Constructs targeting prostate-specific membrane antigen (PSMA) and uses thereof

Abstract

The present application provides constructs comprising an antibody moiety that specifically binds to prostate specific membrane antigen (PSMA) (e.g., PSMA expressed on the surface of a cell). Chimeric antigen receptor (CAR) constructs targeted to human PSMA ("hPSMA") and immune cells (e.g., T cells and NK cells) expressing such CARs are described. Also provided are methods of making and using these constructs.

Description

Attorney Docket No.: 75004-20023.40 CONSTRUCTS TARGETING PROSTATE-SPECIFIC MEMBRANE ANTIGEN (PSMA) AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 594,058, filed on October 30, 2023, U.S. Provisional Application No.63 / 594,404, filed on October 30, 2023, and U.S. Provisional Application No.63 / 596,188, filed on November 3, 2023, the contents of each of which are incorporated herein in their entireties. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (750042002340SEQLIST.xml; Size: 131,537 bytes; and Date of Creation: October 25, 2024) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION

[0003] The present disclosure pertains to polypeptide constructs that specifically bind prostate specific membrane antigen (PSMA) and uses thereof including treating diseases such as cancer using anti-PSMA chimeric antigen receptor (CAR) immune cell therapy and diagnosing diseases. BACKGROUND OF THE INVENTION

[0004] Prostate cancer is the second most common cancer and the second leading cause of cancer-related deaths in American men. Since 2014, the incidence rate has increased by 3% per year overall and by about 5% per year for advanced-stage prostate cancer. There are projected to be over 35,250 deaths from prostate cancer in 2024 in the United States alone (American Cancer Society). Between 35-61% of prostate cancer patients undergoing radical prostatectomy or radical radiotherapy eventually relapse. These patients may respond transiently to androgen deprivation therapy, but a majority will subsequently progress to hormone-refractory disease for which curative systemic therapies are lacking (Perambakam S, et al., Clin. Dev. Immunol.2010; Epub 2011 Jan 5).

[0005] Prostate-Specific Membrane Antigen (PSMA) is a 750 amino acid type II transmembrane glycoprotein that is highly expressed on the surface of prostate tumor cells at all tumor stages and is known to be upregulated in castrate-resistant and metastatic prostate cancers sf-5670772Attorney Docket No.: 75004-20023.40 (Afshar-Oromieh, A. et al., J. Nucl. Med.2016, 57: 79S). In other solid tumors including colon, ovarian, breast, and kidney cancers, elevated PSMA expression has been observed on tumor neovasculature, but not normal vasculature, suggesting a role for PSMA in angiogenesis. Most PSMA expression appears to be restricted to the prostate, but lower-level expression is seen in the brain, kidneys, salivary glands, and small intestine.

[0006] Small molecule PSMA ligands have been used in imaging studies in the detection of metastasis of prostate cancers in lymph nodes and bone. Given the expression pattern of PSMA, it has also been explored as a therapeutic target. Current immunotherapy approaches to target PSMA include peptide, cell, vector or DNA-based vaccines, administration of monoclonal antibodies (mAb) or expression of a DNA-encoded mAb against PSMA (Muthumani, K. et al., Cancer Immunol. Immunother.2017, 66: 1577) and chimeric antigen receptor (CAR)-modified T cells (Junghans, RP et al., Prostate.2016, 76: 1257). Despite the reported expression of PSMA in normal tissues, anti-PSMA toxicities were not observed in the Phase I clinical study using anti-PSMA CAR T cells.

[0007] Accordingly, there remains a need in the art for agents (such as polypeptide constructs) that target PSMA for the diagnosis and / or treatment of cancer. The present application addresses these and other needs.

[0008] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety. BRIEF SUMMARY OF THE INVENTION

[0009] In some embodiments, provided herein is an anti-prostate specific membrane antigen (PSMA) construct comprising an antibody moiety specifically recognizing PSMA (anti-PSMA antibody moiety), wherein the anti-PSMA antibody moiety comprises: (i) a heavy chain variable domain (VH) comprising a complementarity determining region heavy chain (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable domain (VL) comprising a complementarity determining region light chain (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; (ii) a VHcomprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising a CDR-L1 sf-5670772Attorney Docket No.: 75004-20023.40 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19; (iii) a VHcomprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; (iv) a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; or (v) a VHcomprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a FWR-H4 that does not comprise the amino acid sequence of SEQ ID NO: 7; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and a FWR-L3 that does not comprise the amino acid sequence of SEQ ID NO: 13.

[0010] In some embodiments according to (or as applied to) any of the embodiments above, the anti-PSMA antibody moiety comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 29, and a VLcomprising the amino acid sequence of SEQ ID NO: 30; (ii) a VHcomprising the amino acid sequence of SEQ ID NO: 20, and a VLcomprising the amino acid sequence of SEQ ID NO: 21; (iii) a VHcomprising the amino acid sequence of SEQ ID NO: 35, and a VLcomprising the amino acid sequence of SEQ ID NO: 36; (iv) a VHcomprising the amino acid sequence of SEQ ID NO: 25, and a VLcomprising the amino acid sequence of SEQ ID NO: 26; or (v) a VH comprising the amino acid sequence of SEQ ID NO: 40, and a VL comprising the amino acid sequence of SEQ ID NO: 41.

[0011] In some embodiments according to (or as applied to) any of the embodiments above, the anti-PSMA antibody moiety is a full-length antibody, a Fab, a Fab’, a F(ab’)2, an Fv, or a single chain Fv (scFv).

[0012] In some embodiments according to (or as applied to) any of the embodiments above, the anti-PSMA construct is an anti-PSMA chimeric antibody-T cell receptor (TCR) construct 3 sf-5670772Attorney Docket No.: 75004-20023.40 (caTCR) comprising: (a) an extracellular domain comprising the anti-PSMA antibody moiety; and (b) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule. In some embodiments according to (or as applied to) any of the embodiments above, (a) the first TCR- TM and the second TCR-TM are derived from a ^ / ^ TCR or an ^ / ^ TCR; and / or (b) the TCR- associated signaling molecule is selected from the group consisting of CD3^^, CD3^^, and CD3^^.

[0013] In some embodiments according to (or as applied to) any of the embodiments above, the anti-PSMA construct is an anti-PSMA chimeric signaling receptor (CSR) comprising: i) a ligand-binding module comprising the anti-PSMA antibody moiety; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that is capable of providing a co-stimulatory signal to an effector cell, wherein the CSR lacks a functional primary immune cell signaling domain. In some embodiments according to (or as applied to) any of the embodiments above, the transmembrane module of the CSR and the co-stimulatory immune cell signaling module of the CSR are derived from the same molecule. In some embodiments according to (or as applied to) any of the embodiments above, the molecule is selected from the group consisting of CD28, 4-1BB (CD137), OX40, CD30, CD27, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0014] In some embodiments according to (or as applied to) any of the embodiments above, the anti-PSMA construct is an anti-PSMA chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising the anti-PSMA antibody moiety; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments according to (or as applied to) any of the embodiments above, (a) the intracellular signaling domain comprises a primary immune cell signaling sequence derived from CD3^, TCR^, FcR^, FcR^, CD3^, CD3^, CD3^, CD5, CD22, CD79a, CD79b, or CD66d; and / or (b) the intracellular signaling domain further comprise a costimulatory signaling sequence derived from CD30, CD28, 4-1BB, ICOS, or OX40.

[0015] In some embodiments according to (or as applied to) any of the embodiments above, the anti-PSMA construct is conjugated to an effector molecule, wherein the effector molecule is a detectable label or a therapeutic agent selected from the group consisting of: a drug, a toxin, a radioisotope, a protein, a peptide, and a nucleic acid. 4 sf-5670772Attorney Docket No.: 75004-20023.40

[0016] In some embodiments, provided herein is an effector cell that has been genetically modified with one or more nucleic acids encoding: (a) the anti-PSMA caTCR of any one of the embodiments above; (b) the anti-PSMA scFv of any one of the embodiments above; (c) the anti- PSMA CSR of any one of the embodiments above; (d) the anti-PSMA CAR of any one of the embodiments above; or (e) the anti-PSMA caTCR of any one of the embodiments above, wherein: (i) the one or more nucleic acids encoding the anti-PSMA caTCR further encode a CSR comprising a ligand-binding module that binds a target antigen or an scFv that binds a target antigen; or (ii) the effector cell has been genetically modified with one or more additional nucleic acids encoding a CSR comprising a ligand-binding module that binds a target antigen or an scFv that binds a target antigen.

[0017] In some embodiments, provided herein is a method of producing an effector cell, comprising genetically modifying a cell with one or more nucleic acids encoding: (a) the anti- PSMA caTCR of any one of the embodiments above; (b) the anti-PSMA scFv of any one of the embodiments above; (c) the anti-PSMA CSR of any one of the embodiments above; (d) the anti- PSMA CAR of any one of the embodiments above; or (e) the anti-PSMA caTCR of any one of the embodiments above, wherein: (i) the one or more nucleic acids encoding the anti-PSMA caTCR further encode a CSR comprising a ligand-binding module that binds a target antigen or an scFv that binds a target antigen; or (ii) the method further comprises genetically modifying the effector cell with one or more additional nucleic acids encoding a CSR comprising a ligand- binding module that binds a target antigen or an scFv that binds a target antigen .

[0018] In some embodiments according to (or as applied to) any of the embodiments above, the effector cell is an immune cell. In some embodiments according to (or as applied to) any of the embodiments above, the immune cell is a T cell.

[0019] In some embodiments, provided herein is a nucleic acid or a vector encoding the polypeptide portion(s) of the anti-PSMA construct of any one of the embodiments above.

[0020] In some embodiments, provided herein is a host cell comprising the nucleic acid or the vector of any one of the embodiments above.

[0021] In some embodiments, provided herein is a method of producing an anti-PSMA construct, comprising culturing the host cell of any one of the embodiments above under conditions where the anti-PSMA construct is expressed, and recovering the anti-PSMA construct produced by the host cell.

[0022] In some embodiments, provided herein is a pharmaceutical composition comprising the anti-PSMA construct of any one of the embodiments above, the effector cell of any one of the 5 sf-5670772Attorney Docket No.: 75004-20023.40 embodiments above, and / or the nucleic acid or vector of any one of the embodiments above, and a pharmaceutical acceptable carrier.

[0023] In some embodiments, provided herein is a method of treating an individual having a PSMA-associated disease or disorder, comprising: (i) administering to the individual an effective amount of the pharmaceutical composition of any one of the embodiments above; or (ii) administering to the individual the effector cell of any one of the embodiments above.

[0024] In some embodiments, provided herein is a nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising: (i) an scFv that binds human PSMA (hPSMA) and comprises: (a) a complementarity determining region light chain (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a complementarity determining region heavy chain (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (c) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; or (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and a FWR-L3 that does not comprise the amino acid sequence of SEQ ID NO: 13, and a CDR- H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a FWR-H4 that does not comprise the amino acid sequence of SEQ ID NO: 7; (ii) a 6 sf-5670772Attorney Docket No.: 75004-20023.40 spacer domain; (iii) a transmembrane domain; (iv) a costimulatory signaling domain; and (v) a CD3^ signaling domain.

[0025] In some embodiments according to (or as applied to) any of the embodiments above, the scFv comprises: (a) a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30; and a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 29; (b) a VL comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26; and a VH comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25; (c) a VLcomprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 21; and a VHcomprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 20; (d) a VL comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 36; and a VH comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 35; or (e) a VL comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 41; and a VH comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0026] In some embodiments, provided herein is a nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising: (i) an scFv that binds human PSMA (hPSMA) and comprises: (a) a complementarity determining region light chain (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13, a complementarity determining region heavy chain (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7; or (b) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 7 sf-5670772Attorney Docket No.: 75004-20023.40 comprising the amino acid sequence of SEQ ID NO: 115, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113; (ii) a spacer domain; (iii) a transmembrane domain; (iv) a costimulatory signaling domain; and (v) a CD3^ signaling domain; and wherein the nucleic acid molecule further encodes a membrane-bound human IL-12 (mbIL-12).

[0027] In some embodiments according to (or as applied to) any of the embodiments above, the scFv comprises: (a) a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 16; and a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 15; or (b) a VLcomprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 110; and a VH comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 109.

[0028] In some embodiments according to (or as applied to) any of the embodiments above, (a) the scFv comprises the amino acid sequence of any of SEQ ID NOs: 31, 48, 22, 27, 37, and 42; (b) the spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-74; (c) the transmembrane domain is selected from the group consisting of CD3^, CD28TM1, CD28TM2, CD4, CD8TM1, CD8TM2, CD8TM3, 4-1BB, and NKG2D; and / or (d) the costimulatory signaling domain is selected from the group consisting of CD28, CD28gg*, 4-1BB, OX40, and 2B4.

[0029] In some embodiments according to (or as applied to) any of the embodiments above, (a) the scFv comprises the amino acid sequence of any of SEQ ID NOs: 17, 49, and 117; (b) the spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-74; (c) the transmembrane domain is selected from the group consisting of CD3^, CD28TM, CD28TM2, CD4, CD8TM1, CD8TM2, CD8TM3, 4-1BB, and NKG2D; and / or (d) the costimulatory signaling domain is selected from the group consisting of CD28, CD28gg*, 4- 1BB, OX40, and 2B4.

[0030] In some embodiments according to (or as applied to) any of the embodiments above, the CAR comprises the amino acid sequence of any of SEQ ID NOs: 50-54 and 102-106. 8 sf-5670772Attorney Docket No.: 75004-20023.40

[0031] In some embodiments according to (or as applied to) any of the embodiments above, the nucleic acid molecule further encodes a membrane-bound human IL-12 (mbIL-12).

[0032] In some embodiments according to (or as applied to) any of the embodiments above, the CAR comprises the amino acid sequence of any of SEQ ID NOs: 92-101.

[0033] In some embodiments according to (or as applied to) any of the embodiments above, the mbIL-12 comprises the amino acid sequence of SEQ ID NO: 107 or 108.

[0034] In some embodiments, provided herein is an immune cell harboring the nucleic acid molecule of any one of the embodiments above, or a vector comprising nucleic acid molecule of any one of the embodiments above.

[0035] In some embodiments according to (or as applied to) any of the embodiments above, the immune cell further harbors a nucleic acid molecule encoding a mbIL-12.

[0036] In some embodiments, provided herein is an immune cell harboring: (a) a vector comprising the nucleic acid molecule of any one of the embodiments above and a nucleic acid molecule encoding a mbIL-12; or (b) the nucleic acid molecule of any one of the embodiments above, or a vector comprising the nucleic acid molecule of any one of the embodiments above.

[0037] In some embodiments, provided herein is a chimeric antigen receptor (CAR) comprising: (i) an scFv that binds human PSMA (hPSMA) and comprises: (a) a complementarity determining region light chain (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR- L3 comprising the amino acid sequence of SEQ ID NO: 10, a complementarity determining region heavy chain (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR- H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR- H3 comprising the amino acid sequence of SEQ ID NO: 3; (c) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) a CDR- L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, 9 sf-5670772Attorney Docket No.: 75004-20023.40 a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; or (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and a FWR-L3 that does not comprise the amino acid sequence of SEQ ID NO: 13, and a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR- H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a FWR-H4 that does not comprise the amino acid sequence of SEQ ID NO: 7; (ii) a spacer domain; (iii) a transmembrane domain; (iv) a costimulatory signaling domain; and (v) a CD3^ signaling domain.

[0038] In some embodiments according to (or as applied to) any of the embodiments above, the scFv comprises: (a) a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30; and a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 29; (b) a VL comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26; and a VH comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25; (c) a VL comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 21; and a VHcomprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 20; (d) a VLcomprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 36; and a VHcomprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 35; or (e) a VL comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 41; and a VH comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0039] In some embodiments according to (or as applied to) any of the embodiments above, (a) the scFv comprises the amino acid sequence of any of SEQ ID NOs: 31, 48, 22, 27, 37, and 42; 10 sf-5670772Attorney Docket No.: 75004-20023.40 (b) the spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-74; (c) the transmembrane domain is selected from the group consisting of CD3^, CD28TM, CD28TM2, CD4, CD8TM1, CD8TM2, CD8TM3, 4-1BB, and NKG2D; and / or (d) the costimulatory signaling domain is selected from the group consisting of CD28, CD28gg*, 4-1BB, OX40, and 2B4.

[0040] In some embodiments according to (or as applied to) any of the embodiments above, the CAR comprises the amino acid sequence of any of SEQ ID NOs: 50-54 and 102-106.

[0041] In some embodiments, provided herein is an immune cell harboring the CAR of any one of the embodiments above.

[0042] In some embodiments according to (or as applied to) any of the embodiments above, the immune cell further harbors a mbIL-12.

[0043] In some embodiments according to (or as applied to) any of the embodiments above, the immune cell is a T cell.

[0044] In some embodiments, provided herein is a method of treating an individual suffering from a cancer whose cells express PSMA, comprising administering to the individual an effective amount of the immune cell of any one of the embodiments above. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Various aspects of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0046] FIG.1 shows the post-enrichment assessment of hPSMA CAR T cells. T cells expressing ET260-1 and ET260-2 CARs were successfully enriched and no substantial difference in stability between the two scFv CARs was found. J591 CAR was used as a positive control for CAR and PSMA targeting.

[0047] FIGs.2A-2C show the relative killing by hPSMA CAR T cells of PC-3 PSMA- expressing cell lines at 72 hrs at an effector to target ratio (E:T) of 1:4. There was comparable killing by ET260-1 CAR T cells when compared to J591 CAR T cells, while ET260-2 CAR T cells showed suboptimal killing (FIG.2A). Activation as measured by 4-1BB+ cell percentage and IFN^ production (pg / mL) are shown in FIG.2B and FIG.2C, respectively. The IFN^ levels were less pronounced when cocultured with PC-3 PSMA cell lines in the presence of both sf-5670772Attorney Docket No.: 75004-20023.40 ET260-1 and ET260-2 CAR T cells when compared to J591 CAR T cells. In each group, from left to right: mock, ET260-1 CAR, ET260-2 CAR, and J591 CAR.

[0048] FIG.3 shows the post-enrichment assessment of hPSMA CARs with different linker and transmembrane (TM) combinations. All CAR T cells were successfully enriched for truncated CD19 (CD19t). The hPSMA CD28TM CAR had improved stability (as measured by detection of the Fc linker) compared to the original CD4TM construct. Top to bottom: mock, ET260-1 CD4TM CAR, ET260-1 CD28TM2 CAR, ET260-1 CD8TM1 CAR, ET260-1 HL CAR, and J591 CAR.

[0049] FIGs.4A-4C show the functional activity of different linker and TM combinations of the hPSMA CAR. CAR T cells expressing four different hPSMA CAR constructs were cocultured with PC-3 PSMA cell lines at an E:T of 1:4 for 72 hrs. Comparable tumor cell killing activity of hPSMA ET260-1 with the CD4TM, CD28TM and HL CAR T cells was observed when compared to J591 CAR T cells (FIG.4A).4-1BB expression in CAR T cells showed that the hPSMA CD28TM CAR provided improved activation compared to the original CD4TM CAR construct when expressed in T cells, but still suboptimal to T cells expressing the J591 CAR. T cells expressing the hPSMA CAR with the CD28TM showed improved cytokine secretion compared to the T cells expressing the CAR with a CD4TM but still suboptimal to T cells expressing the J591 CAR (4-1BB+ percentage is shown in FIG.4B; IFN^ (pg / mL) is shown in FIG.4C). In each group, from left to right: mock, ET260-1 CD4TM CAR, ET260-1 CD28TM2 CAR, ET260-1 CD8TM1 CAR, ET260-1 HL CAR, and J591 CAR.

[0050] FIG.5 shows the stability of affinity matured scFvs with an HL-CH3-CD28TM2 backbone. Five new scFvs were generated, with clones AMET260-1-56 and AMET260-1-69 showing poor Fc stability. Clones AMET260-1-58 and AMET260-1-67 had detectable and stable Fc staining.

[0051] FIGs.6A-6D show the comparison of T cells expressing hPSMA CAR scFv variants over 72 hrs and 8 days. Killing (FIG.6A) and activation (FIG.6B, FIG.6C) is comparable between T cells expressing AMET260-1-58 and ET260-1 CAR at an E:T of 1:4 over 72 hrs. Cytokine secretion between AMET260-1-58 and ET260-1 CAR T cells was comparable but still suboptimal to J591 CAR T cells.8 day killing (FIG.6D) shows AMET260-1-58 CAR T cells have improved activity against low and high expressing PSMA cell lines at an E:T of 1:8. In each group, from left to right: mock, ET260-1 CAR, AMET260-1-39 CAR, AMET260-1-56 CAR, AMET260-1-58 CAR, AMET260-1-67 CAR, AMET260-1-69 CAR, and J591 CAR. sf-5670772Attorney Docket No.: 75004-20023.40

[0052] FIG.7 shows the comparison of hPSMA CAR scFv variants against cell lines which endogenously express PSMA. AMET260-1-58-expressing CAR T cells showed comparable or improved IFN^ secretion against endogenous and engineered cells lines expressing PSMA at 48 hrs at an E:T of 1:1. In each group, from left to right: mock, ET260-1 CAR, AMET260-1-39 CAR, AMET260-1-56 CAR, AMET260-158 CAR, AMET260-1-67 CAR, AMET260-1-69 CAR, and J591 CAR.

[0053] FIGs.8A-8D show the post-production profile of hPSMA CARs with non-targeting CD19 CAR controls. FIG.8A and FIG.8B show that the CD19 CAR (which carries an EGFR sequence tag) and the PSMA CARs (which carry a CD19 sequence tag) are properly enriched in the transduced T cells. FIG.8C shows the expression of the Fc linker on the surface of primary T cells transduced with CARs. Double transduction of both CAR and membrane-bound IL12 (“mbIL12”) did not significantly impact CAR expression. FIG.8D shows the expression of mbIL12 on the surface of T cells was comparable across the CD19 CAR T cell control and the two hPSMA CAR T cells tested. Top to bottom: CD19 CAR, CD19 CAR + mbIL12, ET260-1 CAR, ET260-1 CAR + mbIL12, AMET260-1-58 CAR, AMET260-1-58 CAR + mbIL12, and J591 CAR.

[0054] FIGs.9A-9D show the in vitro characterization of hPSMA CAR T cells co-expressing mbIL12. Relative killing by hPSMA CAR T cells was determined at an E:T of 1:4 over 72 hrs (FIG.9A). Activation was determined by examining IFNγ release (FIG.9C), 4-1BB (FIG.9B) and CD25 (FIG.9D) expression. hPSMA CAR T cells with mbIL12 had noticeably higher IFNγ expression compared to the single CAR controls (FIG.9C). In each group left to right: CD19 CAR, CD19 CAR + mbIL12, ET260-1 CAR, ET260-1 CAR + mbIL12, AMET260-1-58 CAR, AMET260-1-58 CAR + mbIL12, and J591 CAR.

[0055] FIGs.10A-10C show the in vitro characterization of hPSMA CAR T cells co- expressing mbIL12 over 8 days at an E:T of 1:20. Relative killing (FIG.10A) by hPSMA CAR T cells showed increased activity of ET260-1 CAR T cells co-expressing mbIL12 compared to the single CAR control as well as AMET260-1-58 CAR T cells co-expressing mbIL12. hPSMA CAR T cells with mbIL12 had increased expansion (FIG.10B) against high expressers of PSMA and surpassed expansion of J591 CAR T cells, while the single CAR T cell controls did not. hPSMA CAR T cells co-expressing mbIL12 had significant levels of IFN^ secretion (FIG. 10C) compared to J591 CAR T cells, while the single CAR T cell controls did not secrete cytokine over 8 days. In each group left to right: CD19 CAR, CD19 CAR + mbIL12, ET260-1 sf-5670772Attorney Docket No.: 75004-20023.40 CAR, ET260-1 CAR + mbIL12, AMET260-1-58 CAR, AMET260-1-58 CAR + mbIL12, and J591 CAR.

[0056] FIGs.11A-11C show the in vitro IFN^Rα1 block of J591 and PSMA CAR T cells co- expressing mbIL12. The initial E:T was 1:10 with a processing timepoint after 5 days. Two concentrations of IFN^ blockade were chosen, 10 µg / mL and 100 µg / mL. An isotype control antibody was used at the concentration of 100 µg / mL, while a blank in which no antibodies were present in the coculture, also served as a negative control. Tumor cell killing (FIG.11A) was lowest by the J591 CAR T cells at the highest concentration of the IFN^Rα1 block. This also resulted in lower expression of 4-1BB (FIG.11B) and CD25 levels (FIG.11C) when J591 CAR T cells were compared to the hPSMA CAR T cells with or without mbIL12 co-expression. In each group left to right: CD19 CAR, CD19 CAR + mbIL12, ET260-1 CAR, ET260-1 CAR + mbIL12, AMET260-1-58 CAR, AMET260-1-58 CAR + mbIL12, and J591 CAR.

[0057] FIG.12 shows the activity of ET260-1 CAR and AMET260-1-58 CAR against a PC-3 PIP tumor model. Tumor cells were engrafted subcutaneously in male non-obese diabetic (NOD) severe combined immunodeficiency (SCID) gamma (NSG) mice. Tumors were measured 2x weekly by caliper measurement. On day 21, the mice were sorted into each treatment group (7 mice per group) for an average tumor volume size of 140 mm3. CD19 targeted CAR T cells (with and without co-expressed mbIL12) were used as a control. On day 22, the mice were treated with 1x106CAR T cells via retro-orbital injection.

[0058] FIGs.13A-13D show the in vivo validation using intratibial (i.ti.) injection of LAPC-9 cells as a model to test hPSMA CAR efficacy. On the day of engraftment, LAPC-9-eGFP-ffluc cells (0.15x106) were engrafted in the left hind leg of NSG mice. Mice were imaged on day 13 post-engraftment and sorted into treatment groups. Mice were treated with 1x106CAR T cells intravenously retro-orbitally (i.v.r.o.). The treatment groups were non-targeting CD19 CAR T cells co-expressing an mbIL12 control (n=2) (FIG.13B), ET260-1 CAR T cells co-expressing mbIL12 (n=3) (FIG.13C), and J591 CAR T cells (n=3) (FIG.13D). Transient responses were observed in two out of the 3 mice in the ET260-1 CAR with mbIL12 treated group, meanwhile only one mouse had a transient response in the J591 CAR treated group. Combined data is shown in FIG.13A.

[0059] FIGs.14A-14D show the in vivo validation of C4-2-ffluc i.ti. as a model to test hPSMA CAR efficacy. On the day of engraftment, C4-2-ffluc cells (0.10x106) were engrafted in the left hind leg of NSG mice. Mice were imaged on day 13 post-engraftment and sorted into treatment sf-5670772Attorney Docket No.: 75004-20023.40 groups. Mice were treated with 1x106CAR T cells i.v.r.o. The treatment groups were non- targeting CD19 CAR T cells co-expressing mbIL12 as a control (n=3) (FIG.14B), ET260-1 CAR T cells co-expressing mbIL12 (n=2) (FIG.14C), and J591 CAR T cells (n=3) (FIG.14D). Mice treated with the PSMA CARs showed a rapid decline in tumor flux while mice treated with the CD19 CAR control continued to show an increase in tumor flux. Combined data is shown in FIG.14A.

[0060] FIG.15 shows the amino acid sequence of PSMA scFv(ET260-1)-IgG4(HL-CH3)- CD4TM-41BB-zeta with a signal sequence. The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 92 with signal sequence; SEQ ID NO: 93 without signal sequence)

[0061] FIG.16 shows the amino acid sequence of PSMA(ET260-2)-IgG4(HL-CH3)-CD4TM- 41BB-zeta with a signal sequence. The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 94 with signal sequence; SEQ ID NO: 95 without signal sequence)

[0062] FIG.17 shows the amino acid sequence of PSMA(ET260-1)-IgG4(HL-CH3)- CD28TM2-41BB-zeta with a signal sequence. The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 96 with signal sequence; SEQ ID NO: 97 without signal sequence)

[0063] FIG.18 shows the amino acid sequence of PSMA(ET260-1)-CD8h3-CD8TM1-41BB- zeta with a signal sequence. The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 98 with signal sequence; SEQ ID NO: 99 without signal sequence)

[0064] FIG.19 shows the amino acid sequence of PSMA(ET260-1)-HL-CD28TM2-41BB-zeta with a signal sequence. The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 100 with signal sequence; SEQ ID NO: 101 without signal sequence)

[0065] FIG.20 shows the amino acid sequence of the AMET260-1-39 CAR construct PSMA(ET260-1-39)-IgG4(HL-CH3)-CD28TM2-41BB-zeta with a signal sequence. The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 102 with signal sequence; SEQ ID NO: 50 without signal sequence)

[0066] FIG.21 shows the amino acid sequence of the AMET260-1-56 CAR construct PSMA(ET260-1-56)-IgG4(HL-CH3)-CD28TM2-41BB-zeta with a signal sequence. The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 103 with signal sequence; SEQ ID NO: 51 without signal sequence)

[0067] FIG.22 shows the amino acid sequence of the AMET260-1-58 CAR construct PSMA(ET260-1-58)-IgG4(HL-CH3)-CD28TM2-41BB-zeta with a signal sequence. The various 15 sf-5670772Attorney Docket No.: 75004-20023.40 domains are labelled and indicated by alternating underlining. (SEQ ID NO: 104 with signal sequence; SEQ ID NO: 52 without signal sequence)

[0068] FIG.23 shows the amino acid sequence of the AMET260-1-67 CAR construct PSMA(ET260-1-67)-IgG4(HL-CH3)-CD28TM2-41BB-zeta with a signal sequence. The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 105 with signal sequence; SEQ ID NO: 53 without signal sequence)

[0069] FIG.24 shows the amino acid sequence of the AMET260-1-69 CAR construct PSMA(ET260-1-69)-IgG4(HL-CH3)-CD28TM2-41BB-zeta with a signal sequence. The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 106 with signal sequence; SEQ ID NO: 54 without signal sequence)

[0070] FIG.25 shows the amino acid sequence of membrane bound human IL-12 (mbIL12). The various domains are labelled and indicated by alternating underlining. (SEQ ID NO: 107 with signal sequence; SEQ ID NO: 108 without signal sequence)

[0071] FIGs.26A-26C show the in vitro rechallenge of hPSMA CAR T cells co-expressing mbIL12 with tumor cells, demonstrating that hPSMA CARs with mbIL12 have improved polyfunctionality in recursive tumor challenges in vitro. FIG.26A shows hPSMA CARs with mbIL12 rechallenged with PC-3 PSMAlo(left), PC-3 PSMAhi(center), and PC-3 PIP (right). Tumor cell counts were calculated by flow cytometry. FIG.26B shows fold expansion of T cells after each rechallenge, quantified by flow cytometry. FIG.26C shows secretion of IFN^ by T cells following each rechallenge as determined by ELISA. For PSMAhi: T cells co-expressing ET260-1 CAR and mbIL12 or AMET260-1-58 CAR and mbIL12 exhibited better control of tumor cells (FIG.26A) by the 3rdrechallenge compared to the single CAR T cell controls; hPSMA CAR T cells co-expressing mbIL12 showed increased expansion (FIG.26B) compared to the single hPSMA CAR T cells and surpassed expansion of J591 CAR T cells; and hPSMA CAR T cells co-expressing mbIL12 had higher levels of IFN^ secretion (FIG.26C) compared to J591 CAR.

[0072] FIGs.27A-27C show the testing of hPSMA CARs in vivo against a subcutaneous PC-3 PIP tumor model. FIG.27A shows a schema of the subcutaneous model of PC-3 PIP (2.5e6 per mouse) treated with CAR T cells (1e6 per mouse) 22 days post engraftment. Tumor volume was measured by caliper measurement (L x W x H). FIG.27B shows the average tumor volume of mice (n=6-8 per group). FIG.27C shows the quantification of tumor volume in individual mice per group. sf-5670772Attorney Docket No.: 75004-20023.40

[0073] FIGs.28A-28E show that ET260-1 / mbIL12 T cells display potent activity against C4-2 ffluc intratibial tumors. FIG.28A shows a schema of the intratibial model of C4-2 ffluc (1.5e5 per mouse) treated with CAR T cells (0.5e6 per mouse) 21 days post engraftment. Tumor burden was quantified using bioluminescent imaging. FIG.28B shows the average tumor flux of mice (n=5-6 per group). FIG. 28C shows the Kaplan-Meier survival analysis for the mice treated. FIG.28D shows the quantification of flux from individual mice in each treatment group. FIG. 28E shows the quantification of human CD45+ cells in the peripheral blood of mice treated with T cells at days 7 and 14 post-treatment. DETAILED DESCRIPTION

[0074] The present application provides isolated constructs (referred to herein as “anti-PSMA constructs”) that comprise an antibody moiety (referred to herein as an “anti-PSMA antibody moiety”) that specifically binds to prostate specific membrane antigen, or “PSMA” (e.g., PSMA, such as human PSMA (hPSMA)) expressed on the surface of a cell, such as a cancer cell). The anti-PSMA constructs allow for specific targeting of cells expressing PSMA (e.g., cells expressing PSMA on their surfaces), such as disease cells expressing (or overexpressing) PSMA. When present in a chimeric antigen receptor (CAR) or chimeric antibody-T cell receptor construct (caTCR) expressed by a T cell, the anti-PSMA antibody moiety specifically redirects human T cells to kill target cells (e.g., cancer cells) expressing PSMA. Furthermore, when fused to a detectable label, the anti-PSMA antibody moiety may be used to visualize changes in the number and localization of PSMA-expressing cells. Such information can, in turn, be used to diagnose and / or prognose PSMA-associated diseases or disorders.

[0075] The present application provides constructs (such as isolated constructs) comprising an antibody moiety that specifically binds to PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell). Exemplary constructs include, but are not limited to, e.g., full-length anti- PSMA antibodies, multispecific anti-PSMA constructs (such as a bispecific anti-PSMA antibodies), anti-PSMA chimeric antigen receptors (“CARs”), anti-PSMA chimeric antibody-T cell receptor constructs (caTCRs), anti-PSMA chimeric signaling receptors (CSRs), an anti- PSMA immunoconjugates, as well as other constructs, as described in further detail below. Each of the constructs described herein demonstrates high specificity for human PSMA in native form (e.g., expressed on the surface of a cell, such as a cancer cell).

[0076] The present application also provides nucleic acids that encode the anti-PSMA constructs described herein (or the polypeptide portion(s) thereof). 17 sf-5670772Attorney Docket No.: 75004-20023.40

[0077] Also provided herein are compositions (such as pharmaceutical compositions or formulations) comprising an anti-PSMA construct described herein or an effector cell expressing or associated with anti-PSMA construct described herein (such as a T cell expressing an anti- PSMA CAR, an anti-PSMA caTCR, an anti-PSMA chimeric signaling receptor (CSR), or a combination thereof).

[0078] The present application also provides methods of making and using the anti-PSMA constructs (or effector cells expressing or associated with the anti-PSMA constructs) for treatment, for diagnostic purposes, for prognostic purposes, and for inclusion into kits and articles of manufacture useful for the treatment, diagnosis, and / or prognosis of PSMA-associated diseases and disorders. Definitions

[0079] Before describing the disclosed embodiments in detail, it is to be understood that the present disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0080] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a molecule” optionally includes a combination of two or more such molecules, and the like.

[0081] As used herein “prostate specific membrane antigen” or “PSMA” refers to any native PSMA from any vertebrate source, including mammals such as primates (e.g., humans, non- human primates (e.g., cynomolgus or rhesus monkeys)) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed PSMA as well as any form of PSMA that results from processing in the cell. The term also encompasses naturally occurring variants of PSMA, e.g., splice variants, allelic variants, and isoforms. PSMA is a type II membrane protein originally characterized by the murine monoclonal antibody (mAb) 7E11- C5.3 and is expressed in all forms of prostate tissue (including carcinoma). The PSMA protein has a 3-part structure: a 19-amino-acid internal portion, a 24-amino-acid transmembrane portion, and a 707-amino-acid external portion (e.g., the extracellular domain). An exemplary amino acid sequence for human PSMA is MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNM KAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLL sf-5670772Attorney Docket No.: 75004-20023.40 SYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNY ARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAP GVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVH PIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVT RIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRP RRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMY SLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIAS GRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELAN SIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSER LQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIY DALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA (SEQ ID NO: 43).

[0082] An exemplary amino acid sequence for the extracellular domain of human PSMA is KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKE FGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAF SPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGA KGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYR RGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFST QKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEI VRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIE GNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLG SGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHL TVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLF SAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYA PSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA (SEQ ID NO: 44).

[0083] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of the present application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications 19 sf-5670772Attorney Docket No.: 75004-20023.40 required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of cancer (such as, for example, tumor volume). The methods provided herein contemplate any one or more of these aspects of treatment.

[0084] The terms “recurrence,” “relapse” or “relapsed” refers to the return of a cancer or disease after clinical assessment of the disappearance of disease. A diagnosis of distant metastasis or local recurrence can be considered a relapse.

[0085] The term “refractory” or “resistant” refers to a cancer or disease that has not responded to treatment.

[0086] “Activation,” as used herein in relation to T cells, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions.

[0087] The term “antibody moiety” includes full-length antibodies and antigen-binding fragments thereof. A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in each chain generally comprise three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including CDR-L1, CDR- L2, and CDR-L3, heavy chain (HC) CDRs including CDR-H1, CDR-H2, and CDR-H3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs or FWRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ^, ^, ^, ^, and ^ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (^I heavy chain), IgG2 (^2 heavy chain), IgG3 (^3 heavy chain), IgG4 (^4 heavy chain), IgA1 (^1 heavy chain), or IgA2 (^2 heavy chain).

[0088] The term “antigen-binding fragment” as used herein refers to an antibody fragment including, but not limited to, e.g., a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide 20 sf-5670772Attorney Docket No.: 75004-20023.40 stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0089] The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope (or overlapping epitopes) within an antigen if they exhibit competitive binding for the antigen.

[0090] As used herein, a first antibody moiety “competes” for binding to PSMA with a second antibody moiety when the first antibody moiety inhibits binding of the second antibody moiety to PSMA by at least about 50% (such as at least about any of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolar concentration of the first antibody moiety, or vice versa. A high throughput process for “binning” antibodies based upon their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0091] As use herein, the term “specifically binds” or “is specific for” refers to measurable and reproducible interactions (such as binding between a target and an antibody or an antibody moiety) that are determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody or antibody moiety that specifically binds to a target (which can be an epitope) is an antibody or antibody moiety that binds the target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In some embodiments, an antibody or antibody moiety that specifically binds to an antigen reacts with one or more antigenic determinants of the antigen (for example an epitope on the extracellular domain of PSMA) with a binding affinity that is at least about 10 times its binding affinity for other targets.

[0092] An “isolated” anti-PSMA construct as used herein refers to an anti-PSMA construct that (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, (3) is expressed by a cell from a different species, or (4) does not occur in nature. 21 sf-5670772Attorney Docket No.: 75004-20023.40

[0093] The term “isolated nucleic acid” as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated nucleic acid” (1) is not associated with all or a portion of a polynucleotide in which the “isolated nucleic acid” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

[0094] As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem.252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol.262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. Table 1: CDR Definitions1Residue numbering follows the nomenclature of Kabat et al., J. Biol. Chem.252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991).2Residue numbering follows the nomenclature of Chothia et al., J. Mol. Biol.196:901-917 (1987); Al- Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997).3Residue numbering follows the nomenclature of MacCallum et al., J. Mol. Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008).4Residue numbering follows the nomenclature of Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001).5Residue numbering follows the nomenclature of Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). sf-5670772Attorney Docket No.: 75004-20023.40

[0095] The term “chimeric antibodies” refer to antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a biological activity of interest (e.g., binding to PSMA, such as human PSMA, on the surface of a cell, e.g., a cancer cell) (see U.S. Patent No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0096] The term “semi-synthetic” in reference to an antibody or antibody moiety means that the antibody or antibody moiety has one or more naturally occurring sequences and one or more non-naturally occurring (i.e., synthetic) sequences or amino acids.

[0097] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0098] “Single-chain Fv,” also abbreviated as “sFv” or “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VHand VLdomains which permits the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthün in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).

[0099] The term “diabodies” refers to small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) typically with short linkers (such as about 5 to about 10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen- binding sites. Bispecific diabodies are heterodimers of two “crossover” scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). 23 sf-5670772Attorney Docket No.: 75004-20023.40

[0100] “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FWR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., affinity for the target antigen). In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

[0101] “Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences 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 BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE 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. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0102] The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is one that binds an IgG antibody (a ^ receptor) and includes receptors of the Fc^RI, Fc^RII, and Fc^RIII subclasses, including allelic 24 sf-5670772Attorney Docket No.: 75004-20023.40 variants and alternatively spliced forms of these receptors. Fc^RII receptors include Fc^RIIA (an “activating receptor”) and Fc^RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor Fc^RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor Fc^RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review M. in Daëron, Annu. Rev. Immunol.15:203-234 (1997)). The term includes allotypes, such as Fc^RIIIA allotypes: Fc^RIIIA-Phe158, Fc^RIIIA-Val158, Fc^RIIA-R131 and / or Fc^RIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)).

[0103] The term “FcRn” refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an a-chain noncovalently bound to b2-microglobulin. The multiple functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol.18, 739-766. FcRn plays a role in the passive delivery of immunoglobulin IgGs from mother to young and the regulation of serum IgG levels. FcRn can act as a salvage receptor, binding and transporting pinocytosed IgGs in intact form both within and across cells, and rescuing them from a default degradative pathway.

[0104] The “CH1 domain” of a human IgG Fc region (also referred to as “C1” of “H1” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0105] The “hinge region” is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol.22:161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter- heavy chain S-S bonds in the same positions.

[0106] The “CH2 domain” of a human IgG Fc region (also referred to as “C2” of “H2” domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol.22:161-206 (1985). sf-5670772Attorney Docket No.: 75004-20023.40

[0107] The “CH3 domain” (also referred to as “C2” or “H3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).

[0108] A “functional Fc fragment” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody variable domain) and can be assessed using various assays known in the art.

[0109] An antibody with a variant IgG Fc with “altered” FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity (e.g., Fc^R or FcRn) and / or ADCC activity compared to a parent polypeptide or to a polypeptide comprising a native sequence Fc region. The variant Fc which “exhibits increased binding” to an FcR binds at least one FcR with higher affinity (e.g., lower apparent Kd or IC50 value) than the parent polypeptide or a native sequence IgG Fc. According to some embodiments, the improvement in binding compared to a parent polypeptide is about 3-fold, such as about any of 5, 10, 25, 50, 60, 100, 150, 200, or up to 500-fold, or about 25% to 1000% improvement in binding. The polypeptide variant which “exhibits decreased binding” to an FcR, binds at least one FcR with lower affinity (e.g., higher apparent Kd or higher IC50 value) than a parent polypeptide. The decrease in binding compared to a parent polypeptide may be about 40% or more decrease in binding.

[0110] “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express Fc^RIII only, whereas monocytes express Fc^RI, Fc^RII and Fc^RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No.5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. 26 sf-5670772Attorney Docket No.: 75004-20023.40 Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0111] The polypeptide comprising a variant Fc region which “exhibits increased ADCC” or mediates ADCC in the presence of human effector cells more effectively than a polypeptide having wild type IgG Fc or a parent polypeptide is one which in vitro or in vivo is substantially more effective at mediating ADCC, when the amounts of polypeptide with variant Fc region and the polypeptide with wild type Fc region (or the parent polypeptide) in the assay are essentially the same. Generally, such variants will be identified using any in vitro ADCC assay known in the art, such as assays or methods for determining ADCC activity, e.g. in an animal model etc. In some embodiments, the variant is from about 5-fold to about 100-fold, e.g. from about 25 to about 50-fold, more effective at mediating ADCC than the wild type Fc (or parent polypeptide).

[0112] “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capability are described in US patent No.6,194,551B1 and WO99 / 51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol.164: 4178-4184 (2000).

[0113] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0114] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sf-5670772Attorney Docket No.: 75004-20023.40 sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0115] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0116] An “effective amount” of an anti-PSMA construct or composition as disclosed herein, is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and by known methods relating to the stated purpose.

[0117] The term “therapeutically effective amount” refers to an amount of an anti-PSMA construct or composition as disclosed herein, effective to “treat” a disease or disorder in an individual. In the case of cancer, the therapeutically effective amount of the anti-PSMA construct or composition as disclosed herein can reduce the number of cancer cells; reduce the tumor size or weight; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the anti-PSMA construct or composition as disclosed herein can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In some embodiments, the therapeutically effective amount is an amount that extends the survival of a patient. In some embodiments, the therapeutically effective amount is an amount that improves progression-free survival of a patient.

[0118] As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable 28 sf-5670772Attorney Docket No.: 75004-20023.40 carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and / or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

[0119] The term “label” when used herein refers to a detectable compound or composition which can be conjugated directly or indirectly to the anti-PSMA antibody moiety. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

[0120] The term “chimeric antigen receptor (CAR)” refers to an artificially constructed hybrid single-chain protein or single-chain polypeptide containing a single-chain variable fragment (scFv) as a part of the extracellular antigen-binding domain, linked directly or indirectly to a transmembrane domain (e.g., a TCR transmembrane domain), which is in turn linked directly or indirectly to an intracellular immune cell (e.g., T cell or NK cell) signaling domain. The intracellular signaling domain (ISD) comprises a primary signaling sequence, or primary immune cell signaling sequence, from an antigen-dependent, TCR-associated T cell activation molecule, e.g., a portion of the intracellular domain of CD3^, TCR^, FcR^, FcR^, CD3^, CD3^, CD3^, CD5, CD22, CD79a, CD79b, or CD66d). The ISD can further comprise a co-stimulatory signaling sequence; e.g., a portion of the intracellular domain of an antigen-independent, co- stimulatory molecule such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, or the like. Characteristics of CARs include their ability to redirect immune cell (e.g., T cell or NK cell) specificity and reactivity toward a selected target in either MHC-restricted (in cases of TCR-mimic antibodies) or non-MHC-restricted (in cases of antibodies against cell surface proteins) manners, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives immune cells (e.g., T cells or NK cells) expressing CARs the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.

[0121] There are currently three generations of CARs. The “first generation” CARs are typically single-chain polypeptides composed of a scFv as the antigen-binding domain fused to a transmembrane domain fused to the cytoplasmic / intracellular domain, which comprises a primary immune cell signaling sequence, of a molecule from the T cell receptor (TCR) complex, i.e., an antigen-dependent, TCR-associated T cell activation molecule such as CD3^, TCR^, FcR^, FcR^, CD3^, CD3^, CD3^, CD5, CD22, CD79a, CD79b, or CD66d. The “first 29 sf-5670772Attorney Docket No.: 75004-20023.40 generation” CARs typically have the intracellular domain from the CD3^ chain, which is the primary transmitter of signals from endogenous TCRs. The “first generation” CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T cells through their CD3^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. The “second generation” CARs add intracellular domains from various co- stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the primary immune cell signaling sequence of the CAR to provide additional signals to the T cell. “Second generation” CARs comprise fragments that provide co-stimulation (e.g., CD28 or 4-IBB) and activation (e.g., CD3^). Preclinical studies have indicated that the “second generation” CARs can improve the antitumor activity of T cells. For example, robust efficacy of the “second generation” CAR modified T cells was demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL). The “third generation” CARs comprise those that provide multiple co-stimulation (e.g., CD28 and 4-1BB) and activation (e.g., CD3^).

[0122] As used herein, the term “chimeric antibody-T cell receptor construct” (or caTCR) refers to a functional polypeptide complex comprising two separate polypeptide chains, one including an antibody heavy chain variable region (VH) and an antibody heavy chain constant region (CH), and the other including an antibody light chain variable region (VL) and an antibody light chain constant region (CL). A caTCR as defined herein is therefore a 2-subunit construct, each subunit substantially resembling a cell membrane-anchored antibody heavy chain or light chain that is fused to a transmembrane domain (e.g., a TCR transmembrane domain) and an intracellular immune cell signaling domain. In some embodiments, a caTCR does not include a co-stimulatory domain (e.g., a portion of the intracellular domain of CD3^, CD3^, CD3^, or CD3^). In some embodiments, a caTCR comprises: a) an extracellular domain comprising an antibody moiety and b) a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling module. In some embodiments, an anti-PSMA caTCR comprises: a) an extracellular domain comprising an anti-PSMA antibody moiety that specifically binds to an extracellular region of PSMA or a portion thereof (e.g., SEQ ID NO: 44 or a portion thereof) and b) a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling module.

[0123] A caTCR as defined herein comprises a first polypeptide chain and a second polypeptide chain, in which the first polypeptide chain comprises an antibody VH fused to an antibody CH fused to a transmembrane domain and an intracellular immune cell signaling 30 sf-5670772Attorney Docket No.: 75004-20023.40 domain, and the second polypeptide comprises an antibody VLfused to an antibody CLfused to a transmembrane domain and an intracellular immune cell signaling domain. In some embodiments, the first and second polypeptide chains are linked, such as by a covalent linkage (e.g., peptide or other chemical linkage) or non-covalent linkage. In some embodiments, the anti- PSMA caTCR is a heterodimer comprising the first polypeptide chain and the second polypeptide chain. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked by at least one disulfide bond. The specificity of the anti-PSMA caTCR derives from an antibody moiety that confers binding specificity to an extracellular region of PSMA or a portion thereof (e.g., SEQ ID NO: 44 or a portion thereof).

[0124] The terms “chimeric antibody-T cell receptor (caTCR)” and “antibody-TCR chimeric molecule or construct (abTCR or AbTCR)” are used interchangeably herein. Further descriptions and examples of caTCR and abTCR may be found in, e.g., WO 2017 / 070608 and PCT / US2018 / 029217 (now published as WO 2018 / 200582), the contents of which are incorporated by reference herein in their entirety.

[0125] It is understood that embodiments of the invention described herein include “consisting” and / or “consisting essentially of” embodiments.

[0126] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0127] As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X. I. Anti-PSMA Constructs

[0128] Provided herein are constructs that specifically bind to prostate specific membrane antigen (PSMA) (such as human PSMA (hPSMA)) that comprise an antibody moiety that specifically binds to PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell). Such constructs are also referred to herein as “anti-PSMA constructs.” The specificity of the anti-PSMA construct for PSMA is derived from the anti-PSMA antibody moiety (such as a full-length antibody or antigen-binding fragment thereof) that specifically binds to cell surface- bound PSMA. In some embodiments, the extracellular domain of PSMA comprises the amino acid sequence of SEQ ID NO: 44. sf-5670772Attorney Docket No.: 75004-20023.40

[0129] Anti-PSMA constructs within the scope of the present application include, without limitation, e.g., full-length anti-PSMA antibodies, multispecific anti-PSMA constructs, anti- PSMA chimeric antigen receptor constructs (CARs), anti-PSMA chimeric antibody-T cell receptor (TCR) constructs (caTCRs), anti-PSMA chimeric signaling receptor constructs (CSRs), anti-PSMA immunoconjugates, and others, as described herein below.

[0130] For example, in some embodiments, the anti-PSMA construct (such as an isolated anti- PSMA construct) comprises an anti-PSMA antibody moiety that specifically binds to PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell). In some embodiments, the extent of binding of the anti-PSMA antibody to a non-target polypeptide is less than about 10% of the binding of the anti-PSMA antibody moiety to PSMA as determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of nonlabeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10-4M, alternatively at least about 10-5M, alternatively at least about 10-6M, alternatively at least about 10-7M, alternatively at least about 10-8M, alternatively at least about 10-9M, alternatively at least about 10-10M, alternatively at least about 10-11M, alternatively at least about 10-12M, or less. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide (e.g., PSMA) or epitope on a particular polypeptide (e.g., PSMA) without substantially binding to any other polypeptide or polypeptide epitope.

[0131] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that specifically binds to PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell) and competes for binding to PSMA with a second anti-PSMA antibody (or antibody moiety) that specifically binds PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell) and comprises: (a) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, 32, or 111; (b) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2 or 112; (c) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3, 23, or 113; (d) a CDR-L1 32 sf-5670772Attorney Docket No.: 75004-20023.40 comprising an amino acid sequence of SEQ ID NO: 8, 28, or 114; (e) a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 9 or 115; and (f) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 10, 19 or 116. In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that specifically binds to the same epitope of PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell) as a second anti-PSMA antibody (or antibody moiety) that specifically binds PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell) and comprises (a) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, 32, or 111; (b) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2 or 112; (c) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3, 23, or 113; (d) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 8, 28, or 114; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or 115; and (f) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 10, 19, or 116.

[0132] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, three, four, five, or six complementarity determining region (CDR) sequences selected from the group consisting of: (a) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, 32, or 111, or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (b) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2 or 112, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution) (c) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3, 23, or 113, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (d) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 8, 28, or 114, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or 115, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); and (f) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 10, 19, or 116, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution). sf-5670772Attorney Docket No.: 75004-20023.40

[0133] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises (a) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, 32, or 111, or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (b) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2 or 112, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (c) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 3, 23, or 113, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (d) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 8, 28, or 114, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or 115, or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); and (f) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 10, 19, or 116, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution).

[0134] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, three, four, five, or six complementarity determining region (CDR) sequences selected from the group consisting of: (a) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, 32, or 111; (b) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2 or 112; (c) a CDR-H3 comprising an amino acid sequence set forth in SEQ ID NO: 3, 23, or 113; (d) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 8, 28, or 114; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or 115; and (f) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 10, 19 or 116.

[0135] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises (a) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 1, 32, or 111; (b) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 2 or 112; (c) a CDR- H3 comprising an amino acid sequence of SEQ ID NO: 3, 23, or 113; (d) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 8, 28, or 114; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or 115; and (f) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 10, 19, or 116. In some embodiments, the CDRs are human CDRs. 34 sf-5670772Attorney Docket No.: 75004-20023.40

[0136] The amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NOs: 1-3, 23, 32 and 111-113) are provided in Table 2A below, and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NOs: 8-10, 19, 28, and 114-116) are provided in Table 2B below. Table 2A: Heavy chain CDR sequences of anti-PSMA constructsTable 2B: Light chain CDR sequences of anti-PSMA constructs

[0137] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, three, four, five, six, seven, or eight framework region (FWR) sequences selected from the group consisting of: (a) a FWR-H1 comprising an amino acid sequence of SEQ ID NO: 4, 24, or 118, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (b) a FWR-H2 comprising an amino acid sequence of SEQ ID NO: 5 or 119, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (c) a FWR-H3 comprising an amino acid sequence of SEQ ID NO: 6, 18, or 120, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (d) a FWR-H4 comprising an amino acid sequence of any one of SEQ ID NOs: 7, 33, 38, or 121, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations 35 sf-5670772Attorney Docket No.: 75004-20023.40 (e.g., insertion, deletion, or substitution, such as conservative substitution); (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or 122, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (f) a FWR-L2 comprising an amino acid sequence of SEQ ID NO: 12, 34, or 123, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (g) a FWR-L3 comprising an amino acid sequence of SEQ ID NO: 13, 39, or 124, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); and (h) a FWR-L4 comprising an amino acid sequence of SEQ ID NO: 14 or 125, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution).

[0138] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises (a) a FWR-H1 comprising an amino acid sequence of SEQ ID NO: 4, 24, or 118, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (b) a FWR-H2 comprising an amino acid sequence of SEQ ID NO: 5 or 119, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (c) a FWR-H3 comprising an amino acid sequence of SEQ ID NO: 6, 18, or 120, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (d) a FWR-H4 comprising an amino acid sequence of any one of SEQ ID NOs: 7, 33, 38, or 121, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or 122, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (f) a FWR-L2 comprising an amino acid sequence of SEQ ID NO: 12, 34, or 123, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); (g) a FWR-L3 comprising an amino acid sequence of SEQ ID NO: 13, 39, or 124, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution); and (h) a 36 sf-5670772Attorney Docket No.: 75004-20023.40 FWR-L4 comprising an amino acid sequence of SEQ ID NO: 14 or 125, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid variations (e.g., insertion, deletion, or substitution, such as conservative substitution).

[0139] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, three, four, five, six, seven, or eight framework region (FWR) sequences selected from the group consisting of: (a) a FWR-H1 comprising an amino acid sequence of SEQ ID NO: 4, 24, or 118; (b) a FWR-H2 comprising an amino acid sequence of SEQ ID NO: 5 or 119; (c) a FWR-H3 comprising an amino acid sequence of SEQ ID NO: 6, 18, or 120; (d) a FWR-H4 comprising an amino acid sequence of any one of SEQ ID NOs: 7, 33, 38, or 121; (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or 122; (f) a FWR-L2 comprising an amino acid sequence of SEQ ID NO: 12, 34, or 123; (g) a FWR-L3 comprising an amino acid sequence of SEQ ID NO: 13, 39, or 124; and (h) a FWR-L4 comprising an amino acid sequence of SEQ ID NO: 14 or 125.

[0140] In some embodiments, the anti-PSMA construct comprises (a) a FWR-H1 comprising an amino acid sequence of SEQ ID NO: 4, 24, or 118; (b) a FWR-H2 comprising an amino acid sequence of SEQ ID NO: 5 or 119; (c) a FWR-H3 comprising an amino acid sequence of SEQ ID NO: 6, 18, or 120; (d) a FWR-H4 comprising an amino acid sequence of any one of SEQ ID NOs: 7, 33, 38, or 121; (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or 122; (f) a FWR-L2 comprising an amino acid sequence of SEQ ID NO: 12, 34, or 123; (g) a FWR-L3 comprising an amino acid sequence of SEQ ID NO: 13, 39, or 124; and (h) a FWR-L4 comprising an amino acid sequence of SEQ ID NO: 14 or 125. The amino acid sequences of the framework regions (e.g., SEQ ID NOs: 4, 5, 6, 7, 11-14, 18, 24, 33, 34, 38, 39, and 118-125) are shown in Table 2C. Table 2C: Framework region sequences of anti-PSMA constructssf-5670772Attorney Docket No.: 75004-20023.40

[0141] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, or three CDRs of an antibody heavy chain variable domain (VH) comprising an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 15, 20, 25, 29, 35, 40, or 109. Additionally or alternatively, in some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, or three CDRs of a light chain variable domain (VL) comprising an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 16, 21, 26, 30, 36, 41, or 110. sf-5670772Attorney Docket No.: 75004-20023.40

[0142] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VHcomprising an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of one of SEQ ID NOs: 15, 20, 25, 29, 35, 40, or 109 and / or a VL comprising an amino acid sequence having at least about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 16, 21, 26, 30, 36, 41, or 110. The amino acid sequences of the VH and VLs (e.g., SEQ ID NOs: 15, 16, 20, 21, 25, 26, 29, 30, 35, 36, 40, 41, 109, and 110) are provided in Table 3 below. The CDR sequences are in underlined bold type. Table 3: VHand VLsequences of anti-PSMA constructssf-5670772Attorney Docket No.: 75004-20023.40

[0143] The heavy and light chain variable domains can be combined in pair-wise combinations to generate additional anti-PSMA antibody moieties that can be incorporated into and / or used with the anti-PSMA constructs of the present disclosure.

[0144] In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid substitutions, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti- PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 40 sf-5670772Attorney Docket No.: 75004-20023.40 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR- H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 12, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CDRs are human CDRs. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, or three CDRs of a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and one, two, or three CDRs of a VL domain comprising the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VH domain 41 sf-5670772Attorney Docket No.: 75004-20023.40 having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 15, and / or a VLdomain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15, and a VL domain comprising the amino acid sequence of SEQ ID NO: 16. An anti-PSMA antibody moiety comprising the amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: 16 is alternatively referred to herein as “ET260-1”.

[0145] In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid substitutions, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti- PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 42 sf-5670772Attorney Docket No.: 75004-20023.40 5) amino acid substitutions, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR- H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 18, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 12, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CDRs are human CDRs. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, or three CDRs of a VH domain comprising the amino acid sequence of SEQ ID NO: 20 and one, two, or three CDRs of a VLdomain comprising the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VHdomain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 20, and / or a VL domain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 20, and a VL domain comprising the amino acid sequence of SEQ ID NO: 21. An anti-PSMA antibody sf-5670772Attorney Docket No.: 75004-20023.40 moiety comprising the amino acid sequence of SEQ ID NO: 20 and SEQ ID NO: 21 is alternatively referred to herein as “AMET260-1-39”.

[0146] In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid substitutions, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti- PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR-H1 comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (g) a FWR-L3 44 sf-5670772Attorney Docket No.: 75004-20023.40 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR- H1 comprising the amino acid sequence of SEQ ID NO: 24, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 12, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CDRs are human CDRs. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, or three CDRs of a VHdomain comprising the amino acid sequence of SEQ ID NO: 25 and one, two, or three CDRs of a VL domain comprising the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VH domain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 25, and / or a VL domain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 26. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 25, and a VLdomain comprising the amino acid sequence of SEQ ID NO: 26. An anti-PSMA antibody moiety comprising the amino acid sequence of SEQ ID NO: 25 and SEQ ID NO: 26 is alternatively referred to herein as “AMET260-1-56”.

[0147] In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid substitutions, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a 45 sf-5670772Attorney Docket No.: 75004-20023.40 CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti- PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR- H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11, (f) a FWR-L2 comprising the amino 46 sf-5670772Attorney Docket No.: 75004-20023.40 acid sequence of SEQ ID NO: 12, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CDRs are human CDRs. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, or three CDRs of a VH domain comprising the amino acid sequence of SEQ ID NO: 29 and one, two, or three CDRs of a VL domain comprising the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VH domain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 29, and / or a VL domain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 30. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 29, and a VLdomain comprising the amino acid sequence of SEQ ID NO: 30. An anti-PSMA antibody moiety comprising the amino acid sequence of SEQ ID NO: 29 and SEQ ID NO: 30 is alternatively referred to herein as “AMET260-1-58”.

[0148] In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid substitutions, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti- PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID 47 sf-5670772Attorney Docket No.: 75004-20023.40 NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR- H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 33, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 34, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CDRs are human CDRs. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, or three CDRs of a VH domain comprising the amino acid sequence of SEQ ID NO: 35 and one, two, or three CDRs of a VL domain comprising the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VH domain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino 48 sf-5670772Attorney Docket No.: 75004-20023.40 acid sequence of SEQ ID NO: 35, and / or a VLdomain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 35, and a VL domain comprising the amino acid sequence of SEQ ID NO: 36. An anti-PSMA antibody moiety comprising the amino acid sequence of SEQ ID NO: 35 and SEQ ID NO: 36 is alternatively referred to herein as “AMET260-1-67”.

[0149] In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid substitutions, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (g) a FWR-H4 that does not comprise the amino acid sequence of SEQ ID NO: 7, and (h) a FWR-L3 that does not comprise the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, (g) a FWR-H4 that does not comprise the amino acid sequence of SEQ ID NO: 7, and (h) a FWR-L3 that does not comprise the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid substitutions, (b) 49 sf-5670772Attorney Docket No.: 75004-20023.40 a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (g) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and (h) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, (g) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 38, and (h) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR-H1 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 38 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (g) a 50 sf-5670772Attorney Docket No.: 75004-20023.40 FWR-L3 comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR- H1 comprising the amino acid sequence of SEQ ID NO: 4, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 5, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 6, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 38, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 11, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 12, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 39, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CDRs are human CDRs. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, or three CDRs of a VHdomain comprising the amino acid sequence of SEQ ID NO: 40 and one, two, or three CDRs of a VL domain comprising the amino acid sequence of SEQ ID NO: 41. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VH domain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 40, and / or a VL domain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 41. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 40, and a VLdomain comprising the amino acid sequence of SEQ ID NO: 41. An anti-PSMA antibody moiety comprising the amino acid sequence of SEQ ID NO: 40 and SEQ ID NO: 41 is alternatively referred to herein as “AMET260-1-69”.

[0150] In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111 or a variant thereof comprising up to about 4 (such as about any of 1, 2, 3, or 4) amino acid substitutions, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid sf-5670772Attorney Docket No.: 75004-20023.40 substitutions, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115 or a variant thereof comprising up to about 3 (such as about any of 1, 2, or 3) amino acid substitutions, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, (c) a CDR- H3 comprising the amino acid sequence of SEQ ID NO: 113; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR-H1 comprising the amino acid sequence of SEQ ID NO: 118 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (b) a FWR-H2 comprising the amino acid sequence of SEQ ID NO: 119 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 120 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (d) a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 121 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 122 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 123 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 124 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 125 or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises: (a) a FWR-H1 comprising the amino acid sequence of SEQ ID NO: 118, (b) a FWR- H2 comprising the amino acid sequence of SEQ ID NO: 119, (c) a FWR-H3 comprising the amino acid sequence of SEQ ID NO: 120, (d) a FWR-H4 comprising the amino acid sequence 52 sf-5670772Attorney Docket No.: 75004-20023.40 of SEQ ID NO: 121, (e) a FWR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (f) a FWR-L2 comprising the amino acid sequence of SEQ ID NO: 123, (g) a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 124, and (h) a FWR-L4 comprising the amino acid sequence of SEQ ID NO: 125. In some embodiments, the CDRs are human CDRs. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises one, two, or three CDRs of a VH domain comprising the amino acid sequence of SEQ ID NO: 109 and one, two, or three CDRs of a VL domain comprising the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VHdomain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 109, and / or a VLdomain having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 109, and a VL domain comprising the amino acid sequence of SEQ ID NO: 110. An anti-PSMA antibody moiety comprising the amino acid sequence of SEQ ID NO: 109 and SEQ ID NO: 110 is alternatively referred to herein as “ET260-2”.

[0151] The amino acid sequences of nine exemplary anti-PSMA scFvs are provided Table 4 below. The VL in each scFv is in plain text (i.e., no underline), the VH in each scFv is underlined, and the linker is in italic type. The CDRs are in bold type and underlined bold type. Table 4: anti-PSMA scFv sequences53 sf-5670772Attorney Docket No.: 75004-20023.40

[0152] In some embodiments, the anti-PSMA scFv comprises an amino acid sequence that has at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 17, 22, 27, 31, 37, 42, 48, 49, or 117. sf-5670772Attorney Docket No.: 75004-20023.40

[0153] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that binds human PSMA, mouse PSMA, rat PSMA, cynomolgus monkey PSMA, and / or rhesus PSMA. In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that specifically binds human PSMA (hPSMA). In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that specifically binds to PSMA present on or expressed on the surface of a cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell expresses abnormally high levels of PSMA, as compared to a reference cell. In some embodiments, the reference cell is a cell obtained from or derived from non-diseased (such as non-cancerous) tissue. In some embodiments, the cell that expresses abnormally high levels of PSMA is a cancer cell. In some embodiments, the cancer cell is in a solid tumor. In some embodiments, the cancer cell is a prostate cancer cell, a renal cell cancer cell, a uterine cancer cell, or a liver cancer cell. In some embodiments, the cancer cell is a metastatic cancer cell. A. Anti-PSMA Constructs Comprising Anti-PSMA Antibody Moiety Sequence Variants

[0154] In some embodiments, anti-PSMA constructs of the present application comprise variants (such as amino acid sequence variants) of the anti-PSMA antibody moieties described herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of the anti-PSMA antibody moiety of an anti-PSMA construct. Amino acid sequence variants of an anti-PSMA antibody moiety may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody moiety, or by peptide synthesis. Such modifications include, for example, deletions from, insertions into, and / or substitutions of residues within the amino acid sequences of the anti-PSMA antibody moiety. Any combination of deletion(s), insertion(s), and substitution(s) can be made to arrive at the final anti-PSMA antibody moiety, provided that the final antibody moiety possesses the desired characteristics, e.g., binding to PSMA (such as PSMA expressed on the surface of a cell, e.g., a cancer cell).

[0155] In some embodiments, an anti-PSMA antibody moiety sequence variant comprises one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include the CDRs and / or the framework regions (FWRs). Amino acid substitutions may be introduced into an anti-PSMA antibody moiety of interest and the products screened for a desired activity, e.g., retained / improved binding to PSMA (e.g., cell surface-bound PSMA), decreased immunogenicity, or improved ADCC or CDC, etc. Amino acid sequence insertions include sf-5670772Attorney Docket No.: 75004-20023.40 amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-PSMA antibody moiety with an N-terminal methionyl residue. Other insertional variants of the anti-PSMA antibody moiety include the fusion to the N- or C-terminus of the antibody moiety to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the anti-PSMA antibody moiety.

[0156] In some embodiments, an anti-PSMA antibody moiety sequence variant comprises one or more conservative amino acid substitutions, as shown in Table 5 below. Table 5: Conservative substitutions

[0157] Amino acids may be grouped into different classes according to common side-chain properties: a) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; sf-5670772Attorney Docket No.: 75004-20023.40 b) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c) Acidic: Asp, Glu; d) Basic: His, Lys, Arg; e) Residues that influence chain orientation: Gly, Pro; f) Aromatic: Trp, Tyr, Phe.

[0158] Non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0159] An exemplary substitutional variant is an affinity matured antibody moiety, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant antibody moieties displayed on phage and screened for a particular biological activity (e.g. binding affinity). Alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody moiety affinity. Such alterations may be made in CDR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or specificity determining residues (SDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0160] In some embodiments, one or more CDR sequences provided herein is either unaltered, or contains no more than one, two, three, four, or five amino acid substitutions. In some embodiments a VHand / or VLsequence provided herein is either unaltered, or contains no more than one, two, three, four, or five amino acid substitutions. In some embodiments one or more CDR sequences within a VHand / or VLsequence provided herein is either unaltered, or contains no more than one, two, three, four, or five amino acid substitutions.

[0161] Diversity may be introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody moiety variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted. sf-5670772Attorney Docket No.: 75004-20023.40

[0162] The anti-PSMA antibodies or anti-PSMA antibody moieties may also be identified by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating polypeptide display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0163] In certain phage display methods, repertoires of VHand VLgenes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self as well as self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.5,750,373, and US Patent Publication Nos.2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0164] Anti-PSMA antibody moiety sequence variants can be prepared using phage display to screen libraries for antibodies specific to PSMA (e.g., a cell surface-bound PSMA). The library can be a human scFv phage display library having a diversity of at least 1 x 109(such as at least about any one of 1x109, 2.5x109, 5x109, 7.5x109, 1x1010, 2.5x1010, 5x1010, 7.5x1010, or 1x1011) unique human antibody fragments. In some embodiments, the library is a naïve human library constructed from DNA extracted from human PMBCs and spleens from healthy donors, 58 sf-5670772Attorney Docket No.: 75004-20023.40 encompassing all human heavy and light chain subfamilies. In some embodiments, the library is a naïve human library constructed from DNA extracted from PBMCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library, wherein heavy chain CDR3 (CDR-H3) is completely randomized, with all amino acids (with the exception of cysteine) equally likely to be present at any given position (see, e.g., Hoet, R.M. et al., Nat. Biotechnol.23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 (CDR-H3) of the semi-synthetic human library has a length from about 5 to about 24 amino acids (such as about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids). In some embodiments, the library is a non-human phage display library.

[0165] Phage clones that bind to PSMA (e.g., a cell surface-bound human PSMA) with high affinity can be selected by iterative binding of phage to PSMA, which is bound to a solid support (such as, for example, beads for solution panning or mammalian cells for cell panning), followed by removal of non-bound phage and by elution of specifically bound phage. In an example of solution panning, the PSMA can be biotinylated for immobilization to a solid support. The biotinylated PSMA is mixed with the phage library and a solid support, such as streptavidin- conjugated Dynabeads M-280, and then PSMA-phage-bead complexes are isolated. The bound phage clones are then eluted and used to infect an appropriate host cell, such as E. coli XL1- Blue, for expression and purification.

[0166] In another example of cell panning, mammalian cells expressing cell surface-bound PSMA (such as Jurkat cells expressing human PSMA) are mixed with the phage library, after which the cells are collected and the bound clones are eluted and used to infect an appropriate host cell for expression and purification. The panning can be performed for multiple (such as about any of 2, 3, 4, 5, 6 or more) rounds via solution panning, cell panning, or a combination of both, to enrich for phage clones binding specifically to the PSMA. Enriched phage clones can be tested for specific binding to PSMA by any methods known in the art, including for example ELISA and FACS.

[0167] A useful method of identification of residues or regions of an anti-PSMA antibody moiety that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody moiety with antigen is 59 sf-5670772Attorney Docket No.: 75004-20023.40 affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody moiety complex can be determined to identify contact points between the antibody moiety and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0168] An anti-PSMA antibody moiety provided herein may additionally comprise one or more peptide tag sequences, peptide linker sequences (including self-cleaving linkers), cleavage sites, or other peptide sequences (e.g., signal peptides). B. Full-Length Anti-PSMA Antibodies

[0169] In some embodiments, the anti-PSMA construct provided herein is or comprises a full- length antibody, e.g., a full-length antibody comprising an anti-PSMA antibody moiety, also referred to herein as a “full-length anti-PSMA antibody.” In some embodiments, the full-length antibody is a monoclonal antibody, as described in further detail elsewhere herein.

[0170] In some embodiments, the full-length anti-PSMA antibody comprises an Fc sequence from an immunoglobulin, e.g., a human immunoglobulin such as IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length anti-PSMA antibody comprises an Fc sequence of IgG, e.g., a human IgG, such as any of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the full-length anti-PSMA antibody comprises an Fc sequence of a rabbit, rat, or mouse immunoglobulin. In some embodiments, the full-length anti-PSMA antibody comprises an Fc sequence of a non- human primate (e.g., a rhesus monkey or cynomolgus monkey). In some embodiments, the full- length anti-PSMA antibody comprises an Fc sequence that has been altered or otherwise changed so that it has enhanced antibody dependent cellular cytotoxicity (ADCC) function and / or enhanced complement dependent cytotoxicity (CDC) effector function, as described in further detail elsewhere herein. C. Human and Humanized Anti-PSMA Antibodies and Antibody Moieties

[0171] In some embodiments, the anti-PSMA construct comprises an anti-PSMA antibody moiety that is a human or humanized. Humanized forms of non-human (e.g., murine) antibody moieties are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2, scFv), or other antigen-binding subsequences of full-length antibodies that typically contain minimal sequence derived from non-human immunoglobulin. Humanized sf-5670772Attorney Docket No.: 75004-20023.40 antibody moieties include human immunoglobulins (recipient antibodies) in which residues from one or more CDRs of the recipient are replaced by residues (import residues) from a CDR of a non-human species (donor antibody) such as a mouse, rat, or rabbit antibody having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In some embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992), Verhoeyen et al., Science, 239: 1534-1536 (1988), and U.S. Patent No. 4,816,567.

[0172] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ- line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos.5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852. Alternatively, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); 61 sf-5670772Attorney Docket No.: 75004-20023.40 Neuberger, Nature Biotechnology, 14: 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

[0173] Human antibodies may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147(1): 86-95 (1991). D. Monoclonal Anti-PSMA Antibodies and Antibody Moieties

[0174] In some embodiments, an anti-PSMA construct of the present disclosure comprises a monoclonal anti-PSMA antibody or a monoclonal anti-PSMA antibody moiety. Monoclonal antibodies can be prepared, e.g., using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and Sergeeva et al., Blood, 117(16):4262-4272, using the phage display methods described herein and in the Examples below, or using recombinant DNA methods (see, e.g., US Patent No.4,816,567).

[0175] In a hybridoma method, a hamster, mouse, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro. The immunizing agent can include a polypeptide or a fusion protein of the protein of interest, or a complex comprising at least two molecules. Generally, peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. See, e.g., Goding, Monoclonal Antibodies: Principles and Practice (New York: Academic Press, 1986), pp.59-103. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which prevents the growth of HGPRT-deficient cells. sf-5670772Attorney Docket No.: 75004-20023.40

[0176] In some embodiments, the immortalized cell lines fuse efficiently, support stable high- level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. In some embodiments, the immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al. Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Inc.: New York, 1987) pp.51-63.

[0177] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide. The binding specificity of monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0178] After the desired hybridoma cells are identified, the clones can be sub cloned by limiting dilution procedures and grown by standard methods. Goding, supra. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0179] The monoclonal antibodies secreted by the sub clones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0180] In certain embodiments, the anti-PSMA antibody or antibody moiety is monovalent. Methods for preparing monovalent antibodies are known in the art. One exemplary method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy-chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

[0181] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using any method known in the art. sf-5670772Attorney Docket No.: 75004-20023.40

[0182] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant-domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

[0183] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No.4,816,567. DNA encoding the monoclonal anti-PSMA antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells as described above or PSMA-specific phage clones can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains and / or framework regions in place of the homologous non- human sequences (U.S. Patent No.4,816,567; Morrison et al., supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an anti-PSMA antibody, or can be substituted for the variable domains of one antigen-combining site of an anti-PSMA antibody to create a chimeric bivalent antibody. E. Multispecific Anti-PSMA Constructs

[0184] In some embodiments, the anti-PSMA construct is multispecific. Multispecific anti- PSMA constructs provided herein demonstrate binding specificities for at least two different antigens or two different epitopes (e.g., two different epitopes on the same antigen).

[0185] Multispecific constructs comprising more than two valencies and / or antigen specificities are also contemplated. For example, trispecific antibodies can be prepared. See, e.g., Tutt et al. J. Immunol.147: 60 (1991). Thus, in some embodiments, the multispecific anti-PSMA sf-5670772Attorney Docket No.: 75004-20023.40 construct comprises an anti-PSMA antibody moiety and at least one additional binding moiety, such as an antigen-binding moiety, e.g., an antibody moiety.

[0186] In some embodiments, the multispecific (e.g., bispecific) anti-PSMA construct comprises a) an anti-PSMA antibody moiety (such as described herein) that specifically binds to PSMA (e.g., a cell surface-bound PSMA), and b) a second binding moiety (such as an antigen- binding moiety). In some embodiments, the second binding moiety specifically binds to an epitope on PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell) that does not overlap with the epitope bound by the anti-PSMA antibody moiety. In some embodiments, the second binding moiety specifically binds to a different antigen (i.e., an antigen other than PSMA). In some embodiments, the second binding moiety specifically binds to an antigen on the surface of a cell, such as a cancer cell or an immune cell. In some embodiments, the second binding moiety specifically binds to an antigen on the surface of a lymphocyte, such as a T cell, an NK cell, a neutrophil, a monocyte, a macrophage, or a dendritic cell. In some embodiments, the second binding moiety specifically binds to an effector T cell, such as a cytotoxic T cell (also known as cytotoxic T lymphocyte (CTL) or T killer cell). In some embodiments, the second binding moiety is an antibody moiety.

[0187] In some embodiments, the multispecific anti-PSMA construct comprises a) an anti- PSMA antibody moiety (such as described herein) that specifically binds to PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell), and b) a second binding moiety that binds specifically to CD3. In some embodiments, the second binding moiety is an antibody moiety that binds CD3. In some embodiments, the second antigen-binding moiety is a human, humanized, or semi-synthetic antibody moiety. In some embodiments, the second binding moiety specifically binds to CD3^. In some embodiments, the second binding moiety specifically binds to an agonistic epitope of CD3^. In some embodiments, the term “agonistic epitope” refers to an epitope that, upon binding of the multispecific molecule, optionally upon binding of several multispecific molecules on the same cell, allows said multispecific molecules to activate TCR signaling and induce T cell activation. In some embodiments, the term “agonistic epitope” refers to an epitope that is solely composed of amino acid residues of the epsilon chain of CD3 and is accessible for binding by the multispecific molecule, when presented in its natural context on T cells (i.e. surrounded by the TCR, the CD3^ chain, etc.). In some embodiments, the term “agonistic epitope” refers to an epitope that, upon binding of the multispecific molecule, does not lead to stabilization of the spatial position of CD3^ relative to sf-5670772Attorney Docket No.: 75004-20023.40 CD3^. In some embodiments, the multispecific anti-PSMA construct further comprises at least one (such as at least about any of 2, 3, 4, 5, or more) additional antigen-binding moieties.

[0188] In some embodiments, the multispecific anti-PSMA construct comprises a) an anti- PSMA antibody moiety (such as described herein) that specifically binds to PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell), and b) a second binding moiety that binds specifically to an antigen on the surface of an effector cell, including for example CD3^, CD3^, CD3^, CD3^, CD28, CD16a, CD56, CD68, and GDS2D. In some embodiments, the second binding moiety is an antibody moiety. In some embodiments, the second antigen- binding moiety is a human, humanized, or semi-synthetic antibody moiety. In some embodiments, the multispecific anti-PSMA construct further comprises at least one (such as at least about any of 2, 3, 4, 5, or more) additional antigen-binding moieties.

[0189] In some embodiments, the multispecific anti-PSMA construct comprises a) an anti- PSMA antibody moiety (such as described herein) that specifically binds to PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell), and b) a second binding moiety that binds specifically to a component of the complement system, such as C1q. C1q is a subunit of the C1 enzyme complex that activates the serum complement system. In some embodiments, the second binding moiety is an antibody moiety. In some embodiments, the second antigen- binding moiety is a human, humanized, or semi-synthetic antibody moiety. In some embodiments, the multispecific anti-PSMA construct further comprises at least one (such as at least about any of 2, 3, 4, 5, or more) additional antigen-binding moieties.

[0190] In some embodiments, the multispecific anti-PSMA construct comprises a) an anti- PSMA antibody moiety (such as described herein) that specifically binds to PSMA (e.g., PSMA expressed on the surface of a cell, such as a cancer cell), and b) a second binding moiety that specifically binds to an Fc receptor, e.g., an Fc^ receptor (Fc^R). The Fc^R may be an Fc^RIII present on the surface of natural killer (NK) cells or one of Fc^RI, Fc^RIIA, Fc^RIIBI, Fc^RIIB2, and Fc^RIIIB present on the surface of macrophages, monocytes, neutrophils and / or dendritic cells. In some embodiments, the second binding moiety is an antibody moiety. In some embodiments, the second antigen-binding moiety is a human, humanized, or semi- synthetic antibody moiety. In some embodiments, the second binding moiety that is an Fc region or functional fragment thereof. In some embodiments, “functional fragment” refers to a fragment of an antibody Fc region that is still capable of binding to an FcR, in particular to an Fc^R, with sufficient specificity and affinity to allow an Fc^R bearing effector cell, in particular a macrophage, a monocyte, a neutrophil and / or a dendritic cell, to kill the target cell by cytotoxic 66 sf-5670772Attorney Docket No.: 75004-20023.40 lysis or phagocytosis. A functional Fc fragment is capable of competitively inhibiting the binding of the original, full-length Fc portion to an FcR such as Fc^RI, Fc^RIIA, Fc^RIIBI, Fc^RIIB2, or Fc^RIIIB. In some embodiments, a functional Fc fragment retains at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of its affinity to an Fc^R, such as an activating Fc^R. In some embodiments, the Fc region or functional fragment thereof is an enhanced Fc region or functional fragment thereof. As used herein, “enhanced Fc region” refers to an Fc region that is modified to enhance Fc receptor-mediated effector-functions, in particular antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody- mediated phagocytosis. For example, an Fc region can be altered in a way that leads to an increased affinity for an activating receptor (e.g. Fc^RIIIA (CD16A) expressed on natural killer (NK) cells) and / or a decreased binding to an inhibitory receptor (e.g. Fc^RIIB1 / B2 (CD32B)). In some embodiments, the second antigen-binding moiety is an antibody or antigen-binding fragment thereof that specifically binds to an FcR, in particular to an Fc^R, with sufficient specificity and affinity to allow an Fc^R bearing effector cell, in particular a macrophage, a monocyte, a neutrophil and / or a dendritic cell, to kill the target cell by cytotoxic lysis or phagocytosis. In some embodiments, the multispecific anti-PSMA construct further comprises at least one (such as at least about any of 2, 3, 4, 5, or more) additional antigen-binding moieties.

[0191] In some embodiments, the multispecific anti-PSMA construct allows killing of target cells (such as cancer cells) expressing PSMA on their surfaces. In some embodiments, the multispecific anti-PSMA construct effectively redirects cytotoxic T lymphocytes (CTLs) to lyse target cells (such as cancer cells) expressing (such as overexpressing) PSMA on their surfaces. In some embodiments, the multispecific (e.g., bispecific) anti-PSMA construct has an in vitro EC50value ranging from 10 to 500 ng / ml. In some embodiments, the multispecific (e.g., bispecific) anti-PSMA construct capable of inducing redirected lysis of about 50% of the target cells through CTLs at a ratio of CTLs:target cells of from about 1:1 to about 50:1 (such as from about 1:1 to about 15:1, or from about 2:1 to about 10:1).

[0192] In some embodiments, the multispecific (e.g., bispecific) anti-PSMA construct is capable of cross-linking a stimulated or unstimulated CTL and the target cell (such as a cancer cell) in such a way that the target cell is lysed. This offers the advantage that no generation of target-specific T cell clones or common antigen presentation by dendritic cells is required for the multispecific anti-PSMA construct to exert its desired activity. In some embodiments, a multispecific anti-PSMA construct provided herein is capable of redirecting CTLs to lyse the target cells (such as cancer cells) in the absence of other activating signals. In some 67 sf-5670772Attorney Docket No.: 75004-20023.40 embodiments, the second antigen-binding moiety of the multispecific anti-PSMA construct specifically binds to CD3 (e.g., CD3^), and signaling through CD28 and / or IL-2 is not required for redirecting CTLs to lyse the target cells (e.g., cancer cells).

[0193] Methods for measuring the preference of the multispecific anti-PSMA construct to simultaneously bind to two antigens (e.g., two different antigens on two different cells or, alternatively two different antigens of the same cell) are within the capabilities of a person of ordinary skill in the art. For example, when the second binding moiety of a multispecific anti- PSMA construct specifically binds to CD3, the multispecific anti-PSMA construct may be contacted with a mixture of CD3+ / PSMA- cells and CD3- / PSMA+cells. The number of single cells bound by the multispecific anti-PSMA constructs and the number of cross-linked cells bound by the multispecific anti-PSMA constructs may then be assessed by fluorescence microscopy, fluorescence-activated cell sorting (FACS), and / or other methods known in the art.

[0194] In some embodiments, the multispecific anti-PSMA construct is, for example, a bispecific antibody, a diabody (Db), a single-chain diabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a di-diabody, a tandem scFv, a tandem di-scFv, a tandem tri-scFv, a tri(a)body, a bispecific Fab2, a di-miniantibody, a tetrabody, an scFv-Fc-scFv fusion, a dual-affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, an IgG-scFab, an scFab-ds-scFv, an Fv2-Fc, an IgG-scFv fusion, a dock and lock (DNL) antibody, a knob-into-hole (KiH) antibody (bispecific IgG prepared by the KiH technology), a DuoBody (bispecific IgG prepared by the Duobody technology), a heteromultimeric antibody, or a heteroconjugate antibody. In some embodiments, the multispecific anti-PSMA molecule is a tandem scFv (e.g., a tandem di-scFv). It is to be appreciated that one of ordinary skill in the art could select appropriate features of various multispecific constructs known in the art and combine them with one another to form a further multispecific anti-PSMA construct within the scope of this disclosure.

[0195] Suitable methods for making multispecific constructs (e.g., bispecific antibodies) are well known in the art. For example, the production of bispecific antibodies can based on the co- expression of two immunoglobulin heavy-chain / light-chain pairs, where the two pairs each have different specificities, and upon association result in a heterodimeric antibody (see, e.g., Milstein and Cuello, Nature, 305: 537-539 (1983); WO 93 / 08829, and Traunecker et al., EMBO J.10: 3655 (1991)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is sf-5670772Attorney Docket No.: 75004-20023.40 usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93 / 08829 and in Traunecker et al., EMBO, 10: 3655-3659 (1991). Alternatively, the combining of heavy and light chains can be directed by taking advantage of species-restricted pairing (see, e.g., Lindhofer et al., J. Immunol., 155:219-225 (1995)) and the pairing of heavy chains can be directed by use of “knob-into hole” engineering of CH3 domains (see, e.g., U.S. Pat. No. 5,731,168; Ridgway et al., Protein Eng., 9(7):617-621 (1996)). Multispecific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009 / 089004A1). In yet another method, stable bispecific antibodies can be generated by controlled Fab-arm exchange, where two parental antibodies having distinct antigen specificity and matched point mutations in the CH3 domains are mixed in reducing condition to allow for separation, reassembly, and reoxidation to form highly pure bispecific antibodies. Labrigin et al., Proc. Natl. Acad. Sci., 110(13):5145-5150 (2013). Such antibodies, comprising a mixture of heavy-chain / light-chain pairs, are also referred to herein as “heteromultimeric antibodies.”

[0196] Antibodies or antigen-binding fragments thereof having different specificities can also be chemically cross-linked to generate multispecific heteroconjugate antibodies. For example, two F(ab’)2 molecules, each having specificity for a different antigen, can be chemically linked. Pullarkat et al., Trends Biotechnol., 48:9-21 (1999). Such antibodies have, for example, been proposed to target immune-system cells to unwanted cells (U.S. Patent No.4,676,980), and for treatment of HIV infection. WO 91 / 00360; WO 92 / 200373; EP 03089. It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.4,676,980.

[0197] In some embodiments, multispecific anti-PSMA constructs can be prepared using recombinant DNA techniques. For example, a bispecific antibody can be engineered by fusing two scFvs, such as by fusing them through a peptide linker, resulting in a tandem scFv (such as a tandem di-scFv). The terms “anti-PSMA tandem di-scFv” and “bispecific anti-PSMA antibody” are used interchangeably herein. In some embodiments, the tandem scFv comprises an anti-CD3 scFv to an scFv comprising an anti-PSMA binding moiety described herein, resulting in the redirection of T cells to target cells that express (such as overexpress) PSMA. Additional details regarding the construction and expression of tandem scFvs are provided in, e.g., Mack et al., 69 sf-5670772Attorney Docket No.: 75004-20023.40 Proc. Natl. Acad. Sci., 92:7021-7025 (1995); Brischwein et al., Mol. Immunol., 43(8):1129-1143 (2006). Additional details regarding tandem scFvs of the present disclosure are provided elsewhere herein.

[0198] By shortening the length of a peptide linker between two variable domains, the variable domains can be prevented from self-assembling and forced to pair with domains on a second polypeptide, resulting in a compact bispecific antibody called a diabody (Db). Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448 (1993). The two polypeptides of a Db each comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VHand VLdomains of one polypeptide are forced to pair with the complementary VL and VH domains of another polypeptide, thereby forming two antigen- binding sites. In a modification of this format, the two polypeptides are linked by another peptide linker, resulting in a single chain diabody (scDb). In yet another modification of the Db format, dual-affinity retargeting (DART) bispecific antibodies can be generated by introducing a disulfide linkage between cysteine residues at the C-terminus of each polypeptide, optionally including domains prior to the C-terminal cysteine residues that drive assembly of the desired heterodimeric structure. Veri et al., Arthritis Rheum., 62(7):1933-1943 (2010). Dual-variable- domain immunoglobulins (DVD-Ig™), in which the target-binding variable domains of two monoclonal antibodies are combined via naturally occurring linkers to yield a tetravalent, bispecific antibody, are also known in the art. Gu and Ghayur, Methods Enzymol., 502:25-41 (2012). In yet another format, Dock and Lock (DNL), bispecific antibodies are prepared by taking advantage of the dimerization of a peptide (DDD2) derived from the regulatory subunit of human cAMP-dependent protein kinase (PKA) with a peptide (AD2) derived from the anchoring domains of human A kinase anchor proteins (AKAPs). Rossi et al., Proc. Natl. Acad. Sci., 103:6841-6846 (2006).

[0199] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). This method can also be utilized for the production of antibody homodimers. F. Tandem scFv Constructs

[0200] In some embodiments, the multispecific anti-PSMA construct is a tandem scFv construct (“anti-PSMA tandem scFv”) comprising a first scFv that comprises an anti-PSMA antibody moiety (such as described herein) and a second scFv that binds to a second target. In sf-5670772Attorney Docket No.: 75004-20023.40 some embodiments, the tandem scFv is a di-scFv (comprising two scFv) or a tandem tri-scFv (comprising three scFv). In some embodiments, the anti-PSMA tandem scFv further comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more scFv. In some embodiments, the second scFv specifically binds to PSMA (such as an epitope that does not overlap the epitope bound by the anti-PSMA antibody moiety of the first scFv). In some embodiments, the second scFv specifically binds to another antigen (i.e., an antigen other than PSMA). In some embodiments, the second scFv specifically binds to an antigen on the surface of a cell, such as a cell that expresses PSMA (e.g., a cancer cell). In some embodiments, the second scFv specifically binds to an antigen on the surface of a cell that does not express PSMA. In some embodiments, the second scFv specifically binds to an antigen on the surface of a cytotoxic cell. In some embodiments, the second scFv specifically binds to an antigen on the surface of a lymphocyte, such as a T cell, an NK cell, a neutrophil, a monocyte, a macrophage, or a dendritic cell. In some embodiments, the second scFv specifically binds to an antigen on the surface of an effector T cell, such as a cytotoxic T cell. In some embodiments, the second scFv specifically binds to an antigen on the surface of an effector cell, including for example CD3^, CD3^, CD3^, CD3^, CD28, CD16a, CD56, CD68, and GDS2D. In some embodiments, the first scFv and / or the second scFv is human, humanized, or semi-synthetic.

[0201] In some embodiments, the anti-PSMA tandem scFv comprises a) a first scFv that comprises an anti-PSMA antibody moiety (such as described herein) that specifically binds to PSMA (e.g., a cell surface-bound PSMA), and b) a second scFv that specifically binds to an antigen on the surface of a T cell. In some embodiments, the second scFv specifically binds to an antigen on the surface of an effector T cell, such as a cytotoxic T cell. In some embodiments, the second scFv specifically binds to, e.g., CD3^, CD3^, CD3^, CD3^, CD28, OX40, GITR, CD137, CD27, CD40L, or HVEM. In some embodiments, the second scFv specifically binds to an agonistic epitope on an antigen on the surface of a T cell, wherein the binding of the second scFv to the agonistic epitope enhances T cell activation. In some embodiments, the first scFv and / or the second scFv is human, humanized, or semi-synthetic.

[0202] In some embodiments, the anti-PSMA tandem scFv comprises a) a first scFv that comprises an anti-PSMA antibody moiety (such as described herein) that specifically binds to PSMA (e.g., a cell surface-bound PSMA), and b) a second scFv that specifically binds to CD3^. In some embodiments, the first scFv is fused to the second scFv through linkage with a peptide linker. In some embodiments, the peptide linker is between about 5 to about 20 (such as about any of 5, 10, 15, or 20, including any ranges between these values) amino acids in length. In 71 sf-5670772Attorney Docket No.: 75004-20023.40 some embodiments, the peptide linker comprises (and in some embodiments consists of) the amino acid sequence of any one of SEQ ID NOs: 45-47. In some embodiments, the first scFv is human, humanized, or semi-synthetic. In some embodiments, the second scFv is human, humanized, or semi-synthetic. In some embodiments, both the first scFv and the second scFv are human, humanized, or semi-synthetic.

[0203] In some embodiments, the peptide linker is between about 5 to about 20 amino acids in length (such as about any of 5, 10, 15, or 20, including any ranges between these values). In some embodiments, the peptide linker comprises (such as consists of or consists essentially of) the amino acid sequence SRGGGGSGGGGSGGGGSLEMA (SEQ ID NO: 45), although alternative linkers may be used, such as SSGGGGSGGGGSGGGGS (SEQ ID NO: 46) or SRGGGGSGGGGSGGGGS (SEQ ID NO: 47). In some embodiments, the first scFv and / or the second scFv is human, humanized, or semi-synthetic.

[0204] In some embodiments, the PSMA-binding scFv of an anti-PSMA tandem scFv provided herein binds to PSMA (e.g., a cell surface-bound PSMA) with a Kdbetween about 0.1 pM to about 500 nM (such as about any one of 0.1 pM, 2.5 pM, 1.0 pM, 5 pM, 10 pM, 25 pM, 50 pM, 75 pM, 100 pM, 250 pM, 500 pM, 750 pM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 250 nM, or 500 nM, including any ranges between these values). In some embodiments, the PSMA-binding scFv of an anti-PSMA tandem scFv provided herein binds to PSMA (e.g., a cell surface-bound PSMA) with a Kd between about 1 nM to about 500 nM (such as about any of 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nM, including any ranges between these values). G. Anti-PSMA Chimeric Antigen Receptor (CAR) Constructs (Anti-PSMA CARs)

[0205] A chimeric antigen receptor (CAR) refers to a type of artificial immune cell receptor that is engineered to recognize and bind to a surface antigen. A T cell that expresses a CAR polypeptide is referred to as a CAR T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner. The non-MHC- restricted antigen recognition gives CAR T cells the ability to recognize an antigen independent of antigen processing, thereby bypassing a major mechanism of tumor escape. A CAR can also be expressed by other immune effector cells, including but not limited to natural killer CAR (“CAR NK cells”) and directed NK cell killing to cells expressing the target of the CAR.

[0206] There are various generations of CARs, each of which contains different components. First generation CARs join an antibody-derived scFv to the CD3^ intracellular signaling domain sf-5670772Attorney Docket No.: 75004-20023.40 of the T cell receptor through a spacer region (also called a hinge domain) and a transmembrane domain. Second generation CARs incorporate an additional co-stimulatory domain (e.g., CD28, 4-1BB, or ICOS) to supply a co-stimulatory signal. Third generation CARs contain two co- stimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or OX40) fused with the TCR CD3^ chain. Any generation of CAR is within the scope of the present disclosure.

[0207] The hPSMA CARs described herein are fusion proteins comprising an extracellular domain that recognizes hPSMA (e.g., a single chain fragment (scFv) of an antibody or other antibody fragment), a spacer, a transmembrane domain, at least one co-stimulatory domain and an intracellular domain comprising a signaling domain of the T cell receptor (TCR) complex (e.g., CD3^). A CAR is often fused to a signal peptide at the N-terminus for cell surface expression.

[0208] In some embodiments, the anti-PSMA construct provided herein is a CAR (also referred to herein as an “anti-PSMA CAR” or “hPSMA CAR”) comprising an anti-PSMA antibody moiety (such as an anti-PSMA antibody moiety described herein). As described in further detail elsewhere herein, the present disclosure also provides CAR effector cells (e.g., T cells) that comprise, express, or are associated with an anti-PSMA CAR. Such effector cells are also referred to herein as an “anti-PSMA CAR effector cells”, e.g., “anti-PSMA CAR immune cells”, “anti-PSMA CAR T cells”, “hPSMA CAR immune cells” or “hPSMA CAR T cells”).

[0209] In some embodiments, the anti-PSMA antibody moiety is an scFv (such as a multispecific anti-PSMA scFv, e.g., an anti-PSMA tandem di-scFv). In some embodiments, the scFv comprises heavy and light chain variable regions linked by a peptide linker.

[0210] In some embodiments, an anti-PSMA CAR comprises a) an extracellular domain comprising an anti-PSMA antibody moiety (such as described herein) that specifically binds to PSMA (e.g., a cell surface-bound PSMA) and b) an intracellular signaling domain. In some embodiments, the anti-PSMA CAR comprises a transmembrane domain between the extracellular domain and the intracellular domain. In some embodiments, the anti-PSMA CAR further comprises a spacer. A. Antigen Binding Extracellular Domain

[0211] The antigen binding extracellular domain is the region of a CAR polypeptide that is exposed to the extracellular fluid when the CAR is expressed on the cell surface. The antigen binding extracellular domain is specific to a target antigen (e.g., hPSMA). In some examples, the antigen binding domain comprises a scFv, which includes an antibody heavy chain variable 73 sf-5670772Attorney Docket No.: 75004-20023.40 region (VH) and an antibody light chain variable region (VL). The scFv fragment retains the antigen binding specificity of the parent antibody, from which the scFv fragment is derived. The VHand VLdomains can be in either orientation (i.e., VH-VLor VL-VH). In some examples, the VH and VL are linked via a peptide linker, which can include hydrophilic residues with stretches of glycine and serine for flexibility as well as stretches of glutamate and lysine for improved solubility. In some embodiments, the scFv can comprise humanized VH and / or VL domains. In some examples, a signal peptide can be located at the N-terminus to facilitate cell surface expression.

[0212] The hPSMA scFv can comprise a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0213] In some cases the scFv comprises: (a) a VLhaving at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 16 (“ET260-1” VL) and (b) a VH having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 15 (“ET260-1” VH).

[0214] The hPSMA scFv can comprise: (a) a VL having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 16 (“ET260-1” VL), a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 10; and (b) a VHhaving at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 15 (“ET260-1” VH), a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0215] The VL can be preceded by the VH and can be joined by a linker that includes 5-20 amino acids, preferably G and S. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 45-47.

[0216] For example, the scFv can comprise or consist of: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV 74 sf-5670772Attorney Docket No.: 75004-20023.40 PDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGGS GGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMGS SLYASSDVWGQGTLVTVSS (SEQ ID NO: 17; “ET260-1” scFv) or QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGGS GGGGSGGGGEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWM GIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMGSSLYASS DVWGQGTLVTVSS (SEQ ID NO: 49; “ET260-1 with alternative linker” scFv).

[0217] For example, the scFv can comprise or consist of an amino acid sequence having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of any of: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGGS GGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMGS SLYASSDVWGQGTLVTVSS (SEQ ID NO: 17; “ET260-1” scFv) or QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGGS GGGGSGGGGEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWM GIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMGSSLYASS DVWGQGTLVTVSS (SEQ ID NO: 49; “ET260-1 with alternative linker” scFv).

[0218] The hPSMA scFv can comprise a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0219] In some cases the scFv comprises: (a) a VL having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 30 (“AMET260-1-58” VL); and (b) a VH having at least 90%, 95% or 98% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 29 (“AMET260-1-58” VH). sf-5670772Attorney Docket No.: 75004-20023.40

[0220] The hPSMA scFv can comprise: (a) a VLhaving at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 30 (“AMET260-1-58” VL); a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and (b) a VH having at least 90%, 95% or 98% identical to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 29 (“AMET260-1-58” VH), a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0221] For example, the scFv can comprise or consist of: QSVLTQPPSVSGAPGQRVTISCTGSSSNFGAGYDVHWYQQLPGTAPKLLIYGNSNRPSG VPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGG SGGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPG KGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMG SSLYASSDVWGQGTLVTVSS (SEQ ID NO: 31; “AMET260-1-58” scFv) or QSVLTQPPSVSGAPGQRVTISCTGSSSNFGAGYDVHWYQQLPGTAPKLLIYGNSNRPSG VPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGG SGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLE WMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMGSSLY ASSDVWGQGTLVTVSS (SEQ ID NO: 48; “AMET260-1-58 with alternative linker” scFv).

[0222] For example, the scFv can comprise or consist of an amino acid sequence having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of any of: QSVLTQPPSVSGAPGQRVTISCTGSSSNFGAGYDVHWYQQLPGTAPKLLIYGNSNRPSG VPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGG SGGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPG KGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMG SSLYASSDVWGQGTLVTVSS (SEQ ID NO: 31; “AMET260-1-58” scFv); or QSVLTQPPSVSGAPGQRVTISCTGSSSNFGAGYDVHWYQQLPGTAPKLLIYGNSNRPSG VPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGG SGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLE sf-5670772Attorney Docket No.: 75004-20023.40 WMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMGSSLY ASSDVWGQGTLVTVSS (SEQ ID NO: 48; “AMET260-1-58 with alternative linker” scFv).

[0223] The hPSMA scFv can comprise a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0224] In some cases the scFv comprises: (a) a VL having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 21 (“AMET260-1-39” VL); and (b) a VH having at least 90%, 95% or 98% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 20 (“AMET260-1-39” VH).

[0225] The hPSMA scFv can comprise: (a) a VLhaving at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 21 (“AMET260-1-39” VL); a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19; and (b) a VH having at least 90%, 95% or 98% identical to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 20 (“AMET260-1-39” VH), a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0226] For example, the scFv can comprise or consist of: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLRGYVFGTGTKVTVLGSRGGGGS GGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSMKASDTAMYYCARSMG SSLYASSDVWGQGTLVTVSS (SEQ ID NO: 22; “AMET260-1-39” scFv).

[0227] For example, the scFv can comprise or consist of an amino acid sequence having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLRGYVFGTGTKVTVLGSRGGGGS 77 sf-5670772Attorney Docket No.: 75004-20023.40 GGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSMKASDTAMYYCARSMG SSLYASSDVWGQGTLVTVSS (SEQ ID NO: 22; “AMET260-1-39” scFv).

[0228] The hPSMA scFv can comprise a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23.

[0229] In some cases the scFv comprises: (a) a VLhaving at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 26 (“AMET260-1-56” VL); and (b) a VHhaving at least 90%, 95% or 98% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 25 (“AMET260-1-56” VH).

[0230] The hPSMA scFv can comprise: (a) a VLhaving at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 26 (“AMET260-1-56” VL); a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and (b) a VH having at least 90%, 95% or 98% identical to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 25 (“AMET260-1-56” VH), a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23.

[0231] For example, the scFv can comprise or consist of: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGGS GGGGSGGGGSLEMAEVQMVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMDS SLYASSDVWGQGTLVTVSS (SEQ ID NO: 27; “AMET260-1-56” scFv).

[0232] For example, the scFv can comprise or consist of an amino acid sequence having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV 78 sf-5670772Attorney Docket No.: 75004-20023.40 PDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGGS GGGGSGGGGSLEMAEVQMVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMDS SLYASSDVWGQGTLVTVSS (SEQ ID NO: 27; “AMET260-1-56” scFv).

[0233] The hPSMA scFv can comprise a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0234] In some cases the scFv comprises: (a) a VL having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 36 (“AMET260-1-67” VL); and (b) a VHhaving at least 90%, 95% or 98% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 35 (“AMET260-1-67” VH).

[0235] The hPSMA scFv can comprise: (a) a VL having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 36 (“AMET260-1-67” VL); a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and (b) a VH having at least 90%, 95% or 98% identical to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 35 (“AMET260-1-67” VH), a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0236] For example, the scFv can comprise or consist of: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGNSNRPSG VPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGG SGGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPG KGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMG SSLYASSDVWGRGTLVTVSS (SEQ ID NO: 37; “AMET260-1-67” scFv).

[0237] For example, the scFv can comprise or consist of an amino acid sequence having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of: 79 sf-5670772Attorney Docket No.: 75004-20023.40 QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGAAPKLLIYGNSNRPSG VPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGG SGGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPG KGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMG SSLYASSDVWGRGTLVTVSS (SEQ ID NO: 37; “AMET260-1-67” scFv).

[0238] The hPSMA scFv can comprise a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0239] In some cases the scFv comprises: (a) a VLhaving at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 41 (“AMET260-1-69” VL); and (b) a VHhaving at least 90%, 95% or 98% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 40 (“AMET260-1-69” VH).

[0240] The hPSMA scFv can comprise: (a) a VL having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 41 (“AMET260-1-69” VL); a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; and (b) a VH having at least 90%, 95% or 98% identical to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 40 (“AMET260-1-69” VH), a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3.

[0241] For example, the scFv can comprise or consist of: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSGSKSRTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGGS GGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMGS SLYASSDVWGQGTPVTVSS (SEQ ID NO: 42; “AMET260-1-69” scFv). sf-5670772Attorney Docket No.: 75004-20023.40

[0242] For example, the scFv can comprise or consist of an amino acid sequence having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of: QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGV PDRFSGSKSRTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGGS GGGGSGGGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK GLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSMGS SLYASSDVWGQGTPVTVSS (SEQ ID NO: 42; “AMET260-1-69” scFv).

[0243] The hPSMA scFv can comprise a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113.

[0244] In some cases the scFv comprises: (a) a VLhaving at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 110 (“ET260-2” VL); and (b) a VH having at least 90%, 95% or 98% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 109 (“ET260-2” VH).

[0245] The hPSMA scFv can comprise: (a) a VL having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 110 (“ET260-2” VL); a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116; and (b) a VHhaving at least 90%, 95% or 98% identical to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 109 (“ET260-2” VH), a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113.

[0246] For example, the scFv can comprise or consist of: QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLMYSNNQRPSGV PDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKVTVLGSRGGGG SGGGGSGGGGSLEMAEVQLVQSGAEMKKPGESLKISCKGSGYNFASYWVGWVRQMP sf-5670772Attorney Docket No.: 75004-20023.40 GKGLEWMGTIYPDDSDTRYGPAFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARD SYYGIDVWGQGTLVTVSS (SEQ ID NO: 117; “ET260-2” scFv).

[0247] For example, the scFv can comprise or consist of an amino acid sequence having at least 90%, 95%, 98% or 100% sequence identity to or has no more than 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid sequence of: QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLMYSNNQRPSGV PDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKVTVLGSRGGGG SGGGGSGGGGSLEMAEVQLVQSGAEMKKPGESLKISCKGSGYNFASYWVGWVRQMP GKGLEWMGTIYPDDSDTRYGPAFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARD SYYGIDVWGQGTLVTVSS (SEQ ID NO: 117; “ET260-2” scFv).

[0248] The VLcan precede the VHand a linker comprising the amino acid sequence of SRGGGGSGGGGSGGGGSLEMA (SEQ ID NO: 45), SSGGGGSGGGGSGGGGS (SEQ ID NO: 46) or SRGGGGSGGGGSGGGGS (SEQ ID NO: 47) can be located between the VHdomain and the VLdomain. The VHcan precede the VLand a linker comprising the amino acid sequence of SRGGGGSGGGGSGGGGSLEMA (SEQ ID NO: 45), SSGGGGSGGGGSGGGGS (SEQ ID NO: 46) or SRGGGGSGGGGSGGGGS (SEQ ID NO: 47) can be located between the VH domain and the VL domain.

[0249] An amino acid modification refers to an amino acid substitution, insertion, and / or deletion in a protein or peptide sequence. An “amino acid substitution” or “substitution” refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid. A substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The following are examples of various groupings of amino acids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Amino acids with uncharged polar R groups: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with charged polar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamic acid; 4) Basic amino acids (positively charged at pH 6.0): Lysine, Arginine, Histidine sf-5670772Attorney Docket No.: 75004-20023.40 (at pH 6.0). Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine. B. Transmembrane Domain

[0250] A CAR disclosed herein can contain a transmembrane domain, which can be a hydrophobic alpha helix that spans the membrane. As used herein, a transmembrane domain refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane.

[0251] The transmembrane domain of the anti-PSMA CAR may be derived from a natural source or a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, in some embodiments, the anti- PSMA CAR comprises a transmembrane domain (e.g., at least one transmembrane domain or at least one transmembrane region) derived from, without limitation, the ^, ^, ^, or ^ chain of the T-cell receptor, CD28, CD3^, CD3^, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the anti-PSMA CAR comprises a synthetic transmembrane domain, in which case the transmembrane domain may comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine may be found at each end of a synthetic transmembrane domain. In some embodiments, a short oligo- or polypeptide linker, having a length of, e.g., between about 2 and about 10 amino acids in length (such as about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length) may form the linkage between the transmembrane domain and the intracellular signaling domain of the anti-PSMA CAR. In some embodiments, the linker is a glycine-serine doublet.

[0252] In some embodiments, the anti-PSMA CAR comprises transmembrane domain that naturally is associated with one of the sequences in the anti-PSMA CAR’s intracellular domain. For example, if an anti-PSMA CAR intracellular domain comprises a CD28 co-stimulatory sequence, the transmembrane domain of the anti-PSMA CAR is derived from the CD28 transmembrane domain. In some embodiments, the anti-PSMA CAR comprises a transmembrane domain that has been selected or modified by amino acid substitution to minimize interactions with other members of the receptor complex and / or to avoid binding to the transmembrane domains of the same or different surface membrane proteins.

[0253] The transmembrane domain of a CAR as provided herein can be a CD28TM2 transmembrane domain having the sequence MFWVLVVVGGVLACYSLLVTVAFIIFWV sf-5670772Attorney Docket No.: 75004-20023.40 (SEQ ID NO: 57). Other transmembrane domains can be used including those shown below in Table 6. Table 6: Examples of Transmembrane DomainsC. Spacer Domain

[0254] A CAR described herein can include a spacer located between the PSMA targeting domain (i.e., a hPSMA targeted scFv or variant thereof) and the transmembrane domain. In some embodiments, the spacer connects the extracellular domain and the transmembrane domain of the anti-PSMA CAR. In some embodiments, the spacer connects the intracellular domain and the transmembrane domain of the anti-PSMA CAR. In some embodiments, the spacer domain is any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular domain or the intracellular domain in the polypeptide chain. For example, a spacer domain may comprise up to about 300 amino acids, including for example between about 10 and about 100 amino acids, or between about 25 and about 50 amino acids. Without being bound by theory, the spacer can function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain, or variants thereof. Table 7 below provides various spacers that can be used in the CARs or polypeptides described herein. sf-5670772Attorney Docket No.: 75004-20023.40 Table 7: Examples of Spacer Domains

[0255] Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3 domain (called CH3 or ^CH2) or both a CH3 domain and a CH2 domain. sf-5670772Attorney Docket No.: 75004-20023.40 The immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.

[0256] The hinge / linker region can also comprise an IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO: 66) or ESKYGPPCPPCP (SEQ ID NO: 65). The hinge / linger region can also comprise the sequence ESKYGPPCPPCP (SEQ ID NO: 65) followed by the linker sequence GGGSSGGGSG (SEQ ID NO: 64) followed by IgG4 CH3 (HL-CH3) sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 74). Thus, the entire linker / spacer region can comprise the sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK (SEQ ID NO: 71). In some cases, the spacer has 1, 2, 3, 4, or 5 single amino acid changes (e.g., conservative changes) compared to the amino acid sequence of SEQ ID NO: 71. In some cases, the IgG4 Fc hinge / linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs). D. Intracellular Signaling Domains

[0257] A CAR construct described herein contains one or more intracellular signaling domains (e.g., CD3^, and optionally one or more co-stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell.

[0258] The intracellular signaling domain of the anti-PSMA CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the anti-PSMA CAR is expressed. Effector function of a T cell, for example, may be cytolytic activity or helper activity, including the secretion of cytokines. Thus, in some embodiments, the term “intracellular signaling domain” refers to the portion of an anti-PSMA CAR that transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. In some embodiments, the term “intracellular signaling sequence” refers to any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. sf-5670772Attorney Docket No.: 75004-20023.40

[0259] In some embodiments, the anti-PSMA CAR comprises an intracellular signaling domain that comprises the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement. In some embodiments, the anti-PSMA CAR comprises an intracellular signaling domain that comprises a derivative or variant of the T cell receptor (TCR) and co-receptors, and / or any synthetic sequence that has the same functional capability.

[0260] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary signaling sequences or primary immune cell signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (co-stimulatory signaling sequences).

[0261] Primary signaling sequences, or primary immune cell signaling sequences, regulate primary activation of the TCR complex in a stimulatory way or in an inhibitory way. Primary signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or ITAMs). Thus, in some embodiments, the anti-PSMA CAR comprises one or more ITAMs. In some embodiments, the anti-PSMA CAR comprises a primary immune cell signaling sequence derived from, without limitation, TCR^, FcR^, FcR^, CD3^, CD3^, CD3^, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the anti-PSMA CAR further comprises a co-stimulatory signaling sequence. In some embodiments, the co-stimulatory signaling sequence is a portion of the intracellular domain of a co-stimulatory molecule including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, and the like. In some embodiments, the anti-PSMA CAR comprises more than one co-stimulatory signaling sequence.

[0262] In some embodiments, the anti-PSMA CAR comprises a primary immune cell signaling sequence derived from CD3^. In some embodiments, the anti-PSMA CAR comprises a primary immune cell signaling sequence derived from CD3^ by itself or combined with any other desired intracellular signaling sequence(s) useful in the context of the anti-PSMA CAR provided herein. For example, in some embodiments, the anti-PSMA CAR comprises an intracellular domain that comprises a primary immune cell signaling sequence derived from CD3^ and a co-stimulatory 87 sf-5670772Attorney Docket No.: 75004-20023.40 signaling sequence. In some embodiments, the co-stimulatory signaling sequence is a portion of the intracellular domain of a co-stimulatory molecule including, for example, CD27, CD28, 4- 1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, and the like. In some embodiments, the co-stimulatory signaling sequence is derived from, e.g., CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, and the like. In some embodiments, the anti-PSMA CAR comprises more than one co- stimulatory signaling sequence.

[0263] In some embodiments, the anti-PSMA CAR comprises the intracellular signaling domain that comprises a primary immune cell signaling sequence derived from CD3^ and a co- stimulatory signaling sequence derived from CD28. In some embodiments, the anti-PSMA CAR comprises the intracellular signaling domain that comprises a primary immune cell signaling sequence derived from CD3^ and a co-stimulatory signaling sequence derived from 4- 1BB. In some embodiments, the intracellular signaling domain of the anti-PSMA CAR comprises a primary immune cell signaling sequence derived from CD3^ and co-stimulatory signaling sequences derived from CD28 and 4-1BB.

[0264] In some embodiments, the anti-PSMA CAR comprises a) an extracellular domain comprising an anti-PSMA antibody moiety (such as described herein) that specifically binds to PSMA (e.g., a cell surface-bound PSMA), b) a transmembrane domain, and c) an intracellular signaling domain capable of activating an immune cell. In some embodiments, the intracellular signaling domain comprises a primary immune cell signaling sequence and a co-stimulatory signaling sequence. In some embodiments, the primary immune cell signaling sequence comprises a CD3^ intracellular signaling sequence. In some embodiments, the co-stimulatory signaling sequence comprises a CD28 or 4-1BB intracellular signaling sequence.

[0265] CD3^ is the cytoplasmic signaling domain of the T cell receptor complex. CD3^ contains three ITAMs, which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen. In some cases, CD3^ provides a primary T cell activation signal but not a fully competent activation signal, which requires a co-stimulatory signal.

[0266] Accordingly, in some examples, the CAR polypeptides disclosed herein may further comprise one or more co-stimulatory signaling domains in addition to CD3^. For example, the co-stimulatory domain CD28 and / or 4-1BB can be used to transmit a proliferative / survival signal together with the primary signaling mediated by CD3^. 88 sf-5670772Attorney Docket No.: 75004-20023.40

[0267] The co-stimulatory domain(s) are located between the transmembrane domain and the CD3^ signaling domain. Table 8 includes examples of suitable co-stimulatory domains together with the sequence of the CD3^ signaling domain. Table 8: CD3# Domain and Examples of Co-stimulatory Domains89 sf-5670772Attorney Docket No.: 75004-20023.40

[0268] In some examples, the CD3^ signaling domain comprises an amino acid sequence having at least 90%, at least 95%, at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 75. In such instances, the CD3^ signaling domain has 1, 2, 3, 4, or 5 amino acid changes (preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 75. In other examples, the CD3^ signaling domain is SEQ ID NO: 75.

[0269] In various embodiments: the co-stimulatory domain is selected from the group consisting of: a co-stimulatory domain depicted in Table 8 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4- 1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications is present in the CAR polypeptides described herein.

[0270] In some embodiments, there are two co-stimulatory domains, for example, a CD28 co- stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acid modification are substitutions. In various embodiments, the co-stimulatory domain is amino terminal to the CD3^ signaling domain and a short linker consisting of 2 – 10, e.g., 3 amino acids (e.g., GGG) can be positioned between the co-stimulatory domain and the CD3^ signaling domain.

[0271] The anti-PSMA CAR can comprise or consist of an amino acid sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to any of the following sequences or has 1, 2, 3, 4 or 5 single amino acid substitutions or deletions (preferable deletions are amino terminal or carboxy terminal) compared to and of the following amino acid sequences: SEQ ID NO: 50 (“AMET260-1-39” CAR), SEQ ID NO: 51 (“AMET260-1-56” CAR), SEQ ID NO: 52 (“AMET260-1-58” CAR), SEQ ID NO: 53 (“AMET260-1-67” CAR), SEQ ID NO: 54 (“AMET260-1-69” CAR).

[0272] In some embodiments, the anti-PSMA CAR comprises the amino acid sequence of any one of: SEQ ID NO: 50 (“AMET260-1-39” CAR), SEQ ID NO: 51 (“AMET260-1-56” CAR), sf-5670772Attorney Docket No.: 75004-20023.40 SEQ ID NO: 52 (“AMET260-1-58” CAR), SEQ ID NO: 53 (“AMET260-1-67” CAR), or SEQ ID NO: 54 (“AMET260-1-69” CAR). Preparation of anti-PSMA CAR T cells

[0273] In some cases, the hPSMA CAR (e.g., anti-PSMA CAR) can be produced using a vector in which the CAR open reading frame is followed by a T2A ribosome skip sequence and a truncated EGFR (EGFRt) fragment, which lacks the cytoplasmic signaling tail, or a truncated CD19R (also called CD19t) fragment. In this arrangement, co-expression of EGFRt or CD19t provides an inert, non-immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene-modified cells, as well as efficient cell tracking of the therapeutic T cells in vivo following adoptive transfer. Efficiently controlling proliferation to avoid cytokine storm and off-target toxicity is an important hurdle for the success of T cell immunotherapy. The EGFRt or the CD19t incorporated in the hPSMA CAR lentiviral vector can act as suicide gene to ablate the CAR+ T cells in cases of treatment-related toxicity.

[0274] The CD3^ signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 88) and a truncated EGFR comprising the amino acid sequence of SEQ ID NO: 89 or having at least about 90%, at least about 95%, at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 89. In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to the amino acid sequence of SEQ ID NO: 89.

[0275] Alternatively, the CD3^ signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 88) and a truncated CD19R (also called CD19t) comprising the amino acid sequence of SEQ ID NO: 90 or having at least about 90%, at least about 95%, at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 90.

[0276] Any CAR described herein can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient. The resulting coding region can be inserted into an expression vector and used to transform a suitable expression host cell line. A suitable host cell line includes, for example, a T lymphocyte sf-5670772Attorney Docket No.: 75004-20023.40 (including an autologous T lymphocyte), an NK cell, etc. An expression vector encoding a CAR or polypeptide described herein can be a viral vector. Suitable viral vectors, including lentiviral vectors, are known in the art and can be used in any of the methods described herein. In some aspects, any of the transduced immune cells described herein can be autologous or allogenic. For example, suitable cell populations can include allogenic NK cells, autologous NK cells, allogenic T cells, autologous T cells that harbor a nucleic acid encoding any CAR or polypeptide described herein. Suitable cell populations can also include allogenic NK cells, autogenic NK cells, allogenic T cells, or autogenic T cells expressing any CAR or polypeptide described here.

[0277] Various T cell subsets isolated from the patient can be transduced with a vector for CAR or polypeptide expression. Central memory T cells are one useful T cell subset. Central memory T cells can be isolated from peripheral blood mononuclear cells (PBMC) by selecting for CD45RO+ / CD62L+ cells, using, for example, the CliniMACS® device to immunomagnetically select cells expressing the desired receptors. The cells enriched for central memory T cells can be activated with anti-CD3 / CD28, transduced with, for example, a lentiviral vector that directs the expression of an anti-hPSMA CAR or as well as a non-immunogenic surface marker for in vivo detection, ablation, and potential ex vivo selection. The activated / genetically modified central memory T cells can be expanded in vitro with IL-2 / IL-15 and then cryopreserved. Additional methods of preparing CAR T cells can be found in PCT / US2016 / 043392.

[0278] Methods for preparing useful T cell populations are described in, for example, WO 2017 / 015490 and WO 2018 / 102761. In some cases, it may be useful to use natural killer (NK) cells, e.g., allogenic NK cells derived from peripheral blood or cord blood. In other cases, NK cells can be derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).

[0279] In some embodiments, described herein is a composition comprising the iPSC-derived CAR T cells or CAR NK cells. In some embodiments, a composition comprising iPSC-derived CAR T cells or CAR NK cells has enhanced therapeutic properties. In some embodiments, the iPSC-derived CAR T cells or CAR NK cells demonstrate enhanced functional activity including potent cytokine production, cytotoxicity and cytostatic inhibition of tumor growth, e.g., as activity that reduces the amount of tumor load.

[0280] The CAR can be transiently expressed in a T cell population by an mRNA encoding the CAR. The mRNA can be introduced into the T cells by electroporation (Wiesinger et al.2019 Cancers (Basel) 11:1198). sf-5670772Attorney Docket No.: 75004-20023.40

[0281] In some embodiments, a composition comprising the CAR T cells comprise one or more of helper T cells, cytotoxic T cells, memory T cells, naïve T cells, regulatory T cells, natural killer T cells, or combinations thereof.

[0282] Disclosed herein, amongst other things, are methods of making any CAR T cell described herein by introducing into a T cell a vector comprising a nucleotide sequence encoding the CAR. Disclosed herein, amongst other things, are methods of making a population of T cells and / or NK cells comprising a nucleic acid encoding any CAR or polypeptide described herein. Disclosed herein, amongst other things, are methods of making a population of T cells and / or NK cells expressing any CAR or polypeptide described herein. H. Anti-PSMA Chimeric Antibody-T Cell Receptor (TCR) Constructs (Anti-PSMA caTCRs)

[0283] In some embodiments, the anti-PSMA construct is a chimeric antibody-T cell receptor (TCR) construct (caTCR) comprising an anti-PSMA antibody moiety (such as an anti-PSMA antibody moiety described herein). Such construct is also referred to herein as an “anti-PSMA caTCR.” Exemplary caTCRs are discussed in PCT / US2016 / 058305 (published as WO 2017 / 070608), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the anti-PSMA caTCR specifically bind to PSMA (such as PSMA expressed on the surface of a cell, e.g., a cancer cell) and is capable of recruiting at least one TCR-associated signaling molecule (such as CD3^^, CD3^^, and / or CD3^^).

[0284] As described in further detail below, the present disclosure also provides an effector cell (e.g., T cell) that comprises, expresses, or is associated with an anti-PSMA caTCR. Such effector cells are also referred to herein as an “anti-PSMA caTCR effector cells” e.g., “anti- PSMA caTCR T cells”).

[0285] In some embodiments, the anti-PSMA caTCR comprises a) an antigen-binding module comprising an anti-PSMA antibody moiety (such as described herein) that specifically recognizes PSMA (e.g., cell surface-bound human PSMA (hPSMA)), and b) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) derived from one of the transmembrane domains of a naturally occurring TCR (such as an ^^TCR or a ^^TCR) and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the naturally occurring TCR (such as an ^^TCR or a ^^TCR), wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule (such as CD3^^, CD3^^, and / or CD3^^), and wherein the antibody moiety is sf-5670772Attorney Docket No.: 75004-20023.40 linked to the first and / or second TCRDs. In some embodiments, the first TCR-TM and the second TCR-TM are derived from a ^ / ^ TCR. In some embodiments, the first TCR-TM is derived from a TCR ^ chain and the second TCR-TM is derived from a TCR ^ chain. In some embodiments, the first TCR-TM is derived from a TCR ^ chain and the second TCR-TM is derived from a TCR ^ chain. In some embodiments, the first TCR-TM and the second TCR-TM are derived from an ^ / ^ TCR. In some embodiments, the first TCR-TM is derived from a TCR ^ chain and the second TCR-TM is derived from a TCR ^ chain. In some embodiments, the first TCR-TM is derived from a TCR ^ chain and the second TCR-TM is derived from a TCR ^ chain. In some embodiments, the anti-PSMA caTCR comprises naturally occurring TCR domains. In some embodiments, the anti-PSMA caTCR comprises at least one non-naturally occurring TCR domain. For example, the ^ / ^ TCR, the ^ / ^ TCR, the TCR ^ chain, the TCR ^ chain, the TCR ^ chain, and / or the TCR ^ chain may be naturally occurring or non-naturally occurring. The antigen-binding module of the anti-PSMA caTCR provides the antigen specificity and a TCRM that allows for CD3 recruitment and signaling. In some embodiments, the antigen-binding module is not a naturally occurring T cell receptor antigen-binding moiety. In some embodiments, the antigen-binding module is linked to the N-terminus of a polypeptide chain in the TCRM. In some embodiments, the antigen binding module is an anti-PSMA antibody moiety selected from the group consisting of a Fab, a Fab’, a F(ab’)2, an Fv, or an scFv. The TCRM comprises a transmembrane module derived from the transmembrane domains of one or more TCRs (TCR-TMs), such as an ^ / ^ and / or ^^ TCR, and optionally further comprises one or both of the connecting peptides or fragments thereof of a TCR and / or one or more TCR intracellular domains or fragments thereof. In some embodiments, the TCRM comprises two polypeptide chains, each polypeptide chain comprising, from N-terminus to C-terminus, a connecting peptide, a transmembrane domain, and optionally a TCR intracellular domain. In some embodiments, the TCRM comprises one or more non-naturally occurring TCR domains. For example, in some embodiments, the TCRM comprises one or two non-naturally occurring TCR transmembrane domains. A non-naturally occurring TCR domain may be a corresponding domain of a naturally occurring TCR modified by substitution of one or more amino acids, and / or by replacement of a portion of the corresponding domain with a portion of an analogous domain from another TCR. In some embodiments, the anti-PSMA caTCR comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains together form the antigen-binding module and the TCRM. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and the caTCR is a 94 sf-5670772Attorney Docket No.: 75004-20023.40 multimer, such as a dimer. In some embodiments, the first and second polypeptide chains are covalently linked, such as by a peptide linkage, or by another chemical linkage, such as a disulfide linkage. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked by at least one disulfide bond. In some embodiments, the anti-PSMA caTCR further comprises one or more T cell co-stimulatory signaling sequences. The one or more co- stimulatory signaling sequences can be, individually, all or a portion of the intracellular domain of a co-stimulatory molecule including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, and the like. In some embodiments, the one or more co-stimulatory signaling sequences are between the first TCR-TM and the first TCR intracellular domain and / or between the second TCR-TM and the second TCR intracellular domain. In some embodiments, the one or more co-stimulatory signaling sequences are C- terminal to the first TCRD and / or the second TCRD. In some embodiments, the anti-PSMA caTCR lacks a T cell co-stimulatory signaling sequence. In some embodiments, the caTCR lacks a functional primary immune cell signaling domain of a TCR-associated T cell activation molecule selected from the group consisting of CD3^ (TCR^), FcR^, FcR^, CD3^, CD3^, CD3^, CD5, CD22, CD79a, CD79b, and CD66d. Additionally or alternatively, in some embodiments, the caTCR lacks any primary immune cell signaling sequences. In some embodiments, the anti- PSMA caTCR further comprises a stabilization module comprising a first stabilization domain and a second stabilization domain, wherein the first and second stabilization domains have a binding affinity for each other that stabilizes the anti-PSMA caTCR. In some embodiments, the stabilization module is located between the antigen-binding module and the TCRM. In some embodiments, the anti-PSMA caTCR further comprises a spacer module between any two caTCR modules or domains. In some embodiments, the spacer module comprises one or more peptide linkers connecting two caTCR modules or domains.

[0286] In some embodiments, the anti-PSMA caTCR comprises: a) a first polypeptide chain comprising a first antigen-binding domain comprising VH and CH1 antibody domains and a first T cell receptor domain (TCRD) comprising a first transmembrane domain of a first TCR subunit; and b) a second polypeptide chain comprising a second antigen-binding domain comprising VL and CL antibody domains and a second TCRD comprising a second transmembrane domain of a second TCR subunit, wherein the VH and CH1 domains of the first antigen-binding domain and the VL and CL domains of the second antigen-binding domain form an antigen-binding module that specifically binds to PSMA, and wherein the first TCRD and the 95 sf-5670772Attorney Docket No.: 75004-20023.40 second TCRD form a T cell receptor module (TCRM) that is capable of recruiting at least one TCR-associated signaling module. In some embodiments, the antigen-binding module comprises a disulfide bond between a residue in the CH1 domain and a residue in the CLdomain.

[0287] In some embodiments, the anti-PSMA caTCR comprises: a) a first polypeptide chain comprising a first antigen-binding domain comprising a VH antibody domain and a first TCRD comprising a first transmembrane domain of a first TCR subunit; and b) a second polypeptide chain comprising a second antigen-binding domain comprising a VL antibody domains and a second TCRD comprising a second transmembrane domain of a second TCR subunit, wherein the VHdomain of the first antigen-binding domain and the VLdomain of the second antigen- binding domain form an antigen-binding module that specifically binds to PSMA, wherein the first TCRD and the second TCRD form a T cell receptor module (TCRM) that is capable of recruiting at least one TCR-associated signaling module.

[0288] In some embodiments, the anti-PSMA caTCR comprises a TCRM that comprises a) a first T cell receptor domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and b) a second TCRD comprising a second TCR-TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule. In some embodiments, both of the TCR-TMs are naturally occurring. In some embodiments, at least one of the TCR-TMs is non-naturally occurring. In some embodiments, both of the TCR-TMs are non-naturally occurring. In some embodiments, the first TCR-TM is derived from one of the transmembrane domains of a T cell receptor (such as an ^^ TCR or a ^^ TCR) and the second TCR-TM is derived from the other transmembrane domain of the T cell receptor. In some embodiments, the TCRM allows for enhanced recruitment of the at least one TCR-associated signaling molecule as compared to a TCRM comprising the transmembrane domains of the T cell receptor. Recruitment of TCR-associated signaling molecules can be determined by methods known in the art, such as FACS analysis for TCR-CD3 complex surface expression or co- immunoprecipitation of CD3 subunits with the caTCR.

[0289] In some embodiments, the anti-PSMA caTCR lacks the functional variable domains or functional constant domains of a TCR. In some embodiments, the anti-PSMA caTCR lacks the functional variable domains of a TCR. In some embodiments, the anti-PSMA caTCR lacks the functional constant domains of a TCR. In some embodiments, the anti-PSMA caTCR lacks the functional variable domains and functional constant domains of a TCR. In some embodiments, the anti-PSMA caTCR comprises a portion of the variable domains of a TCR. In some embodiments, the anti-PSMA caTCR comprises a non-functional portion of the variable 96 sf-5670772Attorney Docket No.: 75004-20023.40 domains of a TCR. In some embodiments, the anti-PSMA caTCR lacks the complete variable domains of a TCR. In some embodiments, the anti-PSMA caTCR comprises a portion of the constant domains of a TCR. In some embodiments, the anti-PSMA caTCR comprises a non- functional portion of the constant domains of a TCR. In some embodiments, the anti-PSMA caTCR lacks the complete constant domains of a TCR.

[0290] In some embodiments, the anti-PSMA caTCR does not comprise a functional Ig-like constant domain of a TCR (e.g., the anti-PSMA caTCR may comprise a portion of an Ig-like constant domain of a TCR, so long as it is non-functional). In some embodiments, the anti- PSMA caTCR does not comprise a functional Ig-like constant domain of any of C^, C^, C^, and C^. In some embodiments, the anti-PSMA caTCR does not comprise an Ig-like constant domain of a TCR. In some embodiments, the anti-PSMA caTCR does not comprise an Ig-like constant domain of C^. In some embodiments, the anti-PSMA caTCR does not comprise an Ig-like constant domain of C^. In some embodiments, the anti-PSMA caTCR does not comprise an Ig- like constant domain of C^. In some embodiments, the anti-PSMA caTCR does not comprise an Ig-like constant domain of C^. In some embodiments, the anti-PSMA caTCR does not comprise an Ig-like constant domain of any of C^, C^, C^, and C^.

[0291] In some embodiments, the anti-PSMA caTCR comprises an antigen-binding module that comprises a first antigen-binding domain comprising a VH antibody domain (e.g., a VH antibody domain described herein) and a second antigen-binding domain comprising a VL antibody domain (e.g., a VL antibody domain described herein). In some embodiments, the VH antibody domain and VL antibody domain CDRs are derived from the same anti-PSMA antibody moiety. In some embodiments, some of the VHantibody domain and VLantibody domain CDRs are derived from different anti-PSMA antibody moieties. In some embodiments, the VHantibody domain and / or VLantibody domain are human, humanized, chimeric, semi-synthetic, or fully synthetic.

[0292] In some embodiments, the anti-PSMA caTCR comprises an antigen-binding module described herein linked to a TCRM described herein, optionally including a stabilization module. For example, in some embodiments, the anti-PSMA caTCR comprises the antigen- binding module linked to the N-terminus of one or both of the TCRDs. In some embodiments, the anti-PSMA caTCR comprises a stabilization module between a TCRM and an antigen- binding module. In some embodiments, the anti-PSMA caTCR further comprises a spacer module between any two anti-PSMA caTCR modules or domains. In some embodiments, the spacer module comprises one or more peptide linkers between about 5 to about 70 (such as 97 sf-5670772Attorney Docket No.: 75004-20023.40 about any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any ranges between these values) amino acids in length. In some embodiments, the anti-PSMA caTCR further comprises one or more accessory intracellular domains. In some embodiments, the one or more accessory intracellular domains are carboxy-terminal to the first and / or second TCRD. In some embodiments, the one or more accessory intracellular domains are between the first TCR-TM and the first TCR intracellular domain and / or between the second TCR-TM and the second TCR intracellular domain. In some embodiments, the one or more accessory intracellular domains comprise, individually, a TCR co-stimulatory domain. In some embodiments, the TCR co- stimulatory domain comprises all or a portion of the intracellular domain of an immune co- stimulatory molecule (such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, and the like).

[0293] In some embodiments, the anti-PSMA caTCR comprises a) an antigen-binding module comprising an antibody moiety (such as described herein) that recognizes a cell surface-bound PSMA, and b) a T cell receptor module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) derived from one of the transmembrane domains of a naturally occurring TCR (such as an ^^TCR or a ^^TCR) and a second TCRD comprising a second TCR-TM derived from the other transmembrane domain of the naturally occurring TCR (such as an ^^TCR or a ^^TCR), wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule (such as CD3^^, CD3^^, and / or CD3^^), and wherein the antibody moiety is linked to the first and / or second TCRDs.

[0294] In some embodiments, the anti-PSMA caTCR comprises: a) a first polypeptide chain comprising a first antigen-binding domain comprising VHand CH1 antibody domains and a first T cell receptor domain (TCRD) comprising a first transmembrane domain of a first TCR subunit; and b) a second polypeptide chain comprising a second antigen-binding domain comprising VLand CLantibody domains and a second TCRD comprising a second transmembrane domain of a second TCR subunit, wherein the VH and CH1 domains of the first antigen-binding domain and the VL and CL domains of the second antigen-binding domain form an antigen-binding module that specifically binds to PSMA (e.g., cell surface bound-PSMA), and wherein the first TCRD and the second TCRD form a T cell receptor module (TCRM) that is capable of recruiting at least one TCR-associated signaling module. In some embodiments, the anti-PSMA caTCR comprises an antigen-binding module that comprises a disulfide bond between a residue in the CH1 domain and a residue in the CL domain. 98 sf-5670772Attorney Docket No.: 75004-20023.40

[0295] In some embodiments, the anti-PSMA caTCR comprises: a) a first polypeptide chain comprising a first antigen-binding domain comprising a VHantibody domain and a first TCRD comprising a first transmembrane domain of a first TCR subunit; and b) a second polypeptide chain comprising a second antigen-binding domain comprising a VL antibody domain and a second TCRD comprising a second transmembrane domain of a second TCR subunit, wherein the VH domain of the first antigen-binding domain and the VL domain of the second antigen- binding domain form an antigen-binding module that specifically binds to PSMA (e.g., cell surface bound-PSMA), wherein the first TCRD and the second TCRD form a T cell receptor module (TCRM) that is capable of recruiting at least one TCR-associated signaling module. I. Anti-PSMA Chimeric Stimulatory Receptor (CSR) Constructs (Anti-PSMA CSRs)

[0296] Also provided herein are PSMA-specific chimeric stimulatory receptor constructs, which are alternatively referred to herein as chimeric signaling receptor constructs (i.e., “anti- PSMA CSRs”). Exemplary CSRs are discussed in PCT / US2018 / 029218 (now published as WO 2018 / 200583), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the anti-PSMA CSR is expressed on the surface of an immune cell (such as a T cell). The anti-PSMA CSR binds to PSMA expressed on or associated with the surface of a cell (such as a cancer cell) and, upon binding to PSMA, is capable of stimulating the immune cell on which the anti-PSMA CSR is expressed. An anti-PSMA CSR comprises a PSMA- binding module (e.g. a ligand-binding module comprising an anti-PSMA antibody moiety described herein), a transmembrane (TM) module, and a co-stimulatory immune cell signaling module that allows for stimulating the immune cell in or on which the anti-PSMA CSR is expressed. In some embodiments, the anti-PSMA CSR lacks a functional primary immune cell signaling sequence. In some embodiments, the anti-PSMA CSR lacks a primary immune cell signaling sequence. In some embodiments, the anti-PSMA CSR comprises a single polypeptide chain comprising the PSMA-binding module (e.g. ligand-binding module comprising an anti- PSMA antibody moiety described herein), TM module, and co-stimulatory immune cell signaling module. In some embodiments, the anti-PSMA CSR comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains together form the PSMA-binding module (e.g. ligand-binding module comprising an anti-PSMA antibody moiety described herein), the transmembrane module, and the co-stimulatory signaling module. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and the anti-PSMA CSR is a multimer, such as a dimer. In some embodiments, the first sf-5670772Attorney Docket No.: 75004-20023.40 and second polypeptide chains are covalently linked, such as by a peptide linkage, or by another chemical linkage, such as a disulfide linkage. In some embodiments, the first polypeptide chain and the second polypeptide chain are linked by at least one disulfide bond.

[0297] Also provided are effector cells (such as T cells) expressing an anti-PSMA CSR of the present disclosure. Such effector cells (such as T cells) are produced by introducing (e.g., transducing or transfecting) a nucleic acid encoding an anti-PSMA CSR described herein (or a vector comprising such a nucleic acid) into the effector cell (e.g., T cell).

[0298] Examples of co-stimulatory immune cell signaling domains for use in an anti-PSMA CSR include, but are not limited to, the cytoplasmic sequences of co-receptors of the T cell receptor (TCR), which can act in concert with a caTCR to initiate signal transduction following caTCR engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. Thus, in some embodiments provided is an effector cell (such as a T cell) that expresses a caTCR and an anti-PSMA CSR. Effector cells (such as T cells) expressing a caTCR and an anti-PSMA CSR (i.e., “caTCR plus anti-PSMA CSR effector cells”) are described in further detail below.

[0299] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular (IC) signaling sequence: those that initiate antigen-dependent primary activation through the TCR (referred to herein as “primary T cell signaling sequences”) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (referred to herein as “co-stimulatory T cell signaling sequences”).

[0300] Primary immune cell signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as ITAMs. Examples of ITAM-containing primary immune cell signaling sequences include those derived from CD3^, TCR^, FcR^, FcR^, CD3^, CD3^, CD3^, CD5, CD22, CD79a, CD79b, and CD66d. A “functional” primary immune cell signaling sequence is a sequence that is capable of transducing an immune cell activation signal when operably coupled to an appropriate receptor. “Non-functional” primary immune cell signaling sequences, which may comprise fragments or variants of primary immune cell signaling sequences, are unable to transduce an immune cell activation signal. Thus, in some embodiments, the anti-PSMA CSRs described herein lack a functional primary immune cell signaling sequence, such as a functional signaling sequence comprising an ITAM. In some sf-5670772Attorney Docket No.: 75004-20023.40 embodiments, the anti-PSMA CSRs described herein lack any primary immune cell signaling sequence.

[0301] In some embodiments, the anti-PSMA CSR comprises a co-stimulatory signaling module that comprises (such as consists of or consists essentially of) all or a portion of the intracellular (IC) domain of an immune cell co-stimulatory molecule including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, and the like. In some embodiments, the CSR comprises a fragment of an immune cell co- stimulatory molecule (fCSM), wherein the fCSM comprises the CSR transmembrane (TM) domain and CSR intracellular (IC) co-stimulatory signaling domain.

[0302] In some embodiments, the PSMA-binding module of an anti-PSMA CSR of the present disclosure is an anti-PSMA antibody moiety. In some embodiments, the anti-PSMA antibody moiety is a Fab, a Fab’, a (Fab’)2, an Fv, or a single chain Fv (scFv). In some embodiments, the anti-PSMA antibody moiety comprises the CDRs or variables domains (VHand / or VLdomains) of an antibody moiety specific for PSMA (e.g., PSMA expressed on or associated with the surface of a cell, e.g., a cancer cell), such as any of anti-PSMA antibody moieties described elsewhere herein.

[0303] In some embodiments, the transmembrane module of an anti-PSMA CSR of the present disclosure comprises one or more transmembrane domains derived from, for example, CD28, CD3^, CD3^, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the CSR comprises a fragment of a transmembrane protein (fTMP), wherein the fTMP comprises the CSR transmembrane domain.

[0304] In some embodiments, the anti-PSMA CSR further comprises a spacer module between any of the PSMA-binding module, the transmembrane module, and the co-stimulatory signaling module. In some embodiments, the spacer module comprises one or more peptide linkers connecting two CSR modules. In some embodiments, the spacer module comprises one or more peptide linkers between about 5 to about 70 (such as about any of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70, including any ranges between these values) amino acids in length.

[0305] In some embodiments, the CSR is a bivalent anti-PSMA CSR that comprises two anti- PSMA antibody moieties. In some embodiments, the bivalent anti-PSMA CSR is homobivalent, e.g., comprising two anti-PSMA antibody moieties that comprise identical VH sequences and identical VL sequences. In some embodiments, the bivalent anti-PSMA CSR is heterobivalent, sf-5670772Attorney Docket No.: 75004-20023.40 e.g., comprising two different anti-PSMA antibody moieties, where each anti-PSMA antibody moiety comprises a different VHsequence and / or different VLsequence.

[0306] In some embodiments, the anti-PSMA moiety further comprises a N-terminal signal peptide. In some embodiments, the anti-PSMA moiety comprises a peptide linker and / or a peptide tag.

[0307] The present disclosure also provides effector cells (such as T cells) that express a caTCR or a CAR and an anti-PSMA CSR (such as an anti-PSMA CSR described herein). Such effector cells are also referred to herein as “caTCR plus anti-PSMA CSR effector cells.” In some embodiments, the caTCR plus anti-PSMA CSR effector cell (such as a T cell) comprises a nucleic acid sequence encoding the anti-PSMA CSR operably linked to an inducible promoter, including any of the inducible promoters described herein. In some embodiments, the expression of the anti-PSMA CSR in the caTCR plus anti-PSMA CSR effector cell (such as a T cell) is inducible upon signaling through the caTCR. In some such embodiments, the caTCR plus anti-PSMA CSR effector cell (such as a T cell) comprises a nucleic acid sequence encoding the anti-PSMA CSR operably linked to a promoter or regulatory element that is responsive to signaling through the caTCR. In some embodiments, the nucleic acid sequence encoding the anti-PSMA CSR is operably linked to a nuclear-factor of the activated T-cell (NFAT)-derived promoter. In some embodiments, the NFAT-derived promoter is an NFAT-derived minimal promoter (see for example Durand, D. et al., Molec. Cell. Biol.8, 1715-1724 (1988); Clipstone, NA, Crabtree, GR. Nature.1992357(6380): 695-7; Chmielewski, M., et al. Cancer research 71.17 (2011): 5697-5706; and Zhang, L., et al. Molecular therapy 19.4 (2011): 751-759). In some embodiments, the caTCR expressed by the caTCR plus anti-PSMA CSR effector cell (such as a T cell) is an anti-PSMA caTCR. In some embodiments the caTCR expressed by the caTCR plus anti-PSMA CSR effector cell (such as a T cell) is not an anti-PSMA caTCR and targets a different antigen. Further description of CSRs may be found in US Application No. 62 / 490,578, filed April 26, 2017, which is incorporated by reference herein in its entirety. J. Anti-PSMA Construct Combinations

[0308] Also provided are construct combinations that comprise at least two different anti- PSMA constructs described herein. In some embodiments, the at least two different anti-PSMA constructs are the same format, e.g., at least two different antibodies (e.g., two different full- length IgG antibodies or two different bispecific antibodies), at least two different CARs, at least two different caTCRs, or at least two different CSRs. In some embodiments, the at least two sf-5670772Attorney Docket No.: 75004-20023.40 different anti-PSMA constructs are different formats, e.g., an antibody and a CAR; an antibody and a caTCR; a CAR and a CSR; a caTCR and a CSR, etc.

[0309] In some embodiments, the construct combination comprises an anti-PSMA caTCR and an anti-PSMA CSR (i.e., an “anti-PSMA caTCR + anti-PSMA CSR construct combination”).

[0310] In some embodiments, the anti-PSMA caTCR and the anti-PSMA CSR of a construct combination provided herein are encoded on separate nucleic acids. In some embodiments, the separate nucleic acids are each expressed (e.g., separately) and translated (e.g., separately) in a cell (such as an anti-PSMA effector cell, which is described in further detail elsewhere herein). In some embodiments, the anti-PSMA caTCR and the anti-PSMA CSR of a construct combination provided herein are encoded on the same nucleic acid (e.g., a single nucleic acid). In some embodiments, the single nucleic acid encoding the anti-PSMA caTCR and anti-PSMA CSR construct combination is expressed and translated to generate a single polypeptide which is subsequently processed (e.g., such as cleaved during or following translation) into separate polypeptides, e.g., the anti-PSMA caTCR polypeptide(s) and anti-PSMA CSR polypeptide.

[0311] In some embodiments, a single nucleic acid encoding an anti-PSMA caTCR + anti- PSMA CSR construct combination expresses a polypeptide comprising (from N-terminus to C- terminus) the amino acid sequence(s) of an anti-PSMA caTCR construct, a peptide linker, and the amino acid sequence of an anti-PSMA CSR construct. In some embodiments, a single nucleic acid encoding anti-PSMA caTCR + anti-PSMA CSR construct combination expresses a polypeptide comprising (from N-terminus to C-terminus) the amino acid sequence of an anti- PSMA CSR construct, a peptide linker, and the amino acid sequence(s) of an anti-PSMA caTCR construct. In some embodiments, the nucleic acid further encodes, e.g., one or more peptide linkers, peptide spacers, peptide tags, signal peptides and / or other amino acid sequences. K. Anti-PSMA Constructs Comprising Fc Region Variants

[0312] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-PSMA construct provided herein (e.g., a full-length anti-PSMA antibody), thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced antibody dependent cellular cytotoxicity (ADCC) effector function, often related to binding to Fc receptors (FcRs). In some embodiments, the Fc region variant has decreased ADCC effector function. There are many examples of changes or mutations to Fc sequences that can alter effector function. For example, WO 00 / 42072 and Shields et al. J Biol. Chem. sf-5670772Attorney Docket No.: 75004-20023.40 9(2): 6591-6604 (2001) describe antibody variants with improved or diminished binding to FcRs. The contents of those publications are specifically incorporated herein by reference.

[0313] Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is a mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell (e.g., a cancer cell), whose membrane-surface antigens have been bound by specific antibodies (e.g., an anti-PSMA antibody). The typical ADCC involves activation of NK cells by antibodies. An NK cell expresses CD16 which is an Fc receptor. This receptor recognizes, and binds to, the Fc portion of an antibody bound to the surface of a target cell. The most common Fc receptor on the surface of an NK cell is called CD16 or Fc^RIII. Binding of the Fc receptor to the Fc region of an antibody results in NK cell activation, release of cytolytic granules and consequent target cell apoptosis. The contribution of ADCC to tumor cell killing can be measured with a specific test that uses NK-92 cells that have been transfected with a high-affinity FcR. Results are compared to wild-type NK-92 cells that do not express the FcR.

[0314] In some embodiments, provided is an anti-PSMA construct comprising a variant Fc region that possesses some but not all effector functions, which makes it a desirable candidate for applications in which the half-life of the anti-PSMA construct in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc^R binding (hence likely lacking ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC (i.e., NK cells) express Fc^RIII only, whereas monocytes express Fc^RI, Fc^RII and Fc^RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No.5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No.5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc.); and CytoTox 96™ non-radioactive cytotoxicity assay (Promega). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, 104 sf-5670772Attorney Docket No.: 75004-20023.40 e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0315] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

[0316] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No.6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem.9(2): 6591-6604 (2001)).

[0317] In some embodiments, the anti-PSMA construct (e.g., a full-length anti-PSMA antibody) comprises a variant Fc region comprising one or more amino acid substitutions which improve ADCC. In some embodiments, the variant Fc region comprises one or more amino acid substitutions which improve ADCC, wherein the substitutions are at positions 298, 333, and / or 334 of the variant Fc region (EU numbering of residues). In some embodiments, the anti-PSMA construct (e.g., a full-length anti-PSMA antibody) comprises the following amino acid substitutions in its variant Fc region: S298A, E333A, and K334A.

[0318] In some embodiments, alterations are made in the Fc region of the anti-PSMA construct (e.g., a full-length anti-PSMA antibody) hat result in altered (i.e., either improved or diminished) C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No.6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol.164: 4178-4184 (2000).

[0319] In some embodiments, the anti-PSMA construct (e.g., a full-length anti-PSMA antibody) comprising a variant Fc region comprises one or more amino acid substitutions which increase half-life and / or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-lives and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.). Such antibodies comprise an Fc region with one or more substitutions therein which 105 sf-5670772Attorney Docket No.: 75004-20023.40 improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No.7,371,826).

[0320] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No.5,624,821; and WO 94 / 29351 concerning other examples of Fc region variants.

[0321] Anti-PSMA constructs (such as full-length anti-PSMA antibodies) comprising any of the Fc variants described herein, or combinations thereof, are contemplated. L. Anti-PSMA Constructs Comprising Glycosylation Variants

[0322] In some embodiments, an anti-PSMA construct provided herein is altered to increase or decrease the extent to which the anti-PSMA construct is glycosylated. Addition or deletion of glycosylation sites to an anti-PSMA construct may be conveniently accomplished by altering the amino acid sequence of the anti-PSMA construct or polypeptide portion thereof such that one or more glycosylation sites are created or removed.

[0323] Where the anti-PSMA construct comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an anti-PSMA construct described herein may be made in order to create anti- PSMA construct glycosylation variants with certain improved properties.

[0324] In some embodiments, the anti-PSMA construct (such as a full-length anti-PSMA antibody) comprises an Fc region wherein a carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may improve ADCC function. In some embodiments, the anti-PSMA construct (such as full-length anti-PSMA antibody) has reduced fucose relative to the amount of fucose on the same anti-PSMA construct (e.g., the full-length anti-PSMA antibody) produced in a wild-type CHO cell (e.g., a CHO cell that produces a native glycosylation pattern, such as, a CHO cell containing a native FUT8 gene). In some embodiments, the anti-PSMA construct is one wherein less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon comprise fucose. For example, the amount of sf-5670772Attorney Docket No.: 75004-20023.40 fucose in such an anti-PSMA construct may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. In some embodiments, the anti-PSMA construct is one wherein none of the N-linked glycans thereon comprise fucose, i.e., wherein the anti-PSMA construct is completely without fucose, or has no fucose or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng.87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys.249:533-545 (1986); US Pat Appl No US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as ^-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng.87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0325] In some embodiments, the anti-PSMA construct (such as a full-length anti-PSMA antibody) is a glycosylation variant comprising bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the anti-PSMA construct is bisected by GlcNAc. Such anti-PSMA construct (e.g., a full-length anti-PSMA antibody) glycosylation variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5): 851-861 (2006). In some embodiments, the anti-PSMA construct (such as a full-length anti-PSMA antibody) is a glycosylation variant comprising at least one 107 sf-5670772Attorney Docket No.: 75004-20023.40 galactose residue in the oligosaccharide attached to the Fc region. Such anti-PSMA construct glycosylation variants may have improved CDC function. Such glycosylation variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0326] In some embodiments, the anti-PSMA construct (such as full-length anti-PSMA antibody) glycosylation variant comprises an Fc region capable of binding to an Fc^RIII. In some embodiments, the anti-PSMA construct (such as full-length anti-PSMA antibody) glycosylation variant comprises an Fc region have ADCC activity in the presence of human effector cells or have increased ADCC activity in the presence of human effector cells compared to an anti-PSMA construct (such as a full-length anti-PSMA antibody) comprising a human wild-type IgG1 Fc region. M. Anti-PSMA Constructs Comprising Cysteine Engineered Variants

[0327] In some embodiments, the anti-PSMA construct (such as a full-length anti-PSMA antibody) has been engineered such that one or more amino acid residues are substituted with cysteine residues. In some embodiments, the substituted residues occur at the surface of and / or at solvent-accessible sites of the anti-PSMA construct. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the anti-PSMA construct and may be used to conjugate the anti-PSMA construct to other moieties, such as drug moieties or linker-drug moieties, to create anti-PSMA immunoconjugates (which are described in further detail elsewhere herein). Cysteine engineered anti-PSMA constructs (such as full- length anti-PSMA antibodies) may be generated as described, e.g., in U.S. Pat. No.7,521,541. N. Derivatized Anti-PSMA Constructs

[0328] In some embodiments, the anti-PSMA construct has been further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of an anti-PSMA construct of the present disclosure include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, without limitation, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene sf-5670772Attorney Docket No.: 75004-20023.40 oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to a derivatized anti-PSMA construct may vary, and if more than one polymer is attached, the polymers can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the anti-PSMA construct to be improved, whether the anti-PSMA construct derivative will be used in a therapy under defined conditions, etc.

[0329] In some embodiments, conjugates of an anti-PSMA construct and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the anti-PSMA construct- nonproteinaceous moiety are killed. II. Anti-PSMA Effector Cells

[0330] Provided herein is an effector cell (e.g., an immune cell, such as a T cell, e.g., an ^^ T cell, a ^^ T cell, a cytotoxic T cell, a helper T cell, or a natural killer T cell) that comprises, expresses, or is associated with an anti-PSMA CAR, an anti-PSMA caTCR, an anti-PSMA multispecific construct (e.g., a tandem scFv, such as a tandem di-scFv), an anti-PSMA CSR, or an anti-PSMA construct combination described herein. Such cells are also referred to as “anti- PSMA effector cells.”

[0331] In some embodiments, the anti-PSMA effector cells (also referred to herein as “anti- PSMA immune cells” or “anti-PSMA T cells”) of the present disclosure are able to replicate in vivo, resulting in long-term persistence that can lead to sustained control of a disease associated with PSMA (such as cancer, e.g., prostate cancer (such as hormone-refractory or metastatic prostate cancer), renal cell cancer cell (such as clear cell renal cell cancer), uterine cancer, or liver cancer).

[0332] In some embodiments, the anti-PSMA effector cell (such as a lymphocyte, e.g., a T cell) is an anti-PSMA CAR effector cell that comprises, expresses, or is associated with an anti- PSMA CAR described herein. In some embodiments, the anti-PSMA CAR effector cell further sf-5670772Attorney Docket No.: 75004-20023.40 comprises (such as expresses) a multispecific construct. Such effector cells are referred to herein as “anti-PSMA CAR plus multispecific construct effector cells.” In some embodiments, the expression of the multispecific construct is inducible. In some embodiments, the expression of the multispecific construct is inducible upon signaling by the anti-PSMA CAR. In some embodiments, the multispecific construct is selected from the group consisting of a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual-affinity retargeting (DART) antibody, and a dual variable domain (DVD) antibody. In some embodiments, the multispecific construct is a tandem scFv. Such effector cells are also referred to herein as “anti-PSMA CAR plus tandem scFv effector cells.” In some embodiments the tandem scFv is a tandem di-scFv, e.g., a tandem di-scFv comprising a first scFv and a second scFv, optionally connected by a peptide linker. In some embodiments, the first scFv targets a T cell surface antigen (e.g., CD3 or CD16a), a soluble immunosuppressive agent (e.g., TGF-^ 1 to 4, IL-4, or IL-10), or an immune checkpoint inhibitor. In some embodiments, the second scFv targets a disease-associated antigen. In some embodiments, the disease-associated antigen is an antigen other than PSMA. In some embodiments, the disease-associated antigen is PSMA. In some embodiments, the tandem di- scFv is an anti-PSMA anti-CD3 tandem di-scFv that comprises an antibody moiety (such as described herein) that specifically binds PSMA (such as PSMA expressed on the surface of a cell, e.g., a cancer cell) and a second binding moiety that specifically binds CD3. In some embodiments, the anti-PSMA anti-CD3 tandem di-scFv is encoded by a nucleic acid that is operably linked to an NFAT-derived promoter. In some embodiments, the NFAT-derived promoter is an NFAT-derived minimal promoter. In some embodiments, the anti-PSMA anti- CD3 tandem di-scFv is encoded by a nucleic acid that is operably linked to an IL-2 promoter.

[0333] In some embodiments, the anti-PSMA CAR effector cell further comprises (such as expresses) a CSR (see, e.g., US Application No.62 / 490,578, filed April 26, 2017, which is incorporated by reference herein in its entirety). Such effector cells are referred to as “anti- PSMA CAR plus CSR effector cells.” In some embodiments, the CSR is an anti-PSMA CSR (i.e., a CSR that comprises a PSMA-binding module), e.g., such as described herein. In some embodiments, the CSR binds to a target ligand other than PSMA.

[0334] In some embodiments, the anti-PSMA effector cell (such as a lymphocyte, e.g., a T cell) is an anti-PSMA caTCR effector cell that comprises, expresses, or is associated with an anti- PSMA caTCR described herein. In some embodiments, the anti-PSMA caTCR effector cell comprises (such as expresses) a multispecific construct. Such effector cells are referred to herein as “anti-PSMA caTCR plus multispecific construct effector cells.” In some 110 sf-5670772Attorney Docket No.: 75004-20023.40 embodiments, the expression of the multispecific construct is inducible. In some embodiments, the expression of the multispecific construct is inducible upon signaling by the anti-PSMA caTCR. In some embodiments, the multispecific construct is selected from the group consisting of a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual-affinity retargeting (DART) antibody, and a dual variable domain (DVD) antibody. In some embodiments, the multispecific construct is a tandem scFv. Such effector cells are also referred to herein as “anti- PSMA caTCR plus tandem scFv effector cells.” In some embodiments the tandem scFv is a tandem di-scFv, e.g., a tandem di-scFv comprising a first scFv and a second scFv, optionally connected by a peptide linker. In some embodiments, the first scFv targets a T cell surface antigen (e.g., CD3 or CD16a), a soluble immunosuppressive agent (e.g., TGF-^ 1 to 4, IL-4, or IL-10), or an immune checkpoint inhibitor. In some embodiments, the second scFv targets a disease-associated antigen. In some embodiments, the disease-associated antigen is an antigen other than PSMA. In some embodiments, the disease-associated antigen is PSMA. In some embodiments, the tandem di-scFv is an anti-PSMA anti-CD3 tandem di-scFv that comprises an antibody moiety (such as described herein) that specifically binds PSMA (such as PSMA expressed on the surface of a cell, e.g., a cancer cell) and a second binding moiety that specifically binds CD3. In some embodiments, the anti-PSMA anti-CD3 tandem di-scFv is encoded by a nucleic acid that is operably linked to an NFAT-derived promoter. In some embodiments, the NFAT-derived promoter is an NFAT-derived minimal promoter. In some embodiments, the anti-PSMA anti-CD3 tandem di-scFv is encoded by a nucleic acid that is operably linked to an IL-2 promoter.

[0335] In some embodiments, the anti-PSMA caTCR effector cell comprises (such as expresses) a CSR (see, e.g., US Application No.62 / 490,578, filed April 26, 2017, which is incorporated by reference herein in its entirety). Such effector cells are referred to as “anti- PSMA caTCR plus CSR effector cells.” In some embodiments, the CSR is an anti-PSMA CSR (i.e., a CSR that comprises a PSMA-binding module), e.g., such as described herein. In some embodiments, the CSR binds to a target ligand other than PSMA. In some embodiments, the anti-PSMA caTCR plus CSR effector cell comprises (such as expresses) any of the anti-PSMA caTCR plus anti-PSMA CSR construct combinations described elsewhere herein.

[0336] In some embodiments, the effector cell (such as a lymphocyte, e.g., a T cell) comprises a CAR or a caTCR that does not target PSMA and anti-PSMA multispecific construct (i.e., an anti-PSMA tandem scFv, e.g., an anti-PSMA tandem di-scFv), e.g., such as described herein. In 111 sf-5670772Attorney Docket No.: 75004-20023.40 some embodiments, the effector cell referred to as a “CAR plus anti-PSMA tandem scFv effector cell” or “caTCR plus anti-PSMA tandem scFv effector cell.”

[0337] In some embodiments, the effector cell (such as a lymphocyte, e.g., a T cell) comprises a CAR or a caTCR that does not target PSMA and anti-PSMA CSR (i.e., a CSR that comprises a PSMA-binding module), e.g., such as described herein. In some embodiments, the effector cell referred to as a “CAR plus anti-PSMA CSR effector cell” or “caTCR plus anti-PSMA CSR effector cell.”

[0338] In some embodiments, the anti-PSMA effector cell (such as any of the anti-PSMA effector cells described herein) further co-expresses membrane-bound IL-12 (mbIL-12) (SEQ ID NO: 107 with signal sequence; SEQ ID NO: 108 without signal sequence). In some embodiments, the anti-PSMA effector cell is referred to herein as an “anti-PSMA CAR effector cell + mbIL12”. In some embodiments, the anti-PSMA CAR is any one of the anti-PSMA CARs provided herein. In some embodiments, the mbIL-12 comprises a CD28 transmembrane domain. In some embodiments, the mbIL-12 comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the mbIL-12 comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the anti-PSMA effector cell with mbIL12 co-expression exhibits improved activation, cytokine secretion, expansion, and tumor cell clearance as compared to anti-PSMA effector cell without mbIL12 co-expression.

[0339] Also provided herein are methods of producing the effector cells described herein.

[0340] For example, provided is a method of producing an anti-PSMA CAR effector cell, e.g., an anti-PSMA CAR immune cell or an anti-PSMA CAR T cell that comprises genetically modifying (i.e., transducing or transfecting) a cell (e.g., a T cell, such as an ^^ T cell, a ^^ T cell, a cytotoxic T cell, a helper T cell, or a natural killer T cell) with a nucleic acid, a set of nucleic acids, a vector, or a set of vectors encoding an anti-PSMA CAR.

[0341] In some embodiments, the method comprises genetically modifying an anti-PSMA CAR effector cell with a further nucleic acid, a set of nucleic acids, a vector, or a set of vectors encoding a multispecific construct. In some embodiments, the method of producing an anti- PSMA CAR plus multispecific construct effector cell (such as an “anti-PSMA CAR plus tandem scFv effector cell”) comprises genetically modifying (i.e., transducing or transfecting) a cell (e.g., a T cell, such as an ^^ T cell, a ^^ T cell, a cytotoxic T cell, a helper T cell, or a natural killer T cell) with a nucleic acid, a set of nucleic acids, a vector, or a set of vectors that encode the anti-PSMA caTCR and the multispecific construct. In some embodiments, the expression of the multispecific construct is inducible. In some embodiments, the expression of the sf-5670772Attorney Docket No.: 75004-20023.40 multispecific construct is inducible upon signaling by the anti-PSMA CAR. In some embodiments, the multispecific construct is selected from the group consisting of a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual-affinity retargeting (DART) antibody, and a dual variable domain (DVD) antibody. In some embodiments, the multispecific construct is a tandem scFv. Such effector cells are also referred to herein as “anti-PSMA caTCR plus tandem scFv effector cells.” In some embodiments the tandem scFv is a tandem di-scFv, e.g., a tandem di-scFv comprising a first scFv and a second scFv, optionally connected by a peptide linker. In some embodiments, the first scFv targets a T cell surface antigen (e.g., CD3 or CD16a), a soluble immunosuppressive agent (e.g., TGF-^ 1 to 4, IL-4, or IL-10), or an immune checkpoint inhibitor. In some embodiments, the second scFv targets a disease-associated antigen. In some embodiments, the disease-associated antigen is an antigen other than PSMA. In some embodiments, the disease-associated antigen is PSMA. In some embodiments, the tandem di- scFv is an anti-PSMA anti-CD3 tandem di-scFv that comprises an antibody moiety (such as described herein) that specifically binds PSMA (such as PSMA expressed on the surface of a cell, e.g., a cancer cell) and a second binding moiety that specifically binds CD3. In some embodiments, the anti-PSMA anti-CD3 tandem di-scFv is encoded by a nucleic acid that is operably linked to an NFAT-derived promoter. In some embodiments, the NFAT-derived promoter is an NFAT-derived minimal promoter. In some embodiments, the anti-PSMA anti- CD3 tandem di-scFv is encoded by a nucleic acid that is operably linked to an IL-2 promoter.

[0342] In some embodiments, the method comprises genetically modifying an anti-PSMA CAR effector cell with a further nucleic acid, a set of nucleic acids, a vector, or a set of vectors encoding a CSR that comprises ligand-binding domain that specifically binds to a target ligand and a co-stimulatory signaling domain capable of providing a stimulatory signal to the immune cell. In some embodiments, the method of producing an anti-PSMA CAR plus CSR effector cell comprises genetically modifying (i.e., transducing or transfecting) a cell (e.g., a T cell, such as an ^^ T cell, a ^^ T cell, a cytotoxic T cell, a helper T cell, or a natural killer T cell) with a nucleic acid, a set of nucleic acids, a vector, or a set of vectors that encode the anti-PSMA CAR and the CSR. Further details regarding CSRs are described in US Application No.62 / 490,578, filed April 26, 2017, which is incorporated by reference herein in its entirety. In some embodiments, expression of the CSR is inducible upon signaling through the anti-PSMA CAR. In some embodiments, CSR is an anti-PSMA CSR (i.e., a CSR that comprises a PSMA-binding module), e.g., such as described herein. In some embodiments, the CSR binds to a target ligand other than PSMA. sf-5670772Attorney Docket No.: 75004-20023.40

[0343] Also provided is a method of producing an anti-PSMA caTCR effector cell, e.g., an anti-PSMA caTCR immune cell or an anti-PSMA caTCR T cell that comprises genetically modifying (i.e., transducing or transfecting) a cell (e.g., a T cell, such as an ^^ T cell, a ^^ T cell, a cytotoxic T cell, a helper T cell, or a natural killer T cell) with a nucleic acid, a set of nucleic acids, a vector, or a set of vectors encoding an anti-PSMA caTCR.

[0344] In some embodiments, the method comprises genetically modifying an anti-PSMA caTCR effector cell with a further nucleic acid, a set of nucleic acids, a vector, or a set of vectors encoding a multispecific construct. In some embodiments, the method of producing an anti- PSMA caTCR plus multispecific construct effector cell (such as an “anti-PSMA caTCR plus tandem scFv effector cell”) comprises genetically modifying (i.e., transducing or transfecting) a cell (e.g., a T cell, such as an ^^ T cell, a ^^ T cell, a cytotoxic T cell, a helper T cell, or a natural killer T cell) with a nucleic acid, a set of nucleic acids, a vector, or a set of vectors that encode the anti-PSMA caTCR and the multispecific construct. In some embodiments, the expression of the multispecific construct is inducible. In some embodiments, the expression of the multispecific construct is inducible upon signaling by the anti-PSMA caTCR. In some embodiments, the multispecific construct is selected from the group consisting of a tandem scFv, a diabody (Db), a single chain diabody (scDb), a dual-affinity retargeting (DART) antibody, and a dual variable domain (DVD) antibody. In some embodiments, the multispecific construct is a tandem scFv. Such effector cells are also referred to herein as “anti-PSMA caTCR plus tandem scFv effector cells.” In some embodiments the tandem scFv is a tandem di-scFv, e.g., a tandem di-scFv comprising a first scFv and a second scFv, optionally connected by a peptide...

Claims

Attorney Docket No.: 75004-20023.40 CLAIMS What is claimed is:

1. An anti-prostate specific membrane antigen (PSMA) construct comprising an antibody moiety specifically recognizing PSMA (anti-PSMA antibody moiety), wherein the anti- PSMA antibody moiety comprises: (i) a heavy chain variable domain (VH) comprising a complementarity determining region heavy chain (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR- H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable domain (VL) comprising a complementarity determining region light chain (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; (ii) a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VLcomprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19; (iii) a VHcomprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and a VLcomprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; (iv) a VH comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; and a VL comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10; or (v) a VHcomprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a FWR-H4 that does not comprise the amino acid sequence of SEQ ID NO: 7; and a VL comprising a CDR-L1 comprising the amino acid 202 sf-5670772Attorney Docket No.: 75004-20023.40 sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and a FWR-L3 that does not comprise the amino acid sequence of SEQ ID NO:

13.

2. The anti-PSMA construct of claim 1, wherein the anti-PSMA antibody moiety comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 29, and a VL comprising the amino acid sequence of SEQ ID NO: 30; (ii) a VHcomprising the amino acid sequence of SEQ ID NO: 20, and a VLcomprising the amino acid sequence of SEQ ID NO: 21; (iii) a VHcomprising the amino acid sequence of SEQ ID NO: 35, and a VLcomprising the amino acid sequence of SEQ ID NO: 36; (iv) a VHcomprising the amino acid sequence of SEQ ID NO: 25, and a VLcomprising the amino acid sequence of SEQ ID NO: 26; or (v) a VH comprising the amino acid sequence of SEQ ID NO: 40, and a VL comprising the amino acid sequence of SEQ ID NO:

41.

3. The anti-PSMA construct of claim 1 or 2, wherein the anti-PSMA antibody moiety is a full-length antibody, a Fab, a Fab’, a F(ab’)2, an Fv, or a single chain Fv (scFv). The anti-PSMA construct of any one of claims 1-3, wherein the anti-PSMA construct is an anti-PSMA chimeric antibody-T cell receptor (TCR) construct (caTCR) comprising: (a) an extracellular domain comprising the anti-PSMA antibody moiety; and (b) a TCR module (TCRM) comprising a first TCR domain (TCRD) comprising a first TCR transmembrane domain (TCR-TM) and a second TCRD comprising a second TCR- TM, wherein the TCRM facilitates recruitment of at least one TCR-associated signaling molecule.

5. The anti-PSMA construct of claim 4, wherein: (a) the first TCR-TM and the second TCR-TM are derived from a ^ / ^ TCR or an ^ / ^ TCR; and / or (b) the TCR-associated signaling molecule is selected from the group consisting of CD3^^, CD3^^, and CD3^^. sf-5670772Attorney Docket No.: 75004-20023.40 6. The anti-PSMA construct of any one of claims 1-3, wherein the anti-PSMA construct is an anti-PSMA chimeric signaling receptor (CSR) comprising: i) a ligand-binding module comprising the anti-PSMA antibody moiety; ii) a transmembrane module; and iii) a co-stimulatory immune cell signaling module that is capable of providing a co- stimulatory signal to an effector cell, wherein the CSR lacks a functional primary immune cell signaling domain.

7. The anti-PSMA construct of claim 6, wherein the transmembrane module of the CSR and the co-stimulatory immune cell signaling module of the CSR are derived from the same molecule.

8. The anti-PSMA construct of claim 7, wherein the molecule is selected from the group consisting of CD28, 4-1BB (CD137), OX40, CD30, CD27, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

9. The anti-PSMA construct of any one of claims 1-3, wherein the anti-PSMA construct is an anti-PSMA chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising the anti-PSMA antibody moiety; (b) a transmembrane domain; and (c) an intracellular signaling domain.

10. The anti-PSMA construct of claim 9, wherein: (a) the intracellular signaling domain comprises a primary immune cell signaling sequence derived from CD3^, TCR^, FcR^, FcR^, CD3^, CD3^, CD3^, CD5, CD22, CD79a, CD79b, or CD66d; and / or (b) the intracellular signaling domain further comprise a costimulatory signaling sequence derived from CD30, CD28, 4-1BB, ICOS, or OX40. sf-5670772Attorney Docket No.: 75004-20023.40 11. The anti-PSMA construct of any one of claims 1-3, which is conjugated to an effector molecule, wherein the effector molecule is a detectable label or a therapeutic agent selected from the group consisting of: a drug, a toxin, a radioisotope, a protein, a peptide, and a nucleic acid.

12. An effector cell that has been genetically modified with one or more nucleic acids encoding: (a) the anti-PSMA caTCR of claim 4 or 5; (b) the anti-PSMA scFv of claim 3; (c) the anti-PSMA CSR of any one of claims 6-8; (d) the anti-PSMA CAR of claim 9 or 10; or (e) the anti-PSMA caTCR of claim 4 or 5, wherein: (i) the one or more nucleic acids encoding the anti-PSMA caTCR further encode a CSR comprising a ligand-binding module that binds a target antigen or an scFv that binds a target antigen; or (ii) the effector cell has been genetically modified with one or more additional nucleic acids encoding a CSR comprising a ligand-binding module that binds a target antigen or an scFv that binds a target antigen.

13. A method of producing an effector cell, comprising genetically modifying a cell with one or more nucleic acids encoding: (a) the anti-PSMA caTCR of claim 4 or 5; (b) the anti-PSMA scFv of claim 3; (c) the anti-PSMA CSR of any one of claims 6-8; (d) the anti-PSMA CAR of claim 9 or 10; or (e) the anti-PSMA caTCR of claim 4 or 5, wherein: (i) the one or more nucleic acids encoding the anti-PSMA caTCR further encode a CSR comprising a ligand-binding module that binds a target antigen or an scFv that binds a target antigen; or (ii) the method further comprises genetically modifying the effector cell with one or more additional nucleic acids encoding a CSR comprising a ligand-binding module that binds a target antigen or an scFv that binds a target antigen . The effector cell of claim 12, or the method of claim 13, wherein the effector cell is 205 sf-5670772Attorney Docket No.: 75004-20023.40 an immune cell.

15. The effector cell or the method of claim 14, wherein the immune cell is a T cell. A nucleic acid or a vector encoding the polypeptide portion(s) of the anti-PSMA construct of any one of claims 1-11.

17. A host cell comprising the nucleic acid or the vector of claim 16.

18. A method of producing an anti-PSMA construct, comprising culturing the host cell of claim 17 under conditions where the anti-PSMA construct is expressed, and recovering the anti- PSMA construct produced by the host cell.

19. A pharmaceutical composition comprising the anti-PSMA construct of any one of claims 1-11, the effector cell of claim 12, and / or the nucleic acid or vector of claim 16, and a pharmaceutical acceptable carrier.

20. A method of treating an individual having a PSMA-associated disease or disorder, comprising: (i) administering to the individual an effective amount of the pharmaceutical composition of claim 19; or (ii) administering to the individual the effector cell of claim 12.

21. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising: (i) an scFv that binds human PSMA (hPSMA) and comprises: (a) a complementarity determining region light chain (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a complementarity determining region heavy chain (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; sf-5670772Attorney Docket No.: 75004-20023.40 (b) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (c) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; or (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and a FWR-L3 that does not comprise the amino acid sequence of SEQ ID NO: 13, and a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a FWR-H4 that does not comprise the amino acid sequence of SEQ ID NO: 7; (ii) a spacer domain; (iii) a transmembrane domain; (iv) a costimulatory signaling domain; and (v) a CD3^ signaling domain.

22. The nucleic acid molecule of claim 21, wherein the scFv comprises: (a) a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30; and a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 29; sf-5670772Attorney Docket No.: 75004-20023.40 (b) a VLcomprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26; and a VHcomprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25; (c) a VL comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 21; and a VH comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 20; (d) a VLcomprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 36; and a VHcomprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 35; or (e) a VLcomprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 41; and a VH comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

40.

23. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising: (i) an scFv that binds human PSMA (hPSMA) and comprises: (a) a complementarity determining region light chain (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and a FWR-L3 comprising the amino acid sequence of SEQ ID NO: 13, a complementarity determining region heavy chain (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a FWR-H4 comprising the amino acid sequence of SEQ ID NO: 7; or (b) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 114, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 115, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 116, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 111, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 113; sf-5670772Attorney Docket No.: 75004-20023.40 (ii) a spacer domain; (iii) a transmembrane domain; (iv) a costimulatory signaling domain; and (v) a CD3^ signaling domain; and wherein the nucleic acid molecule further encodes a membrane-bound human IL- 12 (mbIL-12).

24. The nucleic acid molecule of claim 23, wherein the scFv comprises: (a) a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 16, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 16; and a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 15; or (b) a VLcomprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 110; and a VH comprising the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

109.

25. The nucleic acid molecule of claim 21 or 22, wherein: (a) the scFv comprises the amino acid sequence of any of SEQ ID NOs: 31, 48, 22, 27, 37, and 42; (b) the spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-74; (c) the transmembrane domain is selected from the group consisting of CD3^, CD28TM1, CD28TM2, CD4, CD8TM1, CD8TM2, CD8TM3, 4-1BB, and NKG2D; and / or (d) the costimulatory signaling domain is selected from the group consisting of CD28, CD28gg*, 4-1BB, OX40, and 2B4.

26. The nucleic acid molecule of claim 23 or 24, wherein: (a) the scFv comprises the amino acid sequence of any of SEQ ID NOs: 17, 49, and 117; (b) the spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-74; sf-5670772Attorney Docket No.: 75004-20023.40 (c) the transmembrane domain is selected from the group consisting of CD3^, CD28TM, CD28TM2, CD4, CD8TM1, CD8TM2, CD8TM3, 4-1BB, and NKG2D; and / or (d) the costimulatory signaling domain is selected from the group consisting of CD28, CD28gg*, 4-1BB, OX40, and 2B4. The nucleic acid molecule of any one of claims 21, 22, and 25, wherein the CAR comprises the amino acid sequence of any of SEQ ID NOs: 50-54 and 102-106.

28. The nucleic acid molecule of claim 27, wherein the nucleic acid molecule further encodes a membrane-bound human IL-12 (mbIL-12).

29. The nucleic acid molecule of any one of claims 23, 24, and 26, wherein the CAR comprises the amino acid sequence of any of SEQ ID NOs: 92-101.

30. The nucleic acid molecule of claim 28 or 29, wherein the mbIL-12 comprises the amino acid sequence of SEQ ID NO: 107 or 108.

31. An immune cell harboring the nucleic acid molecule of any one of claims 21, 22, 25, and 27, or a vector comprising nucleic acid molecule of any one of claims 21, 22, 25, and 27.

32. The immune cell of claim 31, further harboring a nucleic acid molecule encoding a mbIL-12.

33. An immune cell harboring: (a) a vector comprising the nucleic acid molecule of any one of claims 21, 22, 25, and 27 and a nucleic acid molecule encoding a mbIL-12; or (b) the nucleic acid molecule of any one of claims 23, 24, 26, and 28-30, or a vector comprising the nucleic acid molecule of any one of claims 23, 24, 26, and 28-30.

34. A chimeric antigen receptor (CAR) comprising: (i) an scFv that binds human PSMA (hPSMA) and comprises: (a) a complementarity determining region light chain (CDR-L) 1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-L2 comprising the amino acid 210 sf-5670772Attorney Docket No.: 75004-20023.40 sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a complementarity determining region heavy chain (CDR-H) 1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; (c) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; or (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR- L2 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10, and a FWR-L3 that does not comprise the amino acid sequence of SEQ ID NO: 13, and a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and a FWR-H4 that does not comprise the amino acid sequence of SEQ ID NO: 7; (ii) a spacer domain; (iii) a transmembrane domain; (iv) a costimulatory signaling domain; and (v) a CD3^ signaling domain.

35. The CAR of claim 34, wherein the scFv comprises: 211 sf-5670772Attorney Docket No.: 75004-20023.40 (a) a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 30; and a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 29; (b) a VL comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26; and a VH comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 25; (c) a VL comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 21; and a VHcomprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 20; (d) a VLcomprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 36; and a VH comprising the amino acid sequence of SEQ ID NO: 35, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 35; or (e) a VL comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 41; and a VH comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

40.

36. The CAR of claim 34 or 35, wherein: (a) the scFv comprises the amino acid sequence of any of SEQ ID NOs: 31, 48, 22, 27, 37, and 42; (b) the spacer domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-74; (c) the transmembrane domain is selected from the group consisting of CD3^, CD28TM, CD28TM2, CD4, CD8TM1, CD8TM2, CD8TM3, 4-1BB, and NKG2D; and / or (d) the costimulatory signaling domain is selected from the group consisting of CD28, CD28gg*, 4-1BB, OX40, and 2B4. sf-5670772Attorney Docket No.: 75004-20023.40 37. The CAR of any one of claims 34-36, wherein the CAR comprises the amino acid sequence of any of SEQ ID NOs: 50-54 and 102-106.

38. An immune cell harboring the CAR of any one of claims 34-37.

39. The immune cell of claim 38, further harboring a mbIL-12.

40. The immune cell of any one of claims 31-33, 38, and 39, which is a T cell.

41. A method of treating an individual suffering from a cancer whose cells express PSMA, comprising administering to the individual an effective amount of the immune cell of any one of claims 38-40. sf-5670772

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