Compositions and methods related to tumor activated antibodies targeting PSMA and effector cell antigens

Abstract

Disclosed herein are isolated polypeptide and polypeptide complexes that comprise a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA.

Description

[0001] WSGR Docket No. 52426-778.601

[0002] COMPOSITIONS AND METHODS RELATED TO TUMOR ACTIVATED ANTIBODIES TARGETING PSMA AND EFFECTOR CELL ANTIGENS

[0003] CROSS-REFERENCE

[0004]

[0001] The present application claims the benefit of U.S. Provisional Application No. 63 / 715,037 filed on November 1, 2024, which is incorporated herein by reference in 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 23, 2025 is named 52426-778_601_SL.xml and is 3,014,989 bytes in size.

[0007] SUMMARY

[0008]

[0003] Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes that comprise a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule 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; 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: 3425). 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; 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; 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; 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; 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; X4 is selected from W, L, and M; X5 is selected from I, V, T, K, and R; WSGR Docket No. 52426-778.601

[0009] 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.

[0010]

[0004] 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]

[0005] Disclosed herein, in some embodiments, are isolated polypeptide or polypeptide complexes that comprise a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 - 131 and 214-2519. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-84, 105-131, and 214-2519. 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]

[0006] In some embodiments, the effector cell antigen comprises cluster of differentiation 3 (CD3). In some embodiments, the first antigen recognizing molecule comprises an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 heavy chain variable domain that comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC- CDR1, theHC-CDR2, and the HC-CDR3 comprise 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-CD3 antibody comprises an anti-CD3 light chain variable domain that comprises complementarity determining regions (CDRs): LC- CDR1, LC-CDR2, and LC-CDR3, and wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 comprise amino acid sequences of LC-CDR1: SEQ ID NO: 135, LC-CDR2: GT, and LC-CDR3: SEQ ID NO: 137. In some embodiments, the first antigen recognizing molecule that binds to CD3 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 first antigen recognizing molecule that binds to CD3 comprises a single chain variable fragment (scFv) or a Fab or Fab’ fragment.

[0013]

[0007] In some embodiments, the second antigen recognizing molecule that binds to PSMA comprises an anti-PSMA antibody. In some embodiments, the anti-PSMA anti both' comprises an anti-PSMA heavy chain WSGR Docket No. 52426-778.601 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 amino acid sequences of HC-CDR1: SEQ IDNO: 139, HC-CDR2: SEQ IDNO: 140, andHC-CDR3: SEQ ID NO: 141 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 of the light chain variable domain comprise an amino acid sequence of LC-CDR1 : SEQ ID NO: 142, LC-CDR2: EA, and LC-CDR3: SEQ ID NO: 144. 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: 146, 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: 145. 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: 148, 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: 147. 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: 150, 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: 149. 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.

[0014]

[0008] In some embodiments, the anti-CD3 antibody comprises a single chain variable fragment (scFv) and the anti-PSMA antibody comprises a Fab or a Fab’ . In some embodiments, the scFv and the Fab or Fab’ are connected through a linker and the Fab or Fab’ comprise a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the linker connects the scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects the scFv to the Fab light chain polypeptide. In some embodiments, the linker connects the scFv to the N-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the scFv to the C-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the scFv to the N-term of the Fab light chain polypeptide. In some embodiments, the linker connects the scFv to the C-term of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the N-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the C-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the N-term of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the C-term of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the Fab heavy WSGR Docket No. 52426-778.601 chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the N-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the C-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the N-term of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the C-term of the Fab light chain polypeptide.

[0015]

[0009] In some embodiments, the linker is at least 5 amino acids in length. In some embodiments, the linker is no more than 30 amino acids in length. In some embodiments, the linker is at least 5 amino acids and no more than 30 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 151 (GGGGSGGGGSGGGGS) or SEQ ID NO: 152 (GGGGS).

[0010] In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 153 and SEQ ID NO: 154. In some embodiments, the isolated polypeptide or polypeptide complex is human or humanized.

[0016] [OH] In some embodiments, the isolated polypeptide or polypeptide complex further comprises a peptide that is linked to the anti-CD3 antibody, wherein the peptide impairs binding of the anti-CD3 antibody to CD3. In some embodiments, the isolated polypeptide or polypeptide complex comprises a configuration according to Formula I: P2-L2-A2-A1-L1-P1-H1 wherein Ai comprises the anti-CD3 antibody; Pi comprises the peptide that impairs binding of the anti-CD3 antibody to CD3; Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; A2 comprises the anti-PSMA antibody; P2 comprises the peptide that impairs binding of the anti- PSMA antibody to PSMA; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex comprises a configuration according to Formula I: P2-L2-A2-A1-L1-P1-H1 wherein Ai is the anti-CD3 antibody; Pi is the peptide that impairs binding of the anti-CD3 antibody to CD3; Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi is a half-life extending molecule; A2 is the anti-PSMA antibody; P2 is the peptide that impairs binding of the anti-PSMA antibody to PSMA; and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex comprises a configuration comprising Formula I: P2-L2-A2-A1- L1-P1-H1 wherein Ai comprises the anti-CD3 antibody; Pi comprises the peptide that impairs binding of the anti-CD3 antibody to CD3; Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; A2 comprises the anti-PSMA antibody; P2 comprises the peptide that impairs binding of the anti-PSMA antibody to PSMA; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex comprises a configuration comprising WSGR Docket No. 52426-778.601

[0017] Formula I: P2-L2-A2-A1-L1-P1-H1 wherein Ai is the anti-CD3 antibody; Pi is the peptide that impairs binding of the anti-CD3 antibody to CD3; Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi is a half-life extending molecule; A2 is the anti-PSMA antibody; P2 is the peptide that impairs binding of the anti-PSMA antibody to PSMA; and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex is according to Formula I: P2-L2-A2-A1-L1-P1-H1 wherein Ai is the anti-CD3 antibody; Pi is the peptide that impairs binding of the anti-CD3 antibody to CD3; Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi is a half-life extending molecule; A2 is the anti- PSMA antibody; P2 is the peptide that impairs binding of the anti-PSMA antibody to PSMA; and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.

[0018]

[0012] In some embodiments, the anti-CD3 antibody comprises a scFv and the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide, and the anti-PSMA antibody comprises a Fab or Fab’ and the Fab or Fab’ comprise a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and L2 is bound to the Fab heavy chain polypeptide of A2.

[0019]

[0013] In some embodiments, Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bonding interactions, or a combination thereof. In some embodiments, Pi has less than 70% sequence homology to CD3. In some embodiments, P2 impairs binding of A2to PSMA. In some embodiments, P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bonding interactions, or a combination thereof. In some embodiments, P2 is bound to A2 at or near an antigen binding site. In some embodiments, P2 has less than 70% sequence homology to PSMA. In some embodiments, Pi or P2 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, Pi or P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, Pi or P2 comprises a peptide sequence of at least 16 amino acids in length acids. In some embodiments, Pi or P2 comprises a peptide sequence of no more than 40 amino in length. In some embodiments, Pi or P2 comprises at least two cysteine amino acid residues. In some embodiments, Pi or P2 comprises a cyclic peptide or a linear peptide. In some embodiments, Pi or P2 comprises a cyclic peptide. In some embodiments, Pi or?2 comprises a linear peptide. In some embodiments, Pi comprises at least two cysteine amino acid residues.

[0014] In some embodiments, Li is bound to N-terminus of Ai. In some embodiments, Li is bound to C- terminus of Ai . In some embodiments, L2 is bound to N-terminus of A2. In some embodiments, L2 is bound to C-terminus of A2. In some embodiments, Li or L2 is a peptide sequence having at least 5 to no more than WSGR Docket No. 52426-778.601

[0020] 50 amino acids. In some embodiments, Li or L2 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, Li or L2 is a peptide sequence having at least 10 amino acids. In some embodiments, Li or L2 is a peptide sequence having at least 18 amino acids. In some embodiments, Li or L2 is a peptide sequence having at least 26 amino acids.

[0021]

[0015] In some embodiments, Pi comprises an amino acid sequence according to U1-U2-C- U4-P-U6-U7-U8- U9-U10-U11- U12-C-U14 and Ui is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; U4 is selected from G and W; Ue is selected from E, D, V, and P; U7 is selected from W, L, F, V, G, M, I, and Y; Us is selected from E, D, P, and Q; U9 is selected from E, D, Y, V, F, W, P, L, and Q; U10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; Unis selected from I, Y, F, V, L, T, N, S, D, A, and H; U is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H; and U14 is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S. In some embodiments, Ui is selected from D, Y, F, I, V, and N; U2 is selected from D, Y, L, F, I, and N; U4 is selected from G and W; Ue is selected from E and D; U7 is selected from W, L, F, G, and V; Us is selected from E and D; U9 is selected from E, D, Y, and V; U10 is selected from S, D, Y, T, and I; Un is selected from I, Y, F, V, L, and T; U12 is selected from F, D, Y, L, I, V, A, G, and N; and U14 is selected from D, Y, N, F, I, M, and P. In some embodiments, Ui is selected from D, Y, V, and F; U2 is selected from D, Y, L, and F; U4 is selected from G and W; Ue is selected from E and D; U7 is selected from W, L, G, and F; Us is selected from E and D; U9 is selected from E and D; U10 is selected from S, D, T, and Y; Un is selected from I, Y, V, L, and F; U12 is selected from F, D, Y, G, A, and L; U14 is selected from D, Y, M, and N.

[0022]

[0016] In some embodiments, Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 155-159 and 2520-3418. In some embodiments, Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 155-159, 2520-2546, and 3407-3418. In some embodiments, Pi comprises the amino acid sequence according to SEQ ID NO: 157. In some embodiments, Pi comprises the amino acid sequence according to SEQ ID NO: 158.

[0023]

[0017] In some embodiments, Li or L2 has a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 3419). In some embodiments, Li has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 3420), (GGGS)n (SEQ ID NO: 3421), (GGGGS)n(SEQ ID NO: 3422), and (GSSGGS)n (SEQ ID NO: 3423), wherein n is an integer of at least 1.

[0024]

[0018] In some embodiments, Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to CD3. In some embodiments, P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to PSMA. In some embodiments, the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase (MTSP1), urokinase, or hepsin. In some embodiments, Li or L2 comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence. WSGR Docket No. 52426-778.601

[0025]

[0019] In some embodiments, Li or L2 comprises an amino acid sequence according to SEQ ID NO: 163. In some embodiments, Li or L2 comprises an amino acid sequence according to any one of SEQ ID NOs: 160-191. In some embodiments, Li or L2 comprises an amino acid sequence of Linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 186), Linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 187), Linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 188), or Linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 189), or an amino acid sequence that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 25, Linker 26, Linker 27, or Linker 28.

[0026]

[0020] In some embodiments, Hi comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, Hi comprises albumin. In some embodiments, Hi comprises a fragment crystaHizable (Fc) domain. In some embodiments, the Fc domain comprises one or more knob-in- hole (KIH) mutations. In some embodiments, the Fc domain comprises a first polypeptide and a second polypeptide, wherein the first polypeptide of the Fc domain comprises a hole mutation and the second polypeptide of the Fc domain comprises a knob mutation. In some embodiments, the first polypeptide of the Fc domain comprises T366S, L368A, and Y407V mutations according to EU numbering, and the second polypeptide of the Fc domain comprises a T366W mutation according to EU numbering. In some embodiments, the Fc domain comprises Fc effector function silencing mutations L234A, L235A, and P329G according to EU numbering. In some embodiments, the Fc domain comprises an engineered disulfide. In some embodiments, the Fc domain comprises a Y349C mutation and a S354C mutation according to EU numbering, and wherein the engineered disulfide is formed between two cysteines at the positions of the Y349C mutation and the S354C mutation. In some embodiments, the Fc domain comprises a N297G mutation according to EU numbering. In some embodiments, the N297G mutation removes an N- glycosylation site. In some embodiments, the Fc domain lacks a C-terminal glycine and a C-terminal lysine to reduce heterogeneity. In some embodiments, the Fc domain lacks C-terminal G446 and K447 residues according to EU numbering to reduce heterogeneity. In some embodiments, the Fc domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 203 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 204.

[0027]

[0021] In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, Hi comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab. In some embodiments, the single domain antibody comprises a single domain antibody that binds to albumin. In some embodiments, the single domain antibody is a human or humanized antibody. In some WSGR Docket No. 52426-778.601 embodiments, the single domain antibody is 645gHlgLl. In some embodiments, the single domain antibody is 645dsgH5gL4. In some embodiments, the single domain antibody is 23-13-A01 -sc02. In some embodiments, the single domain antibody is Al 0m3 or a fragment thereof. In some embodiments, the single domain antibody is DOM7r-31. In some embodiments, the single domain antibody is DOM7h-l l-15. In some embodiments, the single domain antibody is Alb-1, Alb- 8, or Alb-23. In some embodiments, the single domain antibody is 10E. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC- CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1 : SEQ ID NO: 192, HC-CDR2: SEQ ID NO: 193, and HC-CDR3: SEQ ID NO: 194. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC- CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 196, HC-CDR2: SEQ ID NO: 197, and HC-CDR3: SEQ ID NO: 198. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 195. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 199. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 200. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 201. In some embodiments, the single domain antibody is SA21.

[0028]

[0022] In some embodiments, the 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 modified amino acid or modified non-natural amino acid comprises a post-translational modification.

[0029]

[0023] In some embodiments, Hi comprises a linking moiety (L3) that connects Hi to Pi. In some embodiments, L3 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 3420), (GGGS)n (SEQ ID NO: 3421), (GGGGS)n(SEQ ID NO: 3422), and (GSSGGS)n (SEQ ID NO: 3423), wherein n is an integer of at least 1. In some embodiments, L3 comprises an amino acid sequence according to GGGGSGGGSGG (SEQ ID NO: 3424).

[0030]

[0024] In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 205 and 206. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 207 and 208. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, WSGR Docket No. 52426-778.601

[0031] 99%, or 100% sequence identity to SEQ ID NOs: 209 and 210. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 211, 212, and 213.

[0032]

[0025] Disclosed herein, in some embodiments, are isolated polypeptide and polypeptide complexes according to Formulall: Lia-Pia wherein: Liacomprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Piato an anti-PSMA antibody and the anti-PSMA antibody is connected to an anti-CD3 antibody, and Pia comprises a peptide that impairs binding of the anti-PSMA antibody to PSMA. In some embodiments, Pia when Lia is uncleaved impairs binding of the anti-PSMA antibody to PSMA. In some embodiments, the anti-PSMA antibody comprises a human or humanized antibody. In some embodiments, Pia has less than 70% sequence homology to PSMA. In some embodiments, Pia comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, Pia comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, Pia comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, Pia comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, Pia comprises at least two cysteine amino acid residues. In some embodiments, Pia comprises a cyclic peptide or a linear peptide. In some embodiments, Pia comprises a cyclic peptide. In some embodiments, Pia comprises a linear peptide.

[0033]

[0026] In some embodiments, Pia 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 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; 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; X9 is 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: 3425). In some embodiments, Pia comprises an amino acid sequence according to SEQ ID NO: 118. In some embodiments, Pia comprises an amino acid sequence according to SEQ ID NO: 115. In some embodiments, Pia comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, Pia comprises an amino acid sequence according to any one of SEQ ID NOs: 85- 94. In some embodiments, Pia comprises an amino acid sequence according to any one of SEQ ID NOs: 95- 104. In some embodiments, Pia comprises an amino acid sequence according to any one of SEQ ID NOs: 105-131. In some embodiments, Pia comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 214-2519.

[0034]

[0027] In some embodiments, Lia comprises an amino acid sequence according to any one of SEQ ID NOs: 144-175. In some embodiments, Liacomprises an amino acid sequence according to SEQ ID NO: 173. WSGR Docket No. 52426-778.601

[0035]

[0028] 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.

[0036]

[0029] 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.

[0037]

[0030] 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 isolated polypeptide or polypeptide complex induces T cell mediated cytotoxicity of tumor cells. 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.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039]

[0031] 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 following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0040]

[0032] FIGs. 1-2 illustrate binding of PSMA Fab to peptides as measured by ELISA.

[0041]

[0033] FIGs. 3-4 illustrate inhibition of PSMA Fab binding to PSMA by peptides of the present disclosure.

[0042]

[0034] FIG. 5 illustrates BLI titration data for PSMA Fab binding to peptide-23.

[0043]

[0035] FIG. 6 illustrates BLI titration data for PSMA Fab binding to peptide-63.

[0044]

[0036] FIG. 7 illustrates BLI titration data for PSMA Fab binding to peptide-69.

[0045]

[0037] FIG. 8 illustrates BLI titration data for PSMA Fab binding to peptide-71.

[0046]

[0038] FIG. 9 illustrates BLI titration data for PSMA Fab binding to peptide-74.

[0047]

[0039] FIG. 10 illustrates BLI titration data for PSMA Fab binding to peptide-76.

[0048]

[0040] FIG. 11 illustrates BLI titration data for PSMA Fab binding to peptide-77.

[0049]

[0041] FIG. 12 illustrates BLI titration data for PSMA Fab binding to peptide-79.

[0050]

[0042] FIG. 13 illustrates BLI titration data for PSMA Fab binding to peptide-80.

[0051]

[0043] FIG. 14 illustrates BLI titration data for PSMA Fab binding to peptide-83.

[0052]

[0044] FIG. 15 illustrates BLI titration data for PSMA Fab binding to peptide-24.

[0053]

[0045] FIG. 16 illustrates BLI titration data for PSMA Fab binding to peptide-107.

[0054]

[0046] FIG. 17 illustrates BLI titration data for PSMA Fab binding to peptide-111.

[0055]

[0047] FIG. 18 illustrates BLI titration data for PSMA Fab binding to peptide-112. WSGR Docket No. 52426-778.601

[0056]

[0048] FIG. 19 illustrates BLI titration data for PSMA Fab binding to peptide-114.

[0057]

[0049] FIG. 20 illustrates BLI titration data for PSMA Fab binding to peptide-115.

[0058]

[0050] FIG. 21 illustrates BLI titration data for PSMA Fab binding to peptide-118.

[0059]

[0051] FIG. 22 illustrates BLI titration data for PSMA Fab binding to peptide-121.

[0060]

[0052] FIG. 23 illustrates BLI titration data for PSMA Fab binding to peptide-126.

[0061]

[0053] FIG. 24 illustrates BLI titration data for PSMA Fab binding to peptide-127.

[0062]

[0054] FIG. 25 illustrates BLI titration data for PSMA Fab binding to peptide-128.

[0063]

[0055] FIG. 26 illustrates BLI titration data for PSMA Fab binding to peptide-129.

[0064]

[0056] FIG. 27 illustrates BLI titration data for PSMA Fab binding to peptide-130.

[0065]

[0057] FIG. 28 illustrates the core sequence motif of anti-PSMA Fab peptide-24 sequences generated using Weblogo 3.7.12

[0066]

[0058] FIG. 29 illustrates binding of Ab-1, PC-5, MSTP1 cleaved PC-5, MMP9 cleaved PC-5, and PC-2 to human PSMA as measured by ELISA.

[0067]

[0059] FIG. 30 illustrates binding of Ab-1, PC-5, MSTP1 cleaved PC-5, MMP9 cleaved PC-5, and PC-2 to human CD3 as measured by ELISA.

[0068]

[0060] FIG. 31 illustrates cell viability for 22Rvl tumor cells treated with Ab-1, PC-5, MSTP1 cleaved PC- 5, and MMP-9 cleaved PC-5.

[0069]

[0061] FIG. 32 illustrates cell viability for LNCaP tumor cells treated with Ab-1, PC-5, MSTP1 cleaved PC-5, and MMP-9 cleaved PC-5.

[0070]

[0062] FIG. 33 illustrates PC-5 pharmacokinetics in cynomolgus monkeys after a single IV bolus or subcutaneous injection.

[0071]

[0063] FIG. 34 illustrates interleukin 2 release in cynomolgus monkeys after a single IV bolus or subcutaneous injection.

[0072]

[0064] FIG. 35 illustrates interleukin 6 release in cynomolgus monkeys after a single IV bolus or subcutaneous injection.

[0073]

[0065] FIG. 36 illustrates interleukin 10 release in cynomolgus monkeys after a single IV bolus or subcutaneous injection.

[0074]

[0066] FIG. 37 illustrates tumor necrosis factor alpha release in cynomolgus monkeys after a single IV bolus or subcutaneous injection.

[0075]

[0067] FIG. 38 illustrates interferon gamma release in cynomolgus monkeys after a single IV bolus or subcutaneous injection.

[0076] DETAILED DESCRIPTION

[0077]

[0068] Multispecific antibodies combine the benefits of different binding specificities derived from two or more antibodies into a single composition. Multispecific antibodies for redirecting T cells to cancers have shown promise in both pre-clinical and clinical studies. This approach relies on binding of one antigen interacting portion of the antibody to a tumor-associated antigen or marker, while a second antigen WSGR Docket No. 52426-778.601 interacting portion can bind to an effector cell antigen on a T cell, such as cluster of differentiation 3 (CD3), which then triggers cytotoxic activity.

[0078]

[0069] One such tumor- associated antigen is PSMA. 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 in humans is encoded by the FOLH1 (folate hydrolase 1) gene. PSMA is a zinc metalloenzyme that resides in membranes. Most of the enzyme resides in the extracellular space. 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.

[0079]

[0070] T cell engagers (TCEs) therapeutics have several benefits including they are not cell therapies and thus can be offered as off-the-shelf therapies as opposed to chimeric antigen receptor T cell (CAR T cell) therapies. While TCE therapeutics have displayed potent anti-tumor activity in hematological cancers, developing TCEs to treat solid tumors has faced challenges due to the limitations of prior TCE technologies, namely (i) overactivation of the immune system leading to cytokine release syndrome (CRS), (ii) on-target, healthy tissue toxi cities and (iii) poor pharmacokinetics (PK) leading to short half-life. CRS arises from the systemic activation of T cells and can result in life-threatening elevations in inflammatory cytokines such as interleukin-6 (IL-6). Severe and acute CRS leading to dose limited toxicities and deaths have been observed upon the dosing of T cell engagers develop using other platforms to treat cancer patients in poor clinical studies. This toxicity restricts the maximum blood levels of T cell engagers that can be safely dosed. T cell engager effectiveness has also been limited because of on-target, healthy tissue toxicity. T cell engagers developed using a platform not designed for tumor-specification activation have resulted in clinicals holds and dose-limiting toxicities resulting from target expression in healthy tissues. T cell engagers have also been limited by short half-lives. T cell engagers quickly reach sub-therapeutic levels after being administered as they are quickly eliminated from the body due to their short exposure half-lives. For this reason, T cell engagers such as blinatumomab are typically administered by a low-dose, continuous infusion pump over a period of weeks to overcome the challenge of a short half-life and to maintain therapeutic levels of drug in the body. A continuous dosing regimen represents a significant burden for patients.

[0080]

[0071] To overcome these challenges associated with the effectiveness of T cell engagers, described herein, are polypeptide or polypeptide complexes that comprise binding domains that selectively bind to an effector cell antigen and PSMA and are selectively activated in the tumor microenvironment. Additionally, the polypeptide or polypeptide complexes described herein comprise a half-life extending molecule. In some embodiments, the polypeptide or polypeptide complexes comprise peptides that impair binding of the PSMA binding domain to PSMA and impair binding of the effector cell antigen binding domain to the effector cell antigen. In some embodiments, the peptides are attached to the binding domains via cleavable linkers that are selectively cleaved in tumor microenvironments such that the PSMA binding domain and the effector cell antigen binding domain are available for binding to their respective targets in tumor microenvironments. WSGR Docket No. 52426-778.601

[0081] Such modifications reduce CRS and on-target healthy tissue toxicity risk, improves stability in the bloodstream and serum half-life prior to activation.

[0082] Certain Definitions

[0083]

[0072] 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.”

[0084]

[0073] 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.

[0085]

[0074] 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).

[0086]

[0075] 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.

[0087]

[0076] 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).

[0088]

[0077] 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 WSGR Docket No. 52426-778.601 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.

[0089]

[0078] 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.

[0090]

[0079] 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 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.

[0091]

[0080] 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 WSGR Docket No. 52426-778.601 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 Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” 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.

[0092]

[0081] 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.

[0093] Isolated Polypeptide or Polypeptide Complexes

[0094]

[0082] Disclosed herein are isolated polypeptide or polypeptide complexes that comprise a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule 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 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: 3425). In some embodiments, Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, and W; X2 is 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; X7is M; X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, and H; X9is WSGR Docket No. 52426-778.601 selected from G, V, A, S, W, E, D, and M; and Xio 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; 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; 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 Xio is selected from F, L, M, and S. In some embodiments, 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; X7 is M; Xs is selected from E, S, T, A, and V; X9 is G; and Xio is selected from F, L, and M.

[0083] 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.

[0095]

[0084] Disclosed herein are isolated polypeptide or polypeptide complexes that comprise a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 -131 and 214-2519. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 - 84, 105-131, and 214-2519. 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.

[0096] Table 1. Peptide Mask Sequences for PSMA Binding Domain WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601

[0097] Table 2. Peptide Mask Sequences for PSMA Binding Domain (Alanine scanning peptides of Peptide-

[0098] 23)

[0099] Table 3. Peptide Mask Sequences for PSMA Binding Domain (Alanine scanning peptides of Peptide-

[0100] 24) WSGR Docket No. 52426-778.601

[0101] Table 4. Peptide Mask Sequences for PSMA Binding Domain (Directed evolution mask optimization)

[0102] Table 5. Phage Panning Results of Peptide-24 Library Sequences. (-) indicates the same amino acid position as in Peptide-24 corresponding position. WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601

[0103] CD3 Binding Domain

[0104]

[0085] In some embodiments, the effector cell antigen comprises cluster of differentiation 3 (CD3). In some embodiments, the first antigen recognizing molecule comprises an anti-CD3 antibody. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 heavy chain variable domain that comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC- WSGR Docket No. 52426-778.601

[0105] CDR1, theHC-CDR2, and the HC-CDR3 comprise 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-CD3 antibody comprises an anti-CD3 light chain variable domain that comprises complementarity determining regions (CDRs): LC- CDR1, LC-CDR2, and LC-CDR3, and wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 comprise amino acid sequences of LC-CDR1: SEQ ID NO: 135, LC-CDR2: GT, and LC-CDR3: SEQ ID NO: 137. In some embodiments, the first antigen recognizing molecule that binds to CD3 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 first antigen recognizing molecule comprises a single chain variable fragment (scFv) or a Fab or Fab’ fragment.

[0106] Table 6. anti-CD3 amino acid sequences (CDRs as determined by IMGT numbering system)

[0107] PSMA Binding Domain

[0108]

[0086] In some embodiments, the second antigen recognizing molecule that binds to PSMA comprises an anti-PSMA antibody. In some embodiments, the anti-PSMA anti both' 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 amino acid sequences of HC-CDR1: SEQ ID NO: 139, HC-CDR2: SEQ ID NO: 140, andHC-CDR3: SEQ ID NO: 141 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 of the light chain variable domain comprise an amino acid sequence of LC-CDR1 : SEQ ID NO: 142, LC-CDR2: EA, and LC-CDR3: SEQ ID NO: 144. 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: 146, and wherein the anti-PSMA light chain variable WSGR Docket No. 52426-778.601 domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 145. 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: 148, and 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: 147. 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: 150, and 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: 149. 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.

[0109] Table 7. Exemplary PSMA Binding Domain Sequences. CDRs determined by IMGT definition WSGR Docket No. 52426-778.601

[0110] Formats and Connections

[0111]

[0087] In some embodiments, the anti-CD3 antibody comprises a single chain variable fragment (scFv) and the anti-PSMA antibody comprises a Fab or a Fab’ . In some embodiments, the scFv and the Fab or Fab’ are connected through a linker and the Fab or Fab’ comprise a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the linker connects the scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects the scFv to the Fab light chain polypeptide. In some embodiments, the linker connects the scFv to the N-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the scFv to the C-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the scFv to the N-term of the Fab light chain polypeptide. In some embodiments, the linker connects the scFv to the C-term of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the N-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the C-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the N-term of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 heavy chain variable domain of the scFv to the C-term of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the N-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the C-term of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the N-term of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD3 light chain variable domain of the scFv to the C-term of the Fab light chain polypeptide.

[0112]

[0088] In some embodiments, the linker is at least 5 amino acids in length. In some embodiments, the linker is no more than 30 amino acids in length. In some embodiments, the linker is at least 5 amino acids and no more than 30 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In WSGR Docket No. 52426-778.601 some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 151 (GGGGSGGGGSGGGGS) or SEQ ID NO: 152 (GGGGS).

[0113] Table 8. Linker sequences

[0114] Antibodies that Bind PSMA and CD3

[0115]

[0089] In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 153 and SEQ ID NO: 154. In some embodiments, the isolated polypeptide or polypeptide complex is human or humanized.

[0116] Table 9. Polypeptide Complexes that Bind to CD3 and PSMA

[0117] Tumor Activated Antibodies that Bind PSMA and CD3

[0118]

[0090] In some embodiments, the isolated polypeptide or polypeptide complex further comprises a peptide that is linked to the anti-CD3 antibody, wherein the peptide impairs binding of the anti-CD3 antibody to CD3. In some embodiments, the isolated polypeptide or polypeptide complex comprises a configuration according to Formula I: P2- 2-A2-A1-L1-P1-H1 wherein Ai comprises the anti-CD3 antibody; Pi comprises the peptide that impairs binding of the anti-CD3 antibody to CD3; Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; A2 comprises the anti-PSMA antibody; P2 comprises the peptide that impairs binding of the anti- WSGR Docket No. 52426-778.601

[0119] PSMA antibody to PSMA; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.

[0120]

[0091] In some embodiments, the anti-CD3 antibody comprises a scFv and the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide, and the anti-PSMA antibody comprises a Fab or Fab’ and the Fab or Fab’ comprise a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and L2 is bound to the Fab heavy chain polypeptide of A2.

[0121] Pi, P2

[0122]

[0092] In some embodiments, Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bonding interactions, or a combination thereof. In some embodiments, Pi has less than 70% sequence homology to CD3. In some embodiments, P2 impairs binding of A2to PSMA. In some embodiments, P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bonding interactions, or a combination thereof. In some embodiments, P2 is bound to A2 at or near an antigen binding site. In some embodiments, P2 has less than 70% sequence homology to PSMA. In some embodiments, Pi or P2 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, Pi or P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, Pi or P2 comprises a peptide sequence of at least 16 amino acids in length acids. In some embodiments, Pi or P2 comprises a peptide sequence of no more than 40 amino in length. In some embodiments, Pi or P2 comprises at least two cysteine amino acid residues. In some embodiments, Pi or P2 comprises a cyclic peptide or a linear peptide. In some embodiments, Pi or P2 comprises a cyclic peptide. In some embodiments, Pi orP2 comprises a linear peptide. In some embodiments, Pi comprises at least two cysteine amino acid residues.

[0123] Li, L2

[0124]

[0093] In some embodiments, Li is bound to N-terminus of Ai. In some embodiments, Li is bound to C- terminus of Ai . In some embodiments, L2 is bound to N-terminus of A2. In some embodiments, L2 is bound to C-terminus of A2. In some embodiments, L2 is bound to C-terminus of A2. In some embodiments, Li or L2 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, Li or L2 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, Li or L2 is a peptide sequence having at least 10 amino acids. In some embodiments, Li or L2 is a peptide sequence having at least 18 amino acids. In some embodiments, Li or L2 is a peptide sequence having at least 26 amino acids. WSGR Docket No. 52426-778.601

[0125] Pi - CD3 Binding Domain Mask

[0126]

[0094] In some embodiments, Pi comprises an amino acid sequence according to U1-U2-C- U4-P-U6-U7-U8- U9-U10-U11- U12-C-U14 and Ui is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; U4 is selected from G and W; Ue is selected from E, D, V, and P; U7 is selected from W, L, F, V, G, M, I, and Y; Us is selected from E, D, P, and Q; U9 is selected from E, D, Y, V, F, W, P, L, and Q; U10 is selected from S, D, Y, T, I, F, V, N, A, P,_L, and H; Unis selected from I, Y, F, V, L, T, N, S, D, A, and H; U is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H; and U14 is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S. In some embodiments, Ui is selected from D, Y, F, I, V, and N; U2 is selected from D, Y, L, F, I, and N; U4 is selected from G and W; Ue is selected from E and D; U7 is selected from W, L, F, G, and V; Us is selected from E and D; U9 is selected from E, D, Y, and V; U10 is selected from S, D, Y, T, and I; Un is selected from I, Y, F, V, L, and T; U12 is selected from F, D, Y, L, I, V, A, G, and N; and U14 is selected from D, Y, N, F, I, M, and P. In some embodiments, Ui is selected from D, Y, V, and F; U2 is selected from D, Y, L, and F; U4 is selected from G and W; Ue is selected from E and D; U7 is selected from W, L, G, and F; Us is selected from E and D; U9 is selected from E and D; U10 is selected from S, D, T, and Y; Un is selected from I, Y, V, L, and F; U12 is selected from F, D, Y, G, A, and L; U14 is selected from D, Y, M, and N.

[0127]

[0095] In some embodiments, Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 155-159 and 2520-3418. In some embodiments, Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 155-159, 2520-2546, and 3407-3418. In some embodiments, Pi comprises the amino acid sequence according to SEQ ID NO: 157. In some embodiments, Pi comprises the amino acid sequence according to SEQ ID NO: 158.

[0128] Table 10. Pi Sequences

[0129] Table 11. CD3 Mask Ala Scan Sequences - Peptide A and Peptide-B WSGR Docket No. 52426-778.601

[0130] Table 12. Clonal Phage Peptide Sequences from the Peptide-B Optimization Library Panning (-) indicates same amino acid as in CD3 scFv Peptide-B corresponding position (e.g. Phage-1 position). WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601

[0131] Table 13. CD3 scFv Mask Peptide-B Optimization WSGR Docket No. 52426-778.601

[0132] Li, L2

[0133]

[0096] In some embodiments, Li or L2 has a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 3419). In some embodiments, Li has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 3420), (GGGS)n(SEQ ID NO: 3421), (GGGGS)n(SEQ ID NO: 3422), and (GSSGGS)n(SEQ ID NO: 3423), wherein n is an integer of at least 1. In some embodiments, Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to CD3. In some embodiments, P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to PSMA. In some embodiments, the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase (MTSP1), urokinase, or hepsin. In some embodiments, Li or L2 comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.

[0134]

[0097] In some embodiments, Li or L2 comprises an amino acid sequence according to SEQ ID NO: 163. In some embodiments, Li or L2 comprises an amino acid sequence according to any one of SEQ ID NOs: 160-191. In some embodiments, Li or L2 comprises an amino acid sequence of Linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 186), Linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 187), Linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 188), or Linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 189), or an amino acid sequence that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 25, Linker 26, Linker 27, or Linker 28. In some embodiments, Li or L2 comprises an amino acid sequence of Linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 189).

[0135] Table 14. Linker Amino Acid Sequences WSGR Docket No. 52426-778.601

[0136] Half-Life Extending Molecule (Hi)

[0137]

[0098] In some embodiments, Hi comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, Hi comprises albumin.

[0138]

[0099] In some embodiments, Hi comprises a fragment crystaHizable (Fc) domain. In some embodiments, the Fc domain comprises one or more knob-in-hole (KIH) mutations. In some embodiments, the Fc domain WSGR Docket No. 52426-778.601 comprises a first polypeptide and a second polypeptide, wherein the first polypeptide of the Fc domain comprises a hole mutation and the second polypeptide of the Fc domain comprises a knob mutation. In some embodiments, the first polypeptide of the Fc domain comprises T366S, L368A, and Y407V mutations according to EU numbering, and the second polypeptide of the Fc domain comprises a T366W mutation according to EU numbering. In some embodiments, the Fc domain comprises Fc effector function silencing mutations L234A, L235A, and P329G according to EU numbering. In some embodiments, the Fc domain comprises an engineered disulfide. In some embodiments, the Fc domain comprises a Y349C mutation and a S354C mutation according to EU numbering, and the engineered disulfide is formed between two cysteines at the positions of the Y349C mutation and the S354C mutation. In some embodiments, the Fc domain comprises aN297G mutation according to EU numbering. In some embodiments, the Fc domain lacks a C-terminal glycine and a C-terminal lysine to reduce heterogeneity. In some embodiments, the Fc domain lacks C-terminal G446 and K447 residues according to EU numbering to reduce heterogeneity.

[0139]

[0100] In some embodiments, the Fc region comprises two polypeptides. In some embodiments, the Fc region has a knobs-into-holes format, wherein the first polypeptide of the Fc region comprises a hole mutation, and wherein second polypeptide of the Fc region comprises a knob mutation. In some embodiments, the first polypeptide of the Fc region comprises mutations T366S, L368A and Y407V according to EU numbering, and wherein the second polypeptide of the Fc region comprises a mutation T366W according to EU numbering. In some embodiments, the first polypeptide of the Fc region comprising the hole mutation further comprises a mutation Y349C according to EU numbering, wherein the second polypeptide of the Fc region comprising the knob mutation further comprises a mutation S354C according to EU numbering, and wherein the Fc region comprises an engineered disulfide bond formed between these two cysteines. In some embodiments, one or both of the polypeptides of the Fc region comprise mutations L234A, L235A, and P329G according to EU numbering. In some embodiments, one or both of the polypeptides of the Fc region comprise the mutation N297G according to EU numbering. In some embodiments, the Fc region lacks one or more effector functions and / or lacks glycosylation. In some embodiments, the effector function is selected from the group consisting of antibody -dependent cellular cytotoxicity (ADCC), antibody -dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), binding to a Fey receptor (FcyR), or any combination thereof. In some embodiments, the Fc region lacks the C-terminal G446 and K447 residues by EU numbering to reduce heterogeneity.

[0140]

[0101] In some embodiments, the Fc domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 203 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 204.

[0141]

[0102] In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, Hi comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35 / CR1. In some embodiments, the serum protein WSGR Docket No. 52426-778.601 comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab. In some embodiments, the single domain antibody comprises a single domain antibody that binds to albumin. In some embodiments, the single domain antibody is a human or humanized antibody. In some embodiments, the single domain antibody is 645gHlgLl. In some embodiments, the single domain antibody is 645dsgH5gL4. In some embodiments, the single domain antibody is 23-13-A01 -sc02. In some embodiments, the single domain antibody is A10m3 or a fragment thereof. In some embodiments, the single domain antibody is DOM7r-31. In some embodiments, the single domain antibody is DOM7h-l l-15. In some embodiments, the single domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single domain antibody is 10E. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC- CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1 : SEQ ID NO: 192, HC-CDR2: SEQ ID NO: 193, and HC-CDR3: SEQ ID NO: 194. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC- CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 196, HC-CDR2: SEQ ID NO: 197, and HC-CDR3: SEQ ID NO: 198. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 195. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 199. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 200. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 201. In some embodiments, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 202. In some embodiments, the single domain antibody is SA21.

[0142]

[0103] In some embodiments, the 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 modified amino acid or modified non-natural amino acid comprises a post-translational modification.

[0143]

[0104] In some embodiments, Hi comprises a linking moiety (L3) that connects Hi to Pi. In some embodiments, L3 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 is a peptide sequence having at least 26 WSGR Docket No. 52426-778.601 amino acids. In some embodiments, L3 has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 3420), (GGGS)n(SEQ ID NO: 3421), (GGGGS)n(SEQ ID NO: 3422), and (GSSGGS)n(SEQ IDNO: 3423), wherein n is an integer of at least 1. In some embodiments, L3 comprises an amino acid sequence according to GGGGSGGGSGG (SEQ ID NO: 3424).

[0144] Table 15. Half-Life Extending Molecule (Hi) Sequences WSGR Docket No. 52426-778.601

[0145] Tumor Activated Antibodies that Bind PSMA and CD3

[0146]

[0105] In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 205 and 206. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 205 and SEQ ID NO: 206. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 207 and 208. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 207 and SEQ ID NO: 208. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 209 and 210. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 209 and SEQ ID NO: 210. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 211, 212, and 213. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs: 211, 212, and 213. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3426 and 3427. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 3426 and SEQ ID NO: 3427. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3428 and 3429. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 3428 and SEQ ID NO: 3429. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3430, 3431, and 3432. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs: 3430, 3431, and 3432. In some WSGR Docket No. 52426-778.601 embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3433 and 3434. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 3433 and SEQ ID NO: 3434.

[0147] Table 16. Polypeptide Complexes that Bind CD3 and PSMA WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601 WSGR Docket No. 52426-778.601

[0148] Cleavage Product

[0149]

[0106] Disclosed herein, in some embodiments, are isolated polypeptide and polypeptide complexes according to Formula II: Lia-Pia wherein: Liacomprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Piato an anti-PSMA antibody and the anti-PSMA antibody is connected to an anti-CD3 antibody, and Pia comprises a peptide that impairs binding of the anti-PSMA antibody to PSMA. In some embodiments, Pia when Lia is uncleaved impairs binding of the anti-PSMA antibody to PSMA. In some embodiments, the anti-PSMA antibody comprises a human or humanized antibody. In some embodiments, Pia has less than 70% sequence homology to PSMA. In some embodiments, Pia comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, Pia comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, Pia comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, Pia comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, Pia comprises at least two cysteine amino acid residues. In some embodiments, Pia comprises a cyclic peptide WSGR Docket No. 52426-778.601 or a linear peptide. In some embodiments, Piacomprises a cyclic peptide. In some embodiments, Piacomprises a linear peptide.

[0150]

[0107] In some embodiments, Pia 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,

[0151] 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; X9 is 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: 3425). In some embodiments, Pia comprises an amino acid sequence according to SEQ ID NO: 118. In some embodiments, Pia comprises an amino acid sequence according to SEQ ID NO: 115. In some embodiments, Pia comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, Pia comprises an amino acid sequence according to any one of SEQ ID NOs: 85- 94. In some embodiments, Pia comprises an amino acid sequence according to any one of SEQ ID NOs: 95- 104. In some embodiments, Pia comprises an amino acid sequence according to any one of SEQ ID NOs: 105-131. In some embodiments, Pia comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 214-2519.

[0152]

[0108] In some embodiments, Liacomprises an amino acid sequence according to any one of SEQ ID NOs: 144-175. In some embodiments, Lia comprises an amino acid sequence according to SEQ ID NO: 173.

[0153] Modified Amino Acids

[0154]

[0109] 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.

[0155] Pharmaceutical Compositions

[0156] [HO] Disclosed herein are pharmaceutical compositions comprising: (i) the isolated polypeptide or polypeptide complex according to any embodiment disclosed herein, and (ii) a pharmaceutically acceptable excipient.

[0157] [Hl] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising an isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule 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,

[0158] 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, WSGR Docket No. 52426-778.601

[0159] 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: 3425).

[0160]

[0112] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising an isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 214-2519.

[0161]

[0113] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising an isolated polypeptide or polypeptide complex according to Formula II: Lia-Pi a wherein: Liacomprises atumor specific protease-cleaved linking moiety that when uncleaved connects Pia to an anti-PSMA antibody and the anti- PSMA antibody is connected to an anti-CD3 antibody, and Pia comprises a peptide that impairs binding of the anti-PSMA antibody to PSMA.

[0162]

[0114] 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.

[0163]

[0115] 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.

[0164]

[0116] 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.

[0165]

[0117] 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 WSGR Docket No. 52426-778.601 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.

[0166]

[0118] 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.

[0167] Isolated Recombinant Nucleic Acid Molecules

[0168]

[0119] Disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0169]

[0120] Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule 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: 3425).

[0170]

[0121] Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 214-2519.

[0171]

[0122] Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide complex according to Formula II: Lia-Piawherein: Liacomprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Piato an anti-PSMA antibody and the anti-PSMA antibody is connected to an anti-CD3 antibody, and Piacomprises a peptide that impairs binding of the anti-PSMA antibody to PSMA.

[0172] Methods of Treatment

[0173]

[0123] 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. WSGR Docket No. 52426-778.601

[0174]

[0124] 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 that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule 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 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: 3425).

[0175]

[0125] 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 that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 -131 and 214-2519.

[0176]

[0126] In some embodiments, the isolated polypeptide or polypeptide complex induces T cell mediated cytotoxicity of tumor cells. 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.

[0177] Production of Antibodies

[0178]

[0127] 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.

[0179]

[0128] 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. WSGR Docket No. 52426-778.601

[0180]

[0129] 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.

[0181]

[0130] 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).

[0182]

[0131] 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.

[0183]

[0132] 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).

[0184]

[0133] 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.

[0185]

[0134] 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., WSGR Docket No. 52426-778.601

[0186] 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).

[0187]

[0135] 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 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.

[0188]

[0136] 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 WSGR Docket No. 52426-778.601 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).

[0189]

[0137] 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.

[0190] Expression Vectors

[0191]

[0138] 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.

[0192]

[0139] Disclosed herein, in some embodiments, are vectors comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule 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: 3425).

[0193]

[0140] Disclosed herein, in some embodiments, are vectors comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 214-2519.

[0194]

[0141] Disclosed herein, in some embodiments, are vectors comprising an isolated recombinant nucleic acid molecules encoding an isolated polypeptide or polypeptide according to Formula II: Lia-Piawherein: Liacomprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Piato an anti- WSGR Docket No. 52426-778.601

[0195] PSMA antibody and the anti-PSMA antibody is connected to an anti-CD3 antibody, and Piacomprises a peptide that impairs binding of the anti-PSMA antibody to PSMA.

[0196]

[0142] 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.

[0197]

[0143] Exemplary insect vectors include pFastBacl, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 MIO, 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.

[0198]

[0144] 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.

[0199]

[0145] Exemplary algae vectors include pChlamy-4 vector or MCS vector.

[0200]

[0146] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG- My c-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-My c-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-My c-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0201]

[0147] 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

[0202]

[0148] 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. WSGR Docket No. 52426-778.601

[0203]

[0149] Disclosed herein, in some embodiments, are host cells comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule 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: 3425).

[0204]

[0150] Disclosed herein, in some embodiments, are host cells comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostate-specific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 214-251.

[0205]

[0151] Disclosed herein, in some embodiments, are host cells comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex according to Formula II: Lia-Piawherein: Liacomprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Piato an anti-PSMA antibody and the anti-PSMA antibody is connected to an anti-CD3 antibody, and Piacomprises a peptide that impairs binding of the anti-PSMA antibody to PSMA.

[0206]

[0152] 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 cell. Examples of bacterial cell include gram-positive bacteria or gram-negative bacteria. Sometimes the gram-negative bacteria is anaerobic, rod-shaped, or both.

[0207]

[0153] 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, WSGR Docket No. 52426-778.601

[0208] Thermodesulfobacteria or Thermotogae. A bacterial cell can be Escherichia coli, Clostridium botulinum, or Coli bacilli.

[0209]

[0154] Exemplary prokaryotic host cells include, but are not limited to, BL21, Maehl™, DH10B™, TOPIO, DH5a, DHIOBac™, OmniMax™, MegaX™, DH12S™, INV110, TOPIOF’, INVaF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™

[0210]

[0155] 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).

[0211]

[0156] 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.

[0212]

[0157] 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.

[0213]

[0158] 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, bovine, porcine, canine, feline or rodent. In some cases, a rodent includes mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, or guinea pig.

[0214]

[0159] Exemplary mammalian host cells include, but are not limited to, 293 A 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, WSGR Docket No. 52426-778.601

[0215] 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.

[0216]

[0160] 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. 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.

[0217] Articles of Manufacture

[0218]

[0161] 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.

[0219]

[0162] 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 materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0220] NUMBERED EMBODIMENTS

[0221]

[0163]

[0222] 1. An isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostatespecific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide WSGR Docket No. 52426-778.601 that impairs binding of the second antigen recognizing molecule 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:

[0223] Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R;

[0224] X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K;

[0225] X3 is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D;

[0226] X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D;

[0227] X5is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H;

[0228] X6is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G;

[0229] X7 is selected from M, L, I, A, V, F, G, and K;

[0230] X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W. G, K, R, and P;

[0231] X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and

[0232] 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: 3425).

[0233] 2. The isolated polypeptide or polypeptide complex of embodiment 1, wherein:

[0234] Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, and W;

[0235] X2is selected from E, V, D, T, S, L, G, P, A, M, and I;

[0236] X3 is selected from K, P, R, I, N, H, V, M, A, and L;

[0237] X4is selected from W, L, M, R, V, Y, and A;

[0238] X5 is selected from I, V, T, K, R, and E;

[0239] Xe is selected from A, E, S, P, Q, T, L, D, M, and V;

[0240] X7is M;

[0241] X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, and H;

[0242] X9 is selected from G, V, A, S, W, E, D, and M; and

[0243] X10 is selected from F, L, M, S, I, V, D, and Q.

[0244] 3. The isolated polypeptide or polypeptide complex of embodiment 1 or 2, wherein:

[0245] Xi is selected from V, E, L, D, I, G, and M;

[0246] X2 is selected from E, V, D, T, S, L, G, P, and A;

[0247] X3 is selected from K, P, R, I, N, H, V, and M;

[0248] X4 is selected from W, L, M, and R;

[0249] X5 is selected from I, V, T, K, R, and E;

[0250] Xe is selected from A, E, S, P, Q, and T;

[0251] X7is M;

[0252] X8is selected from E, S, T, A, V, D, and Q;

[0253] X9is selected from G, V, A, S, and W; and

[0254] X10 is selected from F, L, M, and S.

[0255] 4. The isolated polypeptide or polypeptide complex of any one of embodiments 1 -3, wherein:

[0256] Xi is selected from V, E, L, D, I, and G;

[0257] X2is selected from E, V, D, T, S, and, G; WSGR Docket No. 52426-778.601

[0258] Xs is selected from K, P, R, I, N, and H;

[0259] X4 is selected from W, L, and M;

[0260] X5 is selected from I, V, T, K, and R;

[0261] Xe is selected from A, E, S, and P;

[0262] X7is M;

[0263] Xs is selected from E, S, T, A, and V;

[0264] X9 is G; and

[0265] X10 is selected from F, L, and M.

[0266] 5. 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: 118.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 12. An isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostatespecific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 214-2519.

[0274] 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 214-2519.

[0275] 14. The isolated polypeptide or polypeptide complex of embodiment 12 or 13, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 63.

[0276] 15. The isolated polypeptide or polypeptide complex of embodiment 12 or 13, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 74.

[0277] 16. The isolated polypeptide or polypeptide complex of embodiment 12 or 13, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 77.

[0278] 17. The isolated polypeptide or polypeptide complex of embodiment 12 or 13, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 112. WSGR Docket No. 52426-778.601

[0279] 18. The isolated polypeptide or polypeptide complex of embodiment 12 or 13, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 115.

[0280] 19. The isolated polypeptide or polypeptide complex of embodiment 12 or 13, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 118.

[0281] 20. The isolated polypeptide or polypeptide complex of any one of embodiments 1-19, wherein the effector cell antigen comprises cluster of differentiation 3 (CD3).

[0282] 21. The isolated polypeptide or polypeptide complex of any one of embodiments 1 -20, wherein the first antigen recognizing molecule comprises an anti-CD3 antibody.

[0283] 22. The isolated polypeptide or polypeptide complex of embodiment 21, wherein the anti-CD3 antibody comprises an anti-CD3 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 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-CD3 antibody comprises an anti-CD3 light chain variable domain that comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 comprise amino acid sequences of LC-CDR1: SEQ ID NO: 135, LC-CDR2: GT, and LC-CDR3: SEQ ID NO: 137.

[0284] 23. The isolated polypeptide or polypeptide complex of any one of embodiments 20-22, wherein the first antigen recognizing molecule that binds to CD3 comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 138.

[0285] 24. The isolated polypeptide or polypeptide complex of any one of embodiments 1 -23, wherein the first antigen recognizing molecule comprises a single chain variable fragment (scFv) or a Fab or Fab’ fragment.

[0286] 25. The isolated polypeptide or polypeptide complex of any one of embodiments 1-24, wherein the second antigen recognizing molecule that binds to PSMA comprises an anti-PSMA antibody.

[0287] 26. The isolated polypeptide or polypeptide complex of embodiment 25, 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 amino acid sequences of HC-CDR1: SEQ ID NO: 139, HC-CDR2: SEQ ID NO: 140, andHC-CDR3: SEQ ID NO: 141 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 of the light chain variable domain comprise an amino acid sequence of LC-CDR1: SEQ ID NO: 142, LC-CDR2: EA, and LC-CDR3: SEQ ID NO: 144.

[0288] 27. The isolated polypeptide or polypeptide complex of embodiment 26, 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: 146, and 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: 145. WSGR Docket No. 52426-778.601

[0289] 28. The isolated polypeptide or polypeptide complex of embodiment 26, 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: 148, and 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: 147.

[0290] 29. The isolated polypeptide or polypeptide complex of embodiment 26, 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: 150, and 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: 149.

[0291] 30. The isolated polypeptide or polypeptide complex of any one of embodiments 25-29, wherein the anti-PSMA antibody comprises a single chain variable fragment (scFv), a single domain antibody, a Fab, a Fab’, or a CrossFab.

[0292] 31. The isolated polypeptide or polypeptide complex of any one of embodiments 25-30, wherein the anti-CD3 antibody comprises a single chain variable fragment (scFv) and the anti-PSMA antibody comprises a Fab or a Fab’.

[0293] 32. The isolated polypeptide or polypeptide complex of embodiment 31, wherein the scFv and the Fab or Fab’ are connected through a linker and the Fab or Fab’ comprise a Fab heavy chain polypeptide and a Fab light chain polypeptide.

[0294] 33. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the scFv to the Fab heavy chain polypeptide.

[0295] 34. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the scFv to the Fab light chain polypeptide.

[0296] 35. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the scFv to the N-term of the Fab heavy chain polypeptide.

[0297] 36. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the scFv to the C-term of the Fab heavy chain polypeptide.

[0298] 37. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the scFv to the N-term of the Fab light chain polypeptide.

[0299] 38. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the scFv to the C-term of the Fab light chain polypeptide.

[0300] 39. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 heavy chain variable domain of the scFv to the Fab heavy chain polypeptide.

[0301] 40. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 heavy chain variable domain of the scFv to the Fab light chain polypeptide.

[0302] 41. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 heavy chain variable domain of the scFv to the N-term of the Fab heavy chain polypeptide. WSGR Docket No. 52426-778.601

[0303] 42. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 heavy chain variable domain of the scFv to the C-term of the Fab heavy chain polypeptide.

[0304] 43. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 heavy chain variable domain of the scFv to the N-term of the Fab light chain polypeptide.

[0305] 44. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 heavy chain variable domain of the scFv to the C-term of the Fab light chain polypeptide.

[0306] 45. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 light chain variable domain of the scFv to the Fab heavy chain polypeptide.

[0307] 46. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 light chain variable domain of the scFv to the Fab light chain polypeptide.

[0308] 47. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 light chain variable domain of the scFv to the N-term of the Fab heavy chain polypeptide.

[0309] 48. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 light chain variable domain of the scFv to the C-term of the Fab heavy chain polypeptide.

[0310] 49. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 light chain variable domain of the scFv to the N-term of the Fab light chain polypeptide.

[0311] 50. The isolated polypeptide or polypeptide complex of embodiment 32, wherein the linker connects the anti-CD3 light chain variable domain of the scFv to the C-term of the Fab light chain polypeptide.

[0312] 51. The isolated polypeptide or polypeptide complex of any one of embodiments 32-50, wherein the linker is at least 5 amino acids in length.

[0313] 52. The isolated polypeptide or polypeptide complex of any one of embodiments 32-51, wherein the linker is no more than 30 amino acids in length.

[0314] 53. The isolated polypeptide or polypeptide complex of any one of embodiments 32-52, wherein the linker is at least 5 amino acids and no more than 30 amino acids in length.

[0315] 54. The isolated polypeptide or polypeptide complex of any one of embodiments 32-50, wherein the linker is 5 amino acids in length.

[0316] 55. The isolated polypeptide or polypeptide complex of any one of embodiments 32-50, wherein the linker is 15 amino acids in length.

[0317] 56. The isolated polypeptide or polypeptide complex of any one of embodiments 32-50, wherein the linker comprises an amino acid sequence of SEQ ID NO: 151 (GGGGSGGGGSGGGGS) or SEQ ID NO: 152 (GGGGS).

[0318] 57. The isolated polypeptide or polypeptide complex of any one of embodiments 1-56, wherein the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 153 and SEQ ID NO: 154.

[0319] 58. The isolated polypeptide or polypeptide complex of any one of embodiments 1-57, wherein the isolated polypeptide or polypeptide complex is human or humanized. WSGR Docket No. 52426-778.601

[0320] 59. The isolated polypeptide or polypeptide complex of any one of embodiments 21-58, wherein the isolated polypeptide or polypeptide complex further comprises a peptide that is linked to the anti-CD3 antibody, wherein the peptide impairs binding of the anti-CD3 antibody to CD3.

[0321] 60. The isolated polypeptide or polypeptide complex of embodiment 59, wherein the isolated polypeptide or polypeptide complex comprises a configuration according to Formula I: P2-L2-A2-A1-L1-P1- Hi wherein Ai comprises the anti-CD3 antibody; Pi comprises the peptide that impairs binding of the anti- CD3 antibody to CD3; Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; A2 comprises the anti-PSMA antibody; P2 comprises the peptide that impairs binding of the anti-PSMA antibody to PSMA; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.

[0322] 61. The isolated polypeptide or polypeptide complex of embodiment 60, wherein the anti-CD3 antibody comprises a scFv and the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide; and the anti-PSMA antibody comprises a Fab or Fab’ and the Fab or Fab’ comprise a Fab heavy chain polypeptide and a Fab light chain polypeptide.

[0323] 62. The isolated polypeptide or polypeptide complex of embodiment 61, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2.

[0324] 63. The isolated polypeptide or polypeptide complex of embodiment 61, wherein the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab heavy chain polypeptide of A2.

[0325] 64. The isolated polypeptide or polypeptide complex of embodiment 61, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2.

[0326] 65. The isolated polypeptide or polypeptide complex of embodiment 61, wherein the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and L2 is bound to the Fab heavy chain polypeptide of A2.

[0327] 66. The isolated polypeptide or polypeptide complex of any one of embodiments 60-65, wherein Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi -stacking interactions, or H-bonding interactions, or a combination thereof.

[0328] 67. The isolated polypeptide or polypeptide complex of any one of embodiments 60-66, wherein Pi has less than 70% sequence homology to CD3.

[0329] 68. The isolated polypeptide or polypeptide complex of any one of embodiments 60-67, wherein P2 impairs binding of A2 to PSMA.

[0330] 69. The isolated polypeptide or polypeptide complex of any one of embodiments 60-68, wherein P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi -stacking interactions, or H-bonding interactions, or a combination thereof. WSGR Docket No. 52426-778.601

[0331] 70. The isolated polypeptide or polypeptide complex of any one of embodiments 60-69, wherein P2 is bound to A2 at or near an antigen binding site.

[0332] 71. The isolated polypeptide or polypeptide complex of any one of embodiments 60-70, wherein P2 has less than 70% sequence homology to PS MA.

[0333] 72. The isolated polypeptide or polypeptide complex of any one of embodiments 60-71, Pi orP2 comprises a peptide sequence of at least 10 amino acids in length.

[0334] 73. The isolated polypeptide or polypeptide complex of any one of embodiments 60-72, wherein Pi or P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

[0335] 74. The isolated polypeptide or polypeptide complex of any one of embodiments 60-73, wherein Pi or P2 comprises a peptide sequence of at least 16 amino acids in length acids.

[0336] 75. The isolated polypeptide or polypeptide complex of any one of embodiments 60-74, wherein Pi or P2 comprises a peptide sequence of no more than 40 amino in length.

[0337] 76. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 is 11 amino acids in length.

[0338] 77. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 is 12 amino acids in length.

[0339] 78. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 is 13 amino acids in length.

[0340] 79. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 is 14 amino acids in length

[0341] 80. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 is 15 amino acids in length.

[0342] 81. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 is 16 amino acids in length.

[0343] 82. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 comprises at least two cysteine amino acid residues.

[0344] 83. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 comprises a cyclic peptide or a linear peptide.

[0345] 84. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 comprises a cyclic peptide.

[0346] 85. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi or P2 comprises a linear peptide.

[0347] 86. The isolated polypeptide or polypeptide complex of any one of embodiments 60-75, wherein Pi comprises at least two cysteine amino acid residues.

[0348] 87. The isolated polypeptide or polypeptide complex of any one of embodiments 60-86, wherein Li is bound to N-terminus of Ai. WSGR Docket No. 52426-778.601

[0349] 88. The isolated polypeptide or polypeptide complex of any one of embodiments 60-86, wherein Li is bound to C-terminus of Ai.

[0350] 89. The isolated polypeptide or polypeptide complex of any one of embodiments 60-86, wherein L2 is bound to N-terminus of A2.

[0351] 90. The isolated polypeptide or polypeptide complex of any one of embodiments 60-86, wherein L2 is bound to C-terminus of A2.

[0352] 91. The isolated polypeptide or polypeptide complex of any one of embodiments 60-90, wherein Li or L2 is a peptide sequence having at least 5 to no more than 50 amino acids.

[0353] 92. The isolated polypeptide or polypeptide complex of any one of embodiments 60-91, wherein Li or L2 is a peptide sequence having at least 10 to no more than 30 amino acids.

[0354] 93. The isolated polypeptide or polypeptide complex of any one of embodiments 60-90, wherein Li or L2 is a peptide sequence having at least 10 amino acids.

[0355] 94. The isolated polypeptide or polypeptide complex of any one of embodiments 60-93, Li or L2 is a peptide sequence having at least 18 amino acids.

[0356] 95. The isolated polypeptide or polypeptide complex of any one of embodiments 60-94, wherein Li or L2 is a peptide sequence having at least 26 amino acids.

[0357] 96. The isolated polypeptide or polypeptide complex of any one of embodiments 60-95, wherein Pi comprises an amino acid sequence according to U1-U2-C- U4-P-U6-U7-U8-U9-U10-U11- U12-C-U14 and Ui is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; U4 is selected from G and W; Ue is selected from E, D, V, and P; U7 is selected from W, L, F, V, G, M, I, and Y; Us is selected from E, D, P, and Q; U9 is selected from E, D, Y, V, F, W, P, L, and Q; U10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; Un is selected from I, Y, F, V, L, T, N, S, D, A, and H; U is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H; and U14 is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

[0358] 97. The isolated polypeptide or polypeptide complex of any one of embodiments 60-96, wherein Ui is selected from D, Y, F, I, V, and N; U2 is selected from D, Y, L, F, I, and N; U4 is selected from G and W; Ue is selected from E and D; U7 is selected from W, L, F, G, and V; Us is selected from E and D; U9 is selected from E, D, Y, and V; U10 is selected from S, D, Y, T, and I; Un is selected from I, Y, F, V, L, and T; U is selected from F, D, Y, L, I, V, A, G, and N; and U14 is selected from D, Y, N, F, I, M, and P.

[0359] 98. The isolated polypeptide or polypeptide complex of any one of embodiments 60-97, wherein Ui is selected from D, Y, V, and F; U2 is selected from D, Y, L, and F; U4 is selected from G and W; Ue is selected from E and D; U7 is selected from W, L, G, and F; Us is selected from E and D; U9 is selected from E and D; U10 is selected from S, D, T, and Y; Un is selected from I, Y, V, L, and F; U is selected from F, D, Y, G, A, and L; U14 is selected from D, Y, M, and N.

[0360] 99. The isolated polypeptide or polypeptide complex of any one of embodiments 60-95, wherein Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 155-159 and 2520-3418. WSGR Docket No. 52426-778.601

[0361] 100. The isolated polypeptide or polypeptide complex of any one of embodiments 60-95, wherein Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 155-159, 2520-2546, and 3407- 3418.

[0362] 101. The isolated polypeptide or polypeptide complex of any one of embodiments 60-95, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 156.

[0363] 102. The isolated polypeptide or polypeptide complex of any one of embodiments 60-95, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 157.

[0364] 103. The isolated polypeptide or polypeptide complex of any one of embodiments 60-95, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 158.

[0365] 104. The isolated polypeptide or polypeptide complex of any one of embodiments 60-103, wherein Li or L2has a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 3419).

[0366] 105. The isolated polypeptide or polypeptide complex of any one of embodiments 60-103, wherein Li has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n(SEQ ID NO: 3420), (GGGS)n(SEQ ID NO: 3421), (GGGGS)n(SEQ ID NO: 3422), and (GSSGGS)n(SEQ ID NO: 3423), wherein n is an integer of at least 1.

[0367] 106. The isolated polypeptide or polypeptide complex of any one of embodiments 60-105, wherein Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to CD3.

[0368] 107. The isolated polypeptide or polypeptide complex of any one of embodiments 60-106, wherein P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to PSMA.

[0369] 108. The isolated polypeptide or polypeptide complex of embodiment 107, wherein the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease.

[0370] 109. The isolated polypeptide or polypeptide complex of embodiment 108, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

[0371] 110. The isolated polypeptide or polypeptide complex of embodiment 108, wherein the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.

[0372] 111. The isolated polypeptide or polypeptide complex of embodiment 108, wherein Li or L2 comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.

[0373] 112. The isolated polypeptide or polypeptide complex of any one of embodiments 60-111, wherein Li or L2 comprises an amino acid sequence according to SEQ ID NO: 163.

[0374] 113. The isolated polypeptide or polypeptide complex of any one of embodiments 60-111, wherein Li or L2 comprises an amino acid sequence according to any one of SEQ ID NOs: 160-191.

[0375] 114. The isolated polypeptide or polypeptide complex of any one of embodiments 60-111, wherein Li or L2 comprises an amino acid sequence of Linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 186), Linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 187), Linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 188), or Linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 189), or an amino acid sequence that has 1, 2, or 3 amino WSGR Docket No. 52426-778.601 acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 25, Linker 26, Linker 27, or Linker 28.

[0376] 115. The isolated polypeptide or polypeptide complex of any one of embodiments 60-114, wherein Li or L2 is at least 5 amino acids in length.

[0377] 116. The isolated polypeptide or polypeptide complex of any one of embodiments 60-115, wherein Li or L2 is no more than 30 amino acids in length.

[0378] 117. The isolated polypeptide or polypeptide complex of any one of embodiments 60-116, wherein Li or L2is at least 5 amino acids and no more than 30 amino acids in length.

[0379] 118. The isolated polypeptide or polypeptide complex of any one of embodiments 60-114, wherein Li or L2 is 5 amino acids in length.

[0380] 119. The isolated polypeptide or polypeptide complex of any one of embodiments 60-114, wherein Li or L2is 15 amino acids in length.

[0381] 120. The isolated polypeptide or polypeptide complex of any one of embodiments 60-119, wherein Hi comprises a polymer.

[0382] 121. The isolated polypeptide or polypeptide complex of embodiment 120, wherein the polymer is polyethylene glycol (PEG).

[0383] 122. The isolated polypeptide or polypeptide complex of any one of embodiments 60-119, wherein Hi comprises albumin.

[0384] 123. The isolated polypeptide or polypeptide complex of any one of embodiments 60-119, wherein Hi comprises a fragment crystaHizable (Fc) domain.

[0385] 124. The isolated polypeptide or polypeptide complex of embodiment 123, wherein the Fc domain comprises one or more knob-in-hole (KIH) mutations.

[0386] 125. The isolated polypeptide or polypeptide complex of embodiment 124, wherein the Fc domain comprises a first polypeptide and a second polypeptide, wherein the first polypeptide of the Fc domain comprises a hole mutation and the second polypeptide of the Fc domain comprises a knob mutation.

[0387] 126. The isolated polypeptide or polypeptide complex of embodiment 125, wherein the first polypeptide of the Fc domain comprises T366S, L368A, and Y407V mutations according to EU numbering, and the second polypeptide of the Fc domain comprises a T366W mutation according to EU numbering.

[0388] 127. The isolated polypeptide or polypeptide complex of any one of embodiments 123-126, wherein the Fc domain comprises Fc effector function silencing mutations L234A, L235A, and P329G according to EU numbering.

[0389] 128. The isolated polypeptide or polypeptide complex of any one of embodiments 123-127, wherein the Fc domain comprises an engineered disulfide.

[0390] 129. The isolated polypeptide or polypeptide complex of embodiment 128, wherein the Fc domain comprises a Y349C mutation and a S354C mutation according to EU numbering, and wherein the engineered disulfide is formed between two cysteines at the positions of the Y349C mutation and the S354C mutation. WSGR Docket No. 52426-778.601

[0391] 130. The isolated polypeptide or polypeptide complex of any one of embodiments 123 - 129, wherein the Fc domain comprises aN297G mutation according to EU numbering.

[0392] 131. The isolated polypeptide or polypeptide complex of any one of embodiments 123-130, wherein the Fc domain lacks a C-terminal glycine and a C-terminal lysine to reduce heterogeneity.

[0393] 132. The isolated polypeptide or polypeptide complex of any one of embodiments 123-131, wherein the Fc domain lacks C-terminal G446 and K447 residues according to EU numbering to reduce heterogeneity.

[0394] 133. The isolated polypeptide or polypeptide complex of any one of embodiments 123-132, wherein the Fc domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 203 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 204.

[0395] 134. The isolated polypeptide or polypeptide complex of embodiment 122, wherein the albumin is serum albumin.

[0396] 135. The isolated polypeptide or polypeptide complex of embodiment 122, wherein the albumin is human serum albumin.

[0397] 136. The isolated polypeptide or polypeptide complex of any one of embodiments 60-119, wherein Hi comprises a polypeptide, a ligand, or a small molecule.

[0398] 137. The isolated polypeptide or polypeptide complex of embodiment 136, wherein the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35 / CR1.

[0399] 138. The isolated polypeptide or polypeptide complex of embodiment 137, wherein the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.

[0400] 139. The isolated polypeptide or polypeptide complex of embodiment 137, wherein the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.

[0401] 140. The isolated polypeptide or polypeptide complex of embodiment 137, wherein the serum protein is albumin.

[0402] 141. The isolated polypeptide or polypeptide complex of embodiment 136, wherein the polypeptide is an antibody.

[0403] 142. The isolated polypeptide or polypeptide complex of embodiment 136, wherein the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.

[0404] 143. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody comprises a single domain antibody that binds to albumin.

[0405] 144. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is a human or humanized antibody.

[0406] 145. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is 645gHlgLl. WSGR Docket No. 52426-778.601

[0407] 146. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is 645dsgH5gL4.

[0408] 147. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is 23-13-A01 -sc02.

[0409] 148. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is A10m3 or a fragment thereof.

[0410] 149. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is DOM7r-31.

[0411] 150. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is DOM7h- 11-15.

[0412] 151. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.

[0413] 152. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is 10E.

[0414] 153. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC- CDR1: SEQ ID NO: 192, HC-CDR2: SEQ ID NO: 193, and HC-CDR3: SEQ ID NO: 194.

[0415] 154. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC- CDR1: SEQ ID NO: 196, HC-CDR2: SEQ ID NO: 197, and HC-CDR3: SEQ ID NO: 198.

[0416] 155. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 195.

[0417] 156. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 199.

[0418] 157. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 200.

[0419] 158. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 201.

[0420] 159. The isolated polypeptide or polypeptide complex of embodiment 142, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 202. WSGR Docket No. 52426-778.601

[0421] 160. The isolated polypeptide or polypeptide complex of embodiment 142, wherein the single domain antibody is SA21.

[0422] 161. The isolated polypeptide or polypeptide complex of any one of embodiments 1-160, wherein the polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof.

[0423] 162. The isolated polypeptide or polypeptide complex of embodiment 161, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.

[0424] 163. The isolated polypeptide or polypeptide complex of any one of embodiments 60-162, wherein Hi comprises a linking moiety (L3) that connects Hi to Pi.

[0425] 164. The isolated polypeptide or polypeptide complex of embodiment 163, wherein L3 is a peptide sequence having at least 5 to no more than 50 amino acids.

[0426] 165. The isolated polypeptide or polypeptide complex of embodiment 164, wherein L3 is a peptide sequence having at least 10 to no more than 30 amino acids.

[0427] 166. The isolated polypeptide or polypeptide complex of embodiment 163, wherein L3 is a peptide sequence having at least 10 amino acids.

[0428] 167. The isolated polypeptide or polypeptide complex of embodiment 163, wherein L3 is a peptide sequence having at least 18 amino acids.

[0429] 168. The isolated polypeptide or polypeptide complex of embodiment 163, wherein L3 is a peptide sequence having at least 26 amino acids.

[0430] 169. The isolated polypeptide or polypeptide complex of embodiment 163, wherein L^ has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 3420), (GGGS)n (SEQ ID NO: 3421), (GGGGS)n(SEQ ID NO: 3422), and (GSSGGS)n(SEQ ID NO: 3423), wherein n is an integer of at least 1.

[0431] 170. The isolated polypeptide or polypeptide complex of embodiment 163, wherein L3 comprises an amino acid sequence according to GGGGSGGGSGG (SEQ ID NO: 3424).

[0432] 171. The isolated polypeptide or polypeptide complex of any one of embodiments 1-170, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 205 and 206.

[0433] 172. The isolated polypeptide or polypeptide complex of any one of embodiments 1-170, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 207 and 208.

[0434] 173. The isolated polypeptide or polypeptide complex of any one of embodiments 1-170, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 209 and 210.

[0435] 174. The isolated polypeptide or polypeptide complex of any one of embodiments 1-170, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 211, 212, and 213. WSGR Docket No. 52426-778.601

[0436] 175. The isolated polypeptide or polypeptide complex of any one of embodiments 1 -170, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3426 and 3427.

[0437] 176. The isolated polypeptide or polypeptide complex of any one of embodiments 1 -170, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3428 and 3429.

[0438] 177. The isolated polypeptide or polypeptide complex of any one of embodiments 1 -170, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3430, 3431, and 3432.

[0439] 178. The isolated polypeptide or polypeptide complex of any one of embodiments 1 -170, The isolated polypeptide or polypeptide complex of any one of embodiments 1 -170, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3433 and 3434.

[0440] 179. An isolated polypeptide or polypeptide complex according to Formula II: Lia-Pia wherein: Liacomprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Piato an anti- PSMA antibody and the anti-PSMA antibody is connected to an anti-CD3 antibody, and Pia comprises a peptide that impairs binding of the anti-PSMA antibody to PSMA.

[0441] 180. The isolated polypeptide or polypeptide complex of embodiment 179, wherein Piawhen Lia is uncleaved impairs binding of the anti-PSMA antibody to PSMA.

[0442] 181. The isolated polypeptide or polypeptide complex of embodiment 180, wherein the anti-PSMA antibody comprises a human or humanized antibody.

[0443] 182. The isolated polypeptide or polypeptide complex of any one of embodiments 179-181, wherein Pia has less than 70% sequence homology to PSMA.

[0444] 183. The isolated polypeptide or polypeptide complex of any one of embodiments 179-182, wherein Pia comprises a peptide sequence of at least 10 amino acids in length.

[0445] 184. The isolated polypeptide or polypeptide complex of any one of embodiments 179-183, wherein Pia comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

[0446] 185. The isolated polypeptide or polypeptide complex of any one of embodiments 179-184, wherein Pia comprises a peptide sequence of at least 16 amino acids in length.

[0447] 186. The isolated polypeptide or polypeptide complex of any one of embodiments 179-185, wherein Pia comprises a peptide sequence of no more than 40 amino acids in length.

[0448] 187. The isolated polypeptide or polypeptide complex of any one of embodiments 179-186, wherein Pia comprises at least two cysteine amino acid residues.

[0449] 188. The isolated polypeptide or polypeptide complex of any one of embodiments 179-187, wherein Pia comprises a cyclic peptide or a linear peptide.

[0450] 189. The isolated polypeptide or polypeptide complex of any one of embodiments 179-188, wherein Pia comprises a cyclic peptide. WSGR Docket No. 52426-778.601

[0451] 190. The isolated polypeptide or polypeptide complex of any one of embodiments 179-189, wherein Piacomprises a linear peptide.

[0452] 191. The isolated polypeptide or polypeptide complex of any one of embodiments 179-190, wherein Piacomprises an amino acid sequence according to X1-X2-X3-X4-C-X5-P-X6-W-X7-C-X8-X9-X10, wherein:

[0453] Xi is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R;

[0454] X2is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R, and K;

[0455] X3 is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W, and D;

[0456] X4is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D;

[0457] X5is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H;

[0458] X6is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G;

[0459] X7 is selected from M, L, I, A, V, F, G, and K;

[0460] X8is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P;

[0461] X9is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and

[0462] 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: 3425).

[0463] 192. The isolated polypeptide or polypeptide complex of any one of embodiments 179-191, wherein Piacomprises an amino acid sequence according to SEQ ID NO: 118.

[0464] 193. The isolated polypeptide or polypeptide complex of any one of embodiments 179-191, wherein Piacomprises an amino acid sequence according to SEQ ID NO: 115.

[0465] 194. The isolated polypeptide or polypeptide complex of any one of embodiments 179-191, wherein Piacomprises an amino acid sequence according to SEQ ID NO: 23.

[0466] 195. The isolated polypeptide or polypeptide complex of any one of embodiments 179-191, wherein Piacomprises an amino acid sequence according to any one of SEQ ID NOs: 85-94.

[0467] 196. The isolated polypeptide or polypeptide complex of any one of embodiments 179-191, wherein Piacomprises an amino acid sequence according to any one of SEQ ID NOs: 95-104.

[0468] 197. The isolated polypeptide or polypeptide complex of any one of embodiments 179-191, wherein Piacomprises an amino acid sequence according to any one of SEQ ID NOs: 105-131.

[0469] 198. The isolated polypeptide or polypeptide complex of any one of embodiments 179-191, wherein Piacomprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 214-2519.

[0470] 199. The isolated polypeptide or polypeptide complex of any one of embodiments 179-191, wherein Liacomprises an amino acid sequence according to any one of SEQ ID NOs: 160-191.

[0471] 200. The isolated polypeptide or polypeptide complex of any one of embodiments 179-191, wherein Liacomprises an amino acid sequence according to SEQ ID NO: 189.

[0472] 201. An isolated polypeptide or polypeptide complex that comprises an anti-PSMA antibody 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 amino acid sequences of HC-CDR1: SEQ ID NO: 139, HC-CDR2: SEQ ID NO: 140, and HC-CDR3: SEQ ID NO: 141 and complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC- WSGR Docket No. 52426-778.601

[0473] CDR3 comprise amino acid sequences of LC-CDR1: SEQ ID NO: 142, LC-CDR2: EA, and LC-CDR3: SEQ ID NO: 144; a peptide that comprises the amino acid sequence of SEQ ID NO: 118 and impairs binding of the anti-PSMA antibody to PSMA; an anti-CD3 antibody 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 amino acid sequences ofHC-CDRl: SEQ ID NO: 132, HC-CDR2: SEQ ID NO: 133, and HC-CDR3: SEQ ID NO: 134, and the anti-CD3 antibody comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and wherein the LC-CDR1, the LC- CDR2, and the LC-CDR3 comprise amino acid sequences of LC-CDR1: SEQ ID NO: 135, LC-CDR2: GT, and LC-CDR3: SEQ ID NO: 137; and a peptide that comprises the amino acid sequence according to SEQ ID NO: 156 and impairs binding of the anti-CD3 antibody to CD3.

[0474] 202. The isolated polypeptide or polypeptide complex of embodiment 201, wherein the peptide that comprises the amino acid sequence according to SEQ ID NO: 118 and impairs binding of the anti-PSMA antibody to PSMA is no more than 14 amino acids in length.

[0475] 203. The isolated polypeptide or polypeptide complex of embodiment 201, wherein the peptide that comprises the amino acid sequence of SEQ ID NO: 156 and impairs binding of the anti-CD3 antibody to CD3 is no more than 16 amino acids in length.

[0476] 204. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -203, wherein the isolated polypeptide or polypeptide complex further comprises a half-life extending molecule.

[0477] 205. The isolated polypeptide or polypeptide complex of embodiment 204, wherein the half-life extending molecule comprises the amino acid sequences according to SEQ ID NOs: 192-194.

[0478] 206. The isolated polypeptide or polypeptide complex of embodiment 204, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 195.

[0479] 207. The isolated polypeptide or polypeptide complex of embodiment 204, wherein the half-life extending molecule comprises the amino acid sequences according to SEQ ID NOs: 196-198.

[0480] 208. The isolated polypeptide or polypeptide complex of embodiment 204, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 199.

[0481] 209. The isolated polypeptide or polypeptide complex of embodiment 204, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 200.

[0482] 210. The isolated polypeptide or polypeptide complex of embodiment 204, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 201.

[0483] 211. The isolated polypeptide or polypeptide complex of embodiment 204, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 202.

[0484] 212. The isolated polypeptide or polypeptide complex of embodiment 204, wherein the half-life extending molecule comprises the amino acid sequences according to SEQ ID NOs: 203-204.

[0485] 213. The isolated polypeptide or polypeptide complex of embodiment 204, wherein the isolated polypeptide or polypeptide complex further comprises the amino acid sequence according to SEQ ID NO: 189. WSGR Docket No. 52426-778.601

[0486] 214. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -204, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3426 and 3427.

[0487] 215. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -204, wherein the polypeptide or polypeptide complex comprises the amino acid sequences according to SEQ ID NOs: 3426 and 3427.

[0488] 216. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -204, wherein the polypeptide or polypeptide complex consists of the amino acid sequences according to SEQ ID NOs: 3426 and 3427.

[0489] 217. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -204, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3428 and 3429.

[0490] 218. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -204, wherein the polypeptide or polypeptide complex comprises the amino acid sequences according to SEQ ID NOs: 3428 and 3429.

[0491] 219. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -204, wherein the polypeptide or polypeptide complex consists of the amino acid sequences according to SEQ ID NOs: 3428 and 3429.

[0492] 220. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -204, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3433 and 3434.

[0493] 221. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -204, wherein the polypeptide or polypeptide complex comprises the amino acid sequences according to SEQ ID NOs: 3433 and 3434.

[0494] 222. The isolated polypeptide or polypeptide complex of any one of embodiments 201 -204, wherein the polypeptide or polypeptide complex consists of the amino acid sequences according to SEQ ID NOs: 3433 and 3434.

[0495] 223. A pharmaceutical composition comprising: (a) the isolated polypeptide or polypeptide complex of any one of embodiments 1-222; and (b) a pharmaceutically acceptable excipient.

[0496] 224. An isolated recombinant nucleic acid molecule encoding the isolated polypeptide or polypeptide complex of any one of embodiments 1-222.

[0497] 225. A vector comprising the isolated nucleic acid molecule of embodiment 224.

[0498] 226. A host cell comprising the isolated recombinant nucleic acid molecule of embodiment 224.

[0499] 227. 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 1-223.

[0500] 228. The method of embodiment 227, wherein the isolated polypeptide or polypeptide complex induces T cell mediated cytotoxicity of tumor cells. WSGR Docket No. 52426-778.601

[0501] 229. The method of embodiment 228, wherein the tumor cells express PSMA.

[0502] 230. The method of any one of embodiments 227-229, wherein the cancer is a solid tumor cancer.

[0503] 231. The method of embodiment 230, wherein the cancer is prostate, lung, breast, cervical, ovarian, colorectal, pancreatic or gastric cancer.

[0504] EXAMPLES

[0505] Example 1:

[0506] Equilibrium Binding, via ELISA

[0507]

[0164] 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: 145, 146). Briefly, biotinylated peptides were captured on neutravidin coated plates. PSMA Fab diluted in buffer was then added to the peptide captured plates.

[0508] 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 17-19.

[0509] Binding Inhibition by Peptide Masks

[0510]

[0165] Peptides were further evaluated for their ability to inhibit PSMA Fab (SEQ ID NOs: 145, 146) from binding to the PSMA antigen in a standard ELISA format. Briefly, biotinylated PSMA antigen was captured 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 17-19.

[0511] Kinetic Binding via BLI

[0512]

[0166] Kinetic binding peptides to PSMA Fab (SEQ ID NOs: 145, 146) 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 WSGR Docket No. 52426-778.601 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 18-19.

[0513] Peptide mask properties

[0514]

[0167] 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.

[0515] Table 17.

[0516] Table 18. WSGR Docket No. 52426-778.601

[0517] Table 19.

[0518] Example 2: Sequence Activity Relationships - PSMA Mask Peptides

[0519]

[0168] 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 (SEQ ID NOs: 145, 146) 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 20-21. The tables also designate each alanine mutation as tolerant (T), somewhat tolerant (ST), or intolerant (IT) based on changes in binding inhibition WSGR Docket No. 52426-778.601 ability relative to the non-mutated peptide. Cys-5 and Cys-11 of peptide-23 and peptide- 24 were not mutated.

[0520] Table 20. Competition Binding ELISA - Alanine Scanning Sequences of Peptide-23

[0521] Table 21. Competition Binding ELISA - Alanine Scanning Sequences of Peptide-24

[0522] Example 3. Optimization Phage Library Construction of Peptide-24

[0523]

[0169] 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.

[0524] Example 4: Panning of the optimized phage library of Peptide-24 WSGR Docket No. 52426-778.601

[0525]

[0170] 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 22

[0526] Table 22. SAR guided optimization phage library of peptide-24.

[0527] Example 5: Panning of the optimized phage library

[0528]

[0171] 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 WSGR Docket No. 52426-778.601 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.

[0529]

[0172] Phage panning results of peptide-24 library sequences are shown in Table 5. 2323 clonal phage sequences were identified. A consensus sequence was calculated from all the sequences of Table 5 using WebLogo 3.7. 12. The consensus sequence is shown in FIG. 28.

[0530] Example 6. Optimization Phage Library Construction of Peptide-23

[0531]

[0173] 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.

[0532] Example 7: Panning of the optimized phage library of Peptide-23

[0533]

[0174] 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 23

[0534] Table 23. SAR guided optimization phage library of peptide-23. WSGR Docket No. 52426-778.601

[0535] Example 8: PSMA Polypeptide Complex Binding to PSMA and CD3

[0536]

[0175] Ab-1, PC-5, and PC-2 were evaluated for their ability to bind PSMA as well as CD3 in a standard enzyme linked immunosorbent assay (ELISA) format. Polypeptide complex binding of PSMA or CD3 were measured before and after protease treatment. Briefly, biotinylated antigen was captured on neutravidin coated plates. Polypeptide complex molecules were treated with active matriptase (MTSP1) or MMP9 where indicated. Polypeptide complex molecules diluted in buffer were then added to the antigen coated plates. Bound polypeptide complex was detected using a standard horse radish peroxidase conjugate secondary antibody. The concentration of polypeptide complex required to achieve 50% maximal signal (EC50) was calculated in Graphpad Prism. FIG. 29 shows representative PSMA binding ELISAs. This data is summarized in Table 24. FIG. 30 shows representative CD3 binding ELISAs. This data is summarized in Table 25

[0537]

[0176] The masked polypeptide complex of PC-5 has an EC50 about 110-fold higher than the MSTP1 treated PC-5, and an EC50 about 220-fold higher than MMP9 treated PC-5. The masked polypeptide complex of PC-2 has an EC50 about 4.6-fold higher than the masked PC-5. The masked polypeptide complex of PC-5 has an EC50 about 220-fold higher than Ab-1 and the masked polypeptide complex of PC- 2 has an EC50 about 1,020-fold higher than Ab-1. MTSP1 cleaved PC-5 has an ECso about 2-fold higher than Ab-1, and MMP9 cleaved PC-5 has an ECso equal to Ab-1.

[0538]

[0177] The masked polypeptide complex of PC-5 has an EC50 about 930-fold higher than the MSTP1 treated PC-5 and MMP9 treated PC-5. The masked polypeptide complex of PC-2 has an EC50 about 17.6- fold higher than the masked PC-5. The masked polypeptide complex of PC-5 has an ECso about 930-fold higher than Ab-1. The masked polypeptide complex of PC-2 has an ECso about 16,350 higher than Ab-1. Protease cleaved PC-5 has an ECso equal to Ab-1.

[0539] Table 24. WSGR Docket No. 52426-778.601

[0540] Table 25.

[0541] Example 9: PSMA Polypeptide Complex Mediated Tumor Cell Killing

[0542]

[0178] Ab-1 and PC-5 were evaluated in a functional in vitro tumor cell killing assay using the PSMA positive tumor cell lines 22Rvl and LNCaP. Tumor cell killing was measured using a real time cell analyzer from Acea Biosciences that relies on sensor impedance measurements (cell index) that increased as tumor cells adhere, spread, and expand on the surface of the sensor. Likewise, as the tumor cells were killed the impedance decreased. 25,000 tumor cells were added per well and allowed to adhere overnight. The following day polypeptide complexes titrated in human serum supplemented medium along with 75,000 CD8+ T cells were added to the wells. Cell index measurements were taken every 10 minutes for an additional 96 hours. The cell index times number of hours (tumor cell growth kinetics) was then plotted versus concentration of polypeptide complex where the concentration required to reduce the tumor growth 50% (IC50) was calculated using Graphpad Prism.

[0543]

[0179] The 22Rvl tumor cell line has a PSMA density of about 3000 copies per cell. FIG. 31 shows representative viability data for 22Rvl treated with Ab-1 or PC-5. This data is summarized in Table 26 and shows that the masked PC-5 has an ECso about 4,583-fold higher than MTSP1 cleaved PC-5 and an ECso about 11,000-fold higher than MMP9 cleaved PC-5. The masked PC-5 has an ECso about 14,666-fold higher than Ab-1. MTSP1 cleaved PC-5 has an ECso about 3.2-fold higher than Ab-1, and MMP9 cleaved PC-5 has an ECso about 1.3-fold higher than Ab-1.

[0544]

[0180] The LNCaP tumor cell line has a PSMA density of about 350,000 copies per cell. FIG. 32 shows representative viability data for LNCaP. This data is summarized in Table 27 and shows that masked PC-5 has an ECso about 1,560-fold higher than MTSP1 cleaved PC-5 and an ECso about 2,926-fold higher than MMP9 cleaved PC-5. The masked PC-5 has an ECso about 5,852-fold higher than Ab-1. MTSP1 cleaved PC-5 has an ECso about 3.75-fold higher than Ab-1 and MMP9 cleaved PC-5 has an ECso about 2-fold higher than Ab- 1.

[0545] Table 26. WSGR Docket No. 52426-778.601

[0546] Table 27.

[0547] Example 10: PC-5 in cynomolgus cytokine release

[0548]

[0181] Cytokine release after polypeptide molecule administration by IV bolus and subcutaneous injection was evaluated in cynomolgus monkeys. Briefly, cynomolgus monkeys of approximately 3 kg bodyweight were administered polypeptides as an IV bolus or subcutaneous injection and observed daily for signs of adverse events. No in-life adverse events or cytokine release syndrome were observed. After dosing, blood was collected in K2 EDTA tubes at specific timepoints and processed to plasma. Plasma was stored frozen until analysis. Concentration of polypeptide molecules in plasma was measured via standard ELISA techniques relative to a reference standard diluted in control cynomolgus monkey plasma. The concentration of PC-5 over 7 days is shown in FIG. 33. Plasma samples were analyzed for cytokines using a non-human primate cytometric Thl / Th2 bead array kit from BD biosciences following the manufacturer’s instructions. Interferon gamma, tumor necrosis factor alpha, interleukin 6, interleukin 10, and interleukin 2 levels in plasma were calculated relative to reference standards provided with the bead array kit. Data is seen in FIGs. 34-38. FIG. 35 shows that PC-5 administration resulted in a transient increase in interleukin 6 levels following administration that were restored to baseline levels 7 days after administration.

[0549]

[0182] 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

WSGR Docket No. 52426-778.601CLAIMSWHAT IS CLAIMED IS:

1. An isolated polypeptide or polypeptide complex that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostatespecific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule 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;Xs 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;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: 3425).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;X4is 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 or 2, 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;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;X7is M;X8is selected from E, S, T, A, V, D, and Q;WSGR Docket No. 52426-778.601X9 is selected from G, V, A, S, and W; andXio 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 an 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 that comprises a first antigen recognizing molecule that binds to an effector cell antigen and a second antigen recognizing molecule that binds to prostatespecific membrane antigen (PSMA), wherein the second antigen recognizing molecule is linked to a peptide that impairs binding of the second antigen recognizing molecule to PSMA, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1-131 and 214-2519.13 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 214-2519.14 The isolated polypeptide or polypeptide complex of claim 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 63.WSGR Docket No. 52426-778.60115. The isolated polypeptide or polypeptide complex of claim 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 74.

16. 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 12, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 118.

20. The isolated polypeptide or polypeptide complex of claim 12, wherein the effector cell antigen comprises cluster of differentiation 3 (CD3).

21. The isolated polypeptide or polypeptide complex of claim 12, wherein the first antigen recognizing molecule comprises an anti-CD3 antibody.

22. The isolated polypeptide or polypeptide complex of claim 21, wherein the anti-CD3 antibody comprises an anti-CD3 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 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-CD3 antibody comprises an anti-CD3 light chain variable domain that comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 comprise amino acid sequences of LC-CDR1: SEQ ID NO: 135, LC-CDR2: GT, and LC-CDR3: SEQ ID NO: 137.

23. The isolated polypeptide or polypeptide complex of claim 22, wherein the first antigen recognizing molecule that binds to CD3 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 12, wherein the first antigen recognizing molecule comprises a single chain variable fragment (scFv) or a Fab or Fab’ fragment.

25. The isolated polypeptide or polypeptide complex of claim 12, wherein the second antigen recognizing molecule that binds to PSMA comprises an anti-PSMA antibody.

26. The isolated polypeptide or polypeptide complex of claim 25, 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 amino acid sequences of HC-CDR1: SEQ ID NO: 139, HC-CDR2: SEQ ID NO: 140, and HC- CDR3: SEQ ID NO: 141 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 of the light chain variable domain comprise an amino acid sequence of LC-CDR1: SEQ ID NO: 142, LC-CDR2: EA, and LC-CDR3: SEQ ID NO: 144.WSGR Docket No. 52426-778.60127. The isolated polypeptide or polypeptide complex of claim 26, 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: 146, and 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: 145.

28. The isolated polypeptide or polypeptide complex of claim 26, 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: 148, and 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: 147.

29. The isolated polypeptide or polypeptide complex of claim 26, 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: 150, and 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: 149.

30. The isolated polypeptide or polypeptide complex of claim 25, wherein the anti-PSMA antibody comprises a single chain variable fragment (scFv), a single domain antibody, a Fab, a Fab’, or a CrossFab.

31. The isolated polypeptide or polypeptide complex of claim 25, wherein the anti-CD3 antibody comprises a single chain variable fragment (scFv) and the anti-PSMA antibody comprises a Fab or a Fab’.

32. The isolated polypeptide or polypeptide complex of claim 31, wherein the scFv and the Fab or Fab’ are connected through a linker and the Fab or Fab’ comprise a Fab heavy chain polypeptide and a Fab light chain polypeptide.

33. The isolated polypeptide or polypeptide complex of claim 32, wherein the linker is at least 5 amino acids in length.

34. The isolated polypeptide or polypeptide complex of claim 32, wherein the linker is no more than 30 amino acids in length.

35. The isolated polypeptide or polypeptide complex of claim 12, wherein the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 153 and SEQ ID NO: 154.

36. The isolated polypeptide or polypeptide complex of claim 12, wherein the isolated polypeptide or polypeptide complex is human or humanized.

37. The isolated polypeptide or polypeptide complex of claim 21, wherein the isolated polypeptide or polypeptide complex further comprises a peptide that is linked to the anti-CD3 antibody, wherein the peptide impairs binding of the anti-CD3 antibody to CD3.

38. The isolated polypeptide or polypeptide complex of claim 37, wherein the isolated polypeptide or polypeptide complex comprises a configuration according to Formula I: P2-L2-A2-A1-L1-P1-H1 wherein Ai comprises the anti-CD3 antibody; Pi comprises the peptide that impairs binding of the anti-CD3 antibody toWSGR Docket No. 52426-778.601CD3; Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; A2 comprises the anti-PSMA antibody; P2 comprises the peptide that impairs binding of the anti-PSMA antibody to PSMA; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.

39. The isolated polypeptide or polypeptide complex of claim 38, wherein the anti-CD3 antibody comprises a scFv and the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide; and the anti-PSMA antibody comprises a Fab or Fab’ and the Fab or Fab’ comprise a Fab heavy chain polypeptide and a Fab light chain polypeptide.

40. The isolated polypeptide or polypeptide complex of claim 38, wherein P2 impairs binding of A2to PSMA.

41. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 comprises a peptide sequence of at least 10 amino acids in length.

42. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

43. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 comprises a peptide sequence of at least 16 amino acids in length acids.

44. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 comprises a peptide sequence of no more than 40 amino in length.

45. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 is 11 amino acids in length.

46. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 is 12 amino acids in length.

47. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 is 13 amino acids in length.

48. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 is 14 amino acids in length.

49. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 is 15 amino acids in length.

50. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi or P2 is 16 amino acids in length.

51. The isolated polypeptide or polypeptide complex of claim 38, wherein Pi comprises an amino acid sequence according to U1-U2-C- U4-P-U6-U7-U8-U9-U10-U11- U12-C-U14 and Ui is selected from D, Y, F, I, V, H, L, A, T, S, and P; U2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; U4 is selected from G and W Ue is selected from E, D, V, and P; U7 is selected from W, L, F, V, G, M, I, and Y; Us is selected from E, D P and Q; U9 is selected from E, D, Y, V, F, W, P, L, and Q; U10 is selected from S, D, Y, T, I, F, V, N, A, P _L and H; Un is selected from I, Y, F, V, L, T, N, S, D, A, and H; U is selected from F, D, Y, L, I, V, A, T P, S, G, and H; and Ui4is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.WSGR Docket No. 52426-778.60152. The isolated polypeptide or polypeptide complex of claim 51, wherein Ui is selected from D, Y, F, I, V, and N; U2 is selected from D, Y, L, F, I, and N; U4 is selected from G and W; Ue is selected from E and D; U7 is selected from W, L, F, G, and V; Us is selected from E and D; U9 is selected from E, D, Y, and V; U10 is selected from S, D, Y, T, and I; Un is selected from I, Y, F, V, L, and T; U12 is selected from F, D, Y, L, I, V, A, G, and N; and U14 is selected from D, Y, N, F, I, M, and P.

53. The isolated polypeptide or polypeptide complex of claim 52, wherein Ui is selected from D, Y, V, and F; U2 is selected from D, Y, L, and F; U4 is selected from G and W; Ue is selected from E and D; U7 is selected from W, L, G, and F; Us is selected from E and D; U9 is selected from E and D; U10 is selected from S D, T, and Y; Un is selected from I, Y, V, L, and F; U is selected from F, D, Y, G, A, and L; U14 is selected from D, Y, M, and N.54 The isolated polypeptide or polypeptide complex of claim 38, wherein Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 155-159 and 2520-3418.55 The isolated polypeptide or polypeptide complex of claim 38, wherein Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 155-159, 2520-2546, and 3407-3418.56 The isolated polypeptide or polypeptide complex of claim 38, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 156.57 The isolated polypeptide or polypeptide complex of claim 38, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 157.58 The isolated polypeptide or polypeptide complex of claim 38, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 158.59 The isolated polypeptide or polypeptide complex of claim 38, wherein Li or L2 has a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 3419).60 The isolated polypeptide or polypeptide complex of claim 38, wherein Li has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n(SEQ ID NO: 3420), (GGGS)n(SEQ ID NO: 3421), (GGGGS)n (SEQ ID NO: 3422), and (GSSGGS)n(SEQ ID NO: 3423), wherein n is an integer of at least 1.61 The isolated polypeptide or polypeptide complex of claim 38, wherein Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to CD3.62 The isolated polypeptide or polypeptide complex of claim 38, wherein P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to PSMA.63 The isolated polypeptide or polypeptide complex of claim 38, wherein the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease.64 The isolated polypeptide or polypeptide complex of claim 63, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.65 The isolated polypeptide or polypeptide complex of claim 63, wherein the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.WSGR Docket No. 52426-778.60166. The isolated polypeptide or polypeptide complex of claim 38, wherein Li or L2 comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.

67. The isolated polypeptide or polypeptide complex of claim 38, wherein Li or L2 comprises an amino acid sequence according to SEQ ID NO: 163.

68. The isolated polypeptide or polypeptide complex of claim 38, wherein Li or L2 comprises an amino acid sequence according to any one of SEQ ID NOs: 160-191.

69. The isolated polypeptide or polypeptide complex of claim 38, wherein Li or L2 comprises an amino acid sequence of Linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 186), Linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 187), Linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 188), or Linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 189), or an amino acid sequence that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 25, Linker 26, Linker 27, or Linker 28.

70. The isolated polypeptide or polypeptide complex of claim 38, wherein Li or L2 is at least 5 amino acids in length.

71. The isolated polypeptide or polypeptide complex of claim 38, wherein Li or L2 is no more than 30 amino acids in length.

72. The isolated polypeptide or polypeptide complex of claim 38, wherein Li or L2 is at least 5 amino acids and no more than 30 amino acids in length.

73. The isolated polypeptide or polypeptide complex of claim 38, wherein Hi comprises a fragment crystallizable (Fc) domain.

74. The isolated polypeptide or polypeptide complex of claim 73, wherein the Fc domain comprises one or more knob-in-hole (KIH) mutations.

75. The isolated polypeptide or polypeptide complex of claim 73, wherein the Fc domain comprises a first polypeptide and a second polypeptide, wherein the first polypeptide of the Fc domain comprises a hole mutation and the second polypeptide of the Fc domain comprises a knob mutation.

76. The isolated polypeptide or polypeptide complex of claim 75, wherein the first polypeptide of the Fc domain comprises T366S, L368A, and Y407V mutations according to EU numbering, and the second polypeptide of the Fc domain comprises a T366W mutation according to EU numbering.

77. The isolated polypeptide or polypeptide complex of claim 73, wherein the Fc domain comprises Fc effector function silencing mutations L234A, L235A, and P329G according to EU numbering.

78. The isolated polypeptide or polypeptide complex of claim 73, wherein the Fc domain comprises an engineered disulfide.

79. The isolated polypeptide or polypeptide complex of claim 78, wherein the Fc domain comprises a 349C mutation and a S354C mutation according to EU numbering, and wherein the engineered disulfide is formed between two cysteines at the positions of the Y349C mutation and the S354C mutation.WSGR Docket No. 52426-778.60180. The isolated polypeptide or polypeptide complex of claim 73, wherein the Fc domain comprises a N297G mutation according to EU numbering.

81. The isolated polypeptide or polypeptide complex of claim 73, wherein the Fc domain lacks a C- terminal glycine and a C-terminal lysine to reduce heterogeneity.

82. The isolated polypeptide or polypeptide complex of claim 73, wherein the Fc domain lacks C- terminal G446 and K447 residues according to EU numbering to reduce heterogeneity.

83. The isolated polypeptide or polypeptide complex of claim 73, wherein the Fc domain comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 203 and an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 204.

84. The isolated polypeptide or polypeptide complex of claim 73, wherein the polypeptide is an antibody.

85. The isolated polypeptide or polypeptide complex of claim 84, wherein the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.

86. The isolated polypeptide or polypeptide complex of claim 85, wherein the single domain antibody comprises a single domain antibody that binds to albumin.

87. The isolated polypeptide or polypeptide complex of claim 86, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein theHC-CDRl, theHC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 192, HC-CDR2: SEQ ID NO: 193, and HC-CDR3: SEQ ID NO: 194.

88. The isolated polypeptide or polypeptide complex of claim 86, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein theHC-CDRl, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 196, HC-CDR2: SEQ ID NO: 197, and HC-CDR3: SEQ ID NO: 198.

89. The isolated polypeptide or polypeptide complex of claim 86, wherein the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 195.

90. The isolated polypeptide or polypeptide complex of claim 86, wherein the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 199.

91. The isolated polypeptide or polypeptide complex of claim 86, wherein the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 200.

92. The isolated polypeptide or polypeptide complex of claim 86, wherein the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 201.

93. The isolated polypeptide or polypeptide complex of claim 86, the single domain antibody comprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 202.WSGR Docket No. 52426-778.60194. The isolated polypeptide or polypeptide complex of claim 12, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 205 and 206.

95. The isolated polypeptide or polypeptide complex of claim 12, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 207 and 208.

96. The isolated polypeptide or polypeptide complex of claim 12, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 209 and 210.

97. The isolated polypeptide or polypeptide complex of claim 12, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 211, 212, and 213.

98. The isolated polypeptide or polypeptide complex of claim 12, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3426 and 3427.

99. The isolated polypeptide or polypeptide complex of claim 12, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3428 and 3429.

100. The isolated polypeptide or polypeptide complex of claim 12, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3430, 3431, and 3432.

101. The isolated polypeptide or polypeptide complex of claim 12, The isolated polypeptide or polypeptide complex of claim 1-170, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3433 and 3434.

102. An isolated polypeptide or polypeptide complex that comprises an anti-PSMA antibody 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 amino acid sequences of HC-CDR1: SEQ ID O: 139, HC-CDR2: SEQ ID NO: 140, and HC-CDR3: SEQ ID NO: 141 and complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC- CDR3 comprise amino acid sequences of LC-CDR1: SEQ ID NO: 142, LC-CDR2: EA, and LC-CDR3: SEQ ID NO: 144; a peptide that comprises the amino acid sequence of SEQ ID NO: 118 and impairs binding of the anti-PSMA antibody to PSMA; an anti-CD3 antibody 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 amino acid sequences ofHC-CDRl: SEQ ID NO: 132, HC-CDR2: SEQ ID NO: 133, and HC-CDR3: SEQ ID NO: 134, and the anti-CD3 antibody comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, and wherein the LC-CDR1, the LC-WSGR Docket No. 52426-778.601CDR2, and the LC-CDR3 comprise amino acid sequences of LC-CDR1: SEQ ID NO: 135, LC-CDR2: GT, and LC-CDR3: SEQ ID NO: 137; and a peptide that comprises the amino acid sequence according to SEQ ID NO: 156 and impairs binding of the anti-CD3 antibody to CD3.

103. The isolated polypeptide or polypeptide complex of claim 102, wherein the peptide that comprises the amino acid sequence according to SEQ ID NO: 118 and impairs binding of the anti-PSMA antibody to PSMA is no more than 14 amino acids in length.

104. The isolated polypeptide or polypeptide complex of claim 102, wherein the peptide that comprises the amino acid sequence of SEQ ID NO: 156 and impairs binding of the anti-CD3 antibody to CD3 is no more than 16 amino acids in length.

105. The isolated polypeptide or polypeptide complex of claim 102, wherein the isolated polypeptide or polypeptide complex further comprises a half-life extending molecule.

106. The isolated polypeptide or polypeptide complex of claim 105, wherein the half-life extending molecule comprises the amino acid sequences according to SEQ ID NOs: 192-194.

107. The isolated polypeptide or polypeptide complex of claim 105, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 195.

108. The isolated polypeptide or polypeptide complex of claim 105, wherein the half-life extending molecule comprises the amino acid sequences according to SEQ ID NOs: 196-198.

109. The isolated polypeptide or polypeptide complex of claim 105, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 199.

110. The isolated polypeptide or polypeptide complex of claim 105, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 200.

111. The isolated polypeptide or polypeptide complex of claim 105, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 201.

112. The isolated polypeptide or polypeptide complex of claim 105, wherein the half-life extending molecule comprises the amino acid sequence according to SEQ ID NO: 202.

113. The isolated polypeptide or polypeptide complex of claim 105, wherein the half-life extending molecule comprises the amino acid sequences according to SEQ ID NOs: 203-204.

114. The isolated polypeptide or polypeptide complex of claim 105, wherein the isolated polypeptide or polypeptide complex further comprises the amino acid sequence according to SEQ ID NO: 189.

115. The isolated polypeptide or polypeptide complex of claim 105, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3426 and 3427.

116. The isolated polypeptide or polypeptide complex of claim 105, wherein the polypeptide or polypeptide complex comprises the amino acid sequences according to SEQ ID NOs: 3426 and 3427.

117. The isolated polypeptide or polypeptide complex of claim 105, wherein the polypeptide or polypeptide complex consists of the amino acid sequences according to SEQ ID NOs: 3426 and 3427.WSGR Docket No. 52426-778.601118. The isolated polypeptide or polypeptide complex of claim 105, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3428 and 3429.

119. The isolated polypeptide or polypeptide complex of claim 105, wherein the polypeptide or polypeptide complex comprises the amino acid sequences according to SEQ ID NOs: 3428 and 3429.

120. The isolated polypeptide or polypeptide complex of claim 105, wherein the polypeptide or polypeptide complex consists of the amino acid sequences according to SEQ ID NOs: 3428 and 3429.

121. The isolated polypeptide or polypeptide complex of claim 105, wherein the polypeptide or polypeptide complex comprises amino acid sequences with at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NOs: 3433 and 3434.

122. The isolated polypeptide or polypeptide complex of claim 105, wherein the polypeptide or polypeptide complex comprises the amino acid sequences according to SEQ ID NOs: 3433 and 3434.

123. The isolated polypeptide or polypeptide complex of claim 105, wherein the polypeptide or polypeptide complex consists of the amino acid sequences according to SEQ ID NOs: 3433 and 3434.

124. A pharmaceutical composition comprising: (a) the isolated polypeptide or polypeptide complex of claim 102; and (b) a pharmaceutically acceptable excipient.

125. An isolated recombinant nucleic acid molecule encoding the isolated polypeptide or polypeptide complex of claim 102.

126. A vector comprising the isolated nucleic acid molecule of claim 102.

127. A host cell comprising the isolated recombinant nucleic acid molecule of claim 102.

128. A method of treating cancer in a subject in need thereof comprising administering to the subject the isolated polypeptide or polypeptide complex of claim 102.

129. The method of claim 128, wherein the isolated polypeptide or polypeptide complex induces T cell mediated cytotoxicity of tumor cells.

130. The method of claim 128, wherein the tumor cells express PSMA.

131. The method of claim 128, wherein the cancer is a solid tumor cancer.

132. The method of claim 128, wherein the cancer is prostate, lung, breast, cervical, ovarian, colorectal, pancreatic or gastric cancer.

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