NRP1 antibody compositions and methods of use thereof

Abstract

Provided herein are antibodies, antigen-binding portions thereof, and antibody peptide conjugates that specifically bind to neuropilin-1 (NRP1) and various compositions of such antibodies, antigen-binding portions, and antibody-peptide conjugates. The disclosure also provides nucleic acids encoding the antibodies, antigen-binding portions thereof, or antibody peptide conjugates; cells comprising the nucleic acids; and methods for producing the antibodies, antigen-binding portions thereof, or antibody peptide conjugates. Also provided are methods for using the antibodies or antigen-binding portions thereof in therapeutics and diagnostics for cancer.

Description

Attorney Docket No. 090723-1530315-MDA25-028PCTNRP1 ANTIBODY COMPOSITIONS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No. 63 / 716,129, filed on November 4, 2024, which is incorporated herein by reference in its entirety.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under CAI 00632 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Cancer is a leading cause of death worldwide, accounting for nearly one in six deaths in 2023. Despite significant advances in cancer research, efficacious cancer treatments remain limited. Neuropilin-1 (“NRP1”) is involved in the occurrence and development of a variety of cancers. NRP1 expression by cancer cells is associated with tumor progression, metastasis, and worsened prognosis. It is thus being explored as a diagnostic and therapeutic target in cancer. Development of efficacious antibodies and antibody conjugates against NRP1 for diagnostic and therapeutic use in the identification and treatment of NRP1 positive malignancies may therefore advance the standard of cancer treatment.SUMMARY

[0004] The terms “invention,” “the invention,” “this invention” and “the present invention,” as used in this document, are intended to refer broadly to all of the subject matter of this patent application and the claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent claims below. Covered embodiments of the invention are defined by the claims, not this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of theAttorney Docket No. 090723-1530315-MDA25-028PCT entire specification, any or all figures and each claim. Some of the exemplary embodiments of the present invention are discussed below.

[0005] The present disclosure provides antibodies, antigen-binding fragments thereof, and antibody-peptide conjugates that target NRP1. Also provided are methods and compositions for using the antibodies or antigen-binding portions thereof in cancer therapeutics and diagnostics.

[0006] In one aspect, the disclosure provides antibodies and antigen-binding fragments thereof that target NRP1. In some embodiments, the isolated antibody or antigen-binding fragment that comprises:(a) a heavy chain variable region comprising:(i) a CDRH1 comprising SEQ ID NOs: 11, 14, or 19;(ii) a CDRH2 comprising SEQ ID NOs: 12, 15, 17, or 20; and(iii) a CDRH3 comprising SEQ ID NOs: 13, 16, 18, or 21; and(b) a light chain variable region comprising:(i) a CDRL1 comprising SEQ ID NOs: 22, 25, 30, or 33;(ii) a CDRL2 comprising SEQ ID NOs: 23, 26, 28, or 31; and(iii) a CDRL3 comprising SEQ ID NOs: 24, 27, 29, or 32.

[0007] In some embodiments, the isolated antibody or antigen-binding fragment comprises:(a) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24;(b) a CDRH1 comprising SEQ ID NO: 14, a CDRH2 comprising SEQ ID NO: 15, a CDRH3 comprising SEQ ID NO: 16; a CDRL1 comprising SEQ ID NO: 25, a CDRL2 comprising SEQ ID NO: 26, and a CDRL3 comprising SEQ ID NO: 27;(c) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 17, a CDRH3 comprising SEQ ID NO: 18; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 29;(d) a CDRH1 comprising SEQ ID NO: 19, a CDRH2 comprising SEQ ID NO: 20, a CDRH3 comprising SEQ ID NO: 21; a CDRL1 comprising SEQ ID NO: 30, a CDRL2 comprising SEQ ID NO: 31, and a CDRL3 comprising SEQ ID NO: 32; or(e) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 33, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 24.

[0008] In some embodiments, the isolated antibody or antigen-binding fragment comprises:Attorney Docket No. 090723-1530315-MDA25-028PCT(a) the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 1, and the light chain variable region comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 6;(b) the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 2, and the light chain variable region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 7;(c) the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 8;(d) the heavy chain variable region comprises an amino acid sequence that is at least 87% identical to SEQ ID NO: 4, and the light chain variable region comprises SEQ ID NO: 9; or(e) the heavy chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 5, and the light chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 10.

[0009] In some embodiments, the isolated antibody or antigen-binding fragment comprises:(a) the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6;(b) the heavy chain variable region comprises SEQ ID NO: 2, and the light chain variable region comprises SEQ ID NO: 7;(c) the heavy chain variable region comprises SEQ ID NO: 3, and the light chain variable region comprises SEQ ID NO: 8;(d) the heavy chain variable region comprises SEQ ID NO: 4, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 9; or(e) the heavy chain variable region comprises SEQ ID NO: 5, and a light chain variable region comprises SEQ ID NO: 10.

[0010] In certain embodiments, the isolated antibody or antigen-binding fragment comprises a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24. In certain embodiments, the isolated antibody or antigen-binding fragment comprises the heavy chain variable region comprising SEQ ID NO: 1, and the light chain variable region comprising SEQ ID NO: 6.

[0011] In some embodiments, the isolated antigen-binding fragment is a chimeric antibody or antibody fragment. In some embodiments, the isolated antigen-binding fragment is aAttorney Docket No. 090723-1530315-MDA25-028PCT monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)3 fragment, Fv fragment, or single chain antibody. In some embodiments, the antibody or antigen-binding fragment is a chimeric antibody, bispecific antibody, trispecific antibody, or other multi-specific antibody.

[0012] In some embodiments, the antibody or antigen-binding fragment is conjugated or fused to an imaging agent, a cytotoxic agent, or a radioactive moiety. In some embodiments, the antibody or antigen-binding fragment is conjugated or fused to an imaging agent, and wherein the imaging agent is a fluorophore. In some embodiments, the antibody or antigenbinding fragment is conjugated or fused to a radioactive moiety, and the radioactive moiety is Zr-89, Cu-64, F-18, Y-90, Lu-177, At-211, Ac-225, or Pb-212. In some embodiments, the antibody is an immune conjugate. In some embodiments, the antibody is an antibody-drug conjugate.

[0013] In another aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment of the present disclosure and a pharmaceutically acceptable carrier.

[0014] In another aspect, an isolated nucleic acid is provided that encodes an antibody heavy and / or a light chain variable region of an antibody or antigen-binding fragment of the present disclosure.

[0015] In yet another aspect, a hybridoma or engineered cell is provided that comprises a nucleic acid encoding an antibody or antigen-binding fragment of the present disclosure.

[0016] In another aspect, the disclosure provides a method of making an isolated antibody or antigen-binding fragment of the present disclosure. In some embodiments, the method comprises culturing a hybridoma or engineered cell of the present disclosure under conditions that allow expression of the antibody or antigen-binding fragment and, optionally, isolating the antibody or antigen-binding fragment from the culture.

[0017] In another aspect, the disclosure provides an antibody-peptide conjugate that comprises: (a) an antibody or an antigen-binding fragment of the present disclosure; and (b) an immunogenic peptide. In some embodiments, the immunogenic peptide comprises a native cytomegalovirus (NLV) peptide or a cathepsin G 1 (CGI) peptide. In some embodiments, the immunogenic peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 34. In certain embodiments, the immunogenic peptide comprises SEQ ID NO: 34. In some embodiments, the immunogenic peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 35. In certain embodiments, the immunogenic peptide comprises SEQ ID NO:35.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0018] In some embodiments, the antibody or antigen-binding fragment is conjugated to the immunogenic peptide by a chemical linkage. In some embodiments, the chemical linkage comprises an attachment group and protease cleavable linker. In some embodiments, the attachment group comprises a maleimidocaproyl moiety, and the protease-cleavable linker comprises a valine-citrulline-p-aminocarbamate (VC-PABC) linker.

[0019] In some embodiments, the heavy chain of the antibody or antigen-binding fragment is conjugated to the immunogenic peptide by a recombinant linker. In some embodiments, the recombinant linker comprises a glycine-serine linker sequence and a protease-cleavable linker sequence. In certain embodiments, the glycine-serine linker comprises GS or SEQ ID NO: 36. In some embodiments, the protease-cleavable linker comprises SEQ ID NO: 37. In certain embodiments, the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising SEQ ID NO: 36, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, a second protease-cleavable linker sequence comprising SEQ ID NO:37, and a second copy of the immunogenic peptide. In certain other embodiments, the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising GS, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, and a second protease-cleavable linker sequence comprising SEQ ID NO:37. In certain other embodiments, the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising GS, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, a second protease-cleavable linker sequence comprising SEQ ID NO: 37, a second copy of the immunogenic peptide, and a third protease-cleavable linker sequence comprising SEQ ID NO:37.

[0020] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising:(a) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24;(b) a CDRH1 comprising SEQ ID NO: 14, a CDRH2 comprising SEQ ID NO: 15, a CDRH3 comprising SEQ ID NO: 16; a CDRL1 comprising SEQ ID NO: 25, a CDRL2 comprising SEQ ID NO: 26, and a CDRL3 comprising SEQ ID NO: 27;Attorney Docket No. 090723-1530315-MDA25-028PCT(c) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 17, a CDRH3 comprising SEQ ID NO: 18; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 29;(d) a CDRH1 comprising SEQ ID NO: 19, a CDRH2 comprising SEQ ID NO: 20, a CDRH3 comprising SEQ ID NO: 21; a CDRL1 comprising SEQ ID NO: 30, a CDRL2 comprising SEQ ID NO: 31, and a CDRL3 comprising SEQ ID NO: 32; or(e) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 33, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 24.

[0021] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment wherein:(a) the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 1, and the light chain variable region comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 6;(b) the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 2, and the light chain variable region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 7;(c) the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 8;(d) the heavy chain variable region comprises an amino acid sequence that is at least 87% identical to SEQ ID NO: 4, and the light chain variable region comprises SEQ ID NO: 9; or(e) the heavy chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 5, and the light chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 10.

[0022] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment wherein:(a) the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6;(b) the heavy chain variable region comprises SEQ ID NO: 2, and the light chain variable region comprises SEQ ID NO: 7;(c) the heavy chain variable region comprises SEQ ID NO: 3, and the light chain variable region comprises SEQ ID NO: 8;Attorney Docket No. 090723-1530315-MDA25-028PCT(d) the heavy chain variable region comprises SEQ ID NO: 4, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 9; or(e) the heavy chain variable region comprises SEQ ID NO: 5, and a light chain variable region comprises SEQ ID NO: 10.

[0023] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24. In another aspect, an antibody-peptide conjugate comprises an antibody or antigen-binding fragment wherein: the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6.

[0024] In another aspect, the disclosure provides a pharmaceutical composition comprising an antibody-peptide conjugate of the present disclosure and a pharmaceutically acceptable carrier.

[0025] In another aspect, a method of imaging a cell or tissue expressing NRP1 in a subject is provided. The method comprises the steps of: (a) administering to the subject the isolated antibody or antibody fragment of the present disclosure; and (b) detecting the imaging agent in the subject, thereby imaging the cell or tissue.

[0026] In yet another aspect, a method for diagnosing a subject having or suspected of having a cancer characterized by elevated NRP1 expression is provided. The method comprises: (a) contacting a biological sample with the isolated antibody or antigen-binding fragment of the present disclosure; and (b) detecting an amount of binding of the isolated antibody or antigenbinding fragment as a determination of a presence of NRP-1 in the biological sample.

[0027] In a further aspect, a method of treating a human subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure comprising an antibody or antigen-binding fragment thereof of the present disclosure and a pharmaceutically acceptable carrier. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure comprising an antibody-peptide conjugate of the present disclosure and a pharmaceutically acceptable carrier. In some embodiments, the subject has cancer. In some embodiments, the cancer expresses an elevated level of NRP1 relative to a healthy control tissue. In certain embodiments, the cancer is selected from a group consisting of breast, renal, lung, liver, head, neck, and squamous cell cancer. In some embodiments, the method further comprisesAttorney Docket No. 090723-1530315-MDA25-028PCT administering to the subject a therapeutically effective amount of a small molecule drug. In some embodiments, the isolated antibody or antigen-binding fragment used in the present methods is conjugated to a therapeutic agent. In some embodiments, the therapeutic agent is at least one of a cytotoxic agent, a photosensitizer, a chemotherapeutic agent, or an immunosuppressive agent. In some embodiments, the therapeutic agent is a moiety that specifically binds to an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a natural killer cell. In some embodiments, the therapeutically effective amount of the pharmaceutical composition is administered via intravenous injection, subcutaneous injection, or direct injection into a diseased tissue. In some embodiments, the method further comprises administering a composition that binds to the peptide of the antibody-peptide conjugate. In some embodiments, the composition comprises a peptide-specific antibody, T cell, or chimeric antigen receptor (CAR)-T cell.

[0028] In another aspect, a method of inhibiting the binding of neuropilin- 1 (NRP1) to VEGF or semaforin 3a is provided. The method comprises administering the isolated antibody or antibody fragment of the present disclosure. In some embodiments, administering the isolated antibody or antibody fragment inhibits an immune inhibitory action of a regulatory T cell.

[0029] These and other aspects of the present invention may be realized as shown and described in the following figures and related description.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions, systems, and methods, and to supplement any description(s) of the compositions, systems, and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0031] FIG. 1 is a graph demonstrating the binding of exemplary disclosed mouse NRP1 monoclonal antibody clones to human NRP1.

[0032] FIGS. 2A-2C are sensorgrams demonstrating the validation of two exemplary antibodies. FIGS. 2A-2B show BLI OCTET assay results for the disclosed 5D6 and 13C7 antibodies, respectively, demonstrating the ability of these antibodies to bind to the screening peptide, recombinant human NRP1 (“hNRPl”). FIG. 2C shows the BLI OCTET assay results for control NRP1 antibody 17F6 (Miltenyi) binding to hNRPl.Attomey Docket No. 090723-1530315-MDA25-028PCT

[0033] FIG. 3 is a graph showing the ability of exemplary antibodies to inhibit the binding of VEGF to NRP1. The controls used were no antibody or an isotype control antibody. These data demonstrate that the 5D6 and 15D2 antibodies partially block binding of VEGF to NRP1.

[0034] FIGS. 4A-4B are graphs showing the results of staining assays for determining the isotype of exemplary disclosed NRP1 antibodies. FIG. 4A shows that the isotype of the 5D6, 12A10, and 15D7 antibodies was IgG2. FIG. 4B shows that the isotype of the 7E2, 13C7, and control NRP1 antibody was IgGl. In both figures, the negative controls were unstained (unst) and secondary antibody alone (2°).

[0035] FIG. 5 provides immunofluorescence images of NRP1 positive breast cancer cells (MDA-MB-231 cells) showing that the exemplary NRP1 antibodies were internalized into NRP1 positive breast cancer cells within 5 hours of incubation.

[0036] FIG. 6 are natural and immunofluorescence images demonstrating that the exemplary chimeric 5D6 (“Ch5D6”) antibody was internalized into NRP1 positive breast cancer cells within 4 hours of incubation. In both the left panel (taken after one hour) and right panel (taken after four hours), the images are from left to right: unstained, stained with anti-human IgGl with Alexa Fluor 647, stained with HLA-A2 FITC as a membrane marker, and an image combining the anti-human IgGl and HLA-A2 FITC staining.

[0037] FIG. 7 provides confocal images of NRP1 positive breast cancer cells (MDA-MB- 231) incubated with the NRP1 antibody 5D6 linked to NLV peptide, showing that the antibody is internalized within 1 hour and co-localizes with the lysosomal marker within 3 hours. The images were taken using a Confocal Leica SP8 microscope. The NRP1 antibody conjugate stained red. The controls included DAPI for nuclear staining (blue) and either an anti-EEAl antibody (endosomal marker, stained green in top images) or an anti-LAMP2 antibody (lysosomal marker, stained green in the bottom images). Co-localization of the NRP1 antibody conjugate with the endosomal or lysosomal markers resulted in an orange staining.

[0038] FIGS. 8A-8B are graphs demonstrating that NRP1 positive breast cancer cells (MDA-MB-231) treated with a Ch5D6-CGl antibody-peptide conjugate are susceptible to CGI -specific killing. The tumor cells were untreated (231-) or treated with Ch5D6 antibody, recombinantly conjugated Ch5D6-CGl (Ch5D6-CGl(R)), or chemically conjugated Ch5D6- CG1 (Ch5D6-CGl(C)). FIG. 8A shows tumor killing by CGI -specific cytotoxic T lymphocytes, and FIG. 8B shows tumor killing by CGI -CART cells. The effectortarget ratio (ratio of CGI -specific CTL or CAR to tumor cell) is shown on the x axis.

[0039] FIG. 9 is a graph demonstrating that NLV-specific T cell receptor engineered T (“TCRT”) cells kill breast cancer cells that have been treated with a Ch5D6-NLV antibody-Attorney Docket No. 090723-1530315-MDA25-028PCT peptide conjugate. The tumor cells were untreated (-) or treated with Ch5D6 antibody, Ch5D6 antibody recombinantly conjugated with two copies of the NLV peptide (Ch5D6-NLV.2x), or Ch5D6 antibody recombinantly conjugated with two copies of the NLV peptide (Ch5D6- NLV. lx). The effectortarget ratio (ratio of NLV-specific TCRT cell to tumor cell) is shown on the x axis.

[0040] FIGS. 10A-10B are graphs demonstrating that NLV-specific TCRT cells kill lung and renal cancer cells that have been treated with a Ch5D6-NLV antibody-peptide conjugate. The lung cancer cells (FIG. 10A) and renal cancer cells (FIG. 10B) were untreated (-) or treated with Ch5D6 antibody or Ch5D6 antibody recombinantly conjugated with a single NLV peptide (Ch5D6-NLV.lx). The effectortarget ratio (ratio of NLV-specific TCRT cell to tumor cell) is shown on the x axis.

[0041] FIG. 11 is a schematic diagram of the experimental design to assess the in vivo NLV- specific TCRT cell killing of cancer cells that have been treated with Ch5D6-NLV. MDA-MB- 231 -luciferase cells were pre-incubated ex vivo with Ch5D6-NLV before i.v. injection into NSG mice.

[0042] FIGS. 12A-12B show results from the in vivo experiment depicted in FIG. 11, demonstrating that tumor cells were eliminated within 7 days in mice injected with tumor cells that had been pretreated with Ch5D6-NLV antibody and treated with NLV-specific TCRT cells. FIG. 12A shows bioluminescence images of mice from the experiment taken on days 0, 7, and 14. The MDA-MB-231 -luciferase cells were pre-incubated with PBS (negative control), Ch5D6, or Ch5D6-NLV before injection. The mice were untreated or treated with TCRT cells as indicated. FIG. 12B is a graph of the total flux over time.

[0043] FIGS. 13A-13B are graphs showing the phenotypic characterization of NLV-specific TCRT cells used in the in vivo experiment depicted in FIG. 11. The hCD45 cells as a percentage of total CD45 cells are shown in FIG. 13A, and the tetramer positive cells as a percentage of total hCD45 are shown in FIG. 13B.

[0044] FIG. 14 is a schematic diagram of the experimental design to assess in vivo NLV- specific TCRT cell killing of cancer cells treated with Ch5D6-NLV. In this experiment, MDA- MB-231 -luciferase cells were not pre-incubated ex vivo with Ch5D6-NLV antibody before i.v. injection into NSG mice.

[0045] FIG. 15 show bioluminescence images from the in vivo experiment depicted in FIG. 14, demonstrating that tumor cells were eliminated within 7 days in mice dosed with Ch5D6- NLV and treated with NLV-specific TCRT cells. The images were taken on days 0, 7, 14, andAttorney Docket No. 090723-1530315-MDA25-028PCT21. The mice were dosed with Ch5D6 or Ch5D6-NLV after the injection of the tumor cells and were infused with TCRT cells as indicated.

[0046] FIGS. 16A-16B show phenotypic characterization of the NLV-specific TCRT cells used in the in vivo experiment depicted in FIG. 15. The hCD45 cells as a percentage of total CD45 cells are shown in FIG. 16A, and the tetramer positive cells as a percentage of total hCD45 are shown in FIG. 16B.

[0047] FIGS. 17A-17B show that tumor cell elimination was dependent on NLV-specific TCRT cell dosage. The experiment was performed as shown in FIG. 15. The mice were dosed with either Ch5D6 or Ch5D6-NLV, and the mice were treated with 5 x 106or 1 x 106TCRT cells as indicated in FIG. 17A. The mice treated with 5 x 106TCRT cells showed greater decrease in tumor volume than mice treated with 1 x 106TCRT cells. FIG. 17B is a graph of the total flux over time.

[0048] FIGS. 18A-18B are graphs that show phenotypic characterization of NLV-specific TCRT cells in both 5 x 106TCRT cells and 1 x 106TCRT cells dosage groups from the experiment of FIGS. 17A-17B. The hCD45 cells as a percentage of total CD45 cells are shown in FIG. 18A, and the tetramer positive cells as a percentage of total hCD45 are shown in FIG. 18B. In each graph, at each time point, the left bar is for mice treated with Ch5D6 and 1 x 106TCRT cells; the middle bar is for mice treated with Ch5D6-NLV and 5 x 106TCRT cells; and the right bar is for mice treated with Ch5D6-NLV and 1 x 106TCRT cells.

[0049] FIG. 19 is a schematic diagram of the experimental design to assess in vivo CG1- CAR T cell killing of cancer cells treated with Ch5D6-CGl. MDA-MB-231 -luciferase cells were pre-incubated ex vivo with Ch5D6-CGl antibody before i.v. injection into NSG mice.

[0050] FIGS. 20A-20B show that tumor volume was decreased in mice which received MDA-MB-231 cells incubated with Ch5D6-CGl antibody and were treated with CGI CAR-T cells.

[0051] FIGS. 21A-21B show the results of in vivo experiments repeated with a second cancer cell line, H2023 (non-small cell lung cancer cell line). FIG. 21A shows bioluminescence images from the in vivo experiment depicted in FIG. 14, demonstrating that, within 14 days, mice dosed with Ch5D6-NLV and treated with NLV-specific TCRT cells showed a significant reduction in tumor as compared to the control group. The images were taken on days 0, 9, 14, 21, and 28. The mice were dosed with Ch5D6 (left images) or Ch5D6- NLV (right images) after the injection of the tumor cells and were infused with TCRT cells as indicated. FIG. 21B is a graph of the total flux over time.Attorney Docket No. 090723-1530315-MDA25-028PCTDETAILED DESCRIPTION

[0052] The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.I. Introduction

[0053] Neuropilins are multifunctional cell surface receptors highly conserved in vertebrates. Neuropilin subtype 1 (NRP1) is a transmembrane receptor involved in blood vessel, nerve, and tumor development. NRP1 was originally observed on neurons, but was later found to be expressed by several cell types, including osteoblasts, nerve cells, immune cells, adipocytes, stromal cells, and endothelial cells. NRP1 is also expressed by tumor cells of many cancer types (e.g., breast, renal, lung, prostate) and promotes malignant phenotypes through supporting cell viability, proliferation, migration, metastasis, and cancer cell sternness. NRP1 acts as a co-receptor for multiple ligands, including vascular endothelial growth factor (“VEGF”) family proteins and class 3 and 4 semaphorins (“SEMA3” and “SEMA4” respectively). Vascular endothelial growth factor A (“VEGF A”), in particular, is known to promote tumor cell proliferation through NRP1 interactions. VEGFA-NRP1 interaction leads to downstream guanosine triphosphate binding and activation of Ras homolog gene family, member A, promoting tumor cell proliferation (Liu et al., Chin Med. J., 134(15): 508-517 (2020)). The discovery and development of novel antibodies against NRP1, including chimeric antibodies, as described in this disclosure can be used in the diagnosis and treatment of NRP1 positive malignancies.

[0054] This disclosure provides antibodies and antigen-binding fragments thereof that bind specifically to NRP1. The disclosure also provides antibody-peptide conjugates comprising an NRP1 antibody or antigen-binding fragment thereof and an immunogenic peptide. Also provided herein are various compositions of such antibodies, antigen-binding fragments, or antibody-peptide conjugates; nucleic acids encoding the antibodies, antigen-binding portions, or antibody-peptide conjugates; and associated methods of use. The disclosed NRP1 antibodies are capable of high NRP1 binding affinity and blocking NRP1 ligand (e.g., SEMA3A and VEGF) binding. In addition, the disclosed NRP1 antibody-peptide conjugates are capable of increasing the immune recognition of NRP1 positive tumor cells. In particular, this technologyAttorney Docket No. 090723-1530315-MDA25-028PCT allows the tumors expressing NRP1 to be specifically targeted. The antibody-peptide conjugate is internalized and releases the peptide antigens (e.g. viral, bacterial, non-self) which will be presented by MHC-I and recognized by circulating antigen-specific T cells that pre-exist in the host. The antibodies, antigen-binding fragments, antibody-peptide conjugates, and associated methods provided herein represent a novel approach for treating patients with NRP1 positive malignancies. Thus, also provided herein are methods of using the antibody, antibody fragment, and antibody-peptide conjugate compositions of this disclosure for diagnostic and / or therapeutic purposes, as well as for detecting NRP1 protein.II. Terminology

[0055] A number of terms and concepts are discussed below. They are intended to facilitate the understanding of various embodiments of the present disclosure in conjunction with the rest of the present document and the accompanying figures. These terms and concepts may be further clarified and understood based on the accepted conventions in the fields of the present disclosure, as well as the description provided throughout the present document and / or the accompanying figures. Some other terms can be explicitly or implicitly defined in other sections of this document and in the accompanying figures and may be used and understood based on the accepted conventions in the fields of the present disclosure, the description provided throughout the present disclosure, and / or the accompanying figures. The terms not explicitly defined can also be defined and understood based on the accepted conventions in the fields of the present disclosure and interpreted in the context of the present document and / or the accompanying figures.

[0056] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0057] Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0058] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certainAttorney Docket No. 090723-1530315-MDA25-028PCT elements are also contemplated as “consisting essentially of and “consisting of those certain elements. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0059] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, e.g., In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0060] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0061] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20%; preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Alternatively, in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5- fold, and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0062] As used throughout, the terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide,” “oligonucleotide,” “nucleotides,” or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogsAttorney Docket No. 090723-1530315-MDA25-028PCT thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20, 50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A polynucleotide described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. The following are nonlimiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the doublestranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” or the like is the alphabetical representation of a polynucleotide molecule. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0063] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof, alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0064] The term “identity” or “substantial identity,” as used in the context of a polynucleotide or polypeptide sequence described herein, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be anyAttorney Docket No. 090723-1530315-MDA25-028PCT integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of ordinary skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0065] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0066] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0067] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood wordAttorney Docket No. 090723-1530315-MDA25-028PCT hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Set. USA 89:10915 (1989)).

[0068] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10'5, and most preferably less than about IO'20.

[0069] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0070] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoi somers of the naturally occurring amino acids, unnatural amino acids and chemically modified amino acids. Unnatural amino acids (that is, those that are not naturally found in proteins) are also known in the art, as set forth in, for example, Zhang et al. “Protein engineering with unnatural amino acids,” Curr. Opin. Struct. Biol. 23(4): 581- 587 (2013); Xie et la. “Adding amino acids to the genetic repertoire,” 9(6): 548-54 (2005)); and all references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as unnatural amino acids.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0071] As used herein, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, a side chain can be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel. A side chain can also be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group. Post- translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0072] As used herein, “NRP1” refers to neuropilin subtype 1, a transmembrane receptor involved in blood vessel, nerve, and tumor development. NRP1 is expressed by several cell types, including osteoblasts, nerve cells, immune cells, adipocytes, stromal cells, endothelial cells, and tumor cells.III. Antibodies

[0073] In one aspect, the present disclosure provides antibodies and antigen-binding portions thereof that bind specifically to NRP1 (also known as CD304, BDCA-4, NP1, Nrp, and VEGF165R). As used herein, the term “antibody” encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class. Native antibodies are usually heterotetrametric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH or VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL or VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (1), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. As used herein, the term antibody also encompasses an antibody fragment, for example, an antigen-binding fragment. Antigen-Attorney Docket No. 090723-1530315-MDA25-028PCT binding fragments comprise at least one antigen-binding domain. One example of an antigenbinding domain is an antigen-binding domain formed by a VH-VL dimer. Antibodies and antigen-binding fragments thereof can be described by the antigen to which they specifically bind. For example, as used herein, the terms “NRP1 antibody” and “anti-NRPl antibody” both refer to an antibody or fragment thereof that specifically binds NRP1.

[0074] The term “variable” is used herein to describe certain portions of the antibody domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a P-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the P-sheet structure. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. Each VH and VL generally comprises three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in antigen-binding and confer antigen specificity and binding affinity to the antibody. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD.) CDR sequences on the heavy chain (VH) may be designated as CDRH1, CDRH2, and CDRH3 (alternatively as VHCDR1, VHCDR2, and VHCDR3), while CDR sequences on the light chain (VL) may be designated as CDRL1, CDRL2, and CDRL3 (alternatively as VLCDR1, VLCDR2, and VLCDR3).

[0075] The term “epitope,” as used herein, means a component of an antigen capable of specific binding to an antibody or antigen-binding fragment thereof. Such components optionally comprise one or more contiguous amino acid residues and / or one or more noncontiguous amino acid residues. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter isAttorney Docket No. 090723-1530315-MDA25-028PCT lost in the presence of denaturing solvents. An epitope can comprise amino acid residues that are directly involved in the binding, and other amino acid residues, which are not directly involved in the binding. The epitope to which an antigen-binding protein binds can be determined using known techniques for epitope determination such as, for example, testing for antigen-binding protein binding to antigen variants with different point mutations.

[0076] As used herein, the terms “binds specifically to,” “specifically binds to,” “specific for,” “targets,” “binds selectively to,” and “selective for” NRP1 or an isoform or an epitope of a NRP1 protein, and the like, mean binding that is measurably different from a non-specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that is similar to the target, such as an excess of non-labeled target. In that case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by the excess non-labeled target.

[0077] Provided herein are antibodies and antigen-binding portions thereof that bind specifically to NRP1. The NRP1 antibodies and antigen-binding portions thereof are polypeptides. As used herein, the terms “antigen-binding portion” and “antigen-binding fragment” are used interchangeably to refer to a portion of an antibody polypeptide sequence that binds specifically to NRP1. NRP1 -specific antibodies were identified and tested as described in the Examples below. In some embodiments, the antibodies and antigen-binding portions thereof provided herein may be a chimeric antibody and antigen-binding portions thereof.

[0078] In some embodiments, heavy chain variable region sequences and light chain variable region sequences of the isolated antibody or antibody fragment are one or more of the sequences as set forth in Table 1. The CDR sequences in the variable domains listed in Table 1 are indicated by bold text. In some embodiments, the heavy chain variable region is encoded by a nucleotide sequence that can be translated to produce a polypeptide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to SEQ ID NOs: 1-5. In some embodiments, the light chain variable region is encoded by a nucleotide sequence that can be translated to produce a polypeptide having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NOs: 6-10. In certain embodiments, the isolated antibody or antibody fragment comprises: a heavy chain variable region (VH) having at least 85% sequence identity to SEQ ID NOs: 1-5 and a light chain variable region (VL) having at least 85% sequence identity to SEQ ID NOs: 6-10.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0079] In some embodiments, the heavy chain variable region is a polypeptide sequence having at least 86% identity (e.g., 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1. In some embodiments, the heavy chain variable region is a polypeptide sequence having at least 86% identity (e.g., 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 2. In some embodiments, the heavy chain variable region is a polypeptide sequence having at least 90% identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3. In some embodiments, the heavy chain variable region is a polypeptide sequence having at least 87% identity (e.g., 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4. In some embodiments, the heavy chain variable region is a polypeptide sequence having at least 97% identity (e.g., 98% or 99% identical) to SEQ ID NO: 5.

[0080] In some embodiments, the light chain variable region is a polypeptide sequence having at least 97% identity (e.g., 98% or 99% identical) to SEQ ID NO: 6. In some embodiments, the light chain variable region is a polypeptide sequence having at least 98% identity (e.g., 99% identical) to SEQ ID NO: 7. In some embodiments, the light chain variable region is a polypeptide sequence having at least 99% identity to SEQ ID NO: 8. In some embodiments, the light chain variable region is a polypeptide sequence comprising SEQ ID NO: 9. In some embodiments, the light chain variable region is a polypeptide sequence having at least 97% identity (e.g., 98% or 99% identical) to SEQ ID NO: 10.

[0081] In certain embodiments, the isolated antibody or antibody fragment comprises: a heavy chain variable region (VH) having at least 86% identity (for example, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1 and a light chain variable region (VL) having at least 97% identity (for example, 98% or 99% identical) to SEQ ID NO: 6. In certain embodiments, the isolated antibody or antibody fragment comprises: a VH having at least 86% identity (for example, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 2 and a VL having at least 98% identity (for example, 99% identical) to SEQ ID NO: 7. In certain embodiments, the isolated antibody or antibody fragment comprises: a VH having at least 90% identity (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3 and a VL having at least 99% identity to SEQ ID NO: 8. In certain embodiments, the isolated antibody or antibody fragment comprises: a VH having at least 87% identity (for example, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9. In certain embodiments, the isolated antibody orAttorney Docket No. 090723-1530315-MDA25-028PCT antibody fragment comprises: a VH having at least 97% identity (for example, 98%, or 99% identical) to SEQ ID NO: 5 and a VL having at least 97% identity (for example, 98% or 99% identical) to SEQ ID NO: 10.

[0082] In certain embodiments, the isolated antibody or antibody fragment comprises: a VH comprising SEQ ID NO: 1 and a VL comprising SEQ ID NO: 6. In certain embodiments, the isolated antibody or antibody fragment comprises: a VH comprising SEQ ID NO: 2 and a VL comprising SEQ ID NO: 7. In certain embodiments, the isolated antibody or antibody fragment comprises: a VH comprising SEQ ID NO: 3 and a VL comprising SEQ ID NO: 8. In certain embodiments, the isolated antibody or antibody fragment comprises: a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9. In certain embodiments, the isolated antibody or antibody fragment comprises: a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 10.Table 1. Variable region amino acid sequences of select NRPl-antibodies.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0083] In some embodiments, the antibody or antigen-binding fragment thereof as provided in this disclosure has a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences listed by row in Table 2. In some embodiments, the antibody or antigen-binding fragment thereof has a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences listed by row in Table 2. In some embodiments, the antibody or antigen-binding fragment thereof has a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences and a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences listed by row in Table 2.Table 2. Complementarity determining regions of select NRPl-antibodies.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0084] In some embodiments, provided is an isolated antibody or antigen-binding fragment, comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11, 14, or 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12, 15, or 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13, 16, 18, or 21; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22, 25, 30, or 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23, 26, 28, or 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24, 27, 29, or 32.

[0085] In some embodiments, provided herein is an isolated antibody or antigen-binding fragment, comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0086] In some embodiments, the isolated antibody or antigen-binding fragment comprises: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 14; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 15; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 16; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 25; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 26; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 27.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0087] In some embodiments, the isolated antibody or antigen-binding fragment comprises: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 18; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 29.

[0088] In some embodiments, the isolated antibody or antigen-binding fragment comprises: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 20; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 21; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 30; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 32.

[0089] In some embodiments, provided is an isolated antibody or antigen-binding fragment, comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0090] In some embodiments, provided is an isolated antibody or antigen-binding fragment, comprising: (a) a VH having at least 86% identity (for example, at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1 and comprising (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL having at least 97% identity (for example, at least 98%, or 99% identical) to SEQ ID NO: 6 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0091] In some embodiments, provided is an isolated antibody or antigen-binding fragment, comprising: (a) a VH having at least 86% identity (for example, at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 2 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 14; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 15; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 16; and (b) a VL having at least 98% identity (for example, at least 99% identical) to SEQ ID NO: 7 and comprising (i) a CDRL1 amino acid sequence comprisingAttorney Docket No. 090723-1530315-MDA25-028PCTSEQ ID NO: 25; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 26; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 27.

[0092] In some embodiments, provided is an isolated antibody or antigen-binding fragment, comprising: (a) a VH having at least 90% identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 18; and (b) a VL having at least 99% identity to SEQ ID NO: 8 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 29.

[0093] In some embodiments, provided is an isolated antibody or antigen-binding fragment, comprising: (a) a VH having at least 87% identity (for example, at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4 and comprising(i) a CDRH1 amino acid sequence comprising SEQ ID NO: 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 20; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 21; and (b) a VL comprising SEQ ID NO: 9 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 30; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 32.

[0094] In some embodiments, provided is an isolated antibody or antigen-binding fragment, comprising: (a) a VH having at least 97% identity (for example, at 98%, or 99% identical) to SEQ ID NO: 5 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11;(ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL having at least 97% identity (for example, at least 98%, or 99% identical) to SEQ ID NO: 10 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0095] In certain embodiments, the isolated antibody or antibody fragment comprises: a heavy chain variable region (VH) having at least 86% identity (for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1 and a light chain variable region (VL) having at least 97% identity (for example, 98% or 99% identical) to SEQ ID NO: 6, for instance as found in antibody 5D6 as described herein.

[0096] In certain embodiments, the isolated antibody or antibody fragment comprises: a VH having at least 86% identity (for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,Attorney Docket No. 090723-1530315-MDA25-028PCT95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 2 and a VL having at least 98% identity (for example, 99% identical) to SEQ ID NO: 7, for instance as found in antibody 7E2 as described herein.

[0097] In certain embodiments, the isolated antibody or antibody fragment comprises: a VH having at least 90% identity (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3 and a VL having at least 99% identity to SEQ ID NO: 8, for instance as found in antibody 12A10 as described herein.

[0098] In certain embodiments, the isolated antibody or antibody fragment comprises: a VH having at least 87% identity (for example, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9, for instance as found in antibody 13C7 as described herein.

[0099] In certain embodiments, the isolated antibody or antibody fragment comprises: a VH having at least 97% identity (for example, 97%, 98%, or 99% identical) to SEQ ID NO: 5 and a VL having at least 97% identity (for example, 97%, 98% or 99% identical) to SEQ ID NO: 10, for instance as found in antibody 15D2 as described herein.

[0100] In each case, where a specific amino acid sequence is recited, embodiments comprising a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided.

[0101] As with all peptides, polypeptides, and proteins, including fragments thereof, it is understood that additional modifications in the amino acid sequence of the NRP1 -specific antibodies or antigen-binding fragments thereof described herein, for example, in the heavy chain variable region and / or light chain variable region, can occur that do not alter the nature or function of the antibodies or antigen-binding fragments thereof. Such modifications include conservative amino acids substitutions, such that each recited sequence optionally contains one or more conservative amino acid substitutions. The list provided below identifies groups that contain amino acids that are conservative substitutions for one another; these groups are exemplary as other conservative substitutions are known to those of skill in the art:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); andAttorney Docket No. 090723-1530315-MDA25-028PCT8) Cysteine (C), Methionine (M).

[0102] By way of example, when an aspartic acid at a specific residue is mentioned, also contemplated is a conservative substitution at the residue, for example, glutamic acid. Nonconservative substitutions, for example, substituting a proline with glycine or substituting a lysine with an asparagine, are also contemplated.

[0103] Methods of generating and screening for antibodies and antigen-binding fragments thereof as provided in this disclosure are described in the Examples and are well-known in the art. Methods of further modifying antibodies for enhanced properties (e.g., enhanced affinity, chimerization, humanization) as well as generating antigen-binding fragments, as described herein, are also well-known in the art.

[0104] In some embodiments, the heavy chain variable region and / or the light chain variable region of the isolated antibody or antigen-binding fragment has an identical sequence to the heavy chain variable region and / or the light chain variable region of the antibody produced by the methods described herein and, in the Examples below. In some embodiments, the heavy chain variable region and / or the light chain variable region of the isolated antibody or antigenbinding fragments comprises one or more modifications, e.g., amino acid substitutions, deletions, or insertions.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof provided herein can include a heavy (H) chain variable domain sequence (abbreviated herein as VH or VH), and a light (L) chain variable domain sequence (abbreviated herein as VL or VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (sometimes referred to as a half antibody). In another example, and as described further below, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (scFv, for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric antibodies, which may be produced by the modification of whole antibodies or synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to bind specifically to their respective antigen. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies. The preparation of antibody molecules can be monoclonal or polyclonal. An antibody molecule can also be a human, chimeric, humanized, CDR-grafted, or an in vitro generated antibody. The antibody can haveAttorney Docket No. 090723-1530315-MDA25-028PCT a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from either kappa or lambda light chains.

[0106] Antigen-binding fragments of an antibody molecule are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv) (See, e.g., Bird et al., 1988 Science 242:423-26; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83); and (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0107] In some embodiments, the antibody is a monoclonal antibody. As used herein, the term monoclonal antibody refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are the same or substantially similar and that bind the same epitope(s), except for variants that can normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of yeast clones, phage clones, bacterial clones, mammalian cell clones, hybridoma clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the target, for example, by affinity maturation, to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0108] In some embodiments, the antibody or antigen-binding fragment, e.g., a monoclonal antibody, comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin producing mouse B cell, and further comprises a kappa or lambda light chain constant region. In some embodiments, the light chain constant region (kappa or lambda) is from the same type of light chain (i.e., kappa or lambda) as the light chain variable region that was derived from the immunoglobulin producing mouse B cell; as a non-limiting example, if an IgG-producing mouse B cell comprises a kappa light chain, then the monoclonal antibody that is produced can comprise the light chain variableAttorney Docket No. 090723-1530315-MDA25-028PCT region from the IgG-producing mouse B cell and further comprises a kappa light chain constant region.

[0109] In some embodiments, the antibody or antigen-binding fragment, e.g., a monoclonal antibody, comprises a heavy chain variable region sequence and a light chain variable region sequence that are derived from an immunoglobulin-producing mouse B cell, and further comprises a heavy chain constant region having an IgG isotype (e.g., IgG2 or IgGl), an IgA isotype, an IgM isotype, an IgD isotype, an IgE isotype, or that is derived from an IgG, IgA, IgM, IgD, or an IgE isotype e.g., is a modified IgG2 or IgGl constant region). It will be appreciated by a person of ordinary skill in the art that the different heavy chain isotypes (IgA, IgD, IgE, IgG, and IgM) have different effector functions that are mediated by the heavy chain constant region, and that for certain uses it may be desirable to have an antibody that has the effector function of a particular isotype (e.g., IgG).

[0110] The present disclosure also provides chimeric antibodies. The term chimeric antibody refers to an antibody in which a component of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In some embodiments, the heavy chain variable region and the light chain variable region are derived from a mouse antibody, and the heavy chain constant region and the light chain constant region are derived from a human immunoglobulin producing B cell.[OHl] In some embodiments, the chimeric NRP1 antibody comprises the variable region sequences as described herein (e.g., as disclosed in Tables 1 and 2) and further comprises a heavy chain constant region and / or a light chain constant region that is heterologous to the antibody produced by the methods described herein and, in the Examples, below from which the CDR sequences and / or variable region sequences are derived. For example, in some embodiments, the chimeric NRP1 antibody comprises the variable region sequences of an antibody produced by the methods described herein and in the Examples below, and further comprises a heavy chain constant region and a light chain constant region that is heterologous to the antibody produced by the methods described herein and in the Examples below (e.g., the heavy chain constant region and / or light chain constant region is a wild-type or modified IgGl, IgG2, IgG3, or IgG4 constant region).

[0112] In some embodiments, the antibody or antigen-binding fragment, e.g., a chimeric antibody, comprises a mouse heavy chain variable region and mouse light chain variable region and a native (i.e., wild-type) human IgG, IgA, IgM, IgD, or IgE constant region. In some embodiments, the chimeric antibody comprises a mouse heavy chain variable region and mouseAttorney Docket No. 090723-1530315-MDA25-028PCT light chain variable region and a native human IgGl constant region, a native human IgG2 constant region, a native human IgG3 constant region, a native human IgG4 constant region, a native human IgAl constant region, a native human IgA2 constant region, a native human IgM constant region, or a native human IgD constant region. In some embodiments, the chimeric antibody comprises a mouse heavy chain variable region and mouse light chain variable region, and constant region derived from a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, a human IgG4 constant region, a human IgAl constant region, a human IgA2 constant region, a human IgM constant region, a native human IgD constant region, or a human IgE constant region. In some embodiments, the antibody or antigen-binding fragment, e.g., a chimeric antibody, comprises a heavy chain constant region that comprises one or more modifications (e.g., a S239C mutation in the CH2 region). It will be appreciated by a person of ordinary skill in the art that modifications such as amino acid substitutions can be made at one or more residues within the heavy chain constant region that modulate effector function. In some embodiments, the modification reduces effector function, e.g., results in a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on an effector cell that mediates the effector function. In some embodiments, the modification (e.g., amino acid substitution) prevents in vivo Fab arm exchange, which can introduce undesirable effects and reduce the therapeutic efficacy of the antibody. See, e.g., Silva et al., 2015, J. Biol. Chem. 280:5462-69.

[0113] In some embodiments, the antibody or antigen-binding fragment, e.g., a chimeric antibody, comprises a human IgGl constant region that is derived from IgGl, IgG2, IgG3, or IgG4, and comprises one or more modifications that modulate effector function (e.g., a S259C mutation in the CH2 region). In some embodiments, the antibody or antigen-binding fragment, e.g., a chimeric antibody, comprises a human IgG constant region that is derived from IgGl, IgG2, IgG3, or I

[0114] In some embodiments, the antibody with specified CDRs is an allotype other than the allotype(s) found associated with the antibodies produced by the methods described herein and, in the Examples, below. The antibody may comprise an allotype selected from those listed in Table 3 below.Atorney Docket No. 090723-1530315-MDA25-028PCTT able 3. Human immunoglobulin allotypes.Isotype / type Heavy chains Light chainsIgGl IgG2 IgG3 IgAAllotypes Glm G2m G3m A2m Km1(a) 23(n) 21(gl) 1 12(x) 28(g5) 2 23(f) l l(bO) 317(z) 5(bl)13 (b3)14 (b4) 10 (b5) 15(s) 16(t) 6(c3) 24(c5)26(u) 27 (\ )NB: Alphabetical notation given within brackets. From: Jefferis and Marie-Paule Lefranc, 2009, “Human immunoglobulin allotypes: Possible implications for immunogenicity” mAbs 1(4): 332- -38, incorporated herein by reference . gG4.

[0115] Antibody molecules can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, rat, guinea, pig, human, camel, llama, fish, shark, goat, rabbit, and bovine. Single domain antibodies are described, for example, in International Application Publication No. WO 94 / 04678. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species (e.g., camel, llama, dromedary, alpaca, and guanaco) or other species besides Camelidae.

[0116] In some embodiments, an antigen-binding fragment can also be or can also comprise, e.g., a non-antibody, scaffold protein. These proteins are generally obtained through combinatorial chemistry-based adaptation of preexisting antigen-binding proteins. For example, the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. See, e.g., Ali et al., 1999, J. Biol. Chem.Attorney Docket No. 090723-1530315-MDA25-028PCT274:24066-73. The portion of human transferrin not involved with binding the receptor remains unchanged and serves as a scaffold, like framework regions of antibodies, to present the variant binding sites. The libraries are then screened, as an antibody library is screened, and in accordance with the methods described herein, against a target antigen of interest to identify those variants having optimal selectivity and affinity for the target antigen. See, e.g., Hey et al., 2005, TRENDS Biotechnol. 23(10):514-522.

[0117] One of ordinary skill in the art would appreciate that the scaffold portion of the nonantibody scaffold protein can include, e.g., all or part of the Z domain of S. aureus protein A, human transferrin, human tenth fibronectin type III domain, kunitz domain of a human trypsin inhibitor, human CTLA-4, an ankyrin repeat protein, a human lipocalin (e.g., anticalins, such as those described in, e.g., International Application Publication No. WO2015 / 104406), human crystallin, human ubiquitin, or a trypsin inhibitor from E. elaterium.

[0118] In some embodiments, provided are chimeric antibodies, bispecific antibodies (BsAbs), trispecific or other multi-specific antibodies, or bispecific T cell engager (BiTE), that comprise an anti-NRPl antibody or antigen-binding fragment thereof as described in this disclosure. In some embodiments, the NRP1 -specific antibody or antigen-binding fragment thereof is conjugated to a moiety that specifically binds to an immune cell. In some embodiments, provided is a bispecific antibody comprising an NRP1 -specific antibody or antigen-binding fragment thereof as described herein and an antibody or antigen-binding fragment thereof that specifically binds to an immune cell. In some embodiments, the bispecific antibody comprises an NRP1 -specific antibody or antigen-binding portion thereof and an antibody moiety that specifically binds to T cells. Such a molecule is referred to as a bispecific T cell engager and may induce T cell-mediated cytotoxicity of NRP1 -expressing cancer cells (see, e.g., Zhou et al., 2021, Biomarker Research 9:38). In some embodiments, the bispecific antibody comprises an NRP1 -specific antibody or antigen-binding portion thereof and an antibody moiety that specifically binds to natural killer cells (NK cells). Such a molecule is referred to as a NK cell engager and may induce NK cell-mediated cytotoxicity of NRP1- expressing cancer cells (see, e.g., Demaria et al., 2021, European Journal of Immunology 51(8): 1934-1942).

[0119] In some embodiments, the antibody comprises a NRP1 -specific antibody or antigenbinding fragment thereof as described in this disclosure and an anti-CD3 antibody (i.e. bispecific or multi-specific NRPlxCD3antibodies). Such bispecific or multi-specific NRPlxCD3 antibodies act by simultaneous binding to a tumor-associated antigen (TAA) expressed on tumor cells (i.e. NRP1) and to CD3 on a T cell. Crosslinking of these two cellAttorney Docket No. 090723-1530315-MDA25-028PCT types by the antibody composition allows the formation of an immunological synapse, similar to that of a natural T-cell receptor (TCR) / peptide-major histocompatibility complex (MHC) complex. This synapse results in T-cell activation and thereby the secretion of inflammatory cytokines and cytolytic molecules that are able to kill the tumor cells in the process. It is generally thought that any T cell can serve as an effector cell, regardless of TCR specificity, as TCR signaling for such bispecific or multi-specific CD3 antibodies does not require engagement of the antigen-binding domain of the TCR, but is initiated via CD3 (see Middelburg J. et al., 2021, Overcoming Challenges for CD3-Bispecific Antibody Therapy in Solid Tumors, Cancers (Basel). Vol. 13, Issue 2, Article 287). In some embodiments, provided are NRPlxCD3 bispecific antibodies comprising an anti-CD3 antibody, for example, an anti- CD3s antibody. In some embodiments, the anti-CD3 antibody has a sequence as set forth in SEQ ID NO: 38 below.EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRS KYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFG DSYVSWFAYWGQGTLVTVSS / GKPGSGKPGSGKPGSGKPGSQAVVTQEPSLTVS PGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIGGTNKRAPGVPARFSGS LLGGKAALTISGAQPEDEADYYCALWYSNHWVFGGGTKLTV (SEQ ID NO: 38)

[0120] Any of the NRP1 -specific antibodies or antigen-binding fragments thereof described herein can be modified with covalent and / or non-covalent modifications. Such modifications can be introduced into the antibodies or antigen-binding fragments by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the antibodies or antigen-binding fragments. Recombinant techniques can be used to modify antibodies or antigen-binding fragments thereof. For example, amino acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity. Insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antigen-binding fragment is not significantly altered or impaired compared to the non-modified antibody, or antigen-binding fragment thereof can be made. Such methods are readily apparent to a skilled practitioner in the art and can include site specific mutagenesis of the nucleic acid encoding the antibody or antigen-binding fragment thereof. (Zoller et al., 1982, Nucl. Acids Res. 10:6487-500). In some instances, the NRPl-specific antibodies or antigen-binding fragments may be labeled by a variety of means for use in diagnostic and / or pharmaceutical applications.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0121] In some embodiments, the antibodies or antigen-binding fragments thereof can be conjugated to a heterologous moiety. The heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), a photosensitizer, or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin. In some embodiments, the heterologous moiety is an antibody or antigen-binding fragment thereof that specifically binds to a different target, and such a conjugated antibody is a type of antibody composition that can be referred to as a bispecific antibody. Additional suitable heterologous polypeptides include, e.g., an antigenic tag (e.g., FLAG), polyhistidine, glutathione-S- transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibodies or antigen-binding fragments. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). A suitable photosensitizer (also referred to as a photoabsorber is, for example, IRDye700DX (IR700). Suitable radioactive labels include, e.g.,32P,33P,14C,125I,1311,35S, and3H. Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of di ethylene triamine pentaacetic acid (DTP A) or tetraazacyclododecane-l,4,7, 10-tetraacetic acid (DOTA). Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase. Another labeling technique which may result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically altered by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific antihapten antibodies. Additional acceptable heterologous moieties are described below in Section V.

[0122] In some instances, the NRP1 -specific antibody or antigen-binding fragment thereof may be conjugated to an imaging agent. For example, the NRP1 -specific antibody or antigenbinding fragment thereof may be labelled for use in radionuclide imaging. In particular, the agent may be directly or indirectly labelled with a radioisotope. Examples of radioisotopes that may be used are:277Ac,211At,128Ba,131Ba,7Be,204Bi,205Bi,206Bi,76Br,77Br,82Br,109Cd,47Ca,nC,14C,36C1,48Cr,51Cr,62Cu,64Cu,67Cu,165Dy,155Eu,18F,153Gd,66Ga,67Ga,68Ga,72Ga,Attorney Docket No. 090723-1530315-MDA25-028PCT198Au,3H166HO,mIn,113mIn,115mIn,123I,125I,1311,189Ir,191mIr,192Ir,194Ir,52Fe,55Fe,59Fe,177Lu,150,191m'1910s,109Pd,32P,33P,42K,226Ra,186Re,188Re,82mRb,153Sm,46Sc,47Sc,72Se,75Se,105Ag,22Na,24Na,89Sr,35S,38S,177Ta,96Tc, "mTc,2O1T1,2O2T1,113Sn,117mSn,121Sn,166Yb,169Yb,175Yb,88Y,90Y,62Zn and65Zn. Preferably the radioisotope is131I,125I,123I,inI, "mTc,90Y,186Re,188Re,32P,153Sm,67Ga,2O1T1,77Br, or18F, and is imaged with a photoscanning device. Procedures for labeling biological agents with the radioactive isotopes are generally known in the art.

[0123] Two proteins (e.g., an antibody and a heterologous moiety) can be cross-linked using any of a number of known chemical cross linkers. Examples of such cross linkers are those that link two amino acid residues via a linkage that includes a “hindered” disulfide bond. In these linkages, a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4- succinimidyloxycarbonyl-a-methyl-a(2-pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other. Heterobifunctional reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m- maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate).

[0124] Techniques for conjugating a therapeutic moiety (e.g., any of those discussed in Section V) to an NRP1 -specific antibody or antigen-binding fragment thereof as described herein are well known, see, for example, Amon et aL, 1985, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56; Hellstrom et al., 1987, Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53; Thorpe, 1985, Monoclonal Antibodies '84: :Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506; “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy” In: Monoclonal Antibodies For Cancer Detection And Therapy, (Baldwin et al. eds.), pp. 303-316 (1985), and Thorpe et al., 1982, Immunol. Rev. 62: 119-158. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate (e.g., a bispecific antibody) as described, for example, in U.S. Pat. No. 4,676, 980.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0125] In some embodiments, a radioactive label can be directly conjugated to the amino acid backbone of the antibody. Alternatively, the radioactive label can be included as part of a larger molecule (e.g.,125I in meta-[125I]iodophenyl-N-hydroxysuccinimide ([125I]mIPNHS), which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al., 1997, J. NucL Med. 38: 1221-29) or chelate (e.g., to DOTA or DTP A), which is in turn bound to the protein backbone. Methods of conjugating the radioactive labels or larger molecules / chelates containing them to the antibodies or antigen-binding fragments described herein are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt concentration, and / or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, e.g., U.S. Patent No. 6,001,329).

[0126] Methods for conjugating a fluorescent label (sometimes referred to as a fluorophore) or other heterologous moiety to a protein (e.g., an antibody) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some embodiments, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo- SMCC. Suitable conjugation methods involve incubating an antibody protein or antigenbinding fragment thereof with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly, (2003), Handbook of Radiopharmaceuticals: Radiochemistry and Applications, John Wiley and Sons.

[0127] In some embodiments, the antibodies or antigen-binding fragments can be modified, e.g., with a moiety that improves the stabilization and / or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues. For example, the antibody or antigen-binding fragment can be PEGylated as described in, e.g., Lee et al.* 1999, Bioconjug. Chem. 10(6): 973-78; Kinstler et al., 2002, Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al., 2002, Advanced Drug Delivery Reviews 54:459-476, or HESylated (Fresenius Kabi, Germany) (see, e.g., Pavisic et al., 2010, Int. J. Pharm. 387(1-2): 110-119). The stabilization moiety can improve the stability, or retention of, the antibody (or antigen-binding fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

[0128] In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be glycosylated. In some embodiments, an antibody or antigen-binding fragment thereof described herein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the antibody or antigen-binding fragment has reduced or absent glycosylation.Attorney Docket No. 090723-1530315-MDA25-028PCTMethods for producing antibodies with reduced glycosylation are known in the art and described in, e.g., U.S. Patent No. 6,933,368; Wright etal., 1991, EMBO J. 10(10): 2717-2723; and Co et al., (1993), Mol. Immunol. 30: 1361. An altered glycosylation level may comprise altered fucosylation (See e.g. Golay et al. (2022), Frontiers Immunol. 13:929895, doi: 10.3389 / fimmu.2022.929895) to modulate antibody efficacy. Antibodies may also be partially glycosylated, e.g. partially fucosylated, i.e. having less glycosylation than fully glycosylated antibodies, with certain glycosylation sites being glycosylated and other (potential) glycosylation sites being non-glycosylated.IV. Chimeric antigen receptors

[0129] Also provided herein are chimeric antigen receptors comprising any of the antibodies or antigen-binding fragments described herein. Chimeric antigen receptors (CARs, also known as chimeric T cell receptors) are designed to be expressed in host effector cells, e.g., T cells or NK cells, and to induce an immune response against a specific target antigen and cells expressing that antigen. Adoptive T cell immunotherapy, in which a patient’s own T lymphocytes are engineered to express CARs, has shown great promise in treating hematological malignancies. CARs can be engineered and used as described, for example, in Sadelain et al., 2013, Cancer Discov. 3:388-98. A CAR typically comprises an extracellular target-binding module, a transmembrane (TM) domain, and an intracellular signaling domain (ICD). The CAR domains can be joined via flexible hinge and / or spacer regions. The extracellular target-binding module generally comprises an antibody or antigen-binding fragment thereof. In some instances, multiple binding specificities can be included in the extracellular target-binding module. For example, multiple antibodies or antigen-binding fragments thereof that target different antigens can be included to produce bi-specific, tri- specific, or quad-specific CARs.

[0130] Provided herein are chimeric antigen receptors comprising: (a) an extracellular targetbinding domain comprising an NRP1 -specific antibody or antigen-binding portion thereof; (b) a transmembrane domain; and (c) a signaling domain.

[0131] TM domains are primarily considered a structural requirement, anchoring the CAR in the cell membrane, and are most commonly derived from molecules regulating T cell function, such as CD8 and CD28. The intracellular module typically consists of the T cell receptor CD3(^ chain and one or more costimulatory domains from either the Ig (CD28-like) or TNF receptor (TNFR) superfamilies. CARs containing either CD28 or 4- IBB costimulatoryAttorney Docket No. 090723-1530315-MDA25-028PCT domains have been widely used, to date, and both of them have yielded dramatic responses in clinical trials. CAR domains are discussed in more detail below.

[0132] The extracellular target-binding module of a CAR may comprise an antibody or an antigen-binding fragment thereof that specifically binds a target antigen (e.g., NRP1). In certain embodiments, the extracellular target-binding domain can be a single-chain variable fragment derived from an antibody (scFv), a tandem scFv, a single-domain antibody fragment (VHHS or sdAbs), a single domain bispecific antibody, an intrabody, a nanobody, an immunokine in a single chain format, Fab, Fab’, or (Fab’)2 in a single chain format. In other embodiments, the extracellular target-binding domain can be an antibody moiety that comprises covalently bound multiple chains of variable fragments. The antibody antigen-binding fragment may alternatively comprise a VH-VL dimer that comprises such.

[0133] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising a heavy chain variable region polypeptide sequence having at least 86% identity to SEQ ID NO: 1. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising a heavy chain variable region polypeptide sequence having at least 86% identity to SEQ ID NO: 2. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising a heavy chain variable region polypeptide sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments, a CARNRP1 antigen-binding domain of this disclosure comprises a scFv comprising a heavy chain variable region polypeptide sequence having at least 87% identity to SEQ ID NO: 4. In some embodiments, a CARNRP1 antigen-binding domain of this disclosure comprises a scFv comprising a heavy chain variable region polypeptide sequence having at least 97% identity to SEQ ID NO: 5.

[0134] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising a light chain variable region polypeptide sequence having at least 97% identity to SEQ ID NO: 6. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising a light chain variable region polypeptide sequence having at least 98% identity to SEQ ID NO: 7. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising a light chain variable region polypeptide sequence having at least 99% identity to SEQ ID NO: 8. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising a light chain variable region polypeptide sequence comprising SEQ ID NO: 9. In some embodiments, a CARNRP1 antigen-binding domain of this disclosure comprises a scFvAttorney Docket No. 090723-1530315-MDA25-028PCT comprising a light chain variable region polypeptide sequence having at least 97% identity to SEQ ID NO: 10.

[0135] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a heavy chain variable region (VH) having at least 86% identity to SEQ ID NO: 1 and a light chain variable region (VL) having at least 97% identity to SEQ ID NO: 6. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH having at least 86% identity to SEQ ID NO: 2 and a VL having at least 98% identity (for example, 99% identical) to SEQ ID NO: 7. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH having at least 90% identity to SEQ ID NO: 3 and a VL having at least 99% identity to SEQ ID NO: 8. In some embodiments, a CARNRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH having at least 87% identity to SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH having at least 97% identity to SEQ ID NO: 5 and a VL having at least 97% identity to SEQ ID NO: 10.

[0136] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH comprising SEQ ID NO: 1 and a VL comprising SEQ ID NO: 6. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH comprising SEQ ID NO: 2 and a VL comprising SEQ ID NO: 7. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH comprising SEQ ID NO: 3 and a VL comprising SEQ ID NO: 8. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9. In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 10.

[0137] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11, 14, or 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12, 15, or 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13, 16, 18, or 21; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22, 25, 30, or 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23, 26, 28, or 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24, 27, 29, or 32.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0138] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0139] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 14; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 15; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 16; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 25; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 26; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 27.

[0140] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 18; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 29.

[0141] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 20; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 21; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 30; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 32.

[0142] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 33; (ii) a CDRL2 amino acidAttorney Docket No. 090723-1530315-MDA25-028PCT sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0143] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH having at least 86% identity (for example, at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1 and comprising (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL having at least 97% identity (for example, at least 98%, or 99% identical) to SEQ ID NO: 6 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0144] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH having at least 86% identity (for example, at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 2 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 14; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 15; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 16; and (b) a VL having at least 98% identity (for example, at least 99% identical) to SEQ ID NO: 7 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 25; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 26; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 27.

[0145] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH having at least 90% identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 18; and (b) a VL having at least 99% identity to SEQ ID NO: 8 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 29.

[0146] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH having at least 87% identity (for example, at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 19; (ii) a CDRH2Attorney Docket No. 090723-1530315-MDA25-028PCT amino acid sequence comprising SEQ ID NO: 20; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 21; and (b) a VL comprising SEQ ID NO: 9 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 30; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 32.

[0147] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: (a) a VH having at least 97% identity (for example, at 98%, or 99% identical) to SEQ ID NO: 5 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL having at least 97% identity (for example, at least 98%, or 99% identical) to SEQ ID NO: 10 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0148] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a heavy chain variable region (VH) having at least 86% identity to SEQ ID NO: 1 and a light chain variable region (VL) having at least 97% identity to SEQ ID NO: 6, for instance as found in antibody 5D6 as described herein.

[0149] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH having at least 86% identity to SEQ ID NO: 2 and a VL having at least 98% identity to SEQ ID NO: 7, for instance as found in antibody 7E2 as described herein.

[0150] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH having at least 90% identity to SEQ ID NO: 3 and a VL having at least 99% identity to SEQ ID NO: 8, for instance as found in antibody 12A10 as described herein.

[0151] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH having at least 87% identity to SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9, for instance as found in antibody 13C7 as described herein.

[0152] In some embodiments, a CAR NRP1 antigen-binding domain of this disclosure comprises a scFv comprising: a VH having at least 97% identity to SEQ ID NO: 5 and a VL having at least 97% identity to SEQ ID NO: 10, for instance as found in antibody 15D2 as described herein.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0153] In each case, where a specific amino acid sequence is recited, embodiments comprising a sequence having at least 90% identity to the recited sequence are also provided.

[0154] In some embodiments, the scFv comprises a linker polypeptide between the heavy chain and light chain sequences (e.g., SEQ ID NO: 39 or 40) or any of the other linkers described herein.

[0155] In some embodiments, the extracellular target-binding domains of the CARs provided herein further comprise one or more additional antigen-binding domains (i.e., in addition to the NRP1 -specific antibody or antigen-binding portion thereof, as described above). In some embodiments, the extracellular target-binding domain comprises one additional antigenbinding domain. CARs comprising such an extracellular target-binding domain can be referred to as bi-specific CARs. In some embodiments, the extracellular target-binding domain comprises two additional antigen-binding domains. CARs comprising such an extracellular target-binding domain can be referred to as tri-specific CARs. In some embodiments, the extracellular target-binding domain comprises three additional antigen-binding domain. CARs comprising such an extracellular target-binding domain can be referred to as quad-specific CARs. Each of the one or more additional antigen-binding domains may comprise an antibody or antigen-binding portion thereof. In some embodiments, the one or more additional antigenbinding domains specifically bind to the transferrin receptor.

[0156] The transmembrane (TM) domain of a CAR provided herein may be derived from either a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain is derived from (i.e., comprises at least the transmembrane region(s) of) the a, P, 8, y, or C, chain of the T-cell receptor, CD28, CD3s, CD3(^, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, a transmembrane domain can be chosen based on, for example, the nature of the various other proteins or trans-elements that bind the transmembrane domain or the cytokines induced by the transmembrane domain. In one embodiment, the transmembrane domain is a CD8a transmembrane domain having the sequence set forth in SEQ ID NO: 47. When a transmembrane domain is synthetic, it may comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine may be found at each end of a synthetic transmembrane domain. In some embodiments, a short oligo- or polypeptide linker, having a length of, for example, between about 2 and about 10 (such as about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in length may form the linkage between the transmembrane domain and the intracellular signalingAttorney Docket No. 090723-1530315-MDA25-028PCT domain of a CAR described herein. In some embodiments, the short oligo- or polypeptide linker is a glycine-serine doublet.

[0157] The intracellular signaling domain of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in or is designed to be placed in. An effector function of a T cell may be, for example, cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term “intracellular signaling sequence” is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0158] Examples of intracellular signaling domains for use in the CARs provided herein include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0159] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary signaling sequences) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (costimulatory signaling sequences).

[0160] Primary signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. In some embodiments, the CARs described herein comprise one or more IT AMs.

[0161] Examples of IT AM containing primary signaling sequences that are of particular use in the present disclosure and embodiments thereof include those derived from TCR.^, FcRy, FcRp, CD3y, CD38, CD3s, CD3< CD5, CD22, CD79a, CD79b, and CD66d.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0162] In some embodiments, the CAR comprises a primary signaling sequence derived from CD3(^. For example, the intracellular signaling domain of the CAR can comprise the CD3(^ intracellular signaling sequence by itself or combined with any other desired intracellular signaling sequence(s) useful in the context of the CAR disclosed herein. In some embodiments, the intracellular signaling domain of a CAR provided herein comprises a CD3(^ primary intracellular signaling sequence and a 4- IBB costimulatory signaling sequence. An exemplary CD3(^ sequence is set forth in SEQ ID NO: 44. An exemplary 4-1BB costimulatory signaling sequence is set forth in SEQ ID NO: 43.

[0163] The CARs provided herein may include additional elements, such as a signal peptide to ensure proper export of the fusion protein to the cells surface, a leader sequence (e.g., CD8 leader sequence), and a hinge domain (e.g., CD8 hinge domain) that imparts flexibility to the recognition region and allows strong binding to the targeted moiety. In some embodiments, the CARs provided herein comprise a CD8 hinge domain. An exemplary CD8 hinge domain sequence is set forth in SEQ ID NO: 42. In some embodiments, a spacer domain may be present between any of the domains of the CAR. The spacer domain can be any polypeptide that functions to link two parts of the CAR. A spacer domain may comprise up to about 300 amino acids, including for example about 5 to about 200, about 10 to about 100, or about 25 to about 50 amino acids. Methods of identifying and selecting suitable spacer domains are known in the art.V. Antibody-Peptide Conjugates

[0164] Also provided herein are antibody-peptide conjugates comprising any of the antibodies or antigen-binding fragments described herein and an immunogenic peptide. As used herein, an antibody-peptide conjugate refers to a molecule comprising an antibody and a peptide (e.g., an immunogenic peptide).Immunogenic peptides

[0165] An immunogenic peptide refers to a peptide which interacts with an immune cell to produce an immune response. In some embodiments, the immunogenic peptide comprises a native (i.e., wild-type) cytomegalovirus (NLV) peptide. NLV is an immunodominant peptide derived from amino acids 495-503 of the CMV matrix phosphoprotein pp65. In some embodiments, the immunogenic peptide comprises an amino acid sequence having at least 90% identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 34. In some embodiments, the immunogenic peptide comprises SEQ ID NO: 34.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0166] In some embodiments, the immunogenic peptide comprises a native (i.e., wild-type) cathepsin G 1 (CGI) peptide. CGI is an HLA-A2 presented peptide derived from the azurophil granule protease cathepsin G (CG). In some embodiments, the immunogenic peptide comprises an amino acid sequence having at least 90% identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 35. In some embodiments, the immunogenic peptide comprises SEQ ID NO: 35.

[0167] In some embodiments, the immunogenic peptide is internalized into an NRP1 positive cell to which the antibody-peptide conjugate binds. In some embodiments, the immunogenic peptide is expressed on the cell surface of the NRP1 positive cell. Methods for assessing cell surface peptide expression are well known in the art. In some embodiments, the method of measuring the expression of an immunogenic peptide may include protein-based assays, immunoassays (e.g., ELISA assays), or other suitable methods known by those skilled in the art for protein detection.Linkers and methods of conjugation

[0168] In some embodiments, the antibody-peptide conjugate comprises a linker. Linkers, also referred to as spacers, are flexible molecules or a flexible stretch of molecules that joins or connects two portions, such as domains, of an NRP1 antibody-peptide conjugate according to the present disclosure. A linker may increase the range of orientations that may be adopted by the domains of the NRP1 antibody-peptide conjugate. A linker may be optimized to produce desired effects in the NRP1 antibody-peptide conjugate. Aspects of linker design and considerations are described, for example, in Chen et al. 2013 and Klein et al. 2014. In some embodiments, the antibody-peptide conjugates according to the present disclosure include at least one (such as one, two, three, four, five, etc.} peptide linker. In some embodiments, the antibody-peptide conjugates include at least one (such as one, two, three, four, five, etc. nonpeptide linker. In some embodiments, the antibody-peptide conjugates include at least one peptide linker and at least one non-peptide linker. The antibody-peptide conjugates may also include a plurality of different linkers, including at least one peptide linker, at least one non- peptide linker, or at least one peptide linker and at least one non-peptide linker.

[0169] In some embodiments, the length of a linker may affect the ability of the NRP1 antibody-peptide conjugate to bind to NRP1 on a cell surface. In some embodiments, the linkers used in the NRP1 antibody-peptide conjugates according to the present disclosure are peptide linkers that are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids in length. Depending on length, linker sequence may have various conformations in secondary structure, such as helical, P-strand, coil / bend, and turns. In some instances, a linkerAttorney Docket No. 090723-1530315-MDA25-028PCT sequence may have an extended conformation and function as an independent domain that does not interact with the adjacent protein domains. Linker sequences may be flexible or rigid. Flexible linkers provide a certain degree of movement or interaction between the polypeptide domains and are generally rich in small or polar amino acids such as Gly and Ser (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or all of the amino acid residues of the linker are either Gly or Ser). A rigid linker can be used to keep a fixed distance between the domains and to help maintain their independent functions. Linker attachment can be through an amide linkage (e.g., a peptide bond) or other functionalities known to one of ordinary skill in the art.

[0170] Methods of conjugating a peptide (e.g., an immunogenic peptide) to an antibody or antigen-binding fragment to produce an antibody-peptide conjugate as provided in this disclosure are described in the Examples and are well-known in the art. In some embodiments, the antibody or antigen-binding fragment is conjugated to the immunogenic peptide by a chemical linkage. In some embodiments, the chemical linkage comprises an attachment group (e.g., a spacer) and protease cleavable linker. In some embodiments, the attachment group comprises a maleimidocaproyl moiety, and the protease-cleavable linker comprises a valine- citrulline-p-aminocarbamate (VC-PABC) linker.

[0171] In some embodiments, the antibody or antigen-binding fragment is conjugated to the immunogenic peptide by a recombinant linker. Linker sequences for fusion proteins are described, for example, in Chen et al. 2013, Chen et al. 2014, and Rosemalen et al. 2017, the entire contents of which are herein incorporated by reference. In some embodiments, the recombinant linker comprises a glycine-serine linker sequence and a protease-cleavable linker sequence. In some embodiments, the glycine-serine linker sequence comprises one or more of SGG, GSG, GG, GSGG (SEQ ID NO:55), NGTGGSG (SEQ ID NO:56), G, or GGGGS (SEQ ID NO:57). In certain embodiments, the glycine-serine linker sequence comprises GS or SEQ ID NO: 36.

[0172] In some embodiments, the protease-cleavable linker comprises SEQ ID NO: 37. In certain embodiments, the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising SEQ ID NO: 36, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, a second protease-cleavable linker sequence comprising SEQ ID NO: 37, and a second copy of the immunogenic peptide. In some embodiments, the antibodyconjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising GS, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, and a second protease-cleavable linker sequenceAttorney Docket No. 090723-1530315-MDA25-028PCT comprising SEQ ID NO:37. In some embodiments, the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising GS, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, a second protease-cleavable linker sequence comprising SEQ ID NO: 37, a second copy of the immunogenic peptide, and a third protease-cleavable linker sequence comprising SEQ ID NO:37.Additional linkers

[0173] In some embodiments, the peptide linker comprises a protease recognition site, e.g., a Tobacco Etch Virus (TEV) protease cut site ENLYFQG (SEQ ID NO:58).

[0174] In some embodiments, a non-peptide linker can comprise one or more of a number of known chemical linkers. Exemplary chemical linkers can include one or more units of betaalanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminobexanoic acid (Ahx), PEG multimers, and trioxatricdeacan-succinamic acid (Ttds). In some embodiments, the non-peptide linker comprises one or more units of polyethylene glycol (PEG), which is commonly used as a linker for conjugation of polypeptide domains due to its water solubility, lack of toxicity, low immunogenicity, and well-defined chain lengths. See, e.g., Ramirez-Paz et al. 2018.Antibodies and antibody fragments

[0175] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising a heavy chain variable region polypeptide sequence having at least 86% identity to SEQ ID NO: 1. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising a heavy chain variable region polypeptide sequence having at least 86% identity to SEQ ID NO: 2. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising a heavy chain variable region polypeptide sequence having at least 90% identity to SEQ ID NO: 3. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising a heavy chain variable region polypeptide sequence having at least 87% identity to SEQ ID NO: 4. In some embodiments, the antibody- peptide conjugate comprises an antibody or antigen-binding fragment comprising a heavy chain variable region polypeptide sequence having at least 97% identity to SEQ ID NO: 5.

[0176] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising a light chain variable region polypeptide sequence having at least 97% identity to SEQ ID NO: 6. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising a light chain variableAttorney Docket No. 090723-1530315-MDA25-028PCT region polypeptide sequence having at least 98% identity to SEQ ID NO: 7. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising a light chain variable region polypeptide sequence having at least 99% identity to SEQ ID NO: 8. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising a light chain variable region polypeptide sequence comprising SEQ ID NO: 9. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising a light chain variable region polypeptide sequence having at least 97% identity to SEQ ID NO: 10.

[0177] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a heavy chain variable region (VH) having at least 86% identity to SEQ ID NO: 1 and a light chain variable region (VL) having at least 97% identity to SEQ ID NO: 6. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH having at least 86% identity to SEQ ID NO: 2 and a VL having at least 98% identity to SEQ ID NO: 7. In some embodiments, the antibody- peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH having at least 90% identity to SEQ ID NO: 3 and a VL having at least 99% identity to SEQ ID NO: 8. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigenbinding fragment comprising: a VH having at least 87% identity to SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH having at least 97% identity to SEQ ID NO: 5 and a VL having at least 97% identity to SEQ ID NO: 10.

[0178] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH comprising SEQ ID NO: 1 and a VL comprising SEQ ID NO: 6. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH comprising SEQ ID NO: 2 and a VL comprising SEQ ID NO: 7. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH comprising SEQ ID NO: 3 and a VL comprising SEQ ID NO: 8. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH comprising SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9. In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH comprising SEQ ID NO: 5 and a VL comprising SEQ ID NO: 10.

[0179] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequenceAttorney Docket No. 090723-1530315-MDA25-028PCT comprising SEQ ID NO: 11, 14, or 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12, 15, or 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13, 16, 18, or 21; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22, 25, 30, or 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23, 26, 28, or 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24, 27, 29, or 32.

[0180] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0181] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 14; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 15; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 16; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 25; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 26; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 27.

[0182] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 18; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 29.

[0183] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 20; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 21; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 30; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 32.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0184] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL comprising(i) a CDRL1 amino acid sequence comprising SEQ ID NO: 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0185] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH having at least 86% identity to SEQ ID NO: 1 and comprising (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL having at least 97% identity to SEQ ID NO: 6 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22;(ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0186] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH having at least 86% identity to SEQ ID NO: 2 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 14; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 15; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 16; and (b) a VL having at least 98% identity to SEQ ID NO: 7 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 25; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 26; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 27.

[0187] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH having at least 90% identity to SEQ ID NO: 3 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 18; and (b) a VL having at least 99% identity to SEQ ID NO: 8 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 29.

[0188] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH having at least 87% identity to SEQ ID NO: 4 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 19; (ii) a CDRH2Attorney Docket No. 090723-1530315-MDA25-028PCT amino acid sequence comprising SEQ ID NO: 20; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 21; and (b) a VL comprising SEQ ID NO: 9 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 30; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 32.

[0189] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: (a) a VH having at least 97% identity to SEQ ID NO: 5 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL having at least 97% identity to SEQ ID NO: 10 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0190] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a heavy chain variable region (VH) having at least 86% identity to SEQ ID NO: 1 and a light chain variable region (VL) having at least 97% identity to SEQ ID NO: 6, for instance as found in antibody 5D6 as described herein.

[0191] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH having at least 86% identity to SEQ ID NO: 2 and a VL having at least 98% identity to SEQ ID NO: 7, for instance as found in antibody 7E2 as described herein.

[0192] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH having at least 90% identity to SEQ ID NO: 3 and a VL having at least 99% identity to SEQ ID NO: 8, for instance as found in antibody 12A10 as described herein.

[0193] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH having at least 87% identity to SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9, for instance as found in antibody 13C7 as described herein.

[0194] In some embodiments, the antibody-peptide conjugate comprises an antibody or antigen-binding fragment comprising: a VH having at least 97% identity to SEQ ID NO: 5 and a VL having at least 97% identity to SEQ ID NO: 10, for instance as found in antibody 15D2 as described herein.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0195] In each case, where a specific amino acid sequence is recited, embodiments comprising a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided.VI. Antibody Expression and Purification, Nucleic Acids, Vectors, and Cells

[0196] The NRP1 antibodies and antigen-binding fragments thereof and molecules (e.g., antibody-peptide conjugates) comprising such antibodies and antigen-binding fragments thereof discussed above may be produced by recombinant expression in a human or non-human cell. Antibody-producing cells include non-human cells expressing heavy chains, light chains, or both heavy and light chains; human cells that are not immune cells expressing heavy chains, light chains, or both heavy and light chains; and human B cells that produce heavy chains or light chains, but not both heavy and light chains. The antibodies and antigen-binding fragments thereof of this disclosure may be heterologously expressed, in vitro or in vivo, in cells other than human B cells, such as non-human cells and human cells other than B cells, optionally other than immune cells, and optionally in cells other than cells in a B cell lineage.

[0197] The NRP1 antibodies and antigen-binding fragments thereof and molecules comprising them described herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. For example, a nucleic acid encoding the antibody or antigen-binding fragment thereof can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system, such that it can be maintained in two different organisms, for example, in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.

[0198] Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells that have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072) or Tn5 neo (Southern and Berg, 1982, Mol. Appl. Genet. 1 :327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed or introduced into the same cell by cotransfection (Wigler etal., 1979, Cell 16:77). A second class of vectors utilizes DNA elements that confer autonomously replicating capabilities to an extrachromosomal plasmid. TheseAttorney Docket No. 090723-1530315-MDA25-028PCT vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al., 1982, Proc. Natl. Acad. Set. USA, 79:7147), CMV, polyoma virus (Deans et al., 1984, Proc. Natl. Acad. Set. USA 81 : 1292), or SV40 virus (Lusky & Botchan, 1981, Nature 293:79).

[0199] The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaPC>4 precipitation, liposome fusion, cationic liposomes, electroporation, nucleoporation, viral infection, dextran- mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0200] Appropriate host cells for the expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., Chinese hamster ovary (CHO) and human cell lines), as well as primary cell lines. Also provided herein are populations of any such cells.

[0201] In some embodiments, an antibody or antigen-binding fragment thereof can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals). For example, an antibody can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk as described in, e.g., Houdebine, 2002, Curr. Opin. Biotechnol. 13(6):625-29; van Kuik-Romeijn etal., 2000, Transgenic. Res. 9(2): 155-59; and Pollock etal., 1999, J. Immunol. Methods 231(1-2): 147 -57.

[0202] The antibodies and antigen-binding fragments thereof can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the antibodies or antigen-binding fragments, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression vary with the choice of the expression vector and the host cell and are easily ascertained by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al., 1998, Cytokine 10:319-30). Bacterial expression systems and methods for their use are known in the art (see Ausubel et al., 1988, Current Protocols in Molecular Biology, Wiley & Sons; and Green and Sambrook, 2012, Molecular Cloning— A Laboratory Manual, 4th Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors, and suitable host cells vary depending on a number of factors and may be easily optimized as needed. An antibody (or antigen-binding fragment thereof) described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus,Attorney Docket No. 090723-1530315-MDA25-028PCT and in vitro expression systems (see, e.g., Kaszubska el al., 2000, Protein Expression and Purification 18:213-20). Additional discussion of expression vectors for use in eukaryotic cells (e.g., for treating a subject with cancer), along with suitable delivery systems, is provided in Section IX, below.

[0203] Also provided herein are nucleic acid molecules encoding an NRP1 antibody or antigen-binding portion thereof that binds specifically to NRP1 as described in this disclosure. In some embodiments, the nucleic acid molecules comprise a sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to any one of SEQ ID NOs: 45-54. In some embodiments, the nucleic acid molecules encode an NRP1 antibody or antigen-binding fragment thereof comprising (1) a heavy chain variable region comprising an amino acid sequence that is at least 86% identical (e.g., 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to SEQ ID NO: 1; at least 86% identical (e.g., 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to SEQ ID NO: 2; at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to SEQ ID NO: 3; at least 87% identical (e.g., 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to SEQ ID NO: 4; at least 97% identical (e.g., 98% or 99% identical) to SEQ ID NO: 5 (as shown in Table 1) and (2) a corresponding light chain variable region comprising an amino acid sequence that is at least 97% identical (e.g., 98% or 99% identical) to SEQ ID NO: 6; at least 98% identical (e.g., 99% identical) to SEQ ID NO: 7; at least 99% identical to SEQ ID NO: 8; comprises SEQ ID NO: 9; or is at least 97% identical (e.g., 98% or 99% identical) to SEQ ID NO: 10 (as shown in Table 1)

[0204] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11, 14, or 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12, 15, or 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13, 16, 18, or 21 (as shown in Table 2); and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22, 25, 30, or 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23, 26, 28, or 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24, 27, 29, or 32 (as shown in Table 2).

[0205] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11, 14, or 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12, 15, or 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13, 16, 18,Attorney Docket No. 090723-1530315-MDA25-028PCT or 21; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22, 25, 30, or 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23, 26, 28, or 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24, 27, 29, or 32.

[0206] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0207] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 14; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 15; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 16; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 25; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 26; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 27.

[0208] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 18; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 29.

[0209] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 20; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 21; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 30; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 32.

[0210] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12;Attorney Docket No. 090723-1530315-MDA25-028PCT and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0211] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH having at least 86% identity (for example, at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1 and comprising (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL having at least 97% identity (for example, at least 98%, or 99% identical) to SEQ ID NO: 6 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0212] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH having at least 86% identity (for example, at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 2 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 14; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 15; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 16; and (b) a VL having at least 98% identity (for example, at least 99% identical) to SEQ ID NO: 7 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 25; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 26; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 27.

[0213] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH having at least 90% identity (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 18; and (b) a VL having at least 99% identity to SEQ ID NO: 8 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 29.

[0214] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH having at least 87% identity (for example, at least 88%,Attorney Docket No. 090723-1530315-MDA25-028PCT89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 20; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 21; and (b) a VL comprising SEQ ID NO: 9 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 30; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 32.

[0215] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: (a) a VH having at least 97% identity (for example, at 98%, or 99% identical) to SEQ ID NO: 5 and comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13; and (b) a VL having at least 97% identity (for example, at least 98%, or 99% identical) to SEQ ID NO: 10 and comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 28; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24.

[0216] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: a heavy chain variable region (VH) having at least 86% identity (for example, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1 and a light chain variable region (VL) having at least 97% identity (for example, 98% or 99% identical) to SEQ ID NO: 6, for instance as found in antibody 5D6 as described herein.

[0217] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: a VH having at least 86% identity (for example, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 2 and a VL having at least 98% identity (for example, 99% identical) to SEQ ID NO: 7, for instance as found in antibody 7E2 as described herein.

[0218] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: a VH having at least 90% identity (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 3 and a VL having at least 99% identity to SEQ ID NO: 8, for instance as found in antibody 12A10 as described herein.

[0219] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: a VH having at least 87% identity (for example, 88%, 89%, 90%,Attorney Docket No. 090723-1530315-MDA25-028PCT91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 4 and a VL comprising SEQ ID NO: 9, for instance as found in antibody 13C7 as described herein.

[0220] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising: a VH having at least 97% identity (for example, 98%, or 99% identical) to SEQ ID NO: 5 and a VL having at least 97% identity (for example, 98% or 99% identical) to SEQ ID NO: 10, for instance as found in antibody 15D2 as described herein.

[0221] In each case, where a specific amino acid sequence is recited, embodiments comprising a sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to the recited sequence are also provided.

[0222] The amino acid sequences of the CDRs and framework regions can be determined using various well-known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT), AbM, and observed antigen contacts (“Contact”). In some embodiments, CDRs are determined according to the IMGT definition. See, Brochet et al., 2008, Nucl. Acids Res. 36:W503-508. In some embodiments, CDRs are determined by a combination of Kabat, Chothia, and / or Contact CDR definitions.

[0223] Also provided herein are DNA constructs comprising a promoter that drives expression in a host cell operably linked to a recombinant nucleic acid molecule comprising a nucleotide sequence that encodes an NRP1 specific antibody or antigen-binding fragment thereof.

[0224] Also provided herein are vectors, comprising a DNA construct comprising a promoter that drives expression in a host cell operably linked to a recombinant nucleic acid molecule comprising a nucleotide sequence that encodes an NRP1 specific antibody or antigen-binding fragment thereof.

[0225] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters (e.g., P-actin promoter or EFla promoter), or from hybrid or chimeric promoters (e.g., CMV promoter fused to the P- actin promoter). Promoters from the host cell or related species are also useful herein.

[0226] The term “enhancer” generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5’ or 3’ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain responseAttorney Docket No. 090723-1530315-MDA25-028PCT elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0227] The promoter and / or the enhancer can be inducible (e.g., chemically or physically regulated). A chemically regulated promoter and / or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. A physically regulated promoter and / or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and / or enhancer region can be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the beta-actin promoter, the EFl A promoter, and the retroviral long terminal repeat (LTR).

[0228] The vectors also can include, for example, origins of replication and / or markers. A marker gene can confer a selectable phenotype, e.g, antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the A. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.

[0229] Also provided herein are host cells, including bacterial host cells and eukaryotic host cells, comprising a recombinant nucleic acid molecule encoding an NRP1 antibody or antigenbinding fragment thereof as described in this disclosure. In some embodiments, the nucleicAttorney Docket No. 090723-1530315-MDA25-028PCT acid encodes (1) a heavy chain variable region comprising an amino acid sequence that is at least 86% identical (e.g., 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical) to any one of SEQ ID NOs: 1-5 (as shown in Table 1) and (2) a corresponding light chain variable region comprising an amino acid sequence that is at least 97% identical (e.g., 98% or 99% identical) to any one of SEQ ID NOs: 6-10 (as shown in Table 1).

[0230] In some embodiments, the nucleic acid encodes an isolated antibody or antigenbinding fragment comprising (a) a VH comprising (i) a CDRH1 amino acid sequence comprising SEQ ID NO: 11, 14, or 19; (ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 12, 15, or 17; and (iii) CDRH3 amino acid sequence comprising SEQ ID NO: 13, 16, 18, or 21 (as shown in Table 2); and (b) a VL comprising (i) a CDRL1 amino acid sequence comprising SEQ ID NO: 22, 25, 30, or 33; (ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 23, 26, 28, or 31; and (iii) a CDRL3 amino acid sequence comprising SEQ ID NO: 24, 27, 29, or 32 (as shown in Table 2).

[0231] Also provided herein are host cells that have been engineered to express and secrete an NRP1 antibody or antigen-binding fragment thereof as described in this disclosure. In some embodiments, the cells are suitable for implanting in a patient with cancer. In some embodiments, the cells are animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by a subject’s immune system or by other detrimental factors from the surrounding tissues.

[0232] Also provided herein are immune cells (e.g., T cells) expressing any of the CARs described herein. In some embodiments, the immune cell expresses the CAR on its surface. In some embodiments, the immune cell comprises a nucleic acid encoding the CAR, wherein the CAR is expressed from the nucleic acid and localized to the immune cell surface. In some embodiments, the immune cell is a B-lymphocyte, T-lymphocyte, thymocyte, dendritic cell, natural killer (NK) cell, monocyte, macrophage, granulocyte, eosinophil, basophil, neutrophil, myelomonocytic cell, megakaryocyte, peripheral blood mononuclear cell, myeloid progenitor cell, or a hematopoietic stem cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a cytotoxic T cell, a helper T cell, a natural killer T cell, a suppressorAttorney Docket No. 090723-1530315-MDA25-028PCTT cell, a CD8+T cell, a CD4+T cell, a CD8+ / CD4+T cell, y5 T cell, or a T-regulatory (T-reg) cell.

[0233] In some embodiments, immune cells expressing a CAR provided herein are obtained from a subject. Where the immune cells are used to treat (e.g., according to the treatment methods described herein below) the same subject from which they are obtained, they are referred to as autologous cells. Where they are obtained from a different subject, they are referred to as heterologous cells. Immune cells can be isolated from peripheral blood using techniques well known in the art, include Ficoll density gradient centrifugation followed by negative selection to remove undesired cells. In some embodiments, heterologous immune cells useful for the methods provided herein comprise allogeneic T cells, as described in, e.g., Bedoya et al., 2021, Front. Immunol. 12:640082.

[0234] In vitro methods are also suitable for preparing monovalent antibodies or antigenbinding fragments thereof. Digestion of antibodies to produce antigen-binding fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in International Application Publication No. WO 94 / 29348, U.S. Patent No. 4,342,566, and Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988). Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment yields a fragment, called the F(ab’)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[0235] The Fab fragments produced in antibody digestion can also contain the constant domains of the light chain and the first constant domain of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody hinge region. The F(ab’)2 fragment is a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group.

[0236] One method of producing proteins comprising the provided antibodies or antigenbinding fragments is to link two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyl-oxycarbonyl) or Boc (tert-butyl oxy carbonoyl) chemistry (Applied Biosystems, Inc.; Foster City, CA). ThoseAttorney Docket No. 090723-1530315-MDA25-028PCT of skill in the art readily appreciate that a peptide or polypeptide corresponding to the antibody provided herein, for example, can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of an antibody can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group that is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form an antibody, or fragment thereof. (Grant GA, 1992, Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y.; Bodansky M and Trost B., Ed., 1993, Principles of Peptide Synthesis. Springer Verlag Inc., NY). Alternatively, the peptide or polypeptide can by independently synthesized in vivo. Once isolated, these independent peptides or polypeptides may be linked to form an antibody or antigen-binding fragment thereof via similar peptide condensation reactions.

[0237] For example, enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides, or whole protein domains (Abrahmsen et aL, 1991, Biochemistry, 30:4151). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction (Dawson et al., 1994, Science, 266:776 779). The first step is the chemoselective reaction of an unprotected synthetic peptide a thioester with another unprotected peptide segment containing an amino terminal Cys residue to give a thioester linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site. Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Baggiolini et al., 1992, FEBS Lett. 307:97-101; Clark et al., 1994, J. Biol. Chem. 269: 16075; Clark etal., 1991, Biochemistry 30:3128; Rajarathnam et al., 1994, Biochemistry 33:6623-30).

[0238] Alternatively, unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (nonpeptide) bond (Schnolzer et aL, 1992, Science 256:221). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle et aL, 1992, Techniques in Protein Chemistry IV. Academic Press, New York, pp. 257-267).

[0239] Following expression, the antibodies and antigen-binding fragments thereof can be isolated. An antibody or antigen-binding fragment thereof can be isolated or purified in aAttorney Docket No. 090723-1530315-MDA25-028PCT variety of ways known in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including fast protein liquid chromatography (FPLC), ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein- A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes, 1994, Protein Purification, 3rdedition, Springer-Verlag, New York City, New York. The degree of purification necessary varies depending on the desired use. In some instances, no purification of the expressed antibody or antigen-binding fragments thereof is necessary.

[0240] Methods for determining the yield or purity of a purified antibody or antigen-binding fragment thereof are known in the art and include, e.g., fast protein liquid chromatography (FPLC), Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).VII. Pharmaceutical Compositions and Formulations

[0241] The NRP1 -specific antibodies and antigen-binding portions thereof described herein, as well as the various molecules comprising said antibodies and antigen-binding portions thereof (e.g., antibody-peptide conjugates) are suitable for administration in vitro or in vivo. In some embodiments, the compositions comprise an NRP1 -specific antibody or antigen-binding fragment thereof of the present disclosure and a pharmaceutically acceptable carrier (excipient). In some embodiments, the compositions comprise an antibody-peptide conjugate comprising the NRP1 -specific antibody or antigen-binding fragment thereof and an immunogenic peptide and a pharmaceutically acceptable carrier (excipient). A pharmaceutically acceptable carrier (excipient) is a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. The carrier is selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. The compositions may further comprise a diluent, solubilizer, emulsifier, preservative, and / or adjuvant to be used with the methods disclosed herein. Such compositions can be used, for example, in a subject with cancer that would benefit from any of the NRP1 -specific antibodies or antigen-binding fragments thereof or antibody-peptide conjugates as described herein.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0242] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21Edition, Philip P. Gerbino, ed., Lippincott Williams & Wilkins (2006). In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous and / or intravenous administration. In certain embodiments, the formulation comprises an appropriate amount of a pharmaceutically- acceptable salt to render the formulation isotonic. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington: The Science and Practice of Pharmacy, 22ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014). In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the NRP1 -specific antibody or antibody -peptide conjugatesAttorney Docket No. 090723-1530315-MDA25-028PCT

[0243] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be sterile water for injection, physiological saline solution, buffered solutions like Ringer’s solution, dextrose solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise a pH controlling buffer such phosphate-buffered saline or acetate-buffered saline. In certain embodiments, a composition comprising an NRP1 -specific antibody or antigen-binding fragment thereof or antibody-peptide conjugates disclosed herein can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (see Remington: The Science and Practice of Pharmacy, 22ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014)) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising an NRP1- specific antibody or antigen-binding fragment thereof or antibody-peptide conjugates disclosed herein can be formulated as a lyophilizate using appropriate excipients. In some instances, appropriate excipients may include a cryo-preservative, a bulking agent, a surfactant, or a combination of any thereof. Exemplary excipients include one or more of a polyol, a disaccharide, or a polysaccharide, such as, for example, mannitol, sorbitol, sucrose, trehalose, and dextran 40. In some embodiments, the cryo-preservative may be sucrose or trehalose. In some embodiments, the bulking agent may be glycine or mannitol. In one example, the surfactant may be a polysorbate such as, for example, polysorbate-20 or polysorbate-80.

[0244] In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery (e.g., through injection by intravenous, intraperitoneal, intracerebral (intra- parenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intra-ocular, intraarterial, intraportal, or intralesional routes). Preparations for parenteral administration can be in the form of a pyrogen-free, parenterally acceptable aqueous solution (i.e., water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media) comprising an NRPl-specific antibody or antigen-binding fragment thereof or antibody- peptide conjugates in a pharmaceutically acceptable vehicle. Preparations for parenteral administration can also include non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Parenteral vehicles include sodiumAttomey Docket No. 090723-1530315-MDA25-028PCT chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

[0245] In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. Compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. In certain embodiments, the compositions can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in International Application Publication No. WO / 1994 / 020069, which describes pulmonary delivery of chemically modified proteins. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders are optionally desirable.

[0246] In certain embodiments, the compositions can be selected for topical delivery. Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like are optionally necessary or desirable.

[0247] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. For example, the pH may be 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some instances, the pH of the pharmaceutical composition may be in the range of 6.6-8.5 such as, for example, 7.0-8.5, 6.6-7.2, 6.8-7.2, 6.8- 7.4, 7.2-7.8, 7.0-7.5, 7.5-8.0, 7.2-8.2, 7.6-8.5, or 7.8-8.3. In some instances, the pH of the pharmaceutical composition may be in the range of 5.5-7.5 such as, for example, 5.5-5.8, 5.5-Attorney Docket No. 090723-1530315-MDA25-028PCT6.0, 5.7-6.2, 5.8-6.5, 6.0-6.5, 6.2-6.8, 6.5-7.0, 6.8-7.2, or 6.8-7.5. In some instances, the pH of the pharmaceutical composition may be in the range of 4.0-5.5 such as, for example, 4.0-4.3, 4.0-4.5, 4.2-4.8, 4.5-4.8, 4.5-5.0, 4.8-5.2, or 5.0-5.5.

[0248] In certain embodiments, a pharmaceutical composition can comprise an effective amount of an NRP1 -specific antibody or antigen-binding fragment thereof or an antibody - peptide conjugates in a mixture with non-toxic excipients suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water or other appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0249] Additional pharmaceutical compositions can be selected by one skilled in the art, including formulations involving an NRP1 -specific antibody or antigen-binding fragment thereof or an antibody-peptide conjugates in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, International Application Publication No. WO 1993 / 015722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (see, e.g., U.S. Patent No. 3,773,919; U.S. Patent No. 5,594,091; U.S. Patent No. 8,383,153; U.S. Patent No. 4,767,628; International Application Publication No. WO1998 / 043615, Calo et al., 2015, Eur. Polymer J. 65:252-67 and European Patent No. EP 058,481), including, for example, chemically synthesized polymers, starch based polymers, and polyhydroxyalkanoates (PHAs), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1993, Biopolymers 22:547-56), poly (2-hydroxyethyl- methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277; and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Hsu & Langer, 1985, J. Biomed. Materials Res. 19(4):445-60) or poly-D(-)-3 -hydroxybutyric acid (European Patent No. EP0133988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. (See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-92; European Patent No. EP 036,676; and U.S. Patent Nos. 4,619,794 and 4,615,885).Attorney Docket No. 090723-1530315-MDA25-028PCT

[0250] The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, sterilization is accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0251] In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0252] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried antibody composition and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes are included.

[0253] In certain embodiments, the effective amount of a pharmaceutical composition comprising any of the antibody compositions described herein to be employed therapeutically depends, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, vary depending, in part, upon the molecule delivered, the indication for which a NRP1 -specific antibody or antigen-binding fragment thereof or an antibody-peptide conjugate is being used, the route of administration, and the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient. The clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0254] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate in the formulation used. In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in theAttorney Docket No. 090723-1530315-MDA25-028PCT art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0255] The term “unit dose” or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the effect desired. The actual dosage amount of a composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, a dose may also comprise from about 1 pg / kg / body weight to about 1000 mg / kg / body weight (this such range includes intervening doses) or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 pg / kg / body weight to about 100 mg / kg / body weight, about 5 pg / kg / body weight to about 500 mg / kg / body weight, etc., can be administered.

[0256] In certain embodiments, the NRP1 -specific antibodies or antigen-binding fragments thereof or the antibody-peptide conjugates, can be administered at a dose of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, or 5 mg / kg once every other day at least four times. An exemplary treatment regime may include administration once per day, once per week, twice a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months. In some cases, the treatment comprises administering NRP1- specific antibodies or antigen-binding fragments thereof, or antibody-peptide conjugates, according to one of the aforementioned dosing regimens for a first period and another of the aforementioned dosing regimens for a second period. In some cases, the treatment discontinues for a period of time before the same or a different dosing regimen is resumed. For example, a patient may be on an NRP1 -specific antibody dosing regimen for two weeks, off for a week, on for another two weeks, and so on. Dosage regimens for NRP 1 -specific antibodies or antigenbinding fragments thereof or antibody-peptide conjugates of this disclosure include 0.1 mg / kg body weight, 0.3 mg / kg body weight, 2 mg / kg body weight, 3 mg / kg body weight, or 10 mg / kg via intravenous administration, with the NRP 1 -specific antibodies or antigen-binding fragments thereof or antibody-peptide conjugates being given using one of the followingAttorney Docket No. 090723-1530315-MDA25-028PCT dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks.

[0257] In still another aspect, unit dose forms comprising an NRPl-specific antibody or antigen-binding fragment thereof or an antibody-peptide conjugates as described in this disclosure are provided. A unit dose form can be formulated for administration according to any of the routes described in this disclosure. In one example, the unit dose form is formulated for intravenous or intraperitoneal administration. In still another aspect, pharmaceutical packages comprising unit dose forms of an NRPl-specific antibody or antigen-binding fragment thereof, or of antibody-peptide conjugates, are provided.

[0258] In some instances, the NRP1 antibody or antigen-binding fragment may be an isolated NRPl-specific antibody or antigen-binding fragment thereof as described in this disclosure. The term “isolated,” as used with reference to a protein (or nucleic acid), denotes that the protein (or nucleic acid) is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. Purity and homogeneity are typically determined using analytical chemistry techniques such as electrophoresis (e.g., polyacrylamide gel electrophoresis) or chromatography (e.g., high performance liquid chromatography). In some embodiments, an isolated protein (or nucleic acid) is at least 85% pure, at least 90% pure, at least 95% pure, or at least 99% pure.

[0259] In some instances, the NRPl-specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate may be a formulated into virus-like particles (VLPs). VLPs comprise viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, 2004, Current Opinion in Biotechnology 15:513-7.

[0260] In some instances, the NRP1 antibody or antigen-binding fragment thereof or the antibody-peptide conjugate may be a formulated into subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., 2003, Gene Therapy 10:278-84.VIII. Kits and Packaging

[0261] The NRPl-specific antibodies and antigen-binding fragments thereof or the antibody- peptide conjugates disclosed herein may be used for the preparation of a kit (e.g., a diagnostic test kit or kit for the treatment of a patient). In some embodiments, kits are provided for carrying out any of the methods described herein. The kits of this disclosure may comprise a carrier container being compartmentalized to receive in close confinement one or more containersAttorney Docket No. 090723-1530315-MDA25-028PCT such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method.

[0262] In some embodiments, one of the containers may comprise a NRP1 -specific antibody or antigen-binding fragment thereof or an antibody-peptide conjugate as described in this disclosure that is, or can be, detectably labeled. The kit may also have containers containing buffer(s) and / or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic or fluorescent label. For example, a kit for imaging a tumor in a subject with a NRP1 expressing cancer is provided herein. In some embodiments, the kit comprises a container containing a labeled NRP1 -specific antibody or antigen-binding fragment thereof or an antibody-peptide conjugate. In some embodiments, the kit comprises separate containers containing a NRP1 -specific antibody or antigen-binding fragment thereof or an antibody-peptide conjugate and a detectable label.

[0263] A NRPl-specific antibody or antigen-binding fragment thereof, or antibody-peptide conjugate, as described in this disclosure for use in treating cancer patients may be delivered in a pharmaceutical package or kit to doctors, healthcare providers, treatment facilities, or cancer patients. Such packaging is intended to improve patient convenience and compliance with the treatment plan. Typically, the packaging comprises paper (cardboard) or plastic. In some embodiments, the kit or pharmaceutical package further comprises instructions for use (e.g., for administering according to a method as described herein).

[0264] In some embodiments, a pharmaceutical package or kit comprises unit dose forms of an NRPl-specific antibody or antigen-binding fragment or an antibody-peptide conjugate. In some embodiments, the pharmaceutical package or kit further comprises unit dose forms of one or more of a chemotherapeutic agent, a cytotoxic agent, a radiotherapeutic agent, or an immunotherapeutic agent.

[0265] In one embodiment, the kit or pharmaceutical package comprises a NRPl-specific antibody or antigen-binding fragment or an antibody-peptide conjugate in a defined, therapeutically effective dose in a single unit dosage form or as separate unit doses. The dose and form of the unit dose (e.g., pre-filled syringe, tablet, capsule, immediate release, delayed release, etc.) can be any doses or forms as described herein.

[0266] In one embodiment, the kit or pharmaceutical package includes doses suitable for multiple days of administration, such as one week, one month, or three months.

[0267] In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, kits containing single or multi -chambered pre-filled syringes areAttorney Docket No. 090723-1530315-MDA25-028PCT included. In certain embodiments, kits containing one or more containers of a formulation described in this disclosure are included.IX. Methods of UseA. Methods of Detecting NRP1

[0268] Provided herein are methods of imaging a tumor in a subj ect with a NRP 1 -expressing cancer, the method comprising administering to the subject an isolated antibody or antigenbinding portion thereof that binds specifically to NRP1 that is conjugated to an imaging label and detecting the imaging label in the subject. Imaging methods may be used to assess tumor size and changes in tumor size over or after the course of a treatment administered to the subject. The methods may be useful to assess response of the subject to an administered treatment. In some instances, the methods may be useful to grade the subject’s cancer.

[0269] Methods for detecting the presence of NRP 1 expressing cells in a biological sample are also provided. In some embodiments, the methods include: (a) contacting said sample with a composition comprising an isolated NRP 1 -specific antibody or antigen-binding portion thereof as described in this disclosure; and (b) detecting an amount of binding of the isolated antibody or antigen-binding portion thereof as a determination of the presence of NRP1 expressing cells. In some embodiments, the biological sample comprises a tumor sample. The terms “contacted” and “exposed,” when applied to a biological sample (such as a cell), are used herein to describe the process by which a NRP 1 -specific antibody or antigen-binding fragment thereof is delivered to a biological sample (such as a target cell) or are placed in direct juxtaposition with the biological sample (such as the target cell).

[0270] In some embodiments, NRP1 expression in cancer cells can be examined by using one or more routine biochemical analyses. In some embodiments, NRP1 expression is determined by detecting protein expression using methods such as Western blot analysis, flow cytometry, and immunohistochemistry staining using a NRP 1 -specific antibody or antigenbinding portion thereof as described in this disclosure. Examples of types of immunoassays that can utilize the antibodies according to the present disclosure are competitive and noncompetitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of antigens using the antibodies according to the present disclosure can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation. InAttorney Docket No. 090723-1530315-MDA25-028PCT some instances, a combination of these methods may be used, or additional methods may also be used such as microarray analysis and RT-PCR.

[0271] The antibodies or antigen-binding fragments thereof according to the present disclosure can be bound to many different carriers and used to detect the presence of NRP1 expressing cells. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble for purposes according to the present disclosure. Those skilled in the art will know of other suitable carriers for binding antibodies according to the present disclosure, or will be able to ascertain such, using routine experimentation.

[0272] For purposes of this disclosure, NRP1 protein may be detected by the provided antibodies or antigen-binding fragments thereof when the NRP1 protein is present in biological fluids and tissues from a subject. Any sample containing a detectable amount of NRP1 protein can be used. A sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum or the like; a solid or semi-solid such as tissues, feces, or the like; or, alternatively, a solid tissue such as those commonly used in histological diagnosis.

[0273] In some instances, a threshold amount of NRP1 protein expression is used to characterize NRP1 expression as either high or low. A high level of NRP1 protein expression refers to a measure of NRP1 protein expression above a particular threshold. For example, the threshold may be a normal, an average, or a median amount of NRP1 protein expression as measured in a particular set of samples, referred to as a reference population. In some instances, the reference population may be a population of normal / healthy subjects. In other instances, the reference population may be a population of subjects having a particular type of cancer (the same type of cancer that the subject being assessed has). A low level of NRP1 expression refers to the converse of the above. For example, the threshold may be determined by identifying two distinct subgroups in the reference population by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low.B. Methods of Treatment

[0274] Also provided herein are methods to treat, inhibit, or delay progression of a disease or disorder associated with elevated levels of NRP1, such as cancer. In some embodiments, the subject has cancer.

[0275] Functioning of NRP1 may be reduced by any suitable therapeutic drug or molecule. In some embodiments, such substance is a NRPl-specific antibody or antigen-bindingAttorney Docket No. 090723-1530315-MDA25-028PCT fragment thereof or an antibody-peptide conjugate as described in this disclosure. The methods comprise administering to a subject a therapeutically effective amount of a composition comprising an isolated NRP1 -specific antibody or antigen-binding portion thereof or an antibody-peptide conjugate described herein.

[0276] In one aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient a therapeutically effective amount of a composition comprising an NRPl-specific antibody or antigen-binding portion thereof or an antibody- peptide conjugate as described in this disclosure. The composition may further comprise a pharmaceutically acceptable carrier as described above in Section VII.

[0277] As used throughout, subject can be a vertebrate, more specifically a mammal (e.g., a human, monkey, horse, cat, dog, cow, pig, sheep, camel, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, “patient” or “subject” may be used interchangeably and includes human and veterinary subjects. The NRPl-specific antibody or antigen-binding portion thereof or antibody-peptide conjugate described herein is useful for treating cancer in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary applications. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected to have cancer. In some embodiments, the subject is diagnosed with a cancer.

[0278] As used herein the terms “cancer” and “tumor” are used to indicate malignant tissue. The term “cancer” is also used to refer to the disease associated with the presence of malignant tumor cells in an individual, and the term “tumor” is used herein to refer to a plurality of cancer cells that are physically associated with each other. Cancer cells are malignant cells that give rise to cancer, and tumor cells are malignant cells that can form a tumor and thereby give rise to cancer. The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. The term also encompasses a circulating tumor cell. In certain embodiments, the cancer may originate in the pancreas, colon, rectum, or lung. In other embodiments, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, esophagus, duodenum, lung, small intestine, large intestine, gum, head, kidney, liver, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.

[0279] The subject to be treated by any of the methods described herein may have one of various of different cancers, including, for example, breast cancer, renal cancer, lung cancer, liver cancer, head cancer, neck cancer, or squamous cell carcinoma. In some embodiments,Attorney Docket No. 090723-1530315-MDA25-028PCT the subject may have a primary cancer. In other embodiments, the subject may have metastatic cancer. In some embodiments, the cancer comprises cells that abnormally express NRP1 at a level above basal expression in corresponding normal / non-cancer cells (i.e., an NRP1 expressing cancer). In some embodiments, the subject has relapsed or recurrent cancer. In some embodiments, the subject has cancer that has not responded to other treatments. For example, the subject can have breast cancer that has not responded to chemotherapy and / or radiation.

[0280] “ Treat,” “treatment,” and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. “Treating” or “treatment” may refer to any indicia of success in the treatment or amelioration of cancer. Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject or a symptom thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. “Treating” or “treatment” includes the administration of an agent to impede growth of a cancer, to do one or more of the following: cause a cancer to shrink by weight or volume, delay or prevent metastasis, extend the expected survival time of the subject, or extend the expected time to progression of the tumor, or the like. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a cancer in a subject by administering a pharmaceutical composition as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0281] The term “administer,” as used herein, refers to a method of delivering agents, compounds, or compositions to the desired site of biological action. The pharmaceuticalAttorney Docket No. 090723-1530315-MDA25-028PCT compositions (e.g., as described above) are prepared for administration in a number of ways, including but not limited to injection, ingestion, transfusion, implantation, or transplantation, depending on whether local or systemic treatment is desired, and on the area to be treated. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art. The compositions are administered via any of several routes of administration, including topical, oral, parenteral, intravenous, intra-articular, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intraarterial, intraportal, intracavity, intralesional, transdermal, intradermal, intrahepatical, intrathecal, intracranial, rectal, transmucosal, intestinal, intra-ocular or ocular, otic, nasal, inhalation, or intrabronchial delivery, or any other method known in the art. In some embodiments, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody -peptide conjugate is administered intravenously, or through local injection. In certain embodiments, administered is by bolus injection, continuously by infusion, by sustained release system, or by implantation device. In certain embodiments, individual elements of a combination therapy (as discussed below) may be administered by different routes. In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration. Further discussion on routes of administration and formulations is provided in Section VII.

[0282] In some instances, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate can be administered as an isolated protein. In some instances, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate can be administered via virus-like particles or by subviral dense bodies. Virus-like particles and subviral dense bodies may be formulated as described herein and as known in the art. In some instances, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate can be administered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006 / 110728.

[0283] As used herein, the term “therapeutically effective amount” or effective amount refers to an amount of a therapeutic composition that, when administered to a subject, is effective to treat a disease or disorder such that the symptoms of the disease or disorder are ameliorated, or the likelihood of the disease or disorder developing or progressing is decreased. AAttorney Docket No. 090723-1530315-MDA25-028PCT therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. A suitable dose of a therapeutic composition as described herein, which dose is capable of treating a cancer in a subject, can depend on a variety of factors including the particular therapeutic composition used and whether it is used concomitantly with other therapeutic agents. For example, a different dose of a whole NRP1 -specific antibody may be required to treat a subject with cancer as compared to the dose of an antigen-binding fragment of a NRP1- specific antibody (e.g., Fab’ antibody fragment) required to treat the same subject. Other factors affecting the dose administered to the subject include, e.g., the type or extent of cancer. For example, a subject that has had a previous cancer (e.g., a subject with relapsed or recurrent cancer) may require administration of a different dosage of a NRP1 -specific antibody or antigen-binding fragment thereof than a subject who has not previously had cancer. Generally, a therapeutically effective amount may vary with the subject’s age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the age and general health of the subject, the genetic disposition of the subject, diet, time of administration, the route of administration, and the size (body weight, body surface, or organ size), the rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject also depends upon the judgment of the treating medical practitioner (e.g., doctor or nurse) as described above in Section 0. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. The dosage of the therapeutically effective amount may be adjusted by the individual physician or veterinarian in the event of any complication. In some instances, a therapeutically effective amount may vary from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 20 mg / kg, most preferably from about 0.2 mg / kg to about 2 mg / kg, in one or more dose administrations daily, for one or several days. In some embodiments, the doses can be about 1, about 0.5, about 0.1, about 0.05, or about 0.01 mg / kg, or any intervening dose between about 0.01 mg / kg and 1 mg / kg. In some instances, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody -peptide conjugate is administered for 2 to 5 or more consecutive days. In some instances, the NRPl-specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate is administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0284] The clinician also selects the frequency of dosing, taking into account the pharmacokinetic parameters of the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate in the formulation used. Such pharmacokinetic parameters are well known in the art, / .< ., the rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo- Aragones, 1996, J. Steroid Biochem. Mol. Biol. 58:611-17; Groning, 1996, Pharmazie 51 :337-41; Fotherby, 1996, Contraception 54:59-69; Johnson, 1995, J. Pharm. Sci. 84: 1144-46; Rohatagi, 1995, Pharmazie 50:610-13; Brophy, 1983, Eur. J. Clin. Pharmacol. 24: 103-08; the latest Remington's, supra). In certain embodiments, a clinician administers the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via, for example, an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

[0285] Toxicity and therapeutic efficacy of the NRP1 -specific antibodies or antigen-binding fragments thereof or the antibody-peptide conjugate can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. A NRP1 -specific antibody or antigenbinding fragment thereof or an antibody-peptide conjugate or nucleic acid encoding the NRP1 antibody or antigen-binding fragment thereof or antibody-peptide conjugate that exhibits a high therapeutic index is preferred. While antibody compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such constructs to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.

[0286] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of a NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate can lie generally within a range of circulating concentrations of the NRP1 -specific antibody or antigen-binding fragment or the antibody-peptide conjugate that include the ED50 with little or no toxicity. The dosageAttorney Docket No. 090723-1530315-MDA25-028PCT may vary within this range depending upon the dosage form employed and the route of administration utilized. For NRP1 -specific antibodies or antigen-binding fragments thereof or antibody-peptide conjugates as described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the ECso (i.e., the concentration of the construct - e.g., antibody - which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration is desired, cell culture or animal models can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.

[0287] Suitable human doses of any of the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8): 1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

[0288] In some embodiments, a NRP1 -specific antibody or antigen-binding fragment thereof or an antibody-peptide conjugate described herein can be administered to a subject as a monotherapy. Alternatively, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate can be administered in conjunction with other therapies for cancer (combination therapy). For example, in instances in which the subject is administered an antibody-peptide conjugate, the subject may also be administered a composition that binds to the immunogenic peptide of the antibody-peptide conjugate. In some embodiments, the composition that binds to the immunogenic peptide comprises a peptide-specific antibody (e.g., anti-NLV or anti-CGl), a T cell targeting the immunogenic peptide (e.g., a T cell modified to have a NLV or CGI specific T cell receptor), or a CAR-T cell targeting the immunogenic peptide. The composition can be administered to a subject at the same time, prior to, or after, a second therapy. In some embodiments, the NRPl-specific antibody or antigen-binding fragment thereof or molecule comprising the NRP1 antibody or antigen-binding fragment thereof (e.g., an antibody-peptide conjugate) and the one or more additional active agents are administered at the same time. Optionally, the NRPl-specific antibody or antigen-binding fragment thereof or molecule comprising the NRP1 antibody or antigen-binding fragment thereof (e.g., an antibody-peptide conjugate) is administered first in time and the one or more additional active agents are administered second in time. In some embodiments, the one orAttorney Docket No. 090723-1530315-MDA25-028PCT more additional active agents are administered first in time and the NRP1 -specific antibody or antigen-binding fragment thereof or molecule comprising the NRP1 antibody or antigenbinding fragment thereof (e.g., an antibody-peptide conjugate) is administered second in time. Optionally, the NRP1 -specific antibody or antigen-binding fragment thereof or molecule comprising the NRP1 antibody or antigen-binding fragment thereof (e.g., an antibody-peptide conjugate) and the one or more additional agents are administered simultaneously in the same or different routes. For example, a composition comprising the NRP1 antibody or antigenbinding fragment thereof optionally contains one or more additional agents.

[0289] A NRPl-specific antibody or antigen-binding fragment thereof or an antibody- peptide conjugate as described herein can replace or augment a previously or currently administered therapy. For example, upon treating with a NRP1 -specific antibody or antigenbinding fragment thereof, administration of the one or more additional active agents can cease or diminish, e.g., be administered at lower levels or dosages. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, a previous therapy is maintained until the level of the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate reaches a level sufficient to provide a therapeutic effect.

[0290] In some embodiments, the NRPl-specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate can directly inhibit growth and induce cell death of cancer cells. In some instances, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate may inhibit tumor initiation, e.g., by binding to NRP 1 expressed by cancer stem cells. In some instances, the NRPl-specific antibody or antigenbinding fragment thereof can sensitize cancer cells to other cancer therapies (e.g., chemotherapy). In some instances, treating a subject according to the methods described herein inhibits at least one of formation of a tumor, the proliferation of tumor cells, the growth of tumor cells, survival of tumor cells in circulation, or metastasis of tumor cells in the individual. In another embodiment, treating a subject according to the methods described herein may result in tumor growth stasis, reduction of tumor size and, in some instances, elimination of one or more tumors in the subject.

[0291] In some embodiments, the NRPl-specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate itself may not be therapeutic but may be used to target a therapeutic agent to cancer stem cells or cancer cells. By way of example, in instances in which the subject is administered an antibody-peptide conjugate, the immunogenic peptide may be expressed on the NRP1 positive cells and targeted by a therapeutic agent (e.g., aAttomey Docket No. 090723-1530315-MDA25-028PCT peptide-specific antibody, a T cell, or a CAR-T cell). In such embodiments, the NRP1 -specific antibody or antigen-binding fragment thereof of the antibody -peptide conjugate need only bind specifically to the NRP1 protein. As another example, in instances in which the subject is administered an NRP1 -specific antibody or antigen-binding fragment thereof, the NRP1- specific antibody or antigen-binding fragment thereof may be conjugated to a therapeutic agent (e.g,. as an antibody-drug conjugate).

[0292] In some embodiments, the NRPl-specific antibody or antigen-binding fragment thereof the antibody-peptide conjugate is conjugated to a photosensitizer (also referred to as a photoabsorber). In some instances, the method of treatment comprises using the anti-NRPl antibody or antigen-binding fragment thereof or antibody-peptide conjugate in near infrared photoimmunotherapy (NIR-PIT for the subject. NIR-PIT is a two-part therapy that utilizes a monoclonal antibody (mAb) conjugated to a photoabsorbing dye, such as (but not limited to) IRDye700DX (IR700), which is then activated by NIR light (e.g., see Kobayashi et al., Acc. Chem. Res. 52:2332-2339, 2019). After intravenous injection to the subject, the antibodyphotoabsorber conjugate (APC) binds to NRP1 overexpressed on the surface of cancer cells. Then, NIR light at 690 nm is delivered by laser to excite IR700 on the APC, leading to rapid, highly selective, lethal damage to the cell membrane. In some embodiments, the method of treatment comprises administering an anti-NRPl APC to the subject followed by administering NIR to the subject. The NIR may for instance be applied externally onto the subject, or internally via a catheter or similar device.

[0293] In some embodiments, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate is conjugated to a moiety that specifically binds to an immune cell. In some embodiments, provided is a bispecific antibody comprising an NRP1- specific antibody or antigen-binding fragment thereof or an antibody-peptide conjugate as described herein and an antibody or antigen-binding fragment thereof that specifically binds to an immune cell. In some embodiments, the bispecific antibody comprises an NRPl-specific antibody or antigen-binding portion thereof or an antibody-peptide conjugate and an antibody moiety that specifically binds to T cells. Such a molecule is referred to as a bispecific T cell engager and may induce T cell-mediated cytotoxicity of NRP1 expressing cancer cells (see, e.g., Zhou et al., 2021, Biomarker Research 9:38). In some embodiments, the bispecific antibody comprises an NRPl-specific antibody or antigen-binding portion thereof or an antibody-peptide conjugate and an antibody moiety that specifically binds to natural killer cells (NK cells). Such a molecule is referred to as a NK cell engager and may induce NK cell-Attorney Docket No. 090723-1530315-MDA25-028PCT mediated cytotoxicity of NRP1 -expressing cancer cells (see, e.g., Demaria et al., 2021, European Journal of Immunology 51(8): 1934-1942).

[0294] In certain embodiments, it can be desirable to use a pharmaceutical composition comprising an NRPl-specific antibody or antigen-binding fragment thereof or the antibody- peptide conjugate in an ex vivo manner. In such instances, cells that have been removed from a subject may be exposed to a pharmaceutical composition comprising an NRPl-specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate after which the cells are subsequently implanted back into the subject.

[0295] In another aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient immune cells that express a CAR comprising an NRPl- specific antibody or antigen-binding portion thereof as described herein. Thus, also provided are cancer treatment methods using a CAR comprising a NRPl-specific antibody or antigenbinding fragment thereof as described in this disclosure. In some embodiments, these methods comprise using the CAR to redirect the specificity of an immune effector cell (e.g., a T cell) to target a cancer cell (e.g., a NRP1 expressing cancer cell). Thus, provided herein are methods of stimulating an effector cell-mediated response (such as a T cell-mediated immune response) to a target cell population or tissue comprising cancer cells in a mammal, comprising the step of administering to the mammal an effector cell (such as a T cell) that expresses a CAR as described herein. In some embodiments, “stimulating” an immune cell refers to eliciting an effector cell-mediated response (such as a T cell-mediated immune response), which is different from activating an immune cell. CAR-expressing effector cells as described herein can be infused to a subject in need of treatment (e.g., a cancer patient). In some embodiments, the infused cell is able to kill (or lead to the killing of) cancer cells in the subject. Formulations and methods for making CAR-expressing effector cells and using them in therapeutic methods are known in the art (see, e.g., Feins et al., 2019, Am. J. Hematol. 94(S 1) : S3-S9).

[0296] In another aspect, in instances where the patient is administered an antibody-peptide conjugate, provided is a method of treating a subject with cancer, the method comprising administering to the patient a composition that binds to the immunogenic peptide (e.g., CGI or NLV) of the antibody peptide conjugate. In some embodiments, the composition comprises a peptide-specific antibody (e.g., anti-NLV or anti-CGl). In some embodiments, the composition comprises a peptide-specific T cell. The peptide-specific T cell may be autologous or allogeneic to the recipient subject. Further, the peptide-specific T cell may be natively occurring (e.g., a circulating anti-CGl T cell) or may be engineered to target the immunogenic antigen (e.g., comprise a recombinant T cell receptor). In some embodiments, the compositionAttorney Docket No. 090723-1530315-MDA25-028PCT comprises a peptide-specific CAR-T cell. Thus, also provided are cancer treatment methods composition that binds to the immunogenic peptide of the antibody peptide conjugate as described in this disclosure.

[0297] In another aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient a vector comprising a nucleic acid sequence encoding the NRP1 -specific antibody or antigen-binding fragment thereof as described in this disclosure (e.g., as described in Section VI).

[0298] There are a number of compositions and methods that can be used to deliver the nucleic acid molecules and / or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein.

[0299] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without undesired degradation and include a promoter yielding expression of the nucleic acid molecule and / or adapter polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al., 1997, Retroviruses, Cold Spring Harbor Laboratory Press, which is incorporated by reference herein for the vectors and methods of making them. The construction of replication-defective adenoviruses has been described (Berkner et al., 1987, J. Virology 61 : 1213-20; Massie et al., 1986, Mol. Cell. Biol. 6:2872-83; Haj-Ahmad et al., 1986, J. Virology 57:267-74; Davidson et al., 1987, J. Virology 61 : 1226-39; Zhang et al., 1993, BioTechniques 15:868-72). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. In some instances, the nucleic acid molecules encoding the NRP1 antibodies or antigen-binding fragments thereof or antibody -peptide conjugate can be delivered via virus-like particles.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0300] Non-viral based delivery methods, can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding the adapter polypeptides, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clonetech (Pal Alto, CA), Stratagene (La Jolla, CA), and Invitrogen / Life Technologies (Carlsbad, CA). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5’ and 3’ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

[0301] In another aspect, provided is a method of treating a subject with cancer, the method comprising administering to the patient cells that have been genetically engineered, using methods such as those described herein, to express and secrete an NRP1 -specific antibody or antigen-binding portion thereof or antibody-peptide conjugate as described herein.

[0302] In certain embodiments, a NRP1 -specific antibody or antigen-binding fragment thereof or antibody-peptide conjugate can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by a subject’s immune system or by other detrimental factors from the surrounding tissues.

[0303] In some instances, the provided methods may include administering an NRP1- specific antibody or antigen-binding fragment thereof or an antibody-peptide conjugate and a second form of cancer therapy to the subject. The second form of cancer therapy may include a cytotoxic agent, a chemotherapeutic agent, an immunosuppressive agent (including immune checkpoint inhibitors), or radiation therapy. In some embodiments, the second form of cancer therapy is an antibody (e.g., a monoclonal antibody). For example, in some embodiments, the second form of cancer therapy includes, but is not limited to, sotorasib (AMG 510), adagrasib, sintilimab, necitumumab, nivolumab, trastuzumab, and cetuximab or other chemical andAttorney Docket No. 090723-1530315-MDA25-028PCT biological therapeutics approved by the U.S. Food and Drug Administration or other regulatory authorities for combination therapy.

[0304] The methods and compositions, including combination therapies, enhance the therapeutic or protective effect, and / or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and / or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with both an antibody or antigen-binding fragment and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents (i.e., antibody or antigen-binding fragment or an anti-cancer agent), or by contacting the tissue, tumor, and / or cell with two or more distinct compositions or formulations, wherein one composition provides 1) an antibody or antigenbinding fragment, 2) an anti-cancer agent, or 3) both an antibody or antigen-binding fragment and an anti-cancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, immunotherapy, or radioimmunotherapy. Exemplary anti-cancer agents (also referred to below as therapeutic agents) include chemotherapeutic agents, radiotherapeutic agents, and immunotherapeutic agents, as well as combinations thereof. The terms “contacted” and “exposed,” when applied to a cell in this context, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. In some embodiments, to achieve cell killing, the NRP1 -specific antibody or antigen-binding fragment thereof and the chemotherapeutic or radiotherapeutic agent, are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0305] An antibody or antibody-peptide conjugate may be administered before, during, after, or in various combinations relative to another anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the antibody or antigen-binding fragment is provided to a patient separately from another anticancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such embodiments, it is contemplated that one may provide a patient with the antibody therapy and the anti-cancer therapy within about 6 to 72 hours, about 6 to 48 hours, or about 6 to 24 hours of each other and, more particularly, within about 6-12 hours of each other. In some situations, it may be desirable to extend theAttorney Docket No. 090723-1530315-MDA25-028PCT time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.

[0306] In certain embodiments, a course of treatment will last 1-90 days or more (including intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (including intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (including intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1-7 days, and / or 1-5 weeks, and / or 1-12 months or more, or any time period within these ranges (including intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary.

[0307] In some embodiments, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate can be labeled, conjugated, or fused with a therapeutic agent or diagnostic agent (such as an imaging agent). The linkage can be covalent or noncovalent (e.g., ionic). Such antibodies and antigen-binding fragments are referred to as antibody-drug conjugates (ADC) or immunoconjugates. The antibody drug conjugates are useful for the local delivery of therapeutic agents, particularly cytotoxic or cytostatic agents, i.e., drugs to kill or inhibit tumor cells in the treatment of cancer allows targeted delivery of the drug moiety to tumors, and intracellular accumulation therein, where systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated. Therapeutic agents include but are not limited to toxins, including but not limited to plant and bacterial toxins, small molecules, peptides, polypeptides, and proteins. Genetically engineered fusion proteins, in which genes encoding for an antibody, or antigen-binding fragments thereof including the Fv region, or peptides can be fused to the genes encoding a toxin to deliver a toxin to the target cell are also provided. As used herein, a target cell or target cells are NRP1 positive cells.

[0308] In some embodiments, the NRP1 -specific antibody or antigen-binding fragment thereof or the antibody-peptide conjugate is conjugated to a therapeutic agent. The therapeutic agent may be at least one of a cytotoxic agent, a chemotherapeutic agent, or an immunosuppressive agent. Exemplary therapeutic agents include chemotherapeutic agents, radiotherapeutic agents, and immunotherapeutic agents, as well as combinations thereof. In this way, the NRP1 -specific antibody or antigen-binding fragment thereof delivered to theAttorney Docket No. 090723-1530315-MDA25-028PCT subject can be multifunctional, in that it exerts one therapeutic effect by binding to the NRP1 protein and a second therapeutic effect by delivering a supplemental therapeutic agent.

[0309] The therapeutic agent can act extracellularly, for example by initiating or affecting an immune response, or it can act intracellularly, either directly by translocating through the cell membrane or indirectly by, for example, affecting transmembrane cell signaling. The therapeutic agent is optionally cleavable from the NRP1 -specific antibody or antigen-binding fragment thereof. Cleavage can be autolytic, accomplished by proteolysis, or affected by contacting the cell with a cleavage agent.

[0310] In some embodiments, the therapeutic agent is a cytotoxic agent. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples of toxins or toxin moieties include diphtheria, ricin, streptavidin, and modifications thereof. Additional examples include paclitaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), a photosensitizer combined with NIR-PIT, and anti-mitotic agents (e.g., vincristine and vinblastine). Cytotoxic peptides such as auristatin (antineoplastic) peptides auristatin E (AE) and monomethylauristatin (MMAE), which are synthetic analogs of dolastatin, may also be conjugated to the NRP1- specific antibody or antigen-binding fragment thereof. In some embodiments, the NRP1- specific antibody or antigen-binding fragment thereof may be conjugated to a radioactive metal ion.

[0311] As referred to herein, a chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (such as TARCEVA®, Genentech / OSI Pharm.), bortezomib (such as VELCADE®, Millenium Pharm.), fulvestrant (such as FASLODEX®, AstraZeneca), sutent (such as SU11248, Pfizer), letrozole (such as FEMARA®, Novartis), imatinib mesylate (such as GLEEVEC®, Novartis), PTK787 / ZK222584 (Novartis), oxaliplatin (such as Eloxatin®, Sanofi), 5-fluorouracil (5-FU),Attorney Docket No. 090723-1530315-MDA25-028PCT leucovorin, rapamycin (also known as sirolimus) (such as RAPAMUNE®, Wyeth), lapatinib (such as TYKERB®, GSK572016, GlaxoSmithKline), lonafamib (such as SCH 66336), sorafenib (such as BAY43-9006, Bayer Labs.), capecitabine (such as XELODA®, Roche), docetaxel (such as TAXOTERE®), and gefitinib (such as IRESSA®, Astrazeneca), AG1478, AG1571 (such as SU 5271; Sugen Inc.), alkylating agents such as thiotepa and cyclosphosphamide (such as CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin yi1and calicheamicin Oi1); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin (such as ADRI MYCIN®, including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinicAttorney Docket No. 090723-1530315-MDA25-028PCT acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Trametes Versicolor polysaccharide-K (Krestin, PSK) (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine (cytosine arabinoside, “Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (such as TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ (a Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, IL)), and doxetaxel (such as TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (such as GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine (such as NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0312] Chemotherapeutic agents, as used herein, also refers to (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (such as FARESTON®); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (such as MEGASE®), exemestane (such as AROMASIN®), formestanie, fadrozole, vorozole (such as RIVISOR®), letrozole (such as FEMARA®), and anastrozole (such as ARIMIDEX®); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; (viii) VEGFAttorney Docket No. 090723-1530315-MDA25-028PCT receptor and angiogenesis inhibitors (including ribozymes such as ANGIOZYME®) and a HER2 expression inhibitor; (ix) vaccines such as gene therapy vaccines, for example, ALLOVECTIN-7® vaccine (plasmid / lipid complex containing the DNA sequences encoding HLA-B7 and 132 microglobulin), LEUVECTIN® vaccine (plasmid DNA expression vector encoding interleukin-2 (IL-2) complexed with a lipid delivery vehicle (DMRIE / DOPE)), and VAXID® vaccine (patient-specific naked DNA vaccine); IL-2 or aldesleukin (such as PROLEUKIN®); topoisomerase 1 inhibitors (such as TOPOTECAN®); gonadotropinreleasing hormone antagonists (such as ABARELIX®); (x) anti-angiogenic agents such as bevacizumab (such as AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0313] In some embodiments, the antibodies and antigen-binding fragments thereof or the antibody-peptide conjugates may be conjugated to nucleic acid sequences encoding a microbial flagellin or a flagellin derivative, for instance wherein the flagellin protein sequence serves as an adjuvant and / or modulates a subject’s immune response or antibody efficacy (see, e.g. Lockner c / a / . (2015) Mol. Pharmacol. 12:653-662; doi: 10.1021 / mp500520r).

[0314] In some embodiments, the treatment methods provided herein may further comprise administering an immunosuppressive agent such as an immune checkpoint inhibitor as part of the method. These treatments work by “taking the brakes off’ the immune system (are immunosuppressive), allowing it to mount a stronger and more effective attack against cancer. Several different types of checkpoint inhibitors, targeting different checkpoints or “brakes” on immune cells, are currently in use. Immune checkpoint proteins that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), CXCL9, CXCR5, glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), HLA- DRB 1, ICOS (also known as CD278), HLA-DQA1, HLA-E, indoleamine 2,3 -dioxygenase 1 (IDO1), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG-3, also known as CD223), Mer tyrosine kinase (MerTK), NKG7, 0X40 (also known as CD134), programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1, also known as CD274), PDCD1LG2, PSMB 10, ST A Tl, T cell immunoreceptor with 1g and ITIM domains (TI GIT), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain 1g suppressor of T cell activation (VISTA, also known as C10orf54). In particular, the immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4. Exemplary immunosuppressive agents are PD-1 inhibitors (such as nivolumab and pembrolizumab), PD-L1 inhibitors (such as atezolizumab,Attorney Docket No. 090723-1530315-MDA25-028PCT durvalumab, and avelumab), and CTLA-4 inhibitors (such as ipilimumab). In one example, the second form of cancer therapy comprises a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA4 inhibitor. In some instances, combinations of such inhibitors can be administered. In some instances, the PD-L1 inhibitor, the PD-1 inhibitor, and / or the CTLA4 inhibitor may be an inhibitory antibody that binds specifically to PD-L1, PD-1, or CTLA4, respectively.

[0315] In some instances, the treatment methods provided herein may further comprise administering radiation therapy to the subject. Radiation therapy uses high-energy radiation to shrink tumors and kill cancer cells. X-rays, gamma rays, and charged particles are types of radiation used for cancer treatment. The radiation may be delivered by a machine outside the body (external -beam radiation therapy), or it may come from radioactive material placed in the body near cancer cells (internal radiation therapy, also called brachytherapy). Systemic radiation therapy uses radioactive substances, such as radioactive iodine, that travel in the blood to kill cancer cells.

[0316] In some embodiments of the treatment methods, NRP1 expression (e.g., in cancer cells) can be examined by using one or more routine biochemical analyses before, during, or after treatment. In some embodiments, NRP1 expression is determined by detecting protein expression using methods such as mass spectrometry, Western blot analysis, flow cytometry, or immunohistochemistry staining. In some embodiments, such methods comprise use of a NRP1 -specific antibody or antigen-binding portion thereof (e.g., as described in this disclosure). In some embodiments, NRP1 expression is determined by detecting mRNA levels using methods such as RT-PCR, RNA sequencing, microarray analysis, and Northern blot analysis. In some embodiments, a combination of these methods may be used, or additional methods known in the art may also be used. Suitable methods are described in more detail in Section IX.

[0317] In another aspect, a method of inhibiting the binding of NRP1 to ligands VEGF or semaforin 3a (“SEMA3A”) comprising administering the isolated antibody or antibody fragment of the present disclosure, is provided. Methods for assessing receptor-ligand binding are known in the art and include fluourescent and chemiluminescent-based detection methods, immunoassays (e.g., ELISA assays), or other suitable methods known by those skilled in the art for receptor-ligand binding analysis. In some embodiments, administering the isolated antibody or antibody fragment inhibits an immune inhibitory action of a regulatory T cell.Attorney Docket No. 090723-1530315-MDA25-028PCTC. Diagnostic and Prognostic Methods

[0318] In another aspect, provided are prognostic and diagnostic methods for cancer based on detection and / or quantitation of NRP1 using a NRP1 -specific antibody or antigen-binding fragment as described in this disclosure.

[0319] In some embodiments, provided are methods of assessing eligibility of a subject for inclusion in or exclusion from a clinical trial of or treatment with a NRP1 targeted therapy using a NRP1 antibody or antigen-binding fragment thereof. The method comprises (a) measuring in a tumor sample from a subj ect the amount of NRP 1 ; (b) determining if the subj ect has a cancer characterized as having a high level of NRP 1 expression; and (c) indicating that the subject is eligible for a clinical trial of or treatment with a NRP1 targeted therapy if the subject's cancer is characterized as having a high level of NRP1 expression, i.e., above a predetermined threshold or that the subject is ineligible for a clinical trial of treatment with the NRP1 targeted therapy if the subject’s cancer is characterized as having a low level of NRP 1 expression, i.e., below a predetermined threshold. In some embodiments, the amount of NRP1 in the tumor sample from the subject is measured using a NRPl-specific antibody or antigenbinding fragment thereof as described herein. In some embodiments, the amount of NRP 1 in the sample is measured using a method of detecting NRP1 as described in Section IX. A. In some instances, the threshold level is a median amount of NRP1 determined in a reference population of patients having the same kind of cancer as the subject. In another instance, the threshold level is an optimal amount of NRP1 determined in a reference population of patients having the same kind of cancer as the subject. “Optimal cutoff’ as used herein, refers to the value of a predetermined measure on subjects exhibiting certain attributes that allow the best discrimination between two categories of an attribute. For example, finding a value for an optimal cutoff that allows one to best discriminate between two categories (subgroups) of patients for determining at least one of overall survival, time to disease progression, progression-free survival, and likelihood to respond to treatment (e.g., based on clinical assessment using the RECIST criteria, e.g., Eisenhauer, E.A., et al., 2009, Eur. J. Cancer 45:228-247, or the like as recognized in the medical field). Optimal cutoffs are used to separate the subjects with values lower than or higher than the optimal cutoff to optimize the prediction model, for example, without limitation, to maximize the specificity of the model, maximize the sensitivity of the model, maximize the difference in outcome, or minimize the p-value from hazard ratio or a difference in response.

[0320] In another aspect, provided are methods for assessing responsiveness of a subject with cancer to a NRPl-specific antibody or antigen-binding fragment thereof comprising: (a)Attorney Docket No. 090723-1530315-MDA25-028PCT measuring in a tumor sample from a subj ect the amount of NRP 1 ; (c) determining if the subj ect has a cancer characterized as having a high level of NRP 1 expression; and (d) indicating that the subject is more likely to respond to the NRP 1 antibody or antigen-binding fragment thereof if the subject’s cancer is characterized as having a high level of NRP 1 expression. Conversely, if the subject’s cancer is characterized as having a low level of NRP 1 expression, the subject is less likely to respond to a NRP 1 -specific antibody or antigen-binding fragment thereof. In some instances, the amount of NRP 1 in the tumor sample is measured using a NRP 1 -specific antibody or antigen-binding fragment thereof as described herein. In some embodiments, the amount of NRP1 in the sample is measured using a method of detecting NRP1 as described in Section IX. A.

[0321] In another aspect, provided are methods to diagnose cancer in a subject. Specifically, the diagnosis may be of an NRP 1 -expressing cancer. The method may comprise measuring in a sample from a subject the amount of NRP 1 and diagnosing the subject with cancer if the amount of NRP 1 expression in the sample is high. In some instances, the method may comprise (a) measuring in a tumor sample from a subject the amount of NRP 1 using an NRP1 antibody or antigen-binding fragment thereof; and (c) determining if the subject has a cancer characterized as having a high level of NRP 1 expression. Conversely, if the amount of NRP 1 expression in the sample or the subject’s cancer low level, the subject may not be diagnosed with cancer or may not be diagnosed with an NRP1 expressing cancer. In some instances, the amount of NRP 1 in the tumor sample is measured using a NRP1 -specific antibody or antigenbinding fragment thereof as described herein. In some embodiments, the amount of NRP 1 in the sample is measured using a method of detecting NRP1 as described in Section IX. A.

[0322] In some instances, to diagnose cancer in a subject, or to characterize a subject’s cancer, a biopsy is typically taken from a subject having an abnormal tissue growth, such as a tumor. Samples may be formalin-fixed, paraffin-embedded tissue samples obtained from the subject’s cancer (tumor). In other instances, such as where circulating tumor cells are to be assessed, the sample from the subject is a blood, plasma, or lymph sample. Typically, the tissue or cells of the patient sample reexamined under a microscope in order to confirm the diagnosis and / or assess information about the tumor. In some cases, additional tests may need to be performed on the proteins, DNA, and / or mRNA of the cells in the ample to verify the diagnosis or characterization.

[0323] Also provided are methods of monitoring response of a subject with a NRP1 expressing cancer to cancer therapy. Monitoring a subject (e.g., a human patient) for an improvement in cancer status, as used herein, means evaluating the subject for a change in aAttorney Docket No. 090723-1530315-MDA25-028PCT disease parameter, e.g., a reduction in one or more symptoms of cancer exhibited by the subject. The subject may be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation may include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a cancer described herein. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration.

[0324] The monitoring methods may include administering to the subject a NRP1 -specific antibody or antigen-binding fragment thereof conjugated to an imaging label at a first time point prior to the subject before the subject receives cancer therapy, detecting the imaging label in the subject to obtain a first image of the tumor, administering to the subject a NRP1 -specific antibody or antigen-binding fragment thereof conjugated to an imaging label at a second time point after the subject receives cancer therapy, detecting the imaging label in the subject to obtain a second image of the tumor; and comparing the first image to the second image to determine whether a change in tumor size has occurred. In some instances, the steps of administering to the subject a NRP1 -specific antibody or antigen-binding fragment thereof conjugated to an imaging label at a first time point after the subject receives cancer therapy, detecting the imaging label in the subject to obtain a second image of the tumor; and comparing the first image to the second image to determine whether a change in tumor size has occurred may be repeated at a third time point (or additional time points) after the subject receives cancer therapy.

[0325] In one embodiment, a subject is administered a labeled NRP1 antibody or antigenbinding fragment thereof as described in this disclosure that is conjugated to an imaging agent. The labeled NRP1 antibody or antigen-binding fragment thereof is allowed to incubate in vivo and bind to NRP1 in the subject’s tissues. The imaging label is thereby localized to tumor cells or tissues, and the localized imaging label is detected using an appropriate imaging device as known to those skilled in the art.

[0326] The imaging agent may carry a bioluminescent or chemiluminescent label. Such labels include polypeptides known to be fluorescent, bioluminescent or chemiluminescent, or that act as enzymes on a specific substrate (reagent), or can generate a fluorescent, bioluminescent or chemiluminescent molecule. Examples of bioluminescent orAttorney Docket No. 090723-1530315-MDA25-028PCT chemiluminescent labels include luciferases, aequorin, obelin, mnemiopsin, berovin, a phenanthridinium ester, and variations thereof and combinations thereof. A substrate for the bioluminescent or chemiluminescent polypeptide may also be used in imaging. For example, the chemiluminescent polypeptide can be luciferase and the reagent luciferin. A substrate for a bioluminescent or chemiluminescent label can be administered before, at the same time (e.g., in the same formulation), or after administration of the agent.

[0327] The imaging agent may include a paramagnetic compound, such as a polypeptide chelated to a metal (e.g., a metalloporphyrin). The paramagnetic compound may also include a monocrystalline nanoparticle, e.g., a nanoparticle including a lanthanide (e.g., Gd) or iron oxide; or a metal ion such as a lanthanide. Examples of elements that are useful in magnetic resonance imaging include gadolinium, terbium, tin, iron, or isotopes thereof.

[0328] Whole body imaging techniques using radioisotope labeled agents can be used for locating diseased cells and tissues (e.g., primary tumors and tumors which have metastasized). In some cases, the labeled agents for locating the tumor tissue or cells are administered intravenously. The bio-distribution of the label can be monitored by scintigraphy, and accumulations of the label are related to the presence of NRP1 or other tumor markers. Whole body imaging techniques are described in, e.g., U.S. Patent Nos. 4,036,945 and 4,311,688.

[0329] An image according to this disclosure can be generated by computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS) image, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or bioluminescence imaging (BLI) or equivalent.

[0330] Computer assisted tomography (CAT) and computerized axial tomography (CAT) systems and devices well known in the art can be used to generate an image. (See, for example, U.S. Pat. Nos. 6,151,377; 5,946,371; 5,446,799; 5,406,479; 5,208,581; and 5,109,397.) The imaging methods may also utilize animal imaging modalities, such as MicroCAT™ (ImTek, Inc.).

[0331] Magnetic resonance imaging (MRI) systems and devices well known in the art can be used for imaging. For a description of MRI methods and devices, see, for example, U.S. Pat. Nos. 6,151,377. MRI and supporting devices are commercially available, for example, from Bruker Medical GMBH; Caprius; Esaote Biomedica; Fonar; GE Medical Systems (GEMS); Hitachi Medical Systems America; Intermagnetics General Corporation; Lunar Corp.; MagneVu; Marconi Medicals; Philips Medical Systems; Shimadzu; Siemens; Toshiba America Medical Systems; including imaging systems, by, e.g., Silicon Graphics.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0332] Positron emission tomography imaging (PET) systems and devices well known in the art can be used for imaging. For example, an imaging method of this disclosure may use the system designated Pet VI located at Brookhaven National Laboratory. For descriptions of PET systems and devices, see, for example, U.S. Pat. Nos. 6,151,377. Animal imaging modalities such as micro-PETs (Concorde Microsystems, Inc.) can also be used.

[0333] Single-photon emission computed tomography (SPECT) systems and devices well known in the art can be used for imaging. (See, for example, U.S. Pat. Nos. 6,115,446; 6,072,177; 5,608,221; 5,600,145; 5,210,421; 5,103,098) Imaging methods may also use animal imaging modalities, such as micro-SPECTs.

[0334] Sensitive photon detection systems can be used to detect bioluminescent and fluorescent proteins externally; see for example, Contag, 2000, Neoplasia 2:41-52; and Zhang, 1994, Clin. Exp. Metastasis, 12:87-92. The imaging methods of the disclosure can be practiced using any such photon detection device, for example, an intensified charge-coupled device (ICCD) camera coupled to an image processor. Photo detection devices are also commercially available from Xenogen, Hamamatsue.

[0335] Disclosed herein are materials, compositions, and methods that can be used for, can be used in conjunction with or can be used in preparation for the disclosed embodiments. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compositions may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and a number of modifications that can be made to a number of molecules included in the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.

[0336] Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties. The following description provides further non-limiting examples of the disclosed compositions and methods.Attorney Docket No. 090723-1530315-MDA25-028PCTX. EXEMPLARY EMBODIMENTS

[0337] Embodiment 1. An isolated antibody or antigen-binding fragment thereof, comprising: a) a heavy chain variable region comprising:(i) a CDRH1 comprising SEQ ID NOs: 11, 14, or 19;(ii) a CDRH2 comprising SEQ ID NOs: 12, 15, 17, or 20; and(iii) a CDRH3 comprising SEQ ID NOs: 13, 16, 18, or 21; and b) a light chain variable region comprising:(i) a CDRL1 comprising SEQ ID NOs: 22, 25, 30, or 33;(ii) a CDRL2 comprising SEQ ID NOs: 23, 26, 28, or 31; and(iii) a CDRL3 comprising SEQ ID NOs: 24, 27, 29, or 32.

[0338] Embodiment 2. The isolated antibody or antigen-binding fragment of embodiment 1, wherein the isolated antibody or antigen-binding domain comprises: a) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24; b) a CDRH1 comprising SEQ ID NO: 14, a CDRH2 comprising SEQ ID NO: 15, a CDRH3 comprising SEQ ID NO: 16; a CDRL1 comprising SEQ ID NO: 25, a CDRL2 comprising SEQ ID NO: 26, and a CDRL3 comprising SEQ ID NO: 27; c) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 17, a CDRH3 comprising SEQ ID NO: 18; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 29; d) a CDRH1 comprising SEQ ID NO: 19, a CDRH2 comprising SEQ ID NO: 20, a CDRH3 comprising SEQ ID NO: 21; a CDRL1 comprising SEQ ID NO: 30, a CDRL2 comprising SEQ ID NO: 31, and a CDRL3 comprising SEQ ID NO: 32; or e) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 33, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 24.

[0339] Embodiment 3. The isolated antibody or antigen-binding fragment of embodiment 1 or 2, wherein:Attorney Docket No. 090723-1530315-MDA25-028PCT a. the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 1, and the light chain variable region comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 6; b. the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 2, and the light chain variable region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 7; c. the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 8; d. the heavy chain variable region comprises an amino acid sequence that is at least 87% identical to SEQ ID NO: 4, and the light chain variable region comprises SEQ ID NO: 9; or e. the heavy chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 5, and the light chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 10.

[0340] Embodiment 4. The isolated antibody or antigen-binding fragment of any one of embodiments 1-3, wherein: a. the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6; b. the heavy chain variable region comprises SEQ ID NO: 2, and the light chain variable region comprises SEQ ID NO: 7; c. the heavy chain variable region comprises SEQ ID NO: 3, and the light chain variable region comprises SEQ ID NO: 8; d. the heavy chain variable region comprises SEQ ID NO: 4, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 9; or e. the heavy chain variable region comprises SEQ ID NO: 5, and a light chain variable region comprises SEQ ID NO: 10.

[0341] Embodiment 5. The isolated antibody or antibody fragment of any of embodiments 1- 4, wherein the isolated antibody or antibody fragment comprises a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0342] Embodiment 6. The isolated antibody or antibody fragment of embodiment 5, wherein the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6.

[0343] Embodiment 7. The isolated antibody or antibody fragment of any of embodiments 1- 6 wherein the antibody or antibody fragment is a chimeric antibody or antibody fragment.

[0344] Embodiment 8. The isolated antibody or antigen-binding fragment of any one of embodiments 1-7, wherein the antibody fragment is a monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)s fragment, Fv fragment, or single chain antibody.

[0345] Embodiment 9. The isolated antibody or antigen-binding fragment of any one of embodiments 1-8, wherein the antibody or antibody fragment is a chimeric antibody, bispecific antibody, trispecific antibody, or other multi-specific antibody.

[0346] Embodiment 10. The isolated antibody or antigen-binding fragment of any one of embodiments 1-9, wherein the antibody or antibody fragment is conjugated or fused to an imaging agent, a cytotoxic agent, or a radioactive moiety.

[0347] Embodiment 11. The isolated antibody or antigen-binding fragment of embodiment 10, wherein the antibody or antibody fragment is conjugated or fused to an imaging agent, and wherein the imaging agent is a fluorophore.

[0348] Embodiment 12. The isolated antibody or antigen-binding fragment of embodiment 10, wherein the antibody or antibody fragment is conjugated or fused to a radioactive moiety, and wherein the radioactive moiety is Zr-89, Cu-64, F-18, Y-90, Lu-177, At-211, Ac-225, or Pb-212.

[0349] Embodiment 13. The isolated antibody or antigen-binding fragment of any one of embodiments 1-9, wherein the antibody is an immune conjugate.

[0350] Embodiment 14. The isolated antibody or antigen-binding fragment of any one of embodiments 1-9, wherein the antibody or antibody fragment is an antibody-drug conjugate.

[0351] Embodiment 15. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment of any one of embodiments 1-14 and a pharmaceutically acceptable carrier.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0352] Embodiment 16. An isolated nucleic acid encoding the antibody heavy and / or light chain variable region of the antibody or antigen-binding fragment of any one of embodiments 1-14.

[0353] Embodiment 17. A hybridoma or engineered cell comprising the nucleic acid encoding the antibody or antigen-binding fragment of any one of embodiments 1-14.

[0354] Embodiment 18. A hybridoma or engineered cell comprising the nucleic acid of embodiment 16.

[0355] Embodiment 19. A method of making an isolated antibody or antigen-binding fragment, comprising culturing the hybridoma or engineered cell of embodiment 17 or 18 under conditions that allow expression of the antibody or antigen-binding fragment and, optionally, isolating the antibody or antigen-binding fragment from the culture.

[0356] Embodiment 20. An antibody-peptide conjugate, comprising: a) the antibody or antigen-binding fragment of any one of embodiments 1-9; and b) an immunogenic peptide.

[0357] Embodiment 21. The antibody-peptide conjugate of embodiment 20, wherein the immunogenic peptide comprises a native cytomegalovirus (NLV) peptide or a cathepsin G 1 (CGI) peptide.

[0358] Embodiment 22. The antibody-peptide conjugate of embodiment 20 or 21, wherein the immunogenic peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:34.

[0359] Embodiment 23. The antibody-peptide conjugate of embodiment 20, wherein the immunogenic peptide comprises SEQ ID NO:34.

[0360] Embodiment 24. The antibody-peptide conjugate of embodiment 20 or 21, wherein the immunogenic peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:35.

[0361] Embodiment 25. The antibody-peptide conjugate of embodiment 24, wherein the immunogenic peptide comprises SEQ ID NO:35.

[0362] Embodiment 26. The antibody-peptide conjugate of any one of embodiments 20-25, wherein the antibody or antigen-binding fragment is conjugated to the immunogenic peptide by a chemical linkage.Attomey Docket No. 090723-1530315-MDA25-028PCT

[0363] Embodiment 27. The antibody-peptide conjugate of embodiment 26, wherein the chemical linkage comprises an attachment group and protease cleavable linker.

[0364] Embodiment 28. The antibody-peptide conjugate of embodiment 27, wherein the attachment group comprises a maleimidocaproyl moiety, and the protease-cleavable linker comprises a valine-citrulline- / ?-aminocarbamate (VC-PABC) linker.

[0365] Embodiment 29. The antibody-peptide conjugate of any one of embodiments 20-25, wherein the heavy chain of the antibody or antigen-binding fragment is conjugated to the immunogenic peptide by a recombinant linker.

[0366] Embodiment 30. The antibody-peptide conjugate of embodiment 29, wherein the recombinant linker comprises a glycine-serine linker sequence and a protease-cleavable linker sequence.

[0367] Embodiment 31. The antibody-peptide conjugate of embodiment 29 or 30, wherein the glycine-serine linker sequence comprises GS or SEQ ID NO: 36.

[0368] Embodiment 32. The antibody-peptide conjugate of any one of embodiments 29-31, wherein the protease-cleavable linker comprises SEQ ID NO: 37.

[0369] Embodiment 33. The antibody-peptide conjugate of any one of embodiments 29-32, wherein the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising SEQ ID NO: 36, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, a second protease-cleavable linker sequence comprising SEQ ID NO:37, and a second copy of the immunogenic peptide.

[0370] Embodiment 34. The antibody-peptide conjugate of any one of embodiments 29-32, wherein the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising GS, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, and a second protease- cleavable linker sequence comprising SEQ ID NO:37.

[0371] Embodiment 35. The antibody-peptide conjugate of any one of embodiments 29-32, wherein the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising GS, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, a second protease-Attorney Docket No. 090723-1530315-MDA25-028PCT cleavable linker sequence comprising SEQ ID NO:37, a second copy of the immunogenic peptide, and a third protease-cleavable linker sequence comprising SEQ ID NO:37.

[0372] Embodiment 36. The antibody-peptide conjugate of any one of embodiments 20-35, wherein the antibody or antigen-binding fragment comprises: a) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24; b) a CDRH1 comprising SEQ ID NO: 14, a CDRH2 comprising SEQ ID NO: 15, a CDRH3 comprising SEQ ID NO: 16; a CDRL1 comprising SEQ ID NO: 25, a CDRL2 comprising SEQ ID NO: 26, and a CDRL3 comprising SEQ ID NO: 27; c) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 17, a CDRH3 comprising SEQ ID NO: 18; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 29; d) a CDRH1 comprising SEQ ID NO: 19, a CDRH2 comprising SEQ ID NO: 20, a CDRH3 comprising SEQ ID NO: 21; a CDRL1 comprising SEQ ID NO: 30, a CDRL2 comprising SEQ ID NO: 31, and a CDRL3 comprising SEQ ID NO: 32; or e) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 33, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 24.

[0373] Embodiment 37. The antibody-peptide conjugate of any one of embodiments 20-36, wherein: a) the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 1, and the light chain variable region comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 6; b) the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 2, and the light chain variable region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 7; c) the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 8; d) the heavy chain variable region comprises an amino acid sequence that is at least 87% identical to SEQ ID NO: 4, and the light chain variable region comprises SEQ ID NO: 9; orAttorney Docket No. 090723-1530315-MDA25-028PCT e) the heavy chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 5, and the light chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 10.

[0374] Embodiment 38. The antibody-peptide conjugate of any one of embodiments 20-37, wherein: a) the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6; b) the heavy chain variable region comprises SEQ ID NO: 2, and the light chain variable region comprises SEQ ID NO: 7; c) the heavy chain variable region comprises SEQ ID NO: 3, and the light chain variable region comprises SEQ ID NO: 8; d) the heavy chain variable region comprises SEQ ID NO: 4, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 9; or e) the heavy chain variable region comprises SEQ ID NO: 5, and a light chain variable region comprises SEQ ID NO: 10.

[0375] Embodiment 39. The antibody-peptide conjugate of any one of embodiments 20-38, wherein the isolated antibody or antibody fragment comprises a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24.

[0376] Embodiment 40. The antibody-peptide conjugate of any one of embodiments 20-39, wherein the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6.

[0377] Embodiment 41. A pharmaceutical composition comprising the antibody-peptide conjugate of any one of embodiments 20-40 and a pharmaceutically acceptable carrier.

[0378] Embodiment 42. A method of imaging a cell or tissue expressing neuropilin-1 (NRP1) in a subject, the method comprising:(a) administering to the subject the isolated antibody or antibody fragment of any of embodiment 10 or 11, and(b) detecting the imaging agent in the subject, thereby imaging the cell or tissue.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0379] Embodiment 43. A method for diagnosing a subject having or suspected of having a cancer characterized by elevated neuropilin-1 (NRP1) expression, comprising:(a) contacting a biological sample with the isolated antibody or antigen-binding fragment of any one of embodiments 1-12; and(b) detecting an amount of binding of the isolated antibody or antigen-binding fragment as a determination of a presence of NRP-1 in the biological sample.

[0380] Embodiment 44. A method of treating a human subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 15.

[0381] Embodiment 45. A method of treating a human subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 41.

[0382] Embodiment 46. The method of embodiment 43 or 44, wherein the subject has cancer.

[0383] Embodiment 47. The method of embodiment 46, wherein the cancer expresses an elevated level of neuropilin-1 (NRP1) relative to a healthy control tissue.

[0384] Embodiment 48. The method of embodiment 46 or 47, wherein the cancer is selected from a group consisting of breast, renal, lung, liver, head, neck, and squamous cell cancer.

[0385] Embodiment 49. The method of any one of embodiments 44-48, further comprising administering to the subject a therapeutically effective amount of a small molecule drug.

[0386] Embodiment 50. The method of any one of embodiments 44-49, wherein the isolated antibody or antigen-binding fragment is conjugated to a therapeutic agent.

[0387] Embodiment 51. The method of embodiment 50, wherein the therapeutic agent is at least one of a cytotoxic agent, a photosensitizer, a chemotherapeutic agent, or an immunosuppressive agent.

[0388] Embodiment 52. The method of embodiment 50 or 51, wherein the therapeutic agent is a moiety that specifically binds to an immune cell.

[0389] Embodiment 53. The method of embodiment 52, wherein the immune cell is a T cell.

[0390] Embodiment 54. The method of embodiment 52, wherein the immune cell is a natural killer cell.Attorney Docket No. 090723-1530315-MDA25-028PCT

[0391] Embodiment 55. The method of any one of embodiments 44-54, wherein the therapeutically effective amount of the pharmaceutical composition is administered via intravenous injection, subcutaneous injection, or direct injection into a diseased tissue.

[0392] Embodiment 56. The method of any one of embodiments 45-48, further comprising administering a composition that binds to the peptide of the antibody-peptide conjugate.

[0393] Embodiment 57. The method of embodiment 56, wherein the composition comprises a peptide-specific antibody, T cell, or chimeric antigen receptor (CAR)-T cell.

[0394] Embodiment 58. A method of inhibiting the binding of neuropilin- 1 (NRP1) to VEGF or semaforin 3a, comprising administering the isolated antibody or antibody fragment of any one of embodiments 1-9.

[0395] Embodiment 59. The method of embodiment 58, wherein administering the isolated antibody or antibody fragment inhibits an immune inhibitory action of a regulatory T cell.Attorney Docket No. 090723-1530315-MDA25-028PCTEXAMPLES

[0396] The following examples are offered to illustrate, but not to limit, the present disclosure.EXAMPLE 1. Materials and Methods

[0397] NRP1 Antibodies. The extracellular domain of recombinant human NRP1 antigen (GenScript) was used was used to immunize mice for monoclonal antibody production. Chimeric antibodies were produced by replacing the constant region of the mouse antibodies with the human IgGl Fc from h8F4 / trastuzumab. The mouse and chimeric antibodies were assessed for binding to human NRP1.

[0398] Bio-layer interferometry (BLI) Octet analysis for antibody screening. Each antibody clone was digested with papain, and the Fab fragments were purified and used to measure affinity to recombinant human NRP1 (“hNRPl”). For the BLI assays, peptide antigens were captured on streptavid sensors, Fabs were diluted to 100 nM in lOx kinetics Buffer and loaded for 600 seconds. After loading, the baseline signal was then recorded for 1 minute in lOx kinetics Buffer. The sensors were immersed into wells containing Fab with 100 nM and 20 nM in 10X Kinetics Buffer for 600 seconds (association phase), followed by immersion in 10X Kinetics Buffer for an additional 600 seconds (dissociation phase). The background signal from each analyte-containing well was measured using empty reference sensors coated with same concentrations of BSA and subtracted from the signal obtained with each corresponding mAb loaded sensor. Kinetic analyses were performed at least twice with an independently prepared analyte dilution series. Curve fitting was performed using a 1 : 1 binding model and the ForteBio data analysis software. Mean kon, koff values were determined by averaging all binding curves that matched the theoretical fit with an R2 value of >=0.95.

[0399] In vitro internalization assays of NRP1 antibodies in MB-231 cell line. MB-231 cells were pre-treated with 10 pg / ml chi orhexi dine (CHX) or control vehicle for 1 hour. Cells were seeded in non-tissue-culture (non-TC) treated 96-well plates at 2e6 / ml in 150 pl / well. The plates were placed on ice for 30 minutes. 150 pl of cold antibody solution (a 2X solution was used to result in a final concentration 5 pg / ml of antibody) was added, with or without 1 pg / ml CHX inhibitor, to corresponding wells and incubated for 30 minutes. The cells were then washed twice with ice-cold media and resuspended in media with 2% FBS. After one hour or five hours, 50 pl of cells were collected from each well and transferred to a 96-well plate containing 200 pl of cold FACS buffer. The transferred cells were kept cold and fixed for FACS staining using anti-human secondary antibody. The stained cells were imaged using an Amnis® instrument.Attorney Docket No. 090723-1530315-MDA25-028PCTEXAMPLE 2. NRP1 Antibody Binding Characterization

[0400] Recombinant human NRP1 extracellular domain antigen (GenScript) was used to immunize mice to produce NRP1 monoclonal antibodies. Binding of the resulting mouse antibodies to recombinant human NRP1 was assessed using an enzyme-linked immunosorbent assay (“ELISA”). The ELISA plate was coated with 50 ng of recombinant NRP1 overnight at 4°C. The coated plates were then blocked with 1% bovine serum albumin (“BSA”) for 1 hour at room temperature. The monoclonal antibody sample (5 pg / mL) was added to the plates and incubated for 1 hour at room temperature, before an anti-mouse horseradish peroxidase (“HRP”) conjugated secondary antibody (1 : 1000) was added and incubated for 1 hour at room temperature. Then 3,3',5,5'-Tetramethylbenzidine (“TMB”) was used to detect HRP activity. As shown in FIG. 1, each mouse NRP1 antibody clone exhibited significant binding to human NRP1.

[0401] Octet analysis of NRP1 antibody clones was performed as described in the Example 1. The 5D6 and 13C7 antibodies showed high binding affinity to hNRPl (FIGS. 2A and 2B, respectively), similar to the binding affinity of control NRP1 antibody 17F6 (Miltenyi) to hNRPl (FIG. 2C). Surface plasmon resonance affinity measurements are shown below in Table 4. These results showed the specific and high affinity binding of clone 5D6 to recombinant human NRP1 (K=2.37 nM).Table 4. Surface plasmon resonance affinity measurementsEXAMPLE 3. Identification of Binding Location of NRP1 Antibodies

[0402] Vascular endothelial growth factor (“VEGF”) is known to bind the B1 / B2 extracellular domain of NRP1. ELISA analysis was used to assess the NRP1 binding location of the disclosed mouse NRP1 antibody clones by determining whether the disclosed antibodies block binding of VEGF to the B1 / B2 extracellular domain of NRP1. The ELISA plates were coated with 50 ng of recombinant NRP1 overnight at 4°C. The coated plates were blocked with 1% BSA for 1 hour at room temperature. The monoclonal antibody sample (25 ng) was added, and the plates were incubated for 1 hour at room temperature. Varying concentrations of VEGF and 50 pl of 2 pg / ml anti-VEGF antibody (Avastin) were added, and the plates were incubated for 1 hour at room temperature. Anti-HRP conjugated secondary antibody (1 : 1000) was added,Attorney Docket No. 090723-1530315-MDA25-028PCT the plates were incubated for 1 hour at room temperature, and TMB was used to detect HRP activity. As shown in FIG. 3, clones 5D6 and 15D2 partially block binding of VEGF to NRP1.EXAMPLE 4. Isotype Characterization of Anti-NRPl Antibodies

[0403] To determine the isotype of the disclosed anti-NRPl antibodies, MDA-MB-231 breast cancer cells were stained with antibodies from immunized mice (5D6, 12A10, 15D7, 7E2, or 13C7) or with a control co...

Claims

1. Attorney Docket No. 090723-1530315-MDA25-028PCTWHAT IS CLAIMED IS:

1. An isolated antibody or antigen-binding fragment thereof, comprising: a) a heavy chain variable region comprising:(i) a CDRH1 comprising SEQ ID NOs: 11, 14, or 19;(ii) a CDRH2 comprising SEQ ID NOs: 12, 15, 17, or 20; and(iii) a CDRH3 comprising SEQ ID NOs: 13, 16, 18, or 21; and b) a light chain variable region comprising:(i) a CDRL1 comprising SEQ ID NOs: 22, 25, 30, or 33;(ii) a CDRL2 comprising SEQ ID NOs: 23, 26, 28, or 31; and(iii) a CDRL3 comprising SEQ ID NOs: 24, 27, 29, or 32.

2. The isolated antibody or antigen-binding fragment of claim 1, wherein the isolated antibody or antigen-binding domain comprises: a) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24; b) a CDRH1 comprising SEQ ID NO: 14, a CDRH2 comprising SEQ ID NO: 15, a CDRH3 comprising SEQ ID NO: 16; a CDRL1 comprising SEQ ID NO: 25, a CDRL2 comprising SEQ ID NO: 26, and a CDRL3 comprising SEQ ID NO: 27; c) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 17, a CDRH3 comprising SEQ ID NO: 18; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 29; d) a CDRH1 comprising SEQ ID NO: 19, a CDRH2 comprising SEQ ID NO: 20, a CDRH3 comprising SEQ ID NO: 21; a CDRL1 comprising SEQ ID NO: 30, a CDRL2 comprising SEQ ID NO: 31, and a CDRL3 comprising SEQ ID NO: 32; or e) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 33, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 24.

3. The isolated antibody or antigen-binding fragment of claim 1 or 2, wherein: a. the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 1, and the light chain variable region comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 6;Attorney Docket No. 090723-1530315-MDA25-028PCT b. the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 2, and the light chain variable region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 7; c. the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 8; d. the heavy chain variable region comprises an amino acid sequence that is at least 87% identical to SEQ ID NO: 4, and the light chain variable region comprises SEQ ID NO: 9; or e. the heavy chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 5, and the light chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 10.

4. The isolated antibody or antigen-binding fragment of claim 1 or 2, wherein: a. the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6; b. the heavy chain variable region comprises SEQ ID NO: 2, and the light chain variable region comprises SEQ ID NO: 7; c. the heavy chain variable region comprises SEQ ID NO: 3, and the light chain variable region comprises SEQ ID NO: 8; d. the heavy chain variable region comprises SEQ ID NO: 4, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 9; or e. the heavy chain variable region comprises SEQ ID NO: 5, and a light chain variable region comprises SEQ ID NO: 10.

5. The isolated antibody or antibody fragment of claim 1 or 2, wherein the isolated antibody or antibody fragment comprises a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24.

6. The isolated antibody or antibody fragment of claim 5, wherein the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6.Attorney Docket No. 090723-1530315-MDA25-028PCT7. The isolated antibody or antibody fragment of claim 1 or 2, wherein the antibody or antibody fragment is a chimeric antibody or antibody fragment.

8. The isolated antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody fragment is a monovalent scFv (single chain fragment variable) antibody, divalent scFv, Fab fragment, F(ab’)2 fragment, F(ab’)s fragment, Fv fragment, or single chain antibody.

9. The isolated antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody or antibody fragment is a chimeric antibody, bispecific antibody, trispecific antibody, or other multi-specific antibody.

10. The isolated antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody or antibody fragment is conjugated or fused to an imaging agent, a cytotoxic agent, or a radioactive moiety.

11. The isolated antibody or antigen-binding fragment of claim 10, wherein the antibody or antibody fragment is conjugated or fused to an imaging agent, and wherein the imaging agent is a fluorophore.

12. The isolated antibody or antigen-binding fragment of claim 10, wherein the antibody or antibody fragment is conjugated or fused to a radioactive moiety, and wherein the radioactive moiety is Zr-89, Cu-64, F-18, Y-90, Lu-177, At-211, Ac-225, or Pb-212.

13. The isolated antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody is an immune conjugate.

14. The isolated antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody or antibody fragment is an antibody-drug conjugate.

15. A pharmaceutical composition comprising the isolated antibody or antigenbinding fragment of claim 1 or 2 and a pharmaceutically acceptable carrier.

16. An isolated nucleic acid encoding the antibody heavy and / or light chain variable region of the antibody or antigen-binding fragment of claim 1 or 2.

17. A hybridoma or engineered cell comprising the nucleic acid encoding the antibody or antigen-binding fragment of claim 1 or 2.Attorney Docket No. 090723-1530315-MDA25-028PCT18. A hybridoma or engineered cell comprising the nucleic acid of claim 16.

19. A method of making an isolated antibody or antigen-binding fragment, comprising culturing the hybridoma or engineered cell of claim 17 under conditions that allow expression of the antibody or antigen-binding fragment and, optionally, isolating the antibody or antigen-binding fragment from the culture.

20. An antibody-peptide conjugate, comprising: c) the antibody or antigen-binding fragment of claim 1 or 2; and d) an immunogenic peptide.

21. The antibody-peptide conjugate of claim 20, wherein the immunogenic peptide comprises a native cytomegalovirus (NLV) peptide or a cathepsin G 1 (CGI) peptide.

22. The antibody-peptide conjugate of claim 20, wherein the immunogenic peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:34.

23. The antibody-peptide conjugate of claim 20, wherein the immunogenic peptide comprises SEQ ID NO:34.

24. The antibody-peptide conjugate of claim 20, wherein the immunogenic peptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:35.

25. The antibody-peptide conjugate of claim 24, wherein the immunogenic peptide comprises SEQ ID NO:35.

26. The antibody-peptide conjugate of claim 20, wherein the antibody or antigenbinding fragment is conjugated to the immunogenic peptide by a chemical linkage.

27. The antibody-peptide conjugate of claim 26, wherein the chemical linkage comprises an attachment group and protease cleavable linker.

28. The antibody-peptide conjugate of claim 27, wherein the attachment group comprises a maleimidocaproyl moiety, and the protease-cleavable linker comprises a valine- citrulline- / ?-aminocarbamate (VC-PABC) linker.Attorney Docket No. 090723-1530315-MDA25-028PCT29. The antibody-peptide conjugate of claim 20, wherein the heavy chain of the antibody or antigen-binding fragment is conjugated to the immunogenic peptide by a recombinant linker.

30. The antibody-peptide conjugate of claim 29, wherein the recombinant linker comprises a glycine-serine linker sequence and a protease-cleavable linker sequence.

31. The antibody-peptide conjugate of claim 29, wherein the glycine-serine linker sequence comprises GS or SEQ ID NO: 36.

32. The antibody-peptide conjugate of claim 29, wherein the protease-cleavable linker comprises SEQ ID NO: 37.

33. The antibody-peptide conjugate of claim 29, wherein the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising SEQ ID NO: 36, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, a second protease-cleavable linker sequence comprising SEQ ID NO:37, and a second copy of the immunogenic peptide.

34. The antibody-peptide conjugate of claim 29, wherein the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising GS, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, and a second protease-cleavable linker sequence comprising SEQ ID NO: 37.

35. The antibody-peptide conjugate of claim 29, wherein the antibody-conjugate comprises the antibody or antigen-binding fragment thereof, a glycine-serine linker sequence comprising GS, a first protease-cleavable linker sequence comprising SEQ ID NO: 37, a first copy of the immunogenic peptide, a second protease-cleavable linker sequence comprising SEQ ID NO: 37, a second copy of the immunogenic peptide, and a third protease-cleavable linker sequence comprising SEQ ID NO:37.

36. The antibody-peptide conjugate of claim 20, wherein the antibody or antigenbinding fragment comprises: a) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ IDAttorney Docket No. 090723-1530315-MDA25-028PCTNO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24; b) a CDRH1 comprising SEQ ID NO: 14, a CDRH2 comprising SEQ ID NO: 15, a CDRH3 comprising SEQ ID NO: 16; a CDRL1 comprising SEQ ID NO: 25, a CDRL2 comprising SEQ ID NO: 26, and a CDRL3 comprising SEQ ID NO: 27; c) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 17, a CDRH3 comprising SEQ ID NO: 18; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 29; d) a CDRH1 comprising SEQ ID NO: 19, a CDRH2 comprising SEQ ID NO: 20, a CDRH3 comprising SEQ ID NO: 21; a CDRL1 comprising SEQ ID NO: 30, a CDRL2 comprising SEQ ID NO: 31, and a CDRL3 comprising SEQ ID NO: 32; or e) a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 33, a CDRL2 comprising SEQ ID NO: 28, and a CDRL3 comprising SEQ ID NO: 24.

37. The antibody-peptide conjugate of any one of claims 20-36, wherein: f) the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 1, and the light chain variable region comprising an amino acid sequence that is at least 97% identical to SEQ ID NO: 6; g) the heavy chain variable region comprises an amino acid sequence that is at least 86% identical to SEQ ID NO: 2, and the light chain variable region comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 7; h) the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 3, and the light chain variable region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 8; i) the heavy chain variable region comprises an amino acid sequence that is at least 87% identical to SEQ ID NO: 4, and the light chain variable region comprises SEQ ID NO: 9; orAttorney Docket No. 090723-1530315-MDA25-028PCT j) the heavy chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 5, and the light chain variable region comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 10.

38. The antibody-peptide conjugate of claim 20, wherein: a) the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6; b) the heavy chain variable region comprises SEQ ID NO: 2, and the light chain variable region comprises SEQ ID NO: 7; c) the heavy chain variable region comprises SEQ ID NO: 3, and the light chain variable region comprises SEQ ID NO: 8; d) the heavy chain variable region comprises SEQ ID NO: 4, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 9; or e) the heavy chain variable region comprises SEQ ID NO: 5, and a light chain variable region comprises SEQ ID NO: 10.

39. The antibody-peptide conjugate of claim 20, wherein the isolated antibody or antibody fragment comprises a CDRH1 comprising SEQ ID NO: 11, a CDRH2 comprising SEQ ID NO: 12, a CDRH3 comprising SEQ ID NO: 13; a CDRL1 comprising SEQ ID NO: 22, a CDRL2 comprising SEQ ID NO: 23, and a CDRL3 comprising SEQ ID NO: 24.

40. The antibody-peptide conjugate of claim 20, wherein the heavy chain variable region comprises SEQ ID NO: 1, and the light chain variable region comprises SEQ ID NO: 6.

41. A pharmaceutical composition comprising the antibody-peptide conjugate of claim 20 and a pharmaceutically acceptable carrier.

42. A method of imaging a cell or tissue expressing neuropilin-1 (NRP1) in a subject, the method comprising:(a) administering to the subject the isolated antibody or antibody fragment of claim 10, and(b) detecting the imaging agent in the subject, thereby imaging the cell or tissue.Attorney Docket No. 090723-1530315-MDA25-028PCT43. A method for diagnosing a subject having or suspected of having a cancer characterized by elevated neuropilin-1 (NRP1) expression, comprising:(a) contacting a biological sample with the isolated antibody or antigen-binding fragment of claim 1 or 2; and(b) detecting an amount of binding of the isolated antibody or antigen-binding fragment as a determination of a presence of NRP-1 in the biological sample.

44. A method of treating a human subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 15.

45. A method of treating a human subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 41.

46. The method of claim 43, wherein the subject has cancer.

47. The method of claim 46, wherein the cancer expresses an elevated level of neuropilin-1 (NRP1) relative to a healthy control tissue.

48. The method of claim 46, wherein the cancer is selected from a group consisting of breast, renal, lung, liver, head, neck, and squamous cell cancer.

49. The method of claim 44, further comprising administering to the subject a therapeutically effective amount of a small molecule drug.

50. The method of claim 44, wherein the isolated antibody or antigen-binding fragment is conjugated to a therapeutic agent.

51. The method of claim 50, wherein the therapeutic agent is at least one of a cytotoxic agent, a photosensitizer, a chemotherapeutic agent, or an immunosuppressive agent.

52. The method of claim 50, wherein the therapeutic agent is a moiety that specifically binds to an immune cell.

53. The method of claim 52, wherein the immune cell is a T cell.

54. The method of claim 52, wherein the immune cell is a natural killer cell.Attorney Docket No. 090723-1530315-MDA25-028PCT55. The method of claim 44, wherein the therapeutically effective amount of the pharmaceutical composition is administered via intravenous injection, subcutaneous injection, or direct injection into a diseased tissue.

56. The method of claim 45, further comprising administering a composition that binds to the peptide of the antibody-peptide conjugate.

57. The method of claim 56, wherein the composition comprises a peptide-specific antibody, T cell, or chimeric antigen receptor (CAR)-T cell.

58. A method of inhibiting the binding of neuropilin- 1 (NRP1) to VEGF or semaforin 3a, comprising administering the isolated antibody or antibody fragment of claim 1 or 2.

59. The method of claim 58, wherein administering the isolated antibody or antibody fragment inhibits an immune inhibitory action of a regulatory T cell.

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