Antibodies for targeting PSMA and uses thereof

Abstract

Disclosed herein are isolated polypeptide and polypeptide complex compositions that comprise a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA.

Description

[0001] WSGR Docket No. 52426-777.601

[0002] ANTIBODIES FOR TARGETING PSMA AND USES THEREOF

[0003] CROSS REFERENCE

[0004]

[0001] The present application claims the benefit of U. S. Provisional ApplicationNo. 63 / 715,025 filed on November 1, 2024, which is incorporated herein by reference it its entirety.

[0005] SEQUENCE LISTING

[0006]

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 24, 2025 is named 52426-777_601_SL.xml and is 2,201,047 bytes in size.

[0007] SUMMARY

[0008]

[0003] Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to X1-X2-X3-X4-C-X5-P-X6-W-X7-C-X8-X9-X10, wherein Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R; X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K; X3is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D; X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D; X5is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; X6is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; X7 is selected from M, L, I, A, V, F, G, and K; Xs is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and X10 is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K (SEQ ID NO: 2486).

[0009]

[0004] In some embodiments, Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, and W; X2is selected from E, V, D, T, S, L, G, P, A, M, and I; X3 is selected from K, P, R, I, N, H, V, M, A, and L; X4is selected from W, L, M, R, V, Y, and A; X5 is selected from I, V, T, K, R, and E; X& is selected from A, E, S, P, Q, T, L, D, M, and V; X7 is M; Xs is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, and H; X9 is selected from G, V, A, S, W, E, D, and M; and X10 is selected from F, L, M, S, I, V, D, and Q. In some embodiments, Xi is selected from V, E, L, D, I, G, and M; X2is selected from E, V, D, T, S, L, G, P, and A; X3 is selected from K, P, R, I, N, H, V, and M; X4is selected from W, L, M, and R; X5 is selected from I, V, T, K, R, and E; Xe is selected from A, E, S, P, Q, and T; X7 is M; Xs is selected from E, S, T, A, V, D, and Q; X9 is selected from G, V, A, S, and W; and X10 is selected from F, L, M, and S. In some embodiments, Xi is selected from V, E, L, D, I, and G; X2is selected from E, V, D, T, S, and, G; X3 is selected from K, P, R, I, N, and H; X4is selected from W, L, and M; X5 is selected from I, V, T, K, and R; Xe is selected from A, E, S, and P; X7 is M; Xs is selected from E, S, T, A, and V; X9 is G; and X10 is selected from F, L, and M. WSGR Docket No. 52426-777.601

[0010]

[0005] In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 118. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 115. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 85-94. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 24. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 95-104. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 105-131.

[0011]

[0006] Disclosed herein, in some embodiments, are isolated polypeptide or polypeptide complexes comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 284-2479. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 -84, 105-131, and 284-2479. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 63. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 74. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 77. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 112. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 115.

[0012]

[0007] In some embodiments, the PSMA binding domain comprises an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain that comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 comprise the amino acid sequences of HC-CDR1 : SEQ ID NO: 132, HC-CDR2: SEQ ID NO: 133, and HC-CDR3: SEQ ID NO: 134, and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain that comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 comprise the amino acid sequences of LC-CDR1: SEQ ID NO: 135, LC-CDR2: EA, and LC-CDR3: SEQ ID: 137.

[0013]

[0008] In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139. In some embodiments, the anti-PSMA light chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139, and the anti-PSMA light chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138.

[0014]

[0009] In some embodiments, the anti-PSMA antibody comprises a single chain variable fragment (scFv), a single domain antibody, a Fab, a Fab’, or a CrossFab. In some embodiments, the anti-PSMA antibody WSGR Docket No. 52426-777.601 comprises the Fab or Fab’. In some embodiments, the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139. In some embodiments, the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. In some embodiments, the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 142 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 143.

[0015]

[0010] In some embodiments, the peptide is connected to the PSMA binding domain in a configuration according to Ai-Li-Pi wherein Ai comprises the PSMA binding domain, Pi comprises the peptide that impairs binding of the PSMA binding domain to PSMA, and Li comprises a cleavable linker. In some embodiments, Pi is connected N-terminal to the cleavable linker and Ai is connected C-terminal to the cleavable linker. In some embodiments, Pi is connected C-terminal to the cleavable linker and Ai is connected N-terminal to the cleavable linker.

[0016] [OH] In some embodiments, Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, H-bonding interactions, or a combination thereof. In some embodiments, Pi is bound to Ai at or near an antigen binding site. In some embodiments, Pi becomes unbound from Ai when Li is cleaved by a tumor specific protease, thereby exposing Ai to PSMA. In some embodiments, Pi has less than 75% sequence identity to PSMA. In some embodiments, Pi comprises a de novo amino acid sequence that shares less than 10% sequence identity to PSMA. In some embodiments, Pi comprises a modified amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, Pi does not comprise albumin or an albumin fragment. In some embodiments, Pi does not comprise an albumin binding domain.

[0017]

[0012] In some embodiments, Li is bound to the N-terminus of Ai. In some embodiments, Li is bound to the C-terminus of Ai. In some embodiments, Li is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, Li is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, Li is a peptide sequence having at least 10 amino acids. In some embodiments, Li is a peptide sequence having at least 18 amino acids. In some embodiments, Li is a peptide sequence having at least 26 amino acids. In some embodiments, Li comprises a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 2484). In some embodiments, Li comprises a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 2480), (GGGS)n(SEQ ID NO: 2481), (GGGGS)n(SEQ ID NO: 2482), and (GSSGGS)n(SEQ ID NO: 2483), wherein n is an integer of at least 1. WSGR Docket No. 52426-777.601

[0018]

[0013] In some embodiments, Li comprises an amino acid sequence selected from any one of SEQ ID NOs: 144-175. In some embodiments, Li comprises an amino acid sequence according to SEQ ID NO: 173. In some embodiments, Li is a substrate for a tumor specific protease. In some embodiments, Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to PSMA. In some embodiments, Li comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, or a matrix metalloprotease cleavable amino acid sequence.

[0019]

[0014] In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 274, 276, 280, and 282 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 275, 277, 281, and 283. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 256, 258, 260, 262, 264, 266, 268, 270, 274, 276, 280, and 282 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 257, 259, 261, 263, 265, 267, 269, 271, 275, 277, 281, and 283.

[0020]

[0015] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising the isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein, and a pharmaceutically acceptable excipient.

[0021]

[0016] Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein. Disclosed herein, in some embodiments, are vectors comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein. Disclosed herein, in some embodiments, are host cells comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0022]

[0017] Disclosed herein, in some embodiments, are methods of treating cancer in a subject in need thereof comprising administering to the subject an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein. In some embodiments, the cancer has cells that express PSMA. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is prostate, lung, breast, cervical, ovarian, colorectal, pancreatic, or gastric cancer.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

[0018] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the WSGR Docket No. 52426-777.601 following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0025]

[0019] FIGs. 1-2 illustrate binding of PSMA Fab to peptides as measured by ELISA.

[0026]

[0020] FIGs. 3-4 illustrate inhibition of PSMA Fab binding to PSMA by peptides of the present disclosure.

[0027]

[0021] FIG. 5 illustrates BLI titration data for PSMA Fab binding to peptide-23.

[0028]

[0022] FIG. 6 illustrates BLI titration data for PSMA Fab binding to peptide-63.

[0029]

[0023] FIG. 7 illustrates BLI titration data for PSMA Fab binding to peptide-69.

[0030]

[0024] FIG. 8 illustrates BLI titration data for PSMA Fab binding to peptide-71.

[0031]

[0025] FIG. 9 illustrates BLI titration data for PSMA Fab binding to peptide-74.

[0032]

[0026] FIG. 10 illustrates BLI titration data for PSMA Fab binding to peptide-76.

[0033]

[0027] FIG. 11 illustrates BLI titration data for PSMA Fab binding to peptide-77.

[0034]

[0028] FIG. 12 illustrates BLI titration data for PSMA Fab binding to peptide-79.

[0035]

[0029] FIG. 13 illustrates BLI titration data for PSMA Fab binding to peptide-80.

[0036]

[0030] FIG. 14 illustrates BLI titration data for PSMA Fab binding to peptide-83.

[0037]

[0031] FIG. 15 illustrates BLI titration data for PSMA Fab binding to peptide-24.

[0038]

[0032] FIG. 16 illustrates BLI titration data for PSMA Fab binding to peptide-107.

[0039]

[0033] FIG. 17 illustrates BLI titration data for PSMA Fab binding to peptide-111.

[0040]

[0034] FIG. 18 illustrates BLI titration data for PSMA Fab binding to peptide-112.

[0041]

[0035] FIG. 19 illustrates BLI titration data for PSMA Fab binding to peptide-114.

[0042]

[0036] FIG. 20 illustrates BLI titration data for PSMA Fab binding to peptide-115.

[0043]

[0037] FIG. 21 illustrates BLI titration data for PSMA Fab binding to peptide-118.

[0044]

[0038] FIG. 22 illustrates BLI titration data for PSMA Fab binding to peptide-121.

[0045]

[0039] FIG. 23 illustrates BLI titration data for PSMA Fab binding to peptide-126.

[0046]

[0040] FIG. 24 illustrates BLI titration data for PSMA Fab binding to peptide-127.

[0047]

[0041] FIG. 25 illustrates BLI titration data for PSMA Fab binding to peptide-128.

[0048]

[0042] FIG. 26 illustrates BLI titration data for PSMA Fab binding to peptide-129.

[0049]

[0043] FIG. 27 illustrates BLI titration data for PSMA Fab binding to peptide-130.

[0050]

[0044] FIG. 28 illustrates the core sequence motif of anti-PSMA Fab peptide-24 sequences generated using Weblogo 3.7.12.

[0051] DETAILED DESCRIPTION

[0052]

[0045] Protein-based therapies such as antibodies and bispecific or multispecific antibodies, such as T cell engagers, have proven effective for a variety diseases and disorders. As with any therapy, there is a need to minimize off-target effects of the protein-based therapy in healthy tissue while maintaining activity of the protein-based therapy in disease tissue. One such strategy is to create an inactive form of the protein -based therapy in which a necessary binding site on the protein-based therapy is blocked with a molecule linked to the protein-based therapy, thereby preventing the protein-based therapy from binding or interacting with its WSGR Docket No. 52426-777.601 cognate receptor or target antigen when in healthy tissue. For activating the protein -based therapy in the desired disease-state microenvironment, the molecule is cleaved from the protein-based therapy by a protease that is specific to the disease-state microenvironment. The molecule is then released from the protein-based therapy and the protein-based therapy is free to interact with its cognate receptor or target antigen in the diseased tissue.

[0053]

[0046] Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), or NAAG peptidase is an enzyme that resides in membranes. Human PSMA is highly expressed in the prostate, roughly a hundred times greater than in most other tissues. In some prostate cancers, PSMA is the second-most upregulated gene product, with an 8- to 12-fold increase over levels in noncancerous prostate cells. Disclosed herein are peptides that bind to and impair binding of PSMA binding domains to PSMA. In some embodiments, the peptides are attached to the PSMA binding domain via a cleavable linker that is selectively cleaved in tumor microenvironments such that the PSMA binding domain is available for binding to the PSMA target in tumor microenvironments. The peptides as disclosed herein can be applied to a variety of antibody formats that bind to PSMA to reduce the off-target effects of the antibodies in healthy tissue while maintaining activity of the anti -PSMA in diseased tissue.

[0054] Certain Definitions

[0055]

[0047] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0056]

[0048] The term “antibody” is used in the broadest sense and covers fully assembled antibodies, antibody fragments that can bind antigen, for example, Fab, F(ab’)2, Fv, single chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, and the like.

[0057]

[0049] The term “complementarity determining region” or “CDR” is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region. Accordingly, a CDR is sometimes referred to as hypervariable region. A variable region comprises three CDRs. CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody -producing cells. See, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications , Birch et al., (eds.), pages 137-185 (Wiley-Liss, Inc. 1995). WSGR Docket No. 52426-777.601

[0058]

[0050] The term “Fab” refers to a protein that contains the constant domain of the light chain and the first constant domain (CHI) 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 CHI domain including one or more cysteines from the antibody 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. Fab' fragments are produced by reducing the F(ab’)2 fragment’s heavy chain disulfide bridge. Other chemical couplings of antibody fragments are also known.

[0059]

[0051] A “CrossFab configuration” as used herein is a Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (replaced by each other) (see, e.g., W02009 / 080252A1 and WO2017 / 055388A2). For example, a Fab molecule in a CrossFab configuration may comprise a peptide chain composed of the variable light chain domain and the heavy chain constant domain (VL-CH1 in an N- to C-terminal direction), and a peptide chain composed of the variable heavy chain domain and the light chain constant domain (VH-CL in an N- to C-terminal direction).

[0060]

[0052] A “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, e.g. described in Houston, J. S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with aVH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.

[0061]

[0053] As used herein, the term “percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0062]

[0054] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence WSGR Docket No. 52426-777.601 alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0063]

[0055] The terms “complementarity determining region,” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” are known in the art to refer to non -contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and / or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabatet al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabaf ’ numbering scheme), Al- Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT’ numbering scheme); Honegger A and Pluckthun A, ‘Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and WhiteleggNR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng. 2000 Dec;13(12):819-24 (“AbM’ numbering scheme. In certain embodiments the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0064]

[0056] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0065] Isolated Polypeptide or Polypeptide Complexes

[0066]

[0057] Disclosed herein are isolated polypeptide or polypeptide complexes comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA WSGR Docket No. 52426-777.601 binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to X1-X2-X3- X4-C-X5-P-X6-W-X7-C-X8-X9-X10, whereinXi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R; X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K; X3is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D; X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D; X5is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; X6is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; X7is selected from M, L, I, A, V, F, G, and K; X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W. G, K, R, and P; X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and X10 is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K (SEQ ID NO: 2486).

[0067]

[0058] In some embodiments, Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, and W; X2is selected from E, V, D, T, S, L, G, P, A, M, and I; X3 is selected from K, P, R, I, N, H, V, M, A, and L; X4is selected from W, L, M, R, V, Y, and A; X5 is selected from I, V, T, K, R, and E; X& is selected from A, E, S, P, Q, T, L, D, M, and V; X7 is M; X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, and H; X9 is selected from G, V, A, S, W, E, D, and M; and X10 is selected from F, L, M, S, I, V, D, and Q. In some embodiments, Xi is selected from V, E, L, D, I, G, and M; X2is selected from E, V, D, T, S, L, G, P, and A; X3 is selected from K, P, R, I, N, H, V, and M; X4is selected from W, L, M, and R; X5 is selected from I, V, T, K, R, and E; Xe is selected from A, E, S, P, Q, and T; X7 is M; X8is selected from E, S, T, A, V, D, and Q; X9 is selected from G, V, A, S, and W; and X10 is selected from F, L, M, and S. In some embodiments, Xi is selected from V, E, L, D, I, and G; X2is selected from E, V, D, T, S, and, G; X3 is selected from K, P, R, I, N, and H; X4is selected from W, L, and M; X5 is selected from I, V, T, K, and R; Xe is selected from A, E, S, and P; X7 is M; X8is selected from E, S, T, A, and V; X9 is G; and X10 is selected from F, L, and M.

[0068]

[0059] In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 118. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 115. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 85-94. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 24. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 95-104. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 105-131.

[0069]

[0060] Disclosed herein, in some embodiments, are isolated polypeptide or polypeptide complexes comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 -131 and 284-2479 or the peptide comprises 1, 2, or 3 amino acid substitutions, additions, or deletions relative to an amino acid sequence selected from any one of SEQ ID NOs: 1-131 and 284-2479. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 284-2479. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 -84 and 105-131, and 284-2479. In some WSGR Docket No. 52426-777.601 embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 63. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 74. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 77. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 112. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 115.

[0070] Table 1. Peptide Mask Sequences for PSMA Binding Domain WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601

[0071] Table 2. Peptide Mask Sequences for PSMA Binding Domain (Alanine scanning peptides of Peptide- 23)

[0072] Table 3. Peptide Mask Sequences for PSMA Binding Domain (Alanine scanning peptides of Peptide- 24)

[0073] Table 4. Peptide Mask Sequences for PSMA Binding Domain (Directed evolution mask optimization) WSGR Docket No. 52426-777.601

[0074] PSMA Binding Domain

[0075]

[0061] In some embodiments, the PSMA binding domain comprises an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain that comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 comprise the amino acid sequences of HC-CDR1 : SEQ ID NO: 132, HC-CDR2: SEQ ID NO: 133, and HC-CDR3: SEQ ID NO: 134, and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain that comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 comprise the amino acid sequences of LC-CDR1: SEQ ID NO: 135, LC-CDR2: EA, and LC-CDR3: SEQ ID: 137.

[0076] Table 5. Exemplary PSMA Binding Domain Sequences. CDRs determined by IMGT definition WSGR Docket No. 52426-777.601

[0077]

[0062] In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139. In some embodiments, the anti-PSMA light chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139, and the anti-PSMA light chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138.

[0078]

[0063] In some embodiments, the anti-PSMA antibody comprises a single chain variable fragment (scFv), a single domain antibody, a Fab, a Fab’, or a CrossFab. In some embodiments, the anti-PSMA antibody comprises the Fab or Fab’ . In some embodiments, the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139. In some embodiments, the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. In some embodiments, the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 142 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 143.

[0079] Ai-Li-Pi Configuration

[0080]

[0064] In some embodiments, the peptide is connected to the PSMA binding domain in a configuration according to Ai-Li-Pi wherein Ai comprises the PSMA binding domain, Pi comprises the peptide that impairs binding of the PSMA binding domain to PSMA, and Li comprises a cleavable linker. In some WSGR Docket No. 52426-777.601 embodiments, Pi is connected N-terminal to the cleavable linker and Ai is connected C-terminal to the cleavable linker. In some embodiments, Pi is connected C-terminal to the cleavable linker and Ai is connected N-terminal to the cleavable linker.

[0081]

[0065] In some embodiments, Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, H-bonding interactions, or a combination thereof. In some embodiments, Pi is bound to Ai at or near an antigen binding site. In some embodiments, Pi becomes unbound from Ai when Li is cleaved by a tumor specific protease, thereby exposing Ai to PSMA. In some embodiments, Pi has less than 75% sequence identity to PSMA. In some embodiments, Pi comprises a de novo amino acid sequence that shares less than 10% sequence identity to PSMA. In some embodiments, Pi comprises a modified amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, Pi does not comprise albumin or an albumin fragment. In some embodiments, Pi does not comprise an albumin binding domain.

[0082] Li

[0083]

[0066] In some embodiments, LI is bound to the N-terminus of Al . In some embodiments, Li is bound to the C-terminus of Ai. In some embodiments, Li is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, Li is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, Li is a peptide sequence having at least 10 amino acids. In some embodiments, Li is a peptide sequence having at least 18 amino acids. In some embodiments, Li is a peptide sequence having at least 26 amino acids. In some embodiments, Li comprises a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 2484). In some embodiments, Li comprises a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 2480), (GGGS)n(SEQ ID NO: 2481), (GGGGS)n(SEQ ID NO: 2482), and (GSSGGS)n(SEQ ID NO: 2483), wherein n is an integer of at least 1. In some embodiments, Li comprises an amino acid sequence selected from any one of SEQ ID NOs: 144-175. In some embodiments, Li comprises an amino acid sequence according to SEQ ID NO: 173.

[0084]

[0067] In some embodiments, Li is a substrate for a tumor specific protease. In some embodiments, P i becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to PSMA. In some embodiments, Li comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, or a matrix metalloprotease cleavable amino acid sequence.

[0085] Table 6. Linker Amino Acid Sequences WSGR Docket No. 52426-777.601

[0086] Ai-Li-Pi Sequences

[0087]

[0068] In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 274, 276, 280, and 282 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 275, 277, 281, and 283. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 256, 258, 260, 262, 264, 266, 268, 270, 274, 276, 280, and 282 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 257, 259, 261, 263, 265, 267, 269, 271, 275, 277, 281, and 283.

[0088] Table. 7 Masked Polypeptide Complexes (A-L-P) WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601

[0089] Modified Amino Acids

[0090]

[0069] In some embodiments, the isolated polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof. In some embodiments, the non-natural amino acid comprises a D-amino acid. In some embodiments, the modified amino acid or modified non-natural amino acid comprises a post -translational modification.

[0091] Pharmaceutical Compositions WSGR Docket No. 52426-777.601

[0092]

[0070] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising: (i) the isolated polypeptide or polypeptide complex according to any embodiment disclosed herein, and (ii) a pharmaceutically acceptable excipient.

[0093]

[0071] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to X1-X2-X3-X4-C-X5-P-X6-W-X7-C-X8-X9-X10, wherein Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R; X2 is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K; X3is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D; X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D; X5 is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; Xe is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; X7 is selected from M, L, I, A, V, F, G, and K; Xs is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and X10 is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K (SEQ ID NO: 2486).

[0094]

[0072] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 -131 and 284-2479.

[0073] In some embodiments, the polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety. In some embodiments, the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.

[0095]

[0074] For administration to a subject, the polypeptide or polypeptide complex as disclosed herein, may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservatives, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.

[0096]

[0075] The pharmaceutical composition may be in any suitable form, depending upon the desired method of administration. It may be provided in unit dosage form, may be provided in a sealed container and may be provided as part of a kit. Such a kit may include instructions for use. It may include a plurality of said unit dosage forms. WSGR Docket No. 52426-777.601

[0097]

[0076] The pharmaceutical composition may be adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, or intravenous) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0098]

[0077] Dosages of the substances of the present disclosure can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

[0099] Isolated Recombinant Nucleic Acid Molecules

[0100]

[0078] Disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0101]

[0079] Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to X1-X2-X3-X4-C-X5-P-X6-W-X7-C-X8- X9-X10, wherein Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R; X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K; X3is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D; X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D; X5 is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; Xe is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; X7is selected from M, L, I, A, V, F, G, and K; X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and Xi0is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K (SEQ ID NO: 2486).

[0102]

[0080] Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 -131 and 284-2479.

[0103] Methods of Treatment

[0104]

[0081] Disclosed herein, in some embodiments, are methods of treating cancer in a subject in need thereof comprising administering to the subject an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0105]

[0082] Disclosed herein, in some embodiments, are methods of treating cancer in a subject in need thereof comprising administering to the subject an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to X1-X2-X3-X4-C-X5-P-X6-W-X7-C-X8-X9-X10, wherein Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R; X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K; X3 WSGR Docket No. 52426-777.601 is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D; X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D; X5is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; X6is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; X7is selected from M, L, I, A, V, F, G, and K; X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W. G, K, R, and P; X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and Xio is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K (SEQ ID NO: 2486).

[0106]

[0083] Disclosed herein, in some embodiments, are methods of treating cancer in a subject in need thereof comprising administering to the subject an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 284-2479.

[0107]

[0084] In some embodiments, the cancer has cells that express PSMA. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is prostate, lung, breast, cervical, ovarian, colorectal, pancreatic, or gastric cancer.

[0108] Production of Antibodies

[0109]

[0085] In some embodiments, the antibodies or antigen binding fragments thereof as described herein are produced using any method known in the art to be useful for the synthesis of polypeptides (e.g. , antibodies), in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

[0110]

[0086] In some instances, an antibody or its binding fragment thereof is expressed recombinantly, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0111]

[0087] Alternatively, a nucleic acid molecule encoding an antibody is optionally generated from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.

[0112]

[0088] In some instances, an antibody or its binding fragment is optionally generated by immunizing an animal, such as a mouse, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody is optionally obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246: 1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937). WSGR Docket No. 52426-777.601

[0113]

[0089] In some embodiments, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity are used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

[0114]

[0090] In some embodiments, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879- 5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242: 1038-1041).

[0115]

[0091] In some embodiments, an expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody is transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.

[0116]

[0092] In some embodiments, a variety of host-expression vector systems is utilized to express an antibody, or its binding fragment described herein. Such host-expression systems represent vehicles by which the coding sequences of the antibody is produced and subsequently purified, but also represent cells that are, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or its binding fragment in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an antibody or its binding fragment coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing an antibody or its binding fragment coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an antibody or its binding fragment coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an antibody or its binding fragment coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0117]

[0093] For long term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express an antibody are optionally engineered. Rather than using expression vectors that contain viral origins of replication, host cells are transformed with DNA controlled by WSGR Docket No. 52426-777.601 appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, poly adenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are then allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn are cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody or its binding fragments.

[0118]

[0094] In some instances, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes are employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance are used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O’Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; May 1993, TIB TECH 11(5): 155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30: 147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1). In some instances, the expression levels of an antibody are increased by vector amplification (for a review, see Bebbington and Hentschel, the use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0119]

[0095] In some instances, any method known in the art for purification of an antibody is used, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0120] Expression Vectors WSGR Docket No. 52426-777.601

[0121]

[0096] Disclosed herein, in some embodiments, are vectors comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0122]

[0097] Disclosed herein, in some embodiments, are vectors comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to X1-X2-X3- X4-C-X5-P-X6-W-X7-C-X8-X9-X10, whereinXi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R; X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K; X3is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D; X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D; X5is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; X6is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; X7is selected from M, L, I, A, V, F, G, and K; X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and X10 is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K (SEQ ID NO: 2486).

[0123]

[0098] Disclosed herein, in some embodiments, are vectors comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 284-2479.

[0124]

[0099] In some embodiments, vectors include any suitable vectors derived from either eukaryotic or prokaryotic sources. In some cases, vectors are obtained from bacteria (e.g. E. coli), insects, yeast (e.g. Pichiapastoris), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-l, pFLAGATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0125]

[0100] Exemplary insect vectors include pFastBacl, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 Mi l, pVL1393 M12, FLAG vectors such as pPolh-FLAGl or pPolh-MAT 2, or MAT vectors such as pPolh-MATl, or pPolh-MAT2.

[0126]

[0101] In some cases, yeast vectors include Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLDl Pichi pastoris vector, pGAPZA,B, & C Pichiapastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEFl / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B, & C yeast vector, or pYES3 / CT yeast vector. WSGR Docket No. 52426-777.601

[0127]

[0102] Exemplary algae vectors include pChlamy-4 vector or MCS vector.

[0128]

[0103] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MATl, pCMV-FLAG- MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Mammalian stable expression vector may include pFLAG- CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0129]

[0104] In some instances, a cell-free system is a mixture of cytoplasmic and / or nuclear components from a cell and is used for in vitro nucleic acid synthesis. In some cases, a cell -free system utilizes either prokaryotic cell components or eukaryotic cell components. Sometimes, a nucleic acid synthesis is obtained in a cell-free system based on for example Drosophila cell, Xenopus egg, or HeLa cells. Exemplary cell -free systems include, but are not limited to, E. coli S30 Extract system, E. coli T7 S30 system, or PURExpress®. Host Cells

[0130]

[0105] Disclosed herein, in some embodiments, are host cells comprising an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0131]

[0106] Disclosed herein, in some embodiments, are host cells comprising an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to X1-X2-X3-X4-C-X5-P-X6-W-X7-C-X8-X9-X10, wherein Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R; X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K; X3is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D; X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D; X5is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; X6is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; X7 is selected from M, L, I, A, V, F, G, and K; Xs is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and Xi0is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K (SEQ ID NO: 2486).

[0132]

[0107] Disclosed herein, in some embodiments, are host cells comprising an isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 284-2479.

[0133]

[0108] In some embodiments, a host cell includes any suitable cell such as a naturally derived cell or a genetically modified cell. In some instances, a host cell is a production host cell. In some instances, a host cell is a eukaryotic cell. In other instances, a host cell is a prokaryotic cell. In some cases, a eukaryotic cell includes fungi (e.g., yeast cells), animal cell or plant cell. In some cases, a prokaryotic cell is a bacterial WSGR Docket No. 52426-777.601 cell. Examples of bacterial cell include gram-positive bacteria or gram-negative bacteria. Sometimes the gram-negative bacteria is anaerobic, rod-shaped, or both.

[0134]

[0109] In some instances, gram-positive bacteria include Actinobacteria, Firmicutes or Tenericutes. In some cases, gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres- Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes- Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes or Synergistetes. Other bacteria can be Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria or Thermotogae. A bacterial cell can be Escherichia coli, Clostridium botulinum, or Coli bacilli.

[0135]

[0110] Exemplary prokaryotic host cells include, but are not limited to, BL21, Maehl™, DH10B™, TOPIO, DH5a, DHIOBac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F’, INVaF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0136] [Hl] In some instances, animal cells include a cell from a vertebrate or from an invertebrate. In some cases, an animal cell includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal. In some cases, a fungus cell includes a yeast cell, such as brewer’s yeast, baker’s yeast, or wine yeast. Fungi include ascomycetes such as yeast, mold, filamentous fungi, basidiomycetes, or zygomycetes. In some instances, yeast includes Ascomycota or Basidiomycota. In some cases, Ascomycota includes Saccharomycotina (true yeasts, e.g. Saccharomyces cerevisiae (baker’s yeast)) or Taphrinomycotina (e.g. Schizosaccharomycetes (fission yeasts)). In some cases, Basidiomycota includes Agari corny cotina (e.g. Tremellomycetes) or Pucciniomy cotina (e.g. Microbotryomycetes).

[0137]

[0112] Exemplary yeast or filamentous fungi include, for example, the genus: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeast or filamentous fungi include, for example, the species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru- Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neof ormans, Cryptococcus gattii, or Saccharomyces boulardii.

[0138]

[0113] Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strain such as INVScl.

[0139]

[0114] In some instances, additional animal cells include cells obtained from a mollusk, arthropod, annelid or sponge. In some cases, an additional animal cell is a mammalian cell, e.g., from a primate, ape, equine, WSGR Docket No. 52426-777.601 bovine, porcine, canine, feline or rodent. In some cases, a rodent includes mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, or guinea pig.

[0140]

[0115] Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells , 293 H cells, CHO DG44 cells, CHO-S cells, CH0-K1 cells, FUT8 KO CH0K1, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-l cell line, Flp-In™-Jurkat cell line, FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

[0141]

[0116] In some instances, a mammalian host cell is a stable cell line, or a cell line that has incorporated a genetic material of interest into its own genome and has the capability to express the product of the genetic material after many generations of cell division. In some cases, a mammalian host cell is a transient cell line, or a cell line that has not incorporated a genetic material of interest into its own genome and does not have the capability to express the product of the genetic material after many generations of cell division.

[0142] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells. In some instances, plant cells include a cell from algae. Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.

[0143] Articles of Manufacture

[0144]

[0117] In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle). At least one active agent in the composition is an isolate polypeptide or polypeptide complex of any embodiment disclosed herein. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises the bispecific antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.

[0145]

[0118] Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other WSGR Docket No. 52426-777.601 materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0146] EMBODIMENTS

[0147]

[0119] Embodiment 1. An isolated polypeptide or polypeptide complex comprising a prostatespecific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to Xi- X2-X3-X4-C-X5-P-X6-W-X7-C-X8-X9-X10, wherein: Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R; X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K; X3is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D; X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D; X5is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; X6is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; X7is selected from M, L, I, A, V, F, G, and K; X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and X10 is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K (SEQ ID NO: 2486).

[0148]

[0120] Embodiment 2. The isolated polypeptide or polypeptide complex of embodiment 1, wherein: Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, and W; X2is selected from E, V, D, T, S, L, G, P, A, M, and I; X3 is selected from K, P, R, I, N, H, V, M, A, and L; X4is selected from W, L, M, R, V, Y, and A; X5 is selected from I, V, T, K, R, and E; X& is selected from A, E, S, P, Q, T, L, D, M, and V; X7 is M; X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, and H; X9 is selected from G, V, A, S, W, E, D, and M; and X10 is selected from F, L, M, S, I, V, D, and Q.

[0149]

[0121] Embodiment 3. The isolated polypeptide or polypeptide complex of embodiment 1 or 2, wherein: Xi is selected from V, E, L, D, I, G, and M; X2is selected from E, V, D, T, S, L, G, P, and A; X3 is selected from K, P, R, I, N, H, V, and M; X4is selected from W, L, M, and R; X5 is selected from I, V, T, K, R, and E; X& is selected from A, E, S, P, Q, and T; X7 is M; X8is selected from E, S, T, A, V, D, and Q; X9 is selected from G, V, A, S, and W; and X10 is selected from F, L, M, and S.

[0150]

[0122] Embodiment 4. The isolated polypeptide or polypeptide complex of any one of embodiments 1-3, wherein: Xi is selected from V, E, L, D, I, and G; X2is selected from E, V, D, T, S, and, G; X3 is selected from K, P, R, I, N, and H; X4is selected from W, L, and M; X5 is selected from I, V, T, K, and R; Xe is selected from A, E, S, and P; X7 is M; X8is selected from E, S, T, A, and V; X9 is G; and X10 is selected from F, L, and M.

[0151]

[0123] Embodiment 5. The isolated polypeptide or polypeptide complex of any one of embodiments 1-4, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 118.

[0152]

[0124] Embodiment 6. The isolated polypeptide or polypeptide complex of any one of embodiments 1-4, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 115.

[0153]

[0125] Embodiment 7. The isolated polypeptide or polypeptide complex of any one of embodiments 1-4, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 23. WSGR Docket No. 52426-777.601

[0154]

[0126] Embodiment 8. The isolated polypeptide or polypeptide complex of any one of embodiments 1-4, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 85-94.

[0155]

[0127] Embodiment 9. The isolated polypeptide or polypeptide complex of any one of embodiments 1-4, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 24.

[0156]

[0128] Embodiment 10. The isolated polypeptide or polypeptide complex of any one of embodiments 1-4, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 95-104.

[0157]

[0129] Embodiment 11. The isolated polypeptide or polypeptide complex of any one of embodiments 1-4, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 105-131.

[0158]

[0130] Embodiment 12. An isolated polypeptide or polypeptide complex comprising a prostatespecific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 284-2479.

[0159]

[0131] Embodiment 13. The isolated polypeptide or polypeptide complex of embodiment 12, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 -84, 105-131, and 284-2479.

[0160]

[0132] Embodiment 14. The isolated polypeptide or polypeptide complex of embodiment 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 63.

[0161]

[0133] Embodiment 15. The isolated polypeptide or polypeptide complex of embodiment 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 74.

[0162]

[0134] Embodiment 16. The isolated polypeptide or polypeptide complex of embodiment 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 77.

[0163]

[0135] Embodiment 17. The isolated polypeptide or polypeptide complex of embodiment 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 112.

[0164]

[0136] Embodiment 18. The isolated polypeptide or polypeptide complex of embodiment 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 115.

[0165]

[0137] Embodiment 19. The isolated polypeptide or polypeptide complex of any one of the preceding embodiments, wherein the PSMA binding domain comprises an anti-PSMA antibody.

[0166]

[0138] Embodiment 20. The isolated polypeptide or polypeptide complex of embodiment 19, wherein the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain that comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC- CDR1, the HC-CDR2, and the HC-CDR3 comprise the amino acid sequences of HC-CDR1: SEQ ID NO: 132, HC-CDR2: SEQ ID NO: 133, and HC-CDR3: SEQ ID NO: 134, and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain that comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 WSGR Docket No. 52426-777.601 comprise the amino acid sequences of LC-CDR1 : SEQ ID NO: 135, LC-CDR2: EA, and LC-CDR3: SEQ ID: 137.

[0167]

[0139] Embodiment 21. The isolated polypeptide or polypeptide complex of embodiment 20, wherein the anti-PSMA heavy chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139.

[0168]

[0140] Embodiment 22. The isolated polypeptide or polypeptide complex of embodiment 20 or 21, wherein the anti-PSMA light chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138.

[0169]

[0141] Embodiment 23. The isolated polypeptide or polypeptide complex of any one of embodiments 20 to 22, wherein the anti-PSMA heavy chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139, and the anti-PSMA light chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138.

[0170]

[0142] Embodiment 24. The isolated polypeptide or polypeptide complex of any one of embodiments 19 to 23, wherein the anti-PSMA antibody comprises a single chain variable fragment (scFv), a single domain antibody, a Fab, a Fab’, or a CrossFab.

[0171]

[0143] Embodiment 25. The isolated polypeptide or polypeptide complex of embodiment 24, wherein the anti-PSMA antibody comprises the Fab or Fab’.

[0172]

[0144] Embodiment 26. The isolated polypeptide or polypeptide complex of embodiment 24 or 25, wherein the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139.

[0173]

[0145] Embodiment 27. The isolated polypeptide or polypeptide complex of embodiment 24 or 25, wherein the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.

[0174]

[0146] Embodiment 28. The isolated polypeptide or polypeptide complex of embodiment 24 or 25, wherein the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 142 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 143.

[0175]

[0147] Embodiment 29. The isolated polypeptide or polypeptide complex of any one of the preceding embodiments, wherein the peptide is connected to the PSMA binding domain in a configuration according to Ai-Li-Pi wherein Ai comprises the PSMA binding domain, Pi comprises the peptide that impairs binding of the PSMA binding domain to PSMA, and Li comprises a cleavable linker. WSGR Docket No. 52426-777.601

[0176]

[0148] Embodiment 30. The isolated polypeptide or polypeptide complex of embodiment 29, wherein Pi is connected N-terminal to the cleavable linker and Ai is connected C-terminal to the cleavable linker.

[0177]

[0149] Embodiment 31. The isolated polypeptide or polypeptide complex of embodiment 29, wherein Pi is connected C-terminal to the cleavable linker and Ai is connected N-terminal to the cleavable linker.

[0178]

[0150] Embodiment 32. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 31, wherein Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, H-bonding interactions, or a combination thereof.

[0179]

[0151] Embodiment 33. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 32, wherein Pi is bound to Ai at or near an antigen binding site.

[0180]

[0152] Embodiment 34. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 33, wherein Pi becomes unbound from Ai when Li is cleaved by a tumor specific protease, thereby exposing Ai to PSMA.

[0181]

[0153] Embodiment 35. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 34, wherein Pi has less than 75% sequence identity to PSMA.

[0182]

[0154] Embodiment 36. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 34, wherein Pi comprises a de novo amino acid sequence that shares less than 10% sequence identity to PSMA.

[0183]

[0155] Embodiment 37. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 36, wherein Pi comprises a modified amino acid, or a modified non-natural amino acid, or a combination thereof.

[0184]

[0156] Embodiment 38. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 37, wherein Pi does not comprise albumin or an albumin fragment.

[0185]

[0157] Embodiment 39. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 37, wherein Pi does not comprise an albumin binding domain.

[0186]

[0158] Embodiment 40. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 39, wherein Li is bound to the N-terminus of Ai.

[0187]

[0159] Embodiment 41. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 39, wherein Li is bound to the C-terminus of Ai.

[0188]

[0160] Embodiment 42. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li is a peptide sequence having at least 5 to no more than 50 amino acids.

[0189]

[0161] Embodiment 43. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li is a peptide sequence having at least 10 to no more than 30 amino acids.

[0190]

[0162] Embodiment 44. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li is a peptide sequence having at least 10 amino acids. WSGR Docket No. 52426-777.601

[0191]

[0163] Embodiment 45. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li is a peptide sequence having at least 18 amino acids.

[0192]

[0164] Embodiment 46. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li is a peptide sequence having at least 26 amino acids.

[0193]

[0165] Embodiment 47. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li comprises a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 2484).

[0194]

[0166] Embodiment 48. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li comprises a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 2480), (GGGS)n(SEQ ID NO: 2481), (GGGGS)n(SEQ ID NO: 2482), and (GSSGGS)n (SEQ ID NO: 2483), wherein n is an integer of at least 1.

[0195]

[0167] Embodiment 49. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li comprises an amino acid sequence selected from any one of SEQ ID NOs: 144-175.

[0196]

[0168] Embodiment 50. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li comprises an amino acid sequence according to SEQ ID NO: 173.

[0197]

[0169] Embodiment 51. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 41, wherein Li is a substrate for a tumor specific protease.

[0198]

[0170] Embodiment 52. The isolated polypeptide or polypeptide complex of embodiment 51, wherein Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to PSMA.

[0199]

[0171] Embodiment 53. The isolated polypeptide or polypeptide complex of any one of embodiments 29 to 52, wherein Li comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, or a matrix metalloprotease cleavable amino acid sequence.

[0200]

[0172] Embodiment 54. The isolated polypeptide or polypeptide complex of embodiment 1 or 12, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222,

[0201] 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,

[0202] 266, 268, 270, 274, 276, 280, and 282 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 177, 179, 181, 183, 185, 187, 189, 191, 193,

[0203] 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,

[0204] 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 275, 277, 281, and 283.

[0205]

[0173] Embodiment 55. The isolated polypeptide or polypeptide complex of embodiment 1 or 12, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 256, 258, 260, WSGR Docket No. 52426-777.601

[0206] 262, 264, 266, 268, 270, 274, 276, 280, and 282 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 257, 259, 261, 263, 265, 267, 269, 271, 275, 277, 281, and 283.

[0207]

[0174] Embodiment 56. A pharmaceutical composition comprising: (i) the isolated polypeptide or polypeptide complex of any one of the preceding embodiments; and (ii) a pharmaceutically acceptable excipient.

[0208]

[0175] Embodiment 57. An isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex of any one of embodiments 1-55.

[0209]

[0176] Embodiment 58. A vector comprising the isolated nucleic acid molecule of embodiment 57.

[0210]

[0177] Embodiment 59. A host cell comprising the isolated recombinant nucleic acid molecule of embodiment 57.

[0211]

[0178] Embodiment 60. A method of treating cancer in a subject in need thereof comprising administering to the subject the isolated polypeptide or polypeptide complex of any one of embodiments 1-

[0212]

[0179] Embodiment 61. The method of embodiment 60, wherein the cancer has cells that express

[0213] PSMA.

[0214]

[0180] Embodiment 62. The method of embodiment 60 or 61, wherein the cancer is a solid tumor cancer.

[0215]

[0181] Embodiment 63. The method of any one of embodiments 60 to 62, wherein the cancer is prostate, lung, breast, cervical, ovarian, colorectal, pancreatic, or gastric cancer.

[0216] EXAMPLES

[0217] Example 1:

[0218] Equilibrium Binding via ELISA

[0219]

[0182] Peptides were evaluated for their ability to bind to PSMA Fab in a standard enzyme linked immunosorbent assay (ELISA) format. Specifically, peptides were evaluated for their ability to bind polypeptide sequence PSMA Fab (SEQ ID NOs: 138, 139). Briefly, biotinylated peptides were captured on neutravidin coated plates. PSMA Fab diluted in buffer was then added to the peptide captured plates.

[0220] Bound antibody was detected using a standard horse radish peroxidase conjugate secondary antibody. The concentration of PSMA Fab required to achieve 50% maximal signal (ECso) was calculated using Graphpad Prism software. Exemplary binding curves for PSMA Fab binding to peptides are shown in FIGs. 1-2. ECsoS for PSMA Fab binding to the peptides are shown in Tables 8-10.

[0221] Binding Inhibition by Peptide Masks

[0222]

[0183] Peptides were further evaluated for their ability to inhibit PSMA Fab (SEQ ID NOs: 138, 139) from binding to the PSMA antigen in a standard ELISA format. Briefly, biotinylated PSMA antigen was captured WSGR Docket No. 52426-777.601 on neutravidin coated plates. PSMA Fab fixed at 0.3 nanomolar (nM) concentration was pre-incubated with 0-100 micromolar (pM) titrated peptides. After a short pre-incubati on period, the mixture of titrated peptide with fixed PSMA Fab were added to the PSMA antigen coated plates. After a short incubation on the plates, bound PSMA Fab was detected with a standard horse radish peroxidase conjugated secondary antibody. The concentration of peptide required to reduce the maximum signal by 50% (IC50) was calculated in Graphpad Prism software. FIGs. 3-4 illustrate inhibition of PSMA Fab binding to PSMA by the peptide masks as measured by ELISA. IC50S are provided in Tables 8-10.

[0223] Kinetic Binding, via BLI

[0224]

[0184] Kinetic binding peptides to PSMA Fab (SEQ ID NOs: 138, 139) was evaluated using biolayer interferometry (BLI). Briefly, biotinylated peptides were loaded onto a streptavidin coated Octet® SAX biosensor, quenched in biocytin, and baselined in buffer. PSMA Fab was titrated in solution (100 nM, 50 nM, 25 nM, and 12.5 nM) and associated onto the peptide loaded sensor. After a short association period, sensors were transferred into buffer and the dissociation of bound PSMA Fab was measured. Association and dissociation signals were recorded in real time and analyzed using a 1 : 1 binding model within the instrument software. Analysis using a 1: 1 binding model enabled the calculation of the on and off rate constant as well as affinity, KD. Binding sensorgrams are shown in FIGs. 5-27. Half-life (ti / 2) values obtained from the kinetic binding measurements are provided in Tables 9-10.

[0225] Peptide mask properties

[0226]

[0185] Peptide off rate, binding affinity, and inhibition strength are key design features of peptide masks as described herein. Peptides with the lowest EC50 value and lowest IC50 value with fast off rates, lowest ti / 2 values, are desired. Peptides with high binding affinity and inhibition capacity imply masking strength while a fast off rate ensures that the mask comes off the antibody once the antibody is cleaved by protease.

[0227] Table 8. WSGR Docket No. 52426-777.601

[0228] Table 9.

[0229] Table 10. WSGR Docket No. 52426-777.601

[0230] Example 2: Sequence Activity Relationships - PSMA Mask Peptides

[0231]

[0186] Sequence activity relationships (SAR) were established for peptide-23 and peptide-24 which were found to exhibit strong binding to the PSMA Fab. The sequence activity relationships were established by mutating individual residues within each peptide to alanine and measuring the influence of each mutation on the ability of the peptide to inhibit the ability of the PSMA Fab from binding to PSMA by competition ELISA experiments as described above in Example 1. IC50S for binding inhibition of the alanine scanning sequences are provided in Tables 11-12. The tables also designate each alanine mutation as tolerant (T), somewhat tolerant (ST), or intolerant (IT) based on changes in binding inhibition ability relative to the nonmutated peptide. Cys-5 and Cys-11 of peptide-23 and peptide-24 were not mutated.

[0232] Table 11. Competition Binding ELISA - Alanine Scanning Sequences of Peptide-23

[0233] Table 12. Competition Binding ELISA - Alanine Scanning Sequences of Peptide-24 WSGR Docket No. 52426-777.601

[0234] Example 3. Optimization Phage Library Construction of Peptide-24

[0235]

[0187] Using the peptide sequence activity relationships of Example 2, DNA oligo libraries were constructed where codons encoding critical residues within peptide-24 were minimally mutated, and codons encoding non-critical residues that were tolerated in the alanine scanning mutation studies (e. g. , He- 1 , Ala- 2, Val-3, Gln-6, Asn-8, and Ala-12) were heavily mutated. The resulting oligos were cloned into bacteriophage vectors used to display the SAR guided peptides via fusion to the pill filament of the bacteriophage. The relevant vectors were then used to produce the phage optimization libraries via amplification in bacteria using standard techniques in the field.

[0236] Example 4: Panning of the optimized phage library of Peptide-24

[0237]

[0188] Once the phage optimization libraries were completed, phage libraries were bio-panned using PSMA Fab loaded beads. Multiple rounds of panning were performed where bacteriophage was allowed to bind to PSMA Fab loaded beads, washed, eluted, and amplified. After panning, phage infected bacteria were plated out and colonies picked into 96 well blocks. Clonal phage was then amplified and separated from bacterial cells via centrifugation. Phage containing supernatants were tested in binding ELIS As against PSMA Fab coated plates in the presence or absence of saturating concentrations of PSMA antigen. Phage able to bind PSMA Fab were selected for sequence analysis if the binding signal was reduced in the presence of PSMA. SAR guided optimization phage library panning results of peptide-24 are shown in Table 13.

[0238] Table 13. SAR guided optimization phage library of peptide-24. WSGR Docket No. 52426-777.601

[0239] Example 5: Panning of the optimized phage library

[0240]

[0189] Clonal phage were harvested as crude supernatants and screened via standard ELISAs. Briefly, biotinylated PSMA Fab was captured on neutravidin coated plates. Prior to the addition of clonal phage, wells were incubated with blocking buffer and PSMA antigen or blocking buffer alone. Without washing or aspirating, clonal phage supernatants were then added to the wells and incubated for a short time. Wells were then washed followed by detection of bound phage using ahorse radish conjugated anti-M13 antibody. Clonal phage of interest were then sent for sequence analysis.

[0241]

[0190] Phage panning results of peptide-24 library sequences are shown in Table 14. 2323 clonal phage sequences were identified. A consensus sequence was calculated from all the sequences of Table 14 using WebLogo 3.7. 12. The consensus sequence is shown in FIG. 28.

[0242] Table 14. Phage Panning Results of Peptide-24 Library Sequences. (-) indicates the same amino acid position as in Peptide-24 corresponding position. WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601 WSGR Docket No. 52426-777.601

[0243] Example 6. Optimization Phage Library Construction of Peptide-23

[0244]

[0191] Using the peptide sequence activity relationships of Example B, DNA oligo libraries were constructed where codons encoding critical residues within peptide-23 were minimally mutated, and codons encoding non-critical residues that were tolerated in the alanine scanning mutation studies (e.g., Leu-1, Met- 2, Arg- 8, Ala- 12, Ala- 13, and Glu-14) were heavily mutated. The resulting oligos were cloned into bacteriophage vectors used to display the SAR guided peptides via fusion to the pill filament of the bacteriophage. The relevant vectors were then used to produce the phage optimization libraries via amplification in bacteria using standard techniques in the field.

[0245] Example 7: Panning of the optimized phage library of Peptide-23

[0246]

[0192] Once the phage optimization libraries were completed, phage libraries were bio-panned using PSMA Fab loaded beads. Multiple rounds of panning were performed where bacteriophage was allowed to bind to PSMA Fab loaded beads, washed, eluted, and amplified. After panning, phage infected bacteria were plated out and colonies picked into 96 well blocks. Clonal phage was then amplified and separated from bacterial cells via centrifugation. Phage containing supernatants were tested in binding ELIS As against PSMA Fab coated plates in the presence or absence of saturating concentrations of PSMA antigen. Phage able to bind PSMA Fab were selected for sequence analysis if the binding signal was reduced in the presence of PSMA. SAR guided optimization phage library panning results of peptide-23 are shown in Table 15.

[0247] Table 15. SAR guided optimization phage library of peptide-23. WSGR Docket No. 52426-777.601

[0248]

[0193] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. WSGR Docket No. 52426-777.601CLAIMSWHAT IS CLAIMED IS:

1. An isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to X1-X2-X3-X4-C-X5-P-X6-W-X7- C-X8-X9-X10, wherein:Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R;X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K;X3is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D;X4 is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D;X5is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H;X6is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G;X7 is selected from M, L, I, A, V, F, G, and K;X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W. G, K, R, and P;X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; andX10 is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K (SEQ ID NO: 2486).2 The isolated polypeptide or polypeptide complex of claim 1, wherein:Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, and W;X2is selected from E, V, D, T, S, L, G, P, A, M, and I;X3 is selected from K, P, R, I, N, H, V, M, A, and L;X4 is selected from W, L, M, R, V, Y, and A;X5 is selected from I, V, T, K, R, and E;Xe is selected from A, E, S, P, Q, T, L, D, M, and V;X7is M;X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, and H;X9 is selected from G, V, A, S, W, E, D, and M; andX10 is selected from F, L, M, S, I, V, D, and Q.3 The isolated polypeptide or polypeptide complex of claim 1, wherein:Xi is selected from V, E, L, D, I, G, and M;X2 is selected from E, V, D, T, S, L, G, P, and A;X3 is selected from K, P, R, I, N, H, V, and M;X4 is selected from W, L, M, and R;X5 is selected from I, V, T, K, R, and E;Xe is selected from A, E, S, P, Q, and T;X7is M;X8is selected from E, S, T, A, V, D, and Q;X9 is selected from G, V, A, S, and W; andWSGR Docket No. 52426-777.601Xio is selected from F, L, M, and S.

4. The isolated polypeptide or polypeptide complex of claim 1, wherein:Xi is selected from V, E, L, D, I, and G;X2 is selected from E, V, D, T, S, and, G;X3 is selected from K, P, R, I, N, and H;X4 is selected from W, L, and M;X5 is selected from I, V, T, K, and R;Xe is selected from A, E, S, and P;X7is M;Xs is selected from E, S, T, A, and V;X9 is G; andXio is selected from F, L, and M.5 The isolated polypeptide or polypeptide complex of claim 1, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 118.6 The isolated polypeptide or polypeptide complex of claim 1, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 115.7 The isolated polypeptide or polypeptide complex of claim 1, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 23.8 The isolated polypeptide or polypeptide complex of claim 1, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 85-94.9 The isolated polypeptide or polypeptide complex of claim 1, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 24.10 The isolated polypeptide or polypeptide complex of claim 1, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 95-104.11 The isolated polypeptide or polypeptide complex of claim 1, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 105-131.12 An isolated polypeptide or polypeptide complex comprising a prostate-specific membrane antigen (PSMA) binding domain that is linked to a peptide that impairs binding of the PSMA binding domain to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 284-2479.13 The isolated polypeptide or polypeptide complex of claim 12, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-84, 105-131, and 284-2479.14 The isolated polypeptide or polypeptide complex of claim 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 63.15 The isolated polypeptide or polypeptide complex of claim 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 74.WSGR Docket No. 52426-777.60116. The isolated polypeptide or polypeptide complex of claim 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 77.

17. The isolated polypeptide or polypeptide complex of claim 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 112.

18. The isolated polypeptide or polypeptide complex of claim 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 115.

19. The isolated polypeptide or polypeptide complex of claim 1 or 12, wherein the PSMA binding domain comprises an anti-PSMA antibody.

20. The isolated polypeptide or polypeptide complex of claim 19, wherein the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain that comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, andHC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 comprise the amino acid sequences of HC-CDR1 : SEQ ID NO: 132, HC-CDR2: SEQ ID NO: 133, and HC- CDR3: SEQ ID NO: 134, and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain that comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 comprise the amino acid sequences of LC-CDR1 : SEQ ID NO: 135, LC-CDR2: EA, and LC-CDR3: SEQ ID: 137.

21. The isolated polypeptide or polypeptide complex of claim 20, wherein the anti-PSMA heavy chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139.

22. The isolated polypeptide or polypeptide complex of claim 20, wherein the anti-PSMA light chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138.

23. The isolated polypeptide or polypeptide complex of claim 20, wherein the anti-PSMA heavy chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139, and the anti-PSMA light chain variable domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138.

24. The isolated polypeptide or polypeptide complex of claim 20, wherein the anti-PSMA antibody comprises a single chain variable fragment (scFv), a single domain antibody, a Fab, a Fab’, or a CrossFab.

25. The isolated polypeptide or polypeptide complex of claim 24, wherein the anti-PSMA antibody comprises the Fab or Fab’.

26. The isolated polypeptide or polypeptide complex of claim 24, wherein the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 139.

27. The isolated polypeptide or polypeptide complex of claim 24, wherein the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%,WSGR Docket No. 52426-777.60190%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.

28. The isolated polypeptide or polypeptide complex of claim 24, wherein the anti-PSMA antibody comprises the Fab or Fab’, and the Fab or Fab’ comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 142 and an amino acid sequence with at least at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 143.

29. The isolated polypeptide or polypeptide complex of claim 1 or 12, wherein the peptide is connected to the PSMA binding domain in a configuration according to Ai-Li-Pi wherein Ai comprises the PSMA binding domain, Pi comprises the peptide that impairs binding of the PSMA binding domain to PSMA, and Li comprises a cleavable linker.

30. The isolated polypeptide or polypeptide complex of claim 29, wherein P i is connected N-terminal to the cleavable linker and Ai is connected C-terminal to the cleavable linker.

31. The isolated polypeptide or polypeptide complex of claim 29, wherein P i is connected C-terminal to the cleavable linker and Ai is connected N-terminal to the cleavable linker.

32. The isolated polypeptide or polypeptide complex of claim 29, wherein Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi -stacking interactions, H-bonding interactions, or a combination thereof.

33. The isolated polypeptide or polypeptide complex of claim 29, wherein Pi is bound to Ai at or near an antigen binding site.

34. The isolated polypeptide or polypeptide complex of claim 29, wherein Pi becomes unbound from Ai when Li is cleaved by a tumor specific protease, thereby exposing Ai to PSMA.

35. The isolated polypeptide or polypeptide complex of claim 29, wherein Pi has less than 75% sequence identity to PSMA.

36. The isolated polypeptide or polypeptide complex of claim 29, wherein Pi comprises a de novo amino acid sequence that shares less than 10% sequence identity to PSMA.

37. The isolated polypeptide or polypeptide complex of claim 29, wherein Pi comprises a modified amino acid, or a modified non-natural amino acid, or a combination thereof.

38. The isolated polypeptide or polypeptide complex of claim 29, wherein Pi does not comprise albumin or an albumin fragment.

39. The isolated polypeptide or polypeptide complex of claim 29, wherein Pi does not comprise an albumin binding domain.

40. The isolated polypeptide or polypeptide complex of claim 29, wherein Li is bound to the N-terminus of Ai.

41. The isolated polypeptide or polypeptide complex of claim 29, wherein Li is bound to the C-terminus of Ai.

42. The isolated polypeptide or polypeptide complex of claim 29, wherein Li is a peptide sequence having at least 5 to no more than 50 amino acids.WSGR Docket No. 52426-777.60143. The isolated polypeptide or polypeptide complex of claim 29, wherein Li is a peptide sequence having at least 10 to no more than 30 amino acids.

44. The isolated polypeptide or polypeptide complex of claim 29, wherein Li is a peptide sequence having at least 10 amino acids.

45. The isolated polypeptide or polypeptide complex of claim 29, wherein Li is a peptide sequence having at least 18 amino acids.

46. The isolated polypeptide or polypeptide complex of claim 29, wherein Li is a peptide sequence having at least 26 amino acids.

47. The isolated polypeptide or polypeptide complex of claim 29, wherein Li comprises a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 2484).

48. The isolated polypeptide or polypeptide complex of claim 29, wherein Li comprises a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 2480), (GGGS)n(SEQ ID NO: 2481), (GGGGS)n (SEQ ID NO: 2482), and (GSSGGS)n(SEQ ID NO: 2483), wherein n is an integer of at least 1.

49. The isolated polypeptide or polypeptide complex of claim 29, wherein Li comprises an amino acid sequence selected from any one of SEQ ID NOs: 144-175.

50. The isolated polypeptide or polypeptide complex of claim 29, wherein Li comprises an amino acid sequence according to SEQ ID NO: 173.

51. The isolated polypeptide or polypeptide complex of claim 29, wherein Li is a substrate for a tumor specific protease.

52. The isolated polypeptide or polypeptide complex of claim 51 , wherein P i becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to PSMA.

53. The isolated polypeptide or polypeptide complex of claim 51, wherein Li comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, or a matrix metalloprotease cleavable amino acid sequence.

54. The isolated polypeptide or polypeptide complex of claim 1 or 12, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 274, 276, 280, and 282 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 275, 277, 281, and 283.

55. The isolated polypeptide or polypeptide complex of claim 1 or 12, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 256, 258, 260, 262, 264, 266, 268, 270, 274, 276,WSGR Docket No. 52426-777.601280, and 282 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 257, 259, 261, 263, 265, 267, 269, 271, 275, 277, 281, and 283.

56. A pharmaceutical composition comprising:(i) the isolated polypeptide or polypeptide complex of claim 1 or 12; and(ii) a pharmaceutically acceptable excipient.

57. An isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex of claim 1 or 12.

58. A vector comprising the isolated nucleic acid molecule of claim 57.

59. A host cell comprising the isolated recombinant nucleic acid molecule of claim 57.

60. A method of treating cancer in a subject in need thereof comprising administering to the subject the isolated polypeptide or polypeptide complex of claim 1 or 12.

61. The method of claim 60, wherein the cancer has cells that express PSMA.

62. The method of claim 60, wherein the cancer is a solid tumor cancer.

63. The method of claim 60, wherein the cancer is prostate, lung, breast, cervical, ovarian, colorectal, pancreatic, or gastric cancer.

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