Anti-PSMA Antibodies, Variants, and Uses Thereof

Abstract

The present invention relates to improved anti-PSMA antibodies or antigen-binding fragments thereof, related nucleic acids, vectors, cells, compositions, kits, and methods of use thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 325,330, filed March 30, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference This application incorporates by reference a sequence listing submitted in computer readable form as file SeqList_168276.00301, created on Mar. 27, 2023, and containing 81,222 bytes.

[0003] The present invention relates to improved anti-PSMA antibodies and methods of their use. [Background technology]

[0004] Prostate-specific membrane antigen (PSMA) has been established as a specific marker for prostate epithelial cells, which has laid the foundation for the development of precision medicine targeting prostate cancer. Histological studies have shown that almost all prostate cancers express PSMA, and cancers with higher grade or metastasis, or resistance to androgen suppression therapy, usually express much higher PSMA (Ananias, HJ, et al. (2009) Prostate, 69(10): 1101-8; Wright, GL, Jr., et al. (1995) Urol Oncol, 1(1): 18-28; Wright, GL, Jr., et al. (1996) Urology, 48(2): 326-34; Sweat, SD, et al. (1998) Urology, 52(4): 637-40). PSMA expression levels in normal tissues are 100-1000 times lower than in tumors (Sokoloff, RL, et al. (2000) Prostate, 43(2):150-7), and these normal tissues are usually not easily accessible to circulating antibodies (Troyer, JK et al. (1995) Int J Cancer, 62(5):552-8), further confirming the safety of PSMA-targeted imaging and therapy. In addition to the prostate, PSMA is also highly expressed in angiogenesis in many solid tumors, but is absent in normal blood vessels (Sokoloff, RL, et al. (2000) Prostate, 43(2):150-7). Therefore, PSMA is an ideal marker not only for prostate cancer, but also for angiogenesis-targeted therapy for other solid tumors.

[0005] Antibodies are the most efficient tools for tumor targeting, which can specifically recognize tumor-associated or tumor-specific antigens expressed in tumor cells, paving the way for antibody-based precision medicine, including tumor-targeted imaging and therapy, such as optical, PET, SPECT, or MRI imaging for early tumor detection, antibody-drug conjugates, and radiation therapy for cancer treatment, bispecific or multispecific antibodies, chimeric antigen receptor T cell or NK cell therapy.

[0006] However, the stability of protein drugs such as antibodies is adversely affected by many different factors.Protein degradation and the subsequent decline in protein activity are recurring problems in the pharmaceutical industry.Therefore, there is still a need for antibodies as therapeutic agents with improved stability. Summary of the Invention

[0007] The present disclosure addresses the above needs in several aspects. In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof, or a variant thereof, that specifically binds to PSMA, such as human PSMA. In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a light chain variable region (LCVR) comprising three light chain complementarity determining regions (LCDRs) (LCDR1, LCDR2, and LCDR3), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 13 or 41, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 17; and / or (ii) a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions (HCDRs) (HCDR1, HCDR2, and HCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 25 or 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 33 or 49, and the variant comprises one or more conservative amino acid substitutions in LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, or HCDR3.

[0008] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, HCDR1 comprises the amino acid sequence of SEQ ID NO: 25, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 33.

[0009] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 33.

[0010] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 41, LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 33.

[0011] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49.

[0012] In some embodiments, the light chain variable region comprises one or more of the amino acid sequences of SEQ ID NOs: 5, 7, 11, 15, 19, 39, 43, 61, 63, 65, 67, and 69.

[0013] In some embodiments, the heavy chain variable region comprises one or more of the amino acid sequences of SEQ ID NOs: 21, 23, 27, 31, 35, 47, 51, 53, 55, 57, 59, and 68.

[0014] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:5 or comprises the amino acid sequence of SEQ ID NO:5, and / or the heavy chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:21 or comprises the amino acid sequence of SEQ ID NO:21.

[0015] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 70, or comprises the amino acid sequence of SEQ ID NO: 70, and / or the heavy chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 72, or comprises the amino acid sequence of SEQ ID NO: 72.

[0016] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:71, or comprises the amino acid sequence of SEQ ID NO:71, and / or the heavy chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:73, or comprises the amino acid sequence of SEQ ID NO:73.

[0017] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:5 or comprises the amino acid sequence of SEQ ID NO:5, and / or the heavy chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:74 or comprises the amino acid sequence of SEQ ID NO:74.

[0018] In some embodiments, the light chain variable region and the heavy chain variable region comprise the LCVR and HCVR amino acid sequence pairs of SEQ ID NOs:5 and 21, SEQ ID NOs:70 and 72, SEQ ID NOs:71 and 73, or SEQ ID NOs:5 and 74.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof, or variant thereof, comprises the heavy chain / light chain sequence pair of SEQ ID NOs: 75 and 77, SEQ ID NOs: 76 and 78, SEQ ID NOs: 69 and 68, or SEQ ID NOs: 75 and 79.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof, or variant thereof, comprises at least one of the amino acid sequences of SEQ ID NOs: 3 and 37.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof is selected from IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE, or has wild-type or non-fucosylated immunoglobulin constant and / or variable domains of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgA, or IgE.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, a mixture of monoclonal and / or polyclonal antibodies, a human antibody, a humanized antibody, or a chimeric antibody.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof or variant thereof is conjugated to a cytotoxic agent, a drug, a growth inhibitory agent, a toxin, an enzyme, a tag, a label, a radioisotope, or a nanoparticle.

[0024] In another aspect, the present disclosure also provides bispecific or multispecific antibodies or antigen-binding fragments thereof that bind to two or more different epitopes on the same or different antigens. In some embodiments, one of the epitopes is on human prostate-specific membrane antigen (PSMA), and the bispecific or multispecific antibodies comprise a light chain variable region and / or a heavy chain variable region of an antibody or antigen-binding fragment thereof, or a variant thereof, as described herein.

[0025] In another aspect, the present disclosure further provides an isolated chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antibody or antigen-binding fragment thereof or variant thereof described herein, a transmembrane domain, and an intracellular signaling domain comprising one or more stimulatory domains.

[0026] Also within the scope of the disclosure are (a) nucleic acid molecules encoding the polypeptide chains of an antibody or antigen-binding fragment thereof or variants thereof, a bispecific or multispecific antibody or antigen-binding fragment thereof, or a claimed CAR, as described herein, (b) a vector comprising the nucleic acid molecule described herein, and (c) a cell comprising the nucleic acid molecule or vector described herein.

[0027] In another aspect, the disclosure provides a method of preparing an antibody or antigen-binding fragment thereof. In some embodiments, the method includes (i) obtaining a cell as described herein, (ii) culturing the cell in a medium under conditions that allow expression of a polypeptide encoded by the nucleic acid molecule and assembly of the antibody or fragment thereof, and (iii) purifying the antibody or fragment from the cultured cell or the cell medium.

[0028] In another aspect, the disclosure additionally provides a composition comprising an antibody or antigen-binding fragment thereof or variant thereof, a bispecific or multispecific antibody or antigen-binding fragment thereof, a CAR, a nucleic acid molecule, a vector, or a cell described herein, and optionally a pharma- ceutically acceptable carrier.

[0029] In another aspect, the disclosure additionally provides a kit comprising an antibody or antigen-binding fragment thereof or variant thereof, a bispecific or multispecific antibody or antigen-binding fragment thereof, a CAR, a nucleic acid molecule, a vector, a cell, or a composition described herein.

[0030] In another aspect, the present disclosure also provides a use of a composition described herein for the manufacture of a medicament for the diagnosis, prevention, treatment, or a combination thereof, of a condition resulting from a proliferative disease associated with expression of PSMA in a subject.

[0031] In another aspect, the disclosure further provides a method of preventing or treating a proliferative disease associated with expression of PSMA in a subject. In some embodiments, the method comprises administering to the subject an effective amount of an antibody or antigen-binding fragment thereof or variant thereof, a bispecific or multispecific antibody or antigen-binding fragment thereof, a CAR, a nucleic acid molecule, a vector, a cell, or a composition described herein.

[0032] In some embodiments, the method further comprises administering to the subject a second agent or therapy. In some embodiments, the second agent or therapy comprises an anti-cancer agent. In some embodiments, the second agent or therapy is administered to the subject before, after, or simultaneously with the antibody or antigen-binding fragment thereof or variant thereof, bispecific or multispecific antibody or antigen-binding fragment thereof, CAR, nucleic acid molecule, vector, cell, or composition described herein.

[0033] In another aspect, the disclosure also provides a method of identifying a subject having a disease or condition associated with expression of PSMA. In some embodiments, the method includes: (a) providing a tissue sample from the subject; (b) contacting the sample with an antibody or antigen-binding fragment thereof or variant thereof; and (c) determining binding of the antibody or antigen-binding fragment thereof or variant thereof to the tissue sample, wherein binding of the antibody or antigen-binding fragment thereof or variant thereof to the tissue sample indicates the presence of a disease condition associated with expression of PSMA in the subject.

[0034] In yet another aspect, the present disclosure also provides a method of imaging targeting PSMA. The method comprises administering to a subject in need thereof an antibody or antigen-binding fragment thereof or variant thereof or a composition, wherein the antibody or antigen-binding fragment thereof or variant is conjugated to a reagent. In some embodiments, the reagent comprises a photoactivatable agent, a fluorophore, a radioisotope, a bioluminescent protein, a bioluminescent peptide, a fluorescent tag, a fluorescent protein, a fluorescent peptide, an imaging agent, an enzyme, a nuclear magnetic resonance active reagent, or a nanoparticle.

[0035] In some embodiments, the disease comprises cancer, hi some embodiments, the cancer comprises prostate cancer.

[0036] In some embodiments, the subject is a mammal, for example a human.

[0037] The above summary is not intended to define all aspects of the disclosure, and additional aspects are described in other sections, such as the following detailed description. It should be understood that the entire specification is intended to be related as a unified disclosure, and that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of the specification. Other features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while showing specific embodiments of the present disclosure, are given for illustrative purposes only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0038] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0039] [Figure 1] 1 shows the results of an experiment measuring the affinity of PSMAb and PSMAbLm by capture ELISA. The data showed that the affinity of PSMAbLm was approximately 2.3-fold higher than that of PSMAb (Kd=0.0479 nM (PSMAbLm) vs. Kd=0.1088 nM (PSMAb)). [Diagram 2]Figure 1 shows the ADCC activity of PSMAb-WT, PSMAb-AF, and PSMAbLm-AF against target cells. ADCC was investigated using CD16-Jurkat reporter cells with PSMA+CHO cells as targets. Abbreviations: PSMAb-WT: wild-type PSMAb with normal glycosylation, PSMAb-AF: nonfucosylated PSMAb, PSMAbLm-AF: nonfucosylated PSMAb with an N=>S mutation in the light chain CDR3 region. [Figure 3A-3B] 3 shows the binding of PSMAb and PSMAbLm to LnCap cells, with Figure 3A showing the cell populations and Figure 3B showing the mean fluorescence intensity for each antibody concentration. [Figure 4A-4D] Figure 4A-4C show the internalization of PSMAb and PSMAbLm into LnCap cells. Figure 4A-4C show cell populations with internalized antibody at antibody concentrations of 0.001-0.009 nM, 0.027-0.247 nM, and 0.74-6.67 nM, respectively. Figure 4D shows the statistical fluorescence mean intensity of cells with internalized antibody at different concentrations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The present disclosure relates to compositions and methods for treating and / or preventing cancer, which are based in part on the discovery of optimized antibodies, or antigen-binding fragments thereof, that specifically bind to an epitope in the extracellular domain of PSMA (e.g., human PSMA).

[0041] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to PSMA. The disclosed anti-PSMA antibody or antigen-binding fragment thereof has superior stability and efficacy compared to the parent antibody. Also within the scope of the present disclosure are bispecific antibodies, antibody-drug conjugates, PSMA-targeted imaging agents, chimeric antigen receptors, cells expressing chimeric antigen receptors, and the like.

[0042] In some embodiments, the present disclosure provides a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of PSMA. The antibody or antigen-binding fragment thereof can be formulated as a therapeutic and diagnostic composition that targets PSMA present in the vascularization of prostate cancer and / or other solid tumors. In one embodiment, the composition can comprise an antibody-drug conjugate (ADC), where the antibody or the antibody or antigen-binding fragment thereof targets the drug to the tumor site. In one embodiment, the composition comprises a bispecific antibody. In some embodiments, the bispecific antibody comprises a first antigen-binding arm that specifically binds to PSMA and a second antigen-binding arm that binds to a T cell antigen (e.g., CD3). In one embodiment, the composition is a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof that specifically binds to PSMA.

[0043] In another aspect, the present disclosure also provides an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof, a bispecific antibody, or a chimeric antigen receptor as disclosed herein. Also within the scope of the present disclosure are cells and cell cultures comprising the cells that have been modified to express an antibody or antigen-binding fragment thereof, a bispecific antibody, or a chimeric antigen receptor as disclosed herein.

[0044] The present disclosure additionally provides a method for treating or preventing cancer, including but not limited to prostate cancer. In some embodiments, the method comprises administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof that specifically binds to PSMA. In some embodiments, the method comprises administering to the subject an antibody or antigen-binding fragment thereof, a bispecific antibody, or a chimeric antigen receptor. In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof, a bispecific antibody, or a chimeric antigen receptor. In one embodiment, the method comprises administering to the subject a cell modified to express the antibody or antigen-binding fragment thereof, a bispecific antibody, or a chimeric antigen receptor.

[0045] The present disclosure further provides a method for detecting cancer in a subject. In some embodiments, the method comprises administering to the subject a targeted imaging agent comprising an antibody or antigen-binding fragment thereof that specifically binds to PSMA. In some embodiments, the antibody or antigen-binding fragment thereof can be conjugated to any imaging agent to provide a targeted imaging agent for use in a variety of imaging modalities, including but not limited to PET, SPECT, MRI, or optical imaging.

[0046] antibody In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope in the extracellular portion of PSMA. In some embodiments, the extracellular portion of PSMA comprises the amino acid sequence of SEQ ID NO:1.

[0047] The disclosed antibody or antigen-binding fragment thereof is based in part on the optimization of an anti-PSMA antibody described in WO2017 / 180713A1. The optimization includes the mutation of the "NG" sequence in the CDR3 of the light chain to "SG". Importantly, such optimization eliminates potential deamidation of asparagine (Asn or N). It also confers several superior properties to the disclosed antibody or antigen-binding fragment thereof, including improved stability and efficacy, without adversely affecting antigen-binding affinity. For example, the affinity of the optimized full antibody, as represented by PSMAbLm, shows the same or even slightly higher affinity than its parent antibody PSMAb (e.g., Kd=0.049nM for PSMAbLm compared to Kd=0.1088nM for the antibody of WO2017 / 180713A1).

[0048] The disclosed antibodies or antigen-binding fragments are not limited to the mutation of the "NG" sequence in the CDR3 of the light chain to "SG". The disclosed antibodies or antigen-binding fragments also include any possible mutations, including one or a combination of the mutations listed below: any mutation, including but not limited to, any mutation in the CDR3 of the light chain of Asn to Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr Trp, Tyr, or Val, and / or any mutation, including but not limited to, any mutation in the CDR3 of the light chain of Gly to Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0049] The disclosed antibodies or antigen-binding fragments also include any possible mutations, including one or a combination of the mutations listed below, to remove potential deamidation sites of "NS" in FR3 of the heavy chain: any mutation including but not limited to a mutation of Asn in FR3 to Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, and / or any mutation including but not limited to a mutation of Ser in FR3 of the heavy chain to Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val.

[0050] The disclosed antibodies or antigen-binding fragments also include any possible mutations, including one or a combination of the mutations listed below, to remove potential deamidation sites of "NT": any mutation including but not limited to a mutation of Asn in CDR2 of the light chain to Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr Trp, Tyr, or Val, and / or any mutation including but not limited to a mutation of Thr in CDR2 of the light chain to Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val.

[0051] The disclosed antibodies or antigen-binding fragments also include any possible mutations, including one or a combination of the mutations listed below, to remove potential deamidation sites of "NT" in FR3 of the heavy chain: any mutation including but not limited to a mutation of Asn to Ala, Arg, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val in FR3 of the heavy chain, and / or any mutation including but not limited to a mutation of Thr to Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val in FR3 of the heavy chain.

[0052] The disclosed antibodies or antigen-binding fragments also include any possible mutations, including one or a combination of the mutations listed below, to remove the potential "DG" isomerization motif in CDR2 of the heavy chain: any mutation including but not limited to a mutation of Asp to Ala, Arg, Asn, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val in CDR2 of the heavy chain, and / or any mutation including but not limited to a mutation of Gly to Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val in CDR2 of the heavy chain.

[0053] The disclosed antibodies or antigen-binding fragments also include any possible mutations, including one or a combination of the mutations listed below, to remove the potential isomerization motif of "DS" in the CDR3 of the light chain: any mutation including but not limited to a mutation of Asp to Ala, Arg, Asn, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val in the CDR3 of the light chain, and / or any mutation including but not limited to a mutation of Ser to Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val in the CDR3 of the light chain.

[0054] The disclosed antibodies or antigen-binding fragments also include any possible mutations, including one or a combination of the mutations listed below, to remove the potential "DS" isomerization motif in FR3 of the heavy chain: any mutation including but not limited to a mutation of Asp to Ala, Arg, Asn, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val in FR3 of the heavy chain, and / or any mutation including but not limited to a mutation of Ser to Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr, or Val in FR3 of the heavy chain.

[0055] Also provided in this disclosure are antibody-drug conjugates (ADCs), a platform strategy that arms antibodies for antigen-specific toxic delivery. The rationale is that antibodies, usually tumor-targeting antibodies, are conjugated with ultratoxic drugs to selectively kill tumor cells in a targeted manner while sparing normal tissues. The drugs used in ADCs are mainly classified into two types: auristatins (MMAE, MMAF) (Stephan, JP, et al. (2008) Bioconjug Chem, 19(8): 1673-83; Younes, A., et al. (2010) N Engl J Med, 363(19): 1812-21; Okeley, NM, et al. (2010) Clin Cancer Res, 16(3): 888-970) and maytansinoids (DM1 and DM4) (Lambert, JMet al. (2014). J Med Chem, 57(16): 6949-64; Bender, B., et al. (2014) AAPS J, 16(5): 994-1008; Raufi, A., ASEbrahim et al. (2013) Cancer Manag Res,5:225-33), and the other type is a double-stranded DNA breaker, such as calicheamicin. Due to excessive toxicity, certain drugs cannot be used alone as chemotherapeutic agents; instead, they must be conjugated with antibodies to reduce side effects and improve therapeutic efficacy.

[0056] Bispecific or multispecific antibodies are also within the scope of this disclosure. Bispecific monoclonal antibodies (BsMAb, BsAb) are another strategy to use novel antibodies to create a powerful antitumor weapon. BsAb are artificial proteins that are composed of fragments of two different monoclonal antibodies and thus bind to two different types of antigens. The most widely used application of this approach is in cancer immunotherapy, where BsMAb are engineered to simultaneously bind to cytotoxic cells (using the CD3 like receptor) and targets such as tumor cells to be destroyed (Muller D et al. (2010) BioDrugs, 24(2):89-98; Chames P1 et al. (2009) MAbs, 1(6):539-47). Bispecific T cell engagers (BiTE) and dual affinity retargeting (DART) are examples of small fragment BsAb. A variety of larger BsAbs have also been developed, such as knob-in-hole IgG, CrossMab, TrioMab, DVD Ig, etc. (Kontermann RE et al. (2015) Drug Discov Today, 20(7):838-47). In BsAbs, one arm can be a T-cell or NK cell activating antibody such as anti-CD3 or anti-CD16 antibody, etc., and the other arm can be a tumor-targeting antibody, or both arms target different tumor markers for synergistic inhibition of tumor growth.

[0057] The present disclosure further provides compositions comprising chimeric antigen receptors (CARs). For example, in some embodiments, the compositions are cells genetically modified to express CARs. For example, in some embodiments, T cells or NK cells are genetically engineered to produce CARs on their surface that allow the T cells or NK cells to recognize specific proteins (antigens) on tumor cells. scFvs are the most commonly used receptors for such engineering and have been successfully used in the clinic for cancer treatment (Grupp SA et al. (2013) N Engl J Med, 368(16): 1509-18; Porter DL et al. (2011) N Engl J Med, 365(8): 725-33). The scFvs are fused to intracellular signaling domains via hinge and transmembrane domains. Such molecules result in activation of T cells or NK cells in response to recognition of their target by the scFv. When T cells or NK cells express such CARs, they recognize and kill target cells expressing the target antigen. Several CARs have been developed against tumor-associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trials for the treatment of various cancers.

[0058] In one embodiment, the antibody or antigen-binding fragment thereof comprises the high affinity anti-PSMA scFv Mut-gy1, comprising the amino acid sequence of SEQ ID NO:3.

[0059] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO:5. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL FR1 comprising the amino acid sequence of SEQ ID NO:7. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:9. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL FR2 comprising the amino acid sequence of SEQ ID NO:11. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO:13. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL FR3 comprising the amino acid sequence of SEQ ID NO:15. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO:17. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL FR4 comprising the amino acid sequence of SEQ ID NO:19.

[0060] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:21. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH FR1 comprising the amino acid sequence of SEQ ID NO:23. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH FR2 comprising the amino acid sequence of SEQ ID NO:27. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO:29. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH FR3 comprising the amino acid sequence of SEQ ID NO:31. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:33. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH FR4 comprising the amino acid sequence of SEQ ID NO:35. In one embodiment, the antibody or antigen-binding fragment thereof comprises an scFv linker comprising the amino acid sequence of SEQ ID NO:37.

[0061] For example, in one embodiment, the antibody or antigen-binding fragment thereof comprises an scFv designated herein as Mut-gy1. In one embodiment, the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, Mut-gy1 comprises a light chain comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, Mut-gy1 comprises a VL FR1 comprising the amino acid sequence of SEQ ID NO: 7, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15, a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19, a VH FR1 comprising the amino acid sequence of SEQ ID NO: 23, a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and / or a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment thereof comprises a scFv linker comprising the amino acid sequence of SEQ ID NO: 37.

[0062] In one embodiment, the antibody or antigen-binding fragment thereof comprises one or more mutations. For example, in one embodiment, the antibody or antigen-binding fragment thereof comprises a VL FR2 comprising the amino acid sequence of SEQ ID NO:39, which comprises a V→A point mutation relative to SEQ ID NO:11. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO:41, which comprises a G→E point mutation relative to SEQ ID NO:13. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VL FR4 comprising the amino acid sequence of SEQ ID NO:43, which comprises a V→A point mutation relative to SEQ ID NO:19. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:45, which comprises a S→F point mutation relative to SEQ ID NO:25. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH FR3 comprising the amino acid sequence of SEQ ID NO:47, which comprises a I→V point mutation relative to SEQ ID NO:31. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, which comprises a D→G point mutation relative to SEQ ID NO: 33. In one embodiment, the antibody or antigen-binding fragment thereof comprises a VH FR4 comprising the amino acid sequence of SEQ ID NO: 51, which comprises a G→E point mutation relative to SEQ ID NO: 35.

[0063] In one embodiment, the antibody or antigen-binding fragment thereof comprises an scFv designated herein as Mut-gy1-st. In one embodiment, Mut-gy1-st comprises a VL FR2 comprising the amino acid sequence of SEQ ID NO: 39, a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 45, and a VH FR4 comprising the amino acid sequence of SEQ ID NO: 51. In one embodiment, Mut-gy1-st comprises a VL FR1 comprising the amino acid sequence of SEQ ID NO: 7, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15, a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19, a VH FR1 comprising the amino acid sequence of SEQ ID NO: 23, a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and / or a VH FR4 comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibody or antigen-binding fragment thereof comprises a scFv linker comprising the amino acid sequence of SEQ ID NO: 37.

[0064] In one embodiment, the composition comprises an antibody fragment comprising an scFv designated herein as Mut-gy1-2. In one embodiment, Mut-gy1-2 comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VL FR4 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 45, and a VH FR3 comprising the amino acid sequence of SEQ ID NO: 47. In one embodiment, Mut-gy1-2 comprises a VL FR1 comprising the amino acid sequence of SEQ ID NO: 7, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15, a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL FR4 comprising the amino acid sequence of SEQ ID NO: 43, a VH FR1 comprising the amino acid sequence of SEQ ID NO: 23, a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, and / or a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment thereof comprises a scFv linker comprising the amino acid sequence of SEQ ID NO: 37.

[0065] In one embodiment, the composition comprises an antibody fragment comprising an scFv designated herein as Mut-gy1-3. In one embodiment, Mut-gy1-3 comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:45 and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:49. In one embodiment, Mut-gy1-3 comprises a VL FR1 comprising the amino acid sequence of SEQ ID NO: 7, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VL FR2 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15, a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19, a VH FR1 comprising the amino acid sequence of SEQ ID NO: 23, a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a VH FR2 comprising the amino acid sequence of SEQ ID NO: 27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH FR3 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, and / or a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody or antigen-binding fragment thereof comprises a scFv linker comprising the amino acid sequence of SEQ ID NO: 37.

[0066] In one embodiment, the scFvs described herein can be engineered into another antibody fragment or full length antibody, where an antibody fragment refers to a Fab, Fab', (Fab')2, Fv, scFv-Fc, scFv-CH2, scFv-CH3, or a complete antibody.

[0067] In some embodiments, the antibody or antigen-binding fragment thereof comprises Mut-gy1-2 scFv, herein designated as PSMAbLm. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 68. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain having a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 53 or 55.

[0068] In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57. In one embodiment, the antibody comprises a heavy chain constant region of SEQ ID NO:59.

[0069] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence of SEQ ID NO: 69. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain having a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 61 or 63. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant region of SEQ ID NO: 67.

[0070] In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof, or a variant thereof, that specifically binds to PSMA, such as human PSMA. In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a light chain variable region (LCVR) comprising three light chain complementarity determining regions (LCDRs) (LCDR1, LCDR2, and LCDR3), wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 13 or 41, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 17; and / or (ii) a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions (HCDRs) (HCDR1, HCDR2, and HCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 25 or 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 33 or 49, and the variant comprises one or more conservative amino acid substitutions in LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, or HCDR3.

[0071] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, HCDR1 comprises the amino acid sequence of SEQ ID NO: 25, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 33.

[0072] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 33.

[0073] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 41, LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 33.

[0074] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 49.

[0075] In some embodiments, the light chain variable region comprises one or more of the amino acid sequences of SEQ ID NOs: 5, 7, 11, 15, 19, 39, 43, 61, 63, 65, 67, and 69.

[0076] In some embodiments, the heavy chain variable region comprises one or more of the amino acid sequences of SEQ ID NOs: 21, 23, 27, 31, 35, 47, 51, 53, 55, 57, 59, and 68.

[0077] In some embodiments, the light chain variable region comprises an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO:5 or comprises the amino acid sequence of SEQ ID NO:5, and / or the heavy chain variable region comprises an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO:21 or comprises the amino acid sequence of SEQ ID NO:21.

[0078] In some embodiments, the light chain variable region comprises an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO:70 or comprises the amino acid sequence of SEQ ID NO:70, and / or the heavy chain variable region comprises an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO:72 or comprises the amino acid sequence of SEQ ID NO:72.

[0079] In some embodiments, the light chain variable region comprises an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO:71 or comprises the amino acid sequence of SEQ ID NO:71, and / or the heavy chain variable region comprises an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO:73 or comprises the amino acid sequence of SEQ ID NO:73.

[0080] In some embodiments, the light chain variable region comprises an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO:5 or comprises the amino acid sequence of SEQ ID NO:5, and / or the heavy chain variable region comprises an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO:74 or comprises the amino acid sequence of SEQ ID NO:74.

[0081] In some embodiments, the light chain variable region and the heavy chain variable region comprise the LCVR and HCVR amino acid sequence pairs of SEQ ID NOs:5 and 21, SEQ ID NOs:70 and 72, SEQ ID NOs:71 and 73, or SEQ ID NOs:5 and 74.

[0082] In some embodiments, the antibody or antigen-binding fragment thereof, or variant thereof, comprises the heavy chain / light chain sequence pair of SEQ ID NOs: 75 and 77, SEQ ID NOs: 76 and 78, SEQ ID NOs: 69 and 68, or SEQ ID NOs: 75 and 79.

[0083] In some embodiments, the antibody or antigen-binding fragment thereof, or variant thereof, comprises at least one of the amino acid sequences of SEQ ID NOs: 3 and 37.

[0084] Representative sequence TIFF2025512923000002.tif47170TIFF2025512923000003.tif47170SEQ ID NO: 1 PSMA extracellular domain: KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQL AGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVH EIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYH LTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA SEQ ID NO:2 Mut-gy1 scFv nucleotide sequence: CAGTCTGTGCTGACTCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGTGTCATTATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTCTCATGTACACTGGTACCAGCAGGTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGAAACACCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGGTTCCCTGGCCATCACTGGACTCCAGCCTGAGGATGAGGCTGATTATTATTGTGCAACATGGGATGACAGTCTGAGTGGTGTAATATTCGGCGGAGGGACCAAGGTCACCGTCCTAGGCGGATCCTCTAGGTCAAGTTCCAGCGGCGGCGGTGGCAGCGGAGGCGGCGGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGCCCTGGCCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATCCACCCTCAGTGGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTTTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTACTATTGTGCTAAAGGCCTTACTTGGGGACTCGGTGACAATGATGCTCTCGATATCTGGGGCCCCGGGACCACGGTCACCGTCTCCTCA Sequence number 3 Mut-gy1 scFv amino acid sequence: QSVLTQPPSVSGAPGQSVIISCTGSSSNIGAGSHVHWYQQVPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYCATWDDSLSGVIFGGGTKVTVLGGSSRSSSSGGGGSG GGGEVQLVESGGALAKPGGSLRLSCAASGSTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALDIWGPGTTVTVSS SEQ ID NO: 4 Mut-gy1 VL nucleotide sequence: CAGTCTGTGCTGACTCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGTGTCATTATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGAGGTTCTCATGTACACTGGTACCAGCAGGTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGAAACACCAATC GGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGGTTCCCTGGCCATCACTGGACTCCAGCCTGAGGATGAGGCTGATTATTATTGTGCAACATGGGATGACAGTCTGAGTGGTGTAATATTCGGCGGAGGGACCAAGGTCACCGTCCTA SEQ ID NO:5 Mut-gy1 VL amino acid sequence: QSVLTQPPSVSGAPGQSVIISCTGSSSNIGAGSHVHWYQQVPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYCATWDDSLSGVIFGGGTKVTVL SEQ ID NO:6 Mut-gy1 VL FR1 nucleotide sequence: CAGTCTGTGCTGACTCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGTGTCATTATCTCCTGCACTGGGAGC SEQ ID NO:7 Mut-gy1 VL FR1 amino acid sequence: QSVLTQPPSVSGAPGQSVIISCTGS SEQ ID NO:8 Mut-gy1 VL CDR1 nucleotide sequence: AGCTCCAACATCGGGGCAGGTTCTCAT SEQ ID NO:9 Mut-gy1 VL CDR1 amino acid sequence: SSNIGAGSH SEQ ID NO: 10 Mut-gy1 VL FR2 nucleotide sequence: GTACACTGGTACCAGCAGGTTCCAGGAACAGCCCCCAAACTCCTCATCTAT SEQ ID NO: 11 Mut-gy1 VL FR2 amino acid sequence: VHWYQQVPGTAPKLLIY SEQ ID NO: 12 Mut-gy1 VL CDR2 nucleotide sequence: GGAAACACC SEQ ID NO: 13 Mut-gy1 VL CDR2 amino acid sequence: GNT SEQ ID NO: 14 Mut-gy1 VL FR3 nucleotide sequence: AATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGGTTCCCTGGCCATCACTGGACTCCAGCCTGAGGATGAGGCTGATTATTATTGT SEQ ID NO: 15 Mut-gy1 VL FR3 amino acid sequence: NRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYC SEQ ID NO: 16 Mut-gy1 VL CDR3 nucleotide sequence: GCAACATGGGATGACAGTCTGAGTGGTGTAATA SEQ ID NO: 17 Mut-gy1 VL CDR3 amino acid sequence TIFF2025512923000004.tif4170 (Asn to Ser substitutions are in bold) SEQ ID NO: 18: Mut-gy1 VL FR4 nucleotide sequence TTCGGCGGAGGGACCAAGGTCACCGTCCTA SEQ ID NO: 19 Mut-gy1 VL FR4 amino acid sequence: FGGGTKVTVL SEQ ID NO:20 Mut-gy1 VH nucleotide sequence: GAGGTGCAGCTGGTGGAGTCTGGGGGAGCCCTGGCCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATCCACCCTCAGTGGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAG ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTTTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTACTATTGTGCTAAAGGCCTTACTTGGGGACTCGGTGACAATGATGCTCTCGATATCTGGGGCCCCGGGACCACGGTCACCGTCTCCTCA SEQ ID NO: 21 Mut-gy1 VH amino acid sequence: EVQLVESGGALAKPGGSLRLSCAASGSTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALDIWGPGTTVTVSS SEQ ID NO:22 Mut-gy1 VH FR1 nucleotide sequence: GAGGTGCAGCTGGTGGAGTCTGGGGGAGCCCTGGCCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCT SEQ ID NO: 23 Mut-gy1 VH FR1 amino acid sequence: EVQLVESGGALAKPGGSLRLSCAAS SEQ ID NO:24 Mut-gy1 VH CDR1 nucleotide sequence: GGATCCACCCTCAGTGGCTATGCT SEQ ID NO: 25 Mut-gy1 VH CDR1 amino acid sequence: GSTLSGYA SEQ ID NO: 26 Mut-gy1 VH FR2 nucleotide sequence: ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTT SEQ ID NO: 27 Mut-gy1 VH FR2 amino acid sequence: MHWVRQAPGKGLEWVAV SEQ ID NO:28 Mut-gy1 VH CDR2 nucleotide sequence: ATATCATATGATGGAAGCAATAAA SEQ ID NO:29 Mut-gy1 VH CDR2 amino acid sequence: ISYDGSNK SEQ ID NO: 30 Mut-gy1 VH FR3 nucleotide sequence: TACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTTTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTACTATTGT SEQ ID NO: 31 Mut-gy1 VH FR3 amino acid sequence: YYADSVKGRFTISRDNSKNTLFLQMNSLRPEDTAVYYC SEQ ID NO:32 Mut-gy1 VH CDR3 nucleotide sequence: GCTAAAGGCCTTACTTGGGGACTCGGTGACAATGATGCTCTCGATATC SEQ ID NO: 33 Mut-gy1 VH CDR3 amino acid sequence: AKGLTWGLGDNDALDI SEQ ID NO: 34 Mut-gy1 VH FR4 nucleotide sequence: TGGGGCCCCGGGACCACGGTCACCGTCTCCTCA SEQ ID NO: 35 Mut-gy1 VH FR4 amino acid sequence: WGPGTTVTVSS SEQ ID NO: 36 Mut-gy1 scFv linker nucleotide sequence: GGCGGATCCTCTAGGTCAAGTTCCAGCGGCGGCGGTGGCAGCGGAGGCGGCGGT SEQ ID NO: 37 Mut-gy1 scFv linker amino acid sequence: GGSSRSSSSGGGGSGGGG SEQ ID NO: 38 Mut-gy1 VL FR2 nucleotide sequence (variant): GTACACTGGTACCAGCAGGCTCCAGGAACAGCCCCCAAACTCCTCATCTAT SEQ ID NO: 39 Mut-gy1 VL FR2 amino acid sequence (variant): VHWYQQAPGTAPKLLIY(V=>A) SEQ ID NO: 40 Mut-gy1 VL CDR2 nucleotide sequence (variant): GAAAACACC SEQ ID NO: 41 Mut-gy1 VL CDR2 amino acid sequence (variant): ENT(G=>E) SEQ ID NO: 42 Mut-gy1 VL FR4 nucleotide sequence (variant) TTCGGCGGAGGGACCAAGGCCACCGTCCTA SEQ ID NO: 43 Mut-gy1 VL FR4 amino acid sequence (variant): FGGGTKATVL(V=>A) SEQ ID NO: 44 Mut-gy1 VH CDR1 nucleotide sequence (variant): GGATTCACCCTCAGTGGCTATGCT SEQ ID NO: 45 Mut-gy1 VH CDR1 amino acid sequence (variant): GFTLSGYA(S=>F) SEQ ID NO: 46: Mut-gy1 VH FR3 nucleotide sequence (variant) TACTACGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAACACGCTGTTTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTACTATTGT SEQ ID NO: 47 Mut-gy1 VH FR3 amino acid sequence (variant): YYADSVKGRFTVSRDNSKNTLFLQMNSLRPEDTAVYYC(I=>V) SEQ ID NO: 48 Mut-gy1 VH CDR3 nucleotide sequence (variant): GCTAAAGGCCTTACTTGGGGACTCGGTGACAATGATGCTCTCGGTATC SEQ ID NO: 49 Mut-gy1 VH CDR3 amino acid sequence (variant): AKGLTWGLGDNDALGI(D=>G) SEQ ID NO: 50: Mut-gy1 VH FR4 nucleotide sequence (variant): TGGGGCCCCGAGACCACGGTCACCGTCTCCTCA SEQ ID NO: 51 Mut-gy1 VH FR4 amino acid sequence (variant): WGPETTVTVSS(G=>E) SEQ ID NO:52 PSMAbLm heavy chain nucleotide sequence: SEQ ID NO:53 PSMAbLm amino acid sequence: MEFGLSWVFLVALLRGVQCEVQLVESGGALAKPGGSLRLSCAASGFTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTVSRDNSKNTLFLQMNSLRPEDTAVYYCAKG LTWGLGDNDALDIWGPGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:54 PSMAbLm heavy chain signal peptide nucleotide sequence: ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGT SEQ ID NO:55 PSMAbLm heavy chain signal peptide amino acid sequence: MEFGLSWVFLVALLRGVQC SEQ ID NO:56 PSMAbLm heavy chain variable region nucleotide sequence: GAGGTGCAGCTGGTGGAGTCTGGGGGAGCCCTGGCCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCCTCAGTGGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAG ACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAACACGCTGTTTCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCTGTGTACTATTGTGCTAAAGGCCTTACCTGGGGACTCGGTGACAATGATGCTCTCGATATCTGGGGCCCCGGGACCACGGTCACCGTCTCCTCA SEQ ID NO:57 PSMAbLm heavy chain variable region amino acid sequence: EVQLVESGGALAKPGGSLRLSCAASGFTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTVSRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALDIWGPGTTVTVSS SEQ ID NO:58 PSMAbLm heavy chain constant region nucleotide sequence: GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO:59 PSMAbLm heavy chain constant region amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:60 PSMAbLm light chain nucleotide sequence: ATGGCCTGGTCTCCTCTCCTCCTCACTCTCCTCGCTCACTGCACAGGGTCCTGGGCCCAGTCTGTGCTGACTCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGTGTCATTATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTCTCATGTACACTGGTACCAGCAGGTTCCAGGAACAGCCCCCAAACTCCTCATCTATGAAAACACCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGGTTCCCTGGCCATCACTGGACTCCAGCCTGAGGATGAGGCTGATTATTATTGTGCAACATGGGATGACAGTCTGAGTGGTGTAATATTCGGCGGAGGGACCAAGGCCACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGA SEQ ID NO: 61 Amino acid sequence of the light chain of PSMAbLm: MAWSPLLLTLLAHCTGSWAQSVLTQPPSVSGAPGQSVIISCTGSSSNIGAGSHVHWYQQVPGTAPKLLIYENTNRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYCATWDDSLSGVIFGGGTKATVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO:62 PSMAbLm light chain signal peptide nucleotide sequence: ATGGCCTGGTCTCCTCTCCTCCTCACTCTCCTCGCTCACTGCACAGGGTCCTGGGCC SEQ ID NO: 63 PSMAbLm light chain signal peptide amino acid sequence: MAWSPLLLTLLAHCTGSWA SEQ ID NO:64 PSMAbLm light chain variable region nucleotide sequence: CAGTCTGTGCTGACTCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGTGTCATTATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGAGGTTCTCATGTACACTGGTACCAGCAGGTTCCAGGAACAGCCCCCAAACTCCTCATCTATGAAAACACCAATC GGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGGTTCCCTGGCCATCACTGGACTCCAGCCTGAGGATGAGGCTGATTATTATTGTGCAACATGGGATGACAGTCTGAGTGGTGTAATATTCGGCGGAGGGACCAAGGCCACCGTCCTA SEQ ID NO: 65 PSMAbLm light chain variable region amino acid sequence: QSVLTQPPSVSGAPGQSVIISCTGSSSNIGAGSHVHWYQQVPGTAPKLLIYENTNRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYCATWDDSLSGVIFGGGTKATVL SEQ ID NO:66 PSMAbLm light chain constant region nucleotide sequence: GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTG GAGACCACCACACCCTCCAAACAAAGCAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA SEQ ID NO:67 PSMAbLm light chain constant region amino acid sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 68 PSMAbLm heavy chain amino acid sequence without the signal peptide: EVQLVESGGALAKPGGSLRLSCAASGFTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTVSRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALDIW GPGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 69 PSMAbLm light chain amino acid sequence without the signal peptide: QSVLTQPPSVSGAPGQSVIISCTGSSSNIGAGSHVHWYQQVPGTAPKLLIYENTNRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYCATWDDSLSGVIFGGGTKA TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 70 Mut-gy1-st VL amino acid sequence: QSVLTQPPSVSGAPGQSVIISCTGSSSNIGAGSHVHWYQQAPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYCATWDDSLSGVIFGGGTKVTVL SEQ ID NO: 71 Mut-gy1-2 VL amino acid sequence: QSVLTQPPSVSGAPGQSVIISCTGSSSNIGAGSHVHWYQQVPGTAPKLLIYENTNRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYCATWDDSLSGVIFGGGTKATVL SEQ ID NO: 72 Mut-gy1-st VH amino acid sequence: EVQLVESGGALAKPGGSLRLSCAASGFTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALDIWGPETTVTVSS SEQ ID NO: 73 Mut-gy1-2 VH amino acid sequence: EVQLVESGGALAKPGGSLRLSCAASGFTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTVSRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALDIWGPGTTVTVSS SEQ ID NO: 74 Mut-gy1-3 VH amino acid sequence: EVQLVESGGALAKPGGSLRLSCAASGFTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALGIWGPGTTVTVSS SEQ ID NO: 75 PSMAb1Lm light chain amino acid sequence without the signal peptide (from Mut-gy1): QSVLTQPPSVSGAPGQSVIISCTGSSSNIGAGSHVHWYQQVPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYCATWDDSLSGVIFGGGTKV TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 76 PSMAb1stLm light chain amino acid sequence without signal peptide (from Mut-gy1-st): QSVLTQPPSVSGAPGQSVIISCTGSSSNIGAGSHVHWYQQAPGTAPKLLIYGNTNRPSGVPDRFSGSKSGTSGSLAITGLQPEDEADYYCATWDDSLSGVIFGGGTKV TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 77 PSMAb1Lm heavy chain amino acid sequence without the signal peptide (from Mut-gy1): EVQLVESGGALAKPGGSLRLSCAASGSTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALDIW GPGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 78 PSMAb1stLm heavy chain amino acid sequence without signal peptide (from Mut-gy1-st): EVQLVESGGALAKPGGSLRLSCAASGFTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALDIW GPETTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 79 PSMAb3Lm light chain amino acid sequence without the signal peptide (from Mut-gy1-3): EVQLVESGGALAKPGGSLRLSCAASGFTLSGYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLFLQMNSLRPEDTAVYYCAKGLTWGLGDNDALGIW GPGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0085] In some embodiments, the term "epitope" refers to a region of an antigen that binds to an antibody. It is a region of an antigen that is recognized by a first antibody, and whose binding to that region prevents the binding of a second antibody or other bivalent molecule to that region. The region encompasses a specific core sequence or sequences that are selectively recognized by a class of antibodies. In general, epitopes are composed of local surface structures that can be formed by continuous or non-contiguous amino acid sequences.

[0086] In another embodiment, the terms "selectively recognize," "selectively bind," or "selectively recognize" mean binding of an antibody or other bivalent molecule to an epitope that is at least 2-fold greater, 2-5-fold greater, or 5-fold greater than binding of the bivalent molecule to a non-related epitope, or to an epitope of a non-related bivalent molecule, as determined by techniques known in the art and described herein, such as, for example, ELISA and cold displacement assays.

[0087] In some embodiments, the term "antibody" refers to the structure that constitutes the natural biological form of an antibody. In most mammals, including humans and mice, this form is a tetramer, consisting of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain containing immunoglobulin domains VL and CL, and each heavy chain containing immunoglobulin domains VH, Cγ1, Cγ2, Cγ3, and Cγ4. In each pair, the light and heavy chain variable regions (VL and VH) are together involved in binding to the antigen, and the constant regions (CL, Cγ1, Cγ2, Cγ3, and Cγ4, especially Cγ1, Cγ2, and Cγ3) are involved in the antibody effector functions. In some mammals, such as camels and llamas, full-length antibodies may consist of only two heavy chains, each heavy chain containing immunoglobulin domains VH, Cγ2, and Cγ3. As used herein, "immunoglobulin (Ig)" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have several structural forms, including, but not limited to, full length antibodies, antibody fragments, and individual immunoglobulin domains, including, but not limited to, VH, Cγ1, Cγ2, Cγ3, Cγ4, VL, and CL.

[0088] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0089] In some embodiments, the term "antibody" or "antigen-binding fragment" refers to intact molecules that are capable of specifically interacting with a desired target, as well as functional fragments thereof, such as Fab, scFv-Fc bivalent molecules, F(ab')2, and Fv, respectively. In some embodiments, an antigen-binding fragment is (1) Fab, which is a fragment that contains a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of a whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain, with two Fab's being obtained per antibody molecule. (3) The fragment of an antibody which can be obtained by treating a whole antibody with the enzyme pepsin without subsequent reduction, which is a dimer of two Fab' fragments held together by two disulfide bonds, (Fab'); (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibodies (SCAs or scFvs), which are genetically engineered molecules containing the variable region of a light chain and the variable region of a heavy chain linked by a suitable polypeptide linker as a genetically fused single chain molecule; (6) scFv-Fc, which is created by fusing a single-chain Fv (scFv) with a hinge region from an immunoglobulin (Ig), such as an IgG and Fc region.

[0090] In one embodiment, the antibody provided herein is a monoclonal antibody. In another embodiment, the antigen-binding fragment provided herein is a single chain Fv (scFv), a diabody, a tandem scFv, a scFv-Fc bivalent molecule, a Fab, a Fab', an Fv, or an F(ab')2.

[0091] In one embodiment, the term "bivalent molecule" or "BV" refers to a molecule that can bind to two distinct targets simultaneously. A bivalent molecule is not limited to having two and only two binding domains, but can be a multivalent molecule or a molecule composed of linked monovalent molecules. The binding domains of a bivalent molecule can selectively recognize the same or different epitopes located on the same target or on targets from different species. The binding domains can be linked in any of several ways, including, but not limited to, disulfide bonds, peptide bridges, amide bonds, and other natural or synthetic bonds known in the art (Spatola et al., "Chemistry and Biochemistry of Amino Acids, Peptides and Proteins," B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983) (general review); Morley, JS, "Trends Pharm Sci" (1980) pp. 463-468 (general review); Hudson et al., Int J Pept Prot Res (1979) 14, 177-185; Spatola et al., Life Sci (1986) 38, 1243-1249; Hann, MM, J Chem Soc Perkin Trans I (1982) 307-314; Almquist et al., J Med Chem(1980)23,1392-1398, Jennings-White et al., Tetrahedron Lett(1982)23,2533, Szelke et al.,European Application EP 45665;Chemical Abstracts 97,39405(1982), Holladay,et al.,Tetrahedron Lett (1983) 24, 4401-4404, and Hruby, VJ, Life Sci (1982) 31, 189-199).

[0092] The sequence alignment methods that can be used to achieve the desired sequence alignment include, but are not limited to, pairwise alignment or multiple sequence alignment methods, as will be understood by those skilled in the art. The sequence alignment can be saved in a wide variety of text-based file formats. In one embodiment, this is achieved by using conversion programs and programming packages such as READSEQ, EMBOSS, and BioPerl, BioRuby, and in some embodiments, by converting any format, such as FASTA or GenBank, SwissProt, Entrez, and EMBL formats. It should be understood that those skilled in the art can use any program or storage medium to convert, correct, score, update, and / or save sequences as needed, as will be understood by those skilled in the art.

[0093] In some embodiments, the term "sequence alignment" includes the use of any program or method, as would be understood by one of skill in the art, used to perform nucleic acid or amino acid sequence alignments and generate results that can be readily explored, evaluated, and subjected to mathematical and statistical calculations. In one embodiment, methods for sequence or structure alignment include alignments based on sequence and structural homology, as is well known in the art and understood by one of skill in the art.

[0094] In one embodiment, the term "homology", "homolog", or "homologous" refers to sequence identity, or structural identity, or functional identity. By using the term "homology" and other similar forms, it should be understood that any molecule, whether nucleic acid or peptide, that functions similarly and / or contains sequence identity and / or is structurally preserved to approximate the reference sequence is considered as part of the present invention. In another embodiment, the term "homology", "homolog", or "homologous" indicates that the sequence referred to, whether amino acid sequence or nucleic acid sequence, in any case, exhibits at least 86% identity with the sequence shown. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 90% identity with the sequence shown. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 92% identity with the sequence shown. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 95% identity with the sequence shown. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 95% identity or more with the sequence shown. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 97% identity or more with the sequence shown. In another embodiment, the amino acid or nucleic acid sequence shows 97%-100% identity to the sequence shown. In another embodiment, the amino acid or nucleic acid sequence shows 100% identity to the sequence shown. Similarly, in one embodiment, reference to identity to a particular sequence includes both a direct identity as well as homology to that sequence as defined herein.

[0095] Thus, in one embodiment, the term "non-homologous" refers to an amino acid sequence or a nucleic acid sequence that shows no more than 85% identity with the indicated sequence. In another embodiment, the amino acid sequence or the nucleic acid sequence shows no more than 75% identity with the indicated sequence. In another embodiment, the amino acid sequence or the nucleic acid sequence shows no more than 65-74% identity with the indicated sequence. In another embodiment, the amino acid sequence or the nucleic acid sequence shows no more than 55-64% identity with the indicated sequence. In another embodiment, the amino acid sequence or the nucleic acid sequence shows no more than 45-54% identity with the indicated sequence. In another embodiment, the amino acid sequence or the nucleic acid sequence shows no more than 35-44% identity with the indicated sequence. In another embodiment, the amino acid sequence or the nucleic acid sequence shows no more than 35-44% identity with the indicated sequence. In another embodiment, the amino acid sequence or the nucleic acid sequence shows no more than 15-34% identity with the indicated sequence. In another embodiment, the amino acid sequence or the nucleic acid sequence shows no more than 5-14% identity with the indicated sequence. In another embodiment, the amino acid sequence or the nucleic acid sequence shows no more than 0.1-4% identity with the indicated sequence. In another embodiment, the term "non-homologous" can be used interchangeably with "low sequence similarity."

[0096] In one embodiment, the light chain comprises the CDR1, CDR2, and CDR3 sequences listed above or has greater than 70% homology, e.g., greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, and has, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations, deletions, or insertions.

[0097] In one embodiment, the light chain comprises the FR1, FR2, and FR3 sequences listed above or has greater than 70% homology, e.g., greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, and has, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations, deletions, or insertions.

[0098] In one embodiment, the heavy chain comprises the CDR1, CDR2, and CDR3 sequences listed above or has greater than 70% homology, e.g., greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, and has, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations, deletions, or insertions.

[0099] In one embodiment, the heavy chain comprises the FR1, FR2, and FR3 sequences listed above or has greater than 70% homology, e.g., greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, and has, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations, deletions, or insertions.

[0100] In one embodiment, the full antibody is one of the following types: IgG1, IgG2, IgG3, or IgG4. In another embodiment, the Mut-gy1 scFv was engineered into an IgG1 full antibody.

[0101] In one embodiment, the complete antibody has a constant region of lambda, kappa, or mutated therefrom, hi another embodiment, Mut-gy1 scFv was engineered into a complete antibody using the CL2 constant region.

[0102] In one embodiment, the heavy chain comprises a constant region of SEQ ID NO: 59, or a sequence with greater than 70%, e.g., greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology thereto, and has, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations, deletions, or insertions.

[0103] In one embodiment, the light chain comprises a constant region of SEQ ID NO: 67, or a sequence with greater than 70%, e.g., greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology thereto, and has, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations, deletions, or insertions.

[0104] In one embodiment, there may or may not be a linker peptide between the signal peptide and the variable region, and between the variable and constant regions of the heavy and / or light chains. One example of such a linker peptide is encoded by a restriction enzyme site.

[0105] In one embodiment, the antibody or antigen-binding fragment thereof is displayed on the surface of a yeast cell. In another embodiment, the antibody or antigen-binding fragment thereof is coated onto a nanoparticle surface. In another embodiment, the antibody or antigen-binding fragment thereof is displayed on the surface of a mammalian cell, such as a T cell, NK cell, or other human or other mammalian cell. In yet another embodiment, the antibody or antigen-binding fragment thereof is produced as a secreted protein by yeast, E. coli, or a mammalian cell.

[0106] In one embodiment, the terms "bind", "binding", or grammatical equivalents refer to compositions that have affinity for one another. "Specific binding" is when the binding is selective between two molecules. A particular example of specific binding is that which occurs between an antibody and an antigen. Typically, specific binding has a dissociation constant (KD) of about 1×10 -5 Less than M or about 1 x 10 -6 M or 1×10 -7 Specific binding can be distinguished from non-specific binding when the binding is less than M. Specific binding can be detected, for example, by ELISA, immunoprecipitation, co-precipitation, two-hybrid assay with or without chemical cross-linking, etc. Using appropriate controls, "specific" binding can be distinguished from "non-specific" binding.

[0107] In some embodiments, the antibody or antigen-binding fragment thereof has a modification. The modification is as further defined herein below. In some embodiments, the modification is an N-terminal modification. In another embodiment, the modification is a C-terminal modification. In another embodiment, the modification is in the middle of the protein. In one embodiment, the secretable form of the antibody or antigen-binding fragment comprises an N-terminal modification that allows it to bind to an immunoglobulin (Ig) hinge region. In another embodiment, the Ig hinge region is from, but not limited to, an immunoglobulin hinge region. In some embodiments, the modification is a direct modification on the antibody or antigen-binding fragment thereof. In other embodiments, the modification is an indirect modification cross-linked by one or more other peptides, proteins, chemicals, carbohydrates, or even a secondary antibody.

[0108] Additional post-translational modifications encompassed by the invention include, for example, N- or O-linked carbohydrate chains, N- or C-terminal processing, conjugation of chemical moieties to the amino acid backbone, chemical modifications of N- or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression.

[0109] In one embodiment, the term "polypeptide" refers generally to an antibody, antigen-binding fragment, or variant of the invention.

[0110] In one embodiment, the polypeptide of the invention comprises an amino acid substitution. In one embodiment, the amino acid substitution is conservative. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In another embodiment, the amino acid substitution is not conservative and results in an enhanced activity of the mutant polypeptide compared to the native polypeptide.

[0111] The antibodies or antigen-binding fragments of the present invention can be produced by any synthetic or recombinant process, such as those known in the art. The antibodies or antigen-binding fragments of the present invention can be further modified to alter their biophysical or biological properties by techniques known in the art. For example, a polypeptide can be modified to increase its stability against proteases, or to alter its lipophilicity, solubility, or binding affinity to its natural receptor.

[0112] In some embodiments, antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can in some embodiments be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are incorporated herein by reference in their entireties. See also Porter, RR, Biochem. J., 73:119-126, 1959. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques, can also be used so long as the fragments bind to the antigen recognized by the intact antibody.

[0113] A "variant" of a polypeptide, antibody, or protein of the invention, in one embodiment, refers to an amino acid sequence that is altered by one or more amino acids relative to a reference polypeptide, antibody, or protein. In the present invention, a variant of a polypeptide retains the antibody-binding properties of the reference protein. In another embodiment, a "variant" refers to an antigen-binding fragment of the invention. In yet another embodiment, a variant is a variant of an antigen-binding fragment that retains specificity for a target or marker. A variant may have "conservative" changes, where the substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). In another embodiment, a variant has a conservative amino acid substitution at one or more predicted non-essential amino acid residues. In another embodiment, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge, and in other embodiments, the opposite charge is an example of a "non-conservative substitution". Families of amino acid residues with side chains of similar charge have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be routinely expressed and the functional and / or biological activity of the encoded protein can be determined using the techniques described herein or by routine modification techniques known in the art. Similar minor changes can also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted, inserted, or deleted without losing immunological reactivity can be found using computer programs well known in the art, for example, DNASTAR software.

[0114] In one embodiment, the term "framework region" or "FR" refers to those variable domain residues other than the hypervariable region residues. Framework regions are precisely defined. See, for example, Kabat, EA et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, National Institutes of Health, USA (5th ed. 1991). Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4. In some embodiments, "FR" also refers to the antibody variable region, including the amino acid residues adjacent to or adjacent to the CDR regions, i.e., the regions in the variable region of the antibody that directly interact with the antigen, which function as the recognition elements of the antibody molecule, but outside the CDR regions. In one embodiment, the term "framework region" is intended to mean each domain of the framework separated by the CDRs. In some embodiments, the sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The combined heavy and light chain framework regions of an antibody function to position and align the CDRs for proper binding to the antigen.

[0115] In one embodiment, the term "CDR" or "complementarity determining region" refers to the amino acid residues which comprise the noncontiguous antigen binding sites found in the variable regions of both heavy and light chain polypeptides. In another embodiment, the term "CDR" includes the regions described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), as well as Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996). The amino acids of the CDRs of variable domains were originally defined by Kabat based on sequence variability as consisting of amino acid residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the human heavy chain variable domain (VH), and amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the human light chain variable domain (VL), using the Kabat numbering system for antibody amino acid residues. See Kabat et al., sequences of proteins of immunological interest, US Dept. Health and Human Services, NIH, USA (5th ed. 1991). Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) provided another definition of CDRs based on residues contained in the three-dimensional structural loops of the variable domain regions, which were found to be important in antigen binding activity. Chothia et al. defined the CDRs as consisting of amino acid residues 26-32 (H1), 52-56 (H2), and 95-102 (H3) in the human heavy chain variable domain (VH), and amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the human light chain variable domain (VL).Combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35B (H1), 50-65 (H2), and 95-102 (H3) in human VH, and amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in human VL, based on the Kabat numbering system.

[0116] In some embodiments, "variable region" when used in reference to an antibody or its heavy or light chain is intended to mean the amino terminal portion of the antibody that confers antigen binding to the molecule and is not the constant region. The term is intended to include functional fragments thereof, which maintain some of the full binding functions of the variable region. Thus, the term "heteromeric variable region binding fragment" is intended to mean at least one heavy chain variable region and at least one light chain variable region assembled into a heteromeric complex or functional fragments thereof. Heteromeric variable region binding fragments include, for example, functional fragments such as Fab, F(ab)2, Fv, single chain Fv (scfv), etc. Such functional fragments are well known to those of skill in the art. Thus, the use of these terms in describing functional fragments of heteromeric variable regions is intended to correspond to the definitions well known to those of skill in the art. Such terms are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989), Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.), Huston et al., Cell Biophysics, 22:189-224 (1993), Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989), and Day, ED, Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990).

[0117] In one embodiment, the polypeptide of the present invention is an isoform of an isolated polypeptide. In one embodiment, "isoform" refers to a type of molecule, such as a protein or polypeptide of the present invention, that has slight differences from other isoforms of the same protein or polypeptide. In one embodiment, isoforms are produced from different but related genes, or in another embodiment, they arise from the same gene by alternative splicing. In another embodiment, isoforms are caused by a single nucleic acid polymorphism.

[0118] In one embodiment, the isolated polypeptide of the invention is a fragment of a naturally occurring protein. In one embodiment, a "fragment" refers to a protein or polypeptide that is shorter or contains fewer amino acids than the full-length protein or polypeptide. In another embodiment, a fragment refers to a nucleic acid that is shorter or contains fewer nucleic acids than the full-length nucleic acid. In another embodiment, the fragment is an N-terminal fragment. In another embodiment, the fragment is a C-terminal fragment. In one embodiment, a fragment of the invention is an intrasequence portion of a protein, peptide, or nucleic acid. In another embodiment, the fragment is a functional intrasequence portion of a protein, peptide, or nucleic acid. In another embodiment, the fragment is a functional intrasequence portion of a protein, peptide, or nucleic acid. In another embodiment, the fragment is an N-terminal functional fragment. In one embodiment, the fragment is a C-terminal functional fragment.

[0119] In one embodiment, the term "functional fragment" refers to a modified form of a native or wild-type antibody or polypeptide, yet maintains some degree of biological activity compared to the wild-type. The degree of activity may range from moderate to high compared to the wild-type, where "activity" refers to its native biophysical or biochemical characteristics, such as binding capacity, affinity, half-life, etc.

[0120] In one embodiment, the isolated polypeptide of the invention comprises a derivative of the polypeptide of the invention. "Derivative" should be understood in some embodiments as referring to less than the full length of the native sequence of the protein in question. In some embodiments, a "derivative" may further comprise (at its termini and / or within the sequence itself) a non-native sequence, i.e. a sequence that does not form part of the native protein in question. The term "derivative" also includes within its scope molecular species produced by conjugating chemical groups to amino acid residue side chains of a native protein or fragment thereof, which chemical groups do not form part of the naturally occurring amino acid residues present in the native protein.

[0121] Methods for producing antibodies and antibody fragments are known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[0122] The antibodies can be produced by immunization of various animals, including mice, rats, rabbits, goats, primates, humans, and chickens, with a target antigen, such as PSMA or a peptide fragment of PSMA that contains the anti-PSMA epitope of the invention. In one embodiment, the antibody or antigen-binding fragment is purified before immunization of the animal. In one embodiment, the antibody or antigen-binding fragment of the invention can be purified by methods known in the art, such as gel filtration, ion exchange, affinity chromatography, etc. Polyclonal or monoclonal antibodies can be isolated from serum, ascites, or hybridoma supernatants using affinity chromatography or any of several other techniques known in the art.

[0123] By "purified" it is meant that the monoclonal antibody has been separated from at least some of the proteins normally associated with the monoclonal antibody, and preferably separated from all other cellular material.

[0124] Also provided are chemically modified derivatives of the antibodies of the invention, which may provide additional advantages, such as increased solubility, stability, in vivo or in vitro circulation time, or reduced immunogenicity of the polypeptide (see U.S. Pat. No. 4,179,337). Chemical moieties for derivatization may be selected from water soluble polymers, such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. The antibodies may be modified at random positions within the molecule or at predetermined positions within the molecule and may contain one, two, three, or more attached chemical moieties.

[0125] The polymers can be of any molecular weight, branched or unbranched. For polyethylene glycol, the molecular weight is about 1 kDa to about 100 kDa for ease in handling and manufacturing (the term "about" indicates that in a preparation of polyethylene glycol, some molecules will be higher and some will be lower in weight than the stated molecular weight). Other sizes can be used depending on the desired therapeutic profile (e.g., the desired duration of sustained release, the effect on biological activity (if any), ease of handling, degree or lack of antigenicity, and other known effects of polyethylene glycol on therapeutic proteins or analogs). For example, polyethylene glycol may be about 200, 500, 1000, 1500, 2000, 2560, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000 , 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0126] As mentioned above, polyethylene glycol can have a branched structure. Branched polyethylene glycol is described, for example, in U.S. Patent No. 5,643,575, Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996), Vorobjev et al., Nucleosides Nucleic acids 18:2745-2750(1999), and Caliceti et al., Bioconjug. Chem. 10:638-646(1999), the disclosures of each of which are incorporated herein by reference.

[0127] Polyethylene glycol molecules (or other chemical moieties) should be attached to the antibody with consideration of their effect on the functional or antigenic domains of the antibody. There are several attachment methods available to those skilled in the art, see, for example, EP0401384 (Coupling of PEG to G-CSF), which is incorporated herein, and also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting the PEGylation of GM-CSF using tresyl chloride). For example, polyethylene glycol can be covalently attached through amino acid residues via reactive groups, such as free amino or carboxyl groups. Reactive groups are those to which an activated polyethylene glycol molecule can be attached. Amino acid residues with free amino groups can include, for example, lysine residues and N-terminal amino acid residues, and those with free carboxyl groups can include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. Sulfhydryl groups can also be used as reactive groups to attach polyethylene glycol molecules. In some embodiments, attachment is at an amino group, such as attachment at the N-terminus or lysine group.

[0128] As alluded to above, polyethylene glycol can be attached to a protein, such as an antibody, via linkage to any of several amino acid residues. For example, polyethylene glycol can be linked to a protein via a covalent bond to a lysine, histidine, aspartic acid, glutamic acid, or cysteine ​​residue. One or more reaction chemistries can be used to attach polyethylene glycol to a specific amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of a protein, or to one or more types of amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine, and combinations thereof).

[0129] Glycosylation variants In some embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0130] For example, an aglycosylated antibody (i.e., the antibody lacks glycosylation) can be made. Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0131] Glycosylation of the constant region on N297 may be prevented by mutating the N297 residue to another residue, e.g., N297A, and / or by mutating adjacent amino acids, e.g., 298, to reduce glycosylation on N297.

[0132] Additionally or alternatively, antibodies can be made with altered types of glycosylation, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies described herein, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hanai et al. describes cell lines with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation. PCT Publication No. 03 / 035835 by Presta describes a variant Chinese hamster ovary cell line, Led 3 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, RR et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication No. 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures resulting in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

[0133] Fc region variants The variable regions of the antibodies described herein may be linked (e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc, of any allotype or isoallotype, e.g., for IgG1: Glm, Glml(a), Glm2(x), Glm3(f), Glml7(z); for IgG2: and for K: Km, Kml, Km2, Km3 (see, e.g., Jefferies et al. (2009) mAbs 1:1). In some embodiments, the antibody variable regions described herein are linked to Fc that binds to one or more activating Fc receptors (FcγI, FcγIIa, or FcγIIIa), thereby stimulating ADCC and causing T cell depletion. In some embodiments, the antibody variable regions described herein are linked to an Fc, which causes depletion.

[0134] In some embodiments, the antibody variable regions described herein may be linked to an Fc that typically includes one or more modifications to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Additionally, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or their glycosylation may be altered to alter one or more functional properties of the antibody. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0135] The Fc region encompasses domains derived from the constant region of an immunoglobulin, preferably a human immunoglobulin, including fragments, analogs, variants, mutants, or derivatives of the constant region. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide that is homologous to the immunoglobulin C-terminal region and can include a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, either separately or in combination. In some embodiments, the antibodies of the invention have an Fc region other than that of wild-type IgA1. The antibodies can have an Fc region from that of IgG (e.g., IgG1, IgG2, IgG3, and IgG4) or from that of other classes such as IgA2, IgD, IgE, and IgM. The Fc can be a variant of IgA1.

[0136] The constant region of immunoglobulins is responsible for the important antibody functions of Fc receptor (FcR) binding and complement fixation. There are five major classes of heavy chain constant regions, classified as IgA, IgG, IgD, IgE, and IgM, each with characteristic effector functions designated by their isotype. For example, IgG is separated into four subclasses known as IgG1, IgG2, IgG3, and IgG4.

[0137] Ig molecules interact with multiple classes of cell receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR) specific for the IgG class of antibodies: FcγRI, FcγRII, and FcγRIIL. It has been reported that the critical sequences for binding of IgG to FcγR receptors are located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by its ability to bind to FcR.

[0138] In some embodiments, the Fc region is a variant Fc region that has been modified (e.g., by amino acid substitution, deletion, and / or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate the variant) to provide desired structural features and / or biological activity. For example, modifications may be made in the Fc region to generate an Fc variant with (a) increased or decreased ADCC, (b) increased or decreased CDC, (c) increased or decreased affinity for Clq, and / or (d) increased or decreased affinity for Fc receptors compared to the parent Fc. Such Fc region variants generally contain at least one amino acid modification in the Fc region. Combinations of amino acid modifications may be particularly desirable. For example, the variant Fc region may contain substitutions at, for example, two, three, four, five, etc., of the specific Fc region positions identified herein.

[0139] The variant Fc region may also include sequence modifications in which amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid reactions with other cysteine-containing proteins present in the host cell used to produce the antibodies described herein. Even if the cysteine ​​residues are removed, the single chain Fc domains can still form dimeric Fc domains that are held together non-covalently. In other embodiments, the Fc region may be modified to make it more compatible with the host cell of choice. For example, the PA sequence near the N-terminus of a typical native Fc region may be removed, which may be recognized by digestive enzymes in E. coli, such as proline iminopeptidase. In other embodiments, one or more glycosylation sites in the Fc domain may be removed. Typically, residues that are glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or replaced with non-glycosylated residues (e.g., alanine). In other embodiments, sites involved in interaction with complement, such as Clq binding sites, may be removed from the Fc region. For example, the EKK sequence of human IgG1 may be deleted or replaced. In some embodiments, sites that affect binding to Fc receptors may be removed, preferably sites other than the salvage receptor binding site. In other embodiments, the Fc region may be modified to remove ADCC sites. ADCC sites are known in the art, see, for example, Molec. Immunol. 29(5):633-9 (1992) for ADCC sites in IgG1. Specific examples of variant Fc domains are disclosed, for example, in WO97 / 34631 and WO96 / 32478.

[0140] In one embodiment, the hinge region of the Fc is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of the Fc is altered, e.g., to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcus aureus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is further described in U.S. Patent No. 6,165,745 by Ward et al.

[0141] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al.

[0142] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and / or reduced or abolished CDC. This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0143] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT Publication No. 94 / 29351 by Bodmer et al.

[0144] In yet another example, the Fc region can be modified to increase ADCC and / or increase affinity for Fcγ receptors by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285. , 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T. Other modifications to enhance FcγR and complement interactions include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0145] Fc modifications that increase binding to Fcγ receptors include those at amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, and 327 of the Fc region. , 329, 330, 335, 337, 3338, 340, 360, 373, 376, 379, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439, where the numbering of the residues in the Fc region is the same as the EU index in abat (WO 00 / 42072).

[0146] Other Fc modifications that can be made to the Fc are to reduce or eliminate binding to FcγR and / or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, antibody-dependent cellular phagocytosis (ADCP), and CDC. Exemplary modifications include, but are not limited to, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, numbering according to the EU index. Exemplary substitutions include, but are not limited to, 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, numbering according to the EU index. Fc variants may include 236R / 328R. Other modifications to reduce FcγR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removing glycosylation at position 297 by mutational or enzymatic means, or by production in an organism such as a bacterium that does not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0147] Optionally, the Fc region may include non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720; WO00 / 42072, WO (see WO01 / 58957, WO02 / 06919, WO04 / 016750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925 and WO06 / 020114).

[0148] Fc variants that enhance affinity to the inhibitory receptor FcγRIIb may also be used. Such variants may provide Fc fusion proteins with immunomodulatory activity associated with FcγRIIb cells, including, for example, B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcγRIIb compared to one or more activating receptors. Modifications to alter binding to FcγRIIb include one or more modifications at positions selected from 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332 according to the EU index. Exemplary substitutions for improving FcγRIIb affinity include, but are not limited to, 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to FcγRllb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0149] The affinity and binding properties of an Fc region for its ligand can be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art, including, but not limited to, equilibrium (e.g., ELISA, or radioimmunoassay), or kinetic (e.g., BIACORE analysis), as well as other methods, such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labels of one or more of the components being tested and / or may use a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on the interaction of antibodies and immunogens.

[0150] In some embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region to FcRn. For example, as described in U.S. Pat. No. 6,277,375, one or more of the following residues can be mutated: 252, 254, 256, 433, 435, 436. Specific exemplary substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase biological half-life, the antibody can be modified in the CH1 or CL region to contain a salvage receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M.Other variants that increase Fc binding to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al,, 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, and 434A (Shields et al, Journal of Biological Chemistry,2001,276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 433I, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S(Dall Acqua et al.Journal of Immunology,2002,169:5171-5180,Dall'Acqua et al.,2006,Journal of Biological Chemistry 281:23514-23524). Other modifications to modulate FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671. In some embodiments, hybrid IgG isotypes with specific biological characteristics may be used. For example, IgG1 / IgG3 hybrid variants may be constructed by substituting IgG1 positions in the CH2 and / or CH3 regions with amino acids from IgG3 at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies may be constructed that include one or more substitutions, for example, 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F.In other embodiments described herein, IgG1 / IgG2 hybrid variants may be constructed by substituting IgG2 positions within the CH2 and / or CH3 regions with amino acids from IgG1 at positions where the two isotypes differ. Thus, hybrid variant IgG antibodies may be constructed with a CHAT containing one or more substitutions, for example one or more of the following amino acid substitutions: 233E, 234L, 235L, 236G (referring to the insertion of glycine at position 236), and 321H.

[0151] Furthermore, the binding sites on human IgG1 for FcγRl, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 have been shown to improve binding to FcγRIII. In addition, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A, which have been shown to exhibit enhanced FcγRIIIa binding and ADCC activity (Shields et al., 2001). Other IgG1 variants with strongly enhanced binding to FcγRIIIa have been identified, including variants with S239D / I332E and S239D / I332E / A330L mutations that showed the greatest increase in affinity for FcγRIIIa, reduced FcγRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) translated into greatly enhanced ADCC activity in vitro, with the S239D / I332E variant showing enhanced ability to deplete B cells in monkeys (Lazar et al., 2006). In addition, IgG1 variants containing the L235V, F243L, R292P, Y300L and P396L mutations have been identified that showed enhanced binding to FcγRIIIa in transgenic mice expressing human FcγRIIIa and concomitant enhanced ADCC activity in models of B cell malignancies and breast cancer (Stavenhagen et al., 2007; Nordstrom et al., 2011).Other Fc variants that may be used include: S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, M428L / N434S.

[0152] In some embodiments, an Fc with reduced binding to FcγR is selected. An exemplary Fc with reduced FcγR binding, such as an IgG1 Fc, contains the following three amino acid substitutions: L234A, L235E, and G237A.

[0153] In some embodiments, an Fc with reduced complement fixation is selected. An exemplary Fc with reduced complement fixation, e.g., an IgGl Fc, has two amino acid substitutions: A330S and P331S.

[0154] In some embodiments, an Fc is selected that has essentially no effector function, i.e., reduced binding to FcγR and reduced complement fixation. An exemplary effector-less Fc, e.g., an IgG1 Fc, contains the following five mutations: L234A, L235E, G237A, A330S, and P331S.

[0155] When an IgG4 constant domain is used, it is usually preferred to include the substitution S228P, which mimics the hinge sequence in IgG1, thereby stabilising the IgG4 molecule.

[0156] Fc mutations that increase serum half-life In some embodiments, the Fc region or Fc domain of the anti-HIV gp120-directed antibody comprises an amino acid modification that promotes an increase in the serum half-life of the antigen-binding molecule. Mutations that increase the half-life of antibodies have been reported. In one embodiment, the Fc region or Fc domain of one or both of the CD3-targeting heavy chain and the HIV antigen-targeting heavy chain comprises a methionine to tyrosine substitution at position 252 (EU numbering), a serine to threonine substitution at position 254 (EU numbering), and a threonine to glutamic acid substitution at position 256 (EU numbering). See, e.g., U.S. Patent No. 7,658,921. This type of variant, designated as a "YTE variant", exhibits a 4-fold increased half-life compared to the wild-type version of the same antibody (DalFAcqua, et al., J Biol Chem, 281:23514-24 (2006); Robbie, et al., Antimicrob Agents Chemotherap., 57(12):6147-6153 (2013)). In certain embodiments, the Fc region or domain of one or both of the CD3-targeting heavy chain and the HIV antigen-targeting heavy chain comprises an IgG constant domain comprising one, two, three or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436 (EU numbering). Alternatively, M428L and N434S ("LS") substitutions can increase the pharmacokinetic half-life of the multispecific antigen-binding molecule. In other embodiments, the Fc region or Fc domain of one or both of the CD3-targeting heavy chain and the HIV-antigen-targeting heavy chain comprises an M428L and an N434S substitution (EU numbering). In other embodiments, the Fc region or Fc domain of one or both of the CD3-targeting heavy chain and the HIV-antigen-targeting heavy chain comprises a T250Q and an M428L (EU numbering) mutation. In other embodiments, the Fc region or Fc domain of one or both of the CD3-targeting heavy chain and the HIV-antigen-targeting heavy chain comprises an H433K and an N434F (EU numbering) mutation.

[0157] Fc mutations that enhance effector activity In some embodiments, the Fc region or Fc domain of an anti-HIV gpl20 directed antibody comprises post-translational and / or amino acid modifications that increase effector activity, e.g., have improved FcγIIIa binding and increased antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc region or Fc domain of an anti-HIV gpl20 directed antibody comprises DE modifications in the Fc region (i.e., S239D, and I332E according to EU numbering). In some embodiments, the Fc region or Fc domain of an anti-HIV gpl20 directed antibody comprises DEL modifications in the Fc region (i.e., S239D, I332E, and A330L according to EU numbering). In some embodiments, the Fc region or Fc domain of an anti-HIV gpl20 directed antibody comprises DEA modifications in the Fc region (i.e., S239D, I332E, and G236A according to EU numbering). In some embodiments, the Fc region or Fc domain of the anti-HIV gpl20 directed antibody comprises DEAL modifications in the Fc region (ie, S239D, I332E, G236A, and A330L according to EU numbering). See, e.g., U.S. Patent Nos. 7,317,091, 7,662,925, 8,039,592, 8,093,357, 8,093,359, 8,383,109, 8,388,955, 8,735,545, 8,858,937, 8,937,158, 9,040,041, 9,353,187, 10,184,000, and 10,584,176. Additional amino acid modifications that increase effector activity, for example with improved Fcylla binding and increased antibody-dependent cellular cytotoxicity (ADCC), include, but are not limited to, (EU numbering) F243L / R292P / Y300L / V305I / P396L, S298A / E333A / K334A, or L234Y / L235Q / G236W / S239M / H268D / D270E / S298A in the first Fc domain, and D270E / K326D / A330M / K334E in the second Fc domain. Amino acid mutations that increase Clq binding and complement-dependent cytotoxicity (CDC) include, but are not limited to, (EU numbering) S267E / H268F / S324T or K326W / E333S.Fc region mutations that enhance effector activity are reviewed, for example, in Wang, et al., Protein Cell (2018) 9(1):63-73, and Saunders, Front Immunol. (2019) 10:1296. In other embodiments, the anti-HIV gpl20-directed antibody or antigen-binding fragment thereof has modified glycosylation, which can be introduced, for example, post-translationally or by genetic engineering. In some embodiments, the anti-HIV gpl20-directed antibody or antigen-binding fragment thereof is non-fucosylated, for example, at glycosylation sites present in the antibody or antigen-binding fragment thereof. Most approved monoclonal antibodies are of the IgG1 isotype, with two N-linked biantennary complex-type oligosaccharides attached to the Fc region. The Fc region exerts effector functions of ADCC through its interaction with leukocyte receptors of the FcγR family. A nonfucosylated monoclonal antibody is a monoclonal antibody which has been engineered such that the oligosaccharides in the Fc region of the antibody do not carry any fucose sugar units.

[0158] Multivalent antibodies In one embodiment, the antibody of the present disclosure can be monovalent or multivalent (e.g., bivalent, trivalent, etc.). As used herein, the term "valency" refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds to one target molecule or a specific position or locus on a target molecule. If an antibody is monovalent, each binding site on the molecule specifically binds to a single antigen position or epitope. If an antibody contains more than one target binding site (multivalent), each target binding site can specifically bind to the same or different molecules (e.g., different ligands or different antigens, or different epitopes or positions on the same antigen). See, for example, USPN 2009 / 0130105. In each case, at least one of the binding sites contains an epitope, motif, or domain associated with a DLL3 isoform.

[0159] In one embodiment, the antibody is a bispecific antibody, in which the two chains have different specificities, as described in Millstein et al., 1983, Nature, 305:537-539. Other embodiments include antibodies with additional specificities, such as trispecific antibodies. Other more sophisticated and interchangeable multispecific constructs and methods for their assembly are shown in USPN2009 / 0155255, as well as WO94 / 04690, Suresh et al., 1986, Methods in Enzymology, 121:210, and WO96 / 27011.

[0160] As mentioned above, a multivalent antibody may immunospecifically bind to different epitopes of a desired target molecule, or may immunospecifically bind to both a target molecule and a heterologous epitope, such as a heterologous polypeptide or solid support material. In some embodiments, a multivalent antibody may include a bispecific antibody or a trispecific antibody. Bispecific antibodies also include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several cross-linking techniques.

[0161] In some embodiments, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences, such as immunoglobulin heavy chain constant domains comprising at least part of the hinge, CH2, and / or CH3 regions, using methods well known to those skilled in the art.

[0162] In some embodiments, the isolated anti-HIV antibody is a bispecific antibody comprising a first antigen-binding arm that binds a first antigen and a second antigen-binding arm that binds a second antigen, wherein the first antigen and the second antigen are distinct, and the first antigen-binding arm comprises a heavy chain amino acid sequence and a light chain amino acid sequence as identified above.

[0163] In some embodiments, the second antigen is CD3, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), CD89, CCR5, CD4, CD8, CD28, CD137, CTLA-4, gp41, killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1), killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1), killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR3DL1), Killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1), Killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2), Killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3), Killer cell lectin-like receptor Cl (KLRC1), Killer cell lectin-like receptor C2 (KLRC2), Killer cell lectin-like receptor C3 (KLRC3), Killer cell lectin-like receptor C4 (KLRC4), Killer cell lectin-like Receptor D1 (KLRD1), killer cell lectin-like receptor K1 (KLRK1), natural cytotoxicity-inducing receptor 3 (NCR3 or NKp30), natural cytotoxicity-inducing receptor 2 (NCR2 or NK-p44), natural cytotoxicity-inducing receptor 1 (NCR1 or NK-p46), CD226 (DNAM-1), cytotoxic and regulatory T cell molecule (CRTAM or CD355), signaling lymphocyte activation molecule family member 1 (SLAMF1), CD48 (SLAMF2), lymphocyte antigen 9 (LY9 or SLAMF3), CD244 (2B4 or SLAMF4), CD84 (SLAMF5), SLAM family member 6 (SLAMF6 or NTB-A), SLAM family member 7 (SLAMF7 or CRACC), CD27 (TNFRSF7), semaphorin 4D (SEMA4D or CD100), CD160 (NK1), an immune checkpoint protein (e.g., PD-1, PD-L1, CTLA-4), and a second epitope of gpl20.

[0164] antibody derivative The antibodies provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water soluble polymers.

[0165] Non-limiting examples of water-soluble polymers include, but are not limited to, PEG, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, 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 polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0166] In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (see Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that are not harmful to normal cells but heat the non-proteinaceous moiety to a temperature that kills cells in close proximity to the antibody non-proteinaceous moiety.

[0167] Another modification of the antibodies described herein is PEGylation. Antibodies can be PEGylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To PEGylate an antibody, the antibody, or a fragment thereof, is typically reacted with PEG, such as a reactive ester or aldehyde derivative of PEG, under conditions where one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono(CI-CIO)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be PEGylated is a non-glycosylated antibody. Methods for PEGylating proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

[0168] The present disclosure also encompasses the human monoclonal antibodies described herein conjugated to a therapeutic agent, a polymer, a detectable label, or an enzyme. In one embodiment, the therapeutic agent is a cytotoxic agent. In one embodiment, the polymer is PEG.

[0169] nucleic acid In one embodiment, the present disclosure also provides polynucleic acids that comprise, or alternatively consist of, a nucleic acid sequence encoding a polypeptide chain of the disclosed antibody or antigen-binding fragment thereof or variant thereof. The present disclosure also encompasses polynucleic acids that hybridize under high stringency, or alternatively medium or low stringency hybridization conditions, e.g., as defined above, to a polynucleic acid complementary to a nucleic acid having a polynucleic acid sequence encoding a polypeptide chain of the disclosed antibody or antigen-binding fragment thereof or variant thereof.

[0170] In another embodiment, polynucleic acids are obtained and the nucleic acid sequence of the polynucleic acid can be determined by any method known in the art. Alternatively, polynucleic acids encoding antibodies (including molecules including or alternatively consisting of antibody fragments or variants thereof) are generated from nucleic acids from a suitable source. Although a clone containing nucleic acid encoding a particular antibody is not available, the sequence of the antibody molecule is known, and nucleic acids encoding immunoglobulins can be chemically synthesized with natural or optimized codons for a particular species, or can be obtained from a suitable source (e.g., antibody cDNA libraries, or cDNA libraries generated from any tissue or cell expressing an antibody, such as hybridoma cells selected to express an antibody of the invention, or nucleic acids isolated therefrom, preferably polyA+RNA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning, for example, using oligonucleic acid probes specific for a particular gene to identify cDNA clones from a cDNA library encoding an antibody. The amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0171] In some embodiments, the term "nucleic acid" refers to poly- or oligonucleic acids, such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA) or mimetics thereof. The term should also be understood to include, as equivalents, either analogs of RNA or DNA made from nucleic acid analogs, as well as single (sense or antisense) and double-stranded polynucleic acids, as applicable to the described embodiments. The term includes oligonucleic acids composed of naturally occurring nucleobases, sugars, and covalent internucleoside (backbone) linkages, as well as oligonucleic acids having non-naturally occurring portions that function similarly. Such modified or substituted oligonucleic acids are often preferred over natural forms due to desirable properties, such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases.

[0172] As will be understood by one of skill in the art, fragments or derivatives of a nucleic acid sequence or gene that encodes a protein or peptide can still function in the same manner as the entire wild-type gene or sequence. Similarly, forms of a nucleic acid sequence can have mutations relative to the wild-type sequence and yet still retain the wild-type function of encoding a protein or peptide, or a fragment thereof, that exhibits the same biological effect despite these mutations. Each of these represents a separate embodiment of the present invention.

[0173] The nucleic acid can be produced by any synthetic or recombinant process, such as those known in the art. The nucleic acid can be further modified to change its biophysical or biological properties by techniques known in the art. For example, the nucleic acid can be modified to increase its stability against nucleases (e.g., "end-capping"), or to increase expression levels by codon optimization, or to improve its lipophilicity, solubility, or binding affinity with complementary sequences.

[0174] Methods for modifying nucleic acids to achieve specific purposes have been published in the art, for example, Sambrook et al. (1989). In addition, the nucleic acid sequences of the present invention can include one or more portions of nucleic acid sequences that do not code for a protein of interest. The present invention further provides DNA sequences that code for proteins similar to those encoded by the sequences described herein, but that differ in their codon sequences due to degeneracy of the genetic code or allelic variation (base changes that occur naturally in a species population, which may or may not result in amino acid changes), and can also code for the proteins of the present invention described herein. Mutations in DNA sequences that enhance the activity, half-life, or production of the polypeptides encoded by the DNA sequences, caused by point mutations or induced modifications (including insertions, deletions, and substitutions), are also encompassed by the present invention.

[0175] DNA encoding the antibodies or antigen-binding fragments provided herein is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleic acid probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). Once isolated, the DNA can be placed into an expression vector and then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, yeast cells, or myeloma cells, that do not otherwise produce immunoglobulin proteins, to obtain synthesis of the antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0176] In one embodiment, the nucleic acid molecule encoding scFv Mut-gy1 comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2.

[0177] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO:5. For example, in one embodiment, the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO:4. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VL FR1 comprising the amino acid sequence of SEQ ID NO:7. For example, in one embodiment, the nucleic acid sequence encoding the VL FR1 comprises the nucleotide sequence of SEQ ID NO:6. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VL CDR1 comprising the amino acid sequence of SEQ ID NO:9. For example, in one embodiment, the nucleic acid sequence encoding the VL CDR1 comprises the nucleotide sequence of SEQ ID NO:8. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VL FR2 comprising the amino acid sequence of SEQ ID NO:11. For example, in one embodiment, the nucleic acid sequence encoding the VL FR2 comprises the nucleotide sequence of SEQ ID NO:10. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VL CDR2 comprising the amino acid sequence of SEQ ID NO:13. For example, in one embodiment, the nucleic acid sequence encoding the VL CDR2 comprises the nucleotide sequence of SEQ ID NO:12. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VL FR3 comprising the amino acid sequence of SEQ ID NO: 15. For example, in one embodiment, the nucleic acid sequence encoding the VL FR3 comprises the nucleotide sequence of SEQ ID NO: 14. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 17. For example, in one embodiment, the nucleic acid sequence encoding the VL CDR3 comprises the nucleotide sequence of SEQ ID NO: 16. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VL FR4 comprising the amino acid sequence of SEQ ID NO: 19. For example, in one embodiment, the nucleic acid sequence encoding the VL FR4 comprises the nucleotide sequence of SEQ ID NO: 18.

[0178] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO:21. For example, in one embodiment, the nucleic acid sequence encoding the heavy chain comprises the nucleotide sequence of SEQ ID NO:20. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VH FR1 comprising the amino acid sequence of SEQ ID NO:23. For example, in one embodiment, the nucleic acid sequence encoding VH FR1 comprises the nucleotide sequence of SEQ ID NO:22. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25. For example, in one embodiment, the nucleic acid sequence encoding VH CDR1 comprises the nucleotide sequence of SEQ ID NO:24. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VH FR2 comprising the amino acid sequence of SEQ ID NO:27. For example, in one embodiment, the nucleic acid sequence encoding VH FR2 comprises the nucleotide sequence of SEQ ID NO:26. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VH CDR2 comprising the amino acid sequence of SEQ ID NO:29. For example, in one embodiment, the nucleic acid sequence encoding VH CDR2 comprises the nucleotide sequence of SEQ ID NO:28. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VH FR3 comprising the amino acid sequence of SEQ ID NO: 31. For example, in one embodiment, the nucleic acid sequence encoding the VH FR3 comprises the nucleotide sequence of SEQ ID NO: 30. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33. For example, in one embodiment, the nucleic acid sequence encoding the VH CDR3 comprises the nucleotide sequence of SEQ ID NO: 32. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a VH FR4 comprising the amino acid sequence of SEQ ID NO: 35. For example, in one embodiment, the nucleic acid sequence encoding the VH FR4 comprises the nucleotide sequence of SEQ ID NO: 34. In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding a scFv linker comprising the amino acid sequence of SEQ ID NO: 37. For example, in one embodiment, the nucleic acid sequence encoding the scFv linker comprises the nucleotide sequence of SEQ ID NO: 36.

[0179] For example, in one embodiment, the nucleic acid molecule encodes Mut-gy1. In one embodiment, the nucleic acid molecule encodes Mut-gy1 comprising the amino acid sequence of SEQ ID NO:3. For example, in one embodiment, the nucleic acid sequence encoding Mut-gy1 comprises the nucleotide sequence of SEQ ID NO:2. In one embodiment, the nucleic acid molecule encoding Mut-gy1 comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO:5 and a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO:21. For example, in one embodiment, the nucleotide sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO:4 and the nucleotide sequence encoding the heavy chain comprises the nucleotide sequence of SEQ ID NO:20.

[0180] In one embodiment, the nucleic acid molecule encoding Mut-gy1 is selected from the group consisting of a nucleotide sequence encoding a VL FR1 comprising the amino acid sequence of SEQ ID NO:7, a nucleotide sequence encoding a VL CDR1 comprising the amino acid sequence of SEQ ID NO:9, a nucleotide sequence encoding a VL FR2 comprising the amino acid sequence of SEQ ID NO:11, a nucleotide sequence encoding a VL CDR2 comprising the amino acid sequence of SEQ ID NO:13, a nucleotide sequence encoding a VL FR3 comprising the amino acid sequence of SEQ ID NO:15, a nucleotide sequence encoding a VL CDR3 comprising the amino acid sequence of SEQ ID NO:17, a nucleotide sequence encoding a VL FR4 comprising the amino acid sequence of SEQ ID NO:19, a nucleotide sequence encoding a VH FR1 comprising the amino acid sequence of SEQ ID NO:23, a nucleotide sequence encoding a VH CDR1 comprising the amino acid sequence of SEQ ID NO:25, a nucleotide sequence encoding a VH FR2 comprising the amino acid sequence of SEQ ID NO:27, a nucleotide sequence encoding a VH CDR2 comprising the amino acid sequence of SEQ ID NO:29, a nucleotide sequence encoding a VH FR3 comprising the amino acid sequence of SEQ ID NO:31, a nucleotide sequence encoding a VH CDR3 comprising the amino acid sequence of SEQ ID NO:33, a nucleotide sequence encoding a VH FR4 comprising the amino acid sequence of SEQ ID NO:35, It comprises a nucleotide sequence encoding FR4 and / or a nucleotide sequence encoding an scFv linker comprising the amino acid sequence of SEQ ID NO:37.

[0181] For example, in one embodiment, the nucleotide sequence encoding VL FR1 comprises the nucleotide sequence of SEQ ID NO:6, the nucleotide sequence encoding VL CDR1 comprises the nucleotide sequence of SEQ ID NO:8, the nucleotide sequence encoding VL FR2 comprises the nucleotide sequence of SEQ ID NO:10, the nucleotide sequence encoding VL CDR2 comprises the nucleotide sequence of SEQ ID NO:12, the nucleotide sequence encoding VL FR3 comprises the nucleotide sequence of SEQ ID NO:14, the nucleotide sequence encoding VL CDR3 comprises the nucleotide sequence of SEQ ID NO:16, the nucleotide sequence encoding VL FR4 comprises the nucleotide sequence of SEQ ID NO:18, the nucleotide sequence encoding VH FR1 comprises the nucleotide sequence of SEQ ID NO:22, the nucleotide sequence encoding VH CDR1 comprises the nucleotide sequence of SEQ ID NO:24, the nucleotide sequence encoding VH FR2 comprises the nucleotide sequence of SEQ ID NO:26, the nucleotide sequence encoding VH CDR2 comprises the nucleotide sequence of SEQ ID NO:28, the nucleotide sequence encoding VH FR3 comprises the nucleotide sequence of SEQ ID NO:30, the nucleotide sequence encoding VH CDR3 comprises the nucleotide sequence of SEQ ID NO:32, and the nucleotide sequence encoding VH FR4 comprises the nucleotide sequence of SEQ ID NO:33. The nucleotide sequence encoding FR4 comprises the nucleotide sequence of SEQ ID NO:34 and / or the nucleotide sequence encoding the scFv linker comprises the nucleotide sequence of SEQ ID NO:36.

[0182] In one embodiment, the nucleic acid molecule encodes an antibody or antigen-binding fragment thereof comprising one or more mutations. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a VL FR2 comprising the amino acid sequence of SEQ ID NO:39, which comprises a V→A point mutation relative to SEQ ID NO:11. For example, in one embodiment, the nucleotide sequence encoding the variant VL FR2 comprises the nucleotide sequence of SEQ ID NO:38. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a VL CDR2 comprising the amino acid sequence of SEQ ID NO:41, which comprises a G→E point mutation relative to SEQ ID NO:13. For example, in one embodiment, the nucleotide sequence encoding the variant VL CDR2 comprises the nucleotide sequence of SEQ ID NO:40. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a VL FR4 comprising the amino acid sequence of SEQ ID NO:43, which comprises a V→A point mutation relative to SEQ ID NO:19. For example, in one embodiment, the nucleotide sequence encoding the variant VL FR4 comprises the nucleotide sequence of SEQ ID NO:42. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 45, which comprises an S→F point mutation relative to SEQ ID NO: 25. For example, in one embodiment, the nucleotide sequence encoding the variant VH CDR1 comprises the nucleotide sequence of SEQ ID NO: 44. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a VH FR3 comprising the amino acid sequence of SEQ ID NO: 47, which comprises an I→V point mutation relative to SEQ ID NO: 31. For example, in one embodiment, the nucleotide sequence encoding the variant VH FR3 comprises the nucleotide sequence of SEQ ID NO: 46. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, which comprises a D→G point mutation relative to SEQ ID NO: 33. For example, in one embodiment, the nucleotide sequence encoding the variant VH CDR3 comprises the nucleotide sequence of SEQ ID NO: 48. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a VH FR4 comprising the amino acid sequence of SEQ ID NO: 51, which comprises a G→E point mutation relative to SEQ ID NO: 35.For example, in one embodiment, the nucleotide sequence encoding the variant VH FR4 comprises the nucleotide sequence of SEQ ID NO:50.

[0183] In one embodiment, the nucleic acid molecule encodes an antibody fragment comprising an scFv, herein designated Mut-gy1-st. In one embodiment, the nucleic acid molecule encoding Mut-gy1-st comprises a nucleotide sequence encoding a VL FR2 comprising the amino acid sequence of SEQ ID NO: 39, a nucleotide sequence encoding a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 45, and a nucleotide sequence encoding a VH FR4 comprising the amino acid sequence of SEQ ID NO: 51. In one embodiment, the nucleic acid molecule encoding Mut-gy1-st is selected from the group consisting of a nucleotide sequence encoding a VL FR1 comprising the amino acid sequence of SEQ ID NO:7, a nucleotide sequence encoding a VL CDR1 comprising the amino acid sequence of SEQ ID NO:9, a nucleotide sequence encoding a VL FR2 comprising the amino acid sequence of SEQ ID NO:39, a nucleotide sequence encoding a VL CDR2 comprising the amino acid sequence of SEQ ID NO:13, a nucleotide sequence encoding a VL FR3 comprising the amino acid sequence of SEQ ID NO:15, a nucleotide sequence encoding a VL CDR3 comprising the amino acid sequence of SEQ ID NO:17, a nucleotide sequence encoding a VL FR4 comprising the amino acid sequence of SEQ ID NO:19, a nucleotide sequence encoding a VH FR1 comprising the amino acid sequence of SEQ ID NO:23, a nucleotide sequence encoding a VH CDR1 comprising the amino acid sequence of SEQ ID NO:45, a nucleotide sequence encoding a VH FR2 comprising the amino acid sequence of SEQ ID NO:27, a nucleotide sequence encoding a VH CDR2 comprising the amino acid sequence of SEQ ID NO:29, a nucleotide sequence encoding a VH FR3 comprising the amino acid sequence of SEQ ID NO:31, a nucleotide sequence encoding a VH CDR3 comprising the amino acid sequence of SEQ ID NO:33, a nucleotide sequence encoding a VH FR4 comprising the amino acid sequence of SEQ ID NO:51, It comprises a nucleotide sequence encoding FR4 and / or a nucleotide sequence encoding an scFv linker comprising the amino acid sequence of SEQ ID NO:37.

[0184] In one embodiment, the nucleotide sequence encoding VL FR1 comprises the nucleotide sequence of SEQ ID NO:6, the nucleotide sequence encoding VL CDR1 comprises the nucleotide sequence of SEQ ID NO:8, the nucleotide sequence encoding VL FR2 comprises the nucleotide sequence of SEQ ID NO:38, the nucleotide sequence encoding VL CDR2 comprises the nucleotide sequence of SEQ ID NO:12, the nucleotide sequence encoding VL FR3 comprises the nucleotide sequence of SEQ ID NO:14, the nucleotide sequence encoding VL CDR3 comprises the nucleotide sequence of SEQ ID NO:16, the nucleotide sequence encoding VL FR4 comprises the nucleotide sequence of SEQ ID NO:18, the nucleotide sequence encoding VH FR1 comprises the nucleotide sequence of SEQ ID NO:22, the nucleotide sequence encoding VH CDR1 comprises the nucleotide sequence of SEQ ID NO:44, the nucleotide sequence encoding VH FR2 comprises the nucleotide sequence of SEQ ID NO:26, the nucleotide sequence encoding VH CDR2 comprises the nucleotide sequence of SEQ ID NO:28, the nucleotide sequence encoding VH FR3 comprises the nucleotide sequence of SEQ ID NO:30, the nucleotide sequence encoding VH CDR3 comprises the nucleotide sequence of SEQ ID NO:32, The nucleotide sequence encoding FR4 comprises the nucleotide sequence of SEQ ID NO:50 and / or the nucleotide sequence encoding the scFv linker comprises the nucleotide sequence of SEQ ID NO:36.

[0185] For example, in one embodiment, a composition comprises a nucleic acid molecule encoding an antibody fragment comprising an scFv designated herein as Mut-gy1-2. In one embodiment, the nucleic acid molecule encoding Mut-gy1-2 comprises a nucleotide sequence encoding a VL CDR2 comprising the amino acid sequence of SEQ ID NO:41, a nucleotide sequence encoding a VL FR4 comprising the amino acid sequence of SEQ ID NO:43, a nucleotide sequence encoding a VH CDR1 comprising the amino acid sequence of SEQ ID NO:45, and / or a nucleotide sequence encoding a VH FR3 comprising the amino acid sequence of SEQ ID NO:47.

[0186] In one embodiment, the nucleic acid molecule encoding Mut-gy1-2 is selected from the group consisting of a nucleotide sequence encoding a VL FR1 comprising the amino acid sequence of SEQ ID NO:7, a nucleotide sequence encoding a VL CDR1 comprising the amino acid sequence of SEQ ID NO:9, a nucleotide sequence encoding a VL FR2 comprising the amino acid sequence of SEQ ID NO:11, a nucleotide sequence encoding a VL CDR2 comprising the amino acid sequence of SEQ ID NO:41, a nucleotide sequence encoding a VL FR3 comprising the amino acid sequence of SEQ ID NO:15, a nucleotide sequence encoding a VL CDR3 comprising the amino acid sequence of SEQ ID NO:17, a nucleotide sequence encoding a VL FR4 comprising the amino acid sequence of SEQ ID NO:43, a nucleotide sequence encoding a VH FR1 comprising the amino acid sequence of SEQ ID NO:23, a nucleotide sequence encoding a VH CDR1 comprising the amino acid sequence of SEQ ID NO:45, a nucleotide sequence encoding a VH FR2 comprising the amino acid sequence of SEQ ID NO:27, a nucleotide sequence encoding a VH CDR2 comprising the amino acid sequence of SEQ ID NO:29, a nucleotide sequence encoding a VH FR3 comprising the amino acid sequence of SEQ ID NO:47, a nucleotide sequence encoding a VH CDR3 comprising the amino acid sequence of SEQ ID NO:33, a nucleotide sequence encoding a VH FR4 comprising the amino acid sequence of SEQ ID NO:35, It comprises a nucleotide sequence encoding FR4 and / or a nucleotide sequence encoding an scFv linker comprising the amino acid sequence of SEQ ID NO:37.

[0187] In one embodiment, the nucleotide sequence encoding VL FR1 comprises the nucleotide sequence of SEQ ID NO:6, the nucleotide sequence encoding VL CDR1 comprises the nucleotide sequence of SEQ ID NO:8, the nucleotide sequence encoding VL FR2 comprises the nucleotide sequence of SEQ ID NO:10, the nucleotide sequence encoding VL CDR2 comprises the nucleotide sequence of SEQ ID NO:40, the nucleotide sequence encoding VL FR3 comprises the nucleotide sequence of SEQ ID NO:14, the nucleotide sequence encoding VL CDR3 comprises the nucleotide sequence of SEQ ID NO:16, the nucleotide sequence encoding VL FR4 comprises the nucleotide sequence of SEQ ID NO:42, the nucleotide sequence encoding VH FR1 comprises the nucleotide sequence of SEQ ID NO:22, the nucleotide sequence encoding VH CDR1 comprises the nucleotide sequence of SEQ ID NO:44, the nucleotide sequence encoding VH FR2 comprises the nucleotide sequence of SEQ ID NO:26, the nucleotide sequence encoding VH CDR2 comprises the nucleotide sequence of SEQ ID NO:28, the nucleotide sequence encoding VH FR3 comprises the nucleotide sequence of SEQ ID NO:46, the nucleotide sequence encoding VH CDR3 comprises the nucleotide sequence of SEQ ID NO:32, The nucleotide sequence encoding FR4 comprises the nucleotide sequence of SEQ ID NO:34 and / or the nucleotide sequence encoding the scFv linker comprises the nucleotide sequence of SEQ ID NO:36.

[0188] In one embodiment, the nucleic acid molecule encodes an antibody fragment comprising an scFv designated herein as Mut-gy1-3. In one embodiment, the nucleic acid molecule encoding Mut-gy1-3 comprises a nucleotide sequence encoding a VH CDR1 comprising the amino acid sequence of SEQ ID NO:45, and a nucleotide sequence encoding a VH CDR3 comprising the amino acid sequence of SEQ ID NO:49. In one embodiment, Mut-gy1 comprises a nucleotide sequence encoding a VL FR1 comprising the amino acid sequence of SEQ ID NO:7, a nucleotide sequence encoding a VL CDR1 comprising the amino acid sequence of SEQ ID NO:9, a nucleotide sequence encoding a VL FR2 comprising the amino acid sequence of SEQ ID NO:11, a nucleotide sequence encoding a VL CDR2 comprising the amino acid sequence of SEQ ID NO:13, a nucleotide sequence encoding a VL FR3 comprising the amino acid sequence of SEQ ID NO:15, a nucleotide sequence encoding a VL CDR3 comprising the amino acid sequence of SEQ ID NO:17, a nucleotide sequence encoding a VL FR4 comprising the amino acid sequence of SEQ ID NO:19, a nucleotide sequence encoding a VH FR1 comprising the amino acid sequence of SEQ ID NO:23, a nucleotide sequence encoding a VH CDR1 comprising the amino acid sequence of SEQ ID NO:45, a nucleotide sequence encoding a VH FR2 comprising the amino acid sequence of SEQ ID NO:27, a nucleotide sequence encoding a VH CDR2 comprising the amino acid sequence of SEQ ID NO:29, a nucleotide sequence encoding a VH FR3 comprising the amino acid sequence of SEQ ID NO:31, a nucleotide sequence encoding a VH CDR3 comprising the amino acid sequence of SEQ ID NO:49, and / or a nucleotide sequence encoding a VH FR4 comprising the amino acid sequence of SEQ ID NO:35. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an scFv linker comprising the amino acid sequence of SEQ ID NO:37.

[0189] In one embodiment, the nucleotide sequence encoding VL FR1 comprises the nucleotide sequence of SEQ ID NO:6, the nucleotide sequence encoding VL CDR1 comprises the nucleotide sequence of SEQ ID NO:8, the nucleotide sequence encoding VL FR2 comprises the nucleotide sequence of SEQ ID NO:10, the nucleotide sequence encoding VL CDR2 comprises the nucleotide sequence of SEQ ID NO:12, the nucleotide sequence encoding VL FR3 comprises the nucleotide sequence of SEQ ID NO:14, the nucleotide sequence encoding VL CDR3 comprises the nucleotide sequence of SEQ ID NO:16, the nucleotide sequence encoding VL FR4 comprises the nucleotide sequence of SEQ ID NO:18, the nucleotide sequence encoding VH FR1 comprises the nucleotide sequence of SEQ ID NO:22, the nucleotide sequence encoding VH CDR1 comprises the nucleotide sequence of SEQ ID NO:44, the nucleotide sequence encoding VH FR2 comprises the nucleotide sequence of SEQ ID NO:26, the nucleotide sequence encoding VH CDR2 comprises the nucleotide sequence of SEQ ID NO:28, the nucleotide sequence encoding VH FR3 comprises the nucleotide sequence of SEQ ID NO:30, the nucleotide sequence encoding VH CDR3 comprises the nucleotide sequence of SEQ ID NO:48, and / or the nucleotide sequence encoding VH FR4 comprises the nucleotide sequence of SEQ ID NO:50. The nucleotide sequence encoding FR4 comprises the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the nucleotide sequence encoding the scFv linker comprises the nucleotide sequence of SEQ ID NO:36.

[0190] In some embodiments, the nucleic acid molecule encodes a PSMAb or a polypeptide chain thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO:68. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain having a signal peptide, the heavy chain having a signal peptide comprising the amino acid sequence of SEQ ID NO:53. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain signal peptide comprising the amino acid sequence of SEQ ID NO:55. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:57. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain constant region of SEQ ID NO:59. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO:69. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain having a signal peptide, the light chain having a signal peptide comprising the amino acid sequence of SEQ ID NO:61. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain signal peptide comprising the amino acid sequence of SEQ ID NO:63. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain variable region comprising the amino acid sequence of SEQ ID NO:65. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain constant region of SEQ ID NO:67. In one embodiment, the nucleotide sequence encoding a heavy chain having a signal peptide comprises the nucleotide sequence of SEQ ID NO:52. In one embodiment, the nucleotide sequence encoding a heavy chain signal peptide comprises the nucleotide sequence of SEQ ID NO:54. In one embodiment, the nucleotide sequence encoding a heavy chain variable region comprises the nucleotide sequence of SEQ ID NO:56. In one embodiment, the nucleotide sequence encoding a heavy chain constant region comprises the nucleotide sequence of SEQ ID NO:58. In one embodiment, the nucleotide sequence encoding a light chain having a signal peptide comprises the nucleotide sequence of SEQ ID NO:60. In one embodiment, the nucleotide sequence encoding a light chain signal peptide comprises the nucleotide sequence of SEQ ID NO:62. In one embodiment, the nucleotide sequence encoding a light chain variable region comprises the nucleotide sequence of SEQ ID NO:64.In one embodiment, the nucleotide sequence encoding the light chain constant region comprises the nucleotide sequence of SEQ ID NO:66.

[0191] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having homology to one or more of the nucleotide sequences described herein. For example, in some embodiments, the nucleic acid molecule comprises a nucleotide sequence having 70% or more, 75% or more, 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more homology to the nucleotide sequences described herein.

[0192] In another aspect, the disclosure also provides methods for expressing or producing recombinant proteins of anti-PSMA antibodies, or antigen-binding fragments thereof, using various protein expression systems.

[0193] In one embodiment, the invention also provides transformed cells and their progeny into which a nucleic acid molecule encoding an antibody or antigen-binding fragment has been introduced by means of recombinant DNA techniques in vitro, ex vivo, or in vivo. Eukaryotic or prokaryotic transformed cells can be used to produce recombinant antibodies or antigen-binding fragments thereof for purification or for in situ or secretory expression for various purposes, such as tumor diagnosis or treatment. The transformed cells can be propagated and the introduced nucleic acid transcribed or the encoded protein expressed. It is understood that progeny cells may not be identical to the parent cell, since there may be mutations that occur during replication. Transformed cells include, but are not limited to, prokaryotic and eukaryotic cells, such as bacteria, fungi, plants, insects, and animal (e.g., mammalian, including human) cells. The cells can be in culture, ex vivo in cells, tissues, or organs, or can be in a subject.

[0194] Typically, cell transformation is performed using a vector. The term "vector" refers to, for example, a plasmid, a virus, such as a viral vector, or other vehicle known in the art that can be manipulated by insertion or incorporation of a nucleic acid for genetic manipulation (i.e., a "cloning vector") or can be used to transcribe or translate an inserted polynucleic acid (i.e., an "expression vector"). Such vectors are useful for introducing nucleic acids, including an antibody-encoding nucleic acid operably linked to an expression control element, and expressing the encoded protein in vitro (e.g., in solution or solid phase), in a cell, or in vivo.

[0195] In one embodiment, the expression vector is transferred into a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody or antigen-binding fragment of the invention. Thus, the invention includes a host cell containing a polynucleic acid encoding an antibody of the invention (e.g., a whole antibody, a heavy or light chain thereof, or a portion thereof, or a single chain antibody, or a fragment or variant thereof) operably linked to a heterologous promoter. In other embodiments, for expression of the whole antibody molecule, vectors encoding both the heavy and light chains are co-expressed within the host cell for expression of the whole immunoglobulin molecule.

[0196] A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleic acid coding sequence, may express the antibody molecule of the invention in situ. These include bacteriophage particles engineered to express antibody fragments or variants thereof (single chain antibodies), microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequence; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequence; Examples of suitable recombinant antibody molecules include, but are not limited to, plant cell systems infected with a recombinant human antibody molecule, such as Escherichia coli, or a plant cell line infected with a recombinant human antibody molecule, such as a plant cell line infected with a recombinant human antibody molecule, such as a plant cell line infected with a recombinant human antibody molecule, such as a plant cell line infected with a recombinant human antibody molecule, such as a plant cell line infected with a recombinant human antibody molecule, such as a plant cell line infected with a recombinant human antibody molecule, such as a plant cell line infected with a recombinant human antibody molecule, such as a plant cell line infected with a recombinant human antibody molecule, such as a plant cell line infected with a recombinant human antibody molecule, such as a plant cell line infected with a recombinant human antibody moleculeFor example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio / Technology 8:2 (11990); Bebbington et al., Bio / Techniques 10:169 (1992); Keen and Hale, Cytotechnology 18:207 (1996)). These references are incorporated by reference herein in their entireties.

[0197] A vector or host expression vector used to transform a cell generally contains at least an origin of replication for propagation in a cell. Control elements present in a vector, including expression control elements as shown herein, are included to facilitate transcription and translation. The term "expression control element" is intended to include, at a minimum, one or more components whose presence can affect expression, and can include components other than or in addition to promoters or enhancers, such as leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene or polycistronic messages, splicing signals for introns, maintaining the correct reading frame of a gene to allow in-frame translation of mRNA, polyadenylation signals to provide proper polyadenylation of the transcript of the gene of interest, stop codons, etc.

[0198] The vector may include a selection marker. As known in the art, a "selection marker" refers to a gene that allows for the selection of cells that contain the gene. "Positive selection" refers to a process in which only cells that contain the selection marker survive exposure to the positive selection. Drug resistance is an example of a positive selection marker, where cells that contain the marker survive in a medium containing the selection drug, while cells that do not contain the marker die. Such markers include drug resistance genes such as neo, which confers resistance to G418, hygr, which confers resistance to hygromycin, or puro, which confers resistance to puromycin, among others. Other positive selection marker genes include genes that allow for the identification or screening of cells that contain the marker. These genes include, among others, genes for fluorescent protein (GFP), lacZ genes, alkaline phosphatase genes, and surface markers such as CD8.

[0199] The vector can contain a negative selection marker. "Negative selection" refers to the process in which cells containing the negative selection marker are killed when exposed to an appropriate negative selection agent. For example, cells containing the herpes simplex virus-thymidine kinase (HSV-tk) gene (Wigler et al., Cell 11:223 (1977)) are sensitive to the drug ganciclovir (GANC). Similarly, the gpt gene makes cells sensitive to 6-thioxanthine.

[0200] Mammalian expression systems further include vectors specifically designed for in vivo and ex vivo expression. Such systems include adeno-associated virus (AAV) vectors (U.S. Pat. No. 5,604,090). AAV vectors have been shown to provide expression of factor IX in humans and mice at levels sufficient for therapeutic efficacy (Kay et al., Nat. Genet. 24:257 (2000); Nakai et al., Blood 91:4600 (1998)). Adenoviral vectors (U.S. Pat. Nos. 5,700,470, 5,731,172, and 5,928,944), herpes simplex viral vectors (U.S. Pat. No. 5,501,979), and retroviral (e.g., lentiviral vectors are useful for infecting dividing cells as well as non-dividing cells and foamy viruses) vectors (U.S. Pat. Nos. 5,624,820, 5,693,508, 5,665,577, 6,013,516, and 5,674,703, and WIPO Publication Nos. 92 / 05266 and 92 / 14829), and papillomaviral vectors (e.g., human and bovine papilloma viruses) have all been used in gene therapy (U.S. Pat. No. 5,719,054). Vectors also include cytomegalovirus (CMV)-based vectors (US Pat. No. 5,561,063). Vectors that efficiently deliver genes to cells of the intestinal tract have been developed and can be used (see, for example, US Pat. Nos. 5,821,235, 5,786,340, and 6,110,456). In yeast, vectors that facilitate the integration of foreign nucleic acid sequences into chromosomes, for example, via homologous recombination, are known in the art and can be used. Yeast artificial chromosomes (YACs) are typically used when the nucleic acid to be inserted is too large (e.g., greater than about 12 kb) for more conventional vectors.

[0201] In one embodiment, phagemid vectors for use in the present invention include any available in the art suitable for producing the antibody / antibody template / FR library of the present invention, including phagemid vectors pCB04, pIT1, pIT2, CANTAB6, pComb3HS. Methods for filamentous vector and phagemid construction are described, for example, in U.S. Patent No. 6,054,312 and U.S. Patent No. 6,803,230, each of which is incorporated herein by reference. Bacteriophage display systems, including non-filamentous bacteriophage vectors known as cytoplasmic or lytic phages, can also be utilized, for example, as described in U.S. Patent No. 5,766,905, which is incorporated herein by reference.

[0202] Bacterial expression constructs suitable for use in the present invention include, but are not limited to, commercially available expression constructs pCAL, pUC, pET, pETBlue™ (Novagen), pBAD, pLEX, pTrcHis2, pSE280, pSE380, pSE420 (Invitrogen), pKK223-2 (Clontech), pTrc99A, pKK223-3, pRIT2T, pMC1871, pEZZ 18 (Pharmacia), pBluescript II SK (Stratagene), pALTER-Ex1, pALTER-Ex2, pGEMEX (Promega), pFivE (MBI), pQE (Qiagen), and derivatives thereof, as well as others known in the art. In some embodiments, the constructs may also comprise viruses, plasmids, bacmids, phagemids, cosmids, or bacteriophages.

[0203] The use of liposomes to introduce various compositions, including nucleic acids, into cells is known to those skilled in the art (see, for example, U.S. Pat. Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282). Carriers comprising natural polymers, or derivatives or hydrolysates of natural polymers, as described in WO94 / 20078 and U.S. Pat. No. 6,096,291, are suitable for mucosal delivery of molecules such as polypeptides and polynucleic acids. Piperazine-based amphiphilic cationic lipids useful for gene therapy are also known (see, for example, U.S. Pat. No. 5,861,397). Cationic lipid systems are also known (see, for example, U.S. Pat. No. 5,459,127). Thus, means of delivery of viral and non-viral vectors to cells or tissues in vitro, in vivo, and ex vivo are included.

[0204] In one embodiment, the nucleic acid sequences can be "operably linked", i.e., positioned to ensure the function of the expression control sequences. These expression constructs are typically replicable in the cell either as episomes or as an integral part of the chromosomal DNA of the cell, and may contain an appropriate origin of replication in the respective prokaryotic strain used for expression. In general, the expression constructs contain a selection marker, such as tetracycline resistance, ampicillin resistance, kanamycin resistance, or chlormaphenicol resistance, to facilitate detection and / or selection of those bacterial cells transfected with the desired nucleic acid sequence (see, for example, U.S. Pat. No. 4,704,362). However, these markers are not exclusive, and many other markers can be used, as known to those skilled in the art. In some embodiments, the expression constructs contain both positive and negative selection markers.

[0205] Similarly, reporter genes may be incorporated into the expression construct to facilitate identification of the transcript. Thus, in one embodiment of the present invention, the reporter gene utilized is selected from the group consisting of β-galactosidase, chloramphenicol acetyltransferase, luciferase, and fluorescent proteins.

[0206] Prokaryotic promoter sequences regulate the expression of encoded polynucleic acid sequences and, in some embodiments of the invention, are operably linked to polynucleic acids encoding polypeptides of the invention. In additional embodiments of the invention, these promoters are either constitutive or inducible and provide a means for high and low level expression of the polypeptides of the invention, in some embodiments, for regulated expression of multiple polypeptides of the invention, in some embodiments expressed as a fusion protein.

[0207] Many well-known bacterial promoters may be used, including the T7 promoter system, lactose promoter system, typtophan (Trp) promoter system, Trc / Tac promoter system, beta-lactamase promoter system, tetA promoter system, arabinose-regulated promoter system, phage T5 promoter, or promoter system from phage lambda, as well as others, and include embodiments of the present invention. The promoter typically controls expression, optionally with an operator sequence, and may include, for example, a ribosome binding site sequence, for initiating and completing transcription and translation. According to additional embodiments, the vector may also contain expression control sequences, enhancers that may regulate the transcriptional activity of the promoter, appropriate restriction sites that facilitate cloning of the insert adjacent to the promoter, and other necessary information processing sites such as RNA splice sites, polyadenylation sites, transcription termination sequences, and any other sequences that may facilitate expression of the inserted nucleic acid.

[0208] In another embodiment, the invention includes methods of use of the polynucleic acids, vectors, antibodies, and / or fragments thereof described herein, and / or compositions comprising same, in the treatment, inhibition, or prevention.

[0209] PSMA-targeting antibody-drug conjugates In one embodiment, the invention provides an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioisotope (i.e., a radioconjugate). In certain embodiments, the drug includes, but is not limited to, tubulin inhibitors and DNA cleavage agents, such as maytansinoids, auristatins, dolastatins, and calicheamicins. In another embodiment, the invention further provides methods of using the ADC. In one aspect, the ADC comprises any of the above PSMA antibodies or antibody fragments covalently linked to a cytotoxic agent or a detectable agent.

[0210] The use of antibody-drug conjugates for the localized delivery of cytotoxic or cytostatic agents, i.e., drugs that kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows for targeted delivery of the drug moiety to tumors and their intracellular accumulation therein, but systemic administration of these unconjugated drugs can result in unacceptable levels of toxicity to normal cells, as well as the tumor cells that are sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” Monoclonal Antibodies '84: Biological And Clinical applications, A. Pinchera et al. (ed.s), pp. 475-506). Maximum efficacy with minimum toxicity is thereby sought. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) ibid.).Toxins that have been used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, geldanamycin (Mandler et al. (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al. (1998) Cancer Res. 58:2928; Hinman et al. (1993) Cancer Examples of small molecule toxins include small molecule toxins such as cyclosporine, ...

[0211] Examples of antibody-drug conjugates include ZEVALIN® (ibritumomab tiuxetan, Biogen / Idec), an antibody-radioisotope conjugate composed of a murine IgG1 kappa monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes and an In or Y radioisotope attached by a thiourea linker chelator (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al. (2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al. (2002) J. Clin. Oncol. 20(15):3262-69).

[0212] Additionally, MYLOTARG™ (gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate composed of a human CD33 antibody linked to calicheamicin, was approved in 2000 for the treatment of acute myeloid leukemia by injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos. 4,970,198, 5,079,233, 5,585,089, 5,606,040, 5,693,762, 5,739,116, 5,767,285, 5,773,001).

[0213] Finally, auristatin peptides such as monomethylauristatin E (MMAE), a synthetic analog of dolastatin, have been conjugated to the chimeric monoclonal antibodies cBR96 (specific for Lewis Y in carcinomas) and cAC10 (specific for CD30 in hematological malignancies) (Doronina et al. (2003) Nature Biotechnology 21(7):778-784). cAC10 is under therapeutic development.

[0214] Additionally, chemotherapeutic agents useful for generating ADCs are described herein. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and trichothecenes. See, for example, WO 93 / 21232 published October 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re. Conjugates of antibodies and cytotoxic agents are made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al (1987) Science, 238: 1098. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleic acids with antibodies (WO94 / 11026).

[0215] Conjugates of an antibody and one or more small molecule toxins, such as calicheamicins, maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.

[0216] Maytansinoids: Maytansine compounds suitable for use as maytansinoid drug moieties are well known in the art and can be isolated from natural sources in accordance with known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogs are prepared synthetically in accordance with known methods.

[0217] Exemplary maytansinoid drug moieties include those with modified aromatic rings, such as C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by lithium aluminum hydride reduction of ansamitocin P2), C-20-hydroxy (or C-20-demethyl) + / -C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces, or by dechlorination using LAH), and C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechloro (U.S. Pat. No. 4,294,757) (prepared by acylation using acyl chlorides), as well as those with modifications at other positions.

[0218] Exemplary maytansinoid drug moieties also include C-9-SH (U.S. Pat. No. 4,424,219) (prepared by reaction of maytansinol with H2S or P2S5), C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Pat. No. 4,331,598), C-14-hydroxymethyl or acyloxymethyl (CH2OH or CHOAc) (U.S. Pat. No. 4,450,254) (prepared from Nocardia), C-15-hydroxy / acyloxy (U.S. Pat. No. 4,364,866) (prepared by conversion of maytansinol by Streptomyces), C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (prepared from Trewia nudlflora), C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol with Streptomyces), and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol).

[0219] ADCs containing maytansinoids, methods for making them, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, 6,441,163, and European Patent EP 0425235 B1, the disclosures of which are expressly incorporated herein by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) reported an ADC containing a maytansinoid called DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic to cultured colon cancer cells and exhibited antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127-131 (1992) reported ADCs in which maytansinoids were conjugated via a disulfide linker to the murine antibody A7, which binds to an antigen in a human colon cancer cell line, or to another murine monoclonal antibody, TA.1, which binds to the HER-2 / neu oncogene. The cytotoxicity of the TA.1-maytansonoid conjugates was measured at 3×10 per cell. 5 The A7-maytansinoid conjugates were tested in vitro on the human breast cancer cell line SK-BR-3, which expresses the HER-2 surface antigen. The drug conjugates achieved a similar degree of cytotoxicity as the free maytansinoid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugates showed low systemic cytotoxicity in mice.

[0220] An exemplary maytansinoid embodiment is DM1, where the wavy line indicates the covalent bond to the linker (L) of the antibody-drug conjugate. TIFF2025512923000005.tif56170

[0221] Auristatins and Dolastatins: In some embodiments, the ADC comprises an antibody of the invention conjugated to a dolastatin or dolostatin peptide analogs and derivatives, auristatin (U.S. Pat. Nos. 5,635,483 and 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug moiety can be attached to the antibody via the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety (WO02 / 088172).

[0222] Exemplary auristatin embodiments include the N-terminally linked monomethyl auristatin drug moieties DE and DF and are described in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004, and U.S. Patent Publication No. 2005 / 0238649, the disclosure of which is expressly incorporated by reference in its entirety.

[0223] ADCs using MMAE and MMAF with various linker components have been disclosed (US2005 / 0238649, US08968742).

[0224] An exemplary auristatin embodiment is MMAE, where the wavy line indicates a covalent bond to the linker (L) of the antibody-drug conjugate. TIFF2025512923000006.tif22170

[0225] Another exemplary auristatin embodiment is MMAF, where the wavy line indicates the covalent bond to the linker (L) of the antibody-drug conjugate (US2005 / 0238649). TIFF2025512923000007.tif22170

[0226] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments, for example, according to solution phase synthesis methods well known in the art of peptide chemistry (see E. Schroder and K. Lubke, "The Peptides," volume 1, pp 76-136, 1965, Academic Press). Auristatin / dolastatin drug moieties may be prepared according to the methods of U.S. Pat. No. 5,635,483, U.S. Pat. No. 5,780,588, Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465, Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277, Pettit, GR, et al. Synthesis, 1996, 719-725, Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863, and Doronina (2003) Nat Biotechnol 21(7):778-784.

[0227] Caliche machine: In other embodiments, the ADC comprises an antibody of the invention conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics can produce double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that may be used include, but are not limited to, gamma 1I, alpha 2I, alpha 3I, N-acetyl-gamma 1I, PSAG, and theta 1I (Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998); and the aforementioned U.S. patents to American Cyanamid). Another antitumor agent to which antibodies can be conjugated is QFA, an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, cellular uptake of these agents by antibody-mediated internalization greatly enhances their cytotoxic effects.

[0228] Other cytotoxic agents: Other anti-tumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoicin, vincristine, and 5-fluorouracil, a family of agents known collectively as LL-E33288 conjugates, which are described in U.S. Pat. Nos. 5,053,394, 5,770,710, and esperamicin (U.S. Pat. No. 5,877,296).

[0229] Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. See, e.g., WO 93 / 21232 published Oct. 28, 1993.

[0230] The invention further contemplates ADCs formed between an antibody and a compound with nucleolytic activity (e.g., a DNA endonuclease, such as a ribonuclease or deoxyribonuclease, DNase).

[0231] For selective destruction of tumors, the antibody may contain highly radioactive atoms. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu radioisotopes. When the conjugate is used for detection, it may contain radioactive atoms for scintigraphy tests, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging (MRI)), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0232] Radioactive or other labels can be incorporated into the conjugates by known methods. For example, peptides can be biosynthesized or synthesized by chemical amino acid synthesis using suitable amino acid precursors containing, for example, fluorine-19 instead of hydrogen. Labels such as tc99m or I123, Re186, Re188, and In111 can be attached via cysteine ​​residues in the peptide. Yttrium-90 can be attached via lysine residues. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80:49-57) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

[0233] PSMA-targeting antibody-drug conjugate compounds: The present invention provides, inter alia, an antibody-drug conjugate compound for targeted delivery of drugs. The inventors have discovered that the antibody-drug conjugate compound has potent cytotoxic and / or cytostatic activity against cells expressing PSMA. The antibody-drug conjugate compound comprises an antibody unit covalently linked to at least one drug unit. The drug unit can be covalently linked directly or via a linker unit (-LU-).

[0234] In some embodiments, the antibody-drug conjugate compound has the following formula: Ab-(LU-D)p or a pharma- ceutically acceptable salt or solvate thereof, wherein: Ab is an antibody unit, e.g., a complete antibody of the invention or an antigen-binding fragment thereof derived from Mut-gy1 or a mutated variant thereof, such as PSMAb, (LU-D) is the Linker Unit-Drug Unit moiety, wherein LU- is a linker unit, -D is a drug entity having cytostatic or cytotoxic activity against a target cell, p is an integer from 1 to 20.

[0235] In some embodiments, p ranges from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4.

[0236] In some embodiments, the antibody-drug conjugate compound has the following formula: Ab-(A a -W w -Y y -D) p or a pharma- ceutically acceptable salt or solvate thereof, wherein: Ab is an antibody unit, e.g., a complete antibody of the invention or an antigen-binding fragment thereof derived from Mut-gy1 or a mutated variant thereof, such as PSMAb, -A a -W w -Y y - is a linker unit (LU), wherein -A- is the stretcher unit, a is 0 or 1, each -W- is independently an amino acid unit; w is an integer from 0 to 12, -Y- is a self-immolative spacer unit; y is 0, 1, or 2; -D is a drug entity having cytostatic or cytotoxic activity against a target cell, p is an integer from 1 to 20.

[0237] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, p is in the range of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4. In some embodiments, when w is not zero, y is 1 or 2. In some embodiments, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2 to 12, and y is 1 or 2. In some embodiments, a is 1, and w and y are 0.

[0238] In compositions comprising multiple antibodies, the drug loading is represented by p, the average number of drug molecules per antibody. Drug loading can range from 1 to 20 drugs (D) per antibody. The average number of drugs per antibody in a preparation of a conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of antibody-drug conjugates with respect to p can be determined. In some cases, when p is a specific value from antibody-drug conjugates with other drug loadings, separation, purification, and characterization of homogenous antibody-drug conjugates can be achieved by means such as reverse-phase HPLC or electrophoresis. In an exemplary embodiment, p is 2 to 8.

[0239] The production of antibody-drug conjugate compounds can be achieved by any technique known to those skilled in the art. Briefly, antibody-drug conjugate compounds comprise a complete antibody or an antigen-binding fragment thereof derived from Mut-gy1 or a mutated variant thereof of the present invention as an antibody unit, a drug, and optionally a linker that connects the drug and the binding agent. In a preferred embodiment, the antibody is an antibody or an antigen-binding fragment thereof derived from Mut-gy1 or a variant thereof with a point mutation, as described elsewhere herein. For the covalent attachment of the drug and / or the linker to the binding agent, several different reactions are available. This is often achieved by reaction of amino acid residues of the binding agent, e.g., antibody molecules, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and various moieties of aromatic amino acids. One of the most commonly used non-specific methods of covalent attachment is the carbodiimide reaction, which links the carboxy (or amino) group of a compound to the amino (or carboxy) group of an antibody. In addition, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino groups of compounds to the amino groups of antibody molecules. Also available for coupling drugs to binders is the Schiff base reaction. This method involves periodate oxidation of drugs containing glycol or hydroxy groups, thus forming aldehydes that then react with the binder. The coupling occurs via the formation of a Schiff base with the amino groups of the binder. Isothiocyanates can also be used as coupling agents to covalently bind drugs to binders. Other techniques are known to those skilled in the art and are within the scope of the present invention.

[0240] In some embodiments, an intermediate that is a precursor of the linker is reacted with the drug under appropriate conditions. In some embodiments, a reactive group is used on the drug and / or the intermediate. The product of the reaction between the drug and the intermediate, or the derivatized drug, is then reacted with a complete antibody or an antigen-binding fragment thereof derived from Mut-gy1 or a mutant thereof under appropriate conditions.

[0241] PSMA-targeted CAR-T or CAR-NK Recent developments using chimeric antigen receptor (CAR) modified autologous T cell (CART) therapy, which relies on redirecting T cells to suitable cell surface molecules on cancer cells such as B cell malignancies, have shown promising results in harnessing the power of the immune system to treat B cell malignancies and other cancers (see, e.g., Sadelain et al. Cancer Discovery 3:388-398 (2013)). Clinical results of murine CART19 (i.e., "CTL019") have shown promise in establishing complete remissions in patients suffering from CLL, as well as in pediatric ALL (see, e.g., Kalos et al., Sci Transl Med 3:95ra73 (2011); Porter et al., NEJM 365:725-733 (2011); Grupp et al., NEJM 368:1509-1518 (2013)). In addition to the ability of genetically modified T cells with chimeric antigen receptors to recognize and destroy target cells, successful therapeutic T cell therapy requires that they have the ability to proliferate and persist over time, and further monitor for leukemic cell escape. Variable quality of T cells, whether it is the result of energy, inhibition, or exhaustion, will affect the performance of CAR-transformed T cells, but trained physicians currently have limited control over this. To be effective, CAR-transformed patient T cells must persist and maintain the ability to proliferate in response to the CAR's antigen. ALL patient T cells have been shown to be able to do this with CART19 containing murine scFv (e.g., Grupp et al., NEJM 368:1509-1518 (2013)).

[0242] The present invention addresses controlling immune responses in patients by providing fully human antibody fragments (e.g., scFvs) that bind PSMA incorporated into chimeric antigen receptor (CAR) constructs that redirect engineered T cells to recognize and kill PSMA-positive tumor cells.

[0243] Thus, in one aspect, the invention relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), the CAR comprising an antibody or antigen-binding fragment thereof comprising a PSMA-binding domain, a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain). In one embodiment, the CAR comprises an antibody or antigen-binding fragment thereof comprising a fully human anti-PSMA binding domain as described herein, a transmembrane domain as described herein, and an intracellular signaling domain as described herein (e.g., an intracellular signaling domain comprising a costimulatory domain and / or a primary signaling domain).

[0244] In one embodiment, the encoded human anti-PSMA binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a complete human anti-PSMA binding domain described herein, and / or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a complete human anti-PSMA binding domain described herein, e.g., a complete human anti-PSMA binding domain comprises one or more, e.g., all three LC CDRs, and / or one or more, e.g., all three HC CDRs. In one embodiment, the encoded light chain variable region comprises one, two, three, or all four framework regions described herein. In one embodiment, the encoded heavy chain variable region comprises one, two, three, or all four of the framework regions set forth below. In one embodiment, the encoded fully human anti-PSMA binding domain comprises a human light chain variable region set forth below and / or a human heavy chain variable region set forth below. In one embodiment, the encoded anti-PSMA binding domain is an scFv comprising light and heavy chains of the amino acid sequences set forth below. In an embodiment, the anti-PSMA binding domain (e.g., scFv) comprises a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of a light chain variable region provided below, or a sequence having 95-99% identity to an amino acid sequence set forth below, and / or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but not more than 30, 20, or 10 modifications (e.g., substitutions) of the amino acid sequence of a heavy chain variable region provided below, or a sequence having 95-99% identity to an amino acid sequence set forth below. In one embodiment, the encoded human anti-PSMA binding domain comprises a sequence set forth below, or a sequence having 95-99% identity thereto. In one embodiment, the nucleic acid sequence encoding the human anti-PSMA binding domain comprises a sequence set forth below, or a sequence having 95-99% identity thereto.In one embodiment, the encoded human anti-PSMA binding domain is an scFv, and the light chain variable region is linked to the heavy chain variable region via a linker, e.g., a linker described herein. In one embodiment, the encoded human anti-PSMA binding domain comprises a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4. In another embodiment, the encoded human anti-PSMA binding domain comprises the linker sequence set forth in SEQ ID NO: 37. The light and heavy chain variable regions of the scFv can be, for example, in any of the following configurations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0245] In some embodiments, the anti-PSMA binding domain comprises an antibody or antigen-binding fragment thereof described elsewhere herein. For example, in some embodiments, the anti-PSMA binding domain comprises Mut-gy1, Mut-gy1-st, Mut-gy1-2, Mut-gy1-3, or PSMAbLm, as described elsewhere herein.

[0246] PSMA targeting bispecific antibody Unlike natural antibodies, bispecific antibodies (BsAbs) are artificial antibodies with dual specificity, targeting two different tumor antigens, or targeting one with tumor cells and the other with effector cells that can efficiently recruit immune effector cells to the tumor site and activate them to specifically kill tumor cells. An example of the former, i.e., BsAbs targeting two different tumor-associated antigens, is bH1, which simultaneously binds Her2 and VEGF (Bostrom J1 et al. 2009, Science 323: 1610-4), or ErBb2 / ErBb3 dual-targeting bispecific scFv (Robinson MK et al. 2008, Br J Cancer 99:1415-25). Two tumor-associated antigens may be both expressed on tumor cells, or one may be expressed on tumor cells and the other on tumor-associated cells such as fibroblasts, vascular cells, endothelium, pericytes, or immune cells (macrophages, B cells, T cells, etc.) in the tumor microenvironment. An example of the latter, i.e., a BsAb targeting one tumor-associated antigen and an immune-activatable antigen, is a BsAb with one arm targeting a tumor antigen such as Her2, CD19, or CD123, and the other arm targeting an immune-activatable antigen such as CD3 or CD16, which can associate tumor cells with immune cells such as T cells, NK cells, or macrophages (Kontermann RE, et al. 2015, Drug Discovery Today 20:838-847).

[0247] BsAbs, including anti-CD3 antibodies, bring T cells and tumor cells together, leading to the killing of activated T cells of tumor cells (Muller and Kontermann, BioDrugs 2010;24:89-98; Baeuerle and Reinhardt 2009, Cancer Research 96:4941). Blinatumomab (Bargou et al., Science 2008,321:974-976) is a single-chain antibody construct called BiTE, which induces cytotoxicity by targeting CD19 and CD3. Other antibody fragment-based T cell-engaging bispecifics have been reported (Moore et al. 2011, Blood 117:4542-4551; Baeuerle et al. Current Opinion in Molecular Therapeutics 2009,11:22-30). BiTE™ format is a bispecific single-chain antibody construct that links variable domains from two different antibodies.However, Blinatumomab has a low half-life in vivo and is difficult to manufacture in terms of production and stability.Therefore, there is a need for improved bispecific antibodies that can target T cells to tumor cells and have improved manufacturability.

[0248] BsAbs are hybrid proteins that can be produced by chemical cross-linking, hybridoma technology, or genetic methods. In the chemical cross-linking method, two types of monoclonal antibodies and their fragments were dissociated by a reducing agent to produce monovalent antibodies and their fragments. The resulting BsAbs are constructed through chemical cross-linking of two monovalent antibodies and their fragments from different parent antibodies. This strategy can be used for rapid production of BsAbs on a large scale, but sometimes BsAbs can be inactivated during cross-linking, and it is difficult to guarantee homogeneity of the product. Another strategy for the production of BsAbs is the hybridoma technology, in which an established hybridoma cell line secreting one monoclonal antibody is fused to spleen cells immunized with the other antigen, or two established hybridoma cell lines secreting two different monoclonal antibodies are fused to each other to create a hybrid hybridoma. The resulting hybridomas of the former are called dimeric and tetrameric hybridomas. In general, BsAbs produced by hybridoma technology maintain high bioactivity. However, the procedure is laborious and time-consuming, and it is not easy to isolate BsAbs from other non-active and undesired antibodies produced at the same time. These BsAb formats have encountered another predictable problem: the excessive size and murine components contained in BsAbs may be immunogenic in patients and induce the production of human anti-mouse antibodies (HAMA), preventing the reuse of these BsAbs in clinics. Furthermore, the production and purification of these forms of BsAbs is costly, limiting the application of BsAbs in clinics. The replacement of these traditional methods with recombinant approaches has accelerated progress in this area. Based on the technology of small molecule antibodies, the production of BsAbs by genetic engineering has advantages beyond those mentioned above, such as process stability, large-scale production, low cost, and ease of use. Genetic engineering has led to the development of various small molecule BsAb formats by connecting two different types of scFv. There are three types of BsAb formats classified by different linkages.(1) Miniantibodies are heterodimers assembled by connecting two scFv fragments together with oligomerization domains (e.g., leucine zipper motifs from Fos or Jun transcription factors). (2) Diabodies are non-covalently associated dimmers assembled by two single chains VH1-VL2 and VH2-VL1, both connected by short linkers that are too short to allow pairing between V domains from the same chain. Thus, each chain alone cannot bind to an antigen, but co-expression of two chains (VH1-VL2 and VH2-VL1) results in the assembly of a heterodimeric diabody that can bind to two different antigens. (3) ScBsAb: An interlinker was used to connect two different scFvs with different specificities, and the ScBsAb was expressed in host cells as a single polypeptide. The intralinker between the two domains in the scFv is often (Gly4Ser)3. For the interlinker between two scFvs, there are two strategies to design it. For the purpose of avoiding incorrect pairing between heterologous variable regions, the interlinker is often a short peptide linker with less than 10 amino acid residues, such as Gly4Ser. Another strategy is to select a longer linker for the interlinker. In short, the most important thing to design an interlinker is to ensure proper pairing between the variable domains and protein folding, resulting in the formation of a BsAb that maintains biological activity and stability. In order to facilitate purification and extend plasma half-life, some new properties should be introduced.

[0249] BsAb-mediated immunotherapy plays a promising role in clinical biotherapy of tumors. The tumor-killing effect mediated by BsAb is based on stimulating the immune system, is highly specific to tumors, and is not MHC-restricted. Therefore, BsAb-mediated therapy complements traditional methods such as surgery, radiotherapy, and chemotherapy. BsAb can not only cure tumors, but also stimulate the immune system to provide and maintain immune protection for a long period of time. Based on the results of experiments in mice and in clinics, an optimal BsAb prepared for investigational use should have at least five characteristics: 1): it targets the relevant tumor antigen with high specificity and affinity; 2): it can bind to triggering factors in effector cells-cytotoxic cells, resulting in cross-linking only when the BsAb binds to the tumor antigen; 3): the BsAb can promote effective cytotoxicity and inflammation selectively generated by the corresponding population of leukocytes at the tumor site; 4): the BsAb must be humanized to minimize the induction of human anti-mouse responses after repeated use; and finally, 5): the BsAb should be small enough to penetrate the tumor but also large enough to remain in the circulation for a sufficient period of time.

[0250] Based on these points above, numerous BsAbs have been developed in the past few years to trigger many kinds of immune effector cells and target different tumor cells, including T lymphocytes, NK cells, monocytes, macrophages, neutrophils, LAK cells (lymphokine-activated cytotoxic cells), and TIL cells (tumor-infiltrating lymphocytes), etc. T cells are generally recognized as the main specific cells for immune response. CD3, expressed on the surface of all mature T cells, is a common surface marker on T cells. CD3 binds to TCR non-covalently to form the whole TCR-CD3 complex and participates in the immune response to antigen stimulation. Currently, CD3 is the most widely and successfully used surface trigger molecule on immune effector cells. After the anti-CD3 antibody in the BsAb binds to the CD3 molecule on the surface of T cells, many effects occur to kill tumor cells, including: (1) T cell proliferation and differentiation. First, BsAb can activate resting T cells, resulting in Th cells and Tc cells derived from early effector T cells with CD4+ or CD8+. Second, BsAb can activate a large number of memory cells to proliferate and differentiate into effector T cells that attack and kill tumor cells. The number of effector cells is directly related to the rate of tumor elimination. (2) Cytokine release: CD4+Th cells activated by BsAb can secrete large amounts of IL-2. IL-2 not only stimulates the proliferation of Th cells in autocrine secretion, but also activates naive CD8+T cells to become Tc cells in paracrine secretion, resulting in the expansion of cytotoxicity of Tc cells. In addition, IL-2 is a costimulatory signal for activating T cells. Therefore, IL-2 plays an important role in BsAb-mediated immune effects. Some other cytokines, such as TNF-α and IFN-γ, are produced in the process of T cell activation and can result in a "bystander" effect by inhibiting the growth of "bystander" tumor cells through the intercellular medium.(3) Cytotoxicity: In vitro experiments have shown that when mediated by BsAb, CD8+Tc directly interact with tumor cells, release cytotoxic substances via granule exocytosis, and lyse the target cells, which usually occurs rapidly within 4-6 hours after targeting tumor cells. The main components of the cytotoxic substances are perforin and serine esterase or granzyme. Perforin attacks the plasma membrane and forms ion channels, thus causing the ingress of large amounts of ions and water, resulting in cell lysis and necrosis, while granzyme is similar to lymphotoxin and can activate intracellular DNase, thus causing the lysis of nuclear DNA and resulting in the apoptosis of the target cells.

[0251] Currently, Fv fragments are widely used for the construction of BsAbs because they are the smallest unit with a complete antigen-binding site, small (about one sixth of the total antibody), lack an Fc domain, low immunogenicity, and easily penetrate vascular walls and solid tumors. However, because covalent bonds between the VH and VL domains cannot be generated, Fvs are unstable and prone to dissociation in vivo. To improve the stability of Fv fragments, a polypeptide intralinker between the VH and VL domains is used to form a so-called ScFv. Intralinkers are generally short, flexible peptides with a length of 15 amino acid residues, such as (Gly4Ser)3. In one embodiment of the present invention, an intralinker is used for anti-CD3 scFv and a different intralinker is used for anti-PSMA scFv.

[0252] Bispecific antibodies with standard IgG format can be difficult to produce because they contain four different polypeptide chains. The effectiveness of smaller, more easily produced bispecific molecules has been clinically demonstrated in non-Hodgkin's lymphoma. See, for example, Bargou et al. (2008), Science 321(5891):974-977.

[0253] Due to the short in vivo half-life of this small single chain molecule, chronic administration by continuous intravenous infusion was used to achieve these results. Thus, there is a need in the art for bispecific therapeutics that retain similar therapeutic efficacy, have a format that is less complicated to produce, and have favorable pharmacokinetic properties, including a longer half-life.

[0254] As described herein, bispecific Fc (Bs-Fc) can bind to two different proteins, including the Fc region of an antibody or a portion thereof. Bs-Fc can have favorable pharmacokinetic properties compared to bispecific single chain molecules lacking the Fc region. One protein bound by Bs-Fc can be expressed in immune effector cells, such as T cells, NK cells, neutrophils, or macrophages, and the other protein can be expressed in target cells, e.g., cancer cells, cells infected by pathogens, or cells that mediate disease, such as fibroblasts that cause fibrosis. The Bs-Fc molecules described herein can induce activation of immune effector cells in the presence of target cells and / or killing of target cells in the presence of immune effector cells.

[0255] In some embodiments, the anti-PSMA binding domain comprises an antibody or antigen-binding fragment thereof described elsewhere herein. For example, in some embodiments, the anti-PSMA binding domain comprises Mut-gy1, Mut-gy1-st, Mut-gy1-2, Mut-gy1-3, or PSMAbLm, as described elsewhere herein.

[0256] Treatment method In another aspect, the disclosure further provides a method of preventing or treating a proliferative disease associated with expression of PSMA in a subject. In some embodiments, the method comprises administering to the subject an effective amount of an antibody or antigen-binding fragment thereof or variant thereof, a bispecific or multispecific antibody or antigen-binding fragment thereof, a CAR, a nucleic acid molecule, a vector, a cell, or a composition described herein. In some embodiments, the subject is a mammal, e.g., a human.

[0257] In some embodiments, the method further comprises administering to the subject a second agent or therapy. In some embodiments, the second agent or therapy comprises an anti-cancer agent. In some embodiments, the second agent or therapy is administered to the subject before, after, or simultaneously with the antibody or antigen-binding fragment thereof or variant thereof, bispecific or multispecific antibody or antigen-binding fragment thereof, CAR, nucleic acid molecule, vector, cell, or composition described herein.

[0258] In one embodiment, the invention provides a method of treating a solid tumor with aberrant PSMA expression, such as prostate cancer or a solid tumor with high PSMA expression in angiogenesis, in a subject, comprising targeting high PSMA expressing cells with an antibody or antigen-binding fragment. In some embodiments, the method comprises administering to a subject having a tumor associated with high PSMA expression a composition comprising the antibody or antigen-binding fragment thereof.

[0259] In one embodiment, the antibody or antigen-binding fragment thereof is operably linked to a biologically active agent or combination of such agents, the agent being a toxin, a radioisotope, a nanoparticle, or a bioactive peptide. These tumors include, but are not limited to, prostate cancer, lung cancer, liver cancer, pancreatic cancer, colon cancer, gastric cancer, breast cancer, ovarian cancer, renal cancer, prostate cancer, bladder cancer, melanoma, glioma, etc.

[0260] In one embodiment, provided herein is a method of inhibiting or suppressing a tumor in a subject, hi another embodiment, the method comprises administering an effective amount of an antibody or antigen-binding fragment described herein.

[0261] In another embodiment, provided herein is a method of slowing the progression of a solid tumor in a subject. In yet another embodiment, the method comprises administering to the subject an effective amount of an antibody or antigen-binding fragment thereof provided herein. In another embodiment, the subject mounts an immune response against angiogenesis of the solid tumor, thereby slowing the progression of the solid tumor in the subject.

[0262] In one embodiment, the term "operably linked" refers to the positioning / linking of two or more molecules or sequences in a manner that ensures proper function or expression of the molecules and sequences.

[0263] In one embodiment, the term "therapeutically effective amount" refers to an amount that provides a therapeutic effect for a given condition and administration regimen. In the present invention, a therapeutic effect is the prevention or inhibition of tumor growth, invasion, spread, metastasis or recurrence, or preferably a reduction in tumor burden, or an improvement in patient outcome.

[0264] In one embodiment, the term "preventing or treating" refers to any one or more of delaying the onset of symptoms, reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, alleviating symptoms, reducing secondary symptoms, reducing secondary infections, extending patient survival, preventing disease recurrence, reducing the number or frequency of recurrent episodes, increasing the latency between episodes of symptoms, increasing the time to sustained progression, promoting remission, inducing remission, increasing remission, speeding up recovery, or increasing the effectiveness of or reducing resistance to alternative therapies. In one embodiment, "treating" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose being to prevent or alleviate the targeted pathological condition or disorder.

[0265] In another embodiment, a "symptom" is a manifestation of a disease or pathological condition described above.

[0266] In one embodiment, a composition of the invention comprises a polypeptide, antibody, or antigen-binding fragment of the invention, alone or, in some embodiments, in combination with a second pharma- ceutical active agent, hi another embodiment, the composition further comprises a proteolytic inhibitor, a pharmaceutical carrier, a diluent, and an adjuvant.

[0267] In one embodiment, the term "pharmaceutical active agent" refers to any pharmaceutical agent that fulfills an indicated purpose. In some embodiments, pharmaceutical active agents include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, angiogenesis inhibitors, tumor imaging probes, immunomodulatory agents, or any other tumor therapy and / or imaging drugs / agents, etc.

[0268] In another embodiment, provided herein is a method of delivering a biologically active agent and an antibody or antigen-binding fragment of the invention for the treatment of a tumor in a subject. In another embodiment, the method comprises simultaneous but separate administration of the biologically active agent and the antibody or antigen-binding fragment. In another embodiment, the method comprises separate administration of the biologically active agent and the antibody or antigen-binding fragment.

[0269] In one embodiment, the antibodies or antigen-binding fragments provided herein are themselves "biologically active", meaning that they, even after modification, are capable of exerting the biological action or an enhanced action of their corresponding parent antibody, particularly in terms of binding to the target antigen, inhibiting or even modulating the binding of a ligand to its receptor, particularly in inhibiting antigen-mediated signal transduction, and preventing or treating antigen-mediated diseases. The term "biologically active", when used in connection with any of the biologically active agents described herein, also refers to the ability of the agent to modulate the immune response in a manner that can result in a prophylactic, diagnostic, or therapeutic effect, as will be understood by those skilled in the art. In some embodiments, the agents used to achieve this biological activity include, but are not limited to, cytokines, enzymes, chemokines, radioisotopes, enzymatically active toxins, therapeutic nanoparticles, or chemotherapeutic agents, as will be understood by those skilled in the art.

[0270] In an alternative embodiment, the antibody polypeptides are conjugated or operably linked to enzymes to function for their intended purpose in order to use antibody-dependent enzyme-mediated prodrug therapy (ADEPT). ADEPT can be used by conjugating or operably linking the antibody or Fc fusion to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent) into an active anticancer drug. The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme that can act on a prodrug in such a way as to convert it into its more active cytotoxic form. Additional modifications of the modified molecules provided herein are contemplated herein. For example, the polypeptide / antibody can be conjugated to one of a variety of non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol.

[0271] In another embodiment, the antibodies / polypeptides provided herein are administered with one or more immunomodulatory agents that may increase or decrease the production of one or more cytokines, up- or down-regulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation state of one or more types of immune cells. Immunomodulators include nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (e.g., glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, trimcinolone, azulfidine icosanoids such as prostaglandins, thromboxanes, and leukotrienes; and anthralin. , topical steroids such as calcipotriene, clobetasol, and tazarotene; cytokines such as TGFβ, IFNα, IFNβ, IFNγ, IL-2, IL-4, IL-10; cytokines, chemokines, or receptor antagonists such as BATF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD2O, CD23, CD28, CD40, CD40L, CD44, CD45, CD52, CD64, CD80, CD86, CD147, CD152, complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFN-αIFN-β, IFN-γ., IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9 IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3, MHC, selectins, TGF-β, TNF-α, TNF-β, TNF-R1, antibodies against T-cell receptors, soluble antibodies, and receptor-Fc fusions, including Enbrel® (etanercept), Humira® (adalimumab), Remicade® (infliximab), PD1 antibodies (OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab)) or PD-L1 antibodies (durvalumab, MPDL3280A); species antilymphocyte globulin; 2-amino-6-aryl-5-substituted pyrimidines, anti-idiotypic antibodies against MHC-binding peptides and MHC fragments, azathioprine, brequinar, bromocriptine, cyclophosphamide, cyclosporine A, D-penicillamine, deoxyspergualin, FK506, glutaldehyde, gold, hydroxychloroquine, leflunomide, malononitriloamide (e.g., leflunomide), methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin, and other immunomodulatory molecules such as sulfasasazine.

[0272] In an alternative embodiment, the antibodies of the invention are administered with a cytokine. As used herein, "cytokine" is meant as a general term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines are fibroblast growth factors, prolactin, placental lactogen, tumor necrosis factor-alpha and -beta, Mullerian inhibitory factor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factors such as NGF-beta, platelet growth factors, transforming growth factors (TGFs) such as TGF-alpha and TGF-beta, insulin-like growth factors-I and -II, erythropoietin (EPO), bone morphogenetic factors, interferon-alpha, -beta, and -gamma. Examples of cytokines include interferons such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, tumor necrosis factors such as IL-15, TNF-alpha or TNF-beta, and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0273] Chemotherapeutic agents or other cytotoxic agents can be administered as prodrugs. The term "prodrug" refers to a precursor or derivative form of a pharmacologic active substance that is less cytotoxic to tumor cells compared to the parent drug and can be enzymatically activated or converted to a more active parent form. See, for example, Wilman, 1986, Biochemical Society Transactions, 615th Meeting Belfast, 14:375-382, and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.): 247-267, Humana Press, 1985. Prodrugs that may find use with the compositions and methods provided herein include, but are not limited to, phosphate-containing prodrugs, thiosulfate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouridine prodrugs that can be converted to more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use with the antibodies / polypeptides of the compositions and methods provided herein include, but are not limited to, any of the chemotherapeutic agents described above.

[0274] In some embodiments, any combination of antibody / polypeptide and biologically active agent identified above, i.e., cytokine, enzyme, chemokine, radioisotope, enzymatically active toxin, or chemotherapeutic agent, may be applied. In another embodiment, the antibody / polypeptide may be operably linked to a biologically active agent and used in the methods described herein, or the antibody / polypeptide provided herein may simply be used in combination with a biologically active agent in a manner in which both are administered separately (i.e., not conjugated) to achieve the desired prophylactic, diagnostic, or therapeutic effect.

[0275] Combination therapy Compositions comprising the antibodies, antibody fragments, ADCs, CAR-expressing cells, or bispecific antibodies described herein may be used in combination with other known drugs and therapies. Administered "in combination" as used herein means that two (or more) different therapies are delivered to a subject during the course of the subject's suffering from a disorder, e.g., two or more therapies are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or before the treatments are discontinued for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "co-delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments in either case, the treatments are more effective by combined administration. For example, the second treatment is more effective than would be shown if the second treatment were administered in the absence of the first treatment, e.g., a comparable effect is shown with the second treatment to a lesser extent, or the second treatment reduces symptoms to a greater extent, or a similar situation is shown with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with one treatment delivered in the absence of the other treatment. The effect of the two treatments can be partially additive, fully additive, or greater than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0276] A composition comprising an antibody, antibody fragment, ADC, CAR-expressing cell, or bispecific antibody described herein and at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially. In sequential administration, the CAR-expressing cell described herein can be administered first and the additional agent can be administered second, or the order of administration can be reversed.

[0277] In further aspects, compositions comprising the antibodies, antibody fragments, ADCs, CAR-expressing cells, or bispecific antibodies described herein may be used in treatment regimens in combination with surgery, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506 antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation, or peptide vaccines as described in Izumoto et al. 2008 J Neurosurg 108:963-971.

[0278] In one embodiment, a composition comprising an antibody, antibody fragment, ADC, CAR-expressing cell, or bispecific antibody described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include antiandrogens (androgen antagonists), anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab), antimetabolites (e.g., folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulators such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0279] Common chemotherapy agents considered for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU), and rifampicin (Ricin®). (registered trademark), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubicin HCl), bidine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (Diazepam®), fluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Milotarg,These include paclitaxel (Taxol®), Phoenix (Yttrium 90 / MX-DTPA), pentostatin, polipheprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

[0280] Exemplary antiandrogen (androgen antagonist) agents include bicalutamide, goserelin acetate SR depot.

[0281] Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil Nitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (A medel®, Vercyte®), triethylenemelamine (Hemel®, Hexylen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and decarbazine (DTIC-Dome®).Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®), temozolomide (Temodar® and Temodal®), dactinomycin (also known as actinomycin-D, Cosmegen®), melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®), altretamine (also known as hexamethylmelamine (HMM), Hexylen®), carmustine (BiCNU®), bendamustine (Treanda®, busulfan (Busulfex® and Myleran®), carboplatin (Paraplatin®), lomustine (also known as CCNU, CeeNU®), cisplatin (also known as CDDP, Platinol® and Platinol®-AQ), chlorambucil (Leukeran®), cisplatin (also known as cisplatin, ... Clophosphamide (Cytoxan® and Neosar®), dacarbazine (also known as DTIC, DIC, and imidazole carboxamide, DTIC-Dome®), altretamine (also known as hexamethylmelamine (HMM), Hexylen®), ifosfamide (Ifex®), prednisolone, procarbazine (Matulane®), mechlorethamine (also known as nitrogen mustard, mustine, and methicillin), These include chloroethamine hydrochloride, Mustargen®), streptozocin (Zanosar®), thiotepa (also known as thiophosphoamide, TESPA, and TSPA, Thioplex®), cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®), and bendamustine hydrochloride (Treanda®).

[0282] Exemplary mTOR inhibitors include, for example, temsirolimus, ridaforolimus (formerly known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,3 6-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldyldimethylphosphinic acid, also known as AP23573 and MK8669, and described in PCT Publication No. 03 / 064383), everolimus (Afinitor® or RAD001), rapamycin (AY229 89, Sirolimus®), simapimod (CAS 164301-51-3), emsirolimus, (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055), 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl- ethyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4), and N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartyl-L-serine-, inner salt (SF1126, CAS 936487-67-1), and XL765.

[0283] Exemplary immunomodulatory agents include, for example, afutuzumab (available from Roche®), pegfilgrastim (Neulasta®), lenalidomide (CC-5013, Revlimid®), thalidomide (Thalomid®), actimid (CC4047), and IRX-2 (a mixture of human cytokines including interleukin-1, interleukin-2, and interferon-gamma, CAS951209-71-5, available from IRX Therapeutics).

[0284] Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®), bleomycin (Lenoxane®), daunorubicin (dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®), daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®), mitoxantrone (DHAD, Novantrone®), epirubicin (Ellence™), idarubicin (Idamycin®, Idamycin PFS®), mitomycin C (Mutamycin®), geldanamycin, herbimycin, rabidomycin, and desacetylrabidomycin.

[0285] Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®, and vindesine (Eldisine®), vinblastine (also known as vinblastine sulfate, vincaleukoblastine, and VLB, Alkaban-AQ®, and Velban®), and vinorelbine (Navelbine®).

[0286] Exemplary proteosome inhibitors include bortezomib (Velcade®), carfilzomib (PX-171-007, (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutadiene mido)-pentanamide), marizomib (NPI-0052), ixazomib citrate (MLN-9708), delanzomib (CEP-18770), and O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0287] Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as the GITR fusion proteins described in U.S. Pat. No. 6,111,090, European Patent No. 090505B1, U.S. Pat. No. 8,586,023, PCT Publication Nos. 2010 / 003118 and 2011 / 090754, or those described in, for example, U.S. Pat. No. 7,025,962, European Patent No. 1947183B1, U.S. Pat. No. 7,812,135, U.S. Pat. No. 8,388,967, U.S. Pat. No. 8,591,886, European Patent No. 1866339, PCT Publication No. 2011 / 028683, PCT Publication No. 2013 / 039954, PCT Publication No. 2005 / 007190, PCT Publication No. 2007 / 133822, PCT Publication No. 2005 / 055808, PCT Publication No. 99 / 40196, PCT Publication No. 2001 / 03720, PCT Publication No. 99 / 20758, PCT Publication No. 2006 / 083289, PCT Publication No. 2005 / 115451, U.S. Patent No. 7,618,632, and PCT Publication No. 2011 / 051726.

[0288] In one embodiment, a subject can be administered an agent that reduces or ameliorates a side effect associated with administration of a naked antibody or antigen-binding fragment thereof, ADC, CAR-expressing cell, or bispecific antibody.

[0289] In one embodiment, a subject can be administered an agent that enhances the activity of a naked antibody or antigen-binding fragment thereof, an ADC, a CAR-expressing cell, or a bispecific antibody. For example, in one embodiment, the agent can be an agent that inhibits an inhibitory molecule. An inhibitory molecule, e.g., programmed death 1 (PD1), can, in some embodiments, reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR beta. Inhibition of an inhibitory molecule can optimize the performance of a naked antibody or antigen-binding fragment thereof, an ADC, a CAR-expressing cell, or a bispecific antibody, e.g., by inhibition at the DNA, RNA, or protein level. In an embodiment, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, can be used to inhibit expression of an inhibitory molecule in a CAR-expressing cell or a bispecific Ab-responsive cell. In an embodiment, the inhibitor is an shRNA. In one embodiment, the inhibitor of the inhibitory signal can be, for example, an antibody or antigen-binding fragment thereof that binds to an inhibitory molecule. For example, the agent can be an antibody or antigen-binding fragment thereof that binds to PD1, PD-L1, PD-L2, or CTLA4 (e.g., ipilimumab (also known as MDX-010 and MDX-101, commercially available as Yervoy®, Bristol-Myers Squibb; tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675, 206). In an embodiment, the agent is an antibody or antigen-binding fragment thereof that binds to TIM3. In an embodiment, the agent is an antibody or antigen-binding fragment thereof that binds to LAG3.

[0290] PD1 is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2, have been shown to downregulate T cell activation when bound to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1. Antibodies, antibody fragments, and other inhibitors of PD1, PD-L1, and PD-L2 are available in the art and can be used in combination with the PSMA-targeted naked antibodies or antigen-binding fragments thereof, ADCs, CAR-expressing cells, or bispecific antibodies described herein. For example, Nivolumab (also called BMS-936558 or MDX1106, Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in U.S. Patent No. 8,008,449 and WO2006 / 121168. Pidilizumab (CT-011, Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1 Pidilizumab, another humanized anti-PD1 monoclonal antibody, is disclosed in WO2009 / 101611. Lambrolizumab (also called MK03475, Merck) is a humanized IgG4 monoclonal antibody that binds to PD1.Lambrolizumab and other humanized anti-PD1 antibodies are disclosed in U.S. Patent No. 8,354,509 and WO2009 / 114335. MDPL3280A (Genentech / Roche) is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed in U.S. Patent No. 7,943,743 and U.S. Publication No. 2012 / 0039906. Other anti-PD-L1 binding agents include YW243.55.S70 (heavy and light chain variable regions shown in SEQ ID NOs: 20 and 21 in WO2010 / 077634) and MDX-1 105 (also referred to as BMS-936559, e.g., an anti-PD-L1 binding agent disclosed in WO2007 / 005874). AMP-224 (B7-DCIg, Amplimmune, disclosed, e.g., in WO2010 / 027827 and WO2011 / 066342) is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1. Other anti-PD1 antibodies include AMP514 (Amplimmune), among others, anti-PD1 antibodies disclosed, e.g., in U.S. Patent No. 8,609,089, US2010 / 028330, and / or US2012 / 0114649.

[0291] In some embodiments, the agent that enhances the activity of a CAR-expressing cell can be, for example, a fusion protein comprising a first domain and a second domain, where the first domain is an inhibitory molecule or a fragment thereof, and the second domain is a polypeptide associated with a positive signal, for example, a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the polypeptide associated with a positive signal can comprise, for example, a costimulatory domain of CD28, CD27, ICOS, for example, an intracellular signaling domain of CD28, CD27, and / or ICOS, and / or a primary signaling domain, for example, CD3 zeta, as described herein. In one embodiment, the fusion protein is expressed by the same cell that expressed the CAR. In another embodiment, the fusion protein is expressed by a cell, for example, a T cell that does not express an anti-PSMA CAR.

[0292] Formulation and route of administration The antibodies, ADCs, CAR-expressing cells, or bispecific antibodies of the invention may find use in a wide range of products. In one embodiment, the antibodies, ADCs, CAR-expressing cells, or bispecific antibodies of the invention are therapeutic, diagnostic, or research reagents. In one embodiment, the antibodies, ADCs, CAR-expressing cells, or bispecific antibodies of the invention are therapeutic agents. In some embodiments, the antibodies or antigen-binding fragments thereof, ADCs, CAR-expressing cells, or bispecific antibodies of the invention are used for industrial use. The antibodies of the invention may find use in antibody compositions that are monoclonal or polyclonal. The antibodies of the invention may be agonistic, antagonistic, neutralizing, inhibitory, or stimulatory. In one embodiment, the antibodies or antibody fragments, ADCs, CAR-expressing cells, or bispecific antibodies of the invention are used to kill target cells bearing the target antigen, e.g., cancer cells. In alternative embodiments, the antibodies of the invention are used to block, antagonize, or agonize the target antigen. In an alternative embodiment, the antibodies of the present invention are used to block, antagonize, or agonize the target antigen and kill the target cells bearing the target antigen. In another embodiment, the target cells are tumor cells or their angiogenesis. In one embodiment, angiogenesis plays an important role in blood vessel formation and is considered to be a target for the antibodies or antibody fragments, ADCs, CAR-expressing cells, or bispecific antibodies provided herein.

[0293] The present disclosure further provides a kit comprising one or more compositions of the invention, including pharmaceutical formulations, packaged in a suitable packaging material. In another embodiment, the kit comprises a nucleic acid encoding an antibody or antigen-binding fragment thereof, Car T or Car NK cells, or a bispecific antibody of the invention. In additional embodiments, the kit further comprises a nucleic acid comprising an expression control element, an expression vector, a viral expression vector, an adeno-associated virus expression vector, an adenovirus expression vector, and a retrovirus expression vector. In yet additional embodiments, the kit comprises a cell expressing an antibody or antigen-binding fragment thereof of the invention, such as a Car T cell or a Car NK cell.

[0294] In additional embodiments, the kit comprises a label or packaging insert containing instructions for expressing the antibody or bispecific antibody, or a nucleic acid encoding the antibody, antigen-binding fragment, or bispecific antibody, in a cell in vitro, in vivo, or ex vivo. In yet additional embodiments, the kit comprises a label or packaging insert containing instructions for treating a subject (e.g., a subject having or at risk for having asthma) with an antibody or antigen-binding fragment thereof, ADC, CAR-expressing cell, or bispecific antibody of the invention in vivo or ex vivo.

[0295] As used herein, the term "packaging material" refers to a physical structure that contains the components of the kit. The packaging material can maintain the components sterile and can be made of materials commonly used for such purposes (e.g., paper, cardboard, glass, plastic, foil, ampoules, etc.). The label or package insert can include appropriate written instructions, e.g., for performing the method of the invention, e.g., for treating the common cold, etc. The kit can additionally further include instructions for using the kit components in the method of the invention.

[0296] The instructions can include instructions for carrying out any of the methods of the invention described herein. Thus, the pharmaceutical compositions of the invention can be included in a container, pack, or dispenser together with instructions for administration to a subject. The instructions can further include indications of satisfactory clinical endpoints or any adverse symptoms that may occur, or additional information required by the Food and Drug Administration for use in human subjects.

[0297] In one embodiment, the polypeptides of the invention are administered as part of a vaccine. In some embodiments, the term vaccine should be understood to encompass any immunomodulatory composition, and such vaccines may include, in addition to the polypeptides of the invention, adjuvants, antigens, immunomodulatory compounds, or combinations thereof.

[0298] In some embodiments, the adjuvant includes (A) an aluminum compound (e.g., aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, oxyhydroxide, orthophosphate, sulfate, etc. [see, e.g., chapters 8 and 9 of Ref. 96]) or a mixture of different aluminum compounds having any suitable morphology (e.g., gel, crystalline, amorphous, etc.) and with favorable adsorption; (B) MF59 (5% squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer); (C) liposomes; (D) ISCOMs, which may lack additional detergent; (E) SAF, containing 10% squalane, 0.4% Tween 80, 5% Pluronic-block polymer L121, and thr-MDP, microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion; (F) Ribi™ Adjuvant System (RAS), (Ribi Immunochem), 2% squalene, 0.(G) saponin adjuvants, such as QuilA or QS21, also known as Stimulon™, containing 2% Tween 80 and one or more bacterial cell wall components from the group consisting of monophospholipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (H) chitosan; (I) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (J) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-γ), macrophage colony stimulating factor, tumor necrosis factor, etc.; (K) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL)]; (L) 3d (M) an oligonucleic acid comprising a CpG motif, i.e. containing at least one CG dinucleotide, with 5-methylcytosine optionally used instead of cytosine, (N) a polyoxyethylene ether or polyoxyethylene ester, (O) a polyoxyethylene sorbitan ester surfactant in combination with octoxynol, or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as octoxynol, (P) an immunostimulatory oligonucleic acid (e.g., a CpG oligonucleic acid) and a saponin, (O) an immunostimulatory agent and particles of a metal salt, (R) a saponin and an oil-in-water emulsion, (S) a saponin (e.g., QS21) + 3dMPL + IL12 (optionally + sterol), (T) E.Examples of suitable antimicrobial agents include, but are not limited to, E. coli heat labile enterotoxin ("LT"), or a detoxified mutant thereof, such as the K63 or R72 mutant, (U) cholera toxin ("CT"), or diphtheria toxin ("DT"), or a detoxified mutant of either, (V) double-stranded RNA, (W) a monophosphoryl lipid A mimic, such as an aminoalkyl glucosaminide phosphate derivative, e.g., RC-529, (X) polyphosphazene (PCPP), or (Y) a bioadhesive, such as an esterified hyaluronic acid microsphere, or a mucoadhesive, such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides, and carboxymethylcellulose.

[0299] In some embodiments, administration of the antibody or antigen-binding fragment thereof is intended to reduce the severity of a pathological condition. It should be understood that by the term "reducing the severity of a pathological condition", any reduction via the methods, compounds, and compositions disclosed herein is considered to be encompassed by the present invention. In some embodiments, reduction in severity may include enhanced survival, or in another embodiment, halting disease progression, or in another embodiment, delaying disease progression.

[0300] In one embodiment, the dosage depends on the cellular responsiveness to the administered molecule / compound or composition containing it. In general, the dosage utilized for the above purposes will vary, but will be an effective amount to produce the desired effect as determined by a clinician skilled in the art. As used herein, the term "pharmaceutical effective amount" refers to the amount of the compound described herein that brings about the desired relief of symptoms or other desired phenotype in a patient.

[0301] In one embodiment, the concentration of the antibody or antigen-binding fragment thereof will depend on various factors, including the nature of the condition to be treated, the condition of the patient, the route of administration, and individual tolerability of the composition.

[0302] In some embodiments, any of the compositions of the invention consists essentially of an antibody or antigen-binding fragment thereof described herein. In some embodiments, the term "comprising" refers to the inclusion of the indicated active agent, such as an antibody or antigen-binding fragment thereof of the invention, as well as other active agents, and pharma- ceutically acceptable carriers, excipients, softeners, stabilizers, and the like, as are known in the pharmaceutical arts.

[0303] In some embodiments, the compositions of the invention consist essentially of the polypeptides / polynucleic acids / vectors described herein. In some embodiments, the term "consisting essentially of" refers to a composition in which the only active ingredient of a particular class of agent is the indicated active ingredient, however, other compounds directly involved in the therapeutic effect of the indicated active ingredient may be included. In some embodiments, the term "consisting essentially of" refers to a composition in which the only active ingredient is the indicated active ingredient that targets a particular mechanism or acts through a particular pathway, however, other compounds directly involved in the therapeutic effect of the indicated active ingredient may be included, e.g., have a mechanism of action related to, but not directly related to, the indicated agent. In some embodiments, the term "consisting essentially of" refers to a composition in which the only active ingredient is the indicated active ingredient. However, other compounds not directly involved in the therapeutic effect of the indicated active ingredient may be included, such as for stabilizing, preserving, etc. the formulation. In some embodiments, the term "consisting essentially of" may refer to a component that facilitates the release of the active ingredient. In some embodiments, the term "consisting essentially of" refers to a composition that includes an active ingredient and a pharma- ceutically acceptable carrier or excipient.

[0304] It is understood that the actual amount of antibody or antigen-binding fragment in any particular case will vary depending on the particular composition formulated, the mode of application, and the particular condition and organism being treated. Dosages for a given host can be determined using conventional considerations, for example, by an appropriate conventional pharmacological protocol, for example, by routine comparison of the differential activity of the subject compounds with known agents.

[0305] In one embodiment, the compounds of the present invention can be administered acutely for acute treatment of a temporary condition, or chronically, especially in the case of progressive, recurrent, or degenerative diseases. In one embodiment, one or more compounds of the present invention can be administered simultaneously, or in another embodiment, they can be administered in a staggered manner. In one embodiment, the staggered manner can be determined by the stage or stage of the disease.

[0306] Parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose, sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, such as those based on Ringer's dextrose, electrolyte replenishers, and the like. Examples are sterile liquids such as water and oils, with or without the addition of surfactants and other pharma-ceutically acceptable adjuvants. In general, water, saline, aqueous dextrose, and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[0307] In one embodiment, the route of administration may be parenteral, or a combination thereof. In another embodiment, the route may be intraocular, conjunctival, topical, transdermal, intradermal, subcutaneous, intraperitoneal, intravenous, intraarterial, vaginal, rectal, intratumoral, parcanceral, transmucosal, intramuscular, intravascular, intraventricular, intracranial, inhalation (aerosol), nasal aspirate (spray), intranasal (drops), sublingual, oral, aerosol, or suppository, or a combination thereof. In one embodiment, the dosing regimen is determined by a skilled clinician based on factors such as the exact nature of the condition being treated, the severity of the condition, the age and general physical condition of the patient, weight, and the response of the individual patient.

[0308] For intranasal or inhalation application, a solution or suspension of the compound that is mixed and aerosolized or nebulized in the presence of a suitable carrier is suitable. Such aerosols may include any of the agents described herein.

[0309] For parenteral application, injectable sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories and enemas, are particularly suitable. Ampoules are convenient unit dosages. Such suppositories may contain any of the agents described herein.

[0310] Sustained or direct release compositions can be formulated, for example, in liposomes, or those in which the antibody or antigen-binding fragment is protected with a differentially degradable coating, for example, by microencapsulation, multiple coatings, etc. Such compositions can be formulated for immediate or delayed release. It is also possible to lyophilize the new compounds and use the resulting lyophilizates to prepare products, for example, for injection.

[0311] For liquid preparations, pharma- ceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.

[0312] Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.

[0313] In one embodiment, the compositions of the method of the present invention or the compositions used in the method of the present invention can be administered alone or in a composition. In another embodiment, the compositions of the present invention can be used in admixture with conventional excipients that do not adversely react with the active compounds, i.e., pharma- ceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral), or topical application. In one embodiment, suitable pharma-ceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose, or starch, magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginic acid, hyaluronic acid, collagen, perfume oils, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. In another embodiment, the pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliary substances which do not deleteriously react with the active compounds, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances, etc. In another embodiment, they can also be combined, if desired, with other active agents, such as vitamins.

[0314] Pharmaceutical compositions include "pharmaceutical acceptable" and "physiologically acceptable" carriers, diluents, or excipients. In one embodiment, the terms "pharmaceutical acceptable" and "physiologically acceptable" refer to any formulation that is safe and provides suitable delivery for the desired route of administration of an effective amount of at least one antibody or antigen-binding fragment for use in the present invention. The terms also refer to the use of buffered formulations, as well as the use of a buffered formulation, where the pH is maintained at a specific desired value ranging from pH 4.0 to pH 9.0 according to the stability of the compound and the route of administration. These terms include solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonicity agents, and absorption enhancing or retarding agents that are compatible with pharmaceutical administration. Such formulations can be contained in liquids: emulsions, suspensions, syrups, or elixirs, or in solid forms: tablets (coated or uncoated), capsules (hard or soft), powders, granules, crystals, or microbeads. Supplementary active compounds (eg, preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.

[0315] The pharmaceutical compositions of the present invention include, but are not limited to, nitrogen mustards (e.g., cyclophosphamide and ifosfamide), aziridines (e.g., thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine and streptozocin), platinum complexes (e.g., carboplatin and cisplatin), non-classical alkylating agents (e.g., dacarbazine and temozolamide), folic acid analogs (e.g., methotrexate), purine analogs (e.g., fludarabine and mercaptopurine), adenosine analogs (e.g., cladribine and pentostatin), pyrimidine analogs (e.g., fluorouracil (alone or in combination with leucovorin) and gemcitabine), substituted ureas (e.g., hydroxyurea), antitumor and anticancer agents, and the like. The therapeutic agent may include one or more additional chemotherapeutic agents selected from the group consisting of biologics (e.g., bleomycin and doxorubicin), epipodophyllotoxins (e.g., etoposide and teniposide), microtubule agents (e.g., docetaxel and paclitaxel), camptothecin analogs (e.g., irinotecan and topotecan), enzymes (e.g., asparaginase), cytokines (e.g., interleukin-2 and interferon-α), monoclonal antibodies (e.g., trastuzumab and bevacizumab), recombinant toxins and immunotoxins (e.g., recombinant cholera toxin-B and TP-38), cancer gene therapy, physical therapy (e.g., hyperthermia, radiation therapy, and surgery), and cancer vaccines (e.g., vaccines against telomerase).

[0316] Compositions of the invention (e.g., antibodies, bispecific molecules) can also be administered together with complement. Thus, compositions comprising human antibodies, multispecific or bispecific molecules, and serum or complement are within the scope of the invention. These compositions are advantageous in that the complement is located in close proximity to the human antibodies, multispecific or bispecific molecules. Alternatively, the human antibodies, multispecific or bispecific molecules of the invention and the complement or serum can be administered separately.

[0317] Pharmaceutical compositions can be formulated to be compatible with a particular local or systemic administration route. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by a particular route. Specific non-limiting examples of administration routes for the compositions of the present invention are inhalation or intranasal delivery. Additional routes include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, transdermal (topical), transmucosal, and rectal administration.

[0318] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citric acid, or phosphate; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.

[0319] Pharmaceutical compositions for injection include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars, polyalcohols, for example, mannitol, sorbitol, sodium chloride, can be included in the composition. The inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin, can prolong absorption of the injectable compositions.

[0320] Sterile injectable solution can be prepared by incorporating antibody or its antigen-binding fragment in the required amount in a suitable solvent with one or a combination of the above-mentioned components, followed by filtration sterilization.Generally, dispersion is prepared by incorporating antibody or its antigen-binding fragment into a sterile vehicle that contains a basic dispersion medium and other components as above.In the case of sterile powder for preparing sterile injectable solution, the preparation method includes, for example, vacuum drying and freeze-drying, which produces a powder of active ingredient and any additional desired ingredient from its solution that has been previously sterilized and filtered.

[0321] For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays, inhalation devices (e.g., aspirators), or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams, as generally known in the art.

[0322] The antibodies of the present invention, including subsequences and modified forms and the nucleic acids encoding them, can be prepared with carriers that will protect them against rapid elimination from the body, such as a controlled release formulation or a time delay material, such as glyceryl monostearate or glyceryl stearate. The compositions can also be delivered using implants and microencapsulated delivery systems to achieve sustained or controlled delivery locally or systemically.

[0323] Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for the preparation of such formulations will be apparent to those skilled in the art. Materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes that target cells or tissues using antibodies or viral coating proteins) can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0324] Additional pharmaceutical formulations suitable for compositions for administration in the methods of the present invention are well known in the art (see, for example, Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa., The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, NJ, and Pharmaceutical Principles of Solid Dosage Forms, Technonic Publishing Co., Inc., Lancaster, Pa., (1993)). Pharmaceutical formulations can be packaged in dosage unit form for ease of administration and uniformity of dosage. As used herein, "dosage unit form" refers to physically discrete units suitable as unitary dosages for subjects to be treated, each unit containing a predetermined amount of an antibody or antigen-binding fragment thereof calculated to produce the desired therapeutic effect, together with a pharmaceutical carrier or excipient.

[0325] Although the pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any type of animal. Modifications of pharmaceutical compositions suitable for administration to humans to make compositions suitable for administration to a variety of animals are well understood, and a skilled veterinary pharmacologist can design and implement such modifications with little, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the present invention is contemplated include, but are not limited to, humans and other primates, as well as other mammals.

[0326] It is understood that any amino acid sequence showing sequence, structural, or functional homology to the polypeptides described herein, whether naturally occurring or synthetically obtained by any means, is considered to be part of the invention.

[0327] It should be understood that the reference to any publication, patent application, or issued patent is deemed to be fully incorporated herein by reference in its entirety.

[0328] It should be understood that any assay for measuring a particular activity modulated by a therapeutic compound may be used as a means to determine the efficacy of the compound, in one embodiment, the optimal loading of the compound, in another embodiment, the timing, and dosage, in another embodiment, or a combination thereof.

[0329] How to Detect PSMA It should be understood by one of skill in the art that the antibodies, antigen-binding fragments, or compositions provided herein can be used in diagnostic or therapeutic procedures.

[0330] In one embodiment, provided herein is a method of diagnosing the presence of a tumor or cancerous growth in a subject. In another embodiment, the method comprises sampling a tissue sample isolated from the subject with a composition comprising an antibody or antigen-binding fragment provided herein, whereby specific binding of the antibody or antigen-binding fragment to the tissue sample indicates the presence of a tumor or cancerous growth in the subject.

[0331] In another embodiment, the method further comprises detecting a secondary reagent that specifically binds to the antibody or antigen-binding fragment but does not compete with binding of the antibody or antigen-binding fragment to its target. In another embodiment, the "secondary reagent" is a photoactivatable drug, enzyme, tag, label, fluorophore, radioisotope, bioluminescent protein, bioluminescent peptide, fluorescent tag, fluorescent protein, or fluorescent peptide.

[0332] In one embodiment, the terms "cancer" and "cancerous" refer to or describe, in one embodiment, the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.

[0333] In one embodiment, the term "cancer" includes, but is not limited to, ovarian cancer, breast cancer, glioblastoma, and gastrointestinal cancer, hi another embodiment, the cancer is prostate cancer.

[0334] In another embodiment, "sampling" comprises testing or analyzing the sample using a detection assay that allows for the detection of a secondary reagent that is complexed or conjugated to the antibody or antigen-binding fragment and that emits a detectable "signal" when the antibody or antigen-binding fragment specifically binds to a target. In another embodiment, detection is accomplished using assays routinely used in the art, such as, but not limited to, immunological assays (e.g., immunohistochemistry, ELISA, etc.) or microscopic imaging.

[0335] In one embodiment, the term "labeled" refers to an antibody of the invention having one or more elements, isotopes, or chemical compounds attached to it to allow for detection in a screen. In general, labels are divided into three classes: a) immunolabels, which may be epitopes incorporated as fusion partners recognized by the antibody; b) isotopic labels, which may be radioisotopes or heavy isotopes; and c) small molecule labels, which may include molecules such as biotin that allow for fluorescent and calorimetric dyes or other labeling methods. In one embodiment, an antibody of the invention is labeled with biotin. In other related embodiments, a biotinylated antibody of the invention may be used, for example, as an imaging agent or as a means to identify one or more ligand molecules. In another embodiment, the label may be a nanoparticle that can be detected or visualized when bound to an antibody or antigen-binding fragment. The label may be incorporated into the compound at any position and may be incorporated in vitro or in vivo during protein expression.

[0336] In one embodiment, the conjugates formed by the antibodies or antigen-binding fragments and secondary reagents provided herein are used for a variety of applications, including, but not limited to, flow cytometry, ELISA, Western blotting, immunohistochemistry, membrane assays, and diagnostic and therapeutic methods as further described herein or as routinely applied in the art.

[0337] Imaging of tumors with aberrant PSMA expression In one embodiment, the compositions of the invention are administered to a subject having a disease involving inappropriate expression of a target antigen, protein, or other molecule. For example, in one embodiment, a composition comprising an antibody or antigen-binding fragment thereof that binds to PSMA is administered to detect the presence, abundance, location, or combinations thereof, of PSMA in a subject. Diseases and disorders characterized by abnormal proteins, for example due to changes in the amount of protein present, localization of the protein, post-translational modifications, conformational state, presence of mutant or pathogenic proteins, etc., are within the scope of this disclosure. Similarly, the disease or disorder may be characterized by changes in molecules, including but not limited to polysaccharides and gangliosides. The excess amount may be due to any cause, including but not limited to overexpression at the molecular level, extended or accumulated appearance at the site of action, or increased activity of the protein, compared to normal. Included within this definition are diseases and disorders characterized by a decrease in the protein. This decrease may be due to any cause, including but not limited to, reduced expression at the molecular level, truncated or reduced appearance at the site of action, mutants of the protein, or reduced activity of the protein, compared to normal. Such excess or decrease of protein may be measured relative to the normal expression, appearance, or activity of the protein, and the measurement may play an important role in the development and / or clinical testing of antibodies of the invention.

[0338] In one embodiment, the disclosed antibodies or antigen-binding fragments thereof, when administered to a subject, bind to an antigen expressed in tumor cells, such as prostate cancer cells. In another embodiment, the antibodies or antigen-binding fragments thereof administered to a subject bind to an antigen expressed in the angiogenesis of solid tumors, such as tumors with PSMA-positive angiogenesis, including, but not limited to, lung cancer, liver cancer, pancreatic cancer, colon cancer, gastric cancer, breast cancer, ovarian cancer, renal cancer, prostate cancer, bladder cancer, melanoma, glioma, etc.

[0339] In one embodiment, provided herein is a method of imaging a PSMA-containing tumor. In some embodiments, the method comprises administering to a subject an antibody or antigen-binding fragment thereof described herein, or contacting a tissue sample with the antibody or antigen-binding fragment thereof. In another embodiment, a prostate or other type of solid tumor can be visualized as the antibody or antigen-binding fragment binds to its target, e.g., a PSMA-containing tissue or organ. In some embodiments, the antibody or antigen-binding fragment thereof is operably linked to a secondary reagent. In yet another embodiment, the secondary reagent comprises a photoactivatable agent, an enzyme, a tag, a label, a fluorophore, a radioisotope, a bioluminescent protein, a bioluminescent peptide, a fluorescent tag, a fluorescent protein, or a fluorescent peptide. Non-limiting examples of secondary reagents are provided below.

[0340] In one embodiment, the detectable label or secondary reagent attached thereto includes, but is not limited to, a label such as a fluorescent label (e.g., fluorescein isothiocyanate (FITC), cyanine dyes, etc.), an affinity label (e.g., biotin, avidin, protein A, etc.), an enzyme label (e.g., horseradish peroxidase or alkaline phosphatase), or an isotopic label (e.g., 124I), or any other such detectable moiety that allows for detection and isolation of the antibody.

[0341] Detection methods for identifying binding species within a population of altered variable regions can be direct or indirect and can include, for example, the measurement of light emission, radioisotopes, calorimetric dyes, and fluorochromes. Direct detection includes methods that operate without intermediate or secondary measurement procedures to assess the amount of bound antigen or ligand. Such methods generally use ligands that are themselves labeled, for example, by radioactive, light-emitting, or fluorescent moieties. In contrast, indirect detection includes methods that operate via intermediate or secondary measurement procedures. These methods generally use molecules that react specifically with antigens or ligands and that themselves can be directly labeled or detected by secondary reagents. For example, antibodies specific for a ligand can be detected using a secondary antibody that can interact with the first antibody specific for the ligand, again using the detection methods described above for direct detection. Indirect methods can additionally use detection by enzyme labeling. Furthermore, as a specific example of screening for catalytic antibodies, the disappearance of substrate or the appearance of product can be used as an indirect measure of binding affinity or catalytic activity.

[0342] In some embodiments, the antibody or antigen-binding fragment thereof is labeled with a near-infrared dye. For example, the antibody or antigen-binding fragment thereof can be labeled with IRDye800CW or indocyanine green (ICG).

[0343] In some embodiments, the antibody or antigen-binding fragment thereof is coupled to a macrocyclic chelator useful for conjugating radioactive metal ions to a polypeptide, including, but not limited to, 111In, 177Lu, 90Y, 166Ho, 153Sm, 215Bi, and 225Ac. In some embodiments, the radioactive metal ion associated with a macrocyclic chelator coupled to an antibody of the invention is 111In. In some embodiments, the radioactive metal ion associated with a macrocyclic chelator coupled to an antibody polypeptide of the invention is 90Y. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazaicyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA). In certain embodiments, the macrocyclic chelator is quadrature-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraaza-cyclodo-decane-1,4,7,10-tetraacetic acid. In other particular embodiments, DOTA is coupled to an antibody of the invention via a linker molecule. Examples of linker molecules useful for conjugating macrocyclic chelators such as DOTA to polypeptides are generally known in the art, see, e.g., DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998, Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999, and Zimmerman et al., Nucl. Med. Biol. 26(8):943-50, 1999, which are incorporated by reference in their entireties. Additionally, U.S. Patent Nos. 5,652,361 and 5,756,065, which disclose chelators that can be conjugated to antibodies and methods of making and using them, are incorporated by reference in their entireties.

[0344] definition To aid in the understanding of the detailed description of the compositions and methods according to the present disclosure, some explicit definitions are provided to facilitate a clear disclosure of the various aspects of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0345] As used herein, the term "conjugate" or "conjugation" or "linkage" as used herein refers to the joining of two or more entities to form one entity. Conjugates encompass both peptide-small molecule conjugates as well as peptide-protein / peptide conjugates.

[0346] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicates that there is nucleotide sequence identity to another nucleic acid (or its complementary strand), when optimally aligned with appropriate nucleotide insertions or deletions, of at least about 90%, or at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0347] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 90% sequence identity, or at least 95%, 98%, or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT using predefined gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution will not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0348] Sequence similarity in polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG Version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the region of best overlap between the query and search sequences (Pearson (2000) supra). When comparing the sequences of the present invention to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25: 3389-3402, each of which is incorporated herein by reference.

[0349] As used herein, the term "Kassoc" or "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kdis" or "Kd" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. By way of example, a preferred method for determining the KD of an antibody is to use surface plasmon resonance, preferably using a biosensor system such as a BIACORE system.

[0350] As used herein, the term "detectable label" refers to a detectable molecule, including, but not limited to, a radioisotope, a fluorophore, a chemiluminescent material, a chromophore, an enzyme, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a chromophore, a dye, a metal ion, a metal sol, a ligand (e.g., biotin, avidin, streptavidin or hapten), an intercalating dye, etc. The term "fluorophore" refers to a substance or portion thereof capable of exhibiting fluorescence in the detectable range.

[0351] In many embodiments, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has received or is currently receiving any form of treatment. As used herein, the term "subject" may refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus monkeys and chimpanzees), and humans). The subject may be human or non-human. In a more exemplary aspect, the mammal is a human. As used herein, the phrase "subject in need thereof" or "patient in need thereof" refers to a human or non-human mammal that exhibits one or more symptoms or signs of a disorder (e.g., a neurological disorder, an autoimmune disease, and a cardiovascular disease) and / or has been diagnosed as having an inflammatory disorder. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0352] As used herein, the term "disease" is intended to be generally synonymous with, and is used interchangeably with, the terms "disorder" and "condition" (in medical conditions), in that both reflect an abnormal condition of the human or animal body or parts thereof (e.g., inflammatory disease, cancer) that impairs normal function, is typically manifested by clear signs and symptoms, and reduces the duration or quality of human or animal life.

[0353] The terms "reduce", "reduced", "reduction", "reduce", or "inhibit" are all used herein to generally mean a reduction by a statistically significant amount. However, for the avoidance of doubt, "reduced", "reduction", "reduce", or "inhibit" means a reduction of at least 10% compared to a reference level, including, for example, a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% reduction (e.g., a level that is absent compared to a reference sample), or any reduction between 10-100% compared to a reference level.

[0354] As used herein, the term "agent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein, or portions thereof, e.g., a peptide, etc.), or an extract made from biological material such as a bacterial, plant, fungus, or animal (especially mammalian) cell or tissue. The activity of such an agent may qualify it as a "therapeutic agent," which is a biologically, physiologically, or pharmacologically active substance or substances that act locally or systemically in a subject.

[0355] As used herein, the terms "therapeutic agent," "therapeutic agent," or "therapeutic agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. Beneficial effects include enabling a diagnostic determination; alleviating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or pathological condition.

[0356] The term "therapeutic effect" is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance.

[0357] The term "effective amount", "effective dose", or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of impairment or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dosage" of a drug is an amount of the drug that inhibits the onset or recurrence of a disease when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or at risk of suffering a recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit the onset or recurrence of a disease can be evaluated using a variety of methods known to skilled physicians, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0358] Doses are often expressed relative to body weight. Thus, a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg, etc.) body weight," even if the term "body weight" is not explicitly mentioned.

[0359] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one component useful within the present invention with other components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. A pharmaceutical composition facilitates administration of one or more components of the present invention to an organism.

[0360] "Combination" therapy, as used herein, unless otherwise clear from the context, is meant to include administration of two or more therapeutic agents in a coordinated manner, including but not limited to simultaneous administration. Specifically, combination therapy includes both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that the administration of one therapeutic agent is in some way conditioned on the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a certain period of time. See, for example, Kohrt et al. (2011) Blood 117:2423.

[0361] As used herein...

Claims

1. An antibody or antigen-binding fragment thereof, or a variant thereof, that specifically binds to prostate-specific membrane antigen (PSMA), wherein the antibody or antigen-binding fragment thereof is: a light chain variable region (LCVR) comprising three light chain complementarity determining regions (LCDRs) (LCDR1, LCDR2, and LCDR3), wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 13 or 41, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 17; and / or a heavy chain variable region (HCVR) comprising three heavy chain complementarity determining regions (HCDRs) (HCDR1, HCDR2, and HCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 25 or 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 33 or 49; An antibody or antigen-binding fragment thereof, or a variant thereof, wherein the variant comprises one or more conservative amino acid substitutions in the LCDR1, the LCDR2, the LCDR3, the HCDR1, the HCDR2, or the HCDR3.

2. (a) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 25, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 33; or (b) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 33; or (c) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 41, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 33; or (d) The antibody or antigen-binding fragment thereof or variant thereof of claim 1, wherein the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, the LCDR3 comprises the amino acid sequence of SEQ ID NO: 17, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 45, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and the HCDR3 comprises the amino acid sequence of SEQ ID NO:

49.

3. The antibody or antigen-binding fragment thereof, or variant thereof, according to any one of claims 1 to 2, wherein the light chain variable region comprises one or more of the amino acid sequences of SEQ ID NOs: 5, 7, 11, 15, 19, 39, 43, 61, 63, 65, 67, and 69.

4. The antibody or antigen-binding fragment thereof or variant thereof according to claim 1, wherein the heavy chain variable region comprises one or more of the amino acid sequences of SEQ ID NOs: 21, 23, 27, 31, 35, 47, 51, 53, 55, 57, 59, and 68.

5. (a) the light chain variable region comprises an amino acid sequence at least 75% identical to, or comprises the amino acid sequence of SEQ ID NO: 5, and / or the heavy chain variable region comprises an amino acid sequence at least 75% identical to, or comprises the amino acid sequence of SEQ ID NO: 21; (b) the light chain variable region comprises an amino acid sequence at least 75% identical to, or comprises the amino acid sequence of SEQ ID NO: 70, and / or the heavy chain variable region comprises an amino acid sequence at least 75% identical to, or comprises the amino acid sequence of SEQ ID NO: 72; (c) the light chain variable region comprises an amino acid sequence at least 75% identical to, or comprises the amino acid sequence of SEQ ID NO: 71, and / or the heavy chain variable region comprises an amino acid sequence at least 75% identical to, or comprises the amino acid sequence of SEQ ID NO: 73; or (d) The antibody or antigen-binding fragment thereof or variant thereof according to claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 5 or comprises the amino acid sequence of SEQ ID NO: 5, and / or the heavy chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 74 or comprises the amino acid sequence of SEQ ID NO:

74.

6. The antibody or antigen-binding fragment thereof or variant thereof of claim 1, comprising the heavy chain / light chain sequence pair of SEQ ID NOs: 75 and 77, SEQ ID NOs: 76 and 78, SEQ ID NOs: 69 and 68, or SEQ ID NOs: 75 and 79.

7. The antibody or antigen-binding fragment thereof or variant thereof according to claim 1, comprising at least one of the amino acid sequences of SEQ ID NOs: 3 and 37.

8. 8. A bispecific or multispecific antibody or antigen-binding fragment thereof that binds to two or more different epitopes on the same or different antigens, one of the epitopes being in human prostate-specific membrane antigen (PSMA), the bispecific or multispecific antibody comprising the light chain variable region and / or the heavy chain variable region of the antibody or antigen-binding fragment thereof or variant thereof according to any one of claims 1, 2, and 4 to 7.

9. 10. An isolated chimeric antigen receptor (CAR), comprising the antibody or antigen-binding fragment or variant of any one of claims 1, 2, and 4 to 7, a transmembrane domain, and an intracellular signaling domain comprising one or more stimulatory domains.

10. A nucleic acid molecule encoding a polypeptide chain of the antibody, antigen-binding fragment, or variant thereof according to any one of claims 1, 2, and 4 to 7; a bispecific or multispecific antibody or antigen-binding fragment thereof comprising the light chain variable region and / or the heavy chain variable region of the antibody, antigen-binding fragment, or variant thereof; or an isolated CAR comprising the antibody, antigen-binding fragment, or variant thereof.

11. A vector comprising the nucleic acid molecule of claim 10.

12. A cell comprising the nucleic acid molecule of claim 10 or a vector comprising said nucleic acid molecule.

13. 1. A method for preparing an antibody or antigen-binding fragment thereof, comprising: Obtaining the cells of claim 12; culturing the cells in a culture medium under conditions that allow expression of the polypeptide encoded by the nucleic acid molecule and assembly of the antibody or fragment thereof; and purifying said antibody or fragment from said cultured cells or from said medium of said cells.

14. A composition comprising the antibody or antigen-binding fragment thereof, or variant thereof according to any one of claims 1, 2, and 4 to 7; a bispecific or multispecific antibody or antigen-binding fragment thereof comprising the light chain variable region and / or the heavy chain variable region of the antibody, antigen-binding fragment, or variant thereof; an isolated CAR comprising the antibody, antigen-binding fragment, or variant thereof; a nucleic acid molecule encoding a polypeptide chain of the antibody, antigen-binding fragment, or variant thereof; a vector comprising the nucleic acid molecule; or a cell comprising the nucleic acid molecule; and optionally a pharmaceutically acceptable carrier.

15. A pharmaceutical for use in preventing or treating a proliferative disease associated with expression of PSMA in a subject, the pharmaceutical comprising: the antibody or antigen-binding fragment thereof, or variant thereof according to any one of claims 1, 2, and 4 to 7; a bispecific or multispecific antibody or antigen-binding fragment thereof comprising the light chain variable region and / or the heavy chain variable region of the antibody or antigen-binding fragment or variant thereof; an isolated CAR comprising the antibody or antigen-binding fragment or variant thereof; a nucleic acid molecule encoding a polypeptide chain of the antibody or antigen-binding fragment or variant thereof; a vector comprising the nucleic acid molecule; or a cell comprising the nucleic acid molecule.

16. The method of claim 15, wherein the subject is administered a second drug or therapy.

17. The method of claim 16 , wherein the second drug or therapy comprises an anti-cancer drug.

18. The composition of claim 14, wherein the composition is used for imaging targeted to PSMA, and the antibody or its antigen-binding fragment or variant is conjugated to a reagent.

19. 20. The composition of claim 18, wherein the reagent comprises a photoactivatable drug, a fluorophore, a radioisotope, a bioluminescent protein, a bioluminescent peptide, a fluorescent tag, a fluorescent protein, a fluorescent peptide, an imaging agent, an enzyme, a nuclear magnetic resonance active reagent, or a nanoparticle.

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