer. J. Path. 181:548-559, 2012), and thus, the development of NRP2a-specific antibodies offers the possibility of selectively modulating the function of these isoforms to regulate smooth muscle contractility and tone.

[0131] NRP2 directly contributes to the maintenance of cancer stem cells and to their survival leading to increased tumor initiation, survival, development of chemo- and radio-resistance, 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; Prud'homme et al., Oncotarget 3:921-939, 2012). Thus, the development of NRP2a-specific antibodies offers the possibility to selectively modulate the function of these isoforms to regulate cancer stem cell growth, survival, development of chemo- and radio-resistance, and metastasis.

[0132] Neuropilin-2 is expressed in various cells of the immune system, including lymphoid cells, such as B and T cells, and myeloid cells, such as basophils, eosinophils, monocytes, dendritic cells, NK cells, neutrophils, and tissue-specific macrophages, including macrophages, such as alveolar macrophages. It is also expressed in endothelial and epithelial cells in lung and other tissues, and in muscle cells (see, e.g., Bielenberg et al., Amer. J. Path. 181:548-559, 2012; Aung, et al., PLoS ONE 11(2)e0147358, 2016; Schellenburg et al., Mol. Imm 90:239-244, 2017; and Wild et al., Int. J. Exp. Path. 93:81-103, 2012). NRP2 regulates immune cell activation and migration (see, e.g., Mendes-da-Cruz et al., PLoS ONE 9(7)e103405, 2014). Thus, the development of NRP2a-specific antibodies offers the possibility to selectively modulate the function of these isoforms to regulate immune cell activation and migration, thereby providing or developing anti-inflammatory and immunomodulatory agents against inflammation and autoimmunity.

[0133] Neuropilin-2 also plays an important role in autophagy, endosomal biogenesis, for example by regulating late endosome maturation, a key aspect of phagocytosis and efferocytosis that contribute to the clearance of infected and apoptotic cells, respectively (see, e.g., Diaz-Vera et al., J. Cell. Sci. 130:697-711, 2017; Dutta et al., Cancer Res. 76:418-428, 2016). Thus, the development of NRP2a-specific antibodies offers the possibility to selectively modulate the function of these isoforms to regulate endosomal biogenesis, phagocytosis, efferocytosis, and autophagy.

[0134] Neuropilin-2 is known to be an important player in the pathophysiology of many diseases (e.g., the "NRP2-associated diseases" described herein) and interacts with a broad array of soluble ligands, including semaphorin 3F, VEGF-C and D, and TGF-beta (see, e.g., Tables L1 and L2), as well as an array of cellular receptors and cofactors. NRP2 is also polysialylated on dendritic cells and actively interacts with the chemokine CCL21 and its receptor CCR7 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, soluble, circulating forms of NRP-2 are known (see, e.g., Parker et al., Structure 23(4)677-687, 2015). Thus, given the central role played by NRP2 in the pathophysiology of a wide range of diseases, it is apparent that modulation of the interactions between NRP2 and NRP2 ligands (e.g., the NRP2 ligands from Tables L1 and L2), and their interactions with antibodies to NRP2, to selectively alter the corresponding biological activities, offers a wide range of potential for the treatment of diseases, including NRP2-associated diseases.

[0135] In certain embodiments, the antibody or antigen-binding fragment thereof selectively or preferentially binds to human NRP2a v1 and / or v2 polypeptides, including to human neuropilin-2B (NRP2b) variant 4 (v4) polypeptide and / or human NRP2b variant 5 (v5) polypeptide, relative to other NRP2 isoform polypeptides. That is, in some embodiments, the antibody or antigen-binding fragment thereof does not substantially bind to human NRP2b v4 polypeptide or NRP2b v5 polypeptide. Certain antibodies or antigen-binding fragments thereof also bind to human NRP2a v3 polypeptide, and certain antibodies and antigen-binding fragments thereof do not substantially bind to human NRP2a v3 polypeptide (see, e.g., FIG. 4C). Exemplary amino acid sequences of NRP2 polypeptides are provided in Table N1 below. [Table N1-1] [Table N1-2] [Table N1-3]

[0136] Thus, in certain embodiments, the antibodies or antigen-binding fragments thereof selectively or preferentially bind to human NRP2a v1 and / or v2 polypeptides from Table N1 and do not substantially bind to, for example, human NRP2b v4 polypeptides or human NRP2b v5 polypeptides from Table N1. Certain antibodies or antigen-binding fragments thereof also bind to human NRP2a v3 polypeptides from Table N1, and some antibodies and antigen-binding fragments thereof do not substantially bind to human NRP2a v3 polypeptides from Table N1.

[0137] In certain embodiments, the antibody or antigen-binding fragment thereof binds to human NRP2a v1 and / or v2 polypeptide at a unique epitope in the juxtamembrane domain, i.e., an epitope present in human NRP2a v1 and / or v2, but not present in human NRP2b v4 or NRP2b v5. In some examples, the unique epitope is present in human NRP2a v3, and in some examples, the unique epitope is not present in human NRP2a v3. Exemplary unique juxtamembrane epitopes are provided in Table N2 below. [Table N2]

[0138] Thus, in some embodiments, the antibody or antigen-binding fragment thereof binds to an NRP2a v1 or v2 polypeptide with a sequence or epitope from Table N2. In some embodiments, the epitope comprises at least about 8, 9, 10, 11, or 12 contiguous amino acids of a sequence or epitope from Table N2, e.g., any of SEQ ID NOs:94-104, or a combination thereof. In some embodiments, the antibody or antigen-binding fragment thereof binds to a contiguous epitope comprising an epitope from Table N2. In some embodiments, the antibody or antigen-binding fragment thereof binds to a discontinuous epitope comprising one or more epitopes from Table N2. In certain embodiments, the antibody or antigen-binding fragment thereof binds to SEQ ID NO:100, or about or at least about 8, 9, 10, 11, or 12 contiguous amino acids of SEQ ID NO:100.

[0139] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope in the neuropilin juxtamembrane domain of NRP2a v1, e.g., residues 803-864, 813-864, 823-864, 833-864, 843-864, 853-864; 803-854, 803-844, 803-834, 803-824, and / or 803-814, as defined in SEQ ID NO:94. 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 v2, e.g., residues 803-859, 813-859, 823-859, 833-859, 843-859, 853-859; 803-849, 803-839, 803-829, 803-819, and / or 803-809, as defined in SEQ ID NO:95. In some embodiments, the antibody or antigen-binding fragment thereof binds to at least one epitope in the neuropilin juxtamembrane domain of NRP2a v1 / v2, e.g., residues 818-832, 820-832, 822-832, 824-832, 826-832, 818-830, 818-828, 818-826, and / or 818-824, as defined in SEQ ID NO:95.

[0140] 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 v1 / v2, e.g., residues 818-832, 820-832, 822-832, 824-832, 826-832, or 818-830, 818-828, 818-826, and / or 818-824, as defined in SEQ ID NO: 95. In certain embodiments, the antibody or antigen-binding fragment thereof binds to SEQ ID NO: 100.

[0141] In some embodiments, the antibody or antigen-binding fragment thereof binds to an NRP2a v1 and / or v2 polypeptide, and optionally an NRP2a v3 polypeptide, e.g., a sequence from Table N1 and / or an epitope from Table N2, at about 10 pM to about 500 pM or up to about 50 nM, or at about, at least about, or at about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20 ... or with an affinity of about 10 pM to about 500 pM, about 10 pM to about 400 pM, about 10 pM to about 300 pM, about 10 pM to about 200 pM, about 10 pM to about 100 pM, about 10 pM to about 50 pM, or about 20 pM to about 500 pM, about 20 pM to about 50 pM, or about M~about 400pM, about 20pM to about 300pM, about 20pM to about 200pM, about 20pM to about 100pM, about 20pM to about 50pM, or about 30pM to about 500pM, about 30p M~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 200pM, about 30pM 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.

[0142] In certain embodiments, the binding affinity of the antibody, or antigen-binding fragment thereof, for a human NRP2a v1 or v2 polypeptide (see Table N1) is at least about 1.5 times, 2 times, 4 times, 6 times, 8 times, 10 times, 20 times, 40 times, 60 times, 80 times, 100 times, 200 times, 400 times, 600 times, 800 times, or 1000 times stronger than its binding affinity for an NRP2a v3 polypeptide, an NRP2b v4 polypeptide, and / or an NRP2b v5 polypeptide (see Table N1).

[0143] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to at least one epitope within a region of a human NRP2a polypeptide that binds to or interacts with at least one "NRP2a ligand," which includes any molecule that interacts with or reversibly binds to human NRP2a v1 and / or v2, but does not substantially interact with or bind to NRP2b v4 or NRP2b v5. General examples of NRP2 ligands are provided in Table L1. [Table L1]

[0144] Thus, in certain embodiments, at least one NRP2a ligand is selected from Table L1. Specific examples of "NRP2a ligands" include chemokine (CC motif) ligand 21 (CCL21) polypeptide and CC chemokine receptor type 7 (CCR7) polypeptide. The amino acid sequences of exemplary NRP2a ligands are provided in Table L2 below. [Table L2]

[0145] Thus, in certain embodiments, at least one NRP2a ligand is selected from Table L2, and the anti-NRP2a antibody, or antigen-binding fragment thereof, modulates (e.g., interferes with, inhibits, reduces) binding of human NRP2a v1 and / or v2 polypeptides (e.g., selected from Table N1) to an NRP2a ligand from Table L1 or Table L2, or a biologically active fragment or variant thereof.

[0146] In some embodiments, the at least one NRP2 ligand is selected from CCL21 and its receptor CCR7. CCR7 activity has been implicated in a variety of disease states, including chronic inflammatory conditions (Moschovakis et al., 2012, Eur J Immunol. 42:1949-55), atherosclerosis (Luchtefeld et al., 2010, Circulation 122:1621-28), HIV infection (Evans et al., 2012, Cytokine Growth Factor Rev. 23:151-57), and cancer (Ben-Baruch, 2009, Cell Adhesion Migration 3:328-33). CCR7 activity has been implicated in inflammatory disorders including inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, tissue or organ transplant rejection, asthma, allergic airway inflammation, airway smooth muscle hyperplasia, and fibrotic lung disease (Gomperts et al., 2007, J Leukoc Biol. 82: 449-56; Kawakami et al., 2012, Cell Immunol 2575: 24-32; Saunders et al., 2009, Clin Exp Allergy 39: 1684-92). CCR7 activity has also been implicated in rheumatoid arthritis (Moschovakis et al., 2012, Eur J Immunol. 42: 1949-55). CCR7 activity has been implicated in multiple sclerosis (Aung et al., 2010, J Neuroimmunol. 226:158-64), psoriasis (Fan et al., 2008, Indian J Dermatol Venereol Leprol. 74(5):550; Bose et al., 2013, Am J Pathol, 183(2):413-421), and atherosclerosis (Luchtefeld et al., 2010, Circulation 10 122:1621-28).CCR7 activity has been implicated in HIV infection and other infections, including chronic and latent infections, including Leishmania donovani infection (Evans et al., 2012, Cytokine Growth Factor Rev. 23:151-57).

[0147] Various studies have revealed that CCR7 is expressed in a wide variety of tumor cells, including mantle cell lymphoma (MCL), follicular lymphoma, large B-cell lymphoma, AIDS-related lymphoma, lymphoplasmacytic lymphoma, Burkitt's lymphoma, B-cell acute lymphoblastic leukemia, Hodgkin's disease, adult T-cell leukemia / lymphoma, mycosis fungoides, blast crisis of chronic myeloproliferative syndrome, blast crisis of myelodysplastic syndrome, cancers such as breast cancer, non-small cell lung cancer, melanoma, gastric cancer or squamous cell carcinoma of the head and neck, and colon cancer, including B-cell chronic lymphocytic leukemia, non-Hodgkin's lymphoma, breast cancer cells, and malignant breast tumors (see, for example, WO2007 / 003426). CCR7 also plays a role in lymph node metastasis of various cancers (see, e.g., Viola and Luster, 2008, Annu Rev Pharmacol Toxicol. 48:171-97).

[0148] Thus, anti-NRP2a antibodies that directly modulate either CCL21 binding or CCR7 receptor signaling may be expected to find utility in modulating one or more of these diseases and disorders, including for the treatment of inflammatory disorders, cancer, tissue or organ transplant rejection, airway inflammation, RA, and for the treatment and prevention of latent and persistent infections.

[0149] In certain embodiments, the antibody or antigen-binding fragment thereof is a "blocking antibody" that completely or substantially inhibits binding between a human NRP2a v1 and / or v2 polypeptide and an NRP2a ligand, such as human CCL21 and / or CCR7. In some embodiments, the "blocking antibody" inhibits about or at least about 80-100% (e.g., 80, 85, 90, 95, or 100%) of the theoretical maximum binding between an NRP2a v1 and / or v2 polypeptide and an NRP2a ligand following pre-incubation of the "blocking antibody" with a substantially stoichiometrically equivalent amount of the NRP2a polypeptide. As used herein, "stoichiometrically equivalent amount" refers to a situation in which the number of moles of one substance (e.g., an anti-NRP2a antibody) is equal to or substantially equivalent to the number of moles of at least one other substance (e.g., an NRP2a polypeptide) in a given equation or reaction.

[0150] 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 NRP2a v1 and / or v2 polypeptide and an NRP2a ligand, such as human CCL21 and / or CCR7. In some embodiments, a "partial blocking antibody" inhibits about or at least about 20-80% (e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80%) of the theoretical maximum binding between an NRP2a polypeptide and an NRP2a ligand following pre-incubation of the "partial blocking antibody" with a substantially stoichiometrically equivalent amount of the NRP2a polypeptide.

[0151] In certain embodiments, the antibody or antigen-binding fragment thereof inhibits, blocks, or otherwise reduces binding between an NRP2a v1 or v2 polypeptide and a human CCL21 polypeptide, e.g., in an in vitro binding assay, an in vitro or ex vivo cell-based assay, or in vivo. In some embodiments, the antibody or antigen-binding fragment thereof antagonizes or reduces the theoretical maximum binding between a human NRP2a v1 and / or v2 polypeptide and a human CCL21 NRP2a polypeptide 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%), e.g., after pre-incubation of the anti-NRP2a antibody with a substantially stoichiometrically equivalent amount of the anti-NRP2a polypeptide.

[0152] In some embodiments, the antibody or antigen-binding fragment thereof specifically inhibits, blocks, or otherwise reduces binding (e.g., dimerization) between an NRP2a v1 or v2 polypeptide and a human CCR7 polypeptide, e.g., in an in vitro binding assay, an in vitro or ex vivo cell-based assay, or in vivo. In some embodiments, the antibody or antigen-binding fragment thereof antagonizes or reduces the theoretical maximum binding between a human NRP2a v1 and / or v2 polypeptide and a human CCR7 polypeptide 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%), e.g., after pre-incubation of the anti-NRP2a antibody with a substantially stoichiometrically equivalent amount of the anti-NRP2a polypeptide.

[0153] In some embodiments, the antibody or antigen-binding fragment thereof modulates CCL21 / CCR7-mediated signaling by, for example, 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%) compared to a control. Examples of CCL21 / CCR7-mediated signaling activities include, but are not limited to, induction of immune cell migration, including dendritic cells or mature T cells, inhibition of immature T cells, and induction of tumor or cancer cell migration. Exemplary immune cells and tumor / cancer cells are described herein.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ), wherein 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.

[0155] In some embodiments, the antibody or antigen-binding fragment thereof has an affinity (Kd or EC 50 ), wherein 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.

[0156] In certain embodiments, the antibody or antigen-binding fragment thereof selectively binds to human NRP2a v1 and / or v2 polypeptides (see Table N1) relative to the corresponding mouse NRP2 polypeptide, e.g., where its affinity for human NRP2a v1 and / or v2 polypeptides is significantly stronger than its affinity for the corresponding mouse NRP2 polypeptide, e.g., by about or at least about 2, 5, 10, 20, 30, 40, 50, 100, 500, or 1000 times or more. In certain embodiments, the antibody or antigen-binding fragment thereof selectively binds to human NRP2a v1 and / or v2 polypeptides and does not substantially bind to the corresponding mouse NRP2 polypeptide. Certain exemplary mouse NRP2 polypeptides include Mus musculus NRP2 polypeptides (see, e.g., UniProt O35375).

[0157] In certain embodiments, the antibody or antigen-binding fragment thereof is characterized by or comprises a heavy chain variable region (VH) sequence comprising complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 sequences, and a light chain variable region (VL) sequence comprising complementarity determining regions VLCDR1, VLCDR2, and VLCDR3 sequences. Exemplary VH, VHCDR1, VHCDR2, VHCDR3, VL, VLCDR1, VLCDR2, and VLCDR3 sequences are provided in Tables A1 and A2 below. [Table A1-1] [Table A1-2] [Table A1-3] [Table A2-1] [Table A2-2] [Table A2-3]

[0158] Thus, in certain embodiments, the antibody or antigen-binding fragment thereof comprises: A heavy chain variable region (VH) sequence comprising the complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 sequences selected from Table A1 and variants thereof that specifically bind to a human NRP2a polypeptide or an epitope thereof (e.g., selected from Tables N1, N2); and A light chain variable region (VH) sequence comprising complementarity determining regions VLCDR1, VLCDR2, and VLCDR3 sequences selected from Table A1 and variants thereof that specifically bind to a human NRP2 polypeptide or an epitope thereof (e.g., selected from Tables N1, N2).

[0159] In certain embodiments, the CDR sequences are as follows: VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 130-132, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 133-135, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 136-138, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 139-141, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 142-144, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 145-147, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 148-150, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 151-153, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 154-156, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 157-159, respectively, including variants thereof having a total of 1, 2, 3, 4, 5, or 6 modifications across all of the CDR regions; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 1-3, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 4-6, respectively, including variants thereof; VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 7-9, respectively, and VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 10-12, respectively, including variants thereof; the VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 13-15, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 16-18, respectively, including variants thereof; the VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 19-21, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 22-24, respectively, including variants thereof; the VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 25-27, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 28-30, respectively, including variants thereof; the VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 31-33, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 34-36, respectively, including variants thereof; the VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 37-39, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 40-42, respectively, including variants thereof; the VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 43-45, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 46-48, respectively, including variants thereof; the VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 49-51, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 52-54, respectively, including variants thereof; the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 55-57, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 58-60, respectively, including variants thereof; or The VHCDR1, VHCDR2, and VHCDR3 sequences include SEQ ID NOs: 61 to 63, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences include SEQ ID NOs: 64 to 66, respectively, including variants thereof.

[0160] In certain embodiments, an antibody or antigen-binding fragment thereof (e.g., a variant of the 401 antibody or antigen-binding fragment thereof) comprises CDR consensus sequences, for example, in which the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 13, 127, and GX1X2X3X4X5, respectively (in which X1 is G, A, or S, X2 is Y, F, K, L, or R, X3 is T, A, G, I, L, Q, or V, X4 is D, A, G, K, N, Q, R, or S, and X5 is Y, A, D, E, F, G, H, I, K, L, N, Q, R, S, T, or V), and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 16, 17, and X6X7X8X9X, respectively. 10 X 11 X 12 X 13(wherein X6 is S, A, G, I, L, P, T, or V; X7 is Q, A, G, R, or S; X8 is S, A, H, K, L, Q, or T; X9 is T, F, G, H, I, K, L, N, Q, R, S, V, or Y; and X 10 is H, A, D, E, F, G, I, K, L, N, Q, R, S, T, or Y, and X 11 is V, A, E, F, G, H, I, K, L, N, P, Q, R, S, T, or Y; X 12 is L, A, E, H, I, N, P, Q, S, T, or V, and X 13 is T, A, D, E, F, G, I, K, L, N, Q, R, S, or V) (see Table E7 for definitions of "X" residues); Also included are variants thereof that are affinity matured variants that bind to human NRP2a polypeptides or epitopes thereof (see, e.g., Tables N1, N2), e.g., variants that have 1, 2, 3, 4, 5, or 6 total modifications across all of the CDR regions, ... H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and / or V L Exemplary "modifications" include amino acid substitutions, additions, and deletions.

[0161] In certain embodiments, the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2, including, for example, where the VH sequence has 1, 2, 3, 4, or 5 alterations in one or more framework regions.

[0162] In some embodiments, the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2, including, for example, where the VL sequence has 1, 2, 3, 4, or 5 alterations in one or more framework regions.

[0163] In some embodiments, the VH and VL sequences of the antibody or antigen-binding fragment are as follows: the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 160 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 161; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 162 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 163; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 164 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 165; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 166 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 167; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 168 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 169; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:67 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:68; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:69 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:70; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:71 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:72; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:73 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:74; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:75 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:76; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:77 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:78; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:79 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:80; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:81 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:82; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:83 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:84; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:85 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:86; or The VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:87 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:88.

[0164] 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: 170, L The sequence comprises SEQ ID NO:171 (see Table E7 for definition of "X" residues); V H The sequence comprises SEQ ID NO: 160, L The sequence comprises SEQ ID NO:161; V H The sequence comprises SEQ ID NO: 162, L The sequence comprises SEQ ID NO:163; V H The sequence comprises SEQ ID NO: 164, L The sequence comprises SEQ ID NO:165; V H The sequence comprises SEQ ID NO: 166, L The sequence comprises SEQ ID NO:167; V H The sequence comprises SEQ ID NO: 168, L The sequence comprises SEQ ID NO:169; V H The sequence comprises SEQ ID NO:67, L The sequence comprises SEQ ID NO:68; V H The sequence comprises SEQ ID NO:69, L The sequence comprises SEQ ID NO:70; V H The sequence comprises SEQ ID NO: 71, L The sequence comprises SEQ ID NO:72; V H The sequence comprises SEQ ID NO: 73, L The sequence comprises SEQ ID NO:74; V H The sequence comprises SEQ ID NO: 75, L The sequence comprises SEQ ID NO:76; VH The sequence comprises SEQ ID NO: 77, L The sequence comprises SEQ ID NO:78; V H The sequence comprises SEQ ID NO:79, L The sequence comprises SEQ ID NO:80; V H The sequence comprises SEQ ID NO: 81, L The sequence comprises SEQ ID NO:82; V H The sequence comprises SEQ ID NO: 83, L The sequence comprises SEQ ID NO:84; V H The sequence comprises SEQ ID NO: 85, L The sequence comprises SEQ ID NO:86; or V H The sequence comprises SEQ ID NO:87, L The sequence comprises SEQ ID NO:88.

[0165] Also included are variants thereof, e.g., variants having 1, 2, 3, 4, or 5 modifications in one or more framework regions that bind to a human NRP2a polypeptide or an epitope thereof (see, e.g., Tables N1, N2). Exemplary "modifications" include amino acid substitutions, additions, and deletions.

[0166] For illustrative purposes only, binding interactions between an antibody or antigen-binding fragment thereof, a human NRP2a polypeptide (e.g., NRP2a v1 and / or v2), and / or an NRP2 ligand (e.g., CCL21, CCR7) can be detected and quantified using a variety of routine methods, including Octet and Biacore assays (e.g., coupled to a sensor chip using appropriately tagged soluble reagents), FACS analysis using cells expressing NRP2a polypeptides on the cell surface (either native or recombinant), immunoassays, fluorescent staining assays, ELISA assays, and microcalorimetry approaches such as ITC (isothermal titration calorimetry). See also the Examples.

[0167] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a variant or otherwise modified Fc region, including those that have altered properties or biological activity compared to a wild-type Fc region. Examples of modified Fc regions include those that have a mutated sequence, 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 that are 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 modify the binding characteristics 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, excretion rate, area under the curve (AUC), clearance, C max , t max , C min The Fc region may have altered (e.g., increased, decreased) affinity, immunogenicity, complement fixation or activation, and / or CDC / ADCC / ADCP-related activity, among other properties described herein, as compared to the corresponding wild-type Fc sequence of the antibody or antigen-binding fragment thereof. Included are modified Fc regions of human and / or murine origin.

[0168] 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 hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of CH2 / CH3 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / IgG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA 2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgE / IgA1, IgE / IgA2, IgE / IgD, IgE / Ig E, IgE / IgG1, IgE / IgG2, IgE / IgG3, IgE / IgG4, IgE / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, Ig G1 / IgG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, Ig G3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG In certain embodiments, the antibody comprises a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgM / IgG4, IgG4 / IgM, IgM / IgA1, IgM / IgA2, IgM / IgD, IgM / IgE, IgM / IgG1, IgM / IgG2, IgM / IgG3, IgM / IgG4, IgM / IgM (or a fragment or variant thereof), optionally a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, or IgG4, and / or a CH4 domain from IgE and / or IgM. In certain embodiments, the hinge, CH2, CH3, and CH4 domains are from human Ig.

[0169] Additional examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of CH2 / CH4 domains: IgA1 / IgE, IgA2 / IgE, IgD / IgE, IgE / IgE, IgG1 / IgE, IgG2 / IgE, IgG3 / IgE, IgG4 / IgE, IgM / IgE, IgA1 / IgM, IgA2 / IgM, IgD / IgM, IgE / IgM, IgG1 / IgE, IgM, IgG2 / IgM, IgG3 / IgM, IgG4 / IgM, IgM / IgM (or fragments or variants thereof), optionally a hinge from one or more of IgA1, IgA2, IgD, IgG1, IgG2, IgG3, IgG4, and / or a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In certain embodiments, the hinge, CH2, CH3, and CH4 domains are from a human Ig.

[0170] Particular examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of CH3 / CH4 domains: IgA1 / IgE, IgA2 / IgE, IgD / IgE, IgE / IgE, IgG1 / IgE, IgG2 / IgE, IgG3 / IgE, IgG4 / IgE, IgM / IgE, IgA1 / IgM, IgA2 / IgM, IgD / IgM, IgE / IgM, IgG1 / IgE, IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM. In certain embodiments, the hinge, CH2, CH3, and CH4 domains are from a human Ig.

[0171] Particular examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of hinge / CH2 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 ... gG1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, Ig D / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / I gG4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / I gM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA 1, IgG4 / IgA2, IgG4 / IgD, IgG4 / IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, IgG4 / IgM (or a fragment or variant thereof), optionally comprising a CH3 domain from one or more of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, or IgM, and / or the CH4 domain of IgE and / or IgM. In a particular embodiment, the hinge, CH2, CH3, and CH4 domains are from a human Ig.

[0172] Particular examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of hinge / CH3 domains: IgA1 / IgA1, IgA1 / IgA2, IgA1 / IgD, IgA1 / IgE, IgA1 / IgG1, IgA1 / IgG2, IgA1 / IgG3, IgA1 / IgG4, IgA1 / IgM, IgA2 / IgA1, IgA2 / IgA2, IgA2 / IgD, IgA2 / IgE, IgA2 / Ig G1, IgA2 / IgG2, IgA2 / IgG3, IgA2 / IgG4, IgA2 / IgM, IgD / IgA1, IgD / IgA2, IgD / IgD, IgD / IgE, IgD / IgG1, IgD / IgG2, IgD / IgG3, IgD / IgG4, IgD / IgM, IgG1 / IgA1, IgG1 / IgA2, IgG1 / IgD, IgG1 / IgE, IgG1 / IgG1, IgG1 / IgG2, IgG1 / IgG3, IgG1 / IgG 4, IgG1 / IgM, IgG2 / IgA1, IgG2 / IgA2, IgG2 / IgD, IgG2 / IgE, IgG2 / IgG1, IgG2 / IgG2, IgG2 / IgG3, IgG2 / IgG4, IgG2 / IgM, IgG3 / IgA1, IgG3 / IgA2, IgG3 / IgD, IgG3 / IgE, IgG3 / IgG1, IgG3 / IgG2, IgG3 / IgG3, IgG3 / IgG4, IgG3 / IgM, IgG4 / IgA1, I IgA1, IgA2, IgD, IgE, IgG4 / IgG1, IgG4 / IgG2, IgG4 / IgG3, IgG4 / IgG4, 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 CH4 domain from IgE and / or IgM. In certain embodiments, the hinge, CH2, CH3, and CH4 domains are from a human Ig.

[0173] Some examples include hybrid Fc regions comprising, consisting of, or consisting essentially of the following combinations of hinge / CH4 domains: IgA1 / IgE, IgA1 / IgM, IgA2 / IgE, IgA2 / IgM, IgD / IgE, IgD / IgM, IgG1 / IgE, IgG1 / IgM, IgG2 / IgE, IgG2 / IgM, IgG3 / IgE, IgG3 / IgM, IgG4 / IgE, IgG4 / IgM (or fragments or variants thereof), 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.

[0174] Specific examples of hybrid Fc regions can be found, for example, in WO2008 / 147143, which are derived from a combination of IgG subclasses or a combination of human IgD and IgG.

[0175] Also included are antibodies or antigen-binding fragments thereof having a derivatized or otherwise modified Fc region. In certain aspects, the Fc region may be modified, for example, by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like, as compared to a wild-type or native Fc region. In certain embodiments, the Fc region may comprise a wild-type or native glycosylation pattern, or alternatively, may comprise increased glycosylation as compared to the native form, decreased glycosylation as compared to the native form, or it may be completely deglycosylated. As an example of a modified Fc glycoform, reduced glycosylation of the Fc region reduces binding to the C1q region of the first complement component C1, a decrease in ADCC-related activity, and / or a decrease in CDC-related activity. Certain embodiments thus employ a deglycosylated or non-glycosylated Fc region. For the production of an exemplary non-glycosylated Fc region, see, e.g., WO2005 / 047337. 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 No. 2010 / 0080794). Certain embodiments can include an Fc region in which about 80-100% of the glycoproteins in the Fc region include a mature core sugar structure lacking fructose (see, e.g., U.S. Patent Application No. 2010 / 0255013). Some embodiments include Fc regions that are optimized by substitutions or deletions to reduce levels of fucosylation, e.g., to increase affinity for Fc γRI, Fc γRIa, or Fc γRIIIa, and / or to improve phagocytosis by Fc γRIIa-expressing cells (see U.S. Patent Application Nos. 2010 / 0249382 and 2007 / 0148170).

[0176] As another example of a modified 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: increased ADCC effector activity, increased binding affinity for FcγRIIIA (and certain other FcRs), similar or increased binding specificity for a target of an NRP2a polypeptide, similar or higher binding affinity for a target of an NRP2a polypeptide, and / or similar or lower binding affinity for a mannose receptor compared to a corresponding Fc region comprising complex-type N-glycans (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 an Fc region for FcγR 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 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 attached to the 6-position of the N-acetylglucosamine at the reducing end of the glycan (see, e.g., U.S. Patent Application No. 2010 / 0092997). Certain embodiments may include an IgG Fc region that is glycosylated with at least one galactose moiety connected to each terminal sialic acid moiety by an α-2,6 linkage, optionally where the Fc region has greater anti-inflammatory activity compared to the corresponding wild-type Fc region (see, e.g., U.S. Patent Application No. 2008 / 0206246).Certain of these and related engineered glycosylation approaches, as described herein, have produced substantial enhancements in the ability of an Fc region to selectively bind to an FcR, such as FcγRIII, mediate ADCC, and modify other properties of the Fc region.

[0177] Certain variants, fragments, hybrids, or otherwise modified Fc regions of antibodies or antigen-binding fragments thereof may have altered binding to one or more FcRs and / or corresponding changes in 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 of Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal Fc receptors compared to the corresponding wild-type Fc sequence. In other embodiments, variants, fragments, hybrids, or modified Fc regions may have decreased binding to one or more of Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal Fc receptors compared to the corresponding wild-type Fc sequence. Particular FcRs are described elsewhere herein.

[0178] In some embodiments, the antibody comprises an Fc domain comprising one or more mutations to increase binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal 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 to increase binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal Fc receptors compared to the corresponding wild-type Fc sequence. In some embodiments, the antibody comprises an Fc domain comprising one or more mutations to increase effector function. In some embodiments, at least one antibody comprises an Fc domain selected from human IgG1 and IgG3 comprising one or more mutations to increase effector function.

[0179] In some embodiments, the antibody is a blocking antibody comprising an Fc domain with high effector activity. In some embodiments, the blocking antibody comprises an Fc domain selected from human IgG1 and IgG3 comprising one or more mutations to increase effector function. In some embodiments, the antibody is a partial blocking antibody comprising an Fc domain with high effector activity. In some embodiments, the partial blocking antibody comprises an Fc domain selected from human IgG1 and IgG3 comprising one or more mutations to 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 to increase effector function.

[0180] In some embodiments, the antibody comprises an Fc domain comprising one or more mutations to reduce binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal 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 to reduce binding to one or more Fcγ receptors, Fcα receptors, Fcε receptors, and / or neonatal Fc receptors compared to the corresponding wild-type Fc sequence. In some embodiments, the antibody comprises an Fc domain comprising one or more mutations to reduce effector function. In some embodiments, the antibody comprises an Fc domain selected from human IgG2 and IgG4 comprising one or more mutations to reduce effector function.

[0181] 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 to reduce effector function. In some embodiments, the antibody is a partial blocking antibody comprising an Fc domain with reduced effector activity. In some embodiments, the partial blocking antibody comprises an Fc domain selected from human IgG2 and IgG4 comprising one or more mutations to 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 to reduce effector function.

[0182] 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 Nos. 2009 / 0017023, 2009 / 0010921, and 2010 / 0203046; and WO2000 / 42072 and WO2004 / 016750. Particular 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 decrease binding to the inhibitory receptor FcγRIIb. These mutations can be combined to obtain double and triple mutant variants with further improvements in binding to FcR. Particular embodiments include the S298A / E333A / K334A triple mutant, which has increased binding to FcγRIIIa, decreased binding to FcγRIIb, and increased ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604, 2001; and Presta et al., Biochem Soc Trans. 30:487-490, 2002). See also engineered Fc glycoforms with increased binding to FcR, as disclosed in Umana et al., supra; and U.S. Patent No. 7,662,925. Some embodiments include an Fc region that includes 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. Application Nos. 2009 / 0163699 and 20060173170).

[0183] Certain variants, fragments, hybrids, or modified Fc regions may have altered effector functions compared to the corresponding wild-type Fc sequence. For example, such Fc regions may have increased complement 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. As merely one illustrative example, the Fc region may include deletions or substitutions in complement binding sites, such as C1q binding sites, and / or deletions or substitutions in ADCC sites. Examples of such deletions / substitutions are described, for example, in U.S. Patent No. 7,030,226. Many Fc effector functions, such as ADCC, can be assayed according to routine techniques in the art (see, e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7:1-26, 1978). Useful effector cells for such assays include, but are not limited to, natural killer (NK) cells, macrophages, and other peripheral blood mononuclear cells (PBMCs). Alternatively, or in addition, certain Fc effector functions can be assessed in vivo, for example, by using animal models described in Clynes et al. PNAS. 95:652-656, 1998.

[0184] 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. As an example, modifications to an Fc region that alter its ability to bind to FcRn can alter its half-life in vivo. 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. Pat. Nos. 7,217,797 and 7,732,570; and U.S. Patent Application Nos. US 2010 / 0143254 and 2010 / 0143254.

[0185] Additional non-limiting examples of modifications to alter stability or half-life include substitutions / deletions at 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, substitution with arginine or aspartic acid at position 385, or serine, substitution at position 386 with threonine or proline, substitution at position 387 with arginine or proline, substitution at position 389 with proline, asparagine, or serine, substitution at position 428 with methionine or threonine, substitution at position 434 with tyrosine or phenylalanine, substitution at position 433 with histidine, arginine, lysine, or serine, and / or substitution at position 436 with histidine, tyrosine, arginine, or threonine, including any combination thereof. Such modifications optionally increase the affinity of the Fc region for FcRn, thereby increasing the half-life as compared to the corresponding wild-type Fc region.

[0186] Certain variant hybrids, or modified Fc regions, may have altered solubility compared to the corresponding wild-type Fc sequence. In certain embodiments, such Fc regions may have increased solubility compared to the corresponding wild-type Fc sequence. In other embodiments, variant hybrids, or modified Fc regions may have decreased solubility compared to the corresponding wild-type Fc sequence. Solubility can be measured, for example, in vitro (e.g., under physiological conditions) according to routine techniques in the art. Exemplary solubility measurements are described elsewhere herein.

[0187] Additional examples of variants include IgG Fc regions with conservative or non-conservative substitutions (as described elsewhere herein) at one or more of heavy chain positions 250, 314, or 428, or any combination thereof, such as positions 250 and 428, or 250 and 314, or 314 and 428, or 250, 314, and 428 (see, e.g., US Patent Application Publication No. 2011 / 0183412). In certain embodiments, the residue at position 250 is substituted with glutamic acid or glutamine, and / or the residue at position 428 is substituted with leucine or phenylalanine. As another illustrative example of an IgG Fc variant, any one or more of the amino acid residues at positions 214-238, 297-299, 318-322, and / or 327-331 may be used as suitable targets for modification (e.g., conservative or non-conservative substitution, deletion). In a particular embodiment, the IgG Fc variant CH2 domain comprises amino acid substitutions at positions 228, 234, 235, and / or 331 (see, e.g., human IgG4 with Ser228Pro and Leu235Ala mutations) to attenuate effector functions of the Fc region (see, e.g., 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 Ed., National Institutes of Health, Bethesda, Md. (1991). Certain of these and related embodiments optionally have altered (e.g., increased, decreased) FcRn binding and / or serum half-life, without reduced effector function, such as ADCC or CDC-related activity.

[0188] Additional examples include variant Fc regions that include one or more amino acid substitutions at positions 279, 341, 343, or 373 of a 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 can be conservative or non-conservative, or can include non-natural amino acids or mimetics, as described herein. Alone or in combination with these substitutions, certain embodiments may also use a variant Fc region that includes 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, 244L, 245L, 246L, 247L, 248L, 249L, 250L, 251L, 252L, 253L, 254L, 255L, 256L, 257L, 258L, 259L, 260L, 261L, 262L, 263L, 264L, 265L, 266L, 267L, 268L, 269L, 270L, 271L, 272L, 273L, 274L, 275L, 276L, 277L, 278L, 279L, 280L, 281L, 282L, 283L, 284L, 285L, 286L, 287L, 288L, 289L, 290L, 291L, 292L, 293L, 294L, 295L, 296L, 297L, 298L, 299L, 298L, 299L, 300L, 300L, 45R, 247A, 247D, 247E, 247F, 247M, 247N, 247Q, 247R, 247S, 247T, 247W, 247Y, 248F, 248P, 248Q, 248W, 249L, 249M, 249N, 249P, 249Y, 251H, 251I, 251W, 254D, 254E, 254F, 254G, 254H, 254I, 254K, 254L, 254M, 254N, 254P, 254Q, 254R , 254V, 254W, 254Y, 255K, 255N, 256H, 256I, 256K, 256L, 256V, 256W, 256Y, 257A, 257I, 257M, 257N, 257S, 258D, 260 S, 262L, 264S, 265K, 265S, 267H, 267I, 267K, 268K, 269N, 269Q, 271T, 272H, 272K, 272L, 272R, 279A, 279D, 279F, 27 9G, 279H, 279I, 279K, 279L, 279M, 279N, 279Q, 279R, 279S, 279T, 279W, 279Y, 280T, 283F, 283G, 283H, 283I, 283K, 2 83L, 283M, 283P, 283R, 283T, 283W, 283Y, 285N, 286F, 288N, 288P, 292E, 292F, 292G, 292I, 292L, 293S, 293V, 301W,304E, 307E, 307M, 312P, 315F, 315K, 315L, 315P, 315R, 316F, 316K, 317P , 317T, 318N, 318P, 318T, 332F, 332G, 332L, 332M, 332S, 332V, 332W, 339D , 339E, 339F, 339G, 339H, 339I, 339K, 339L, 339M, 339N, 339Q, 339R, 339S , 339W, 339Y, 341D, 341E, 341F, 341H, 341I, 341K, 341L, 341M, 341N, 341P , 341Q, 341R, 341S, 341T, 341V, 341W, 341Y, 343A, 343D, 343E, 343F, 343 G, 343H, 343I, 343K, 343L, 343M, 343N, 343Q, 343R, 343S, 343T, 343V, 343 W, 343Y, 373D, 373E, 373F, 373G, 373H, 373I, 373K, 373L, 373M, 373N, 373 Q, 373R, 373S, 373T, 373V, 373W, 375R, 376E, 376F, 376G, 376H, 376I, 376 L, 376M, 376N, 376P, 376Q, 376R, 376S, 376T, 376V, 376W, 376Y, 377G, 37 7K, 377P, 378N, 379N, 379Q, 379S, 379T, 380D, 380N, 380S, 380T, 382D, 38 2F, 382H, 382I, 382K, 382L, 382M, 382N, 382P, 382Q, 382R, 382S, 382T, 38 2V, 382W, 382Y, 385E, 385P, 386K, 423N, 424H, 424M, 424V, 426D, 426L, 42 7N, 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 above, the numbering of residues in the heavy chain is:The numbering is from the EU index (Kabat et al., supra). Such variant Fc regions typically confer an altered effector function or altered serum half-life to the antibody to which the variant Fc region is operatively attached. Preferably, the altered effector function is an increase in ADCC, a decrease in ADCC, an increase in CDC, a decrease in CDC, an increase in Clq binding affinity, a decrease in Clq binding affinity, an increase in FcR (preferably FcRn) binding affinity, or a decrease in FcR (preferably FcRn) binding affinity, compared to a corresponding Fc region lacking such amino acid substitutions.

[0189] Additional examples are 221st, 222nd, 224th, 227th, 228th, 230th, 231st, 223rd, 233rd, 234th, 235th, 236th, 237th, 238th, 239th, 240th, 241st, 243rd, 244th, 245th, 246th, 24th 7th, 249th, 250th, 258th, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 275th, 276th, 278th, 280th, 281st, 283rd, 2 and / or 428 (see, e.g., U.S. Pat. No. 7,662,925). In certain embodiments, the variant Fc Region comprises at least one amino acid substitution selected from the group consisting of: P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274 T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L32 8M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y. In other specific embodiments, the variant Fc region comprises at least one amino acid substitution selected from the group consisting of: V264I, F243L / V264I, L328M, I332E, L328M / I332E, V264I / I332E, S298A / I332E, S239E / I332E, S239Q / I332E, S239E, A330Y, I332D,L328I / I332E, L328Q / I332E, V264T, V240I, V266I, S239D, S239D / I332D, S239D / I332E, S239D / I332N, S239D / I332Q, S239E / I332D, S239E / I332N, S239E / I332Q, S239N / I332D, S239N / I332E, S239Q / I332D, A330Y / I332E, V264I / A330Y / I332E, A330L / I332E, V264I / A330L / I332E, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239T, V240M, V264Y, A330I, N325T, L328D / I332E, L328V / I332E, L328T / I332E, L328I / I332E, S239E / V264I / I332E, S239Q / V264I / I332E, S239E / V264I / A330Y / I332E, S239D / A330Y / I332E, S239N / A330Y / I332E, S239D / A330L / I 332E, S239N / A330L / I332E, V264I / S298A / I332E, S239D / S298A / I332E, S239N / S298A / I332E, S239D / V264I / I332E, S239D / V264I / S298A / I 332E, S239D / V264I / A330L / I332E, S239D / I332E / A330I, P230A, P230A / E233D / I332E, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N 276L, Y278T, V302I, E318R, S324D, S324I, S324V, K326I, K326T, T335D, T335R, T335Y, V240I / V266I, S239D / A330Y / I332E / L234I, S239D / A330Y / I332E / L235D, S239D / A330Y / I332E / V240I, S239D / A330Y / I332E / V264T, S239D / A330Y / I332E / K326E, and S239D / A330Y / I332E / K326T. In a more specific embodiment, the variant Fc region comprises a series of substitutions selected from the group consisting of: N297D / I332E,F241Y / F243Y / V262T / V264T / N297D / I332E, S239D / N297D / I332E, S239E / N2 97D / I332E, S239D / D265Y / N297D / I332E, S239D / D265H / N297D / I332E, V264 E / N297D / I332E, Y296N / N297D / I332E, N297D / A330Y / I332E, S239D / D265V / N297D / I332E, S239D / D265I / N297D / I332E, and N297D / S298A / A330Y / I332E. In certain embodiments, the variant Fc region comprises an amino acid substitution at position 332 (EU index, see 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 FcγR, increased stability, and / or increased solubility compared to the corresponding wild-type Fc region.

[0190] Further examples include variant Fc regions that include one or more of the following amino acid substitutions: 224N / Y, 225A, 228L, 230S, 239P, 240A, 241L, 243S / L / G / H / I, 244L, 246E, 247L / A, 252T, 254T / P, 258K, 261Y, 265V, 266A, 267G / N, 268N, 269K / G, 273A, 276D, 278H, 279L, 280L, 281L, 282L, 283L, 284L, 285L, 286L, 287L, 288L, 289L, 290L, 291L, 292L, 293L, 294L, 295L, 296L, 297L, 298L, 299L, 300L, 301L, 302L, 303L, 304L, 305L, 306L, 307L, 308L, 309L, 310L, 311L, 312L, 313L, 314L, 315L, 316L, 317L, 318L, 319L, 320L, 321L, 322L, 323L, 324L, 325L, 326L, 327L, 328L, 329L, 330L, 331L, 332L, 333L, 334L 79M, 280N, 283G, 285R, 288R, 289A, 290E, 291L, 292Q, 297D, 299A, 300H, 301C, 304G, 305A, 306I / F, 311R, 312N, 315D / K / S, 320R, 322E, 323A, 324T, 325S, 326E / R, 332T, 333D / G, 335I, 338R, 339T, 340Q, 341E, 342R , 344Q, 347R, 351S, 352A, 354A, 355W, 356G, 358T, 361D / Y, 362L, 364C, 365Q / P, 370R, 372L, 377V, 378T, 3 83N, 389S, 390D, 391C, 393A, 394A, 399G, 404S, 408G, 409R, 411I, 412A, 414M, 421S, 422I, 426F / P, 428T, 4 30K, 431S, 432P, 433P, 438L, 439E / R, 440G, 441F, 442T, 445R, 446A, 447E, optionally the variant has altered recognition of Fc ligands and / or altered effector function compared to the parent Fc polypeptide, where the numbering of the residues is that of the EU index in Kabat et al.Specific examples of these and related embodiments include variant Fc regions that contain, or consist of, 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, G 341E 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, L3 06I, K326R and Q362L;(14) H224Y, P230S, V323A, E333D, K338R and S364C;(15) T335I, K414M and P445R;(16) T335I and K414M;(17) P247A, E258K, D280N, K288R, N297D, T299A, K322E, Q342R, S354A and L365P;(18) H268N, V279M, A339T, N361D and S426P;(19) C261Y, K290E, L306F, Q311R, E333G and Q438L;(20) E283G, N315K, E333G, R344Q, L365P, and S442T; (21) Q347R, N361Y, and K439R; (22) S239P, S254P, S267N, H285R, N315S, F372L, A378T, N390D, Y391C, F404S, E430K, L432P, and K447E; and (23) E269G, Y278H, N325S, and K370R, where the numbering of the residues is that of the EU index as in Kabat et al. (see, e.g., U.S. Patent Application No. 2010 / 0184959).

[0191] A variant Fc region can also have one or more mutated hinge regions, e.g., as described in U.S. Patent Application Publication No. 2003 / 0118592. For example, one or more cysteines in the hinge region can be deleted or replaced 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 a reduced ability to dimerize compared to the wild-type Ig hinge region.

[0192] In certain embodiments, the Fc region comprises, consists of, or consists essentially of Fc from human IgG1 or IgG4 (see, e.g., Allberse and Schuurman, Immunology. 105:9-19, 2002), or a fragment or variant thereof. Table F1 below provides exemplary sequences (CH1, hinge (underlined), CH2, and CH3 regions) from human IgG1 and IgG4. Examples of variant IgG4 sequences that can be used are described, for example, in Peters et al., JBC. 287:24525-24533, 2012, and include substitutions at C227, C230, C127 (e.g., C127S), and C131 (e.g., C131S). Other variants that can be used include the L445P substitution in IgG4 (denoted as IgG4-2), or the D356E and L358M substitutions in IgG1 (denoted as IgG1m(zf)). [Table P1]

[0193] As described above, antibodies with an altered Fc region typically have altered (e.g., improved, increased, decreased) pharmacokinetic properties compared to the corresponding wild-type Fc region. Examples of pharmacokinetic properties include stability or half-life, bioavailability (fraction of drug absorbed), tissue distribution, volume of distribution (the apparent volume into which a drug is distributed immediately after it is injected intravenously 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 removed from the body), elimination rate (rate of infusion required to balance elimination), area under the curve (AUC or exposure; integral of the concentration-time curve, after a single dose or at steady state), clearance (volume of plasma cleared 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 the drug reaches before the next dose is administered), and fluctuation (peak-to-trough fluctuation within one dosing interval at steady state).

[0194] In certain embodiments, the antibody or antigen-binding fragment thereof is incubated for 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 at about pH 7.4, at about physiological pH, at about 25° C. or room temperature, and / or at about 37° C. or human body temperature (e.g., in vivo, in serum, in a given tissue, in a given species, such as rat, mouse, monkey, or human, etc.). In some embodiments, the half-life is about 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, 36, 40, 48, 50, 60, 70, 72, 80, 84, 90, 96, 120, 144 hours or more, or about 1 week, or about 2 weeks, or about 3 weeks, or about 4 weeks, or about 5 weeks, or about 6 weeks or more, or any intervening half-life, including all ranges therebetween.

[0195] 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. mIn some embodiments, the antibody or antigen-binding fragment thereof has a T of about 60° C. or greater. m has.

[0196] 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, platinums, type I topoisomerase inhibitors, type II topoisomerase inhibitors, vinca alkaloids, and taxanes. Specific examples of cytotoxic or chemotherapeutic agents include, but are not limited to, cyclophosphamide, cilengitide, lomustine (CCNU), melphalan, procarbazine, carmustine (BCNU), enzastaurin, busulfan, daunorubicin, doxorubicin, gefitinib, erlotinib, idarubicin, temozolomide, epirubicin, mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin, camptothecin, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, temsirolimus, everolimus, vincristine, vinblastine, vinorelbine, vindesine , CT52923, paclitaxel, imatinib, dasatinib, sorafenib, pazopanib, sunitonib, vatalanib, geftinib, erlotinib, AEE-788, dichloroacetic acid, tamoxifen, fasudil, SB-681323, semaxanib, donepezil, galantamine, memantine, rivastigmine, tacrine, rasigline, naltrexone, lubiprostone, safinamide, istradefylline, pimavanserin, pitolisant, isradipine, pridopidine (ACR16), tetrabenazine, bexarotene, glatirimer acetate, fingolimod, and mitoxantrone, including pharma- ceutical acceptable salts and acids thereof. Further examples of cytotoxic or chemotherapeutic agents include alkylating agents such as thiotepa, cyclophosphamide (CYTOXAN™), and the like; alkylsulfonates such as busulfan, improsulfan, and piposulfan, and the like; aziridines such as benzodopa, carboquone, meturedopa, and uredopa, and the like; ethyleneimine and methylamelamine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine;Nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard, etc.; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.; antibiotics, such as aclacinomycin, actinomycin, azaserine, bleomycin, cactinomycin , calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, donorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, etc.; Antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calsterone, dromostanolone propionate, enocitabine, 5-FU ... Pitiostanol, mepitiostane, testolactone, etc.; antiadrenal drugs, such as aminoglutethimide, mitotane, trilostane, etc.; folic acid supplements, such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; deformamine; demecolcine; diaziquone; erformitin; 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; triazicon; 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 doxetaxel (TAXOTERE®, Rhone-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; vinorelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS 2000; difluoromethylorutin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin), etc; ONTAK™ (denileukin diftitox); esperamicin; capecitabine; and pharma- ceutical acceptable salts, acids, or derivatives of any of the above.

[0197] The antibodies or antigen-binding fragments thereof may be used in any of the compositions, methods, and / or kits described herein and may be combined with one or more of the immunotherapeutic agents described herein.

[0198] Additional Therapeutic Agents and Compositions In certain embodiments, the antibody or antigen-binding fragment thereof is used in combination with one or more additional therapeutic agents, including immunotherapeutic agents, chemotherapeutic agents, hormonal therapeutic agents, and kinase inhibitors.

[0199] Immunotherapeutic Agents. Certain embodiments employ one or more cancer immunotherapeutic agents. In certain instances, the immunotherapeutic agent modulates the immune response of a subject, for example, to increase or maintain a cancer-associated or cancer-specific immune response, thereby resulting in increased immune cell inhibition or reduction of cancer cells. Exemplary immunotherapeutic agents include polypeptides, such as antibodies and antigen-binding fragments thereof, ligands, and small peptides, and mixtures thereof. Also included as immunotherapeutic agents are small molecules, cells (e.g., immune cells, such as T cells), various cancer vaccines, gene therapy or other polynucleotide-based agents, 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 immune checkpoint modulators, cancer vaccines, oncolytic viruses, cytokines, and cell-based immunotherapies.

[0200] In certain embodiments, the cancer immunotherapeutic agent is an immune checkpoint modulator. Particular examples include "antagonists" of one or more inhibitory immune checkpoint molecules, and "agonists" of one or more stimulatory immune checkpoint molecules. In general, immune checkpoint molecules are components of the immune system that either up-signal (co-stimulatory molecules) or down-signal, and targeting them has therapeutic potential in cancer, as cancer cells can interfere with 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, immune checkpoint modulators (e.g., antagonists, agonists) "bind" or "specifically bind" to one or more immune checkpoint molecules, as described herein.

[0201] In certain embodiments, the immune checkpoint modulator is a polypeptide or peptide. The terms "peptide" and "polypeptide" are used interchangeably herein, however, in certain instances, the term "peptide" can refer to shorter polypeptides, such as 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, including all integers and ranges therebetween (e.g., 5-10, 8-12, 10-15). Polypeptides and peptides can be composed of natural and / or unnatural amino acids, as described herein.

[0202] 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.

[0203] In some embodiments, the agent is or includes a "ligand" of an immune checkpoint molecule, e.g., a natural ligand. 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 a target molecule or target protein. Thus, certain agents are naturally protein ligands that bind to immune checkpoint molecules and generate a signal. Also included are "modified ligands," e.g., protein ligands that are fused to a pharmacokinetic modifier, such as an Fc region derived from an immunoglobulin.

[0204] The binding properties 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 an epitope thereof, with an equilibrium dissociation constant that is, or ranges from, about ≦10-7 to about 10-8 M. In some embodiments, the equilibrium dissociation constant is, or ranges from, about ≦10-9 M to about ≦10-10 M. In certain illustrative embodiments, the polypeptide has an affinity (Kd or EC) for a target described herein (to which it specifically binds) of about, at least about, 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 ).

[0205] In some embodiments, the agent is a "small molecule," which refers to an organic compound of synthetic or biological origin (biomolecule), but is typically not a polymer. Organic compounds refer to a large class of chemical compounds whose molecules contain carbon, typically excluding those that contain only carbonates, simple oxides of carbon, or cyanides. "Biomolecules" generally refer to organic molecules produced by living organisms, including large macromolecular molecules (biopolymers), such as peptides, polysaccharides, and nucleic acids, as well as small molecules, such as primary and secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins, and hormones. "Polymer" generally refers to a large molecule or macromolecule composed of repeating structural units, typically connected by covalent chemical bonds.

[0206] In certain embodiments, small molecules have a molecular weight of about 1000-2000 daltons or less, typically between about 300-700 daltons, including about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, 1000, or 2000 daltons or less.

[0207] Certain small molecules can have the "specific binding" characteristic described herein for polypeptides, such as antibodies. For example, in some embodiments, a small molecule has a binding affinity (Kd or ECd) of about, at least about, 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 to a target, such as an immune checkpoint molecule. 50 ) and specifically binds to the antibody.

[0208] In some embodiments, the immune checkpoint modulator is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules. Exemplary inhibitory immune checkpoint molecules include programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), programmed death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), V domain Ig suppressor of T-cell activation (VISTA), attenuator of B and T lymphocytes (BTLA), CD160, and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0209] 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 that belongs to the immunoglobulin superfamily and is 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 activation of T cells, 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 that promotes apoptosis in 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. Pat. Nos. 8,008,449; 8,993,731; 9,073,994; 9,084,776; 9,102,727; 9,102,728; 9,181,342; 9,217,034; 9,387,247; 9,492,539; 9,492,540; and U.S. Application Nos. 2012 / 0039906; 2015 / 0203579).

[0210] In some embodiments, the agent is a PD-L1 antagonist or inhibitor. As described 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), as well as antigen-binding fragments thereof (see, e.g., U.S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; and 9,439,962).

[0211] In some embodiments, the agent is a PD-L2 antagonist or inhibitor. As described 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, e.g., its binding to the PD-1 receptor.

[0212] 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 an antigen-presenting cell. General examples of CTLA-4 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to CTLA-4. Particular examples include the antibodies ipilimumab and tremelimumab, and antigen-binding fragments thereof. At least part of the activity of ipilimumab is believed to be mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) killing of suppressor Tregs expressing CTLA-4.

[0213] In some embodiments, the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor. IDO and TDO are tryptophan catabolic enzymes with immune-inhibitory 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. Common examples of IDO and TDO antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to IDO or TDO (see, e.g., Platten et al., Front Immunol. 5:673, 2014) and reduce or inhibit one or more immune-inhibitory activities. Specific examples of IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carbolines (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Seridan, Nature Biotechnology. 33:321-322, 2015). Specific examples of TDO antagonists or inhibitors include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).

[0214] In some embodiments, the agent is a TIM-3 antagonist or inhibitor. T cell immunoglobulin domain and mucin domain 3 (TIM-3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 also acts as a negative regulator of Th1 / Tc1 function by 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., Li et al., Acta Oncol. 54:1706-13, 2015). Common examples of TIM-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to TIM-3 and reduce or inhibit one or more of its immunosuppressive activities.

[0215] In some embodiments, the agent is a LAG-3 antagonist or inhibitor. Lymphocyte activation gene 3 (LAG-3) is expressed on activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. It negatively regulates 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 has been reported to play a role in Treg suppressive function (see, e.g., Huang et al., Immunity. 21:503-13, 2004). LAG3 also maintains CD8+ T cells in a tolerant state and maintains CD8 T cell exhaustion in combination with PD-1. General examples of LAG-3 antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to LAG-3 and inhibit one or more of its immunosuppressive activities. Specific examples include the antibody BMS-986016, and antigen-binding fragments thereof.

[0216] 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 expressed primarily on hematopoietic cells and highly expressed in the tumor microenvironment where it suppresses T-cell activation and induces Foxp3 expression, which suppresses anti-tumor T-cell responses (see, e.g., Lines et al., Cancer Res. 74:1924-32, 2014). Common 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.

[0217] In some embodiments, the agent is a BTLA antagonist or inhibitor. B and T lymphocyte attenuation factor (BTLA; CD272) expression is induced during T cell activation, and it inhibits T cells through interaction with tumor necrosis family receptors (TNF-R) and the B7 family of cell surface receptors. BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM). The BTLA-HVEM complex negatively regulates T cell immune responses, for example, by inhibiting the function of human CD8+ cancer-specific T cells (see, e.g., Derle et al., J Clin Invest 120:157-67, 2009). Common 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.

[0218] In some embodiments, the agent is an HVEM antagonist or inhibitor, e.g., an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD 160. Common examples of HVEM antagonists or inhibitors include antibodies or antigen-binding fragments or small molecules that specifically bind to HVEM and optionally reduce HVEM / BTLA and / or HVEM / CD 160 interactions, thereby reducing one or more of the immunosuppressive activities of HVEM.

[0219] In some embodiments, the agent is a CD160 antagonist or inhibitor, e.g., an antagonist or inhibitor that specifically binds to CD160 and interferes with its interaction with HVEM. 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.

[0220] In some embodiments, the agent is a TIGIT antagonist or inhibitor. T cell Ig and ITIM domain (TIGIT) is a co-inhibitory receptor found on the surface of various lymphoid cells and suppresses anti-tumor immunity, for example, via Treg (Kurtulus et al., J Clin Invest. 125:4053-4062, 2015). Common examples of TIGIT antagonists or 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., Johnston et al., Cancer Cell. 26:923-37, 2014).

[0221] In certain embodiments, the immune checkpoint modulator is an agonist of one or more stimulatory immune checkpoint molecules. Exemplary stimulatory immune checkpoint molecules include OX40, CD40, glucocorticoid-induced TNFR family related gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and herpes virus entry mediator (HVEM).

[0222] In some embodiments, the agent is an OX40 agonist. OX40 (CD134) promotes the proliferation of effector and memory T cells and inhibits 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). Because OX40 signaling affects both T cell activation and survival, it plays an important role in initiating anti-tumor immune responses in lymph nodes and in maintaining anti-tumor immune responses in the tumor microenvironment. Common examples of OX40 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to OX40 and increase one or more of its immunostimulatory activities. Specific examples include OX86, OX-40L, Fc-OX40L, GSK3174998, MEDI0562 (humanized OX40 agonist), MEDI6469 (murine OX4 agonist), and MEDI6383 (OX40 agonist), and antigen-binding fragments thereof.

[0223] 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). On APCs, ligation leads to upregulation of costimulatory molecules, potentially bypassing the need for T cell help in antitumor immune responses. CD40 agonist treatment plays a key role in APC maturation and their migration from tumors to lymph nodes, resulting in elevated antigen presentation and T cell activation. Anti-CD40 agonist antibodies generate substantial responses and durable anticancer immunity in animal models, an effect that is at least partially mediated by cytotoxic T cells (see, e.g., Johnson et al. Clin Cancer Res. 21:1321-1328, 2015; and Vonderheide and Glennie, Clin Cancer Res. 19:1035-43, 2013). 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.

[0224] In some embodiments, the agent is a GITR agonist. Glucocorticoid-induced TNFR family related gene (GITR) increases T cell proliferation, inhibits the suppressive activity of Tregs, and prolongs the survival of T effector cells. GITR agonists have been shown to promote anti-tumor responses through loss of Treg lineage stability (see, e.g., Schaer et al., Cancer Immunol Res. 1:320-31, 2013). These diverse mechanisms indicate that GITR plays a key role in initiating immune responses in lymph nodes and maintaining immune responses in tumor tissues. Its ligand is GITRL. Common examples of GITR agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to GITR and increase one or more of its immunostimulatory activities. Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and antigen-binding fragments thereof.

[0225] 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. Common examples of CD137 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD137 and increase one or more of its immunostimulatory activities. Specific examples include CD137 (or 4-1BB) ligands (see, e.g., Shao and Schwarz, J Leukoc Biol. 89:21-9, 2011) and the antibody utomirumab, including antigen-binding fragments thereof.

[0226] In some embodiments, the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific proliferation of naive T cells and contributes to 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., J Immunol. 191:4174-83, 2013). General examples of CD27 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD27 and increase one or more of its immunostimulatory activities. Specific examples include CD70, as well as the antibodies valilumab and CDX-1127 (1F5), including antigen-binding fragments thereof.

[0227] In some embodiments, the agent is a CD28 agonist. CD28 is constitutively expressed on CD4+ T cells and on some CD8+ T cells. Its ligands include CD80 and CD86, the stimulation of which increases T cell proliferation. Common examples of CD28 agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to CD28 and increase one or more of its immunostimulatory activities. Specific examples include CD80, CD86, the antibody TAB08, and antigen-binding fragments thereof.

[0228] In some embodiments, the agent is a CD226 agonist. CD226 shares a ligand with TIGIT and is an opposing stimulatory receptor, and CD226 engagement enhances T cell activation (see, e.g., Kurtulus et al., J Clin Invest. 125:4053-4062, 2015; Bottino et al., J Exp Med. 1984:557-567, 2003; and Tahara-Hanaok 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.

[0229] In some embodiments, the agent is an HVEM agonist. Herpes virus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF receptor superfamily. HVEM is found on a variety of cells, including T cells, APCs, and other immune cells. Unlike other receptors, HVEM is expressed at high levels on resting T cells and is downregulated upon activation. HVEM signaling has been shown to play a key role in the early stages of T cell activation and during the proliferation of tumor-specific lymphocyte populations in lymph nodes. Common examples of HVEM agonists include antibodies or antigen-binding fragments or small molecules or ligands that specifically bind to HVEM and increase one or more of its immunostimulatory activities.

[0230] In certain embodiments, the cancer immunotherapeutic agent is a cancer vaccine. Exemplary cancer vaccines include Oncophage, human papillomavirus HPV vaccines, such as Gardasil or Cervarix, hepatitis B vaccines, such as Engelix-B, RecombiVax HB, or Twinrix, and Sipulcel-T (Provenge). In some embodiments, the cancer vaccine comprises or utilizes one or more cancer antigens, or cancer-associated d antigens.Exemplary cancer antigens include, but are not limited to, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD19, CD20, CD22, CD23 (IgE receptor), MAGE-3 C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A) VEGFR-1, VEGFR-2, VEGFR-3, NRP2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimena, Chin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin αvβ3, integrin α5β1, folate receptor 1, transmembrane glycoprotein NMB, fibroblast activation protein alpha (FAP), glycoprotein 75, TAG-72, MUC1, MUC16 (or CA-125), phosphatidylserine, prostate-specific membrane antigen (PSMA), NR-LU-13 antigen, TRAIL-R1, tumor necrosis factor receptor superfamily member 10b (TNFRSF10B or TRAIL-R2), SLAM family member 7 (SLAMF7), EGP40 pan-carcinoma antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), phosphatase of regenerating liver 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin.

[0231] In certain embodiments, the cancer immunotherapeutic agent is an oncolytic virus. Oncolytic viruses are viruses that preferentially infect and kill cancer cells. Included are natural and man-made or engineered oncolytic viruses. Most oncolytic viruses are engineered for tumor selectivity, but there are natural 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™), Oncoline (H101), Peraleorep (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.

[0232] 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).

[0233] In certain embodiments, the cancer immunotherapeutic agent is a cell-based immunotherapy, e.g., 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 certain embodiments, the CAR modified T cells are targeted against CD-19 (see, e.g., Maude et al., Blood. 125:4017-4023, 2015).

[0234] In certain instances, the cancer being 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 being treated. In some embodiments, the cancer antigen is CD19, human Her2 / neu, Her1 / EGF receptor (EGFR), Her3, A33 antigen, B7H3, CD5, CD20, CD22, CD23 (IgE receptor), MAGE-3 C242 antigen, 5T4, IL-6, IL-13, vascular endothelial growth factor VEGF (e.g., VEGF-A), VEGFR-1, VEGFR-2, CD30, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, HLA-DR, CTLA-4, NPC-1C, tenascin, vimentin, insulin-like growth factor 1 receptor (IGF-1R), alpha-fetoprotein, insulin-like growth factor 1 (IGF-1), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), guanylyl cyclase C, NY-ESO-1, p53, survivin, integrin 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-carcinoma antigen, B-cell activating factor (BAFF), platelet-derived growth factor receptor, glycoprotein EpCAM (17-1A), programmed death-1, protein disulfide isomerase (PDI), phosphatase of regenerating liver 3 (PRL-3), prostatic acid phosphatase, Lewis-Y antigen, GD2 (disialoganglioside expressed on tumors of neuroectodermal origin), glypican-3 (GPC3), and mesothelin.

[0235] Additional exemplary cancer antigens include 5T4, 707-AP, 9D7, AFP, AlbVDC 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, CA19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8 / m, cathepsin B, cathepsin L, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, colactocin-like protein, Collage XXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE- 4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, hepsin, Her2 / neu, HERV-K-MEL, HLA-A*0201-R1 7I, HLA-A1 1 / m, HLA-A2 / m, HNE, homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1 R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205 / m, KK-LC-1, K-Ras / m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B1 7, 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, Sp17, SSX-1, SSX-2 / HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF-beta, 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. Particular 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.

[0236] 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, MelanA (e.g., MelanA 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 1 730), WT1 (e.g., WT1 by accession number NM_000378), PRAME (e.g., PRAME by accession number NM_0061 15), EGFRI (epidermal growth factor receptor 1) (e.g., EGFRI (epidermal growth factor receptor 1) by accession number AF288738), MUC1, mucin-1 (e.g., mucin-1 by accession number NM_002456), SEC61 G (e.g., SEC61 G by accession number NM_014302), hTERT (e.g., hTERT accession number NM_198253), 5T4 (e.g., 5T4 by accession number NM_006670), TRP-2 (e.g., TRP-2 by accession number NM_001 922), STEAP1 (six transmembrane epithelial antigen of prostate 1), PSCA, PSA, PSMA, and the like.

[0237] In some embodiments, the cancer antigen is selected from PCA, PSA, PSMA, STEAP, and optionally including MUC-1, 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 including MUC-1, fragments, variants, and derivatives thereof.

[0238] In some cases, cancer antigens include idiotypic antigens associated with cancer or tumor diseases, particularly lymphoma or lymphoma-related diseases, for example, where the idiotypic antigen is an immunoglobulin idiotype of a lymphatic blood cell or a T cell receptor idiotype of a lymphatic blood cell.

[0239] 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 (TIL), and peptide-induced T cells.

[0240] One of skill in the art will understand that the various cancer immunotherapeutic agents described herein can be used in combination with any one or more of the various anti-NRP2a antibodies (including antigen-binding fragments thereof) described herein, in accordance with any one or more of the methods or compositions described herein.

[0241] 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 1 or type II), anti-microtubule agents, among others.

[0242] 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).

[0243] Examples of antimetabolites include antifolates (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogues (e.g., acitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacytidine, 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.

[0244] Examples of anti-microtubule agents include taxanes (eg, paclitaxel and docetaxel) and vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

[0245] One of skill in the art will understand that the various chemotherapeutic agents described herein can be used in combination with any one or more of the various anti-NRP2a antibodies (including antigen-binding fragments thereof) described herein in accordance with any one or more of the methods or compositions described herein.

[0246] Hormonal Therapeutics. Certain embodiments employ at least one hormonal therapeutic agent. General examples of hormonal therapeutic agents include hormonal agonists and hormonal antagonists. Specific examples of hormonal agonists include progestogens (progestins), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factors, VEGF-derived angiogenic and lymphangiogenic factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factors (FGF), galectins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgens, estrogens, CCL21, and somatostatin analogs. Examples of hormone antagonists include hormone synthesis inhibitors, such as aromatase inhibitors and gonadotropin releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), including analogs thereof. Also included are hormone receptor antagonists, such as selective estrogen receptor modulators (SERMs; e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0247] Also included are hormone pathway inhibitors, such as antibodies directed against hormone receptors. Examples include inhibitors of the IGF receptor (e.g., IGF-IR1), such as cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, and lobatumumab; inhibitors of vascular endothelial growth factor receptor 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3) or their ligands, such as alacizumab pegol, bevacizumab, icrucumab, ramucirumab, and the like; inhibitors of TGF-beta receptors R1, R2, and R3, such as fresolimumab and methelimumab; c-Me inhibitors of the EGF receptor, such as cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomzotuximab, and zalutumumab; inhibitors of the FGF receptor, such as aprtumab ixadotin and bemarituzumab; inhibitors of CCR7; and inhibitors of the PDGF receptor, such as olaratumab and tobetumab.

[0248] One of skill in the art will understand that the various hormonal therapy agents described herein can be used in combination with any one or more of the various anti-NRP2a antibodies (including antigen-binding fragments thereof) described herein in accordance with any one or more of the methods or compositions described herein.

[0249] Kinase inhibitors. Certain embodiments employ at least one kinase inhibitor, including a tyrosine kinase inhibitor. Examples of kinase inhibitors include, but are not limited to, adavosertib, afantinib, 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, serotin ... Exemplary PI3 kinase inhibitors include alpelisib, buparlisib, copanlisib, CUDC-907, dactolisib, duvelisib, GNE-477, idelasib, IPI-549, LY294002, ME-401, perifosine, PI-103, pictilisib, PWT33597, RP6503, taselisib, umbralisib, voxtalisib, wortmannin, and XL147.

[0250] One of skill in the art will understand that the various kinase inhibitors described herein can be used in combination with any one or more of the various anti-NRP2a antibodies (including antigen-binding fragments thereof) described herein, in accordance with any one or more of the methods or compositions described herein.

[0251] Methods of Use and Therapeutic Compositions The present disclosure relates in part to the discovery that certain human neuropilin 2 (NRP2) polypeptides, specifically NRP2a v1 and v2, contain unique juxtamembrane domain sequences that mediate binding interactions with NRP2a v1 / v2 ligands, such as CCL21 and / or CCR7. Such CCL21 / CCR7 interaction domains are not found in human NRP2b v4 and NRP2b v5 polypeptides, and are only partially present in human NRP2a v3. Thus, antibodies that selectively bind human NRP2a v1 and / or v2, as compared to NRP2b v4 and NRP2b v5, and inhibit or otherwise interfere with the binding between NRP2a v1 / v2 and CCL21 / CCR7, can be used to modulate downstream signaling events in these pathways. Such antibodies can be used as a sole therapy in the treatment of diseases, including NRP2-related diseases, or in combination with other therapeutic agents described herein.

[0252] Certain embodiments therefore include a method of treating, ameliorating symptoms, and / or slowing the progression of a disease or condition in a subject in need thereof, comprising administering to the subject at least one antibody or antigen-binding fragment thereof that binds to a human NRP2a v1 and / or v2 polypeptide as described herein. In some examples, the antibody or antigen-binding fragment thereof modulates (e.g., antagonizes) binding between an NRP2a v1 and / or v2 polypeptide and CCL21 and / or CCR7. In some embodiments, the anti-NRP2a antibody, or antigen-binding fragment thereof, selectively binds to NRP2a variant 1 and / or 2 isoforms and does not substantially bind to NRP2b v4 and v5 isoforms. In some embodiments, the antibody or antigen-binding fragment thereof modulates signaling activity resulting from the interaction between an NRP2a v1 and / or v2 polypeptide and an NRP2a ligand, such as CCL21 / CCR7.

[0253] Some embodiments include administering an anti-NRP2a antibody or antigen-binding fragment thereof in an amount and frequency sufficient to achieve a steady state concentration, or average circulating concentration, of the anti-NRP2a antibody or antigen-binding fragment thereof 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.

[0254] In certain embodiments, the disease or condition is an NRP2-associated disease or condition. In some embodiments, the disease or condition associated with NRP2 is selected from one or more of cancer, and diseases and pathways associated with cancer, including cancer cell growth, initiation, migration, adhesion, invasion, chemotherapy resistance, and / or metastasis; diseases associated with inflammation, autoimmunity, and related inflammatory diseases, diseases associated with inappropriate immune cell activation or migration, such as graft-versus-host disease (GVHD); diseases associated with lymphatic development, lymphangiogenesis, and lymphatic damage, including edema, lymphedema, secondary lymphedema, inappropriate fat absorption and accumulation, excessive fat accumulation, and vascular permeability; diseases associated with infection, including latent infection; allergic disorders / diseases, diseases associated with allergic responses, such as chronic obstructive pulmonary disease (COPD), and / or chronic obstructive pulmonary disease (COPD). OPD), neutrophilic asthma, antineutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis, systemic lupus erythematosus, rheumatoid arthritis, inflammasome-associated diseases, and skin-associated neutrophil-mediated diseases such as pyoderma gangrenosum; diseases associated with granulomatous inflammatory diseases, including sarcoidosis and granulomas; diseases associated with fibrosis, including fibrotic diseases, fibrosis, endothelial-mesenchymal transition (EMT), and wound healing; diseases associated with 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 neurological diseases, peripheral nervous system remodeling, and pain perception; diseases associated with bone development and bone remodeling.

[0255] In some embodiments, the disease is cancer. Certain embodiments thus include a method of treating, ameliorating symptoms, or inhibiting progression of cancer in a subject in need thereof, comprising administering to the subject at least one antibody or antigen-binding fragment thereof (anti-NRP2a antibody) that specifically binds to a human NRP2a v1 and / or v2 polypeptide and modulates (e.g., interferes with) binding of a human NRP2a polypeptide to a human NRP2a ligand (e.g., an NRP2a ligand from Table N2, such as CCL21 and / or CCR7). Certain embodiments include reducing or preventing the re-emergence of cancer, e.g., metastatic cancer and / or chemotherapy-resistant cancer, in a subject in need thereof, wherein administration of the therapeutic composition allows for the generation of immune memory to the cancer.

[0256] In some examples, the anti-NRP2a antibody or antigen-binding fragment thereof reduces cancer initiation, cancer cell migration, adhesion, or cancer cell metastasis by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to an untreated control. In some examples, the anti-NRP2a antibody or antigen-binding fragment thereof reduces cancer-mediated lymphangiogenesis 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, the anti-NRP2a antibody, or antigen-binding fragment thereof, inhibits or reduces the rate of migration or movement of cancer or migratory cells (e.g., cancer or immune cells isolated from a biopsy or other sample grown in vitro) 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, the anti-NRP2a antibody, or antigen-binding fragment thereof, reduces the invasiveness of a cancer (e.g., cancer cells isolated from a biopsy or other sample grown in vitro) 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.

[0257] In some embodiments, the anti-NRP2a antibody, or antigen-binding fragment thereof, enhances the sensitivity of a cancer to an additional agent (e.g., a chemotherapeutic agent, a hormonal therapy agent, and / or 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, the anti-NRP2a antibody or antigen-binding fragment thereof enhances the anti-tumor and / or immune stimulatory 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.

[0258] Also included are combination therapies for treating cancer, including methods of treating, ameliorating symptoms, or inhibiting progression of cancer in a subject in need thereof, comprising administering to the subject at least one antibody or antigen-binding fragment thereof that specifically binds to human NRP2a v1 and / or v2 polypeptides (anti-NRP2a antibodies) in combination with at least one additional agent, such as a cancer immunotherapy, chemotherapeutic, hormonal therapy, and / or kinase inhibitor. Exemplary cancer immunotherapy, chemotherapeutic, hormonal therapy, and kinase inhibitors are described elsewhere herein.

[0259] In some instances, the anti-NRP2a antibody, or antigen-binding fragment thereof, and the at least one additional agent are administered separately, e.g., in separate therapeutic compositions and at the same or different times, hi some embodiments, the anti-NRP2a antibody, or antigen-binding fragment thereof, and the at least one additional agent are administered simultaneously as part of the same therapeutic composition.

[0260] Certain methods employ one or more anti-NRP2a antibodies, or antigen-binding fragments thereof, as part of (i.e., in addition to) a combination treatment regimen. Exemplary combination regimens are provided in Table M1 below. [Table M1]

[0261] In some embodiments, the methods and therapeutic compositions described herein (e.g., anti-NRP2a antibodies, alone or in combination with at least one additional agent) increase the median survival of a subject by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or more. In certain embodiments, the methods and therapeutic compositions described herein (e.g., anti-NRP2a antibodies, alone or in combination with at least one additional agent) increase the median survival of a subject by 1 year, 2 years, 3 years, or more. In some embodiments, the methods and therapeutic compositions described herein (e.g., anti-NRP2a antibodies, alone or in combination with a cancer immunotherapeutic agent) increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more. In certain embodiments, the methods or therapeutic compositions described herein increase progression-free survival by 1 year, 2 years, 3 years, or more.

[0262] In certain embodiments, the methods and therapeutic compositions described herein (e.g., anti-NRP2a antibodies, alone or in combination with at least one additional agent) are sufficient to cause tumor regression, as indicated by a statistically significant reduction in the amount of viable tumor, e.g., at least a 10%, 20%, 30%, 40%, 50% or greater reduction in tumor mass, or by altered (e.g., reduced with statistical significance) scan dimensions. In certain embodiments, the methods and therapeutic compositions described herein (e.g., anti-NRP2a antibodies, alone or in combination with at least one additional agent) are sufficient to cause stable disease. In certain embodiments, the methods and therapeutic compositions described herein (e.g., anti-NRP2a antibodies, alone or in combination with at least one additional agent) are sufficient to cause a clinically relevant reduction in symptoms of a particular disease indication known to those of skill in the art.

[0263] In some embodiments, the anti-NRP2a antibody increases, complements, or otherwise enhances the anti-tumor and / or immunostimulatory activity of the cancer immunotherapeutic agent compared to the cancer immunotherapeutic agent alone, hi some embodiments, the anti-NRP2a antibody enhances the anti-tumor and / or immunostimulatory activity of the cancer immunotherapeutic agent by about, or at least about, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more compared to the cancer immunotherapeutic agent alone.

[0264] The methods and therapeutic compositions described herein can be used in the treatment of any of a variety of cancers or tumors. In some embodiments, the cancer is a primary cancer, i.e., a cancer that develops at an anatomical site where tumor progression begins and results in a cancerous mass. In some embodiments, the cancer is a secondary or metastatic cancer, i.e., a cancer that has spread from the primary site or tissue of origin to one or more different sites or tissues. In some embodiments, the cancer expresses or overexpresses NRP2.In some embodiments, the subject or patient is diagnosed with melanoma (e.g., metastatic melanoma), epithelial or epithelial derived tumors, 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 or HCC), sarcoma, B cell malignancies, breast cancer (e.g., estrogen receptor positive (ER+), estrogen receptor negative (ER-), Her2 positive (Her2+), Her2 negative (Her2-); or combinations thereof, e.g., ER+ / Her2+, ER+ / Her2-, ER- / Her2+, or ER- / Her2-; or estrogen receptor negative, progesterone receptor negative, and HER2 breast cancer, which is negative for gliomas (triple negative), ovarian cancer, colorectal cancer, gliomas (e.g., astrocytoma, oligodendroglioma, ependymoma, or choroid plexus papilloma), glioblastoma multiforme (e.g., giant cell glioblastoma or gliosarcoma), 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, Neck cancer, cervical cancer, testicular cancer, thyroid cancer, gastric cancer, virus-induced tumors, e.g., papillomavirus-induced carcinomas (e.g., cervical carcinoma, cervical cancer), adenocarcinoma, herpes virus-induced tumors (e.g., Burkitt's lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1-induced and HTLV-2-induced lymphomas, acoustic neuroma, lung cancer (e.g., lung carcinoma, bronchial carcinoma), small cell lung carcinoma, pharyngeal cancer, anal carcinoma, glioblastoma, rectal carcinoma, lymphangioma, astrocytoma, brain tumor, retinoblastoma, basal cell tumor, brain metastasis, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease, pituitary tumor, mycosis fungoides, carcinoid, neurinoma, spinal cord tumor, Burkitt's lymphoma, laryngeal cancer, kidney cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin's syndrome The patient has one or more of the following cancers: lymphoma, urethral cancer, CUP syndrome, head and neck tumor, oligodendroglioma, vulvar cancer, intestinal cancer, colon carcinoma, esophageal cancer (e.g., esophageal carcinoma), warts, small intestinal tumor, craniopharyngioma, ovarian carcinoma, reproductive organ tumor, ovarian cancer (e.g., ovarian carcinoma), pancreatic cancer (e.g., pancreatic carcinoma), endometrial carcinoma, liver metastasis, penile cancer, tongue cancer, gallbladder cancer, leukemia, plasmacytoma, and eyelid tumor.

[0265] In some embodiments, the cancer or tumor is a metastatic cancer, eg, a metastatic cancer that expresses NRP2a v1 and / or v2, as described above. In addition to the above cancers, exemplary metastatic cancers include, but are not limited to, bladder cancer that has metastasized to bone, liver, and / or lungs; breast cancer that has metastasized to bone, brain, liver, and / or lungs; colorectal cancer that has metastasized to the liver, lungs, and / or peritoneum; kidney cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or lungs; lung cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or other pulmonary sites; melanoma that has metastasized to bone, brain, liver, lungs, and / or skin / muscle; ovarian cancer that has metastasized to the liver, lungs, and / or peritoneum; pancreatic cancer that has metastasized to the liver, lungs, and / or peritoneum; prostate cancer that has metastasized to the adrenal glands, bone, liver, and / or lungs; gastric cancer that has metastasized to the liver, lungs, and / or peritoneum; thyroid cancer that has metastasized to the bone, liver, and / or lungs; and uterine cancer that has metastasized to the bone, liver, lungs, peritoneum, and / or vagina, among others.

[0266] In some embodiments, for example, where the cancer immunotherapeutic agent is a PD-1 or PD-L1 antagonist or inhibitor, the subject has one or more biomarkers that make them suitable for PD-1 or PD-L1 inhibitor therapy (e.g., increased PD-1 or PD-L1 levels in cells, such as cancer cells or cancer-specific CTLs). For example, in some embodiments ... hi CTLA-4 hi As another example, in some embodiments, the subject has an increased fraction of circulating tumor reactive (e.g., PD-1 + CD11a hi CD8 + ) have increased levels of Bim (mediator of B-cell lymphoma 2 interaction (Bcl2 interaction)) in T cells, and occasionally have metastatic melanoma (see, e.g., Dronca et al., JCI Insight. May 5;1(6):e86014, 2016).

[0267] Certain specific combinations include an anti-NRP2a antibody and a PD-L1 antagonist or inhibitor, such as atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), for the treatment of a cancer selected from one or more of colorectal cancer, melanoma, breast cancer, non-small cell lung carcinoma, bladder cancer, and renal cell carcinoma.

[0268] Some particular combinations include an anti-NRP2a antibody and a PD-1 antagonist, such as nivolumab, for treating a cancer selected from one or more of Hodgkin's lymphoma, melanoma, non-small cell lung cancer, hepatocellular carcinoma, renal cell carcinoma, and ovarian cancer.

[0269] Certain specific combinations include an anti-NRP2a antibody and a PD-1 antagonist, such as pembrolizumab, for treating a cancer selected from one or more of melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, and urothelial carcinoma.

[0270] Certain specific combinations include an anti-NRP2a antibody and a CTLA-4 antagonist, such as ipilimumab and tremelimumab, for the treatment of a cancer selected from one or more of melanoma, prostate cancer, lung cancer, and bladder cancer.

[0271] Some particular combinations include an anti-NRP2a antibody and an IDO antagonist, such as indoximod (NLG-8189), 1-methyl-tryptophan (1MT), β-carboline (norharman; 9H-pyrido[3,4-b]indole), rosmarinic acid, or epacadostat, for the treatment of metastatic breast cancer and brain cancer, optionally selected from one or more of glioblastoma multiforme, glioma, gliosarcoma, or malignant brain tumor.

[0272] Certain specific combinations include anti-NRP2a antibodies and the cytokine INF-α for treating melanoma, Kaposi's sarcoma, and hematological cancers. Also included is the combination of anti-NRP2a antibodies and IL-2 (e.g., aldesleukin) for treating metastatic renal cancer or metastatic melanoma.

[0273] Some particular combinations include anti-NRP2a antibodies and T cell-based adoptive immunotherapy, including, for example, CAR-modified T cells targeted against CD-19, for treating hematological cancers, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B cell neoplasms (see, e.g., Maude et al., 2015, supra; Lorentzen and Straten, Scand J Immunol. 82:307-19, 2015; and Ramos et al., Cancer J. 20:112-118, 2014).

[0274] The method for treating cancer can be combined with other therapeutic modalities.For example, the combination treatment described herein can be administered to a subject before, during, or after other therapeutic interventions, including symptomatic treatment, radiation therapy, surgery, transplantation, hormonal therapy, photodynamic therapy, antibiotic therapy, or any combination thereof.Symptomatic treatment includes administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment, and vomiting, and administration of anticonvulsants to reduce seizures.Radiation therapy includes whole brain irradiation, fractionated radiation therapy, and radiosurgery, such as stereotactic radiosurgery, which can be further combined with conventional surgery.

[0275] Certain embodiments include the use of anti-NRP2a antibodies described herein to modulate lymphangiogenesis and treat related lymphatic diseases or associated conditions, such as lymphedema or tumor metastasis. The lymphatic system consists of a network of interconnected capillaries, collecting vessels, and lymph nodes that absorb, collect, and transport fluids and proteins filtered from the vascular system. This system provides an important homeostatic function: in humans, lymphatic vessels return >4 liters of fluid and a significant amount of protein per day into the large vein in the neck.

[0276] Lymphatic vascular insufficiency (lymphedema) results in the accumulation of excess fluid in the interstitium (edema). Although lymphedema is typically not life-threatening, it has serious health consequences, including pain, immobility, fibrosis, inflammation, fatty tissue accumulation, and tissue damage. Because the lymphatic system is also a critical component of the immune response, lymphedema is typically accompanied by an increased risk of infection and other immune system problems.

[0277] Lymphangiogenesis is the formation of new lymphatic vessels from pre-existing lymphatic vessels and is associated with a variety of pathological conditions including metastatic dissemination, graft rejection (e.g., cornea, kidney, and heart), type 2 diabetes, obesity, hypertension, and lymphedema (e.g., Alitalo et al. Nature 438:946-953, 2005; Karaman et al. J Clin Invest 124:922-928, 2014; Kim et al., J Clin Invest 124:936-942, 2014; Maby-El Hajjami et al., Histochem Cell Biol 130:1063-107, 2008; Machnik et al., Nat Med 15:545-552; Mortimer et al., 2014. J Clin Invest 124:915-921; Skobe et al., J Clin Invest 124:915-921, 2014). (see, e.g., et al., 2009. Nat Med 15:993-994).

[0278] Lymphatic invasion in and around the primary tumor compared with vascular invasion is a prognostic marker of aggressiveness in various types of cancer. Lymphatic vessel growth has also been implicated in graft rejection (Dietrich, T., et al., J Immunol 184:535-539, 2010; Hall et al., Arch Otolaryngol Head Neck Surg 129:716-719, 2003.; Maula et al., Cancer Res 63:1920-1926, 2003; Miyata et al., J Urol 176:348-353, 2006; Saad et al., Mod Pathol 19:1317-1323, 2006; Schoppmann et al., Ann Surg 240:306-312, 2004; Zeng et al., Prostate 65:222-230, 2005).

[0279] Anti-lymphangiogenic agents are useful, for example, to treat debilitating diseases of the eye in which lymphatic vessel growth is the primary reason for corneal graft rejection and a major contributor to the angiogenesis associated with age-related macular degeneration (Dietrich, T., et al., J Immunol 184:535-539, 2010). In particular, penetrating keratinocyte transplantation is the most common form of solid tissue transplantation, with approximately 40,000 corneal transplants performed annually in the United States. The success rate of penetrating keratinocyte transplantation is as high as 90% for uncomplicated primary grafts performed in avascular, low-risk beds. However, the rejection rate of corneal grafts placed in high-risk, vascularized host beds is extremely high (70%-90%). Thus, certain embodiments include anti-NRP2a antibodies to inhibit lymphangiogenesis, thereby promoting graft survival and inhibiting angiogenesis.

[0280] Anti-lymphangiogenic drugs are also useful for the treatment of dry eye disease. Significant upregulation of lymphatic stimulators (e.g., VEGF-C, VEGF-D, and VEGFR-3) and selective growth of lymphatic vessels without concomitant growth of blood vessels have been demonstrated in corneas with dry eye disease (Goyal et al., Arch Ophthalmol 128:819-824, 2010). Dry eye disease is an immune-mediated disorder that affects approximately 5 million Americans. It severely impacts vision-related quality of life, and symptoms can be debilitating. Current treatment options for dry eye disease are limited, mostly palliative, and expensive. Some embodiments thus include anti-NRP2a antibodies as lymphangiogenesis inhibitors for the treatment of dry eye disease.

[0281] Metastasis is responsible for the majority (90%) of deaths from solid tumors (Gupta and Massague, Cell 127, 679-695, 2006). The complex process of metastasis involves a series of different steps, including detachment of tumor cells from the primary tumor, intravasation of tumor cells into lymphatic or blood vessels, and extravasation and growth of tumor cells in secondary sites. Analysis of regional lymph nodes in many tumor types suggests that lymphatic structures are important pathways for the dissemination of human cancers. Moreover, the presence of tumor cells in lymph nodes is the most important adverse prognostic factor in almost all carcinomas. While such metastasis was previously thought to involve exclusively the passage of malignant cells along pre-existing lymphatic vessels near the tumor, recent experimental studies and clinical pathological reports (see, e.g., Achen et al., Br J Cancer 94, 1355-1360, 2006 and Nathanson, Cancer 98, 413-423, 2003) suggest that lymphangiogenesis can be induced by solid tumors and can facilitate tumor expansion. Some embodiments thus include the use of anti-NRP2a antibodies to target lymphatic vessels and lymphangiogenesis as a therapeutic strategy to limit the occurrence of cancer metastasis.

[0282] Thus, the methods and compositions described herein can be used to inhibit the activity of lymphopoietin factors, thereby treating transplant rejection, dry eye disease, tumor metastasis, lymphedema, and related inflammatory conditions.

[0283] Some embodiments include the use of anti-NRP2 antibodies described herein to modulate smooth muscle contractility and treat associated conditions. Reduced smooth muscle (SM) contractility in the bladder can result from a number of etiologies, including partial obstruction secondary to benign prostatic hyperplasia (BPH), posterior urethral valves, diabetes mellitus, multiple sclerosis, spinal cord injury, or idiopathic causes. (See, e.g., Drake et al., Nat Rev Urol. 11(8):454-464, 2014). In conditions such as BPH or posterior urethral valves, the bladder contracts against an obstructed outlet. The initial response is adaptive and involves a compensatory phase of SM hypertrophy that allows increased force generation to overcome the increased outlet resistance. If demands exceed the adaptive capacity of the bladder, contractile performance becomes less efficient, residual volume increases, the bladder remodels, and ultimately leads to a loss of detrusor contractility as the bladder compensates. (See, e.g., Zderic et al., J Cell Mol Med. 16(2):203-217, 2012). The prevalence of inactive detrusor function has been reported to be as high as 48% in adults (Osman et al., Eur Urol; 65(2):389-398, 2014). Furthermore, existing pharmacological treatments for restoration of SM contraction, such as muscarinic agonists or cholinesterase inhibitors, have shown limited efficacy and adverse effects (Barendrecht et al., BJU Int. 99(4):749-752, 2007).

[0284] Recent studies have identified bladder smooth muscle as a major site of NRP2 expression, demonstrating inhibition of RhoA and cytoskeletal stiffness, and observed increased contractility of bladder SM strips from mice with ubiquitous or smooth muscle-specific deletion of NRP2 in vivo when compared with tissue from NRP2-intact littermate controls (see, e.g., Bielenberg et al., Am. J. Pathol. 181 548-559, 2012; and Vasquez et al., JCI Insight 2(3)e90617, 2017).

[0285] Furthermore, recent studies have shown that targeting NRP2 in bladders undergoing decompensation has the potential to restore contractility despite ongoing obstruction. (Vasquez et al., JCI Insight 2(3)e90617, 2017). These findings argue that the NRP2 axis represents a potentially novel pharmacological target for the restoration of SM contractility in partial bladder outlet obstruction syndrome and provide an important therapeutic opportunity for the development of antibody-based modulators of NRP2 function.

[0286] Pharmacological management of reduced detrusor contractility to date has focused on stimulating parasympathetic activity to enhance bladder contractility and reducing outflow resistance to facilitate bladder emptying (Chancellor et al., Urology 72(5)966-967, 2008). However, an analysis of 10 randomized clinical trials of parasympathomimetic drugs in patients with poorly contractile bladders revealed either worsening of symptoms or a lack of significant improvement (Barendrecht et al., BJU Int. 99(4)749-752, 2007). The increase in contractility following NRP2 deletion in decompensated bladders suggests that NRP2 may be a useful target for alleviating reduced detrusor contractility under conditions of chronic obstruction.

[0287] Given the role of NRP2 in this process, certain embodiments include the use of anti-NRP2 antibodies described herein to modulate smooth muscle contractility, including, for example, in the treatment of reduced smooth muscle (SM) contractility in the bladder, more specifically, in the treatment of syndromes associated with partial bladder outlet obstruction syndrome. Some embodiments thus include a method of treating, ameliorating symptoms, or inhibiting progression of partial bladder outlet obstruction syndrome in a subject in need thereof, comprising administering to the subject at least one antibody, or antigen-binding fragment thereof, described herein, that specifically binds to a human NRP2a polypeptide. Certain embodiments include a method of modulating (e.g., increasing, decreasing) smooth muscle contractility in a subject in need thereof, comprising administering to the subject an anti-NRP2a antibody, or antigen-binding fragment thereof, as described herein. Certain embodiments include a method of treating, ameliorating symptoms, and / or decreasing progression of reduced smooth muscle contractility in a subject in need thereof, comprising administering to the subject an anti-NRP2a antibody, or antigen-binding fragment thereof, as described herein.

[0288] NRP2 expression has been associated with the development of fibrosis. Some embodiments thus include the use of anti-NRP2a antibodies described herein to treat fibrosis, e.g., tissue fibrosis. Examples of tissue fibrosis include fibrosis selected from the group consisting of liver fibrosis, renal fibrosis, pulmonary fibrosis, skin fibrosis, cardiovascular fibrosis, and gastrointestinal fibrosis, among other fibrotic diseases. Examples of liver fibrosis include cirrhosis, ischemia-reperfusion, post-liver transplant injury, necrotizing hepatitis, hepatitis B, hepatitis C, primary biliary cirrhosis, and primary sclerosing cholangitis. In some aspects, cirrhosis is associated with induction by alcohol, drugs, and / or other chemicals. Examples of renal fibrosis include proliferative glomerulonephritis, sclerosing glomerulonephritis, nephrogenic fibrosing dermopathy, diabetic nephropathy, renal tubulointerstitial fibrosis, and focal segmental glomerulosclerosis. Examples of pulmonary fibrosis include pulmonary interstitial fibrosis, drug-induced sarcoidosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, diffuse alveolar injury disease, pulmonary hypertension, and neonatal bronchopulmonary dysplasia. Examples of skin fibrosis include scleroderma, keloid scarring, psoriasis, hypertrophic scarring, and pseudoscleroderma. Examples of cardiovascular fibrosis include atherosclerosis, coronary restenosis, congestive cardiomyopathy, heart failure, heart transplantation, and myocardial fibrosis. Examples of gastrointestinal fibrosis include collagenous colitis, villous atrophy, crypt hyperplasia, polyp formation, Crohn's fibrosis, gastric ulcer healing, and scarring after abdominal adhesive surgery. Also included are fibrotic conditions resulting from bone-related fibrotic diseases and rheumatoid pannus formation.

[0289] Methods for identifying subjects with one or more of the diseases or conditions described herein are known in the art. In some embodiments, a subject is selected for treatment based on having and / or having a disease associated with increased levels or expression of at least one NRP2a ligand, such as CCL21 and / or CCR7, and / or their encoding mRNA, compared to healthy controls. For example, in some embodiments, the level of at least one NRP2a ligand in a diseased subject, cell, or tissue is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold or more of the level of at least one NRP2a ligand in a healthy control. In some embodiments, a subject is selected for treatment based on having and / or having a cancer with an increased level or expression of at least one NRP2a ligand and / or its encoding mRNA compared to a non-cancerous control cell or tissue. For example, in some embodiments, the level of at least one NRP2a ligand in the cancer cell or tissue is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold or more of the level of NRP2a ligand in a non-cancerous control or standard. Thus, certain embodiments include a method of selecting a subject for treatment, comprising (i) detecting an increased expression level of at least one NRP2a ligand and / or its encoding mRNA in the subject compared to a control or reference, and (ii) administering to the subject a therapeutic composition comprising at least one anti-NRP2a antibody or antigen-binding fragment thereof, as described herein. In certain embodiments, the NRP2a ligand is CCL21 and / or CCR7.

[0290] In some embodiments, a subject is selected for treatment based on having and / or having an increased circulating or serum level of an NRP2a polypeptide, such as a soluble NRP2a polypeptide (e.g., selected from Table N1), compared to the levels of a healthy or matched control population of subjects. For example, in certain embodiments, the circulating or serum level is about or at least about 10, 20, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000 pM of soluble NRP2a polypeptide. or a circulating or serum level of about 30-50, 50-100, 100-2000, 200-2000, 300-2000, 400-2000, 500-2000, 600-2000, 700-2000, 800-2000, 900-2000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 pM of a soluble NRP2a polypeptide.

[0291] In certain embodiments, a subject is selected for treatment based on having and / or having a disease associated with increased levels or expression of an NRP2a polypeptide (optionally selected from Table N1) and / or its encoding mRNA compared to healthy controls (e.g., an NRP2-related disease). In some embodiments, the NRP2a polypeptide is an NRP2a variant 1 or variant 2 isoform, or a fragment thereof. For example, in certain embodiments, the level of NRP2a polypeptide in a diseased subject, cell, or tissue is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more of the level of NRP2a polypeptide in a healthy control. In some embodiments, a subject is selected for treatment based on having and / or having a cancer that has increased levels or expression of an NRP2a polypeptide (e.g., selected from Table N1) and / or its encoding mRNA compared to a control cell or tissue, and optionally compared to a non-cancerous cell or tissue of the same type as the cancer. For example, in some embodiments, the level of NRP2a polypeptide in the cancer cell or tissue is about or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more the level of NRP2a polypeptide in a non-cancerous control or standard. Some embodiments thus include a method of selecting a subject for treatment comprising: (i) detecting an increased expression level of an NRP2a polypeptide and / or its encoding mRNA in the subject compared to a control or reference; and (ii) administering to the subject a therapeutic composition comprising at least one anti-NRP2a antibody or antigen-binding fragment thereof, as described herein.

[0292] In some embodiments, the subject has and / or is selected for treatment based on having a disease associated with increased levels or expression of NRP2a (e.g., variants 1 and / or 2 of Table N1) or an altered ratio of NRP2a:NRP2b expression compared to a healthy control or a matched control or a population of subjects. In some embodiments, the subject has significantly higher expression or levels of NRP2a compared to a healthy control or a matched control or a population of subjects. In some embodiments, the level of NRP2a is increased by about or at least about 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% compared to a healthy control or a matched control or a population of subjects. Certain embodiments therefore include a method of selecting a subject for cancer treatment comprising: (i) detecting an increased expression level of NRP2a in the subject compared to a control or reference; and (ii) administering to the subject a therapeutic composition comprising an anti-NRP2a antibody or antigen-binding fragment thereof described herein.

[0293] In some embodiments, a population of healthy controls or matched controls or subjects includes the average range for age-matched samples of the same type of diseased or non-diseased cells or tissues that contain a particular characteristic, such as drug resistance, metastatic potential, invasiveness, genetic signatures (e.g., p53 mutations, PTEN deletion, IGFR expression), and / or expression patterns.

[0294] As noted above, for in vivo use for the treatment or testing of disease in humans or non-human mammals, the agents described herein will typically be incorporated into one or more therapeutic or pharmaceutical compositions prior to administration, including veterinary therapeutic compositions.

[0295] Thus, certain embodiments relate to therapeutic compositions comprising at least one antibody or antigen-binding fragment thereof that specifically binds to a human NRP2a v1 and / or v2 polypeptide, as described herein. In some examples, the therapeutic or pharmaceutical composition comprises one or more of the agents described herein in combination with a pharma- ceutical or physiologically acceptable carrier or excipient. Certain therapeutic compositions further comprise at least one cancer immunotherapeutic agent, as described herein.

[0296] Some therapeutic compositions (and certain methods utilize) include only one anti-NRP2a antibody or antigen-binding fragment thereof. Certain therapeutic compositions (and certain methods utilize) include a mixture of at least two, three, four, or five different anti-NRP2a antibodies or antigen-binding fragments thereof.

[0297] For example, certain therapeutic compositions comprise at least two anti-NRP2 antibodies, a first antibody or antigen-binding fragment thereof that specifically binds to at least one first epitope of a human NRP2a polypeptide, and a second antibody or antigen-binding fragment thereof that specifically binds to at least one second epitope of the same or a different human NRP2 polypeptide, where the at least one first epitope is different from the at least one second epitope. In some embodiments, the first and second antibodies or antigen-binding fragments thereof specifically and non-competitively bind to the same domain of the NRP2 polypeptide. In some embodiments, a first anti-NRP2 antibody or antigen-binding fragment thereof selectively binds to a first epitope that is specific for the NRP2a isoform (e.g., variants 1 and / or 2 of Table N1), and a second anti-NRP2 antibody or antigen-binding fragment thereof selectively binds to a second epitope that is specific for the NRP2b isoform (e.g., variants 4 and / or 5 of Table N1) or is common to both the NRP2a and NRP2b isoforms (e.g., located in a common, surface-exposed a1, a2, b1, b2 or c domain shared by full-length NRP2a and NRP2b).

[0298] In some embodiments, the first and second antibodies or antigen-binding fragments thereof specifically and non-competitively bind to different domains of an NRP2 polypeptide. In some embodiments, the first antibody modulates signaling activity between an NRP2a polypeptide and at least one NRP2a ligand, e.g., CCL21 and / or CCR7.

[0299] In some embodiments, the first and second antibodies or antigen-binding fragments thereof are both blocking antibodies, e.g., with respect to at least two different NRP2 ligands. In some embodiments, the first and second antibodies or antigen-binding fragments thereof are both partially blocking antibodies, e.g., with respect to at least two different NRP2 ligands. In some examples, the first and second antibodies or antigen-binding fragments thereof are both non-blocking antibodies, e.g., with respect to at least two different NRP2 ligands.

[0300] In some examples, the first antibody or antigen-binding fragment thereof is a blocking antibody and the second antibody or antigen-binding fragment thereof is a partially blocking antibody. In certain examples, the first antibody or antigen-binding fragment thereof is a blocking antibody and the second antibody or antigen-binding fragment thereof is a non-blocking antibody.

[0301] In some embodiments, the first and second antibodies or antigen-binding fragments thereof both comprise an IgG Fc domain with high effector function in humans, e.g., an IgG1 or IgG3 Fc domain. In some embodiments, the first and second antibodies or antigen-binding fragments thereof both comprise an IgG Fc domain with low effector function in humans, e.g., an IgG2 or IgG4 Fc domain.

[0302] In some examples, the first antibody or antigen-binding fragment thereof comprises an IgG Fc domain with high effector function in humans, e.g., an IgG1 or IgG3 Fc domain, and the second antibody or antigen-binding fragment thereof comprises an IgG Fc domain with low effector function in humans, e.g., an IgG2 or IgG4 Fc domain.

[0303] In certain embodiments, a therapeutic composition comprising an agent, such as an anti-NRP2a antibody or other polypeptide agent, is substantially pure on a protein or weight-by-weight basis, e.g., the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein or weight-by-weight basis.

[0304] In some embodiments, the antibodies (e.g., anti-NRP2a antibodies) or other polypeptide agents provided herein do not form aggregates, have desirable solubility, and / or have an immunogenicity profile suitable for use in humans, as described herein and known in the art. Thus, in some embodiments, therapeutic compositions comprising a polypeptide agent (e.g., anti-NRP2a antibodies) are substantially free of aggregates. For example, certain compositions comprise less than about 10% (protein basis) high molecular weight aggregated protein, or less than about 5% high molecular weight aggregated protein, or less than about 4% high molecular weight aggregated protein, or less than about 3% high molecular weight aggregated protein, or less than about 2% high molecular weight aggregated protein, or less than about 1% high molecular weight aggregated protein. Some compositions comprise a polypeptide agent (e.g., an antibody, such as an anti-NRP2a antibody) that is at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% monodisperse relative to its apparent molecular weight.

[0305] In some embodiments, a polypeptide agent, such as an antibody (e.g., an anti-NRP2a antibody), is concentrated to about or at least about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6, 0.7, 0.8, 0.9, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11, 12, 13, 14, or 15 mg / ml and formulated for biotherapeutic use.

[0306] To prepare a therapeutic or pharmaceutical composition, an effective or desired amount of one or more agents is mixed with any pharmaceutical carrier or excipient known to those skilled in the art to be appropriate for the particular agent and / or mode of administration. Pharmaceutical carriers can be liquid, semi-liquid, or solid. Solutions or suspensions used for parenteral, intradermal, intraocular, subcutaneous, direct instillation into the bladder, or topical application can contain, for example, sterile diluents (such as water), saline (e.g., phosphate buffered saline; PBS), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents (such as benzyl alcohol and methylparabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates, phosphates, etc.). If administered intravenously (e.g., by IV infusion), suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0307] Administration of the agents described herein, in pure form or in suitable therapeutic or pharmaceutical compositions, can be via any of the accepted modes of administration of agents to accomplish similar utilities. Therapeutic or pharmaceutical compositions can be prepared by combining the agent-containing composition with suitable physiologically acceptable carriers, diluents, or excipients, and can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmacoactive ingredients (including other small molecules described elsewhere herein) and / or suitable excipients, such as salts, buffers, and stabilizers, may, but need not, be present in the composition.

[0308] Administration can be accomplished by a variety of different routes, including oral, parenteral, nasal, intravenous, intraocular, intradermal, intramuscular, subcutaneous, placement in the bladder, or topical. The preferred mode of administration will depend on the nature of the condition being treated or prevented. Certain embodiments include administration by IV infusion.

[0309] Carriers can include, for example, pharma- ceutical or physiologically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as polysorbate 20 (TWEEN®), polyethylene glycol (PEG), and poloxamer (PLURONICS®), and the like.

[0310] In some embodiments, the agent or agents can be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particles or liposomes may further comprise other therapeutic or diagnostic agents.

[0311] The exact dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known test protocols or by testing the composition in model systems known in the art and extrapolating therefrom. Controlled clinical trials can also be carried out. The dose can also vary with the severity of the condition to be alleviated. Pharmaceutical compositions are generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition can be administered once or divided into multiple smaller doses administered at regular intervals. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs.

[0312] Typical routes of administration of these and related therapeutic or pharmaceutical compositions thus include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, ocular, rectal, vaginal, and intranasal. The term "parenteral" as used herein includes subcutaneous injection, intravenous, injection into the bladder, intramuscular, intrasternal injection, or infusion techniques. The therapeutic or pharmaceutical compositions according to certain embodiments of the present disclosure are formulated so that the active ingredients contained therein are bioavailable upon administration of the composition to a subject or patient. The composition administered to a subject or patient may take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit and a container of a medicament described herein in aerosol form may hold multiple dosage units. Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered typically contains a therapeutically effective amount of an agent described herein for the treatment of the disease or condition of interest.

[0313] The therapeutic or pharmaceutical composition may be in solid or liquid form. In one embodiment, the carrier is particulate, so that the composition is, for example, in tablet or powder form. The carrier may be liquid, so that the composition is, for example, an oral oil, an injectable liquid or an aerosol, which is useful, for example, in inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered herein as either solid or liquid. Particular embodiments include sterile injectable solutions.

[0314] As a solid composition for oral administration, the pharmaceutical composition may be formulated into powder, granules, gels, compressed tablets, pills, capsules, chewing gum, wafers, or the like. Such solid compositions typically contain one or more inert diluents or edible carriers. Also, one or more of the following may be present: binders, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginic acid, sodium alginate, primogel, corn starch, and the like; lubricants, such as magnesium stearate or stereotex; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavorings, such as peppermint, methyl salicylate, or orange flavoring; and coloring agents. When the pharmaceutical composition is in the form of a capsule, for example a gelatin capsule, it may contain, in addition to the above types of materials, a liquid carrier, such as polyethylene glycol or oil.

[0315] The therapeutic or pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, gel, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the compound, one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included.

[0316] Liquid therapeutic or pharmaceutical compositions, whether they are in solution, suspension or other similar form, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, etc., fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol or other solvents that can serve as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and agents for adjusting tonicity, such as sodium chloride or glucose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0317] Liquid therapeutic or pharmaceutical compositions intended for either parenteral, ocular, or oral administration should contain an amount of drug so that a suitable dosage is obtained. Typically, this amount is at least 0.01% of the drug of interest in the composition. When intended for oral administration, this amount can vary to be between 0.1 and about 70% by weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain between about 4% and about 75% of the drug of interest. In certain embodiments, therapeutic or pharmaceutical compositions and preparations are prepared such that a parenteral dosage unit contains between 0.01 and 10% by weight of the drug of interest before dilution.

[0318] The therapeutic or pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment, or gel base. The base may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Viscosifiers may be present in therapeutic or pharmaceutical compositions for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device.

[0319] The therapeutic or pharmaceutical composition may be intended for rectal administration, for example, in the form of a suppository that dissolves in the rectum and releases the drug. Compositions for rectal administration may contain an oleaginous base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0320] Therapeutic or pharmaceutical compositions may contain various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition may contain a material that forms a coating shell around the active ingredient. The material that forms the coating shell is typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. Therapeutic or pharmaceutical compositions in solid or liquid form may contain a component that binds to the drug and thereby assists in the delivery of the compound. Suitable components that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0321] The therapeutic or pharmaceutical composition may consist essentially of a dosage unit that can be administered as an aerosol. The term "aerosol" is used to mean a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas, or by a suitable pump system that dispenses the active ingredient. The aerosol may be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient. Delivery of the aerosol includes the necessary containers, activators, valves, subcontainers, and the like, which may together form a kit. Those skilled in the art can determine the preferred aerosol without undue experimentation.

[0322] The compositions described herein may be prepared with carriers that protect the agents against rapid elimination from the body, such as slow release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, as well as biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and others known to those skilled in the art.

[0323] Therapeutic or pharmaceutical compositions may be prepared by methodologies well known in the pharmaceutical arts. For example, therapeutic or pharmaceutical compositions intended to be administered by injection may contain one or more salts, buffers and / or stabilizers with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with drugs to facilitate the dissolution or homogeneous suspension of the drug in an aqueous delivery system.

[0324] Therapeutic or pharmaceutical compositions may be administered in a therapeutically effective amount, which may vary depending on a variety of factors, including the activity of the particular compound used; the metabolic stability and length of action of the compound; the age, weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject being treated. In some examples, the therapeutically effective daily dose is about 0.001 mg / kg (i.e., about 0.07 mg) to about 100 mg / kg (i.e., about 7.0 g) (for a 70 kg mammal); preferably, the therapeutically effective dose is about 0.01 mg / kg (i.e., about 0.7 mg) to about 50 mg / kg (i.e., about 3.5 g) (for a 70 kg mammal); more preferably, the therapeutically effective dose is about 1 mg / kg (i.e., about 70 mg) to about 25 mg / kg (i.e., about 1.75 g) (for a 70 kg mammal). In some embodiments, the therapeutically effective dose is administered weekly, biweekly, or monthly. In certain embodiments, the therapeutically effective dose is administered weekly, biweekly, or monthly, for example, at a dose of about 1-10 or 1-5 mg / kg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg.

[0325] The combination therapy described herein may include administration of a single pharmaceutical dosage formulation containing an anti-NRP2a antibody and an additional therapeutic agent (e.g., immunotherapeutic agent, chemotherapeutic agent, hormonal therapy agent, kinase inhibitor), as well as administration of a composition containing an anti-NRP2a antibody and an additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, the anti-NRP2a antibody described herein and the additional therapeutic agent can be administered together to a subject in a single oral dosage composition, such as a tablet or capsule, or each agent administered in a separate oral dosage formulation. Similarly, the anti-NRP2a antibody described herein and the additional therapeutic agent can be administered together to a subject in a single parenteral administration composition, such as in saline or other physiologically acceptable solution, or each agent can be administered in a separate parenteral administration formulation. As another example, for cell-based therapy, the anti-NRP2a antibody can be mixed with the cells prior to administration, can be administered as part of a separate composition, or both. When separate dosage formulations are used, the compositions can be administered at essentially the same time, i.e., simultaneously, or at separately staggered times, i.e., sequentially and in any order; combination treatment is understood to include all these regimens.

[0326] Also included are patient care kits comprising: (a) at least one antibody or antigen-binding fragment thereof that specifically binds to a human NRP2a variant 1 and / or variant 2 polypeptide (anti-NRP2a antibody), as described herein; and, optionally, (b) at least one additional therapeutic agent (e.g., an immunotherapeutic agent, a chemotherapeutic agent, a hormonal therapy agent, a kinase inhibitor). In certain kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0327] The kits herein may also include one or more additional therapeutic agents or other components appropriate or desired for the indication being treated or for the desired diagnostic application. The kits herein may also include one or more syringes or other components necessary or desirable to facilitate the intended mode of delivery (e.g., stents, implantable depots, etc.).

[0328] In some embodiments, the patient care kit includes separate containers, dividers, or compartments for the composition and the informational material. For example, the composition can be included in a bottle, vial, or syringe, and the informational material can be included in association with the container. In some embodiments, the separate elements of the kit are included in a single, undivided container. For example, the composition is included in a bottle, vial, or syringe having attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each including one or more unit dosage forms (e.g., dosage forms described herein) of an anti-NRP2a antibody, and optionally at least one additional therapeutic agent. For example, the kit includes a plurality of syringes, ampoules, foil packets, or blister packs, each including a single unit dose of an anti-NRP2a antibody, and optionally at least one additional therapeutic agent. The containers of the kit can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.

[0329] The patient care kit optionally includes a device suitable for administration of the composition, such as a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses a metered dose of the agent. Also included are methods of providing the kit, such as by combining the components described herein.

[0330] Drug release assays and bioassays and analytical assays for product specifications, diagnostics, and reagents Also included are bioassays related to anti-NRP2a antibodies and related agents, such as therapeutic and diagnostic reagents. Examples include bioassays and analytical assays that measure purity, biological activity, affinity, solubility, pH, endotoxin levels, among others, many of which are described herein. Also included are assays that establish dose-response curves and / or provide one or more basis for comparison between different batches of antibodies. Batch comparisons can be based on any one or more of chemical characterization, biological characterization, and clinical characterization. Also included are methods for evaluating the potency, stability, pharmacokinetics, and immunogenicity of selected antibodies. Among other uses, these and other methods can be used for lot release testing of biological or chemical agents, including the anti-NRP2a antibodies described herein.

[0331] Certain embodiments include the use of bioaffinity assays. Such assays can be used to assess, for example, the binding affinity between an anti-NRP2a antibody and at least one NRP2 ligand (e.g., an NRP2a ligand, such as CCL21 and / or CCR7), including its ability to interfere with the interaction between a human NRP2a polypeptide and at least one NRP2a ligand, or other cellular binding partners. Certain exemplary binding affinity assays may utilize ELISA assays or protein-protein interaction assays, such as the NanoBiT® Protein:Protein Interaction System (Promega), among other protein complementation assays and approaches described herein and known in the art. Certain assays utilize high performance receptor binding chromatography (see, e.g., Roswall et al., Biologicals. 24:25-39, 1996). Other exemplary binding affinity assays may utilize surface plasmon resonance (SPR)-based technology. Examples include BIACore technologies, certain of which integrate SPR technology with microfluidic systems to monitor molecular interactions in real time at concentrations in the pM-mM range, and KINEXA™ assays, which provide accurate measurements of binding specificity, binding affinity, and binding kinetics / rate constants.

[0332] Certain embodiments relate to immunoassays for evaluating or optimizing the immunogenicity of anti-NRP2a antibodies. Examples include ex vivo human cell assays and in vitro immunoenzymatic assays to provide useful information on the immunogenic potential of therapeutic proteins. Ex vivo cell response assays can be used, for example, to reproduce cellular cooperation between antigen-presenting cells (APCs) and T cells, thereby measuring T cell activation after contact with a protein of interest. Certain in vitro enzymatic assays may utilize a collection of recombinant HLA-DR molecules covering a large proportion of the relevant human population, and may include automated immunoenzymatic assays to test binding of peptides (derived from fragmentation of therapeutic proteins) to HLA-D...

Claims

1. An antibody, or antigen-binding fragment thereof, that binds to a neuropilin-2A (NRP2a) variant 1 (v1) or variant 2 (v2) polypeptide via an epitope comprising a sequence selected from SEQ ID NOs: 94 to 104, wherein the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (VH) sequence comprising complementarity determining regions VHCDR1, VHCDR2, and VHCDR3 sequences, and a light chain variable region (VL) sequence comprising complementarity determining regions VLCDR1, VLCDR2, and VLCDR3 sequences, wherein: The VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 13, 127, and GX1X2X3X4X5 (X1 is G, A, or S; X2 is Y, F, K, L, or R; X3 is T, A, G, I, L, Q, or V; X4 is D, A, G, K, N, Q, R, or S; and X5 is Y, A, D, E, F, G, H, I, K, L, N, Q, R, S, T, or V), and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 16, 17, and X6X7X8X9X10X11X12X13 (X6 is S, A, G, I, L, P, T, or V, X7 is Q, A, G, R, or S, X8 is S, A, H, K, L, Q, or T, X9 is T, F, G, H, I, K, L, N, Q, R, S, V, or Y, X10 is H, A, D, E, F, G, I, K, L, N, Q, R, S, T, or Y, X11 is V, A, E, F, G, H, I, K, L, N, P, Q, R, S, T, or Y, X12 is L, A, E, H, I, N, P, Q, S, T, or V, and X13 is T, A, D, E, F, G, I, K, L, N, Q, R, S, or V); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 130-132, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 133-135, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 136-138, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 139-141, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 142-144, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 145-147, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 148-150, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 151-153, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 154-156, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 157-159, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 4-6, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 7-9, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 10-12, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 13-15, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 16-18, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 19-21, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 22-24, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs:25-27, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs:28-30, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 31-33, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 34-36, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 37-39, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 40-42, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 43-45, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 46-48, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 49-51, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 52-54, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 55-57, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 58-60, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions); or An antibody, or antigen-binding fragment thereof, wherein the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 61-63, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 64-66, respectively (including variants thereof having a total of 1, 2, or 3 alterations across all of the CDR regions).

2. the VH sequence comprises SEQ ID NO: 170 and the VL sequence comprises SEQ ID NO: 171; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 160 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 161; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 162 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 163; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 164 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 165; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 166 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 167; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 168 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 169; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:67 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:68; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:69 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:70; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 71 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 72; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 73 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 74; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 75 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 76; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 77 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 78; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:79 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:80; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 81 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 82; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 83 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 84; the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 85 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 86; or 2. The antibody, or antigen-binding fragment thereof, of claim 1, wherein the VH sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 87 and the VL sequence is at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO:

88.

3. 2. The antibody or antigen-binding fragment thereof of claim 1, which does not substantially bind to human neuropilin-2B (NRP2b) variant 4 (v4) polypeptide and / or human NRP2b variant 5 (v5) polypeptide, wherein optionally the binding affinity of the antibody or antigen-binding fragment thereof for human NRP2a v1 or v2 polypeptide is at least about 1.5-fold, 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, 200-fold, 400-fold, 600-fold, 800-fold, or 1000-fold stronger than its binding affinity for NRP2a v3 polypeptide, the NRP2b v4 polypeptide, and / or the NRP2b v5 polypeptide.

4. optionally blocks or otherwise reduces binding between said NRP2a v1 or v2 polypeptide and a chemokine (C-C motif) ligand 21 (CCL21) polypeptide in an in vitro binding assay, an in vitro or ex vivo cell-based assay, or in vivo, optionally blocks or otherwise reduces binding between said NRP2a v1 or v2 polypeptide and said CCL21 polypeptide by about or at least about 20-100% or more (optionally about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100% or more) compared to a control or reference; optionally blocks or otherwise reduces binding (including dimerization) between said NRP2a v1 or v2 polypeptide and a C-C chemokine receptor type 7 (CCR7) polypeptide in an in vitro binding assay, an in vitro or ex vivo cell-based assay, or in vivo, optionally blocks or otherwise reduces binding (including dimerization) between said NRP2a v1 or v2 polypeptide and said CCR7 polypeptide by about or at least about 20-100% or more (optionally about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100% or more) compared to a control or reference; and / or 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof modulates (optionally antagonizes) signaling activity between the NRP2a v1 or v2 polypeptide and the CCL21 and / or CCR7 polypeptide, optionally by about or at least about 20-100% or more (optionally about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, or 100% or more) compared to a control or reference, and optionally the signaling activity includes induction of migration of immune cells (optionally dendritic cells or mature T cells), and the antibody or antigen-binding fragment thereof reduces the signaling activity; and / or the signaling activity includes induction of tumor cell migration, and the antibody or antigen-binding fragment thereof reduces the signaling activity.

5. 2. The antibody of claim 1, or an antigen-binding fragment thereof, comprising an IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), or IgM Fc domain, optionally a human Fc domain, or hybrids and / or variants thereof.

6. 6. The antibody or antigen-binding fragment thereof of claim 5, comprising an IgG Fc domain with high effector function in humans, optionally an IgG1 or IgG3 Fc domain, or an IgG Fc domain with low effector function in humans, optionally an IgG2 or IgG4 Fc domain.

7. 6. The antibody of claim 5, or an antigen-binding fragment thereof, optionally comprising an IgG1 or IgG4 Fc domain selected from SEQ ID NOs: 109-111.

8. The antibody or antigen-binding fragment thereof according to claim 1, which is a monoclonal antibody and / or a humanized antibody.

9. 2. The antibody or antigen-binding fragment thereof of claim 1, which 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.

10. A therapeutic composition comprising a pharmaceutically acceptable carrier and an antibody, or antigen-binding fragment thereof, according to any one of claims 1 to 9.

11. The therapeutic composition of claim 10 for use in the treatment of a neuropilin 2a (NRP2a)-associated disease or condition, wherein the disease or condition is selected from cancer, an inflammatory disease, an autoimmune disease, a lymphatic disease or associated condition, a fibrotic disease, and a disease associated with reduced smooth muscle contractility.

12. The therapeutic composition for use according to claim 11, wherein the disease is cancer, optionally wherein the cancer expresses or overexpresses NRP2, and optionally wherein the cancer exhibits NRP2-dependent growth, NRP2-dependent adhesion, NRP2-dependent migration, and / or NRP2-dependent invasion.

13. A therapeutic composition for use as described in claim 12, for administration in combination with at least one additional agent selected from one or more of a cancer immunotherapy agent, a chemotherapy agent, a hormonal therapy agent, and a kinase inhibitor.

14. 14. The therapeutic composition for use according to claim 13, wherein the cancer immunotherapeutic agent is selected from one or more of an immune checkpoint modulator, a cancer vaccine, an oncolytic virus, a cytokine, and a cell-based immunotherapy.

15. 15. The therapeutic composition of claim 14, wherein the chemotherapeutic agent is selected from one or more of an alkylating agent, an antimetabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent.

16. 13. The therapeutic composition for use according to claim 12, wherein 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.

17. The cancer is (a) bladder cancer that has metastasized to the bone, liver, and / or lung; (b) breast cancer that has metastasized to the bone, brain, liver, and / or lungs; (c) colorectal cancer that has metastasized to the liver, lung, and / or peritoneum; (d) renal cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or lungs; (e) lung cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or other lung sites; (f) melanoma that has metastasized to bone, brain, liver, lung, and / or skin / muscle; (g) ovarian cancer that has metastasized to the liver, lung, and / or peritoneum; (h) pancreatic cancer that has 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 that has metastasized to the liver, lung, and / or peritoneum; (l) thyroid cancer that has metastasized to the bone, liver, and / or lung; and (m) uterine cancer that has metastasized to the bone, liver, lung, peritoneum, and / or vagina 13. The therapeutic composition for use according to claim 12, wherein the cancer is a metastatic cancer selected from one or more of:

18. A bioassay system comprising the antibody, or antigen-binding fragment thereof, of any one of claims 1 to 9, and a host cell line expressing a human NRP2a polypeptide on the cell surface.

19. A detection system comprising a cell expressing a human neuropilin 2a (NRP2a) polypeptide, at least one NRP2a ligand, and an antibody or antigen-binding fragment thereof described in any one of claims 1 to 9, which regulates the interaction between the NRP2a polypeptide and the at least one NRP2a ligand.

20. 10. A cell composition comprising an engineered cell population, wherein at least one cell comprises one or more polynucleotides encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the cells are capable of growing in serum-free medium.

21. 10. A cell growth device comprising: the antibody, or antigen-binding fragment thereof, of any one of claims 1 to 9; an engineered cell population, wherein at least one cell comprises one or more polynucleotides encoding said antibody or antigen-binding fragment thereof; at least about 10 liters of serum-free growth medium; and a sterile container.

Images