EA046641B1 - PSMA AGENTS AND THEIR USES - Google Patents

Description

List of sequences

The currently pending application contains a sequence listing provided electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy of the list created on April 30, 2019 is named JBI5156WOPCTl_SL.txt and is 272,443 bytes in size.

Technical field

The invention provided herein relates to monoclonal antibodies that immunospecifically bind to prostate-specific membrane antigen (PSMA), multispecific antibodies that immunospecifically bind to PSMA and cluster of differentiation 3 (CD3), and methods for producing and using the disclosed antibodies.

Prerequisites for creating the invention

Prostate cancer is the second most common cancer in men worldwide and the sixth leading cause of cancer-related death. Worldwide, there are approximately 1,100,000 new cases and 300,000 deaths each year, accounting for 4 percent of all cancer deaths. It is estimated that out of every 6 men, 1 in 6 men will be diagnosed with the disease during their lifetime. In the United States, more than 90% of prostate cancer cases are detected at a local or regional stage. In these early stages, the 5-year survival rate approaches 100%. However, when cancer metastasizes, the 5-year survival rate drops to 28%, and there remains a need for effective treatments for advanced prostate cancer.

Prostate-specific membrane antigen (PSMA) is a type II membrane protein that is highly expressed in prostatic intraepithelial neoplasia (PIN), a condition in which some prostate cells begin to look and behave abnormally, and in primary and metastatic prostate cancers [Bostwick DG , Pacelli A, Blute M, Roche P, Murphy GP. Prostate-specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma: a study of 184 cases. Cancer 1998;82(11):22562261.]. PSMA expression in cancer tissues correlates with disease stage and Gleason score [Kawakami M, Nakayama J. Increased expression of the prostate-specific membrane antigen gene in prostate cancer revealed by in situ hybridization. Cancer Res 1997;57(12):2321-2324]. PSMA expression is also higher in prostate cancer cells from patients with hormone-resistant prostate cancer [Wright GL Jr, Grob BM, Haley C, Grossman K, Newhall K, Petrylak D, Troyer J, KongarbaA, Schellhammer PF, Moriarty R. Increased expression of prostate-specific membrane antigen after antiandrogen therapy. Urology 1996;48(2):326-334.], and increased PSMA expression is an independent marker of disease relapse [Mitsiades CS, Lembessis P, Sourla A, Milathianakis S, Tsintavis A, Koutsieris M. Molecular stage according to RT-PCR analysis for PSA and PSMA in peripheral blood and bone marrow samples is an independent predictor of time to biochemical failure after radical prostatectomy for clinically localized prostate cancer. Clin Exp Metastasis 2004;21(6):495-505.]. High levels of PSMA expression correlate with early prostate-specific antigen (PSA) recurrence during surgical treatment of prostate cancer. PSMA expression levels correlate with aggressive disease capacity and thus strongly support PSMA as an excellent target for prostate cancer characterization and subsequent therapy.

Current treatments for prostate cancer include surgery, radiation therapy, and hormonal therapy. If prostate cancers grow despite lowering testosterone levels through hormone therapy, treatment options are limited. Typically, hormonal therapy is sequentially added to the cancer vaccine sipuleucel-T, a radiopharmaceutical agent (such as radium-223 chloride), secondary hormonal therapeutics (such as abiraterone or enzalutamide), and/or chemotherapeutic agents (docetaxel and cabazitaxel). Although each of these treatments can slow the growth of the cancer over several months and reduce the symptoms caused by the disease, eventually the disease becomes resistant to them. This highlights the need for improved treatments and effective therapies for advanced PSMA-expressing prostate cancer.

Summary of the invention

Provided herein are antibodies that immunospecifically bind to Pan troglodytes (chimpanzees), Macaca fascicularis (cynomolgus, cynomolgus, cynomolgus) and/or human PSMA, and antigen-binding fragments thereof. In addition, related polynucleotides capable of encoding the proposed PSMA-specific antibodies and antigen binding fragments, cells expressing the proposed antibodies and antigen binding fragments, as well as corresponding vectors and detectably labeled antibodies and antigen binding fragments are described. Moreover, methods of using the proposed antibodies and antigen-binding fragments are described. For example, PSMA-specific antibodies and antigen-binding fragments can be used to diagnose or monitor the progression, reversal or stability of cancer expression.

- 1 046641 current PSMA; to determine whether to treat a patient's cancer; or to determine whether a subject has a cancer expressing PSMA and therefore will respond to treatment with a PSMA-specific anticancer drug, such as the multispecific (bispecific, trispecific, etc.) anti-PSMA and anti-CD3 antibodies described herein .

Also provided herein are multispecific antibodies that immunospecifically bind PSMA and CD3, and multispecific antigen binding fragments thereof. In addition, related polynucleotides capable of encoding the proposed anti-PSMA x CD3 multispecific antibodies, cells expressing the proposed antibodies, as well as corresponding vectors and detectably labeled multispecific antibodies are described. Moreover, methods of using the proposed multispecific antibodies are described. For example, anti-PSMA x CD3 multispecific antibodies can be used to diagnose or monitor the progression, reversal, or stability of PSMA-expressing cancers; to determine whether to treat a patient's cancer; or to determine whether a subject has a PSMA-expressing cancer and therefore will respond to treatment with a PSMA-specific anticancer drug, such as the anti-PSMA x CD3 multispecific antibodies described herein.

Psma-specific antibodies

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA to Pan troglodytes, Macaca fascicularis and/or humans, containing heavy chain complementarity determining regions 1 (HCDR1), HCDR2 and HCDR3 with SEQ ID NO : 31, 42 and 43 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 31, 42 and 43, respectively, and light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 with SEQ ID NO: 11, 12 and 13 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 25, 26 and 27, respectively.

The invention provides an isolated antibody and its fragments that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 25, 26 and 27, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 28, 29 and 30 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 20, 21 and 22, respectively.

The invention provides an isolated antibody and its fragments that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 20, 21 and 22, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 23, 12 and 24 respectively.

The invention provides an isolated antibody and its fragments that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 14, 15 and 16, respectively, or with SEQ ID NO: 14, 15 and 16 respectively.

The invention provides an isolated antibody and its fragments that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 14, 15 and 16, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 17, 18 and 19 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 36, 37 and 38, respectively.

The invention provides an isolated antibody and its fragments that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 36, 37 and 38, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 39, 40 and 41 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 122, 123 and 124, respectively.

The invention provides an isolated antibody and its fragments that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NO: 122, 123 and 124, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 23, 12, and 24 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA to Pan troglodytes, Macaca fascicularis and/or humans, containing heavy chain complementarity determining regions 1 (HCDR1), HCDR2 and HCDR3 with SEQ ID NO : 31, 44 and 45 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 31, 44 and 45, respectively, and light chain complementarity determining region 1 (LCDR1),

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LCDR2 and LCDR3 with SEQ ID NO: 46, 29 and 27, respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA to Pan troglodytes, Macaca fascicularis and/or humans, containing heavy chain complementarity determining regions 1 (HCDR1), HCDR2 and HCDR3 with SEQ ID NO : 36, 37 and 48 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 36, 37 and 48, respectively, and light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 with SEQ ID NO: 49, 50 and 51 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA to Pan troglodytes, Macaca fascicularis and/or humans, containing heavy chain complementarity determining regions 1 (HCDR1), HCDR2 and HCDR3 with SEQ ID NO : 36, 37 and 52 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 36, 37 and 52, respectively, and light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 with SEQ ID NO: 49, 50 and 51 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA to Pan troglodytes, Macaca fascicularis and/or humans, containing heavy chain complementarity determining regions 1 (HCDR1), HCDR2 and HCDR3 with SEQ ID NO : 8, 9 and 10 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 8, 9 and 10, respectively, and light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 with SEQ ID NO: 11, 12 and 13 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA to Pan troglodytes, Macaca fascicularis and/or humans, containing heavy chain complementarity determining regions 1 (HCDR1), HCDR2 and HCDR3 with SEQ ID NO : 31, 32 and 33 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 31, 32 and 33, respectively, and light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 with SEQ ID NO: 34, 12 and 35, respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA to Pan troglodytes, Macaca fascicularis and/or humans, containing heavy chain complementarity determining regions 1 (HCDR1), HCDR2 and HCDR3 with SEQ ID NO : 53, 54 and 55 respectively.

The invention provides an isolated antibody and fragments thereof that specifically bind PSMA from Pan troglodytes, Macaca fascicularis and/or humans, containing HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 53, 54 and 55, respectively, and light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 with SEQ ID NO: 23, 12 and 35, respectively.

The invention also provides an isolated antibody and fragments thereof that specifically bind Pan troglodytes, Macaca fascicularis and/or human PSMA containing the specific amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 described herein.

The invention also provides an isolated antibody and fragments thereof that specifically bind Pan troglodytes, Macaca fascicularis and/or human PSMA containing the specific variable heavy chain (VH) and variable light chain (VL) amino acid sequences described herein.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody that specifically binds Pan troglodytes, Macaca fascicularis and/or human PSMA and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody that specifically binds Pan troglodytes, Macaca fascicularis and/or human PSMA, and fragments thereof containing a first PSMA-specific binding domain and a second CD3-specific binding domain containing specific HCDR1 amino acid sequences , HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, VH, VL, heavy chain or light chain, as described herein.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 31, 42, 43, 11, 12 and 13 respectively.

The invention also provides an isolated bispecific antibody to PSMA/CD3 and its fragment

- 3 046641 ments that contain a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 26, 27, 28, 29 and 30 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 36, 37, 38, 39, 40 and 41 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 20, 21, 22, 23, 12 and 24 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 14, 15, 16, 17, 18 and 19 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 122, 123, 124, 23, 12 and 24 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 31, 44, 45, 46, 29 and 47 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 36, 37, 48, 49, 50 and 51 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 36, 37, 52, 49, 50 and 51 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 8, 9, 10, 11, 12 and 13 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 31, 32, 33, 34, 12 and 35 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 53, 54, 55, 23, 12 and 35 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 130, 27, 28, 29 and 30 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 130, 27, 131, 29 and 132 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 130, 27, 28, 133 and 132 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 134, 27, 28, 29 and 30 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and

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LCDR3 with SEQ ID NO: 25, 135, 27, 28, 29 and 136 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 135, 27, 28, 29 and 30 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 135, 27, 131, 29 and 132 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 135, 27, 28, 133 and 132 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 134, 27, 28, 29 and 136 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 134, 27, 131, 29 and 132 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 134, 27, 28, 133 and 132 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof that comprise a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 25, 137, 27, 28, 133 and 132 respectively.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 74 and a variable the light chain (VL) region of SEQ ID NO: 61, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 74 and a variable the light chain (VL) region of SEQ ID NO: 61, and the second domain contains VH of SEQ ID NO: 152 and VL of SEQ ID NO: 153.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 66 and a variable the light chain (VL) region of SEQ ID NO: 67, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 66 and a variable the light chain (VL) region of SEQ ID NO: 67, and the second domain contains VH of SEQ ID NO: 152 and VL of SEQ ID NO: 153.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 64 and a variable the light chain (VL) region of SEQ ID NO: 65, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 64 and a variable the light chain (VL) region of SEQ ID NO: 65, and the second domain contains VH of SEQ ID NO: 152 and VL of SEQ ID NO: 153.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising the heavy chain variable region (VH) of SEQ ID

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NO: 62 and a light chain variable region (VL) of SEQ ID NO: 63, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 62 and a variable the light chain (VL) region of SEQ ID NO: 63, and the second domain contains VH of SEQ ID NO: 152 and VL of SEQ ID NO: 153.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 75 and a variable the light chain (VL) region of SEQ ID NO: 76, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 75 and a variable the light chain (VL) region of SEQ ID NO: 76, and the second domain contains VH of SEQ ID NO: 152 and VL of SEQ ID NO: 153.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 160 and a variable the light chain (VL) region of SEQ ID NO: 65, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105.

The invention also provides an isolated anti-PSMA/CD3 bispecific antibody and fragments thereof comprising a first PSMA-specific binding domain and a second CD3-specific binding domain, the first domain comprising a heavy chain variable region (VH) of SEQ ID NO: 160 and a variable the light chain (VL) region of SEQ ID NO: 65, and the second domain contains VH of SEQ ID NO: 152 and VL of SEQ ID NO: 153.

The invention also provides an immunoconjugate comprising an antibody or antigen-binding site thereof of the invention coupled to a therapeutic agent or imaging agent.

The invention also provides a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.

The invention also provides a polynucleotide encoding VH antibodies, VL antibodies, or VH and VL antibodies according to the invention.

The invention also provides a vector containing a polynucleotide that encodes a VH antibody, a VL antibody, or a VH and VL antibody of the invention.

The invention also provides a host cell containing a vector of the invention.

The invention also provides a method for producing an antibody of the invention, comprising culturing a host cell of the invention under antibody expression conditions and isolating the antibody produced by the host cell.

The invention also provides a method of treating PSMA overexpression syndrome and/or cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of an isolated antibody of the invention for a time sufficient to treat the cancer.

The invention also provides a kit containing an antibody of the invention.

Brief description of graphic materials

In fig. Figure 1 shows titration curves for the binding of PSMA compounds selected by phage panning to LNCaP cells.

In fig. Figure 2 shows titration curves for the binding of phage-panned compounds to HEK cells expressing chimpanzee PSMA.

In fig. Figure 3 shows titration curves for the binding of phage-panned anti-PSMA compounds to HEK cells expressing cynomolgus PSMA.

In fig. Figure 4 shows the amino acid sequence of SP34 with sequential numbering. The CDRs in the definition of AbM (K.R. Abhinandan and A.S. Martin, 2008. Mol. Immunol. 45, 3832-3839) are underlined. Ser230 is the last HC residue in papain-digested Fab. Residues 231-455 are from IGHG3_MOUSE (mouse IgG3 isoform 2).

In fig. 5 shows human framework adapted (HFA) versions of V _H (SEQ ID NOs: 104, 102, 115 and 116, respectively, in order of appearance) and V _L (SEQ ID NOs: 103, 117 and 105, respectively, in order of appearance). The numbering is sequential; The CDRs in the AbM definition are underlined; residues different from SP34 are highlighted in bold; back mutations in HFA variants are shown in bold and underlined. The figure shows the VH and VL sequences for SP34 in SEQ ID NO: 128 and 129, respectively.

In fig. 6 shows the binding of HFA variants of SP34 to primary human T cells.

In fig. 7 shows the binding of HFA variants of SP34 to primary cynomolgus T cells.

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In fig. 8 shows that HFA variants of SP34 activate primary human T cells in vitro. Negative controls are shown in white and positive controls are shown in black.

In fig. 9 shows that HFA variants of SP34 activate primary cynomolgus T cells in vitro. Negative controls are shown in white and positive controls are shown in black. The non-CD3e cross-reactive antibody G11 served as an additional negative control.

In fig. 10A and 10B show the correlation of binding and activation by HFA variants of SP34. CD69 mean binding and mean fluorescence intensity (MFI) values are plotted against each other for human (Figure 10A) and cynomolgus monkey (Figure 10B).

In fig. 11A-11F show titration curves for binding of anti-PSMA X CD3 bispecific antibodies to LNCaP cells.

In fig. 12 shows titration curves for anti-PSMA X CD3 bispecific antibodies binding to PSMA-expressing chimpanzee HEK cells.

In fig. 13 shows titration curves for anti-PSMA X CD3 bispecific antibodies binding to PSMA-expressing cynomolgus HEK cells.

In fig. 14 shows titration curves for anti-PSMA X CD3 bispecific antibodies binding to human PSMA-expressing HEK cells.

In fig. 15 shows anti-PSMA X CD3 bispecific antibody titration curves for human PSMA-expressing HEK cells in a T cell-mediated chromium toxicity assay.

In fig. 16 shows a comparison of the medium and high affinity CD3 arms of the PSMA X CD3 bispecific antibodies in a T cell mediated chromium toxicity assay for HEK cells expressing human PSMA.

In fig. 17 shows titration curves for anti-PSMA X CD3 bispecific antibodies for LNCaP cells in a T cell-mediated chromium toxicity assay.

In fig. 18 shows anti-PSMA X CD3 bispecific antibody titration curves for PSMA-expressing cynomolgus HEK cells in a T cell-mediated chromium toxicity assay.

In fig. 19A, 19B and 19C show titration curves for PS3B27 and control bispecific antibodies for PSMA-expressing HEK cells and human and cynomolgus LNCaP cells in a caspase 3/7 T cell-mediated toxicity assay.

In fig. 20 shows T cell activation of PS3B27.

In fig. 21 shows the prevention of tumorigenesis in HEK293-PSMA xenografts treated with PS3B27 or control bispecific antibodies in NSG mice humanized with human peripheral blood mononuclear cells (PBMCs).

In fig. 22 shows the average body weights of NSG mice humanized with PBMCs bearing HEK293-PSMA xenografts treated with PS3B27 and a control bispecific antibody.

In fig. 23 shows the effectiveness of PS3B27 and control bispecific antibodies in preventing tumorigenesis in a mixture of HEK293-PSMA/T cell xenografts in male CD1 nude mice.

In fig. 24 shows the body weight of male CD1 nude mice bearing HEK293-PSMA/T cell xenografts treated with PS3B27 and control bispecific antibodies.

In fig. 25 shows the general structure of PSMB83 (also known as PSMM84) bound to the human PSMA ECD homodimer.

In fig. 26 shows a close-up view of the major interactions of PSMA with the light chain of PSMB83 (also known as PSMM84).

In fig. 27 shows a close-up view of the major interactions of PSMA with the heavy chain of PSMB83 (also known as PSMM84).

In fig. 28 shows a comparison of epitope residues of PSMB83 (also known as PSMM84) in the human (SEQ ID NO: 3), mouse (SEQ ID NO: 157), and cynomolgus (macaque) (SEQ ID NO: 2) PSMA sequences. Epitope residues are shaded and sequence divergence is indicated by underlining.

In fig. Figure 29 shows the remains of the paratope PSMB83 (also known as PSMM84). The CDRs are underlined and the remains of the paratope are shaded. The figure discloses SEQ ID NOs 158 and 159, respectively, in order.

In fig. 30 shows a map of the direct contacts established between PSMA and PSMM83 (also known as PSMM84). Van der Waals interactions are shown as dashed lines, and hydrogen bonds are represented as solid lines with arrows pointing to the backbone atoms.

In fig. 31 shows the expression levels of anti-PSMA Fab clones derived from PSMB83 compared to the expression of the parent PSMB83. A graph was constructed of the dependence of the initial luminescence values on the logarithmic concentration.

In fig. 32 shows the binding of PSMB83-derived anti-PSMA Fab clones to human PSMA compared with binding of parent PSMB83. A graph was constructed of the dependence of the initial luminescence values on the logarithmic concentration.

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In fig. 33 shows the binding to cynomolgus PSMA of anti-PSMA Fab clones derived from PSMB83 compared to the binding of parent PSMB83. A graph was constructed of the dependence of the initial luminescence values on the logarithmic concentration.

In fig. 34 shows the binding of anti-PSMA Fab clones derived from PSMB83 to human PSMA compared to binding of parent PSMB83. Raw luminescence values were normalized to Fab expression levels.

In fig. 35 shows the binding of anti-PSMA Fab clones derived from PSMB83 to cynomolgus PSMA compared with binding of parent PSMB83. Raw luminescence values were normalized to Fab expression levels.

In fig. 36 shows affinity matured subset of PSMAxCD3 bispecific antibodies binding to LNCAP cells.

In fig. 37 shows affinity matured subset of PSMAxCD3 bispecific antibodies binding to LNCAP cells.

In fig. 38 shows the results of PSMA-negative affinity matured PSMAxCD3 bispecific antibodies binding to PCZ cells.

In fig. 39 shows the results of affinity matured bispecific antibodies to PSMAxCD3 in a functional cell killing assay.

In fig. 40 shows the antitumor efficacy of PS3B79 in the LnCAP AR.TB human prostate cell xenograft model in T cell humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice weekly and results were presented as mean tumor volume expressed in mm ³ ± SEM (*, p < 0.0001).

In fig. 41 demonstrates the antitumor efficacy of PS3B90 in the LnCAP AR.TB human prostate cell xenograft model in T cell humanized NSG mice. Subcutaneous LnCAP AR.TB tumors were measured twice weekly and results were presented as mean tumor volume expressed in mm ³ ± SEM (*, p < 0.001).

In fig. 42 shows the effect of PS3B72 (PSMA x CD3) on an established patient-derived LuCaP 86.2 prostate xenograft model in T-cell humanized NSG mice. Subcutaneous LnCAP 86.2 tumors were measured twice weekly and results were presented as mean tumor volume expressed in mm ³ ± SEM (*, p < 0.0001).

Detailed Description of the Invention

All publications, including, without limitation, patents and patent applications cited herein, are incorporated herein by reference as if fully set forth herein.

It should be understood that the terms used herein are intended only for the purpose of describing specific embodiments and are not intended to be limiting. All technical and scientific terms used herein, unless otherwise noted, have the same meaning as would be understood by one of ordinary skill in the art to which the invention relates.

Described herein are exemplary methods and materials, although any methods and materials similar or equivalent to those described herein may be used to test the present invention in practice. In describing and stating the claims of the present invention, the following terms will be used.

When used in this specification and in the accompanying claims, the singular number includes the plural unless the content of the text clearly indicates otherwise. Thus, for example, a reference to a cell includes a combination of two or more cells and the like.

The terms specific binding, or specifically binds, or binds refer to the binding of an antibody to an antigen or epitope within an antigen with greater affinity than to other antigens. Typically, an antibody binds to an antigen or epitope within an antigen with an equilibrium dissociation constant (KD) of about 5x10 ^{-8 M} or less, for example, about 1x 10 ^{-9 M} or less, about 1x10 ^{-10 M} or less, about 1x10 ^{-11 M} or less or about 1x10 ^{-12 M} or less, typically with a KD that is at least one hundred times lower than its KD of binding to a nonspecific antigen (eg, BSA, casein). The dissociation constant can be measured using standard procedures. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to the same antigen from other species (homologues) such as humans or monkeys, such as Macaca fascicularis ( cynomolgus macaque, macaques) or Pan troglodytes (chimpanzees). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two different antigens or two different epitopes.

Antibodies means in a broad sense and includes immunoglobulin molecules, including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other

- 8 046641 a modified configuration of an immunoglobulin molecule that contains an antigen-binding site of the required specificity. Full-length antibody molecules consist of two heavy chains (HC) and two light chains (LC), linked by disulfide bonds, as well as their multimers (for example, IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (consisting of CH1, hinge, CH2 and CH3 domains). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), between which framework regions (FRs) are located. Each of VH and VL consists of three CDRs and four FR segments, arranged in an amino-to-carboxy-terminal direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

Complementarity determining regions (CDRs) are antigen binding sites in an antibody. CDRs can be defined using various terms: (i) Complementarity determining regions (CDRs), three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3), are based on sequence variability (Wu and Kabat ( 1970) J Exp Med 132:211-50; Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. National Institutes of Health, Bethesda, Md., 1991). (ii) The term hypervariable regions, HVR or HV, three in VH (H1, H2, H3) and three in VL (L1, L2, L3), refers to regions of antibody variable domains that are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk (1987) Mol Biol 196:901-17). The international database ImMunoGeneTics (IMGT) (http://www_imgt_org) provides standardized numbering and definition of antigen-binding sites. The agreement between the definitions of CDR, HV and IMGT is described in Lefranc et al., (2003) Dev Comparat Immunol 27:55-77. The terms CDR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as used herein include CDRs determined by any of the methods described above by Kabat, Chothia or IMGT, unless expressly stated otherwise in the specification.

Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can fall into five main classes - IgA, IgD, IgE, IgG and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. The light chains of antibodies from any vertebrate species, based on the amino acid sequences of their constant domains, can be classified into one of two clearly distinct types, namely kappa (k) and lambda (λ).

The terms antibody fragments and antigen binding fragments refer to the part of an immunoglobulin molecule that retains the antigen binding region of the heavy chain and/or light chain, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining region (LCDR) 1 , 2 and 3, heavy chain variable region (VH) or light chain variable region (VL). Antibody fragments or antigen binding fragments include the well known Fab, F(ab')2, Fd and Fv fragments, as well as domain antibodies (dAbs) containing a single VH domain. The VH and VL domains can be linked together via a synthetic linker to form various types of single-chain antibody configurations, in which the VH/VL domains can be paired intramolecularly or intermolecularly in cases where the VH and VL domains are expressed by separate single-chain antibody constructs to form a monovalent antigen-binding site, such as single chain Fv (scFv) or diabody; they are described, for example, in international patent publications No. WO 1998/44001, WO 1988/01649, WO 1994/13804 and WO 1992/01047.

The term monoclonal antibody refers to a population of antibodies with the same amino acid composition in each heavy and each light chain, except for possible known changes such as the removal of the C-terminal lysine from the heavy chain of the antibody. Monoclonal antibodies generally bind to a single antigenic epitope, with the exception of bispecific monoclonal antibodies, which bind to two distinct antigenic epitopes. Within an antibody population, monoclonal antibodies can have heterogeneous glycosylation. A monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. Bispecific antibody is included within the term monoclonal antibody.

The term isolated antibody refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigen specificities (eg, an isolated antibody that specifically binds PSMA is substantially free of antibodies that specifically bind antigens other than PSMA). In the case of PSMA x CD3 bispecific antibodies, the bispecific antibody specifically binds both PSMA and CD3 and is substantially free of antibodies that specifically bind antigens other than PSMA and CD3. The term isolated antibody covers antibodies isolated so that they are of a higher degree of purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% pure. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

The term humanized antibody refers to an antibody in which the antigen binding regions are derived from non-human species and the variable region framework regions are derived from

- 9 046641 human immunoglobulin sequences. A humanized antibody may contain substitutions in the framework regions, such that the framework region may not be an exact copy of the expressed human immunoglobulin or human germline immunoglobulin gene sequences.

The term human antibody refers to an antibody having heavy and light chain variable regions in which both framework and antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region or part of a constant region, then the constant region is also derived from sequences of human origin.

A human antibody contains heavy or light chain variable regions that are derived from sequences of human origin if the antibody variable regions are derived from a system that uses germline human immunoglobulin or rearranged immunoglobulin genes. Such examples of systems are phage-displayed human immunoglobulin gene libraries and transgenic non-human animals, such as mice or rats, carrying human immunoglobulin loci, as described herein. A human antibody may contain amino acid differences compared to human germline immunoglobulin or rearranged immunoglobulin genes, due, for example, to naturally occurring somatic mutations or intentional insertion of substitutions into the framework or antigen binding site, or both. Typically, the antibody is human in amino acid sequence of at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence encoded by human germline immunoglobulin genes or rearranged immunoglobulin genes. In some cases, the human antibody may contain consensus framework sequences derived from human framework sequence assays, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 included in immunoglobulin gene libraries human displayed on phage, for example, as described in Shi et al., (2010) J Mol Biol 397:385-96 and international patent publication No. WO 2009/085462.

Human antibodies derived from human immunoglobulin sequences can be produced using systems such as phage display, including synthetic CDRs and/or synthetic scaffolds, or can be subjected to in vitro mutagenesis to improve antibody properties, allowing the production of antibodies that are not germline expressed. human lineage in vivo.

Antibodies in which the antigen-binding sites are derived from non-human species do not meet the definition of a human antibody.

The term recombinant refers to DNA, antibodies and other proteins that are obtained, expressed, created or isolated by recombinant methods, where segments from different sources are combined to produce recombinant DNA, antibodies or proteins.

The term epitope refers to the portion of an antigen to which an antibody specifically binds. Typically, epitopes are composed of chemically active (such as polar, nonpolar, or hydrophobic) surface moieties such as amino acid or polysaccharide side chains, and may have specific three-dimensional structure characteristics as well as specific charge characteristics. An epitope can be formed from continuous and/or discontinuous amino acids forming a conformational spatial unit. In the case of a discontinuous epitope, amino acids from different parts of the linear antigen sequence come close to each other in 3-dimensional space through the folding of the protein molecule. The epitope of an antibody depends on the methodology used to identify the epitope.

The term paratope refers to the part of the antibody to which the antigen specifically binds. The paratope may be linear or discrete, formed by the spatial relationships between amino acids of the antibody that are not in a continuous sequence, as opposed to the interaction of linear amino acid sequences. The terms light chain paratope and heavy chain paratope, or amino acid residues of a light chain paratope and amino acid residues of a heavy chain paratope, mean the residues of the light and heavy chains of an antibody that are in contact, respectively, with the antigen, or in general, the paratope residues of an antibody mean those amino acids of the antibody that are in contact with the antigen .

The term bispecific refers to an antibody that specifically binds two different antigens or two different epitopes within a single antigen. The bispecific antibody may be cross-reactive with other related antigens, for example the same antigen from other species (homologues) such as humans or monkeys, for example Macaca fascicularis (cynomolgus macaque) or Pan troglodytes, or may bind an epitope that is present in two or more different antigens.

The term multispecific refers to an antibody that specifically binds to two or more different antigens or two or more different epitopes within a single antigen. A multispecific antibody may have cross-reactivity with other related antigens, for example the same antigen from other species (homologues) such as human or monkey,

- 10 046641 for example, Macaca fascicularis (cynomolgus, macaque) or Pan troglodytes, or can bind an epitope that is present in two or more different antigens.

The term variant refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as one or more substitutions, insertions or deletions.

The term vector refers to a polynucleotide that is capable of duplication within a biological system, or can be transferred between such systems. Vector polynucleotides typically contain some functional elements, such as origins of replication, polyadenylation signals, or selectable markers, to facilitate duplication or maintenance of these polynucleotides in a biological system such as a cell, virus, animal, plant, as well as in reconstituted biological systems using biological components capable of vector duplication. The vector polynucleotide may be a DNA or RNA molecule or a hybrid thereof, single-stranded or double-stranded.

The term expression vector refers to a vector that can be used in a biological system or a reconstituted biological system to direct translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

The term polynucleotide means a molecule containing a chain of nucleotides covalently linked through a sugar phosphate backbone or other equivalent covalent chemical structure. Double-stranded and single-stranded DNA and RNA are typical examples of polynucleotides.

The term polypeptide or protein refers to a molecule that contains at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides containing less than 50 amino acid residues may be called peptides.

Flow cytometry is a technology used to determine the physical and chemical characteristics of particles in a fluid as they pass through at least one laser. Cell components are fluorescently labeled and then excited with a laser to emit light at different wavelengths (Adan, et al, Critical Reviews in Biotechnology (2016) 1549-7801).

An anti-idiotypic (anti-Id) antibody is an antibody that recognizes antigenic determinants (eg, paratope or CDR) of the antibody. A method for producing or preparing an anti-idiotypic antibody is generally known to those skilled in the art. (Lathey, J. et al Immunology 1986 57(1):29-35). An anti-idiotypic antibody may be antigen-blocking or non-blocking. An antigen-blocking anti-Id antibody can detect free antibody in a sample (eg, anti-PSMA, anti-CD3, or the PSMA x CD3 bispecific antibody of the invention described herein). A non-blocking anti-idiotypic antibody can be used to detect total antibody (free, partially bound to an antigen, or fully bound to an antigen) in a sample. An anti-idiotypic antibody can be produced by immunizing an animal with the antibody to which the anti-idiotypic antibody is produced. In some embodiments described herein, an anti-idiotypic antibody is used to detect the level of therapeutic antibodies (eg, anti-PSMA, anti-CD3, or PSMA x CD3 bispecific antibodies of the invention described herein) in a sample.

The anti-I antibody can also be used as an immunogen to induce an immune response in another animal, producing a so-called anti-anti-I antibody. An antibody to an anti-idiotypic antibody may be identical in epitopes to the primary mAb that induced the formation of the anti-idiotypic antibody. Thus, by using antibodies to idiotypic mAb determinants, other clones expressing antibodies of identical specificity can be identified. Anti-Id antibodies can be subjected to variations (thus producing anti-Id antibody variants) and/or derivatives by any suitable technique as described elsewhere herein with respect to PSMA or CD3 specific binding antibodies or PSMA x CD3 bispecific antibodies .

PSMA refers to prostate-specific membrane antigen. The amino acid sequence of Pan troglodytes (also called chimpanzees) PSMA is shown in SEQ ID NO: 1. The extracellular domain spans residues 44-750, the transmembrane domain spans residues 20-43, and the cytoplasmic domain spans residues 1-19 in SEQ ID NO: 1. The amino acid sequence of PSMA Macaca fascicularis (also called cynomolgus, macaque or cynomolgus macaque) is shown in SEQ ID NO: 2. The extracellular domain spans residues 44-750, the transmembrane domain spans residues 20-43, and the cytoplasmic domain spans residues 1-19 in SEQ ID NO: 2. The amino acid sequence of mature human PSMA is shown in SEQ ID NO: 3. The extracellular domain spans residues 44-750, the transmembrane domain spans residues 20-43, and the cytoplasmic domain spans residues 1-19 in SEQ ID NO: 3.

CD3 refers to the T cell antigen receptor. As used herein, the term CD3-specific refers to antibodies that specifically bind to the T cell receptor complex. In particular, antibodies bind to the polypeptide POP-epsilon. which together with CD3

- 11 046641 gamma, -delta and -zeta, as well as heterodimers of T-cell receptors alpha/beta and gamma/delta, forms the T-cell receptor-CD3 complex. This complex plays an important role in linking antigen recognition to several intracellular signal transduction pathways. The CD3 complex mediates signal transduction, leading to T cell activation and proliferation. CD3 is required for immune response.

The term in combination with means that two or more therapeutic agents are administered together to a subject in mixtures, simultaneously as separate agents, or sequentially as separate agents in any order.

The terms overexpress, overexpressed, and overexpressed interchangeably refer to a sample, such as a cancer cell, malignant cell, or cancer tissue, that has measurably higher levels of PSMA compared to a reference sample. Overexpression may be caused by gene amplification or increased transcription or translation. Expression and overexpression of a protein in a sample can be measured using known assays, such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassay on live or lysed cells. Expression and overexpression of a polynucleotide in a sample can be measured, for example, using fluorescence in situ hybridization, Southern blotting, or PCR techniques. A protein or polynucleotide is overexpressed when the level of the protein or polynucleotide in a sample is at least 1.5 times higher than the reference sample. The choice of reference sample is well known.

The term sample refers to the collection of similar fluids, cells or tissues isolated from the body of a patient, as well as fluids, cells or tissues located within the patient. Examples of samples are biological fluids such as blood, serum and serous fluids, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucous secretions of secretory tissues and organs, vaginal secretions, ascites fluids such as associated with non-solid tumors, fluids in the pleura, pericardium, peritoneum, abdominal and other body cavities, fluids collected by bronchial lavage, liquid solutions in contact with the subject or biological source, such as cell and organ culture media, including conditioned environment of cells and organs, washing fluids, etc., tissue biopsies, fine needle aspirates, or tumor tissue after surgical resection.

The term cancer cell or tumor cell refers to a cancerous, precancerous or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes. These changes do not necessarily affect the supply of new genetic material. Although transformation can be caused by infection with a transforming virus and the insertion of a new genomic nucleic acid or the uptake of an exogenous nucleic acid, it can also occur spontaneously or after exposure to a carcinogen, resulting in mutation of an endogenous gene. Transformation/malignancy is manifested by morphological changes, cell immortalization, loss of growth control, lesion formation, proliferation, malignancy, tumor-specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, etc., in vitro, in vivo and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)). Unless otherwise indicated, any numerical values, such as concentration or concentration range, described herein are to be understood as modified in all cases by the term about. Thus, the numerical value typically includes ±10% of the specified value. For example, a concentration of 1 mg/ml includes from 0.9 mg/ml to 1.1 mg/ml. Likewise, the concentration range from 1% to 10% (w/v) includes from 0.9% (w/v) to 11% (w/v). As used herein, the use of a numeric range explicitly includes all possible subranges, all individual numeric values within that range, including integers within such ranges, and fractional values, unless the context clearly requires otherwise.

The term effector antigens defines antigens from cells of the immune system that can stimulate or initiate cytotoxicity, phagocytosis, antigen presentation, and cytokine release. Such effector antigens are derived from, for example, but not limited to, T cells and natural killer (NK) cells. Examples of suitable specificities for effector antigens include, but are not limited to, CD3 or CD3 subunits such as CD3ε for T cells and CD16 for NK cells. Such cell surface molecules of effector cells are suitable for mediating cell killing. Effector cells are cells of the immune system that can stimulate or initiate cytotoxicity, phagocytosis, antigen presentation, and cytokine release. Such effector cells include, for example, but are not limited to, T cells, natural killer (NK) cells, granulocytes, monocytes, macrophages, dendritic cells, and antigen presenting cells. Examples of suitable effector cell specificities include, but are not limited to, CD2, CD3 and CD3 subunits such as CD3e, CD5, CD28 and other T cell receptor (TCR) components; CD16, CD16A, CD25, CD38, CD44, CD56, CD69, CD94, CD335

- 12 046641 (NKp46), CD336, (NKp44), CD337 (NKp30), NKp80, NKG2C and NKG2D, DNAM, NCR for NK cells; CD18, CD64 and CD89 for granulocytes; CD18, CD32, CD64, CD89 and mannose receptor for monocytes and macrophages; CD64 and mannose receptor for dendritic cells; as well as CD35. In some embodiments of the invention, these specificities, i.e. cell surface effector cell molecules are suitable for mediating cell killing by binding bispecific or multispecific molecules to such cell surface molecule and thereby inducing cytolysis or apoptosis.

The terms anti-PSMA x CD3 bispecific antibody, anti-PSMA/CD3 antibody, anti-PSMA x CD3 bispecific antibody, or anti-PSMA/CD3 antibody refer to a molecule containing at least one binding domain that specifically binds PSMA and at least one binding domain specifically binding CD3. Domains that specifically bind PSMA and CD3 are usually VH/VL pairs. The anti-PSMA x CD3 bispecific antibody may be monovalent in terms of its binding to either PSMA or CD3.

The term valency refers to the presence in a molecule of a set number of antigen-specific binding sites. Thus, the terms monovalent, divalent, tetravalent and hexavalent refer to the presence of one, two, four and six antigen-specific binding sites in the molecule, respectively. The term multivalent refers to the presence of two or more antigen-specific binding sites in a molecule.

The term antigen-specific CD4+ or CD8+ T cell refers to a CD4+ or CD8+ T cell activated by a specific antigen or its immunostimulatory epitope.

The term patient includes any human or non-human animal. The term non-human animal includes all vertebrates, such as mammals and non-mammalian animals such as primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise noted, the terms patient or subject are used interchangeably.

The numbering of amino acid residues in the antibody constant region in the text of the description is given in accordance with the EU catalog, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) unless explicitly stated otherwise.

This document uses traditional one- and three-letter amino acid codes as shown in Table 1. 1.

Table 1

Amino acid Three letter code Single letter code Alanin Ala A Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Gin E Glutamine Glu Q Glycine Gly G G istidine His H

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The present invention provides antibodies and fragments thereof that specifically bind PSMA, and multispecific antibodies that specifically bind PSMA and CD3, and fragments thereof. The present invention provides polypeptides and polynucleotides encoding the antibodies of the invention, or nucleic acids complementary thereto, vectors, host cells, and methods for their production and use.

Antibodies and antibody fragments that bind to PSMA bind to the target chimpanzee antigen. In one embodiment, the antibodies and fragments thereof bind to human and macaque PSMA target antigens with affinities within 5-fold of each other. In other words, the fold difference in antibody binding is less than 5. In this case, an identical antibody molecule can be used both for preclinical evaluation of the safety, activity and/or pharmacokinetic profile of PSMA in primates and as a drug in humans. In other words, the same PSMA-specific molecule can be used in preclinical animal studies as well as in human clinical studies. This leads to very comparable results and significantly higher predictive power of animal studies compared to species-specific surrogate molecules. Because the PSMA domain is cross-species specific, i.e., reacts with human and macaque antigens, the antibody or fragments thereof of the invention can be used both for preclinical evaluation of the safety, activity and/or pharmacokinetic profile of these binding domains in primates, and in identical form as a drug for people.

The present invention also provides multispecific antibodies that specifically bind to PSMA. According to the invention, a bispecific, ie bifunctional, antibody can be used to act on two different therapeutic targets or to perform two different functions. Such antibodies can be used, for example, to recruit an immune effector cell, such as a T or NK cell, to a particular target cell. Various molecules based on antibody fragments are known and are being studied, for example, for the treatment of malignant neoplasms. The multispecific antibody of the invention may be a trispecific antibody for dual targeting of tumor cells - these are trifunctional structures that can be configured to target two different targets/epitopes on a tumor cell and with a third functionality of binding with high affinity to either T cells or NK cells. Trispecific antibodies that target two different tumor epitopes and interact with T or NK cells lyse tumor cells that express both targets. Such molecules can be prepared using antibody formats known in the art and described in detail herein. (WO 20151842071, WO 2015158636, WO 2010136172, WO 2013174873). In an embodiment of the trispecific antibody of the invention, the multispecific antibody may be specific for PSMA and a second distinct antigen on the same or a different tumor cell and additionally specific for an effector cell, particularly a T cell or NK cell.

The present invention also provides a bispecific antibody to PSMA x effector antigen. In one embodiment, the effector antigen for the anti-PSMA x effector antigen bispecific antibody is CD3. In the present invention, it has been discovered that it is possible to obtain a bispecific antibody to PSMA x CD3, in which the identical molecule can be used in preclinical animal studies as well as in clinical studies

- 14 046641 research and even in therapy in humans. This is due to the identification of a PSMA x CD3 bispecific antibody that, in addition to binding to human PSMA and human CD3, respectively, also binds to homologs of chimpanzee and macaque antigens. The anti-PSMA x CD3 bispecific antibody of the invention can be used as a therapeutic agent against various diseases, including, but not limited to, cancer. In view of the above, there is no need to construct a surrogate target bispecific antibody to PSMA x CD3 for testing in phylogenetically distant (from human) species. As a result, an identical molecule can be used in preclinical animal studies as it is intended to be administered to humans in clinical trials and once registered and approved by the Medical Products Administration. The ability to use the same molecule in preclinical animal studies as it is subsequently administered to humans virtually eliminates, or at least greatly reduces, the risk that data obtained from preclinical animal studies will have limited applicability to humans. In short, obtaining preclinical safety data in animals using the same molecule that will actually be administered to humans largely ensures that the data is applicable to a human-relevant scenario. In contrast, in traditional approaches using surrogate molecules, said surrogate molecules must be molecularly tailored to the animal system of interest used for preclinical safety assessment. Thus, the molecule used in human therapy actually differs in sequence, and probably also in structure, from the surrogate molecule used in the preclinical study, in terms of pharmacokinetic parameters and/or biological activity, resulting in data obtained from the preclinical study in animals. limited applicability/tolerability to humans. The use of surrogate molecules requires the design, production, purification and characterization of an entirely new construct. This results in additional development costs, and obtaining this molecule takes time. In general, surrogates need to be developed separately in addition to the actual drug that will be used in human therapy, so two lines of development need to be pursued for two molecules. Thus, the main advantage of the PSMA x CD3 bispecific antibody of the invention exhibiting the cross-species specificity described herein is that the identical molecule can be used for therapeutic agents in humans and preclinical studies in animals.

Another major advantage of the antibody and multispecific antibody of the invention is its applicability for preclinical testing in a variety of primates. The behavior of a drug candidate in animals should ideally be an indicator of the expected behavior of that drug candidate when administered to humans. Thus, the data obtained from such a preclinical study should inherently have high predictive power in humans. However, as we know from the tragic result of a recent phase I clinical trial for TGN1412 (an anti-CD28 monoclonal antibody), the drug candidate may act differently in primates than in humans: Although preclinical animal studies of the antibody in cynomolgus monkeys reported no adverse effects observed, or only limited adverse effects were observed, with the administration of this antibody, six human patients developed multiple organ failure (Lancet 368 (2006), 2206-7). The results of these critical adverse effects suggest that limiting preclinical testing to only one species (non-chimpanzee primates) may not be sufficient. The fact that the described antibody and multispecific antibody specifically bind chimpanzee and cynomolgus monkey PSMA may help overcome the problems encountered in the above-mentioned case. Accordingly, the present invention provides means and methods for minimizing differences in the types of effects during the development and testing of drugs for humans.

In the case of the antibody and multispecific antibody according to the invention, it is also no longer necessary to adapt the test animal to the drug candidate intended for administration to humans, for example to create transgenic animals. The cross-species specificity of the PSMA antibody or multispecific antibody of the invention allows the antibody to be directly used for preclinical testing in non-chimpanzee primates without any genetic manipulation of the animals. As is well known to those skilled in the art, approaches in which a study animal is adapted to a drug candidate always carry the risk that the results obtained in a preclinical safety study are less representative and predictable for humans due to modification of the animal. For example, in transgenic animals, the proteins encoded by the transgenes are often highly overexpressed. Thus, data obtained on the biological activity of an antibody to a given protein antigen may be limited in its predictive value for individuals in whom the protein is expressed at much lower and physiological levels.

An additional advantage of using an antibody according to the invention exhibiting

- 15 046641 interspecies specificity is the fact that the use of chimpanzees, as an endangered species, for animal research can be avoided. Chimpanzees are the closest relatives of humans and were recently grouped into the family Apes based on genomic sequencing data (Wildman et al., PNAS 100 (2003), 7181). Therefore, the data obtained for chimpanzees are essentially considered to be highly predictive for humans. However, due to the species' endangered status, the number of chimpanzees that can be used for medical experiments is severely limited. Thus, as stated above, keeping chimpanzees for animal testing is expensive and ethically problematic. The use of the antibody according to the invention avoids both ethical problems and financial burden during preclinical research without compromising quality, i.e. applicability of the obtained data from animal studies. In light of this, the use of an antibody or multispecific antibody according to the invention that specifically binds PSMA provides a suitable alternative for studies in chimpanzees.

Another additional advantage of the antibody or multispecific antibody of the invention that specifically binds PSMA is the ability to recover multiple blood samples when used in preclinical animal studies, such as animal pharmacokinetic studies. When using a non-chimpanzee primate, multiple blood samples can be obtained much more easily than when using lower animals such as mice. The collection of multiple blood samples allows for continuous testing of blood parameters to determine the biological effects induced by the antibody or multispecific antibody of the invention that specifically binds the PSMA of the invention. In addition, collecting multiple blood samples allows the researcher to evaluate the pharmacokinetic profile of the antibody or multispecific antibody of the invention that specifically binds PSMA, as defined herein. In addition, potential side effects that may be induced by said antibody or a multispecific antibody of the invention that specifically binds PSMA, as reflected in blood parameters, can be measured in different blood samples collected during administration of said antibody.

This allows the potential toxicity profile of a PSMA binding antibody or multispecific antibody of the invention to be determined as defined herein.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated PSMA-specific binding antibody or antibody fragment thereof has one, two, three, four, or five of the following properties:

a) binds the extracellular domain (ECD) of Pan troglodytes PSMA with an equilibrium dissociation constant (KD) of 25 nM or less, with KD measured using the ProteOn XPR36 system at +25°C,

b) binds LNCaP cells with a calculated EC ₅₀ of 20 nM or less and binds HEK cells expressing Macaca fascicularis PSMA with a calculated EC ₅₀ of 40 nM or less, with a difference in calculated EC ₅₀ between binding of LNCaP cells and binding of HEK cells expressing PSMA Macaca fascicularis, is less than 5 times, and the calculated EC ₅₀ is measured in a whole cell binding assay at 0°C using flow cytometry,

c) binds recombinant ECD of human PSMA (SEQ ID NO: 7), Pan troglodytes (SEQ ID NO: 4) and Macaca fascicularis (SEQ ID NO: 5) with an equilibrium dissociation constant (KD) of 12 nM or less, the KD being measured with using ProteOn XPR36 surface plasmon resonance analysis at +25°C;

d) demonstrates T cell-mediated killing of LNCaP cells, C42 cells, HEK cells expressing human PSMA, or HEK cells expressing Macaca fascicularis PSMA when paired with a bispecific antibody with anti-CD3 antibody CD3B219, and T cell-mediated killing is measured by chromium-51 release or by caspase 3/7 activation assay, or

e) recognizes a conformational epitope, wherein the epitope consists of residues I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307 and K324-P326 of human PSMA (SEQ ID NO: 3).

Examples of such antibodies or fragments thereof are anti-PSMA antibodies PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129,

PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344,

PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362,

PSMB363 and PSMB365 described in this document.

In some embodiments described herein, and in some embodiments of each and every numbered embodiment listed below, an isolated anti-PSMA antibody or PSMA-specific binding fragment thereof binds chimpanzee PSMA ECD with an equilibrium dissociation constant (KD) of about 30 nM or less, with KD measured using the ProteOn XPR36 system at +25°C as described in Example 8. Proteon SPR affinity assays include assays performed at room temperature (e.g., 25°C or near) in which an antibody capable of binding chimpanzee PSMA is captured on a Proteon sensor chip by an anti-Fc antibody (e.g.

- 16 046641 (Jackson ImmunoResearch Laboratory, cat. No. 109-005-098) to a level of about 100 RU, followed by the introduction of recombinant PSMA ECD and collection of association and dissociation data at a flow rate of 50 μl/min.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, an isolated anti-PSMA antibody or a PSMA-specific binding fragment thereof binds LNCaP cells with an EC ₅₀ of 20 nM or less and binds cynomolgus PSMA-expressing HEK cells with an EC ₅₀ of 40 nM or less, wherein the difference in calculated EC ₅₀ between binding of LNCaP cells and binding of cynomolgus PSMA-expressing HEK cells is less than 5-fold, and cell binding is measured by FACS as described in Example 7. Whole-cell binding assays by FACS were performed at a density of 200,000 cells per well for 1 hour on ice. The amount of antibody bound to whole cells is detected using a labeled secondary antibody, such as RPE-conjugated mouse anti-human kappa light chain antibody (Life Technologies, cat. no. MH10514) using a FACS flow cytometer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated antibody or fragment thereof specifically binds to human, chimpanzee PSMA, and cynomolgus PSMA ECD with an equilibrium dissociation constant ( _KD ) 12 nM or less, with KD measured using the ProteOn XPR36 system at +25°C as described in Example 8. Proteon SPR affinity assays include assays performed at room temperature (e.g., 25°C C or about) in which an antibody capable of binding chimpanzee PSMA is captured on a Proteon sensor chip with an anti-Fc antibody (e.g., (Jackson ImmunoResearch Laboratory, cat. no. 109-005-098) to a level of about 100 RU, followed by the introduction of a recombinant ECD PSMA and association and dissociation data acquisition at a flow rate of 50 μL/min.

The measured affinity of a particular antibody/PSMA interaction may change when measured under different conditions (eg, osmolarity, pH). Thus, measurements of affinity and other binding parameters (eg, _KD , _Kon , _Koff ) are typically performed under standardized conditions and using a standardized buffer solution, such as the buffer solution described herein. Those skilled in the art will appreciate that the inherent error in affinity measurements, such as those using Biacore 3000 or ProteOn equipment (measured as standard deviation (SD)), can typically be 5-33% for measurements made within typical detection limits . Therefore, the term about in the context of K _D reflects the typical standard deviation in the analysis. For example, a typical SOS for _KD equal to 1 x 10 ^-9 M is up to 0.33 x 10 ^-9 M.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, an isolated antibody or fragment thereof that specifically binds PSMA exhibits T cell-mediated killing of PSMA-expressing human LNCaP cells, C42 cells. , PSMA-expressing HEK cells or PSMA-expressing cynomolgus HEK cells when paired with a bispecific antibody with an anti-CD3 antibody CD3B219, wherein T cell-mediated killing is measured by chromium-51 release and the target cells are cultured with pre-activated T cells in the ratio 5:1 for 18-24 hours or by caspase 3/7 activation assay as in Example 6. In some embodiments, an isolated antibody or fragment thereof that specifically binds PSMA demonstrates T cell-mediated killing of PSMA-expressing LNCaP and C42- cells human cells with an EC ₅₀ of about 0.3-0.5 nM or less and 0.12-0.03 nM or less, respectively, when pairing the bispecific antibody with the anti-CD3 antibody CD3B219, wherein T cell-mediated killing is measured with using a caspase 3/7 activation assay as in Example 9. Cells expressing the target PSMA are cultured with pre-activated T cells in a 1:3 ratio for 18-24 hours, and cleavage of the added caspase 3/7 substrate produces a fluorescent DNA dye with fluorescence limited to the cell nucleus.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, an isolated antibody or fragment thereof that specifically binds PSMA recognizes a conformational epitope, wherein the epitope consists of residues I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307 and K324-P326 according to X-ray crystallography data described in example 13.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA-specific binding antibody or fragment thereof of the invention comprises HCDR1, HCDR2 and HCDR3 contained within the severe variable region chains (VH) with SEQ ID

- 17 046641

NO: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79 160, 138, 139 or 140, with HCDR1, HCDR2 and HCDR3 determined by Chothia, Kabat or IMGT.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA-specific binding antibody or fragments thereof of the invention comprise LCDR1, LCDR2 and LCDR3 contained within a light chain variable region (VL) with SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 76, 78, 142, 143 or 144, with LCDR1, LCDR2 and LCDR defined by Chothia, Kabat or IMGT.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, a PSMA-specific binding antibody in accordance with the invention comprises:

HCDR1 with SEQ ID NO: 8, 14, 20, 25, 31, 36, 46, 53 or 122;

HCDR2 with SEQ ID NO: 9, 15, 21, 26, 32, 37, 42, 44, 54, 123, 130, 134, 135 or 137; And

HCDR3 with SEQ ID NO: 10, 16, 22, 27, 33, 38, 43, 45, 48, 52, 55, 124.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, a PSMA-specific binding antibody in accordance with the invention comprises:

LCDR1 with SEQ ID NO: 11, 17, 23, 28, 34, 39, 46, 49 or 131;

LCDR2 with SEQ ID NO: 12, 18, 29, 40, 50 or 133; And

LCDR3 with SEQ ID NO: 13, 19, 24, 30, 35, 41, 47, 51, 132 or 136.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises: HCDR1 of SEQ ID NO: 8, 14, 20 , 25, 31, 36, 53 or 122;

HCDR2 with SEQ ID NO: 9, 15, 21, 26, 32, 37, 42, 44, 54, 123, 130, 134, 135 or 137;

HCDR3 with SEQ ID NO: 10, 16, 22, 27, 33, 38, 43, 45, 48, 51, 52, 55 or 124;

LCDR1 with SEQ ID NO: 11, 17, 23, 28, 34, 39, 46, 49 or 131;

LCDR2 with SEQ ID NO: 12, 18, 29, 40, 50 or 133; And

LCDR3 with SEQ ID NO: 13, 19, 24, 30, 35, 41, 47, 51, 132 or 136.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, and HCDR3 with

SEQ ID NO: 8, 9 and 10 respectively;

SEQ ID NO: 14, 15 and 16 respectively;

SEQ ID NO: 20, 21 and 22 respectively;

SEQ ID NO: 25, 26 and 27 respectively;

SEQ ID NO: 25, 130 and 27 respectively;

SEQ ID NO: 25, 134 and 27 respectively;

SEQ ID NO: 25, 135 and 27 respectively;

SEQ ID NO: 25, 137 and 27 respectively;

SEQ ID NO: 31, 32 and 33 respectively;

SEQ ID NO: 36, 37 and 38 respectively;

SEQ ID NO: 31, 42 and 43 respectively;

SEQ ID NO: 31, 44 and 45 respectively;

SEQ ID NO: 36, 37 and 48 respectively;

SEQ ID NO: 36, 37 and 52 respectively;

SEQ ID NO: 53, 54 and 55 respectively; or

SEQ ID NO: 122, 123, and 124 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises LCDR1, LCDR2, and LCDR3 with

SEQ ID NO: 11, 12 and 13 respectively;

SEQ ID NO: 17, 18 and 19 respectively;

SEQ ID NO: 23, 12 and 24 respectively;

SEQ ID NO: 28, 29 and 30 respectively;

SEQ ID NO: 28, 29 and 136 respectively;

SEQ ID NO: 28, 133 and 132 respectively;

SEQ ID NO: 34, 12 and 35 respectively;

SEQ ID NO: 39, 40 and 41 respectively;

SEQ ID NO: 46, 29 and 47 respectively;

- 18 046641

SEQ ID NO: 49, 50 and 51 respectively;

SEQ ID NO: 23, 12 and 35 respectively; or

SEQ ID NO: 131, 29 and 132 respectively;

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 8, 9, 10, 11, 12 and 13 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 14, 15, 16, 17, 18 and 19 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 20, 21, 22, 23, 12 and 24 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 122, 123, 124, 23, 12 and 24 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 26, 27, 28, 29 and 30 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 31, 32, 33, 34, 12 and 35 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 36, 37, 38, 39, 40 and 41 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 36, 37, 38, 49, 50 and 51 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 31, 42, 43, 11, 12 and 13 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 31, 44, 45, 46, 29 and 47 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 36, 37, 52, 49, 50 and 51 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 53, 54, 55, 23, 12 and 35 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 130, 27, 28, 29 and 30 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 130, 27, 131, 29 and 132 respectively.

In some embodiments of the invention described herein, and in some embodiments of each and every numbered embodiment,

- 19 046641 below, the PSMA specific binding antibody according to the invention contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 130, 27, 28, 133 and 132, respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 29 and 30 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 135, 27, 131, 29 and 132 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 29 and 136 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 29 and 30 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 135, 27, 131, 29 and 132 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 29 and 136 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 134, 27, 131, 29 and 132 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 134, 27, 28, 133 and 132 respectively.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 137, 27, 28, 133 and 132 respectively. In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises a heavy chain variable region (VH) of SEQ ID NO: 60, 62 , 64, 66, 68, 70, 72, 74, 75, 77, 79, 160, 138, 139, or 140.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA-specific binding antibody of the invention comprises a light chain variable region (VL) of SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 76, 78, 142, 143, or 144.

The sequences of VH, VL, HCDR and LCDR of examples of antibodies that specifically bind PSMA according to the invention are shown in table. 2.

In table 2 provides a summary of examples of some of the PSMA-specific antibodies described herein.

- 20 046641

table 2

CDR sequences (determined by Kabat) of mAbs obtained by phage panning against human PSMA (SEQ ID NO:)

ID FAB CDR (SEQ ID NO: ) CDR1 CDR2 CDR3 PSMB129 H C NAWIS (8) WINPESGRANYAQKF QG (9) ELYYLVYSTYYYAFDY (10) L.C. RASQSIDRWLN (eleven) AASSLQS (12) QQSPRYPLT (13) PSMB130 H C SYDIS (14) GIIPIEGTANYAQKFQ G (15) DYPAGYGFDY (16) L.C. RASQSVSSSYLA (17) GASSRAT (18) QQYGSSPLT (19) PSMB128 H C SDWMS (20) AISGNGGSTEYADSV KG (21) DPYYYYDGDSYYGMD V (22) L.C. RASQSISSYLN (23) AASSLQS (12) QQSYSTP (24) PSMB127 H C SDAMH (25) EISGSGGYTNYADSV KG (26) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASNRAT (29) QQRSNWPLT (thirty) PSMB124 H C SYAIS (31) WISPYNGNANYAQKF QG (32) DSDRSYNLDY (33)

- 21 046641

L.C. RASQSISGWLN (34) AASSLQS (12) QQSYSTPLT (35) PSMB123 Η WITH SYWIG (36) IIYPGDSDTRYSPSFQG (37) GLPIWYLDY (38) L.C. RASQSVASDLA (39) FASNRAT (40) QQSITWPFT (41) PSMB122 Η WITH SYAIS (31) WIIPYNGNANYAQKF QG (42) VNSAALVWERLDY (43) L.C. RASQSIDRWLN (eleven) AASSLQS (12) QQSPRYPLT (13) PSMB123 Η WITH SYAIS (31) GIIPIFGTANYAQKFQ G (44) ASRVWHASYGYLDY (45) L.C. RASQSVSKWLA (46) DASNRAT (29) QQRFTAPWT (47) PSMB120 Η WITH SYWIG (36) IIYPGDSDTRYSPSFQG (37) GWAYDRGLDY (48) L.C. KSSQSVLYSSNNNKNY L.A. (49) WASTRES (50) QQYYSTPLT (51) PSMB119 Η WITH SYWIG (36) IIYPGDSDTRYSPSFQG (37) AYHYSKGLDY (52) L.C. KSSQSVLYSSNNNKNY L.A. (49) WASTRES (50) QQYYSTPLT (51) PSMB126 Η WITH DYAIS (53) RIDPIEGTANYAQKFQ G (54) DRYYYDGVYWYSDYF DY (55)

- 22 046641

L.C. RASQSISSYLN (23) AASSLQS (12) QQSYSTPLT (35) PSMB87 Η WITH SYWIS (122) IIYPGDSYTRYSPSFQG (123) DYEWELFDSRLDY (124) L.C. RASQSISSYLN (23) AASSLQS (12) QQSYSTP (24) PSMB344 NS SDAMH (25) EISGSGGYTNYADSM KG (130) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASNRAT (29) QQRSNWPLT (30) PSMB345 NS SDAMH (25) EISGSGGYTNYADSM KG (130) DSYDSSLYVGDYFDY (27) L.C. RASQSVSNYLA (131) DASNRAT (29) QQRRNWPLT (132) PSMB346 NS SDAMH (25) EISGSGGYTNYADSM KG (130) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASYRAT (133) QQRRNWPLT (132) PSMB347 NS SDAMH (25) EISGSGGYTNYADSM KS (134) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASNRAT (29) QQRSNWPLT (thirty) PSMB349 NS SDAMH (25) EISGSGGYTNYADSLK G(135) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASNRAT (29) QQRGNWPLT (136) PSMB358 NS SDAMH (25) EISGSGGYTNYADSLK G (135) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASNRAT (29) QQRSNWPLT (30) PSMB359 NS SDAMH EISGSGGYTNYADSLK DSYDSSLYVGDYFDY

- 23 046641

(25) G(135) (27) L.C. RASQSVSNYLA (131) DASNRAT (29) QQRRNWPLT (132) PSMB360 NS SDAMH (25) EISGSGGYTNYADSLK G(135) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASYRAT (133) QQRRNWPLT (132) PSMB361 NS SDAMH (25) EISGSGGYTNYADSM KS (134) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASNRAT (29) QQRGNWPLT (136) PSMB362 NS SDAMH (25) EISGSGGYTNYADSM KS (134) DSYDSSLYVGDYFDY (27) L.C. RASQSVSNYLA (131) DASNRAT (29) QQRRNWPLT (132) PSMB363 NS SDAMH (25) EISGSGGYTNYADSM KS (134) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASYRAT (133) QQRRRNWPLT (132) PSMB365 NS SDAMH (25) EISGSGGYTNYADSLK S(137) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASYRAT (133) QQRRNWPLT (132)

In some embodiments, a PSMA-specific antibody or antigen binding fragment thereof is provided comprising a heavy chain comprising CDR1, CDR2 and CDR3 of any of the antibodies described in Table. 2. In some embodiments, a PSMA-specific antibody or antigen binding fragment thereof is provided comprising a heavy chain comprising CDR1, CDR2 and CDR3 of any of the antibodies described in Table. 2, and a light chain containing CDR1, CDR2 and CDR3 of any of the antibodies described in table. 2. In some embodiments described herein, a PSMA-specific antibody or antigen binding fragment thereof competes for binding to PSMA with an antibody or antigen binding fragment containing a heavy chain containing CDR1, CDR2 and CDR3 of any one of the antibodies described in table. 2, and a light chain containing CDR1, CDR2 and CDR3 of any one of the antibodies described in table. 2.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 14, 15, 16, 17, 18 and 19 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 84 and a light chain (LC) of SEQ ID NO: 85.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 20, 21, 22, 23, 12 and 24 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

- 24 046641

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 86 and a light chain (LC) of SEQ ID NO: 87.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 31, 42, 43, 11, 12 and 13 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 74 and VL of SEQ ID NO: 61.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 96 and a light chain (LC) of SEQ ID NO: 83.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 26, 27, 28, 29 and 30 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 88 and a light chain (LC) of SEQ ID NO: 89.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 122, 123, 124, 23, 12 and 24 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 160 and VL of SEQ ID NO: 65.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 125 and a light chain (LC) of SEQ ID NO: 91.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 8, 9, 10, 11, 12 and 13 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 82 and a light chain (LC) of SEQ ID NO: 83.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 31, 32, 33, 34, 12 and 35 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 70 and VL of SEQ ID NO: 71.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 92 and a light chain (LC) of SEQ ID NO: 93.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 36, 37, 38, 39, 40 and 41 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 72 and VL of SEQ ID NO: 73.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 94 and a light chain (LC) of SEQ ID NO: 95.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 31, 44, 45, 46, 29 and 47 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 75 and VL of SEQ ID NO: 76.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 97 and a light chain (LC) of SEQ ID NO: 98.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 36, 37, 48, 49, 50 and 51 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 77 and VL of SEQ ID NO: 78.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 99 and a light chain (LC) of SEQ ID NO: 100.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 36, 37, 52, 49, 50 and 51 respectively.

- 25 046641

In some embodiments, the antibody comprises VH of SEQ ID NO: 79 and VL of SEQ ID NO: 78.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 101 and a light chain (LC) of SEQ ID NO: 100.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 53, 54, 55, 23, 12 and 35 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 90 and a light chain (LC) of SEQ ID NO: 91.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 130, 27, 28, 29 and 30 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 145 and a light chain (LC) of SEQ ID NO: 89.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 130, 27, 131, 29 and 132 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 142.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 145 and a light chain (LC) of SEQ ID NO: 148.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 130, 27, 28, 133 and 132 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 143.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 145 and a light chain (LC) of SEQ ID NO: 149.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 29 and 30 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 146 and a light chain (LC) of SEQ ID NO: 89.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 29 and 136 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 144.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 147 and a light chain (LC) of SEQ ID NO: 150.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 29 and 30 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 147 and a light chain (LC) of SEQ ID NO: 89.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 135, 27, 131, 29 and 132 respectively.

- 26 046641

In some embodiments, the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 143.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 147 and a light chain (LC) of SEQ ID NO: 148.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 133 and 132 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 147 and a light chain (LC) of SEQ ID NO: 149.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 29 and 136 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 144.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 146 and a light chain (LC) of SEQ ID NO: 150.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 134, 27, 131, 29 and 132 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 67.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 139 and a light chain (LC) of SEQ ID NO: 142.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 134, 27, 28, 133 and 132 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 143.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 146 and a light chain (LC) of SEQ ID NO: 149.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 25, 137, 27, 28, 133 and 132 respectively.

In some embodiments, the antibody comprises VH of SEQ ID NO: 141 and VL of SEQ ID NO: 143.

In some embodiments, the antibody comprises a heavy chain (HC) of SEQ ID NO: 151 and a light chain (LC) of SEQ ID NO: 149.

In some embodiments, the antibody binds human PSMA ECD with an equilibrium dissociation constant ( _KD ) of less than about 100 nM, in some cases less than about 50 nM, such as less than about 12 nM, wherein the _KD is measured using a ProteOn XPR36 system at +25°C .

In some embodiments, the antibody binds cynomolgus PSMA ECD with an equilibrium dissociation constant ( _KD ) of less than about 100 nM, in some cases less than about 50 nM, such as less than about 12 nM, wherein the _KD is measured using a ProteOn XPR36 system at +25° WITH.

In some embodiments, the antibody is of the IgG4 isotype, which in some cases contains the S228P, F234A, and L235A substitution in the heavy chain compared to wild-type IgG4.

In some embodiments, the antibody contains VH of SEQ ID NO: 74 and VL of SEQ ID NO: 61 and is of the IgG4 isotype, in some cases containing substitutions S228P, F234A and L235A compared to wild-type IgG4.

In some embodiments, the antibody is a multispecific antibody, such as a PSMA/CD3 bispecific antibody.

The antibody is suitable for use in therapy, for example in the treatment of cancer.

The antibody is suitable for use in therapy, for example in the treatment of a solid tumor.

The antibody is suitable for use in therapy, for example in the treatment of prostate cancer or castration-resistant prostate cancer.

The antibody is suitable for use in therapy, for example, in the treatment of prostatic intraepithelial neoplasia.

- 27 046641

The antibody is suitable for use in therapy, for example in the treatment of colorectal cancer.

The antibody is suitable for use in therapy, for example, in the treatment of clear cell renal cell carcinoma.

The antibody is suitable for use in therapy, for example in the treatment of gastric cancer.

The antibody is useful for use in therapy, for example in the treatment of renal cell carcinoma (RCC) (eg, clear cell renal carcinoma or papillary cell renal carcinoma) or metastatic lesions thereof.

The antibody is suitable for use in therapy, for example in the treatment of bladder cancer.

The antibody is suitable for use in therapy, for example in the treatment of breast cancer.

The antibody is suitable for use in therapy, for example in the treatment of kidney cancer.

The antibody is suitable for use in therapy, for example, in the treatment of a neovascular disorder, such as, for example, cancer characterized by the growth of a solid tumor.

The antibody is suitable for use in therapy, for example, in the treatment of a neovascular disorder, such as, for example, clear cell kidney cancer (CCRC), colorectal cancer, breast cancer, bladder cancer, lung cancer and pancreatic cancer, as well as various other types of cancer other than prostate cancer, including, without limitation, kidney, urothelial, lung, colon, breast and liver adenocarcinoma.

The IgG class in humans is divided into four isotypes: IgG1, IgG2, IgG3 and IgG4. They have more than 95% homology in the amino acid sequence of the Fc regions, but have significant differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcgRs) on the surface of effector immune cells, such as natural killer cells and macrophages, leading to phagocytosis and lysis of target cells. In CDC, antibodies destroy target cells by triggering a cascade of complement reactions on the cell surface. Antibodies described herein include antibodies with the described variable domain features in combination with any of the IgG isotypes, including modified versions in which the Fc sequence is modified to affect various effector functions.

In many therapeutic antibody applications, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions may be deleterious and potentially pose a safety concern by causing toxicity unrelated to the mechanism of action. Effector functions can be altered by engineering Fc regions to reduce their binding to FcgR or complement factors. The binding of IgG to the activating (FcgRI, FcgRIIa, FcgRIIIa and FcgRIIIb) and inhibitory (FcgRIIb) FcgR receptors or to the first complement component (C1q) depends on residues located in the hinge region and the CH2 domain. Mutations have been introduced into IgG1, IgG2 and IgG4 to reduce or eliminate Fc functions. Antibodies described herein may include these modifications.

In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function compared to the parent Fc; (b) reduced affinity for Fcg RI, Fcg RIIa, Fcg RIIb, Fcg RIIIb and/or Fcg RIIIa; (c) reduced affinity for FcgRI; (d) reduced affinity for FcgRIIa; (e) reduced affinity for FcgRIIb; (f) reduced affinity for FcgRIIIb or (g) reduced affinity for FcgRIIIa.

In some embodiments, the antibodies or antigen-binding fragments are IgG or derivatives thereof, such as the IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments, in which the antibody is of the IgG4 isotype, the antibody contains substitutions S228P, L234A, and L235A in the Fc region. In some embodiments, in which the antibody is of the IgG1 isotype, the antibody contains the S228P, L234A, and L235A substitutions in its Fc region. Antibodies described herein may include these modifications. In some embodiments, the antibody is of the IgG1 isotype.

In some embodiments, the antibody is of the IgG4 isotype, which in some cases contains the S228P substitution in the heavy chain compared to wild-type IgG4.

In some embodiments, the antibody is of the IgG1 isotype, optionally containing heavy chain substitutions L234A, G237A, P238S, H268A, V309L, A330S, and P331S compared to wild-type IgG1.

In addition to the described PSMA-specific antibodies and antigen-binding fragments, polynucleotide sequences capable of encoding the described antibodies and antigen-binding fragments are also provided. In addition, vectors are provided containing the disclosed polynucleotides, as well as cells expressing PSMA-specific antibodies or antigen binding fragments provided herein. In addition, cells capable of expressing the described vectors are described. These cells may be mammalian cells (eg, 293F cells, CHO cells), insect cells (eg, Sf7 cells), yeast cells, plant cells, or bacterial cells (eg, E. coli). The described antibodies can also be produced by hybridoma cells.

- 28 046641

Homologous antibodies

Variants of antibodies that specifically bind PSMA in accordance with the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, containing the amino acid sequences VH, VL or VH and VL shown in table. 3 are within the scope of the invention. For example, variants may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions in VH and/or VL to the extent that homologous antibodies have retained or have improved functional properties compared to the original antibodies. In some embodiments, the sequence identity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to the VH or VL amino acid sequence of the invention. Optionally, any change to the variant from the parent antibody is not performed within the CDR of the variant.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 60, 62, 64. 66, 68, 70, 72, 74, 75, 77, 79, 160, 138, 139 or 140, wherein VH optionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VL of SEQ ID NO: 61, 63, 65. 67, 69, 71, 73, 76, 78, 142, 143 or 144, wherein VL optionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 60 and VL of SEQ ID NO: 60 NO: 61, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions.

Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments of the invention described herein, and in some embodiments of each and every one of the numbered embodiments,

- 29 046641 numbered below, the PSMA specific binding antibody according to the invention comprises VH of SEQ ID NO: 70 and VL of SEQ ID NO: 71, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 72 and VL of SEQ ID NO: 73, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 74 and VL of SEQ ID NO: 74 NO: 61, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions.

Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 75 and VL of SEQ ID NO: 76, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 77 and VL of SEQ ID NO: 78, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 79 and VL of SEQ ID NO: 79 NO: 78, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 160 and VL of SEQ ID NO: 160 NO: 65, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 138 NO: 67, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions.

Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 138 NO: 142, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments of the invention described herein, and in some

- 30 046641 In each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 143, wherein VH, VL, or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 139 NO: 67, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 140 NO: 144, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 140 NO: 67, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions.

Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 140 NO: 142, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 140 NO: 143, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 139 NO: 144, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 139 NO: 142, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 139 NO: 143, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions.

Optionally, any replacements are not performed within the scope of the CDR.

- 31 046641

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VH of SEQ ID NO: 141 and VL of SEQ ID NO: 141 NO: 143, wherein VH, VL or both VH and VL optionally contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any replacements are not performed within the scope of the CDR.

Homologous antibodies that specifically bind PSMA in accordance with the invention described herein, and in some embodiments of any and all of the numbered embodiments listed below, have one, two, three, four, or five of the following properties:

a) binds the ECD of PSMA Pan troglodytes with an equilibrium dissociation constant (KD) of 25 nM or less, with KD measured using the ProteOn XPR36 system at +25°C,

b) binds LNCaP cells with a calculated EC ₅₀ of 20 nM or less and binds HEK cells expressing Macaca fascicularis PSMA with a calculated EC ₅₀ of 40 nM or less, with a difference in calculated EC ₅₀ between binding of LNCaP cells and binding of HEK cells expressing PSMA Macaca fascicularis, is less than 5 times, and the calculated EC ₅₀ is measured in a whole cell binding assay at 0°C using flow cytometry,

c) binds recombinant ECD of human PSMA (SEQ ID NO: 7), Pan troglodytes (SEQ ID NO: 4) and Macaca fascicularis (SEQ ID NO: 5) with an equilibrium dissociation constant (KD) of 12 nM or less, the KD being measured with using ProteOn XPR36 surface plasmon resonance analysis at +25°C;

d) demonstrates T cell-mediated killing of LNCaP cells, C42 cells, HEK cells expressing human PSMA, or HEK cells expressing Macaca fascicularis PSMA when paired with a bispecific antibody with anti-CD3 antibody CD3B219, and T cell-mediated killing is measured by chromium-51 release or by caspase 3/7 activation assay or

e) recognizes a conformational epitope, wherein the epitope consists of residues I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307 and K324-P326 of human PSMA (SEQ ID NO: 3)

Antibodies with conservative modifications

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises VHs containing HCDR1, HCDR2 and HCDR3 sequences, and VLs that contain the sequences LCDR1, LCDR2 and LCDR3, wherein one or more of the CDR sequences contains established amino acid sequences based on the antibodies described herein (for example, the antibodies shown in Table 2) or conservative modifications thereof, and wherein the antibodies retain the desired functional properties parent antibodies that specifically bind PSMA according to the invention.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 20, 21, 22, 23, 12 and 24, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 31, 32, 33, 34, 12 and 35 respectively

- 32 046641 and their conservative modifications.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NO: 31, 42, 43, 11, 12 and 13, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 31, 44, 45, 46, 29 and 47, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 36, 37, 48, 49, 50 and 51, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 36, 37, 52, 49, 50 and 51, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 53, 54, 55, 23, 12 and 35, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 130, 27, 28, 29 and 30, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 130, 27, 131, 29 and 132, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 130, 27, 28, 133 and 132, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 134, 27, 28, 29 and 30, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 135, 27, 28, 29 and 136, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 135, 27, 28, 29 and 30, respectively, and conservative modifications thereof.

In some embodiments of the invention described herein, and in some

- 33 046641 In each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 135, 27, 131, 29 and 132, respectively, and their conservative modifications.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 135, 27, 131, 29 and 132, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 134, 27, 28, 29 and 136, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 134, 27, 131, 29 and 132, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 134, 27, 28, 133 and 132, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 25, 137, 27, 28, 133 and 132, respectively, and conservative modifications thereof.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA specific binding antibody of the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 with SEQ ID NOs: 122, 123, 124, 23, 12 and 24, respectively, and conservative modifications thereof.

Antibodies with conservative modifications in accordance with the invention described herein, and in some embodiments, any and all of the numbered embodiments listed below, have one, two, three, four, or five of the following properties:

a) binds the ECD of PSMA Pan troglodytes with an equilibrium dissociation constant (KD) of 25 nM or less, with KD measured using the ProteOn XPR36 system at +25°C,

b) binds LNCaP cells with a calculated EC ₅₀ of 20 nM or less and binds HEK cells expressing Macaca fascicularis PSMA with a calculated EC ₅₀ of 40 nM or less, with a difference in calculated EC ₅₀ between binding of LNCaP cells and binding of HEK cells expressing PSMA Macaca fascicularis, is less than 5 times, and the estimated EC50 is measured in a whole cell binding assay at 0°C using flow cytometry,

c) binds to recombinant ECD PSMA from humans (SEQ ID NO: 7), Pan troglodytes (SEQ ID NO: 4) and Macaca fascicularis (SEQ ID NO: 5) with an equilibrium dissociation constant (KD) of 12 nM or less, and KD measured using ProteOn XPR36 surface plasmon resonance assay at +25°C;

d) demonstrates T cell-mediated killing of LNCaP cells, C42 cells, HEK cells expressing human PSMA, or HEK cells expressing Macaca fascicularis PSMA when paired with a bispecific antibody with anti-CD3 antibody CD3B219, and T cell-mediated killing is measured by chromium-51 release or by caspase 3/7 activation assay or

e) recognizes a conformational epitope, wherein the epitope consists of residues I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307 and K324-P326 of human PSMA (SEQ ID NO: 3).

The term conservative modification refers to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody containing the amino acid sequences. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative substitutions are substitutions in which an amino acid is replaced by an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains

- 34 046641 chains (for example, aspartic acid, glutamic acid), basic side chains (for example, lysine, arginine, histidine), non-polar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g. phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g. glycine, alanine, valine, leucine , isoleucine, serine, threonine), amides (eg, asparagine, glutamine), beta-branched side chains (eg, threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Additionally, any native residue in the polypeptide can be replaced with alanine, according to a method previously described as alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions in the antibodies of the invention can be performed by known methods, for example, by PCR-mediated mutagenesis (US Pat. No. 4,683,195). Alternatively, variant libraries can be generated, for example, by using random (NNK) or non-random codons, for example DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The characteristics of the resulting antibody variants can be examined using the assays described herein.

Immunoconjugates

The term immunoconjugate means an antibody of the invention conjugated to one or more heterologous molecules.

In some embodiments, an antibody of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, is conjugated to one or more cytotoxic agents. Examples of such cytotoxic agents include chemotherapeutic agents or drugs, growth inhibitory agents, toxins (eg, protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof) and radioactive isotopes.

In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody of the invention is conjugated to one or more drugs, such as a maytansinoid (see, for example, US Pat. Nos. 5,208,020, 5,416,06)); auristatin, for example the monomethyl auristatin drug groups DE and DF (MMAE and MMAF) (see, for example, US Patent Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatin, calicheamicin or derivatives thereof (see, for example, US 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; Hinman et al., (1993) Cancer Res 53:3336-3342 and Lode et al., (1998) Cancer Res 58:2925-29 28); an anthracycline such as daunomycin or doxorubicin (see, for example, Kratz et al., (2006) Current Med. Chem 13:477-523; Jeffrey et al., (2006) Bioorganic & Med Chem Letters 16:358-362; Torgov et al., (2005) Bioconj Chem 16:717-721; Nagy et al., (2000) Proc Natl Acad Sci USA 97:829-834; Bioorg & Med. 1529:-1532 (2002); King et al., (2002) J Med Chem 45:4336-4343; and US Pat. No. 6,630,579), methotrexate, vindesine, taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel.

In some embodiments, the immunoconjugate comprises an antibody of the invention described herein conjugated to an enzymatically active toxin or fragment thereof, such as diphtheria toxin A chain, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain , Abrin A-chain, Modexin A-chain, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), omordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin , restrictocin, fenomycin, enomycin and tricothecenes.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, an antibody is conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available to obtain radioconjugates. Examples include At211, 1131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When used for radioconjugate detection, it may contain a radioactive atom for scintigraphic studies, such as tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123 again. iodine-131, indium-11, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of the antibody of the invention described herein and a cytotoxic agent can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), succinimidyl-4-tNmaleimidomethyl)cyclohexane-1 -carboxylate (SMCC), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as

- 35 046641 bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate) and compounds with two active fluorine groups ( such as 1,5-difluoro-2,4-dinitrobenzene). For example, the immunotoxin ricin can be prepared as described in Vitetta et al., (1987) Science 238: 1098. Carbon-14 labeled l-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating a radionuclide to an antibody. See for example WO94/11026. The linker may be a cleavable linker that promotes release of the cytotoxic drug into the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., (1992) Cancer Res 52: 127-131; US Pat. No. 5,208,020).

Immunoconjugates or ADCs can be prepared with cross-linker reagents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo -KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, Illinois, USA).

One embodiment of the invention, and in some embodiments of each and every one of the numbered embodiments listed below, is an immunoconjugate comprising a PSMA-specific binding antibody of the invention coupled to a therapeutic agent or imaging agent.

Another embodiment of the invention, and in some embodiments of each and every one of the numbered embodiments listed below, is an immunoconjugate comprising a CD3-specific binding antibody of the invention coupled to a therapeutic agent or imaging agent.

Another embodiment of the invention, and in some embodiments of each and every one of the numbered embodiments listed below, is an immunoconjugate comprising an anti-PSMA/CD3 bispecific antibody of the invention coupled to a therapeutic agent or imaging agent.

Creation of monospecific antibodies according to the invention

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA-specific binding antagonist antibodies of the invention are human.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the PSMA-specific binding antagonist antibodies of the invention are humanized.

Monospecific antibodies of the invention described herein (eg, antibodies that specifically bind PSMA) can be generated using a variety of technologies. For example, the hybridoma method of Kohler and Milstein, Nature 256:495, 1975, can be used to generate monoclonal antibodies. In the hybridoma method, a mouse or other animal host, such as a hamster, rat or monkey, is immunized with PSMA or human chimpanzee or macaque CD3 or fragments PSMA or CD3, such as the extracellular domains of PSMA or CD3, followed by fusion of splenocytes from immunized animals with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986 )). Colonies arising from single cells of the immortalized hybridoma are screened based on the production of antibodies with desired properties, such as binding specificity, cross-reactivity or lack thereof, and affinity for the antigen.

A variety of animal hosts may be used to produce anti-PSMA antibodies in accordance with the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below. For example, Balb/c mice can be used to generate murine antibodies to human PSMA. Antibodies obtained from Balb/c mice and other non-human animals can be humanized using a variety of technologies to create sequences that are more similar to human sequences.

Examples of humanization techniques involving selection of human acceptor scaffolds are known and include CDR grafting (US Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), surface modification (Padlan, (1991) Mol Immunol 28:489-499 ), modification of the surface of specificity-determining residues (US Patent Publication No. 2010/0261620), adaptation of a human scaffold (US Patent No. 8,748,356) or superhumanization (US Patent No. 7,709, 226). In these methods, the CDRs of the parent antibodies are transferred to human scaffolds, which can be selected based on their general homology to the parent scaffolds, similarity in CDR length, or identity of the canonical structure, or a combination thereof.

- 36 046641

Humanized antibodies can be further optimized to improve their selectivity or affinity for the desired antigen by the inclusion of altered scaffold supporting residues while maintaining binding affinity (back mutations) by techniques such as those described in International Patent Publications Nos. WO1090/007861 and WO1992/22653, or by introducing a variation into any of the CDRs, for example, to improve the affinity of the antibody.

To obtain human antibodies against a target protein, transgenic animals carrying human immunoglobulin (Ig) loci in their genome, such as mice or rats, can be used, which are described, for example, in US patent No. 6150584, international patent publication No. WO99/45962, international patent publications No. WO2002/066630, WO2002/43478,

WO2002/043478 and WO1990/04036, Lonberg et al (1994) Nature 368:856-9; Green et al (1994) Nature Genet. 7:13-21; Green & Jakobovits (1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int Rev Immunol 13:65-93; Bruggemann et al., (1991) Eur J Immunol 21:1323-1326; Fishwild et al., (1996) Nat Biotechnol 14:845-851; Mendez et al., (1997) Nat Genet 15:146-156; Green (1999) J Immunol Methods 231:11–23; Yang et al., (1999) Cancer Res 59:1236-1243; Bruggemann and Taussig (1997) Curr Opin Biotechnol 8:455–458. Endogenous immunoglobulin loci in such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the animal's genome through homologous or nonhomologous recombination, using transchromosomes, or using minigenes. To obtain human antibodies directed against a selected antigen using the technology described above, companies such as Regeneron (http://_www_regeneron_com), Harbor Antibodies (http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc. can be contacted. (OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com), Trianni (http://_www.trianni_com) and Ablexis (http://_www_ablexis_com).

Human antibodies can be selected from a phage display library, wherein the phage is engineered to express human immunoglobulins or portions thereof, such as Fab, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., (2000) J Mol Biol 296 :57-86; Krebs et al., (2001) J Immunol Meth 254:67-84; Vaughan et al., (1996) Nature Biotechnology 14:309-314; Sheets et al., (1998) PITAS (USA) 95:6157-6162; Hoogenboom and Winter (1991) J Mol Biol 227:381; Marks et al., (1991) J Mol Biol 222:581). Antibodies of the invention can be isolated, for example, from a phage display library expressing the variable regions of the heavy and light chains of the antibody as fusion proteins with the bacteriophage pIX coat protein, as described in Shi et al., (2010) J Mol Biol 397:385 -96 and in international patent publication No. WO 09/085462). Libraries can be screened for phage binding to human and/or cynomolgus PSMA or CD3, and the resulting positive clones can be further characterized; Fabs can be isolated from clone lysates and expressed as full-length IgG. Such methods of using phage display to isolate human antibodies are described, for example, in US patents No. 5223409, 5403484, 5571698, 5427908, 5580717, 5969108, 6172197, 5885793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

The production of immunogenic antigens and the production of monoclonal antibodies can be accomplished using any suitable technique, such as the production of a recombinant protein. Immunogenic antigens can be administered to animals in the form of purified protein or protein mixtures, including whole cells or cellular or tissue extracts, or the antigen can be formed de novo in the animal from nucleic acids encoding the antigen or part thereof.

Creation of multispecific PSMA x CD3 antibodies according to the invention

Multispecific PSMA x CD3 antibodies of the invention (e.g., bispecific antibodies that contain a first PSMA-specific binding domain and a second CD3-specific binding domain) can be generated by combining PSMA-binding VH/VL domains and CD3-binding VH/VL domains, isolated and described in this document. Alternatively, PSMA x CD3 bispecific antibodies can be constructed using VH/VL domains from publicly available anti-PSMA and anti-CD3 monospecific antibodies and/or by combining the PSMA or CD3 binding VH/VL domains specified herein with publicly available ones PSMA or CD3 binding VH/VL domains.

Examples of anti-PSMA antibodies that can be used to construct anti-PSMA x CD3 bispecific molecules include, for example, the antibodies described herein and in Table 1. 2. For example, the VH/VL domains of the anti-PSMA antibodies of the invention may be included in the bispecific antibodies containing the CD3 binding VH/VL domains described herein and in Table. 5. For example, the VH/VL domains of the CD3 antibodies CD3B217 and CD3B219 described herein can be used to generate PSMAC x D3 bispecific antibodies. In addition to the description and characterization of the CD3B217 and CD3B219 antibodies provided herein, a more detailed description of the antibodies can be found in US Patent Application Published No. 2016-0068605 A1, which is incorporated herein by reference.

Likewise, examples of anti-CD3 antibodies that can be used to construct PSMA x CD3 bispecific molecules are, for example, those described in international patents

- 37 046641 publications No. WO 2005/048935, WO 2004/106380 and WO 2015095392. These CD3 VH/VL domains can be incorporated into bispecific antibodies that contain the PSMA binding VH/VL domains described herein and in table. 2. For example, the VH/VL domains of the anti-PSMA antibodies PSMB119,

PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129,

PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344,

PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362,

PSMB363 and PSMB365 described herein can be used to generate bispecific antibodies to PSMAxCD3.

Generated anti-PSMA x CD3 bispecific antibodies can be tested for their binding to PSMA and CD3 and for their desired functional characteristics, such as T Cell-mediated killing of cells expressing PSMA (eg, LNCaP).

The bispecific antibodies of the invention comprise antibodies having a full-length antibody structure.

The term full-length antibody means an antibody having two full-length antibody heavy chains and two full-length antibody light chains. The heavy chain (HC) of a full-length antibody consists of the well-known heavy chain variable and constant domains VH, CH1, hinge region, CH2 and CH3. The full-length antibody light chain (LC) consists of the well-known VL and CL light chain variable and constant domains. A full-length antibody may lack C-terminal lysine (K) in either one or both heavy chains.

The term Fab arm or hemimolecule refers to a single heavy chain-light chain pair that specifically binds to an antigen.

The full-length bispecific antibodies of the invention described herein, and in some embodiments of any and all of the numbered embodiments listed below, can be generated, for example, by exchanging Fab arms (or exchanging hemimolecules) between two monospecific divalent antibodies by introducing into the interaction surface Heavy chain CH3 substitutions in each hemimolecule promote the formation of a heterodimer from two antibody hemimolecules having different specificities, either in vitro in a cell-free environment or using coexpression. The Fab arm exchange reaction is the result of disulfide isomerization and dissociation-association reactions of CH3 domains. The disulfide bonds of the heavy chains in the hinge regions of the original monospecific antibodies are restored. The resulting free cysteines of one of the original monospecific antibodies form a disulfide bond of heavy chains with the cysteine residues of the second molecule of the original monospecific antibody, and at the same time the CH3 domains of the original antibodies are released and reformation occurs by dissociation-association. The CH3 domains of Fab arms can be designed to allow heterodimerization rather than homodimerization. The resulting product is a bispecific antibody having two Fab arms, or hemimolecules, each of which binds to a different epitope, i.e. an epitope on PSMA and an epitope on CD3.

The term homodimerization refers to the interaction of two heavy chains having identical CH3 amino acid sequences. The term homodimer refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

The term heterodimerization refers to the interaction of two heavy chains having non-identical CH3 amino acid sequences. Heterodimer refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

In some embodiments, bispecific antibodies include constructs such as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), ridge-to-hole (Genentech), CrossMAb (Roche), and electrostatically coupled (Chugai, Amgen, NovoNordisk, Oncomed), LUZ-Y (Genentech), chain exchange engineered domain antibody (SEEDbody) (EMD Serono), Biclonic (Merus), and DuoBody (Genmab A/S).

To obtain full-length bispecific antibodies according to the invention, Triomab quadroma technology can be used. Triomab technology promotes the exchange of Fab arms between two parent chimeric antibodies, one parent mAb having IgG2a and a second parent mAb having rat IgG2b constant regions, to produce chimeric bispecific antibodies.

To obtain full-length bispecific antibodies, a projection-to-trench strategy can be used (see, for example, international publication No. WO 2006/028936). Briefly, selected amino acids forming the interface between the CH3 domains in human IgG can be mutated at positions that affect the interactions of the CH3 domains, promoting heterodimer formation. An amino acid with a short side chain (the valley) is introduced into the heavy chain of an antibody that specifically binds to the first antigen, and an amino acid with a long side chain (the overhang) is introduced into the heavy chain of an antibody that specifically binds to the second antigen. After co-expression of two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with the trench and the heavy chain with the knob. Examples of pairs of substitutions in CH3 that form a knob and a trough are the following (indicated as a modified position in the first CH3 domain of the first

- 38 046641 heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S,

F405W/T394S and T366W/T366S_L368A_Y407V.

To obtain full-length bispecific antibodies according to the invention, CrossMAb technology can be used. CrossMAb antibodies, in addition to the use of a knob-to-valley Fab arm exchange strategy in the promoter, have an exchange of CH1 and CL domains in one half of the arms to ensure correct pairing of the light chain of the resulting bispecific antibody (see, for example, US Patent No. 8,242,247) .

To obtain full-length bispecific antibodies of the invention, other retargeting strategies can be used by exchanging variable or constant or both domains between the heavy chain and the light chain or within the heavy chain in bispecific antibodies (either in one or both arms). Such exchanges include, for example, VH-CH1 exchanges with VL-CL, VH with VL, CH3 with CL and CH3 with CH1, as described in patent publication numbers WO 2009/080254, WO 2009/080251, WO 2009/018386 and WO 2009/080252.

Other strategies can be used, such as promoting heavy chain heterodimerization using electrostatic interactions by introducing substitutions of positively charged residues on one CH3 surface and negatively charged residues on the other CH3 surface, as described in US Patent Publication No. US 2010/0015133; US Patent Publication No. US 2009/0182127; US Patent Publication No. US 2010/028637 or US Patent Publication No. US 2011/0123532. In other strategies, heterodimerization can be promoted by the following substitutions (modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain is indicated): L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F40 5A_Y407V, L351Y_Y407A/T366A_K409F, L351Y_Y407A/ T366V_K409F, Y407A/T366A_K409F or

T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W, as described in US Patent Publication No. US 2012/0149876 or US Patent Publication No. US 2013/0195849.

To obtain bispecific antibodies according to the invention, the LUZ-Y technology can be used. In this technology, a leucine zipper type sequence is appended to the C-termini of the CH3 domains to control heterodimer assembly from the parent mAbs, which is removed after purification as described by Wranik et al., (2012) J Biol Chem 287(52): 42221-9.

SEEDbody technology can be used to produce the bispecific antibodies of the invention. To promote heterodimerization, SEEDbody antibodies in their constant domains have the replacement of selected IgG residues with IgA residues, as described in US Patent No. US 20070287170.

The present invention also provides a multispecific, multifunctional antibody that specifically binds to PSMA.

In accordance with the invention, such a multispecific multifunctional antibody that specifically binds to PSMA can be a trispecific antibody for dual targeting of tumor cells - these are trifunctional structures that can be configured to target two different targets/epitopes on a tumor cell and on a third high affinity binding functionality to either T cells or NK cells. Trispecific antibodies that target two different tumor epitopes and interact with T or NK cells lyse tumor cells that express both targets. Such molecules can be prepared using antibody formats known in the art and described in detail herein. (WO 20151842071, WO 2015158636, WO 2010136172, WO 2013174873). In a trispecific embodiment, the antigen binding polypeptide is bispecific to PSMA and a second distinct antigen on a tumor cell and additionally specific to an effector cell, particularly a T cell or NK cell.

In some embodiments described herein, and in some embodiments of any and all of the numbered embodiments listed below, bispecific antibodies can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 region of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions to ensure isomerization of the disulfide bond, in accordance with the methods described in international patent publication No. WO 2011/131746 (DuoBody Technology). In these methods, a first monospecific divalent antibody (eg, an anti-PSMA antibody) and a second monospecific divalent antibody (eg, an anti-CD3 antibody) are designed to have certain substitutions in the CH3 domain that provide heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to render the cysteines in the hinge region susceptible to disulfide bond isomerization; thus obtaining a bispecific antibody by exchanging Fab arms. Incubation conditions can be optimally returned to non-reducing conditions. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE),

- 39 046641 glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol. For example, incubation for at least 90 minutes at a temperature of at least 20° C. in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH level of 5-8, e.g. pH=7.0 or at pH=7.4.

In some embodiments described herein, and in some embodiments of any and all of the numbered embodiments listed below, an isolated bispecific antibody comprising a first PSMA-specific binding domain and a second CD3-specific binding domain comprises at least one substitution in the constant domain of the CH3 antibody.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, at least one substitution in the constant domain of the CH3 antibody is a 409R, F405L, or F405L and R409K substitution, where the residue numbering corresponds to the index EU.

Antibody domains and numbering are well known. The term asymmetric refers to non-identical substitutions in two CH3 domains in two separate heavy chains of an antibody. The CH3 region of IgG1 typically consists of residues 341-446 on IgG1 (residue numbering follows the EU catalogue).

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated anti-PSMA x CD3 bispecific antibody comprises an F405L substitution in the first heavy chain (HC1) of the antibody and a 409 R substitution in the second heavy chain (HC2) antibody.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the isolated anti-PSMA x CD3 bispecific antibody contains the S228P substitution in HC1 and the S228P, F405L, and R409K substitutions in HC2, wherein the antibody belongs to the IgG4 isotype.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, HC1 comprises a first PSMA-specific binding domain and HC2 comprises a second CD3-specific binding domain.

In some embodiments described herein, the bispecific antibody of the invention contains at least one, two, three, four, five, six, seven, or eight asymmetric substitutions in HC1 and HC2 at residue positions 350, 366, 368, 370, 399, 405, 407 or 409 if the numbering of the residues corresponds to the EU index.

In some embodiments described herein, a bispecific antibody of the invention contains at least one, two, three, or four asymmetric substitutions in HC1 and HC2 at residue positions 350, 370, 405, or 409, if the residue numbering corresponds to the EU index.

In some embodiments described herein, the bispecific antibody of the invention contains at least one asymmetric substitution in HC1 and HC2 at residue positions 405 or 409, if the residue numbering corresponds to the EU index.

In some embodiments described herein, a bispecific antibody of the invention contains a 409R or F405L substitution in HC1 and a 409R or F405L substitution in HC2 if the residue numbering corresponds to the EU index.

In some embodiments described herein, the bispecific antibody of the invention contains the F405L substitution in HC1 and the 409 R substitution in HC2.

In some embodiments described herein, a bispecific antibody of the invention contains at least one asymmetric substitution in HC1 and HC2 at residue positions 366, 368, 370, 399, 405, 407, or 409 if the residue numbering corresponds to the EU index.

In some embodiments described herein, there is an amino acid substitution other than Lys, Leu, or Met at position 409 in HC1 and an amino acid substitution other than Phe at position 405 in HC2.

In some embodiments described herein, there is an amino acid substitution other than Phe at position 405 in HC1 and an amino acid substitution other than Lys, Leu, or Met at position 409 in HC2.

In some embodiments described herein, there is an amino acid substitution other than Lys, Leu, or Met at position 409 in HC1 and an amino acid substitution other than Phe, Arg, or Gly at position 405 in HC2.

In some embodiments described herein, there is an amino acid substitution other than Phe, Arg, or Gly at position 405 in HC1 and an amino acid substitution other than Lys, Leu, or Met at position 409 in HC2 CH3.

In some embodiments described herein, HC1 CH3 contains Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, and HC2 contains an amino acid other than Phe at position 405 and contains Lys at position 409 .

In some embodiments described herein, HC1 has the amino acid

- 40 046641 a lot other than Phe at position 405 and a Lys at position 409, and HC2 has a Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, HC1 has a Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, and HC2 has a substitution other than Phe, Arg, or Gly at position 405 and Lys at position 409.

In some embodiments described herein, HC1 has a substitution other than Phe, Arg, or Gly at position 405 and Lys at position 409, and HC2 has Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409.

In some embodiments described herein, HC1 has Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, and HC2 has Leu at position 405 and Lys at position 409.

In some embodiments described herein, HC1 has Leu at position 405 and Lys at position 409, and HC2 has Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409.

In some embodiments described herein, HC1 has Phe at position 405 and Arg at position 409, and HC2 has an amino acid other than Phe, Arg, or Gly at position 405 and Lys at position 409.

In some embodiments described herein, HC1 has an amino acid other than Phe, Arg, or Gly at position 405 and Lys at position 409, and HC2 has Phe at position 405 and Arg at position 409.

In some embodiments described herein, HC1 has Phe at position 405 and Arg at position 409, and HC2 has Leu at position 405 and Lys at position 409.

In some embodiments described herein, HC1 has Leu at position 405 and Lys at position 409, and HC2 has Phe at position 405 and Arg at position 409.

In some embodiments described herein, HC1 has Phe at position 405 and Lys at position 409, and HC2 has Leu at position 405 and Arg at position 409.

In some embodiments described herein, HC1 has Leu at position 405 and Arg at position 409, and HC2 has Phe at position 405 and Lys at position 409.

In some embodiments described herein, HC1 has an amino acid other than Lys, Leu, or Met at position 409, and HC2 has Lys at position 409, Thr at position 370, and Leu at position 405.

In some embodiments described herein, HC1 has a Lys at position 409, a Thr at position 370, and a Leu at position 405, and HC2 has an amino acid other than Lys, Leu, or Met at position 409.

In some embodiments described herein, HC1 has Arg at position 409 and HC2 has Lys at position 409, Thr at position 370, and Leu at position 405.

In some embodiments described herein, HC1 has Lys at position 409, Thr at position 370, and Leu at position 405, and HC2 has Arg at position 409.

In some embodiments described herein, HC1 has Lys at position 370, Phe at position 405, and Arg at position 409, and HC2 has Lys at position 409, Thr at position 370, and Leu at position 405.

In some embodiments described herein, HC1 has Lys at position 409, Thr at position 370, and Leu at position 405, and HC2 has Lys at position 370, Phe at position 405, and Arg at position 409.

In some embodiments described herein, HC1 has an amino acid other than Lys, Leu, or Met at position 409, and HC2 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr , in position 407.

In some embodiments described herein, HC1 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407, and HC2 has an amino acid other than Lys, Leu, or Met , in position 409.

In some embodiments described herein, HC1 has an amino acid other than Lys, Leu, or Met at position 409 and HC2 has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 .

In some embodiments described herein, HC1 has an Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and HC2 has an amino acid other than Lys, Leu, or Met at position 409.

In some embodiments described herein, HC1 has an amino acid other than Lys, Leu, or Met at position 409 and HC2 has Gly, Leu, Met, Asn, or Trp at position 407.

In some embodiments described herein, HC1 has a Gly, Leu, Met, Asn, or Trp at position 407 and HC2 has an amino acid other than Lys, Leu, or Met at position 409.

In some embodiments described herein, HC1 has a Tyr in half

- 41 046641 407 and an amino acid other than Lys, Leu or Met at position 409, and HC2 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and Lys in position 409.

In some embodiments described herein, HC1 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and Lys at position 409, and HC2 has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409.

In some embodiments described herein, HC1 has Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, and HC2 has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp, at position 407 and Lys at position 409.

In some embodiments described herein, HC1 has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and Lys at position 409, and HC2 CH3 has Tyr at position 407 and an amino acid different from Lys, Leu or Met, at position 409.

In some embodiments described herein, HC1 CH3 has Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, and HC2 has Gly, Leu, Met, Asn, or Trp at position 407 and Lys at position 409.

In some embodiments described herein, HC1 has Gly, Leu, Met, Asn, or Trp at position 407 and Lys at position 409, and HC2 has Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409.

In some embodiments described herein, HC1 has Tyr at position 407 and Arg at position 409, and HC2 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and Lys at position 409.

In some embodiments described herein, HC1 has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and Lys at position 409, and HC2 has Tyr at position 407 and Arg at position 409.

In some embodiments described herein, HC1 has Tyr at position 407 and Arg at position 409, and HC2 has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and Lys at position 409 .

In some embodiments described herein, HC1 has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and Lys at position 409, and HC2 has Tyr at position 407 and Arg at position 409 .

In some embodiments described herein, HC1 CH3 has Tyr at position 407 and Arg at position 409, and HC2 CH3 has Gly, Leu, Met, Asn, or Trp at position 407 and Lys at position 409.

In some embodiments described herein, HC1 has Gly, Leu, Met, Asn, or Trp at position 407 and Lys at position 409, and HC2 has Tyr at position 407 and Arg at position 409.

In some embodiments described herein, HC1 has an amino acid other than Lys, Leu, or Met at position 409 and HC2 has (i) an amino acid other than Phe, Leu, and Met at position 368, or (ii) Trp at position 370, or (iii) an amino acid other than Asp, Cys, Pro, Glu or Gln at position 399.

In some embodiments described herein, HC1 has (i) an amino acid other than Phe, Leu, and Met at position 368, or (ii) Trp at position 370, or (iii) an amino acid other than Asp, Cys, Pro, Glu or Gln, at position 399, and HC2 has an amino acid other than Lys, Leu or Met, at position 409.

In some embodiments described herein, HC1 has Arg, Ala, His, or Gly at position 409 and HC2 has (i) Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser , Thr, Val or Trp at position 368, or (ii) Trp at position 370, or (iii) Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399.

In some embodiments described herein, HC1 has (i) Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) Trp at position 370, or (iii) Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg, or Tyr at position 399, and HC2 has Arg, Ala, His, or Gly in position 409.

In some embodiments described herein, HC1 has Arg at position 409 and HC2 has (i) Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) Trp at position 370, or (iii) Phe, His, Lys, Arg, or Tyr at position 399.

In some embodiments described herein, HC1 has (i) Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) Trp at position 370, or (iii) Phe , His, Lys, Arg, or Tyr at position 399, and HC2 has Arg at position 409.

In some embodiments described herein, HC1 contains the 409 R substitution or the F405L substitution, and HC2 contains the 409 R substitution or the F405L substitution if the residue numbering corresponds to the EU index.

In some embodiments described herein, HC1 contains the F405L substitution and HC2 contains the 409 R substitution.

- 42 046641

Typically, substitutions are introduced into a molecule, such as an antibody constant domain, at the DNA level using standard methods.

The antibodies of the invention can be constructed into a variety of known antibody forms.

In some embodiments, the bispecific antibody of the present invention is a diabody or cross-body.

In some embodiments, the bispecific antibodies include recombinant dual-targeting IgG-like molecules, wherein each of the two sides of the molecule contains a Fab fragment or a portion of a Fab fragment of at least two different antibodies; IgG fusion molecules in which full-length IgG antibodies are fused to an additional Fab fragment or parts of a Fab fragment; Fc fusion molecules in which single chain Fv molecules or stabilized diabodies are fused to heavy chain constant domains, Fc regions or portions thereof; Fab fusion molecules, in which different Fab fragments are fused to each other; ScFv-based heavy chain antibodies and diabodies (eg, domain antibodies, nanobodies) in which different single-chain Fv molecules, or different diabodies, or different heavy chain antibodies (eg, domain antibodies, nanobodies) are fused to each other, or to another protein or carrier molecule.

In some embodiments, the recombinant dual-targeting IgG-like molecules include dual-targeting (DT)-Ig molecules (GSK/Domantis), two-in-one antibody (Genentech), and mAb2 (F-Star).

In some embodiments, the IgG fusion molecules include dual variable domain (DVD)-Ig molecules (Abbott), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche).

In some embodiments, the Fc fusion molecules include ScFv/Fc fusion proteins (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), dual-affinity retargeting antibodies (Fc-DART) (MacroGenics).

In some embodiments, the Fab fusion bispecific antibodies include F(ab)2 (Medarex/AMGEN), dual action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), divalent bispecific antibody (Biotecnol ) and Fab-Fv (UCB-Celltech). ScFv-based antibodies, diabodies, and domain antibodies include bispecific T-cell activator (BITE) (Micromet), tandem diabody (Tandab) (Affimed), dual-affinity retargeting antibodies (DART) (MacroGenics), single-chain diabody (Academic), TCR -like antibodies (AIT, ReceptorLogics), hybrids of ScFv and human serum albumin (Merrimack), and COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), dual-targeting domain antibodies having only the heavy chain. Various bispecific antibody formats have been described, for example in Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276 and Nunez-Prado et al., (2015) Drug Discovery Today 20(5):588-594.

Polynucleotides, vectors and host cells

Also described are isolated polynucleotides encoding antibodies or antigen-binding fragments that immunospecifically bind to PSMA. Isolated polynucleotides capable of encoding the variable domain segments provided herein can be incorporated into the same or different vectors for the production of antibodies or antigen-binding fragments.

Polynucleotides encoding recombinant antigen-binding proteins are also included within the scope of the description. In some embodiments, the described polynucleotides (and the peptides they encode) include a leader sequence. Any leader sequence known in the art can be used. The leader sequence may include, without limitation, a restriction site or a translation initiation site.

The PSMA-specific antibodies or antigen-binding fragments described herein include variants having single or multiple amino acid substitutions, deletions or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described PSMA-specific antibodies or antigen-binding fragments . In the context of the present invention, unless otherwise specified, the following notations are used to describe mutations: i) an amino acid change at a given position is written, for example, as K409R, which means a change from lysine to arginine at position 409; and ii) for specific variants, specific three-letter or one-letter codes are used, including Xaa and X codes, to designate any amino acid residue. Thus, the replacement of lysine with arginine at position 409 is designated as K409R, and the replacement of lysine at position 409 with any amino acid residue is designated as K4O9X. The deletion of lysine at position 409 is designated K409*. If a particular amino acid residue may vary between peptide isotypes or variants, and the substitution affects that residue in each isotype or variant or in any of the isotypes or variants, the substitution is designated as, for example, 409 R, which means that the amino acid corresponding to position 409 , replaced by arginine. Variants containing single or multiple amino acid substitutions, deletions or additions can be generated by those skilled in the art.

Such variants may include: (a) variants in which one or more amino acid

- 43 046641 residues are replaced by conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to or removed from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d ) variants in which the polypeptide is fused to another peptide or polypeptide, such as a fusion partner, a protein tag, or other chemical functional group capable of imparting beneficial properties to the polypeptide, such as an epitope for an antibody, a polyhistidine sequence, a biotin residue, or the like. Antibodies or antigen binding fragments described herein may include variants in which amino acid residues of one type are replaced by corresponding residues of another type (at conserved or non-conserved positions). In other embodiments, amino acid residues at non-conserved positions are replaced with conservative or non-conservative residues. Techniques for obtaining such variants, including genetic (deletions, mutations, etc.), chemical and enzymatic, are known to those skilled in the art.

PSMA-specific antibodies or antigen-binding fragments described herein can be of several antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is an IgG1, IgG2, IgG3, or IgG4 isotype, preferably an IgG1 or IgG4 isotype. The specificity of an antibody or antigen-binding fragment thereof is determined in large part by the amino acid sequence and the location of the CDRs. Therefore, CDRs from one isotype can be transferred to another isotype without changing antigen specificity. In an alternative embodiment, techniques have been established to cause hybridomas to switch from producing one antibody isotype to another (isotype switching) without changing antigen specificity. Accordingly, such antibody isotypes are included within the scope of the disclosed antibodies or antigen binding fragments.

In addition, vectors are provided containing the polynucleotides described herein. The vectors may be expression vectors. Therefore, recombinant expression vectors containing the sequence encoding the polypeptide of interest are considered to be within the scope of this specification. The expression vector may contain one or more additional sequences, such as, but not limited to, regulatory sequences (eg, promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide range of host cells are widely known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), as well as other bacterial, yeast and viral vectors.

Recombinant expression vectors included within the scope of the disclosure include synthetic, genomic or cDNA-derived nucleic acid fragments that encode at least one recombinant protein that can be operably linked to suitable regulatory elements. Such regulatory elements may include a transcription promoter, sequences encoding suitable sites for binding of mRNA to the ribosome, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, may also include one or more non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer associated with the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (eg, essential ribosome binding sites), polyadenylation site, splice donor and acceptor sites, or transcription termination sequences. In addition, an origin of replication may be inserted to provide the ability to replicate in the host cell.

Transcription and translation control sequences in expression vectors intended for transformation of vertebrate cells can be obtained from viral sources. Examples of vectors that can be constructed are described in Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the antibody or antigen binding fragment coding sequence is placed under the control of an effective constitutive promoter, such as, for example, the promoters of the following genes:

hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta actin, human myosin, human hemoglobin, human muscle creatine and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include, but are not limited to, cytomegalovirus (CMV) immediate-early promoter, SV40 early and late promoters, mouse mammary tumor virus (MMTV) promoter, Maloney leukemia virus (LTR) long terminal repeats, human immunodeficiency virus (HIV) , Epstein Barr virus (EBV), Rous sarcoma virus (RSV) and other retroviruses and the herpes simplex virus thymidine kinase promoter. In one embodiment, the coding sequence of the PSMA-specific antibody or antigen-binding fragment thereof is under induction control

- 44 046641 protein promoter, for example metallothionein promoter, tetracycline inducible promoter, doxycycline inducible promoter, promoters containing one or more interferon-stimulated response elements (ISRE), protein kinase R 2',5'oligoadenylate synthase promoters, Mx, ADAR1 genes, etc. .P.

The vectors described herein may contain one or more internal ribosome entry sites (IRES). Incorporation of an IRES sequence into fusion vectors may be useful for enhancing the expression of certain proteins. In some embodiments, the vector system may include one or more polyadenylation sites (eg, SV40), which may be upstream or downstream of any of the above nucleic acid sequences. The vector components may be ligated together or arranged to provide optimal spatial separation for expression of the gene products (ie, by introducing spacer nucleotides between open reading frames (ORFs)), or otherwise arranged. Regulatory elements, such as the IRES motif, can also be positioned to provide optimal spacing for expression.

Vectors may contain selectable markers well known in the art. Selectable markers include positive and negative selection markers, antibiotic resistance genes (eg, neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene), glutamate synthase genes, HSV-TK , HSV-TK derivatives for ganciclovir selection or the bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleotide sequence encoding the selectable marker or cloning site may be located upstream or downstream of the nucleotide sequence encoding the polypeptide or cloning site of interest.

The vectors described herein can be used to transform various cells with genes encoding the described antibodies or antigen binding fragments. For example, vectors can be used to generate cells that produce a PSMA-specific antibody or antigen binding fragment. Therefore, another aspect of the invention describes host cells transformed with vectors containing a nucleotide sequence encoding an antibody or antigen binding fragment thereof that specifically binds to PSMA, for example, the antibodies or antigen binding fragments described and exemplified herein.

Numerous methods for introducing foreign genes into cells are known in the art and can be used to construct recombinant cells to implement the described methods in accordance with the various embodiments described and exemplified herein. The technique used must ensure stable transfer of the heterologous gene sequence into the host cell in such a way that the heterologous gene sequence can be inherited and expressed by the cell's progeny, and in such a way that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques that can be used include, but are not limited to, chromosome transfer (e.g., cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (for example, recombinant DNA viruses, recombinant RNA viruses), etc. (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells can also be used to transform cells.

Cells suitable for use in the expression of PSMA-specific antibodies or antigen binding fragments described herein preferably include eukaryotic cells, more preferably cells derived from a plant, rodent or human, such as, but not limited to, the NSO, CHO, CHOK1 cell lines , perC.6, Tk-ts13, BHK, HEK293 cells, cOs7, T98G, CV-1/EBNA, L cells, C127, 3T3, HeLa, NS1, myeloma cells Sp2/0, BHK, etc. In addition, antibody expression can be achieved using hybridoma cells. Methods for producing hybridomas are well known in the art.

Cells transformed with the expression vectors described herein can be selected or screened for recombinant expression of the antibodies or antigen binding fragments described herein. Recombination-positive cells are expanded and screened for subclones that exhibit a desired phenotype, such as high expression levels, enhanced growth characteristics, or the ability to produce proteins with desired biochemical properties, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to intrinsic properties of a given subclone or due to mutation. Mutations can be accomplished by the use of chemical agents, UV light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of these methods.

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Pharmaceutical compositions/administration

The invention provides pharmaceutical compositions containing the antibodies of the invention described herein and a pharmaceutically acceptable carrier. For therapeutic use, it is possible to prepare the antibodies of the invention in the form of pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. The term carrier refers to the diluent, adjuvant, excipient or carrier medium with which the antibody of the present invention is administered. Such carrier media may be liquids such as water and oils, including oils of mineral, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, a 0.4% saline solution and a 0.3% glycine solution can be used. These solutions are sterile and essentially free of solid particles. They can be sterilized using well known standard sterilization techniques (eg filtration). The compositions may contain pharmaceutically acceptable excipients necessary to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizing, thickening, wetting and coloring agents, etc. The concentration of antibodies of the invention in such a pharmaceutical composition may vary from less than about 0.5%, typically at least about 1%, and up to 15 or 20% by weight, and may be selected based primarily on the dose required, fluid volumes, viscosity values, etc. .d. according to the specific route of administration chosen. Suitable carrier media and formulations including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.

The route of administration for therapeutic use of the antibodies of the invention may be any suitable route of delivery of the antibody to the host, such as parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), in the form of a composition in a tablet, capsule, solution, powder, gel, particle; and administering the antibody contained in a syringe, implanted device, osmotic pump, cartridge, micropump, or other means that are well known in the art and obvious to the skilled person. Localized administration can be achieved, for example, by delivery to a tumor, joint, bronchi, abdominal cavity, capsule, cartilage, cavity, cerebellum, cerebral ventricle, colon, cervix, stomach, liver, myocardium, bone, pelvis, pericardium, abdominal cavity , pleura, prostate, lungs, rectum, kidney, retina, spine, joint capsule, chest, uterus, vessel, inside the bladder, damaged tissue, vaginally, rectally, buccally, sublingually, intranasally or transdermally.

Antibodies of the invention described herein, and in some embodiments, any and all of the numbered embodiments listed below, can be administered to a subject by any suitable route, for example, parenterally by intravenous (IV) infusion or bolus injection, intramuscularly, subcutaneously, or intraperitoneally . The intravenous infusion may last for, for example, 15, 30, 60, 90, 120, 180 or 240 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

The dose administered to a subject is sufficient to attenuate or at least partially inhibit the disease being treated (therapeutically effective amount), and may sometimes range from 0.005 mg to about 100 mg/kg, such as from about 0.05 mg to about 30 mg/kg, or from about 5 mg to about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or, for example, about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but can be even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose may also be administered, for example 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's body surface area, for example 500, 400, 300, 250, 200 or 100 mg/ ^m2 For treatment a patient can usually be given 1 to 8 doses (eg, 1, 2, 3, 4, 5, 6, 7, or 8), but 9, 10, 11, 12, 13, 14, 15, 16, 17 can be given , 18, 19, 20 or more doses.

Administration of antibodies in accordance with the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or more. In addition, repeated courses of treatment in the form of long-term administration are possible. When repeated administration, the same dose or a different dose may be administered. For example, the antibodies of the invention described herein can be administered at a dose of 8 mg/kg or 16 mg/kg at weekly intervals for 8 weeks, followed by administration at a dose of 8 mg/kg or 16 mg/kg every two weeks for an additional 16 weeks followed by 8 mg/kg or 16 mg/kg every four weeks by intravenous infusion.

For example, antibodies in the methods of the invention described herein and in some

- 46 046641 embodiments of each and every one of the numbered embodiments listed below can be administered as a daily dose in an amount of about 0.1-100 mg/kg, for example, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or alternatively in at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after the start of treatment, or any combination thereof using single or divided doses every 24, 12, 8, 6, 4 or 2 hours , or any combination thereof.

Antibodies in the methods of the invention described herein, and in some embodiments of any and all of the numbered embodiments listed below, may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of an event during cancer progression, and/or reduce the risk of recurrence in cancer. case of cancer remission.

Antibodies of the invention may be lyophilized for storage and reconstituted in a suitable vehicle before use. This method has been proven effective for standard protein preparations and can also be used with well-known lyophilization and reconstitution techniques.

Methods of using PSMA-specific antibodies

PSMA is a prostate cancer-associated cell membrane antigen often overexpressed in prostatic intraepithelial neoplasia (PIN), a condition in which some prostate cells begin to look and behave abnormally; primary and metastatic prostate cancer; and in newly formed vessels of other solid tumors (eg, breast, lung, bladder, kidney). PSMA expression levels correlate with disease progression and Gleason scores. PSMA expression is increased in metastatic disease, hormonally refractory cases, and higher grade lesions and is further enhanced in androgen-insensitive tumors.

Blockade of PSMA can inhibit or reduce the growth of PSMA-expressing cancer cells and tumors in a subject. It may also have antiangiogenic activity due to the expression of PSMA in tumor neovasculature (Milowsky, et al. 2007). PSMA is highly expressed in prostatic intraepithelial neoplasia, the most advanced precursor of prostate carcinoma, and therefore PSMA blockade may modulate the progression of PIN to prostate cancer.

One embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, is a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of an antibody according to the invention that specifically binds PSMA.

Another embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, is a method of inhibiting the formation or growth of neovascularization of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a PSMA-specific binding antibody, in in accordance with the invention.

Another embodiment of the invention described herein, and in some embodiments of each and every one of the numbered embodiments listed below, is a method of inhibiting the progression of a precancerous condition in a subject, comprising administering to the subject a therapeutically effective amount of an antibody of the invention that specifically binds PSMA.

Another embodiment described herein, and some embodiments of each and every one of the numbered embodiments listed below, provides a method of treating cancer by administering to a subject in need thereof an antibody of the invention that specifically binds PSMA described herein.

One embodiment of the invention described herein, and in some embodiments of any and all of the numbered embodiments listed below, is a method of inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of a bispecific antibody that specifically binds PSMA x CD3 , according to the invention.

Another embodiment described herein, and in some embodiments of each and every one of the numbered embodiments listed below, is a method of inhibiting the formation or growth of neovascularization of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a bispecific antibody that specifically binds PSMA x CD3, in accordance with the invention.

Another embodiment of the invention described herein and in some

- 47 046641 embodiments of each and every one of the numbered embodiments listed below is a method of inhibiting the progression of a precancerous condition in a subject, comprising administering to the subject a therapeutically effective amount of a bispecific antibody of the invention that specifically binds PSMA x CD3.

Another embodiment of the invention described herein, and some embodiments of each and every one of the numbered embodiments listed below, provides a method of treating cancer by administering to a subject in need thereof a bispecific antibody of the invention that specifically binds PSMA x CD3 in accordance with the invention.

Examples of antibodies that can be used in the methods of the invention are PSMA specific binding antibodies and PSMA x CD3 bispecific antibodies as described herein.

Examples of anti-PSMA antibodies that may be monospecific or may be part of bispecific anti-CD3 antibodies include antibodies PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363 and PSMB365, having the VH and VL amino acid sequence and characteristics described herein.

In some embodiments, the antibody that specifically binds PSMA and is used in the methods of the invention described herein is PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB130, PSMB344, PSMB345, PSMB346, PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363 and PSMB365. The amino acid sequences VH and VL of these antibodies are shown in table. 2.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 160 and VL of SEQ ID NO: 65.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 79 and VL of SEQ ID NO: 78.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 77 and VL of SEQ ID NO: 78.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 75 and VL of SEQ ID NO: 76.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 74 and VL of SEQ ID NO: 61.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 72 and VL of SEQ ID NO: 73.

In some embodiments, an antibody that specifically binds PSMA and used in the methods of the invention described herein comprises VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

In some embodiments, an antibody that specifically binds PSMA and used in the methods of the invention described herein comprises VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 70 and VL of SEQ ID NO: 71.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 67.

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In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 142.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 143.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 67.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 144.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 67.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 142.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 143.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 144.

In some embodiments, the PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 142.

In some embodiments, an antibody that specifically binds PSMA and used in the methods of the invention described herein comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 143.

In some embodiments, a PSMA-specific binding antibody used in the methods of the invention described herein comprises VH of SEQ ID NO: 141 and VL of SEQ ID NO: 143. In some embodiments, a PSMA/CD3 bispecific antibody that contains the first PSMA-specific binding domain and the second CD3-specific binding domain used in the methods of the invention described herein comprise VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63 in the first domain, as well as VH of SEQ ID NO: 104 and VL with SEQ ID NO: 105 in the second domain.

In some embodiments, a PSMA/CD3 bispecific antibody that contains a first PSMA-specific binding domain and a second CD3-specific binding domain used in the inventive methods described herein comprises VH of SEQ ID NO: 64 and VL of SEQ ID NO: 64 ID NO: 65 in the first domain, as well as VH with SEQ ID NO: 104 and VL with SEQ ID NO: 105 in the second domain.

In some embodiments, a PSMA/CD3 bispecific antibody that contains a first PSMA-specific binding domain and a second CD3-specific binding domain used in the inventive methods described herein comprises VH of SEQ ID NO: 66 and VL of SEQ ID NO: 66 and VL of SEQ ID NO: 66 ID NO: 67 in the first domain, as well as VH with SEQ ID nO: 104 and VL with SEQ ID NO: 105 in the second domain.

In some embodiments, a PSMA/CD3 bispecific antibody that contains a first PSMA-specific binding domain and a second CD3-specific binding domain used in the inventive methods described herein comprises VH of SEQ ID NO: 75 and VL of SEQ ID NO: 75 and VL of SEQ ID NO: 75 ID NO: 76 in the first domain, as well as VH with SEQ ID NO: 104 and VL with SEQ ID NO: 105 in the second domain.

In some embodiments, a PSMA/CD3 bispecific antibody that contains a first PSMA-specific binding domain and a second CD3-specific binding domain used in the inventive methods described herein comprises VH of SEQ ID NO: 74 and VL of SEQ ID NO: 74 and VL of SEQ ID NO: 74 ID NO: 61 in the first domain, as well as VH with SEQ ID NO: 104 and VL with SEQ ID NO: 105 in the second domain.

In some embodiments, a PSMA/CD3 bispecific antibody that contains a first PSMA-specific binding domain and a second CD3-specific binding domain,

- 49 046641 used in the methods of the invention described herein contains VH with SEQ ID NO: 160 and VL with SEQ ID NO: 65 in the first domain, as well as VH with SEQ ID NO: 104 and VL with SEQ ID NO: 105 in the second domain.

The cancer may be a hyperproliferative condition or disorder, a solid tumor, neovascularization, a soft tissue tumor, or a metastatic lesion.

The term cancer is intended to include all types of cancerous growths or oncogenic processes, tissue metastasis or malignant transformation of cells, tissues or organs, regardless of the type of histopathology or stage of invasiveness. Examples of cancer include solid tumors, hematologic malignancies, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignancies, such as sarcomas, and carcinomas (including adenocarcinomas and squamous cell carcinomas) in various organ systems, such as lesions of the prostate, liver, lung, breast, lymphatic system, gastrointestinal (eg, colon intestine) and genitourinary tract (eg, kidneys, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as many types of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung carcinoma, small intestinal cancer, and esophageal cancer. Squamous cell carcinomas include cancers of, for example, the lung, esophagus, skin, head and neck, mouth, anus, and cervix.

In one embodiment, the cancer is prostate cancer.

Metastatic lesions of the above cancers can also be treated or prevented using the methods and antibodies of the invention described herein.

Examples of cancers whose growth can be inhibited or reduced by the antibodies of the invention described herein include cancers that can overexpress PSMA. Examples of cancers include prostate cancer, or prostatic intraepithelial neoplasia, colorectal cancer, gastric cancer, clear cell kidney cancer, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, kidney cancer, neovascular disorder, clear cell renal cell carcinoma (SPCC), pancreatic cancer and other cancers other than prostate cancer, including, without limitation, kidney cancer, urothelial cancer and liver adenocarcinoma. Persistent or recurrent malignancies can be treated with the antibodies of the invention described herein.

Examples of other types of cancer that can be treated with antibodies of the invention described herein are anal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, fallopian tube carcinoma, vaginal carcinoma, carcinoma vulva, malignant melanoma of the skin or eye, astrocellular esophageal cancer, testicular cancer, ovarian cancer, pancreatic cancer, rectal cancer, uterine cancer, primary central nervous system lymphoma; central nervous system (CNS) tumor, cervical cancer, choriocarcinoma, rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, eye cancer; intraepithelial neoplasm, kidney cancer, laryngeal cancer, liver cancer; small cell lung cancer, neuroblastoma, oral cavity cancer (eg, lip, tongue, mouth and pharynx), nasopharyngeal cancer, retinoblastoma, rhabdomyosarcoma, respiratory system cancer, sarcoma, thyroid cancer, urinary system cancer, hepatocarcinoma, anal cancer, fallopian carcinoma tubes, vaginal carcinoma, vulvar carcinoma, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, tumor angiogenesis, spinal cord cancer, brainstem glioma, adenoma pituitary gland, Kaposi's sarcoma, Merkel cell carcinoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers, including asbestos-induced cancers, and other carcinomas and sarcomas, and combinations of these cancers.

Patients having cancer, including metastatic cancer, which expresses PSMA, can be treated with antibodies of the invention described herein. The cancer may be prostate cancer, or prostatic intraepithelial neoplasia, colorectal cancer, gastric cancer, clear cell kidney cancer, bladder cancer, lung cancer, squamous cell carcinoma, glioma, breast cancer, kidney cancer, neovascular disorder, clear cell renal cell carcinoma (CLRC) ), pancreatic cancer and other cancers other than prostate cancer, including, without limitation, kidney cancer, urothelial cancer and liver adenocarcinoma.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the subject has a solid tumor.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the subject has prostate blastoma.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is colorectal cancer.

- 50 046641

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is gastric cancer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is lung cancer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is bladder cancer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a squamous cell carcinoma.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the cancer is clear cell renal cell carcinoma (CCRC).

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is breast cancer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a glioma.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is prostate cancer or castration-resistant prostate cancer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is kidney cancer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is pancreatic cancer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a liver adenocarcinoma.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the cancer is a neovascular disorder.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the cancer is kidney cancer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a urothelial cancer.

In some embodiments described herein, and in some embodiments of each and every one of the numbered embodiments listed below, the solid tumor is a brain cancer.

In some embodiments described herein, the subject has a tumor that expresses PSMA.

In some embodiments described herein, the subject has tumor-infiltrated T lymphocytes (TILs) in the tumor tissue.

The term increase in number refers to a statistically significant increase in a subject compared to a control. An increase in number, for example, refers to a statistically significant increase in the number of TILs in a subject (eg, a patient) before and after treatment with a PSMA antibody or other therapeutic agent.

In some embodiments described herein, the subject has increased interferon-gamma (IFN-γ) expression or activity.

In some embodiments described herein, the subject received treatment with an anti-PSMA antibody.

In some embodiments described herein, the subject is resistant to treatment with an anti-PSMA antibody.

Any of the PSMA antibodies or PSMA x CD3 bispecific antibodies of the invention described herein can be used in the methods of the invention.

- 51 046641

The antibodies and fragments thereof described herein can also be administered as part of combination therapy, i.e. in combination with other therapeutic agents relevant for the treatment of the disease or condition. Accordingly, in one embodiment, the antibody drug is for combination with one or more additional therapeutic agents, such as a chemotherapeutic agent. In some embodiments, the other therapeutic agent is a radiopharmaceutical (such as radium-223 chloride), secondary hormonal therapeutics (such as abiraterone or enzalutamide), and/or chemotherapeutic agents (docetaxel and cabazitaxel). Such combined administration may be simultaneous, separate or sequential, in any order. For concurrent administration, the agents may be administered as a single composition or as separate compositions, respectively, a chemotherapeutic agent (e.g., docetaxel, carboplatin, fludarabine), abiraterone, hormonal therapy (e.g., flutamide, bicalutamide, nilutamide, cyproterone acetate, ketoconazole, aminoglutethimide, abarelix, degarelix, leuprolide, goserelin, triptorelin, buserelin, ARN-509), serine or tyrosine kinase inhibitor (eg, PI3-kinase inhibitor SF1126,) (eg, lapatanib), multiple kinase inhibitor (eg, sorafenib, sunitinib), VEGF inhibitor (eg, bevacizumab), TAK-700, cancer vaccine (eg, BPX-101, PEP223), listeria vaccine, lenalidomide, TOK-001, IGF-1 receptor inhibitor (eg, cixumumab), TRC105, Aurora kinase inhibitor A (eg, MLN8237), proteasome inhibitor (eg, bortezomib), OGX011, radioimmunotherapy (eg, HuJ591-GS), HDAC inhibitor (eg, valproic acid, SB939, LBH589), hydroxychloroquine, mTOR inhibitor (eg, everolimus), dovitinib lactate, diindolylmethane, efavirenz, OGX-427, genistein, IMC-3G3, bafetinib, CP-675,206, radiation therapy, surgery, or a combination thereof.

In one embodiment, a method is provided for treating a disorder in a subject involving cells expressing BCMA, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a bispecific antibody or fragment, such as the anti-BCMA x CD3 bispecific antibody described herein, and carrying out radiation therapy. In one embodiment, a method of treating or preventing cancer is provided, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a bispecific antibody or fragment, such as the anti-BCMA x CD3 antibody described herein, and administering radiation therapy. Radiation therapy is offered, which may include radiation or appropriate administration of radiopharmaceuticals to the patient. The radiation source may be external or internal to the patient being treated (radiation therapy may, for example, be external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be practically used for these methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine- 123, iodine-131 and indium-111.

The antibodies of the invention described herein can be administered in combination with a vaccine.

Examples of vaccines are immunogenic agents such as cancer cells, purified tumor antigens (including recombinant proteins, antigen epitopes, peptides and carbohydrate molecules), tumor antigens delivered to the patient through gene therapy, cells and cells transfected with genes encoding immune-promoting cytokines. Examples of vaccines that can be used include peptides of melanoma antigens, such as gp100 peptides, MAGE, Trp-2, MART1 and/or tyrosinase antigens, or tumor cells transfected to express the cytokine GM-CSF, DNA-based vaccines, vaccines listeria-based, RNA-based and viral transduction-based vaccines, prostate peptides or antigens (eg PSMA, STEAP1, PSCA), sipuleucel-T cancer vaccine or lung cancer antigen peptides. A cancer vaccine can be preventative or therapeutic.

Many experimental tumor vaccination strategies have been developed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice of Oncology Fifth Edition). In one of these strategies, a vaccine is prepared using autologous or allogeneic tumor cells. These cell-based vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al., (1993) Proc Natl Acad Sci U.S.A. 90: 3539-43.).

Antibodies of the invention described herein can be administered in combination with one or a set of recombinant proteins and/or peptides expressed in or on a tumor to induce an immune response to these proteins. The immune system usually accepts these proteins as self-antigens and therefore tolerates them. The tumor antigen may also include telomerase protein, which is required for chromosome telomere synthesis and is expressed at greater than 85%

- 52,046,641 types of cancer in humans, but only in a limited number of somatic tissues (Kim et al., (1994) Science 266: 2011-2013). Tumor antigens may also be neoantigens expressed in or on cancer cells as a result of somatic mutations that alter the protein sequence or create fusion proteins from two unrelated sequences (eg, bcr-abl in the Philadelphia chromosome) or idiotypes from B-cell tumors. Tumor antigens may be antigenic epitopes of prostate-specific antigen (PSA), mesothelin, prostate-specific membrane antigen (PSMA), synovial sarcoma X2 (SSX2), NKX3.1, prostate acid phosphatase (PAP), or epidermal growth factor receptors, or peptides specific for EGFR variants, such as the known EGFRvIII, overexpressed on tumor cells.

Other tumor vaccines may include proteins from viruses implicated in human cancers, such as human papillomaviruses (HPV), hepatitis viruses (HBV and HCV), Kaposi's sarcoma herpesvirus (KHSV), and Epstein-Barr virus (EBV). Another form of specific tumor antigens that can be used in combination with the antibodies of the invention described herein are purified heat shock proteins (HSPs) isolated from the tumor tissue itself. HSPs contain protein fragments from tumor cells and are highly effective when delivered to antigen presenting cells to induce antitumor immunity (Suot and Srivastava (1995) Science 269:1585-1588; Tamura et al., (1997) Science 278:117-120) .

Dendritic cells (DCs) are potent antigen-presenting cells that can be used to prime specific responses to antigen. DC can be obtained ex vivo and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al., (1998) Nature Medicine 4: 328-332). DCs can also be genetically transformed to express these tumor antigens. DCs have also been fused directly to tumor cells for immunization purposes (Kugler et al., (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be effectively combined with the antibodies of the invention described herein to activate more potent responses against tumors.

In some embodiments described herein, PSMA specific binding antibodies of the invention or PSMA x CD3 bispecific antibodies of the invention are administered in combination with a tumor vaccine that contains a prostate-specific antigen peptide fragment or a vector encoding a prostate-specific antigen peptide fragment. specific antigen.

The antibodies of the invention described herein can be administered in combination with standard cancer treatment.

The antibodies of the invention described herein can be administered in combination with standard courses of chemotherapy for cancer treatment. In these cases, it is possible to reduce the dose of the chemotherapeutic agent administered (Mokyr et al., (1998) Cancer Research 58: 5301-5304).

In some embodiments described herein, antibodies of the invention can be administered in combination with one or more other antibody molecules, chemotherapy, other anticancer therapy (eg, targeted anticancer therapy or oncolytic drugs), cytotoxic agents, cytokines, surgical and /or radiation procedures.

Examples of cytotoxic agents that can be administered in combination with antibodies of the invention described herein include hormone inhibitors, anti-microtubulin agents, topoisomerase inhibitors, antimetabolites, mitosis inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalation agents, agents that interfere with signal transduction pathways, proapoptotic agents, proteasome inhibitors, and irradiation (eg, local or systemic body irradiation).

Standard therapeutic agents include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N-pentoxycarbonyl-5 -deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomes for injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomes for injection (DaunoXome®), dexamethasone , docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol ®), methotrexate (Folex®), mitoxantrone (Novantrone®), paclitaxel (Taxol®), Phoenix (yttrium-90/MX-DTPA), pentostatin, polypheprosan-20 implant with carmustine (Gliadel®), tamoxifen citrate (Nolvadex® ), teniposide (Vumon®), 6thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), vinorelbine (Navelbine®), ibrutinib, idealisib and brentuximab vedotin.

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Examples of alkylating agents include mustard gas derivatives, ethyleneimine derivatives, alkyl sulfonates, 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®), pipobromane (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylene thiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine ( BiCNU®), lomustine (CeeNU®) and streptozocin (Zanosar®) and dacarbazine (DTIC-Dome®). Additional examples of alkylating agents include oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysine and melphalan, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalene®); 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®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazolecarboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine (HMM), Hexalene®); Ifosfamide (Ifex®); Prednimustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechlorethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine hydrochloride (Treanda®).

Examples of anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (Lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposomes, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin.

Examples of vinca alkaloids that can be used in combination with the antibodies of the invention include vinorelbine tartrate (Navelbine®), vincristine (Oncovin®) and vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincalakeoblastine and VLB, AlkabanAQ® and Velban®); and vinorelbine (Navelbine®).

Examples of proteasome inhibitors that can be used in combination with the antibodies of the invention are bortezomib (Velcade®); carfilzomib (PX-171-007, (8)-4-methyl-I-((8)-1-((8)-4methyl-1-(^)-2-methyloxiran-2-yl)-1-oxopentane -2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-(^)-2-(2morpholinoacetamido)-4-phenylbutanamido)-pentanamide); marizomib (NPI-0052); ixazomib citrate (MFN-9708); delanzomib (CEP-18770); and O-methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N[(1S)-2-[(2R)-2-methyl-2-oxiranyl] -2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

In some embodiments described herein, antibodies of the invention are administered in combination with a serine or tyrosine kinase inhibitor (eg, a receptor tyrosine kinase (RTK) inhibitor). Examples of tyrosine kinase inhibitors include an epidermal growth factor (EGF) pathway inhibitor (eg, epidermal growth factor receptor (EGFR) inhibitor), a vascular endothelial growth factor (VEGF) pathway inhibitor (eg, vascular endothelial growth factor receptor (VEGFR) inhibitor (eg , VEGFR-1 inhibitor, VEGFR-2 inhibitor, VEGFR-3 inhibitor)), platelet-derived growth factor (PDGF) pathway inhibitor (eg, platelet-derived growth factor receptor (PDGFR) inhibitor (eg, PDGFR-β inhibitor)), inhibitor RAF-1, KIT inhibitor and RET inhibitor. In some embodiments, the second therapeutic agent is axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib ( Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT® , SU11248), toceranib (PALLADIA®), vadetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®) , panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), alemtuzumab (CAMRATH®), gemtuzumab ozogamicin (MYLOTARG®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, AVT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, - 26483327, MGCD265, DCC2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, XL228, AEE788, AG-490, AST-6, BMS -599626, CUDC-101, PD153035, pelitinib (EKB-569), vadetanib (zactima), WZ3146, WZ4002, WZ8040, AVT-869 (linifanib), AEE788, AP24534 (ponatinib), AV-951 (tivozanib), axitinib, BAY 73-4506 (regorafenib), brivanib alaninate (BMS-582664), brivanib (BMS-540215),

- 54 046641 cediranib (AZD2171), CHIR-258 (dovitinib), CP 673451, CYC116, E7080, Ki8751, mazitinib (AB1010), MGCD-265, motesanib diphosphate (AMG-706), MP-470, OSI-930, pazopanib hydrochloride, PD173074, sorafenib tosylate (Bay 43-9006), SU 5402, TSU-68 (SU6668), vanib, XL880 (GSK1363089, EXEL-2880). The tyrosine kinase inhibitors selected are sunitinib, erlotinib, gefitinib or sorafenib. In some embodiments, the EGFR inhibitor is an EGFRc-Met bispecific antibody (EM-1 mAb) comprising the heavy and light chains of SEQ ID NOs: 249, 250, 251 and 252 (US2014/0141000).

In some embodiments, the antibodies of the invention are administered in combination with vascular endothelial growth factor (VEGF) receptor inhibitors, including bevacizumab (Avastin®), axitinib (Inlyta®); Brivanib alaninate (BMS-582664, ^)-(^)-1-(4-(4-fluoro-2-methyl-III-indol-5-yloxy)-5methylpyrrolo[2.1-1T][1.2, 4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); Sorafenib (Nexavar®); Pazopanib (Votrient®); Sunitinib malate (Sutent®); Cediranib (AZD2171, CAS 288383-20-1); Vargatef (BIBF1120, CAS 928326-83-4); Foretinib (GSK1363089); Telatinib (BAY57-9352, CAS 332012-40-5); Apatinib (YN968D1, CAS 811803-05-1); Imatinib (Gleevec®); Ponatinib (AP24534, CAS 943319-70-8); Tivozanib (AV951, CAS 475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Vatalanib dihydrochloride (PTK787, CAS 212141-51-0); Brivanib (BMS-540215, CAS 649735-46-6); Vandetanib (Caprelsa® or AZD6474); Motesanib diphosphate (AMG706, CAS 857876-30-3, H-(2,3-dihydro-3,3dimethyl-N-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide, described in PCT publication No. WO 02/066470); Dovitinib dilactic acid (TKI258, CAS 852433-84-2); Linifanib (ABT869, CAS 796967-16-3); Cabozantinib (XL184, CAS 849217-68-1); Lestaurtinib (CAS 111358-88-4); N-[5-[[[5-(1,1dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-amino-1 -((4-((3-methoxyphenyl)amino)pyrrolo [2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3 -ol (BMS690514); ^(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3a α, 5β, 6aa)octahydro-2methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8); 4-methyl-3-[[1-methyl6-(3-pyridinyl)-III-pyrazolo[3,4-1]pyrimidin-4-yl]amino]-H-[3-(trifluoromethyl)phenyl]benzamide (BHG712 , CAS 940310-85-0); and aflibercept (Eylea®).

Examples of VEGF inhibitors include a monoclonal antibody that binds to the same epitope as the anti-VEGF monoclonal antibody A4.6.1 produced by the hybridoma ATCC HB 10709; recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al., (1997) Cancer Res 57:4593-4599. In one embodiment, the anti-VEGF antibody is bevacizumab (BV), also known as rhuMAb VEGF or AVASTIN®. It contains mutated human IgG1 framework regions and antigen-binding complementarity-determining regions from a mouse monoclonal antibody to hVEGF (A.4.6.1), which blocks the binding of human VEGF to its receptors. Bevacizumab and other humanized anti-VEGF antibodies are further described in US Patent No. 6,884,879. Additional anti-VEGF antibodies include the G6 or B20 series antibodies (eg, G6-31, B20-4.1), as described in International Patent Publication Nos. WO 2005/012359 and WO 2005/044853. for additional antibodies, see US Patent Nos. 7,060,269, 6,582,959, 6,703,020, 6,054,297, WO 98/45332, WO 96/30046, WO 94/10202, EP 0666868B1, US Patent Applications Published Nos. 2006009360, US 20050186208, US 20030206899, US 20030190317, US 20030203409 and US 20050112126; and in Popkov et al., (2004) Journal of Immunological Methods 288: 149-164. Other antibodies include antibodies that bind to a functional epitope on human VEGF containing residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103 and C104, or alternatively containing residues F17, Y21, Q22, Y25, D63, 183 and Q89.

In some embodiments described herein, antibodies of the invention are administered in combination with a PI3K inhibitor. In one embodiment, the PI3K inhibitor is an inhibitor of the delta and gamma isoforms of PI3K. In one embodiment, the PI3K inhibitor is an inhibitor of PI3K beta isoforms. Examples of PI3K inhibitors, which can be used, for example, in Wo 2010/036380, Wo 2010/006086, Wo 09/114870, Wo 05/113556, GSK 2126458, GDC-0980, GDC-0941, Sanofi XL147, XL756, XL147, XL147, XL147, XL147, XL147, XL147 , PF-46915032, BKM 120, CAL-101, CAL 263, SF1126, PX-886, and a dual PI3K inhibitor (eg, Novartis BEZ235).

In some embodiments described herein, antibodies of the invention are administered in combination with an mTOR inhibitor, for example, one or more mTOR inhibitors selected from one or more of rapamycin, temsirolimus (TORISEL®), AZD8055, BEZ235, BGT226, XL765 , PF-4691502, GDC0980, SF1126, OSI-027, GSK1059615, KU-0063794, WYE-354, Palomid 529 (P529), PF-04691502, or PKI-587, 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, 35K)-1,18-dihydroxy19.30 -dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26 ,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669 and described in PCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimoda (CAS 164301-51-3); emsirolimus, (5-{2,4-bis-[(3S)-3-methylmorpholin-4-yl]-pyrido[2,3-d]pyrimidin-7-yl}-2

- 55 046641 methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3pyridinyl)-4-methylpyrido[2,3D]pyrimidin-7(8H)-one (PF04691502, CAS 1013101 -36-4); and N2-[1,4-guoKCO-4[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]-butyl]-P-arginylglycyl- P-aaspartyl-P-serine (SEQ ID NO: 237), internal salt (SF1126, CAS 936487-67-1) and XL765.

In some embodiments described herein, antibodies of the invention are administered in combination with a BRAF inhibitor, for example, GSK2118436, RG7204, PLX4032, GDC-0879, PLX4720 and sorafenib tosylate (Bay 43-9006).

In some embodiments described herein, antibodies of the invention are administered in combination with an immunomodulatory agent. Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy effects in a variety of cancers by blocking immunoinhibitory signals and providing patients with an effective antitumor response. In some embodiments, antibodies of the invention are administered in combination with an anti-PD1 antibody (eg, nivolumab) and an anti-PDL antibody (eg, MDX-1105) or an anti-CTLA4 antibody (eg, Ipilimumab). The ability of agonistic anti-CD40 antibodies (referred to as aCD40) or CD40 ligand to stimulate immune responses and target tumors indicates that such reagents hold promise as anti-cancer immunotherapies. In some embodiments, antibodies of the invention are administered in combination with an anti-CD40 antibody (eg, SGN-40, CP-870,893) or an anti-CD40L antibody (eg, BG9588).

In some embodiments described herein, antibodies of the invention are administered in combination with a BTK inhibitor.

In some embodiments described herein, antibodies of the invention, administered in combination with a MEK inhibitor, are used in the treatment of prostate cancer, melanoma, colorectal cancer, non-small cell lung cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer gland, hematologic malignancy, or renal cell carcinoma. In some embodiments, the tumor tissue or cancer cell has a BRAF mutation (eg, a BRAF V600 E mutation), a wild-type BRAF mutation, a wild-type KRAS mutation, or an activating KRAS mutation. Cancer may be at an early, intermediate or advanced stage. Any MEK inhibitor can be used in combination, including ARRY142886, G02442104 (also known as GSK1120212), RDEA436, RDEA119/BAY 869766, AS703026, G00039805 (also known as AZD-6244 or selumetinib), BIX 02188, BIX 02189, CI -1040 ( PD184352), PD0325901, PD98059, U0126, GDC-0973 (methanol, [3,4-difluoro-2-[(2-fluoro-4iodophenyl)amino]phenyl][3-hydroxy-3-(25)-2-piperidinyl -1-azetidinyl]-), G-38963, G02443714 (also known as AS703206), or a pharmaceutically acceptable salt or solvate thereof. Additional examples of MEK inhibitors are described in WO 2013/019906, WO 03/077914, WO 2005/121142, WO 2007/04415, WO 2008/024725 and WO 2009/085983.

In some embodiments described herein, antibodies of the invention are administered in combination with a JAK2 inhibitor, for example, CEP-701, INCB18424, CP-690550 (tasocitinib).

In some embodiments described herein, antibodies of the invention are administered in combination with paclitaxel or a paclitaxel agent, for example, TAXOL®, with protein-bound paclitaxel (for example, ABRAXANE®). Examples of paclitaxel agents include nanoparticle albumin-bound paclitaxel (ABRAXANE, marketed by Abraxis Bioscience), docosahexaenoic acid-bound paclitaxel (DHA-paclitaxel, Taxoprexin, marketed by Protarga), polyglutamate-bound paclitaxel (PG-paclitaxel, paclitaxel polyglumex, CT -2103, XYOTAX, marketed by Cell Therapeutic), tumor activated prodrug (TAP), ANG105 (Angiopep-2 linked to three paclitaxel molecules, marketed by ImmunoGen), paclitaxel-EC-1 (paclitaxel linked to erbB2 recognition peptide EC -1; see Li et al., Biopolymers (2007) 87:225-230) and glucose-conjugated paclitaxel (eg, 2'paclitaxel methyl-2-glucopyranosylsuccinate, see Liu et al., (2007) Bioorganic & Medicinal Chemistry Letters 17:617-620).

In some embodiments described herein, antibodies of the invention are administered in combination with cellular immunotherapy (eg, Provenge (eg, Sipuleucel)) and optionally in combination with cyclophosphamide.

Examples of therapeutic agents that can be used in combination with antibodies of the invention for the treatment of prostate cancer include a chemotherapeutic agent (e.g., docetaxel, carboplatin, fludarabine), abiraterone, hormonal therapy (e.g., flutamide, bicalutamide, nilutamide, cyproterone acetate, ketoconazole, aminoglutethimide, abarelix, degarelix, leuprolide, goserelin, triptorelin, buserelin), tyrosine kinase inhibitor (eg, dual kinase inhibitor (eg, lapatanib), multiple kinase inhibitor (eg, sorafenib, sunitinib), VEGF inhibitor (eg, bevacizumab), TAK- 700, cancer vaccine (for example, BPX-101,

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PEP223), lenalidomide, TOK-001, IGF-1 receptor inhibitor (eg, cixumumab), TRC105, Aurora A kinase inhibitor (eg, MLN8237), proteasome inhibitor (eg, bortezomib), OGX-011, radioimmunotherapy (eg, HuJ591- GS), HDAC inhibitor (eg, valproic acid, SB939, LBH589), hydroxychloroquine, mTOR inhibitor (eg, everolimus), dovitinib lactate, diindolylmethane, efavirenz, OGX-427, genistein, IMC-3G3, bafetinib, CP-675,206, ARN -509, radiation therapy, surgery, or a combination of both.

Examples of therapeutic agents that can be used in combination with antibodies of the invention for the treatment of pancreatic cancer include a chemotherapeutic agent, e.g., paclitaxel or a paclitaxel agent (e.g., a paclitaxel composition such as TAXOL, an albumin-stabilized paclitaxel nanoparticle composition (e.g., ABRAXANE) or a liposomal composition of paclitaxel); gemcitabine (eg, gemcitabine alone or in combination with AChP107-11); other chemotherapeutic agents such as oxaliplatin, 5-fluorouracil, capecitabine, rubitecan, epirubicin hydrochloride, NC-6004, cisplatin, docetaxel (eg, TAXOTERE), mitomycin C, ifosfamide; interferon; tyrosine kinase inhibitor (eg, EGFR inhibitor (eg, erlotinib, panitumumab, cetuximab, nimotuzumab)); HER2/neu receptor inhibitor (eg, trastuzumab); dual kinase inhibitor (eg, bosutinib, saracatinib, lapatinib, vandetanib); multiple kinase inhibitor (eg, sorafenib, sunitinib, XL184, pazopanib); VEGF inhibitor (eg, bevacizumab, AV-951, brivanib); radioimmunotherapy (eg XR303); cancer vaccine (eg GVAX, survivin peptide); a COX-2 inhibitor (eg, celecoxib); IGF-1 receptor inhibitor (eg, AMG 479, MK-0646); mTOR inhibitor (eg, everolimus, temsirolimus); IL-6 inhibitor (eg CNTO 328); cyclin-dependent kinase inhibitor (eg, P276-00, UCN-01); a compound targeting energy metabolism modification (AEMD) (eg, CPI-613); HDAC inhibitor (eg, vorinostat); TRAIL receptor-2 (TR-2) agonist (eg, conatumumab); MEK inhibitor (eg AS703026, selumetinib, GSK1120212); Raf/MEK dual kinase inhibitor (eg, RO5126766); signaling cascade inhibitor (eg MK0752); monoclonal antibody-to-antibody fusion protein (eg, L19IL2); curcumin; HSP90 inhibitor (eg tanespimycin, STA-9090); rIL-2; denileukin diftitox; topoisomerase 1 inhibitor (eg, irinotecan, PEP02); statin (eg simvastatin); factor VIIa inhibitor (eg, PCI-27483); AKT inhibitor (eg, RX-0201); hypoxia-activated prodrug (eg, TH-302); metformin hydrochloride, a gamma secretase inhibitor (eg, R04929097); ribonucleotide reductase inhibitor (eg 3-AR); immunotoxin (eg HuC242-DM4); PARP inhibitor (eg, KU0059436, veliparib); CTLA-4 inhibitor (eg, CP-675,206, ipilimumab); AdV-tk therapy; proteasome inhibitor (eg, bortezomib (Velcade), NPI-0052); thiazolidinedione (eg pioglitazone); NPC1C; Aurora kinase inhibitor (eg R763/AS703569), CTGF inhibitor (eg FG-3019); siG12D LODER; and radiation therapy (eg, tomotherapy, stereotactic radiation therapy, proton therapy), surgery, and combinations thereof. In certain embodiments, a combination of paclitaxel or a paclitaxel agent with gemcitabine can be used with the antibodies of the invention.

Examples of therapeutic agents that can be used in combination with antibodies of the invention for the treatment of small cell lung cancer include a chemotherapeutic agent, for example, etoposide, carboplatin, cisplatin, oxaliplatin, irinotecan, topotecan, gemcitabine, liposomal SN-38, bendamustine, temozolomide, belotecan, NK012, FR901228, flavopiridol); tyrosine kinase inhibitor (eg, EGFR inhibitor (eg, erlotinib, gefitinib, cetuximab, panitumumab)); multiple kinase inhibitor (eg, sorafenib, sunitinib); VEGF inhibitor (eg, bevacizumab, vandetanib); a cancer vaccine (eg GVAX); Bc1-2 inhibitor (for example, oblimersen sodium, AVT-263); a proteasome inhibitor (eg, bortezomib (Velcade), NPI-0052), paclitaxel, or a paclitaxel agent; docetaxel; IGF-1 receptor inhibitor (eg, AMG 479); HGF/SF inhibitor (eg AMG 102, MK-0646); chloroquine; Aurora kinase inhibitor (eg, MLN8237); radioimmunotherapy (eg TF2); HSP90 inhibitor (eg tanespimycin, STA-9090); mTOR inhibitor (eg everolimus); bispecific antibody to Ep-CAM/CD3 (for example, MT110); CK-2 inhibitor (eg CX-4945); HDAC inhibitor (eg, belinostat); SMO antagonist (belinostat, BMS 833923); peptide cancer vaccine and radiation therapy (eg, intensity modulated radiation therapy (IMRT), hypofractionated radiation therapy, hypoxia-guided radiation therapy), surgery, and combinations thereof.

Examples of therapeutic agents that can be used in combination with antibodies of the invention for the treatment of non-small cell lung cancer include a chemotherapeutic agent (eg, vinorelbine, cisplatin, docetaxel, pemetrexed disodium, etoposide, gemcitabine, carboplatin, liposomal SN-38, TLK286, temozolomide , topotecan, pemetrexed disodium, azacitidine, irinotecan, tegafur-gimeracil-oteracil-potassium, sapacitabine); tyrosine kinase inhibitor (eg, EGFR inhibitor (eg, erlotinib, gefitinib, cetuximab, panitumumab, necitumumab, PF-00299804, nimotuzumab, RO5083945)), MET inhibitor (eg, PF-02341066, ARQ 197), PI3K kinase inhibitor (eg, XL147 , GDC-0941), Raf/MEK dual kinase inhibitor (e.g. RO5126766), inhibitor

- 57 046641 dual PI3K/mTOR kinase (e.g. XL765), SRC inhibitor (e.g. dasatinib), dual inhibitor (e.g. BIBW 2992, GSK1363089, ZD6474, AZD0530, AG-013736, lapatinib, MEHD7945A, linifanib), multiple kinase inhibitor (eg sorafenib, sunitinib, pazopanib, AMG 706, XL184, MGCD265, BMS-690514, R935788), VEGF inhibitor (eg endostar, endostatin, bevacizumab, cediranib, BIBF 1120, axitinib, tivozanib, AZD2171), cancer vaccine ( eg, BLP25 liposome vaccine, GVAX, recombinant DNA and adenovirus expressing L523S protein), Bcl-2 inhibitor (eg, oblimersen sodium), proteasome inhibitor (eg, bortezomib, carfilzomib, NPI0052, MLN9708), paclitaxel or paclitaxel agent, docetaxel, IGF-1 receptor inhibitor (eg, cixutumumab, MK-0646, OSI 906, CP-751,871, BIIB022), hydroxychloroquine, HSP90 inhibitor (eg, tanespimycin, STA-9090, AUY922, XL888), mTOR inhibitor (eg, everolimus, temsirolimus , ridaforolimus), Ep-CAM/CD3 bispecific antibody (eg, MT110), CK-2 inhibitor (eg, CX-4945), HDAC inhibitor (eg, MS 275, LBH589, vorinostat, valproic acid, FR901228), DHFR inhibitor ( eg, pralatrexate), retinoid (eg, bexarotene, tretinoin), antibody-drug conjugate (eg, SGN-15), bisphosphonate (eg, zoledronic acid), cancer vaccine (eg, belagenpumatucel-L), low molecular weight heparin (LMWH) ) (eg, tinzaparin, enoxaparin), GSK1572932A, melatonin, talactoferrin, dimesna, topoisomerase inhibitor (eg, amrubicin, etoposide, carenitcin), nelfinavir, cilengitide, ErbB3 inhibitor (eg, MM-121, U3-1287), survivin inhibitor (eg e.g. YM155, LY2181308), eribulin mesylate, COX-2 inhibitor (e.g. celecoxib), pegfilgrastim, Polo kinase 1 inhibitor (e.g. BI 6727), TRAIL receptor 2 (TR-2) agonist (e.g. CS- 1008), CNGRC peptide conjugate (SEQ ID NO: 225) and TNF-alpha, dichloroacetate (DCA), HGF inhibitor (e.g. SCH 900105), SAR240550, PPAR-gamma agonist (e.g. CS-7017), gamma secretase inhibitor (eg, RO4929097), epigenetic therapy (eg, 5-azacytidine), nitroglycerin, MEK inhibitor (eg, AZD6244), cyclin-dependent kinase inhibitor (eg, UCN-01), cholesterol-Esh1, antitubulin agent (eg, E7389) , farnesyl-OH-transferase inhibitor (eg, lonafarnib), immunotoxin (eg, BB-10901, SS1 (dsFv) PE38), fondaparinux, vascular disrupting agent (eg, AVE8062), PD-L1 inhibitor (eg, MDX-1105, MDX-1106), beta-glucan, NGR-hTNF, EMD 521873, MEK inhibitor (eg, GSK1120212), epothilone analog (eg, ixabepilone), spindle kinesin inhibitor (eg, 4SC-205), telomere-targeting agent (eg, KML-001), P70 pathway inhibitor (eg, LY2584702), AKT inhibitor (eg, MK-2206), angiogenesis inhibitor (eg, lenalidomide), Notch signaling inhibitor (eg, OMP-21M18), EGFR bispecific antibody EM-1 /c-Met as described in US 2014/0141000 A1, radiation therapy, surgery and combinations thereof.

Examples of therapeutic agents that can be used in combination with antibodies of the invention for the treatment of ovarian cancer include a chemotherapy agent (eg, paclitaxel or a paclitaxel agent; docetaxel; carboplatin; gemcitabine; doxorubicin; topotecan; cisplatin; irinotecan, TLK286, ifosfamide, olaparib, oxaliplatin , melphalan, pemetrexed disodium, SJG-136, cyclophosphamide, etoposide, decitabine); Ghrelin antagonist (eg, AEZS-130), immunotherapy (eg, APC8024, Oregovomab, ORT-821), tyrosine kinase inhibitor (eg, EGFR inhibitor (eg, erlotinib), dual kinase inhibitor (eg, E7080), multiple kinase inhibitor (eg, , AZD0530, JI-101, sorafenib, sunitinib, pazopanib), ON 01910.Na), VEGF inhibitor (eg, bevacizumab, BIBF 1120, cediranib, AZD2171), PDGFR inhibitor (eg, IMC-3G3), paclitaxel, topoisomerase inhibitor ( eg, carenitcin, irinotecan), FIDAC inhibitor (eg, valproate, vorinostat), folate receptor inhibitor (eg, farletuzumab), angiopoietin inhibitor (eg, AMG 386), epothilone analogue (eg, ixabepilone), proteasome inhibitor (eg, carfilzomib) , IGF-1 receptor inhibitor (eg, OSI 906, AMG 479), PARP inhibitor (eg, veliparib, AG014699, iniparib, MK-4827), Aurora kinase inhibitor (eg, MLN8237, ENMD2076), angiogenesis inhibitor (eg, lenalidomide) , DFIFR inhibitor (eg, pralatrexate), radioimmunotherapy agent (eg, Hu3S193), statin (eg, lovastatin), topoisomerase-1 inhibitor (eg, NKTR-102), cancer vaccine (eg, p53 synthetic long peptide vaccine, from autologous OS-DC), mTOR inhibitor (eg, temsirolimus, everolimus), BCR/ABL inhibitor (eg, imatinib), ET-A receptor antagonist (eg, ZD4054), TRAIL receptor-2 (TR-2) agonist ( e.g. CS-1008), hGf/SF inhibitor (e.g. AMG 102), EGEN-001, Polo kinase 1 inhibitor (e.g. BI 6727), gamma secretase inhibitor (e.g. RO4929097), Wee-1 inhibitor ( e.g., MK-1775), antitubulin agent (e.g., vinorelbine, E7389), immunotoxin (e.g., denileukin diftitox), SB-485232, vascular disrupting agent (e.g., AVE8062), integrin inhibitor (e.g., EMD 525797), spindle kinesin inhibitor (eg, 4SC-205), Revlimid, HER2 inhibitor (eg, MGAH22), ErrB3 inhibitor (eg, MM121), radiation therapy, and combinations thereof.

Examples of therapeutic agents that can be used in combination with antibodies of the invention to treat kidney cancer, such as renal cell carcinoma (RCC) or metastatic RCC, include an immune-based strategy (eg, interleukin-2 or interferon-α), targeting agent

- 58 046641 (eg VEGF inhibitor, eg anti-VEGF monoclonal antibody, eg bevacizumab (Rini et al., (2010) J Clin Oncol 28(13): 2137-2143)); a VEGF tyrosine kinase inhibitor such as sunitinib, sorafenib, axitinib and pazopanib (reviewed in Pal et al., (2014) Clin Advances in Hematology & Oncology 12(2): 90-99); an RNAi inhibitor, or an inhibitor of a downstream mediator of the VEGF signaling pathway, such as a mammalian target of rapamycin (mTOR) inhibitor, such as everolimus and temsirolimus (Hudes et al., (2007) N Engl J Med 356(22): 2271-2281, Motzer et al., (2008) Lancet 372: 449-456).

PSMA-specific antibody kits

Described herein are kits comprising the described PSMA-specific antibodies or antigen binding fragments thereof. The described kits can be used to implement methods of using PSMA-specific antibodies or antigen binding fragments proposed herein, or other methods known to those skilled in the art. In some embodiments, the kits described may include antibodies or antigen binding fragments described herein and reagents for use in detecting the presence of PSMA in a biological sample. Accordingly, the disclosed kits may include one or more of the antibodies or antigen binding fragments thereof described herein, a vessel for storing the antibody or fragment when not in use, instructions for use of the antibody or fragment, the antibody or fragment immobilized on a solid support, and/ or detectably labeled forms of the antibody or fragment as described herein.

One embodiment of the invention is a kit containing an antibody of the invention that specifically binds PSMA.

Another embodiment of the invention is a kit containing a PSMA x CD3 bispecific antibody that contains a first PSMA specific binding domain and a second CD3 specific binding domain of the invention.

The kit can be used for therapeutic applications and as diagnostic kits.

The kit can be used to detect the presence of PSMA, CD3, or PSMA and CD3 in a biological sample.

In some embodiments, the kit contains an antibody of the invention described herein and reagents for detecting the antibody. The kit may contain one or more of the following elements: instructions for use; other reagents, such as a label, a therapeutic agent or an agent used for chelating or otherwise combining, a labeling antibody, or a therapeutic agent, or a radioprotective composition; devices or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers and devices or other materials for administration to a patient.

In some embodiments, the kit contains an antibody of the invention contained in a container and instructions for use of the kit.

In some embodiments, the antibody in the kit is labeled.

In some embodiments, the kit contains an anti-PSMA antibody PSMB119, PSMB120, PSMB121, PSMB122, PSMB123, PSMB87, PSMB126, PSMB127, PSMB128, PSMB129, PSMB130, PSMB120, PSMB121, PSMB122, PSMB123, PSMB127, PSMB128, PSMB34, 4, PSMB345, PSMB346 , PSMB347, PSMB349, PSMB358, PSMB359, PSMB360, PSMB361, PSMB362, PSMB363 and PSMB365.

In some embodiments, the kit contains a bispecific antibody to PSMA x CD3, PS3B22, PS3B23, PS3B25, PS3B27, PS3B28, or PS3B30.

Methods for detecting PSMA or PSMA and CD3

One embodiment of the invention described herein is a method for detecting PSMA in a sample, comprising obtaining a sample, contacting the sample with a PSMA-specific binding antagonist antibody of the invention, and detecting the PSMA-bound antibody in the sample.

One embodiment of the invention described herein is a method for detecting PSMA and CD3 in a sample, comprising obtaining the sample, contacting the sample with a bispecific antibody that contains a first PSMA-specific binding domain and a second CD3-specific binding domain. , according to the invention and detection of antibodies associated with PSMA and CD3 in the sample.

In some embodiments described herein, the sample can be obtained from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (for example, surgically excised tumor tissue, biopsy specimens, including those obtained using fine-needle aspiration biopsy), histological preparations, etc.

Antibodies of the invention described herein associated with PSMA or PSMA and CD3 can be detected by known methods. Examples of methods include directly labeling antibodies using fluorescent or chemiluminescent labels or radiolabels, or attaching an easily detectable functional group to the antibodies of the invention, such as

- 59 046641 biotin, enzymes or epitope tags. Examples of labels and functional groups are ruthenium, 111In-DOTA, nnn-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes and Alexafluor® dyes.

The antibodies of the invention can be used in a variety of assays to detect PSMA or PSMA and CD3 in a sample. Examples of assays are Western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS), or ELISA.

Embodiments

1) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment (i) binds to cells expressing recombinant Pan troglodytes PSMA, wherein cell binding is measured by flow cytometry, and (ii) binds to the recombinant extracellular domain of PSMA Pan troglodytes (SEQ ID NO: 4) with an affinity of about 30 nM or less, the affinity being measured using a Proteon XPR36 surface plasmon resonance assay.

2) An antibody according to embodiment 1 that has one, two, three or four of the following properties:

a) binds LNCaP cells with a calculated _{EC50 of} 20 nM or less and binds HEK cells expressing PSMA Macaca fascicularis with a calculated EC50 _of 40 nM or less, with a difference in calculated EC5o between binding of LNCaP cells and binding of HEK cells expressing PSMA Macaca fascicularis is less than 5 times, and the calculated EC ₅₀ is measured in a whole cell binding assay at 0°C using flow cytometry;

b) binds to recombinant ECD of human PSMA (SEQ ID NO: 7), Pan troglodytes (SEQ ID NO: 4) and Macaca fascicularis (SEQ ID NO: 5) with an equilibrium dissociation constant ( _KD ) of 12 nM or less, and KD measured using ProteOn XPR36 surface plasmon resonance assay at +25°C;

c) demonstrates T cell-mediated killing of LNCaP cells, C42 cells, human PSMA-expressing HEK cells, or Macaca fascicularis PSMA-expressing HEK cells when paired with the anti-CD3 bispecific antibody CD3B219, with T cell-mediated killing measured by chromium release 51 or using a caspase 3/7 activation assay; or

d) recognizes a conformational epitope, wherein the epitope consists of residues I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307 and K324-P326 of human PSMA (SEQ ID NO: 3).

3) The antibody of embodiment 2 comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively.

4) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NO: 20, 21, 22, 23, 12 and 24, respectively.

5) The antibody of embodiment 2 comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively.

6) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NO: 36, 37, 38, 39, 40 and 41, respectively.

7) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively.

8) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NOs: 31, 44, 45, 46, 29 and 47, respectively.

9) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NOs: 36, 37, 48, 49, 50 and 51, respectively.

10) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NOs: 36, 37, 52, 49, 50 and 51, respectively.

11) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NO: 8, 9, 10, 11, 12 and 13, respectively.

12) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NO: 31, 32, 33, 34, 12 and 35, respectively.

13) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NOs: 53, 54, 55, 23, 12 and 35, respectively.

14) The antibody of embodiment 2 containing the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NO: 122, 123, 124, 23, 12 and 24, respectively.

15) The antibody of embodiment 2 comprising a heavy chain variable region (VH) of SEQ ID NO: 60, 62, 64, 66, 68, 70, 72, 74, 75, 77, 79, or 160.

16) The antibody of embodiment 15 comprising a light chain variable region (VL) with

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SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 61, 76 or 78.

17) The antibody of embodiment 15, comprising VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

18) The antibody of embodiment 15, comprising VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

19) The antibody of embodiment 15, comprising VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

20) The antibody of embodiment 15, comprising VH of SEQ ID NO: 72 and VL of SEQ ID NO: 73.

21) The antibody of embodiment 15, comprising VH of SEQ ID NO: 74 and VL of SEQ ID NO: 61.

22) The antibody of embodiment 15, comprising VH of SEQ ID NO: 75 and VL of SEQ ID NO: 76.

23) The antibody of embodiment 15, comprising VH of SEQ ID NO: 77 and VL of SEQ ID NO: 78.

24) The antibody of embodiment 15, comprising VH of SEQ ID NO: 79 and VL of SEQ ID NO: 78.

25) The antibody of embodiment 15, comprising VH of SEQ ID NO: 160 and VL of SEQ ID NO: 65.

26) The antibody of embodiment 15, comprising VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

27) The antibody of embodiment 15, comprising VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69. The antibody of embodiment 15, comprising VH of SEQ ID NO: 70 and VL of SEQ ID NO: 71.

28) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 31, 42, 43, 11, 12 and 13, respectively.

29) The antibody of embodiment 28, wherein the antibody comprises VH of SEQ ID NO: 74 and VL of SEQ ID NO: 61.

30) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 14, 15, 16, 17, 18 and 19, respectively.

31) The antibody of embodiment 30, wherein the antibody comprises VH of SEQ ID NO: 62 and VL of SEQ ID NO: 63.

32) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 26, 27, 28, 29 and 30, respectively.

33) The antibody of embodiment 32, wherein the antibody comprises VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

34) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 36, 37, 38, 39, 40 and 41, respectively.

35) The antibody of embodiment 34, wherein the antibody comprises VH of SEQ ID NO: 72 and VL of SEQ ID NO: 73.

36) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 122, 123, 124, 23, 12 and 24, respectively.

37) The antibody of embodiment 36, wherein the antibody comprises VH of SEQ ID NO: 160 and VL of SEQ ID NO: 65.

38) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 8, 9, 10, 11, 12 and 13, respectively.

39) The antibody of embodiment 38, wherein the antibody comprises VH of SEQ ID NO: 60 and VL of SEQ ID NO: 61.

40) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 20, 21, 22, 23, 12 and 24, respectively.

41) The antibody of embodiment 40, wherein the antibody comprises VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65.

42) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 31, 32, 33, 34, 12 and 35, respectively.

43) The antibody of embodiment 42, wherein the antibody comprises VH of SEQ ID NO: 70 and VL of SEQ ID NO: 71.

44) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 31, 44, 45, 46, 29 and 47, respectively.

45) The antibody of embodiment 44, wherein the antibody comprises VH of SEQ ID NO: 75 and VL of SEQ ID NO: 76.

46) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 36, 37, 48, 49, 50 and 51, respectively.

47) The antibody of embodiment 46, wherein the antibody comprises VH of SEQ ID NO: 77 and VL of

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SEQ ID NO: 78.

48) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 36, 37, 52, 49, 50 and 51, respectively.

49) The antibody of embodiment 48, wherein the antibody comprises VH of SEQ ID NO: 79 and VL of SEQ ID NO: 78.

50) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 53, 54, 55, 23, 12 and 35, respectively.

51) The antibody of embodiment 50, wherein the antibody comprises VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69.

52) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 130, 27, 28, 29 and 30, respectively.

53) The antibody of embodiment 52, wherein the antibody comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 67.

54) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 130, 27, 131, 29 and 132, respectively.

55) The antibody of embodiment 54, wherein the antibody comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 142.

56) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 130, 27, 28, 133 and 132, respectively.

57) The antibody of embodiment 56, wherein the antibody comprises VH of SEQ ID NO: 138 and VL of SEQ ID NO: 143.

58) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 134, 27, 28, 29 and 30, respectively.

59) The antibody of embodiment 58, wherein the antibody comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 167.

60) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 135, 27, 28, 29 and 136, respectively.

61) The antibody of embodiment 60, wherein the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 144.

62) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 135, 27, 28, 29 and 30, respectively.

63) The antibody of embodiment 62, wherein the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 167.

64) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 135, 27, 131, 29 and 132, respectively.

65) The antibody of embodiment 64, wherein the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 142.

66) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 135, 27, 28, 133 and 132, respectively.

67) The antibody of embodiment 66, wherein the antibody comprises VH of SEQ ID NO: 140 and VL of SEQ ID NO: 143.

68) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 134, 27, 28, 29 and 136, respectively.

69) The antibody of embodiment 68, wherein the antibody comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 144.

70) An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 134, 27, 131, 29 and 132, respectively.

71) The antibody of embodiment 70, wherein the antibody comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 142.

72) Isolated recombinant antibody to PSMA or its antigen-binding fragment, with

- 62 046641 containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 133 and 132, respectively.

73) The antibody of embodiment 72, wherein the antibody comprises VH of SEQ ID NO: 139 and VL of SEQ ID NO: 143.

74) An isolated recombinant anti-PSMA antibody or antigen binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 25, 137, 27, 28, 133 and 132, respectively.

75) The antibody of embodiment 74, wherein the antibody comprises VH of SEQ ID NO: 141 and VL of SEQ ID NO: 143.

76) The antibody of any one of embodiments 1-75, which is human or humanized.

77) The antibody of embodiment 76, wherein the antibody is of the IgG4 or IgG1 isotype.

78) The antibody of embodiment 77, comprising one, two, three, four, five, six, seven, eight, nine, or ten substitutions in the Fc of the antibody.

79) The antibody of embodiment 77, comprising:

a) replacement of L234A, L235A, G237A, P238S, H268A, A330S and P331S;

b) replacements V234A, G237A, P238S, H268A, V309L, A330S and P331S;

c) replacements F234A, L235A, G237A, P238S and Q268A;

d) replacement of L234A, L235A or L234A and L235A;

e) replacement of F234A, L235A or F234A and L235A; or

f) replacement of V234A, with the numbering of the residues corresponding to the EU catalogue.

80) The antibody of embodiment 79 containing the substitutions S228P, F234A and L235A, with the residue numbering corresponding to the EU catalogue.

81) The antibody of any one of embodiments 1-80, which is bispecific.

82) The antibody of embodiment 76 that specifically binds PSMA and specifically binds CD3, CD5, CD28, CD16, CD16A, CD25, CD38, CD44, CD56, CD69, CD94, CD335 (NKp46), CD336, (NKp44), CD337 ( NKp30), NKp80, NKG2C and NKG2D, DNAM, NCR, CD18, CD89, CD18, CD32, CD64, CD64 and CD35.

83) A pharmaceutical composition comprising the antibody of embodiment 1-82 and a pharmaceutically acceptable carrier.

84) A polynucleotide encoding a VH antibody of embodiment 15, a VL antibody of embodiment 16, or a VH antibody and a VL antibody of embodiment 15 or 16.

85) A polynucleotide encoding a VH antibody, a VL antibody, or a VH antibody and a VL antibody according to any one of embodiments 28-75.

86) A vector containing the polynucleotide of embodiment 84.

87) A vector containing the polynucleotide of embodiment 85.

88) A host cell containing the vector of embodiment 86.

89) A host cell containing the vector of embodiment 87.

90) The method for producing an antibody according to Embodiment 1, comprising culturing a host cell according to Embodiment 89 under antibody expression conditions and isolating the antibody produced by the host cell.

91) A method of treating a cancer in a subject, comprising administering a therapeutically effective amount of the isolated antibody of any one of embodiments 1-82 to a subject in need thereof for a time sufficient to treat the cancer.

92) The method according to embodiment 91, wherein the malignancy is a solid tumor, a malignant tumor, or tumor neoplasms.

93) The method of embodiment 92, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, clear cell kidney cancer, bladder cancer, lung cancer, squamous cell cancer, glioma, breast cancer, kidney cancer, neovascular disorder, clear cell renal cell carcinoma (CCRC), pancreatic cancer, kidney cancer, urothelial cancer, or liver adenocarcinoma.

94) The method of embodiment 93, wherein the prostate cancer is refractory prostate cancer, prostate intraepithelial neoplasia, androgen-independent prostate cancer, malignant prostate cancer.

95) The method according to any one of claims 90-94, wherein the antibody is administered in combination with a second therapeutic agent.

96) The method of embodiment 95, wherein the second therapeutic agent is a conventional drug for treating a solid tumor or malignant tumor or tumor neoplasm.

97) The method of embodiment 96, wherein the second therapeutic agent is

- 63 046641 is a hormone inhibitor, microtubule antimicrobial agent, kinase inhibitor, immunomodulatory agent, topoisomerase inhibitor, antimetabolite, mitosis inhibitor, alkylating agent, anthracycline, vinca alkaloid, intercalating agent, agent capable of influencing the signal transduction pathway, apoptosis stimulating agent, proteasome inhibitor or irradiation.

98) The method of embodiment 96, wherein the second therapeutic agent is a vaccine.

99) The method of embodiment 98, wherein the vaccine is a polypeptide or fragment thereof, or DNA or RNA encoding a polypeptide or fragment thereof expressed on tumor cells.

100) The method of embodiment 99, wherein the polypeptide is PSMA, mesothelin, EGFR or EGFR RvIII.

101) The method of embodiment 95, wherein the second therapeutic agent is administered simultaneously, sequentially, or separately.

102) The method of any one of embodiments 91-101, wherein the subject has received or is receiving radiation therapy treatment.

103) The method according to any one of claims 91-101, in which the subject has undergone or will undergo surgery.

104) The method of any one of embodiments 46-58, wherein the isolated antibody comprises VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67.

105) The antibody of any one of embodiments 1-82 for use in therapy.

106) Binding of an anti-idiotypic antibody to the antibody of any one of embodiments 1-82.

107) A bispecific antibody comprising a first domain that specifically binds PSMA and a second domain that specifically binds CD3, the first domain comprising:

a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 31, 42, 43, 11, 12 and 13, respectively;

b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 14, 15, 16, 17, 18 and 19, respectively;

c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 26, 27, 28, 29 and 30, respectively;

d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 36, 37, 38, 39, 40 and 41, respectively;

e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 122, 123, 124, 23, 12 and 24, respectively;

f) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 8, 9, 10, 11, 12, and 13, respectively;

g) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 14, 15, 16, 17, 18 and 19, respectively;

h) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 20, 21, 22, 23, 12 and 24, respectively;

i) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 31, 32, 33, 34, 12, and 35, respectively;

j) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 31, 44, 45, 46, 29 and 47, respectively;

k) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 36, 37, 48, 49, 50 and 51, respectively;

l) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 53, 54, 55, 23, 12, and 35, respectively;

m) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 130, 27, 28, 29 and 30, respectively;

n) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 130, 27, 131, 29 and 132, respectively;

o) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 130, 27, 28, 133, and 132, respectively;

p) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 29 and 30, respectively;

q) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 29 and 136, respectively;

r) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 29 and 30, respectively;

s) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 135, 27, 131, 29 and 132, respectively;

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t) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 133, and 132, respectively;

u) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 29 and 136, respectively;

v) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 134, 27, 131, 29 and 132, respectively;

w) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 133, and 132, respectively; or

x) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 137, 27, 28, 133, and 132, respectively.

108) The bispecific antibody of embodiment 107, wherein the first domain comprises:

a) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 31, 42, 43, 11, 12 or 13, respectively, and VH with SEQ ID NO: 74 and VL with SEQ ID NO: 61;

b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 14, 15, 16, 17, 18 or 19, respectively, and VH with SEQ ID NO: 62 and VL with SEQ ID NO: 63;

c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 26, 27, 28, 29 or 30, respectively, and VH with SEQ ID NO: 66 and VL with SEQ ID NO: 67;

d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 36, 37, 38, 39, 40 or 41, respectively, and VH with SEQ ID NO: 72 and VL with SEQ ID NO: 73;

e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 122, 123, 124, 23, 12 or 24, respectively, and VH with SEQ ID NO: 160 and VL with SEQ ID NO: 65;

f) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 8, 9, 10, 11, 12, and 13, respectively, and VH with SEQ ID NO: 60 and VL with SEQ ID NO: 61;

g) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 14, 15, 16, 17, 18 and 19, respectively, and VH with SEQ ID NO: 62 and VL with SEQ ID NO: 63;

h) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 20, 21, 22, 23, 12 and 24, respectively, and VH with SEQ ID NO: 64 and VL with SEQ ID NO: 65;

i) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 31, 32, 33, 34, 12, and 35, respectively, and VH with SEQ ID NO: 70 and VL with SEQ ID NO: 71;

j) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 31, 44, 45, 46, 29 and 47, respectively, and VH with SEQ ID NO: 75 and VL with SEQ ID NO: 76;

k) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 36, 37, 48, 49, 50 and 51, respectively, and VH with SEQ ID NO: 77 and VL with SEQ ID NO: 78;

1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 53, 54, 55, 23, 12, and 35, respectively, and VH with SEQ ID NO: 68 and VL with SEQ ID NO: 69;

m) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 130, 27, 28, 29, and 30, respectively, and VH with SEQ ID NO: 138 and VL with SEQ ID NO: 67;

n) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 130, 27, 131, 29 or 132, respectively, and VH with SEQ ID NO: 138 and VL with SEQ ID NO: 142;

o) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 130, 27, 28, 133, and 132, respectively, and VH with SEQ ID NO: 138 and VL with SEQ ID NO: 143;

p) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 29, and 30, respectively, and VH with SEQ ID NO: 139 and VL with SEQ ID NO: 167;

q) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 29, and 136, respectively, and VH with SEQ ID NO: 140 and VL with SEQ ID NO: 144;

r) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 29, and 30, respectively, and VH with SEQ ID NO: 140 and VL with SEQ ID NO: 167;

s) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 135, 27, 131, 29, and 132, respectively, and VH with SEQ ID NO: 140 and VL with SEQ ID NO: 142;

t) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 135, 27, 28, 133, and 132, respectively, and VH with SEQ ID NO: 140 and VL with SEQ ID NO: 143;

u) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 29 and 136, respectively; and VH having SEQ ID NO: 139 and VL having SEQ ID NO: 144;

v) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 134, 27, 131, 29, and 132, respectively, and VH with SEQ ID NO: 139 and VL with SEQ ID NO: 142;

w) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 134, 27, 28, 133, and 132, respectively, and VH with SEQ ID NO: 139 and VL with SEQ ID NO: 143; or

x) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 with SEQ ID NO: 25, 137, 27, 28, 133, and 132, respectively, and VH with SEQ ID NO: 141 and VL with SEQ ID NO: 143.

109) An isolated anti-PSMA/CD3 bispecific antibody containing a first domain that (i) binds to cells expressing recombinant Pan troglodytes PSMA, wherein cell binding is measured by flow cytometry and (ii) binds to the extracellular domain of recom

- 65 046641 binant PSMA Pan troglodytes (SEQ ID NO: 4) with an affinity of about 30 nM or less, the affinity being measured using a Proteon surface plasmon resonance assay specifically binding PSMA and a second domain specifically binding CD3.

110) The anti-PSMA x CD3 bispecific antibody of embodiment 109, wherein the antibody

a) binds LNCaP cells with an estimated _{EC50 of} 20 nM or less and binds HEK cells expressing Macaca fascicularis PSMA with an estimated EC5 of ₀ 40 nM or less, with a difference in estimated EC50 between binding of LNCaP cells and binding of HEK cells expressing PSMA Macaca fascicularis is less than 5 times, and the calculated EC ₅₀ is measured in a whole cell binding assay at 0°C using flow cytometry,

b) binds the ECD of recombinant PSMA from humans (SEQ ID NO: 7), Pan troglodytes (SEQ ID NO: 4) and Macaca fascicularis (SEQ ID NO: 5) with an equilibrium dissociation constant (KD) of 12 nM or less, wherein KD measured by surface plasmon resonance analysis using the ProteOn XPR36 system at +25°C;

c) demonstrates T cell-mediated killing of LNCaP cells, C42 cells, HEK cells expressing human PSMA, or HEK cells expressing Macaca fascicularis PSMA, with T cell-mediated killing measured by chromium-51 release or caspase 3 activation assay /7, or

d) recognizes a conformational epitope, wherein the epitope consists of residues I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307 and K324-P326 of human PSMA (SEQ ID NO: 3).

111) The anti-PSMA x CD3 bispecific antibody of embodiment 109, wherein the antibody binds to T cells.

112) The PSMA x CD3 bispecific antibody of embodiment 109, wherein the first domain comprises

a) heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3 with SEQ ID NOs: 14, 15 and 16, respectively; and light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 with SEQ ID NOs: 17, 18 and 19, respectively;

b) HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 20, 21 and 22, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 23, 12 and 24, respectively;

c) HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 25, 26 and 27, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 28, 29 and 30, respectively;

d) HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 31, 44 and 45, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 46, 29 and 47, respectively;

e) HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 31, 42 and 43, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 11, 12 and 13, respectively; or

f) HCDR1, HCDR2 and HCDR3 with SEQ ID NOs: 122, 123 and 124, respectively, and LCDR1, LCDR2 and LCDR3 with SEQ ID NOs: 23, 12 and 24, respectively.

113) The anti-PSMA x CD3 bispecific antibody of embodiment 109, wherein the first domain contains HCDR1, HCDR2 and HCDR3 with

a) SEQ ID NO: 14, 15 and 16 respectively;

b) SEQ ID NO: 20, 21 and 22 respectively;

c) SEQ ID NO: 25, 26 and 27 respectively;

d) SEQ ID NO: 31, 44 and 45 respectively;

e) SEQ ID NO: 31, 42 and 43 respectively; or

f) SEQ ID NO: 122, 123 and 124 respectively.

114) The anti-PSMA x CD3 bispecific antibody of embodiment 109, wherein the first domain contains LCDR1, LCDR2 and LCDR3 with

a) SEQ ID NO: 17, 18 and 19 respectively;

b) SEQ ID NO: 23, 12 and 24 respectively;

c) SEQ ID NO: 28, 29 and 30 respectively;

d) SEQ ID NO: 46, 29 and 47 respectively;

e) SEQ ID NO: 11, 12 and 13 respectively; or

f) SEQ ID NO: 23, 12 and 24 respectively.

115) The anti-PSMA x CD3 bispecific antibody of embodiment 109, wherein

a) the first domain contains a heavy chain variable region (VH) of SEQ ID NO: 62 and a light chain variable region (VL) of SEQ ID NO: 63, and the second domain contains a VH of SEQ ID NO: 104 and a VL of SEQ ID NO: : 105

b) the first domain contains VH of SEQ ID NO: 64 and VL of SEQ ID NO: 65, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105;

c) the first domain contains VH of SEQ ID NO: 66 and VL of SEQ ID NO: 67, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105;

d) the first domain contains VH of SEQ ID NO: 75 and VL of SEQ ID NO: 76, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105;

- 66 046641

e) the first domain contains VH of SEQ ID NO: 74 and VL of SEQ ID NO: 61, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105;

f) the first domain contains VH of SEQ ID NO: 160 and VL of SEQ ID NO: 65, and the second domain contains VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105.

116) The anti-PSMA x CD3 bispecific antibody of embodiment 109, comprising a first heavy chain (HC1), a first light chain (LC1), a second heavy chain (HC2) and a second light chain (LC2), wherein HC1 and LC1 contain amino acid sequences with

a) SEQ ID NO: 84 and 85 respectively;

b) SEQ ID NO: 86 and 87 respectively;

c) SEQ ID NO: 88 and 89 respectively;

d) SEQ ID NO: 125 and 91 respectively;

e) SEQ ID NO: 94 and 95 respectively; or

f) SEQ ID NO: 96 and 83 respectively.

117) The anti-PSMA x CD3 bispecific antibody of embodiment 116, wherein HC2 and LC2 comprise SEQ ID NOs: 110 and 111, respectively.

118) The anti-PSMA CD3 bispecific antibody of embodiment 109, comprising HC1, LCI, HC2 and LC2 with

a) SEQ ID NO: 84, 85, 110 and 111 respectively;

b) SEQ ID NO: 86, 87, 110 and 111 respectively;

c) SEQ ID NO: 88, 89, 110, 111 respectively;

d) SEQ ID NO: 125, 91, 110 and 111 respectively;

e) SEQ ID NO: 94, 95, 110 and 111 respectively;

f) SEQ ID NO: 96, 83, 110 and 111 respectively.

119) The anti-PSMA x CD3 bispecific antibody of any one of embodiments 109-118, which is human or humanized.

120) The anti-PSMA x CD3 bispecific antibody of embodiment 119, which is an IgG1, IgG2, IgG3, or IgG4 isotype.

121) The anti-PSMA x CD3 bispecific antibody of embodiment 120, wherein the antibody is of the IgG1 or IgG4 isotype.

122) The anti-PSMA x CD3 bispecific antibody of any one of embodiments 120 or 121, comprising one, two, three, four, five, six, seven, eight, nine, or ten substitutions in the Fc of the antibody.

123) The anti-PSMA x CD3 bispecific antibody of embodiment 121, comprising:

a) replacement of L234A, L235A, G237A, P238S, H268A, A330S and P331S;

b) replacements V234A, G237A, P238S, H268A, V309L, A330S and P331S;

c) replacements F234A, L235A, G237A, P238S and Q268A;

d) replacement of L234A, L235A or L234A and L235A;

e) replacement of F234A, L235A or F234A and L235A;

f) replacement of V234A; or

g) substitutions S228P, F234A and L235A, with the numbering of residues corresponding to the EU index.

124) The anti-PSMA x CD3 bispecific antibody of any one of embodiments 109-123, comprising at least one substitution in the constant domain of the CH3 antibody.

125) The anti-PSMA x CD3 bispecific antibody of embodiment 124, wherein the substitution in the constant domain of the CH3 antibody is a 409 R, F405L, or F405L/R409K substitution, wherein the residue numbering corresponds to the EU index.

126) The anti-PSMA x CD3 bispecific antibody of embodiment 124, wherein the antibody comprises

a) substitution F405L in HC1 and substitution 409 R in HC2, and the antibody belongs to the IgG1 isotype; b) substitutions V234A, G237A, P238S, H268A, V309L, A330S, P331S and F405L in HC1 and V234A, G237A, P238S, H268A, V309L , A330S, P331S and 409 R in HC2, and the antibody is of the IgG1 isotype; or

c) substitution S228P in HC1 and substitution S228P, F405L and R409K in HC2, the antibody being of the IgG4 isotype.

127) A pharmaceutical composition comprising the anti-PSMA x CD3 bispecific antibody of embodiment 109-126 and a pharmaceutically acceptable carrier.

128) A polynucleotide encoding an anti-PSMA x CD3 bispecific antibody in HC1, LC1, HC2, or LC2 of embodiment 118.

129) A vector containing a polynucleotide encoding HC1, LC1, HC2, LC2, HC1 and LC1 or HC2 and LC2 according to embodiment 128.

130) An isolated host cell containing the vector of embodiment 129.

131) A method for producing an anti-PSMA x CD3 bispecific antibody according to embodiment 118, comprising culturing a host cell according to embodiment 130 under antibody expression conditions and isolating and purifying the anti-PSMA x CD3 bispecific antibody produced by the host cell.

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132) The method of producing a bispecific anti-PSMA x CD3 antibody according to embodiment 118, comprising:

a) combining a monospecific divalent antibody PSMA having two identical HC1 and two identical LC1, and a monospecific divalent antibody CD3 having two identical HC2 and two identical LC2, in a mixture with a molar ratio of about 1:1;

b) introducing a reducing agent into the mixture;

c) incubating the mixture for about ninety minutes to about six hours;

d) removing the reducing agent; And

e) purifying a CD3 x PSMA bispecific antibody that contains HC1, LC1, HC2 and LC2.

133) The method of embodiment 132, wherein the reducing agent is 2-mercaptoethanolamine (2-MEA).

134) The method of embodiment 133, in which

h) 2-MEA is present at a concentration of from about 25 mM to about 75 mM; And

i) incubation is carried out at a temperature of from about 25°C to about 37°C.

135) A method of treating a cancer in a subject, comprising administering a therapeutically effective amount of an isolated anti-CD3 x PSMA bispecific antibody according to any one of embodiments 109-126 to a subject in need thereof for a time sufficient to treat the cancer.

136) The method according to embodiment 135, wherein the malignancy is a solid tumor, a malignant tumor, or tumor neovasculature.

137) The method of embodiment 136, wherein the solid tumor is prostate cancer, colorectal cancer, gastric cancer, clear cell kidney cancer, bladder cancer, lung cancer, squamous cell cancer, glioma, breast cancer, kidney cancer, neovascular disorder, clear cell renal cell carcinoma (CCRC), pancreatic cancer, kidney cancer, urothelial cancer, or liver adenocarcinoma.

138) The method of embodiment 137, wherein the prostate cancer is refractory prostate cancer, prostate intraepithelial neoplasia, androgen-independent prostate cancer, malignant prostate cancer.

139) The method of any one of embodiments 135-138, wherein the antibody is administered in combination with a second therapeutic agent.

140) The method of embodiment 139, wherein the second therapeutic agent is a conventional drug for treating a solid tumor or malignant tumor or neovascularization of a tumor.

141) The method of embodiment 139, wherein the second therapeutic agent is a hormone inhibitor, a microtubule antimicrobial agent, a topoisomerase inhibitor, an antimetabolite, a mitosis inhibitor, an alkylating agent, an anthracycline, a vinca alkaloid, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an apoptosis-promoting agent, a proteasome inhibitor, or radiation.

142) The method of embodiment 139, wherein the second therapeutic agent is a vaccine.

143) The method of embodiment 142, wherein the vaccine is a polypeptide or fragment thereof, or DNA or RNA encoding a polypeptide or fragment thereof expressed on tumor cells.

144) The method of embodiment 143, wherein the polypeptide is PSMA, mesothelin, EGFR or EGFR RvIII.

145) The method of embodiment 139, wherein the second therapeutic agent is administered simultaneously, sequentially, or separately.

146) The method of any one of embodiments 135-145, wherein the subject has received or is receiving radiation therapy treatment.

147) The method according to any one of claims 135-145, in which the subject has undergone or will undergo surgery.

148) The method of any one of embodiments 135-145, wherein the first domain of the anti-PSMA x CD3 bispecific antibody comprises VH of SEQ ID NO: 66 and VL or SEQ ID NO: 67, and the second domain of the anti-PSMA x CD3 bispecific antibody comprises VH with SEQ ID NO: 104 and VL with SEQ ID NO: 105.

149) The antibody of any one of embodiments 109-126 for use in therapy.

150) Binding of an anti-idiotypic antibody to the antibody of any one of embodiments 109-126.

Example 1. Materials.

Establishment of PSMA cell lines.

Expression vectors representing full-length chimpanzee PSMA (H2Q3K5_PANTR, SEQ

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ID NO: 1) or full-length cynomolgus PSMA (EHH56646.1, SEQ ID NO: 2) were generated for use as screening tools in the evaluation of anti-PSMA lead antibodies using a proprietary expression vector with a CMV promoter using standard molecular biology techniques . Vectors were transiently transfected into HEK293F cells in suspension using standard methods. Transfected 293F suspension cells were placed in growth medium with serum to become adherent and selected for stable plasmid integration. Single cell populations were selected by serial dilutions and PSMA surface receptor expression was quantified by FACS using an affinity purified rabbit polycolonal antibody (PSMAL antibody (Centre) (catalog no. OAAB02483, Aviva Systems Biology) as the R-conjugated primary antibody -PE anti-rabbit secondary antibody (catalog no. 111-116-144, Jackson ImmunoResearch Laboratories, Inc.) and rabbit polyclonal IgG (catalog no. SC-532, Santa Cruz Biotechnology) as isotype control).

To select for PSMA-positive cells, cell lines expressing human PSMA were generated using a lentivirus (Genecopoeia, Cat. No. EX-G0050-Lv105-10) containing full-length human PSMA (FOLH1_HUMAN, SEQ ID NO: 3) and puromycin. PSMA-negative HEK293F (ATCC) cells were transduced with lentiviral particles to overexpress human PSMA. Following transduction, cells positively expressing PSMA and the resistance marker were selected by pooled cell treatment, grown in DMEM+10% HI FBS (Life Technologies), and supplemented with varying concentrations of puromycin (Life Technologies).

In addition to HEK-derived cell lines, several commercial cell lines were used for phage panning, binding, and cell toxicity assays. LNCaP clone FGC cells (ATCC Cat. No. CRL-1740) are commercially available human prostate cancer cell lines. C4-2B cells were originally generated at MD Anderson, and are derived from LNCaP FGCs grown in vivo and metastasized to the bone marrow (Thalmann, et al., 1994, Cancer Research, 54, 2577-81).

Preparation of soluble ECD protein PSMA.

Recombinant chimpanzee PSMA extracellular domain (ECD) protein (chimpanzee PSMA ECD, SEQ ID NO: 4) was generated for panning and evaluation of anti-PSMA leader antibodies using a proprietary expression vector with a CMV promoter using standard molecular biology techniques. Chimpanzee PSMA ECD gene fragment (amino acids 44-750 from SEQ ID NO: 1) with N-terminal signal sequence (SEQ ID NO: 56), N-terminal avidin tag (SEQ ID NO: 57) and 6-histidine tags (SEQ ID NO : 58) was cloned using a proprietary expression vector with a CMV promoter using standard molecular biology techniques and transiently expressed in 293Expi cells (Invitrogen). cDNA was obtained using gene synthesis methods (US Patent No. 6670127; US Patent No. 6,521,427). Supernatants were collected and purified by centrifugation. Proteins were purified using a two-step purification method: 1) IMAC purification using a HisTrap HP column (GE Healthcare) and 2) size exclusion purification (Superdex 200, Ge Healthcare) where the elution buffer is Dulbecco's phosphate-buffered saline, calcium, magnesium ( Thermofisher, No. 14040), containing 0.5 mM ZnCl ₂ to stabilize PSMA dimerization. Fractions containing the protein of interest were pooled and protein concentration was determined by A280. This material was used for binding and affinity measurements and designated PSMG8.

Chimpanzee PSMA ECDs were also biotinylated for panning. The BirA plasmid, which was cotransfected into mammalian cells into biotinylated proteins containing an avidin tag, was produced independently. The BirA coding region (SEQ ID NO: 59) was fused with a mouse IgG heavy chain signal peptide (SEQ ID NO: 80) and an ER retention signal (KDEL (KDEL is described as SEQ ID NO: 156) added to the C terminus to produce BirA (SEQ ID NO: 112) The constructed gene was cloned into an expression vector under the control of the CMV promoter. To obtain the biotinylated PSMA antigen, PSMA plasmid DNA was added in 4-fold excess (w/w) to the BirA plasmid in the transfection mixture.

Biotinylation of the chimpanzee PSMA ECD protein was performed using an avidin tag by cotransfecting a BirA expression construct, and the resulting secreted protein was purified using a two-step purification process: 1) IMAC purification using a HisTrap HP column (GE Healthcare) and 2) size exclusion purification (Superdex 200, Ge Healthcare), where the elution buffer is Dulbecco's phosphate buffered saline calcium magnesium (Thermofisher, no. 14040) containing 0.5 mM ZnCl ₂ to stabilize PSMA dimerization. The protein was tested for endotoxin before being used in phage panning studies.

Recombinant PSMA extracellular domain (ECD) protein (cynomolgus PSMA ECD, SEQ ID NO: 5), corresponding to amino acids 44-750 SEQ ID NO: 2 with N-terminal signal (SEQ ID NO: 56), N-terminal avidin (SEQ ID NO: 57) and 6-histidine (SEQ ID NO: 58) tags were cloned and expressed as previously described for chimpanzee PSMA ECD. Biotinylation of the cynomolgus PSMA ECD protein was performed using an avidin tag by cotransfecting a BirA expression construct, and the resulting secreted protein was purified by a two-step column purification

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IMAC HisTrap HP (GE Healthcare) and MonoAvidin columns. The protein was tested for endotoxin before being used in phage panning studies. This material was also used for binding and affinity measurements and designated PSMG1.

A second recombinant cynomolgus PSMA ECD protein (cynomolgus Fc PSMA, SEQ ID NO: 6) with IgG1 Fc (SEQ ID NO: 81) was cloned and expressed using a proprietary expression vector with a CMV promoter using standard molecular biology techniques. Cynomolgus Fc PSMA protein was transiently expressed in 293HEK-expi cells. The product of transient transfection of PSMG3 in HEK293.Expi cells was collected 5 days after transfection, clarified by centrifugation (30 min, 6000 rpm) and filtered (0.2 μm polyethersulfone membrane, Corning). The relative amount of IgG was determined using an Octet instrument (ForteBio) using purified known IgG (same isotype) added to the spent medium to generate a standard curve.

The clarified supernatant with cynomolgus PSMA Fc was loaded onto an equilibrated (dPBS, pH 7.2) HiTrap MabSelect Sure Protein A column (GE Healthcare) at a relative concentration of ∼30 mg protein per ml resin. After loading, the column was washed in dPBS at pH 7.2, and the protein was eluted using 10 column volumes of 0.1 M sodium acetate, pH 3.5. Peak fractions were pooled, neutralized with 2 M Tris, pH 7, and filtered (0.2 μm). The neutralized protein sample was dialyzed against 3 changes of dPBS containing Ca2+, Mg2+ and 0.5 mM ZnCl2, pH 7.2, overnight at 4°C. The next day, the sample was removed from dialysis, filtered (0.2 μm), and protein concentration was determined by absorbance at 280 nm on a BioTek SynergyHTTM spectrophotometer. The quality of the purified proteins was assessed by SDS-PAGE electrophoresis and analytical size exclusion HPLC (Dionex HPLC system). Endotoxin levels were measured using the LAL test (Pyrotell-T, Associates of Cape Cod). Purified proteins were stored at 4°C.

Recombinant protein human PSMA extracellular domain (ECD) (Human PSMA ECD, SEQ ID NO: 7), corresponding to amino acids 44-750 from SEQ ID NO: 3 with N-terminal avidin and 6-histidine tags (SEQ ID NO: 58) was cloned, expressed and purified as previously described for chimpanzee and cynomolgus monkey PSMA ECD proteins.

Example 2. Identification of Fab to chimpanzee and human PSMA.

Panning with recombinant protein. Solution panning of de novo human Fab-pIX libraries [Shi, L., et al J Mol Biol, 2010. 397(2): p. 385-396. WO 2009/085462], consisting of VH1-69, 3-23 and 5-51 heavy chain libraries paired with four human germline VL gene libraries (A27, B3, L6, 012), was performed using an alternating panning approach with one cycle of phage capture on streptavidin beads (Invitrogen, cat. no. 112.05D, lot no. 62992920) coated with biotinylated chimpanzee PSMA ECD according to the manufacturer's protocol, followed by phage capture on ProtG beads (Invitrogen, cat. no. 10003D) coated with Java PSMA macaque Fc according to the manufacturer's protocol, followed by phage capture on Sera-mag Double Speed magnetic neutravidin beads (Thermo, cat. no. 7815-2104-011150) coated with biotinylated chimpanzee PSMA ECD according to the manufacturer's protocol. As a result of panning, two selected compounds were obtained: PSMB18 and PSMB25.

Whole-cell Fab panning for PSMA. Additional panning experiments were performed on whole cells using the Round 1 output of the chimpanzee ECD panning experiments described above or only the resulting de novo phage libraries as input. Briefly, phage was produced by helper phage infection and concentrated by PEG/NaCl precipitation according to standard protocols known in the art. Phage libraries were prepurified on untransfected parent HEK293F cells overnight at 4°C with gentle rocking. After PEG/NaCl precipitation, precleared libraries were incubated with PSMA-expressing HEK293 cells or LNCAP cells with gentle rocking for 2 h at 4°C. Removal of unbound phage and isolation of phage-bound cells was performed using a Ficoll gradient, and after several washing steps, cells bearing bound phage were incubated with 1 ml of E. coli TG-1 culture at 37°C for 30 minutes without shaking. The resulting mixture was plated on lysogeny broth plates containing carbenicillin and 1% glucose and grown overnight at 37°C. The process was then repeated for subsequent rounds of panning.

Conversion of phage Fab-pIX to Fab-His to produce E. coli supernatants. The resulting selected compounds from Fab-pIX phages were converted to Fab-His using a standard procedure. Plasmid DNA was isolated from E. coli used in phage panning (Plasmid Plus Maxi Kit, Qiagen, cat. no. 12963) and digested with restriction enzymes NheI/SpeI. The resulting fragments are 5400 and 100 bp in size. separated on an agarose gel with a concentration of 0.8% and a fragment of 5400 bp. gel purified (MinElute PCR Purification Kit, Qiagen, cat. no. 28006). The purified band of 5400 bp. was self-ligated using T4 ligase, and the resulting product (encoding the Fab-his fusion) was again transformed into E. coli strain TG-1 and cloned. Fab-His supernatants were generated from clones by overnight induction of cultures with 1 mM IPTG. After centrifugation of the sample

- 70 046641 culture incubated overnight, the clarified supernatants were ready for use in subsequent analyses. To determine the relative expression levels of the different Fab-his supernatants, kappa ELISA (Southern Biotech, cat. no. 2061-05) was performed on serially diluted supernatants. All clones tested showed similar Fab-his expression (data not shown).

Cellular binding of Fab-his hybrids from E. coli. A cell-based binding assay was developed to evaluate the binding ability of individual Fab-his hybrids from E. coli supernatants to cells expressing PSMA. Individual Fab clones were isolated from round 3 of all panning experiments after pIX excision. Fab clones were tested for binding to HEK cells expressing chimpanzee and cynomolgus PSMA, and to human PSMA on LNCaP cells. Briefly, PSMA-expressing cells were aliquoted into a V-bottom plate (CoStar 3357) at a density of 200,000 per well and incubated with (100 μl) Fab-expressing supernatants for 1 h on ice. Cells were washed twice with PBS containing 2% FBS and stained with RPE-conjugated mouse anti-human kappa light chain antibody (Life Technologies, cat. no. MH10514) for 1 hour on ice. Cells were washed twice with PBS containing 2% FBS and resuspended in 100 μl of the same wash buffer. The plates were read on a BD FACS flow cytometer. FACS data were analyzed in FlowJo software by gating a normal cell population in real time using forward and side scatter and then analyzing cells within this gate for PE staining. Average fluorescence intensity (AFI) was calculated and exported to Microsoft Excel. Fab clones that exhibited >3-fold background binding for all three PSMA species (cynomolgus, chimpanzee, and human) and did not exhibit binding to the HEK293 cell line were labeled as tentatively positive. The Fab was sequenced and transferred for cloning into a mammalian expression vector for rescreening. True positives were selected from the binding of Fab supernatants expressed by mammalian cells to cell lines expressing PSMA.

Preparation of Mammalian Fabs. In-Fusion HD Cloning (ClonTech, cat. no. 638918) was used to convert E. coli Fab into mammalian-expressed Fab according to the manufacturer's protocol. Briefly, the nucleotide sequences of clones that have passed the primary screen and are to be translated into mammalian Fab format are loaded into InFu Primer Finder v1.2.3 (in-house developed software), which generates a list of isotype-specific PCR primers used to generate the PCR. -fragments for In-Fusion cloning into the huKappa_muIgGSP and huG1 Fab expression vectors. These vectors are proprietary vectors with CMV promoters based on pcDNA3.1. Following the In-fusion process, E. coli clones were isolated, sequence verified, and transfected into HEK293 cells using standard protocols. Mammalian anti-PSMA Fab to confirm binding to PSMA-expressing cell lines was prepared by collecting 20 ml of supernatants 5 days after transfection.

Re-screening of selected variants from whole-cell panning in mammalian cell supernatant format. Confirmation of the presence of Fab supernatants expressed by mammalian cells was performed using a whole cell binding assay described previously. Fab binding to chimpanzee, cynomolgus and human PSMA cells (LNCaP) was examined and reverse screened for lack of binding to the parental HEK cell line. In table 3 shows the profile of selected compounds with respect to binding of mammalian Fab supernatant to PSMA-expressing cells. Many selected compounds from E. coli supernatants are not supported by proteins expressed in mammals. PSMB47 showed high binding to cynomolgus monkey PSMA-expressing cells and some binding to chimpanzee PSMA-expressing cells, but no binding to human PSMA-expressing LNCaP cells. PSMB55 showed a similar profile, but with some binding to LNCaP cells. PSMB68-PSMB79 binds to LNCaP cells but not to cells expressing chimpanzee or cynomolgus PSMA. Mammalian Fab supernatants PSMB51, PSMB55, and PSMB56 bound to all three cell lines. PSMB49, PSMB50 and PSMB53 show greater binding to chimpanzee or cynomolgus macaque cells. M58 showed negligible binding to chimpanzee and cynomolgus macaque cells.

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Table 3

Profile of Selected Mammalian Fab Protein Binding Compounds to PSMA Expressing Cells as Measured by Geo-SIF (Average Fluorescent Intensity) Measurement

Fab protein ID (DNA Fab ID) Cynomolgus macaque Chimpan ze LNCaP Original NEK PSMB10 (PSMM10) 244 81.6 - 248 PSMB11 (PSMM11) 19 6.6 - 8.14 PSMB12 (PSMM12) 31.6 8.05 - 12.6 PSMB13 (PSMM13) 57.8 18.2 - 50.5 PSMB14 (PSMM14) 32.6 13.1 - 22.2 PSMB15 (PSMM15) 40.4 18.5 - 38 PSMB16 (PSMM16) 175 220 - 6.39

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PSMB17 (PSMM17) 34.9 22.4 - 40.1 PSMB18 (PSMM18) 696 439 - 8.71 PSMB19 (PSMM19) 53.7 - 5.15 4.47 PSMB20 (PSMM20) 5.75 - 5.85 41.3 PSMB21 (PSMM21) 94.4 - 20.7 372 PSMB22 (PSMM22) 9.07 - 7.92 54.9 PSMB23 (PSMM23) 16.4 - 6.66 164 PSMB24 (PSMM24) 14.6 9.6 4.09 3.96 PSMB25 (PSMM25) 15.2 11.3 16.9 4.09 PSMB26 (PSMM26) 9.48 - 7.26 114 PSMB27 (PSMM27) 20 - 7.56 136 PSMB28 (PSMM28) 29.7 - 8.88 302 PSMB29 (PSMM29) 6.87 - 5.7 72.8 PSMB30 (PSMM30) 5.16 - 4.58 45 PSMB31 (PSMM31) 5.99 - - 25.5 PSMB32 (PSMM32) 4.81 - - 27.1 PSMB33 (PSMM33) 5.14 - - 40.1 PSMB34 (PSMM34) 17.9 - - 107 PSMB35 (PSMM35) 58.5 - - 231 PSMB36 (PSMM36) 5.05 - - 6.96 PSMB37 (PSMM37) 23.4 - - 178 PSMB38 (PSMM38) 4.05 - - 7.7 PSMB39 (PSMM39) 10.2 - - 166 PSMB40 (PSMM40) 66.9 - - 348 PSMB41 (PSMM41) 5.39 - - 12 PSMB42 (PSMM42) 7.35 - - 25.8

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PSMB43 (PSMM43) 8.73 - - 7.18 PSMB44 (PSMM44) 12.6 - - 48.9 PSMB45 (PSMM45) 22.4 - - 43.1 PSMB46 (PSMM46) 3.88 - - 5.29 PSMB47 (PSMM48) 101 25.5 3.46 2.85 PSMB48 (PSMM49) 2.72 3.18 2.68 2.72 PSMB49 (PSMM50) 51.6 22 3.22 3.48 PSMB51 (PSMM52) 285 231 41.5 2.68 PSMB52 (PSMM53) 39.2 6.89 2.67 2.56 PSMB53 (PSMM54) 27.6 17.8 4 2.6 PSMB54 (PSMM55) 2.7 2.75 2.65 2.79 PSMB55 (PSMM56) 226 180 17.2 2.58 PSMB56 (PSMM57) 95.6 34.7 24.5 2.52 PSMB57 (PSMM58) 19.8 AND 3.26 2.68 PSMB58 (PSMM59) 121 192 25.3 2.67 PSMB59 (PSMM60) 4.96 9.69 6.04 3 PSMB60 (PSMM61) 2.28 3.07 87.3 4.64 PSMB61 (PSMM62) 2.1 3.16 135 2.98 PSMB62 (PSMM63) 7.17 4.43 54.9 9.09 PSMB63 (PSMM64) 2.07 2.95 27 2.82 PSMB64 (PSMM65) 2.39 3.26 70.5 3.05 PSMB65 (PSMM66) 2.3 3.13 32.4 4.25 PSMB66 (PSMM67) 2.14 3 24.6 2.83 PSMB67 (PSMM68) 2.23 2.95 21 2.95 PSMB68 (PSMM69) 5.44 - 134 35.3 PSMB69 (PSMM70) 2.29 3.38 25.5 3.35

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PSMB70 (PSMM71) 2.22 3.49 15.5 3.26 PSMB71 (PSMM72) 2.54 4.4 18.5 3.07 PSMB72 (PSMM73) 2.13 3.53 227 3.02 PSMB73 (PSMM74) 2.97 4.13 125 11.1 PSMB74 (PSMM75) 120 - 178 132 PSMB75 (PSMM76) 2.99 3.04 173 7.89 PSMB76 (PSMM77) 3.75 3.99 138 3.95 PSMB77 (PSMM78) 4.68 3.96 144 4.71 PSMB78 (PSMM79) 25.2 - 378 24.4 PSMB79 (PSMM80) 38.4 - 512 157 PSMB80 (PSMM81) 19.6 18.6 20.9 6.61 PSMB81 (PSMM82) 2.63 2.06 4.07 2.69 PSMB82 (PSMM83) 2.79 2.23 4.11 2.76 PSMB83 (PSMM84) 2.59 2.28 4.09 2.74 PSMB84 (PSMM85) 750 729 192 3.15 PSMB85 (PSMM86) 2.84 2.59 2.33 3.24 PSMB86 (PSMM87) 224 176 31.7 2.82 PSMB87 (PSMM88) 2.63 2.27 4.23 2.91 PSMB88 (PSMM89) 37.7 29.7 30.3 7.6 PSMB89 (PSMM90) 27.1 27.3 53.2 39.5 PSMB90 (PSMM91) 26.7 24.7 47.1 36.4 PSMB91 (PSMM92) 8.97 6.16 13 6.63 PSMB92 (PSMM93) 20 16.5 57.1 50 PSMB93 (PSMM94) 5.13 9.62 2.5 3.66 PSMB94 (PSMM95) 5.12 2.67 2.22 3.57 PSMB95 (PSMM96) 8.9 8.82 13.4 11.4

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PSMB96 (PSMM97) 2.4 3.25 2.53 4.03 PSMB97 (PSMM98) 2.57 4.73 2.52 3.7 PSMB99 (PSMM100) 9.95 2.4 2.39 4.03 PSMB100 (PSMM101) 4.03 2.52 2.33 3.37 PSMBIOO(PSMMIOI) 3.5 2.86 2.48 4.57 PSMB101 (PSMM102) 5.49 3.18 2.23 3.33 PSMB102 (PSMM103) 2.4 2.42 2.16 3.2 PSMB103 (PSMM104) 3.52 3.26 2.58 4.44 PSMB104 (PSMM105) 2.15 2.5 2.34 3.95 PSMB105 (PSMM106) 2.03 2.39 2.18 3.39 PSMB106 (PSMM107) 2 2.4 2.27 3.59 PSMB107 (PSMM108) 2 2.47 2.33 3.49 PSMB108 (PSMM109) 2 2.58 2.28 3.46 PSMB109 (PSMM110) 321 326 34.9 6.H PSMB110 (PSMM111) 2.3 2.31 2.31 3.4 PSMB111 (PSMM112) 2.32 2.31 - 3.21 PSMB112 (PSMM113) 6.28 5.7 2.71 3.28 PSMB113 (PSMM114) 2.82 2.95 2.32 3.29 PSMB114 (PSMM115) 2.78 2.47 4.3 3.14 PSMB115 (PSMM116) 2.66 2.59 2.2 3.14 PSMB46 (PSMM117) 4.54 3.18 2.21 4.79 PSMB67 (PSMM118) 3.95 4.3 3 6.13 PSMB74 (PSMM119) 7.94 13 3.16 12.5 PSMB78 (PSMM120) 5.08 4.79 22.3 6.82 PSMB81 (PSMM121) 3.66 3.83 3.05 5.H PSMB82 (PSMM122) 15.1 28.4 10.8 24.3

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PSMB83 (PSMM123) 37.5 42.1 3.04 4.88 PSMB85 (PSMM124) 34.6 52.9 20.7 46.8 PSMB87 (PSMM125) 4.23 3.74 2.26 4.73 PSMB89 (PSMM126) 51.8 53.1 11.7 6.27 PSMB90 (PSMM127) 42.8 30.2 7.74 5.99 PSMB91 (PSMM128) 3.9 27.6 2.37 4.32 PSMB92 (PSMM129) 45.7 37.3 12.1 7.4 PSMB93 (PSMM130) 5.13 7.85 4,P 7.82 PSMB94 (PSMM131) 3.67 3.23 2.32 4.72 PSMB95 (PSMM132) 4.05 3.64 2.56 5.57 PSMB96 (PSMM133) 3.91 4.54 2.37 4.65 PSMB97 (PSMM134) 3.22 3.16 4.08 4.22 PSMB98 (PSMM135) 15.6 12.7 2.22 4.21 PSMB99 (PSMM136) 4.08 3.26 2.22 5.04 PSMB100 (PSMM137) 5.24 3.82 2.16 4.83 PSMB101 (PSMM138) 3.84 3.14 2.23 4.52 PSMB102 (PSMM139) 4.51 3.82 2.23 4.59 PSMB103 (PSMM140) 6.81 4.27 2.21 5.41 PSMB104 (PSMM141) 7.52 4.35 2.26 4.39 PSMB105 (PSMM142) 5.03 AND 2 4.87 7.28 PSMB106 (PSMM143) 3.87 3.8 2.73 4.9 PSMB107 (PSMM144) z, z 3.35 2.3 4.64 PSMB108 (PSMM145) 6.78 3.83 2.33 4.98 PSMB110 (PSMM146) 4.03 3.23 2.28 5.3 PSMB111 (PSMM147) 3.71 3.26 2.36 5,I PSMB112 (PSMM148) 4.54 3.26 2.26 4.86 PSMB113 (PSMM149) 84.3 104 51.7 94.2 PSMB114 (PSMM150) 3.31 3.26 2.21 5.14 PSMB115 (PSMM151) 3.55 3.43 2.3 4.21

Dose-response curves for Fabs expressed in mammals. After positive binding of mammalian-expressed Fab clones was confirmed as pure Fab supernatants to PSMA-expressing cell lines, supernatants were normalized for protein concentration using Octet or protein gel, and dose-response curves were generated to confirm PSMA binding using the protocol described previously . In fig. 1-3 show titration curves for selected

- 77 046641 compounds that demonstrated binding to all three PSMA-expressing cells. In fig. Figure 1 shows titration curves for PSMA compounds selected by phage panning and LNCaP cells. In fig. Figure 2 shows titration curves for phage-panned anti-PSMA compounds and HEK cells expressing chimpanzee PSMA. In fig. Figure 3 shows titration curves for phage-panned anti-PSMA compounds and HEK cells expressing cynomolgus PSMA. PSMG5 (cynomolgus PSMA, GenBank: EH56646.1) or PSMG9 (chimpanzee PSMA, NCBI reference sequence: XP_016777253.1) was cloned into a mammalian expression vector between HindII and EcoRI sites under the control of the CMV promoter to generate the cell line. The engineered DNA was transfected into 293 F cells using Lipofectamine LTX followed by geneticin selection to select for PSMA positive (PSMG5 or PSMG9) cells. After selection, cells were screened and sorted using anti-PSMA (Aviva, cat. no. OAAB02483-PSMG9) or anti-PSMA (PSMB18Janssen Innal) using FACS. FACS sorted PSMG5 clones 11, 23, 25 and 32 and PSMG9 clones 2, 10, 11, 12, 20 and 24 were selected and stored for screening. The sequences of PSMG5 and PSMG9 are presented below. The binding profiles of the selected compounds were compared between cell lines expressing different PSMA species. PSMB51 supernatant was used as a positive control in the experiments. Some selected compounds were deprioritized due to N-linked glycosylation sites in the CDR, binding to the PSMA-negative parental HEK cell line, or lack of binding to PSMA-positive cell lines. Eleven selected Fabs remained, and 10 selected compound sequences were cloned into human IgG4 PAA heavy chain constructs and used to generate anti-PSMA x CD3 bispecific antibodies. These selected compounds demonstrated cross-species binding within 3-fold of each other and were translated into a bispecific antibody format to test for redirection of T cells killing PSMA-positive targets.

Panning antigens for each selected compound are shown in Table. 4.

PSMG5 (SEQ Ш NO: 126)

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSSEATNITP

KHNMKAFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELTHY DVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPAGYENVSDIVPPFSAFSPQGMPEGDLV YVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGATGVILYSDPDD YFAPGVKSYPDGWNL PGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGMAEAVGLP SIPVHPIGYYDAQKLLEKMGGSASPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHST SEVTRIYNVIGTLRGAVEPDRYVILGGGHRDSWVFGGIDPQSGAAVVHEIVRSFGMLKKE GWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYIN ADSSIEGNYTLRVDCT PLMYSLVYNLTKELESPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQR LGIASGRARYTKNWETNKFSSYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVF ELANSVVLPFDCRDYAVVLRKYADKIYNISMKHPQEMKTYSVSFDSLFSAVKNFTEIAS KFS ERLRDFDKSNPILLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGES FPGIYDALFDIESKVDPSQAWGEVKRQISIATFTVQAAAETLSEVA

PSMG9 (SEQ Ш NO: 127)

MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITP

KHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHY DVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVLDIVPPFSAFSPQGMPEGDLV YVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPAD YFAPGVKSYPDGW NLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRHGIAEAVGLPS IPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTN EVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEG WRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAY INADSSIEGNYTLRVDCTPL MYSLVYNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLG lASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFEL ANSIVLPFDCRDYAVVLRKYADKIYNISMKHPQEMKTYSVSFDSLFSAVKNFTEIAS KFT ERLQDFDKSNPILLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPG IYDALFDIESKVDPSKAWGDVKRQISVAAFTVQAAAETLSEVA

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Table 4

Antigen for each of the compounds selected by phage panning

Round 1 antigen Antigen round 2-3 Selected connections Identification of selected compounds EVA PSMA chimpanzee ECD PSMA of the cynomolgus macaque 2 PSMB18, PSMB25 EVA PSMA chimpanzee HEKs expressing PSMA in chimpanzees 9 PSMB49, PSMB51, PSMB56, PSMB58, PSMB109, PSMB55, PSMB84, PSMB83 LNCaP HEKs expressing PSMA in chimpanzees 2 PSMB86, PSMB80

Preparation of mAb to PSMA. A total of 12 clones that demonstrated binding to all three PSMA-expressing cells were ultimately converted to an IgG4 mAb having Fc isotype substitutions S228P, F234A, and L235A (PAA) through restriction cloning. Briefly, constructs corresponding to Fab clones that passed the initial screen were digested with HindIII and ApaI. The gel-purified fragments were ligated into a proprietary expression vector with the CMV promoter to generate human IgG4-PAA expression. This made it possible to quickly obtain bispecific antibodies. A previously described in-house expression vector was used to express the heavy and light chains for each PSMA mAb, with both vectors transiently cotransfected into the 293Expi or CHO cell lines to express the mAbs. The CDR sequences of interspecies PSMA positive Fabs obtained by phage panning are shown in Table 1 below. 5. The VH and VL sequences of the selected Fabs are given below in table. 6. The sequences of the Fab-derived mAb heavy and light chains are shown in Table. 7.

Table 5

CDR sequences (as determined by Kabat) of antibodies after phage panning (corresponding SEQ ID NOs are listed in parentheses)

IDFAB CDR (SEQ ID NO:) CDR1 CDR2 CDR3 PSMB58 N WITH SYWIS (122) IIYPGDSYTRYSPSF QG (123) DYEWELFDSRLDY (124) L.C. RASQSISSYLN (23) AASSLQS (12) QQSYSTP (24) PSMB10 9 N WITH NAWIS (8) WINPESGRANYAQK FQG (9) ELYYLVYSTYYYAFD Y (10) L.C. RASQSIDRWLN (eleven) AASSLQS (12) QQSPRYPLT (13) PSMB86 N WITH SYDIS (14) GIIPIEGTANYAQKF QG (15) DYPAGYGFDY (16) L.C. RASQSVSSSYLA (17) GASSRAT (18) QQYGSSPLT (19)

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PSMB84 Η WITH SDWMS (20) AISGNGGSTEYADS VKG (21) DPYYYYDGDSYYGM D.V. (22) L.C. RASQSISSYLN (23) AASSLQS (12) QQSYSTP (24) PSMB83 Η WITH SDAMH (25) EISGSGGYTNYADS VKG (26) DSYDSSLYVGDYFDY (27) L.C. RASQSVSSYLA (28) DASNRAT (29) QQRSNWPLT (thirty) PSMB56 Η WITH SYAIS (31) WISPYNGNANYAQ KFQG (32) DSDRSYNLDY (33) L.C. RASQSISGWLN (34) AASSLQS (12) QQSYSTPLT (35) PSMB55 Η WITH SYWIG (36) IIYPGDSDTRYSPSF QG (37) GLPIWYLDY (38) L.C. RASQSVASDLA (39) FASNRAT (40) QQSITWPFT (41) PSMB51 Η WITH SYAIS (31) WIIPYNGNANYAQK FQG (42) VNSAALVWERLDY (43) L.C. RASQSIDRWLN (eleven) AASSLQS (12) QQSPRYPLT (13) PSMB49 Η WITH SYAIS (31) GIIPIFGTANYAQKF QG (44) ASRVWHASYGYLDY (45) L.C. RASQSVSKWLA (46) DASNRAT (29) QQRFTAPWT (47) PSMB25 Η WITH SYWIG (36) IIYPGDSDTRYSPSF QG (37) GWAYDRGLDY (48) L.C. KSSQSVLYSSNNNKN WASTRES QQYYSTPLT

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YLA (49) (50) (51) n s SYWIG (36) IIYPGDSDTRYSPSF QG (37) AYHYSKGLDY (52) L.C. KSSQSVLYSSNNNKN YLA (49) WASTRES (50) QQYYSTPLT (51) N WITH DYAIS (53) RIDPIEGTANYAQKF QG (54) DRYYYDGVYWYSDY FDY (55) L.C. RASQSISSYLN (23) AASSLQS (12) QQSYSTPLT (35)

A monospecific anti-PSMA antibody, PSMB119, was generated containing the VH and VL regions, having VH of SEQ ID NO: 79 and VL of SEQ ID NO: 78 and an IgG4 constant region with substitutions S228P, F234A and L235A. A monospecific anti-PSMA antibody PSMB120 was generated containing the VH and VL regions, having VH at SEQ ID NO: 77 and VL at SEQ ID NO: 78 and an IgG4 constant region with substitutions S228P, F234A and L235A. A monospecific anti-PSMA antibody PSMB121 was generated containing the VH and VL regions, having VH at SEQ ID NO: 75 and VL at SEQ ID NO: 76 and an IgG4 constant region with substitutions S228P, F234A and L235A. A monospecific anti-PSMA antibody, PSMB122, was generated containing the VH and VL regions, having VH at SEQ ID NO: 74 and VL at SEQ ID NO: 61 and an IgG4 constant region with substitutions S228P, F234A and L235A. A monospecific anti-PSMA antibody PSMB123 was generated containing the VH and VL regions, having VH at SEQ ID NO: 72 and VL at SEQ ID NO: 73 and an IgG4 constant region with substitutions S228P, F234A and L235A. PSMB124, a monospecific anti-PSMA antibody, was generated containing the VH and VL regions, having VH at SEQ ID NO: 70 and VL at SEQ ID NO: 71 and an IgG4 constant region with substitutions S228P, F234A and L235A. A monospecific anti-PSMA antibody, PSMB126, was generated containing the VH and VL regions, having VH of SEQ ID NO: 68 and VL of SEQ ID NO: 69 and an IgG4 constant region with substitutions S228P, F234A and L235A. PSMB127, a monospecific anti-PSMA antibody, was generated containing the VH and VL regions, having VH at SEQ ID NO: 66 and VL at SEQ ID NO: 67 and an IgG4 constant region with substitutions S228P, F234A and L235A. A monospecific anti-PSMA antibody PSMB128 (Alt. Fab ID: PSMB84) was created containing the VH and VL regions, having VH with SEQ ID NO: 64 and VL with SEQ ID NO: 65 and an IgG4 constant region with substitutions S228P, F234A and L235A. A monospecific anti-PSMA antibody PSMB129 was generated containing the VH and VL regions, having VH at SEQ ID NO: 60 and VL at SEQ ID NO: 61 and an IgG4 constant region with substitutions S228P, F234A and L235A.

A monospecific anti-PSMA antibody PSMB130 was generated containing the VH and VL regions, having VH at SEQ ID NO: 62 and VL at SEQ ID NO: 63 and an IgG4 constant region with substitutions S228P, F234A and L235A.

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Table 6

Sequences of VH and VL Fab to PSMA

ID FAB Amino acid VH sequence SEQ ID NO Amino acid subsequence VL SEQ ID NO PSMB10 9 QVQLVQSGAEVKKPGSSVKVSC K ASGGTF S S YAIS W VRQ APGQG LEWMGWISPYNGNANYAQKFQ GRVTITADESTSTAYMELSSLRS EDTAVYYCARVNSAALVWERL DYWGQGTLVTVSS 60 DIQMTQSPSSLSASV GDRVTITCRASQSID RWLNWYQQKPGKA PKLLIYAASSLQSGV PSRFSGSGSGTDFTL TISSLQPEDFATYYC QQSPRYPLTFGQGTK VEIL 61 PSMB86 QVQLVQSGAEVKKPGSSVKVSC KASGGTFKSYDISWVRQAPGQG LEWMGGIIPIEGTANYAQKFQGR VTITADESTSTAYMELSSLRSEDT AVYYCARDYPAGYGFDYWGQG TLVTVSS 62 EIVLTQSPGTLSLSPG ERATLSCRASQSVSS SYLAWYQQKPGQAP RLLIYGASSRATGIP DRFSGSGSGTDFTLT ISRLEPEDFAVYYCQ QYGSSPLTFGQGTK VEIL 63 PSMB84 EVQLLESGGGLVQPGGSLRLSCA ASGFTFDSDWMSWVRQAPGKG LEWVSAISGNGGSTEYADSVKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDPYYYYDGDSYY GMD VWGQGTL VT VS S 64 DIQMTQSPSSLSASV GDRVTITCRASQSISS YLNWYQQKPGKAP KLLIYAASSLQSGVP SRFSGSGSGTDFTLTI S SLQPEDF AT YYCQQ SYSTPLTFGQGTKVE IK 65 PSMB83 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSVKG 66 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP 67

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RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS RLLIYDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRSNWPLTFGQGTK VEIL PSMB80 QVQLVQSGAEVKKPGSSVKVSC KASGGTFDDYAISWVRQAPGQG LEWMGRIDPIEGTANYAQKFQG RVTITADESTSTAYMELSSSLRSE DTAVYYCARDRYYYDGVYWYS D YFD YWGQGTL VT VS S 68 DIQMTQSPSSLSASV GDRVTITCRASQSISS YLNWYQQKPGKAP KLLIYAASSLQSGVP SRFSGSGSGTDFTLTI S SLQPEDF AT YYCQQ SYSTPLTFGQGTKVE IK 69 PSMB58 EVQLVQSGAEVKKPGESLKISCK GSGYSFTSYWISWVRQMPGKGL EWMGIIYPGDSYTRYSPSFQGQV TISADKSISTAYLQWSSLKASDT AMYYCARDYEWELFDSRLDYW GQGTLVTVSS 160 DIQMTQSPSSLSASV GDRVTITCRASQSISS YLNWYQQKPGKAP KLLIYAASSLQSGVP SRFSGSGSGTDFTLTI S SLQPEDF AT YYCQQ SYSTPLTFGQGTKVE IK 65 PSMB56 QVQLVQSGAEVKKPGSSVKVSC K ASGGTF S S YAIS W VRQ APGQG LEWMGWISPYNGNANYAQKFQ GRVTITADESTSTAYMELSSLRS EDT AVYYC ARD SDRS YNLD YW GQGTLVTVSS 70 DIQMTQSPSSLSASV GDRVTITCRASQSIS GWLNWYQQKPGKA PKLLIYAASSLQSGV PSRFSGSGSGTDFTL TIS SLQPEDF AT YYC QQSYSTPLTFGQGTK VEIL 71 PSMB55 EVQLVQSGAEVKKPGESLKISCK GSGYSFTSYWIGWVRQMPGKGL EWMGIIYPGDSDTRYSPSFQGQV TISADKSISTAYLQWSSLKASDT 72 EIVLTQSPATLSLSPG ERATLSCRASQSVAS DLAWYQQKPGQAP RLLIYFASNRATGIP 73

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AMYYCARGLPIWYLDYWGQGT LVTVSS ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QSITWPFTFGQGTKV EIK PSMB51 QVQLVQSGAEVKKPGSSVKVSC K ASGGTF S S YAIS W VRQ APGQG LEWMGWIIPYNGNANYAQKFQ GRVTITADESTSTAYMELSSLRS EDTAVYYCARVNSAALVWERL DYWGQGTLVTVSS 74 DIQMTQSPSSLSASV GDRVTITCRASQSID RWLNWYQQKPGKA PKLLIYAASSLQSGV PSRFSGSGSGTDFTL TISSLQPEDFATYYC QQSPRYPLTFGQGTK VEIL 61 PSMB49 QVQLVQSGAEVKKPGSSVKVSC K ASGGTF S S YAIS W VRQ APGQG LEWMGGIIPIFGTANYAQKFQGR VTITADESTSTAYMELSSLRSEDT AVYYCARASRVWHASYGYLDY WGQGTLVTVSS 75 EIVLTQSPATLSLSPG ERATLSCRASQSVSK WLAWYQQKPGQAP RLLIYDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRFTAPWTFGQGTK VEIL 76 PSMB25 EVQLVQSGAEVKKPGESLKISCK GSGYSFTSYWIGWVRQMPGKGL EWMGIIYPGDSDTRYSPSFQGQV TISADKSISTAYLQWSSLKASDT AMYYCARGWAYDRGLDYWGQ GTLVTVSS 77 DIVMTQSPDSLAVSL GERATINCKSSQSVL YS SNNKNYL AW YQ QKPGQPPKLLIYWAS TRESGVPDRFSGSGS GTDFTLTISSLQAED VAVYYCQQYYSTPL TFGQGTKVEIK 78 PSMB18 EVQLVQSGAEVKKPGESLKISCK GSGYSFTSYWIGWVRQMPGKGL EWMGIIYPGDSDTRYSPSFQGQV TISADKSISTAYLQWSSLKASDT AMYYCARAYHYSKGLDYWGQ 79 DIVMTQSPDSLAVSL GERATINCKSSQSVL YS SNNKNYL AW YQ QKPGQPPKLLIYWAS TRESGVPDRFSGSGS 78 GTLVTVSS GTDFTLTISSLQAED VAVYYCQQYYSTPL TFGQGTKVEIK

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Table 7

Sequences of mAb heavy and light chains with corresponding SEQ ID NO

Aphid identifier Amino acid heavy chain sequence SEQ ID NO Amino acid subsequence light chain SEQ ID NO PSMB129 (FAB PSMB109 ) QVQLVQSGAEVKKPGSSV K VSCK ASGGTF S S YAIS W V RQAPGQGLEWMGWISPYN GNANYAQKFQGRVTITAD ESTSTAYMELSSLRSEDTA VYYCARVNSAALVWERL DYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGL YS LSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLP S SIEKTISKAKGQPREPQ VY TLPPSQEEMTK NQVSLTCL VKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLS LSLGK 82 DIQMTQSPSSLSASVGD RVTITCRASQSIDRWLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSPRYPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDNA LQSGNSQESVTEQDSKD STYSLSSTLTLSK ADYE KHKVYACEVTHQGLSS PVTKSFNRGEC 83 PSMB130 QVQLVQSGAEVKKPGSSV 84 EIVLTQSPGTLLSLSPGER 85

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(FAB PSMB86) KVSCKASGGTFKSYDISW VRQAPGQGLEWMGGIIPIE GTANYAQKFQGRVTITAD ESTSTAYMELSSLRSEDTA VYYCARDYPAGYGFDYW GQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSL S SWT VPS S SLGTKT YTCN VDHK PSNTKVDKRVESKY GPPCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKG FYPSDIAVEW ESNGQPENN YKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSL GK ATLSCRASQSVSSSYLA WYQQKPGQAPRLLIYG ASSRATGIPDRFSGSGSG TDFTLTISRLEPEDFAVY YCQQYGSSPLTFGQGTK VEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLN NFYPREAKVQWKVDNA LQSGNSQESVTEQDSKD STYSLSSTLTLSKADYE KHKVYACEVTHQGLSS P VTKSFNRGEC PSMB128 (FAB PSMB84) EVQLLESGGGLVQPGGSLR LSCAASGFTFDSDWMSWV RQAPGKGLEWVSAISGNG GSTEYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTA VYYCARDPYYYYDGDSY YGMD VWGQGTL VT VS SA STKGPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQ 86 DIQMTQSPSSLSASVGD RVTITCRASQSISSYLNW YQQKPGKAPKLLIYAAS SLQSGVPSRFSGSGSGT DFTLTIS SLQPEDF AT YY CQQSYSTPLTFGQGTKV EIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNF YPREAKVQWKVDNAL QSGNSQESVTEQDSKDS 87

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SSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEAA GGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREP QVYTLPPSQEE MTKNQVS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQE GN VF SC S VMHEALHNHYT QKSLSLSLGK TYSLSSTLTLSKADYEK HKVYACEVTHQGLS SP VTKSFNRGEC PSMB127 (FAB PSMB83) EVQLLESGGGLVQPGGSLR LSCAASGFTFKSDAMHWV RQAPGKGLEWVSEISGSGG YTNYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAV YYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGL YSLSSVVTVP SSSLGTKTY TCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLP S SIEKTISKAKGQPREPQ VY 88 EIVLTQSPATLSLSPGER ATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGT DFTLTIS SLEPEDF A VYY CQQRSNWPLTFGQGTK VEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLN NFYPREAKVQWKVDNA LQSGNSQESVTEQDSKD E KHKVYACEVTHQGLSS P VTKSFNRGEC 89

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TLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLS LSLGK PSMB127 DNA ATGGGCTGGGGTGGACCTGCTATTCTCTGA TGGCAGCCAAGTATAGCAGCGCGCGA GGTTCAGCTGGGGAGGAGGAGTCGCGCTCTCTCTGACTGCTTC TGGCTCACCTCAAGTC CGACGCCACTGGGGGGCAGCGCCCCCCCCCCCCCCGGAAGGAAGGAAGGGGGGGGGGGGGCGCGCGCTCTACCCACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC TCGGGGACACTCAAGAACACACACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC AGACTCCCAGCTCCCCCCCCTACTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGGGGC ACCC TGGTCACCGTTTTTTTCTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC TGCTCCCCCCCCCCCCCCCCCCCCAGCAGCAGGGGGGGGGGGAGGAGGACTAC TTCCCCCCCGGGGGGGGT GTCGTGGAA CTCAGCCCCCCCCCGCGCGCGCCCCCCCCGCGCGTCCTCCTCAGTCAGGGA CTCTACTCCAGCGTGGTGCAGCAGCAGGGGGGGGGGGGGGGGGGGGGACCCCCCCCCCCCCCCCCCCCCCCCCCCCCC AACGTAGATAGATCAAGCCCCAGCAACACACACAAGGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGAGGAGGATGGTGGTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC CCAGCCCCCCCCCCCCCCCCCCecaAcAACAAG 161 atggcctgggtgtggaccctgctgttc ctgatggccgccgcccagagcatcc aggccgagatcgtgctgacccagag ccccgccaccctgagcctgagcccc ggcgagcgggccaccctgagctgc cgggccagccagagcgtgagcagc tacctggcctggtaccagcagaagcc cggccaggccccggctgctgat c tacgacgccagcaaccgggccacc ggcatccccgcccggttcagcggca gcggcagcggcaccgacttcaccct gaccatcagcagcctggagcccgag gacttcgccgtgtactactgccagca gcggagcaactggcccctgaccttc ggccagggcaccaaggtggagatc aagcgtacggtggctgcaccatctgt tcatcttcccgccatctgatgagca gttgaaatctggaactgcctctgttgtg tgcctgctgaataacttctatcccaga gaggccaaagtacagtggaaggtgg ataacgccctccaatcgggtaactcc caggagagtgtcacagagcaggaca gcaaggacagcacctacagcctcag cagcaccctgacgctgagcaaag ca gactacgagaaacacaaagtctacg cctgcgaagtcacccatcagggcctg agctcgcccgtcacaaagagcttcaa caggggagagtgt 162

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gacactctcatgatctcccggacccctga ggtcacgtgcgtggtggtggacgtgagc caggaagaccccgaggtccagttcaact ggtacgtggatggcgtggaggtgcataat gccaagacaaagccgcgggaggagca gttcaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaggactggct g aacggcaaggagtacaagtgcaaggtct ccaacaaaggcctcccgtcctccatcgag aaaaccatctccaaagccaaagggcagc cccgagagccacaggtgtacaccctgcc cccatcccaggaggagatgaccaagaac caggtcagcctgacctgcctggtcaaagg cttctaccccagcgacatcgccgtggagt gggaga gcaatgggcagccggagaaca actacaagaccacgcctcccgtgctggac tccgacggctccttcttcctctacagcagg ctaaccgtggacaagagcaggtggcagg aggggaatgtcttctcatgctccgtgatgc atgaggctctgcacaaccactacacacag aagagcctctccctgtctctgggtaaa P SMB 126 (FAB PSMB80) QVQLVQSGAEVKKPGSSV KVSCKASGGTFDDYAISW VRQAPGQGLEWMGRIDPI EGTANYAQKFQGRVTITA DESTSTAYMELSSLRSEDT AVYYCARDRYYYDGVYW YSD YFD YWGQGTL VTVS S ASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVL QSS GLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEA 90 DIQMTQSPSSLSASVGD RVTITCRASQSISSYLNW YQQKPGKAPKLLIYAAS SLQSGVPSRFSGSGSGT DFTLTIS SLQPEDF AT YY CQQSYSTPLTFGQGTKV EIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNF YPREAKVQWKVDNAL QSGNSQESVTEQDSKDS TYSLSSTLTLS KADYEK HKVYACEVTHQGLS SP VTKSFNRGEC 91

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AGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSD GSFFLYS RLTVDKSRWQE GN VF SC S VMHEALHNHYT QKSLSLSLGK PSMB124 (FAB PSMB56) QVQLVQSGAEVKKPGSSV KVSCKASGGTFSSYAISWV RQAPGQGLEWMGWISPYN GNANYAQKFQGRVTITAD ESTSTAYMELSSLRSEDTA VYYCARDSDRSYNLDYW GQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSL S S VVT PS S SLGTKT YTCN VDHKPSNTKVDKRVESKY GPPCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPP SQEEMTK NQVSLTCLVKG FYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSR 92 DIQMTQSPSSLSASVGD RVTITCRASQSISGWLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSYSTPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDNA LQSGNSQESVTEQDSKD STYSLSSTLTLSK ADYE KHKVYACEVTHQGLSS PVTKSFNRGEC 93

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LTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSL GK PSMB123 (FAB PSMB55) EVQLVQSGAEVKKPGESL KISCKGSGYSFTSYWIGWV RQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADK SISTAYLQWSSLKASDTAM YYCARGLPIWYLDYWGQ GTLVTVSSASTKGPSVFPL APCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSV VTVPSSSLGT KTYTCNVDH KPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVV DVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKE YKCKVSNKGLPS SIEKTISK AKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGF YPS DIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 94 A KVYACEVTHQGLSS PVTKSFNRGEC 95 PSMB122 (FAB PSMB51) QVQLVQSGAEVKKPGSSV K VSCK ASGGTF S S YAIS W V RQAPGQGLEWMGWIIPYN GNANYAQKFQGRVTITAD ESTSTAYMELSSLRSEDTA VYYCARVNSAALVWERL DYWGQGTLVTVSSASTKG 96 DIQMTQSPSSLSASVGD RVTITCRASQ SIDRWLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTIS SLQPEDF AT YYCQQSPRYPLTFGQGT KVEIKRTVAAPSVFIFPP 83

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PSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTV LHQ DWLNGKEYKCKVSNKGLP S SIEKTISKAKGQPREPQ VY TLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLS LSLGK SDEQLKSGTASVVCLLN NFYPREAKVQWKVDNA LQSGNSQESVTEQDSKD STYSLSSTLTLSKADYE KHKVYACEVTHQGLSS PVTKSFNRGEC PSMB121 (FAB PSMB49) QVQLVQSGAEVKKPGSSV K VSCK ASGGTF S S YAIS W V RQAPGQGLEWMGGIIPIFG TANYAQKFQGRVTITADES TSTAYMELSSLRSEDTAVY YCARASRVWHASYGYLD YWGQGTLVTVSSASTKGP SVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLY S LSSVVTVPSSSLGTKTYT CNVDHKPSNTKVDKRVES KYGPPCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQ 97 EIVLTQSPATLSLSPGER ATLSCRASQSVSKWLA WYQQKPGQAPRLLIYD ASNRATGIPARFSGSGS GTDFTLTIS SLEPEDF AV YYCQRFTAPWTFGQG TKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLL NNFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKHK VYACEVTHQGLS SPVTKSFNRGEC 98

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FNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLS LGK PSMB120 (FAB PSMB25) EVQLVQSGAEVKKPGESL KISCKGSGYSFTSYWIGWV RQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADK SISTAYLQWSSLKASDTAM YYCARGWAYDRGLDYWG QGTLVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLV KD YFPEP VT VS WNS GALTS GVHTFP AVLQS SGLYSLS S V VPS QVSLTCLVKGFY PSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLG K 99 DIVMTQSPDSLAVSLGE RATINCKS SQS VL YS SN NKNYLAWYQQKPGQPP KLLIYWASTRESGVPDR FSGSGSGTDFTLTISSLQ AEDVAVYYCQQYYSTP LTFGQGTKVEIKRTVAAA PSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKV QWKVDNALQSGNSQES VTEQDSKDSTYSL SSTL TLSKADYEKHKVYACE VTHQGLSSPVTKSFNRG EC 100

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PSMB119 EVQLVQSGAEVKKPGESL 101 DIVMTQSPDSLAVSLGE 100 (FAB PSMB18) KISCKGSGYSFTSYWIGWV RQMPGKGLEWMGIIYPGD SDTRYSPSFQGQVTISADK SISTAYLQWSSLKASDTAM YYCARAYHYSKGLDYWG QGTLVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLV KD YFPEP VT VS WNS GALTS GVHTFP AVLQS SGL YSLS S VVT VPS SLGTKT YTCNVD HKPSNTKVDKRVESKYGP PCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVV VDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFY PSDI AVEWESNGQPENNY KTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLG K RATINCKS SQS VL YS SN NKNYLAWYQQKPGQPP KLLIYWASTRESGVPDR FSGSGSGTDFTLTISSLQ AEDVAVYYCQQYYSTP LTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKV QWKVDNALQSGNSQES VTEQDSKDSTYSLSSTL TLSKADYEKH KVYACE VTHQGLSSPVTKSFNRG EC PSMB87 (FAB PSMB58) EVQLVQSGAEVKKPGESL KISCKGSGYSFTSYWISWV RQMPGKGLEWMGIIYPGD SYTRYSPSFQGQVTISADK SISTAYLQWSSLKASDTAM YYC ARD YEWELFD SRLD Y WGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALG CL VKD YFPEP VTVSWNSG 125 DIQMTQSPSSLSASVGD RVTITCRASQSISSYLNW YQQKPGKAPKLLIYAAS SLQSGVPSRFSGSGSGT DFTLTIS SLQPEDF AT YY CQQSYSTPLTFGQGTKV EIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNF YPREAKVQWKVDNAL 91

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ALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTKTYT CNVDHKPSNTKVDKRVES KYGPPCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSI EKTISKAKGQ PREPQVYTL PPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLS LGK QSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEK HKVYACEVTHQGLS SP VTKSFNRGEC

Interactions of the parent mAbs PSMB123 (Fab PSMB55), PSMB127 (Fab PSMB83), and PSMB130 (Fab PSMB86) with human, chimpanzee, and cynomolgus PSMA ECDs were measured by surface plasmon resonance (SPR) using the ProteOn XPR36 system (BioRad), as previously described for recombinant chimpanzee PSMA ECD. A summary of the binding affinities for each of these mAbs to human, chimpanzee, and cynomolgus PSMA ECDs is shown in Table 1. 18. These mAbs bind to targets with affinities similar to bispecific antibodies.

Table 18

Binding of monoclonal antibodies to recombinant human, chimpanzee and cynomolgus PSMA ECD by Proteon

Human KD (nM) Chimpanzee KD (nM) Cynomolgus macaque KD (nM) PSMB123 (Fab PSMB55) 9.03±1.20 14.3±3.33 66.7±8.60 PSMB127 (Fab PSMB83) 12.0±2.05 12.8±1.83 6.68±0.45 PSMB130 (Fab PSMB86) 29.6±2.43 31.7±7.48 >300

Example 3 Human Adaptation of Anti-CD3 Antibody SP34 Mouse anti-human CD3 antibody SP34 was humanized by a human adaptation method (Fransson, et al, JMB, 2010 398(2):214-31).

The VH and VL sequences of SP34 are shown in FIG. 4 below, with residues 1-215 of the light chain and residues 1-230 of the heavy chain obtained directly from the electron density map, and residues 231-455 obtained from IGHG3_MOUSE (mouse IgG3 isoform 2).

VH SP34 (SEQ ID NO: 128)

EVKLLESGGGLVQPKGSLKLSCAASGTFNTYAMNWVRQAPGKGLEWVARIRS

KYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSA

VL SP34 (SEQ ID NO: 129)

QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTN

KRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL

Four different heavy chains were combined with three different light chains, resulting in 12 humanized variants.

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SP34 humanization and affinity maturation.

Human germline selection.

For the study, we chose a matrix of human sequences of variable regions of four heavy and three light chains. The selection of human sequences was based solely on overall sequence similarity to SP34 in the framework region (FR). During the selection, neither CDR sequences nor their length or canonical structures were taken into account.

The closest matches for the heavy chain were the human germline IGHV372 and IGHV3-73. Another germline, IGHV3-23, was chosen due to its high frequency in the human B cell repertoire.

The closest matches for the light chain were the human germlines IGLV743 (also known as 7a), IGLV7-46 (also known as 7b) and IGLV1-51 (also known as 1b). IGLV7-46 is almost identical to IGLV7-43, but has the advantage of having Ala at position 2, i.e. as in SP34.

The J regions chosen were: IGHJ1 for the heavy chain; IGLJ3 for lambda light chain.

Back mutations.

To maintain the conformation of CDR-H3, it was necessary to conserve residues in several framework regions in VL, the most significant of which are Val38, Gly48, and Gly51. These back mutations were added to the humanization plan.

The Asn at position 57 of the heavy chain was maturationally truncated to Gly to provide the necessary flexibility and potentially increase stability (by reducing local structural stress associated with the non-glycine residue) without affecting binding.

Several other considerations were present in developing the humanization plan. First, in the human germ lines IGLV7-46 and IGLV7-43, a Trp residue is introduced at position 59 with an undesirable oxidative potential. In the other two germ lines, Gly is found at this position, which corresponds to the mouse sequence. Therefore, in both variants, IGLV7-46 and IGLV7-43 retained Gly59. Finally, Ala at position 49 in VH may be important. Also, the residue at position 99 (Val in SP34) may affect antigen binding. To test these provisions, back mutations were introduced in some variants (Fig. 5).

HFA matrix.

The Human Framework Adaptation Matrix (HFA) (Table 8) consists of four VH options and three VL options (Fig. 5). For the purposes of HFA, the CDR definition from AbM is used (K.R. Abhinandan and A.S. Martin, 2008. Mol. Immunol. 45, 3832-3839).

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VH options:

CD3H141 (SEQ ID NO: 104): IGHV3-72*01 with mouse CDR+Gly49Ala

EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRS KYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVS WF AYWGQGTL VT VS S

CD3H142 (SEQ ID NO: 102): IGHV3-23*01 with mouse CDR+Ser49Ala

EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRS KYNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHGNFGNSYVS WF AYWGQGTL VT VS S

CD3H143 (SEQ ID NO: 115): IGHV3-23*01 with mouse CDR+Ser49Ala,

Ala99Val

EVQLLESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRS KYNNYATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKHGNFGNSYVS WF AYWGQGTL VT VS S

CD3H144 (SEQ ID NO: 116): IGHV3-73*01 with mouse CDR+Asn57Gly

EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVGRIRS KYNGYATYYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVS WF AYWGQGTL VT VS S

VL options:

CD3L63 (SEQ ID NO: 103): IGLV7-46*01 with mouse CDR+F38V, A48G, Y51G,

W59G

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTN KRAPGTPARFSGSLLGGKAALTLLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL

CD3L64 (SEQ ID NO: 117): IGLV1-51*O1 with mouse CDR+Y38V, L48G, Y51G

QSVLTQPPSVSAAPGQKVTISCRSSTGAVTTSNYANWVQQLPGTAPKGLIGGTNK RAPGIPDRFSGSKSGTSATLGITGLQTGDEADYYCALWYSNLWVFGGGTKLTVL

CD3L66 (SEQ ID NO: 105): IGLV7-43*01 with mouse CDR+F38V, A48G, Y51G, W59G

QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTN KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

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Table 8

CD3 Heavy and Light Chain Matrix (All were generated using IgG1-AA Fc containing L234A, L235A and F405L)

CD3L63 (LV7-46/W59G) SEQIDNO: 103 CD3L64 (LV1-51) SEQ ID NO: 117 CD3L66 (LV7-43/W59G) SEQ ID NO: 105 CD3H141 (HV3-72+ G49A) SEQIDNO: 104 CD3B143 CD3B144 CD3B146 CD3H142 (HV3-23+ S49A) SEQIDNO: 102 CD3B147 CD3B148 CD3B150 CD3H143 (HV3-23 +S49A, A99V) SEQIDNO: 115 CD3B151 CD3B152 CD3B154 CD3H144 (VH3-73 with G49) SEQIDNO: 116 CD3B155 CD3B156 CD3B158

The amino acid sequences were reverse translated into DNA and cDNA was obtained by gene synthesis methods (US Patent No. 6,670,127; US Patent No. 6,521,427). Heavy chain variable regions (HC) were subcloned into human IgG1-AA Fc containing the L234A, L235A and F405L mutations using a proprietary expression vector with a CMV promoter using standard molecular biology techniques. Light chain (LC) variable regions were subcloned into human lambda constant regions (λ) using a proprietary expression vector with a CMV promoter using standard molecular biology techniques. The resulting plasmids were transfected into Expi293F cells (Invitrogen) and mAb expression was performed. Purification was performed using standard methods on a protein A column (hiTrap MAbSelect SuRe column). After elution, the pools were dialyzed into D-PBS, pH 7.2. The sequences of VH and VL antibodies are shown in table. 9.

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Table 9

VH and VL sequences of anti-CD3 antibodies

mAb NS Amino acid subsequence VH SEQ ID NO: L.C. Amino acid subsequence VL SEQ ID NO: CD3B 143 CD3H1 41 EVQLVESGGGLVQP GGSLRLSCAASGFTF NTYAMNWVRQAPG KGLEWVARIRSKYN NYATYYAASVKGRF TISRDDSKNSLYLQ MNSLKTEDTAVYYC ARHGNFGNSYVSWF AYWGQGTLVTVSS 104 CD3L 63 QAVVTQEPSLTVS PGGTVTLTCRSST GAVTTSNYANWV QQKPGQAPRGLIG GTNKRAPGTPARF SGSLLGGKAALTL SGAQPEDEAEYYC ALWYSNLWVFGG GTKLTVL 103 CD3B 144 CD3H1 41 EVQLVESGGGLVQP GGSLRLSCAASGFTF NTYAMNWVRQAPG KGLEWVARIRSKYN NYATYYAASVKGRF TISRDDSKNSLYLQ MNSLKTEDTAVYYC ARHGNFGNSYVSWF AYWGQGTLVTVSS 104 CD3L 64 QSVLTQPPSVSAA PGQKVTISCRSSTG AVTTSNYANWVQ QLPGTAPKGLIGG TNKRAPGIPDRFSG SKSGTSATLGITGL QTGDEADYYCAL WYSNLWVFGGGT KLTVL 117 CD3B 146 CD3H1 41 EVQLVESGGGLVQP GGSLRLSCAASGFTF NTYAMNWVRQAPG KGLEWVARIRSKYN NYATYYAASVKGRF TISRDDSKNSLYLQ MNSLKTEDTAVYYC ARHGNFGNSYVSWF AYWGQGTLVTVSS 104 CD3L 66 QTWTQEPSLTVS PGGTVTLTCRSST GAVTTSNYANWV QQKPGQAPRGLIG GTNKRAPGTPARF SGSLLGGKAALTL SGVQPEDEAEYYC ALWYSNLWVFGG GTKLTVL 105 CD3B 147 CD3H1 42 EVQLLESGGGLVQP GGSLRLSCAASGFTF NTYAMNWVRQAPG KGLEWVARIRSKYN NYATYYADSVKGRF TISRDNSKNTLYLQ MNSLRAEDTAVYY CAKHGNFGNSYVS 102 CD3L 63 QAVVTQEPSLTVS PGGTVTLTCRSST GAVTTSNYANWV QQKPGQAPRGLIG GTNKRAPGTPARF SGSLLGGKAALTL SGAQPEDEAEYYC ALWYSNLWVFGG 103

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WFAYWGQGTLVTV ss GTKLTVL CD3B 148 CD3H1 42 EVQLLESGGGLVQP GGSLRLSCAASGFTF NTYAMNWVRQAPG KGLEWVARIRSKYN NYATYYADSVKGRF TISRDNSKNTLYLQ MNSLRAEDTAVYY CAKHGNFGNSYVS WFAYWGQGTLVTV SS 102 CD3L 64 QSVLTQPPSVSAA PGQKVTISCRSSTG AVTTSNYANWVQ QLPGTAPKGLIGG TNKRAPGIPDRFSG SKSGTSATLGITGL QTGDEADYYCAL WYSNLWVFGGGT KLTVL 117 CD3B 150 CD3H1 42 EVQLLESGGGLVQP GGSLRLSCAASGFTF NTYAMNWVRQAPG KGLEWVARIRSKYN NYATYYADSVKGRF TISRDNSKNTLYLQ MNSLRAEDTAVYY CAKHGNFGNSYVS WFAYWGQGTLVTV SS 102 CD3L 66 QTWTQEPSLTVS PGGTVTLTCRSST GAVTTSNYANWV QQKPGQAPRGLIG GTNKRAPGTPARF SGSLLGGKAALTL SGVQPEDEAEYYC ALWYSNLWVFGG GTKLTVL 105 CD3B 151 CD3H1 43 EVQLLESGGGLVQP GGSLRLSCAASGFTF NTYAMNWVRQAPG KGLEWVARIRSKYN NYATYYADSVKGRF TISRDNSKNTLYLQ MNSLRAEDTAVYY CVKHGNFGNSYVS WFAYWGQGTLVTV SS 115 CD3L 63 QAVVTQEPSLTVS PGGTVTLTCRSST GAVTTSNYANWV QQKPGQAPRGLIG GTNKRAPGTPARF SGSLLGGKAALTL SGAQPEDEAEYYC ALWYSNLWVFGG GTKLTVL 103 CD3B 152 CD3H1 43 EVQLLESGGGLVQP GGSLRLSCAASGFTF NTYAMNWVRQAPG 115 CD3L 64 QSVLTQPPSVSAA PGQKVTISCRSSTG AVTTSNYANWVQ 117

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KGLEWVARIRSKYN NYATYYADSVKGRF TISRDNSKNTLYLQ MNSLRAEDTAVYY CVKHGNFGNSYVS WFAYWGQGTLVTV ss QLPGTAPKGLIGG TNKRAPGIPDRFSG SKSGTSATLGITGL QTGDEADYYCAL WYSNLWVFGGGT KLTVL CD3B 154 CD3H1 43 EVQLLESGGGLVQP GGSLRLSCAASGFTF NTYAMNWVRQAPG KGLEWVARIRSKYN NYATYYADSVKGRF TISRDNSKNTLYLQ MNSLRAEDTAVYY CVKHGNFGNSYVS WFAYWGQGTLVTV SS 115 CD3L 66 QTWTQEPSLTVS PGGTVTLTCRSST GAVTTSNYANWV QQKPGQAPRGLIG GTNKRAPGTPARF SGSLLGGKAALTL SGVQPEDEAEYYC ALWYSNLWVFGG GTKLTVL 105 CD3B 155 CD3H1 44 EVQLVESGGGLVQP GGSLKLSCAASGFTF NTYAMNWVRQASG KGLEWVGRIRSKYN GYATYYAASVKGRF TISRDDSKNTAYLQ MNSLKTEDTAVYYC TRHGNFGNSYVSWF AYWGQGTLVTVSS 116 CD3L 63 QAVVTQEPSLTVS PGGTVTLTCRSST GAVTTSNYANWV QQKPGQAPRGLIG GTNKRAPGTPARF SGSLLGGKAALTL SGAQPEDEAEYYC ALWYSNLWVFGG GTKLTVL 103 CD3B 156 CD3H1 44 EVQLVESGGGLVQP GGSLKLSCAASGFTF NTYAMNWVRQASG KGLEWVGRIRSKYN GYATYYAASVKGRF TISRDDSKNTAYLQ MNSLKTEDTAVYYC TRHGNFGNSYVSWF AYWGQGTLVTVSS 116 CD3L 64 QSVLTQPPSVSAA PGQKVTISCRSSTG AVTTSNYANWVQ QLPGTAPKGLIGG TNKRAPGIPDRFSG SKSGTSATLGITGL QTGDEADYYCAL WYSNLWVFGGGT KLTVL 117

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CD3B CD3H1 EVQLVESGGGLVQP GGSLKLSCAASGFTF NTYAMNWVRQASG KGLEWVGRIRSKYN GYATYYAASVKGRF 116 CD3L QTWTQEPSLTVS PGGTVTLTCRSST GAVTTSNYANWV QQKPGQAPRGLIG GTNKRAPGTPARF 105 158 44 66 TISRDDSKNTAYLQ SGSLLGGKAALTL MNSLKTEDTAVYYC SGVQPEDEAEYYC TRHGNFGNSYVSWF ALWYSNLWVFGG AYWGQGTLVTVSS GTKLTVL

Example 4: Endogenous cellular binding of humanized anti-CD3 antibody hits to primary T cells.

The resulting panel of anti-CD3 antibodies was tested for binding to cell surface CD3ε on primary human T cells. To do this, antibody binding from expression supernatants was visualized using a polyclonal secondary antibody to human immunoglobulin and analyzed by flow cytometry. Briefly, the binding of anti-CD3 antibodies to cell surface CD3ε was analyzed by flow cytometry using negative selection-purified human T lymphocytes (Biological Specialty, Colmar, USA). Expression supernatants or purified antibodies were normalized to 10 μg/ml in media or FACS buffer (BD BioSciences), respectively. Aliquots of 2x105 cells were added to the wells of a 96-well round bottom plate (CoStar) for labeling. Antibodies in the expression supernatant were added to the cells and incubated for 45 min at 4°C. After centrifugation at 1300 rpm for 3 min and removal of the supernatant, 50 μl of secondary anti-human IgG antibody (H+L) Alexa Fluor 647 (Life technologies Inc.) was incubated with cells at a final concentration of 10 μg/ml for 30 min. at 4°C in a place protected from direct light. Then washed and resuspended in 30 μl FACS buffer solution (BD BioSciences). Sample collection was performed on the Intellicyt HTFC system using ForeCyt software. Prior to binding assays, viable single cell selection was performed using green or red live/dead cell fixative dye (Life Technologies Inc.) and area and height parameters for forward/side scatter, respectively. Graphs were generated in GraphPad Prism version 5 software using mean fluorescence intensity values.

Although a series of titrations were performed, FIG. 6 shows intermediate concentrations for clarity. Two in-house phage-derived therapeutic antibodies with the same Fc:G11 region (HC SEQ ID NO: 118, LC SEQ ID NO: 119) were used as controls, and a non-cynomolgus cross-reacting agonist antibody was used as a positive control. , and CD3B94 (HC-SEQ ID NO: 120, LC - SEQ ID NO: 121) a non-binding/non-agonist antibody was used to assess non-specific binding. The commercial SP34 antibody was not used for comparison in this study because it is a murine antibody and the use of a different secondary detection reagent would not allow direct comparison of the variants studied.

The data demonstrated a matrix of binding potential in a panel of humanized anti-CD3 antibody hits, where two antibodies (CD3B144, CD3B152) showed complete loss of binding to human T cells. The remaining antibodies showed some range of binding potential, which can be broadly categorized as strongly binding and weakly binding, using G11 antibody binding as the threshold. Based on these parameters, seven strongly binding and seven weakly binding antibodies were identified in the panel of variants (Fig. 6).

Anti-CD3 antibody hit binding assays were then performed on primary cynomolgus CD4 T cells to assess persistence of cross-reactivity. Purified CD4+ T cells from the peripheral blood of cynomolgus monkeys (Zen Bio, Triangle Research Park, USA) were used. The study protocols were similar to those described above. Because the G11 antibody does not cross-react to cynomolgus CD3ε, CD3B124, an in-house developed chimeric antibody derived from SP34, having the VH and VL of SP34, a mouse scaffold, and a human IgG1 Fc, was used as a positive control in this assay (Fig. 7). Interestingly, several variants showed reduced binding potential compared to that observed in human cells. These included the strongly binding antibodies CD3B150, CD3B151, and CD3B154, in which binding was reduced, and several weakly binding antibodies in which binding above background was no longer detectable. This loss of binding was not associated with any specific immunoglobulin chain, and this may indicate that a combination of heavy and mild plays a role in the loss of cross-reactivity

- 102 046641 chains. In combination, these analyzes allowed the identification of variants that retain cross-reactivity to human and cynomolgus CD3ε.

Example 5: Functional analysis of humanized anti-CD3 antibody hits in primary T cells.

Binding analysis showed that the panel of anti-CD3 antibody hits exhibited a range of binding to human and cynomolgus T cells. To examine the ability of each variant to induce activation through cross-linking with CD3ε, primary T cells were cultured overnight in the presence of bead-conjugated antibody. The next day, cells were harvested and labeled with anti-CD69 antibody to measure activation (Fig. 8). Humanized anti-CD3 antibodies were bound to protein A-coated magnetic beads (SpheroTech, Lake Forest, USA) by overnight incubation with 10 μg/ml antibody. The next day, 2 x 10 ⁵ primary human T cells were seeded onto round-bottom culture plates in triplicate and 2 x 10 ⁵ coated beads were added. After overnight incubation at 37°C, cells were harvested and labeled with anti-CD69 antibody Alexa Fluor® 488 (clone FN50; Biolegend) to assess the increase in this activation marker. Sample collection and analysis were performed as described above for binding. Several negative controls were used, including T cells alone, T cells with uncoated beads, and T cells with beads coated with an isotype control (CD3B94). All of these controls exhibited similar mean fluorescence intensity values comparable to unstained T cells, indicating that the background in this assay was low. Several positive controls were used for comparison, including OKT3 (US 5929212) and commercial antibodies SP34-2.

The humanized anti-CD3 antibody hits were further tested for their ability to activate primary cynomolgus CD4+ T cells (Zen Bio, Triangle Research Park, USA) in the same assay (Fig. 9). The anti-CD69 antibody FN50 has been described as cross-reactive with a non-human protein and can therefore be used to test the activation of these cells.

The human and cynomolgus activation data correlated with the binding data in that the hit panel exhibited a range of activation potentials. A number of strong binding molecules have demonstrated the ability to activate human T cells to an equal or greater extent than the commercial SP34-2 antibody. Several variants showed lower activation potential than SP34-2, and some binding antibodies showed no evidence of CD69 stimulation. Failure to activate was observed only in variants that exhibited no or weak binding, and all variants with strong binding exhibited some level of activation, indicating a correlation between binding and activation potential in both humans (Fig. 10A) and case of the cynomolgus monkey (Fig. 10B).

To generate bispecific antibodies with PSMA-specific antibodies, two anti-CD3 antibodies, CD3B146 and CD3B147, with high and medium affinities, respectively, were selected. These two anti-CD3 antibodies were produced in the IgG4 PAA GenMab format (Labrijn et al, 2013), in which the targeting parent mAb (PSMA) contains the 409R GenMab mutation (the native amino acid for IgG4) and the killing parent mAb (CD3) contains the F405L GenMab and the mutation R409K. Monospecific anti-CD3 antibody was expressed as IgG4 having Fc substitutions S228P, F234A, L235A, F405L and R409K (arm to CD3) (EU numbering) in the Fc regions. Heavy chain variable regions (HC) were subcloned into human IgG4-PAA Fc containing mutations S228P, F234A, L235A, F405L and R409K using a proprietary expression vector with a CMV promoter using standard molecular biology techniques. Light chain (LC) variable regions were subcloned into human lambda constant regions (λ) using a proprietary expression vector with a CMV promoter using standard molecular biology techniques. The resulting plasmids were transfected into Expi293F cells (Invitrogen) and mAb expression was performed. Anti-CD3 antibodies were purified by standard purification methods using a protein A column with an elution buffer of 100 mM NaAc, pH 3.5, and a neutralization buffer of 2 M Tris, pH 7.5, and 150 mM NaCl. The mAbs were desalted using a PD10 column (Sephadex G25M) and the pools were dialyzed into D-PBS at pH 7.2.

A monospecific anti-CD3 antibody CD3B217 was generated containing the VH and VL regions, having VH at SEQ ID NO: 102 and VL at SEQ ID NO: 103, and an IgG4 constant region with substitutions S228P, F234A, L235A, F405L and R409K. CD3B217 contains a heavy chain of SEQ ID NO: 108 and a light chain of SEQ ID NO: 109. A monospecific CD3 antibody CD3B219 was generated containing the VH and VL regions, having VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105 , and the IgG4 constant region with substitutions S228P, F234A, L235A, F405L and R409K. CD3B219 contains a heavy chain of SEQ ID NO: 110 and a light chain of SEQ ID NO: 111.

Example 6. Preparation of bispecific antibodies to PSMA x CD3.

To generate PSMA x CD3 bispecific antibodies, two parent mAbs are required, one specific for the targeting arm (eg, PSMA) and the other specific for the effector arm (eg, CD3). The PSMA mAb was recombined with the medium affinity arms of CD3B217 (VH SEQ ID NO: 102, VL SEQ ID NO: 103) and the high affinity CD3 arms of CD3B219 (VH SEQ ID NO: 104, VL SEQ ID NO: 105). These parent mAbs are in the GenMab format (Labrijn et al, 2013), in which

- 103 046641 the targeting parent mAb (PSMA) contains the 409R GenMab mutation (the native amino acid for IgG4), and the killing parent mAb (CD3) contains the F405L GenMab mutation and the R409K mutation. Monospecific anti-CD3 antibody was expressed as IgG4 having Fc substitutions S228P, F234A, L235A, F405L and R409K (arm to CD3) (EU numbering) in the Fc regions. Monospecific antibodies were expressed in HEK cell lines under the control of CMV promoters.

PSMA and CD3 parent antibodies were purified using a Protein A column with an elution buffer of 100 mM NaAc pH 3.5 and a neutralization buffer of 2 M Tris pH 7.5 and 150 mM NaCl. The mAb was desalted using a PD10 column (Sephadex G25M) and dialyzed in D-PBS buffer at pH 7.2.

After purification, the parent anti-PSMA antibodies were mixed with the desired parent anti-CD3 antibody under reducing conditions in 75 mM cysteamine-HCl and incubated at 31°C for 4 h. Recombination reactions were based on molar ratios, where a given amount of PSMA (e.g., 10 mg or ~67.8 nanomoles) was combined with anti-CD3 antibody (eg, ~71.8 nanomoles), with anti-CD3 antibody added at a 6% excess of anti-PSMA antibody. Concentrations of anti-PSMA antibody stock solutions varied from 0.8 to 6 mg/ml, and recombination reaction mixture volumes were varied for each mating. The recombination products were then dialyzed against PBS to remove the reducing agent. Bispecific antibody reactions were performed with an excess of anti-CD3 antibody (ratio) to minimize the amount of unreacted parent anti-PSMA antibody remaining after recombination. After partial reconstitution of the parent mAbs, the reducing agent was removed by dialysis overnight in PBS.

The final bispecific antibodies obtained along with the parent mAbs (i.e. PSMA, CD3 or Null) used in the recombination reactions are listed in Table. 10 and the sequences are listed in table. 11 and 12.

Selected appropriate PSMA antibodies were also combined with the non-killing arm (Null) to generate negative controls for testing purposes. For control bispecific antibodies, B2M1, anti-RSV antibody was generated, purified in IgG4 PAA format (VH with SEQ ID NO: 106, VL with SEQ ID NO: 107) and combined with either the anti-CD3 arm CD3B219-F405L, R409K to produce CD3B288 (CD3 X null), or with shoulders to PSMA, PSMB162, PSMB126, PSMB130 to obtain PS3B37, PS3B39 and PS3B40, respectively (PSMAxnull).

Table 10

Obtained bispecific antibodies to PSMA x CD3

Bispecific antibody SHOULDER 1 SEQIDNO: SHOULDER 2 SEQ Ш NO: (VH;VL IgG4PAA F405L, R409K) NS L.C. NS L.C. PS3B2 PSMB120 (FAB PSMB25) 99 100 CD3B217 108 109 PS3B3 PSMB121 (FAB PSMB49) 97 98 CD3B217 108 109 PS3B4 PSMB122 (FAB PSMB51) 96 83 CD3B217 108 109 PS3B5 PSMB123 (FAB PSMB55) 94 95 CD3B217 108 109

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PS3B7 PSMB87 (FAB PSMB58) 125 91 CD3B217 108 109 PS3B8 PSMB126 (FAB PSMB80) 90 91 CD3B217 108 109 PS3B9 PSMB127 (FAB PSMB83) 88 89 CD3B217 108 109 PS3B10 PSMB128 (FAB PSMB84) 86 87 CD3B217 108 109 PS3B11 PSMB129 (FAB PSMB109) 82 83 CD3B217 108 109 PS3B12 PSMB130 (FAB PSMB86) 84 85 CD3B217 108 109 PS3B19 PSMB119 (FAB PSMB18) 101 100 CD3B219 110 111 PS3B20 PSMB120 (FAB PSMB25) 99 100 CD3B219 110 111 PS3B21 PSMB121 (FAB PSMB49) 97 98 CD3B219 110 111 PS3B22 PSMB122 (FAB PSMB51) 96 83 CD3B219 110 111 PS3B23 PSMB123 94 95 CD3B219 110 111

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(FAB PSMB55) PS3B24 PSMB124 (FAB PSMB56) 92 93 CD3B219 110 111 PS3B25 PSMB87 (FAB PSMB58) 125 91 CD3B219 110 111 PS3B26 PSMB126 (FAB PSMB80) 90 91 CD3B219 110 111 PS3B27 PSMB127 (FAB PSMB83) 88 89 CD3B219 110 111 PS3B28 PSMB128 (FAB PSMB84) 86 87 CD3B219 110 111 PS3B29 PSMB129 (FAB PSMB109) 82 83 CD3B219 110 111 PS3B30 PSMB130 (FAB PSMB86) 84 85 CD3B219 110 111

A monospecific anti-CD3 antibody CD3B217 was created containing the VH and VL regions, having VH with SEQ ID NO: 102 and VL with SEQ ID NO: 103, and an IgG4 constant region with S228P substitutions,

F234A, L235A, F405L and R409K. CD3B217 contains a heavy chain of SEQ ID NO: 108 and a light chain of SEQ ID NO: 109. A monospecific CD3 antibody CD3B219 was generated containing the VH and VL regions, having VH of SEQ ID NO: 104 and VL of SEQ ID NO: 105 , and the IgG4 constant region with substitutions S228P, F234A, L235A, F405L and R409K. CD3B219 contains a heavy chain of SEQ ID NO: 110 and a light chain of SEQ ID NO: 111. As a control, a monospecific RSV antibody derived from B21M was generated containing the VH and VL regions having the VH and VL of SEQ ID NO: 106 and VL SEQ ID NO: 107, and an IgG4 constant region with S228P, F234A, L235A or F234A, L235A, R409K, F405L to partner as the null arm of either the CD3 or PSMA bispecific antibody.

The PS3B2 bispecific antibody contains the CD3-binding mAb arm CD3B217-F405L, R409K and the PSMA-binding mAb arm PSMB120.

Bispecific antibody PS3B3 contains the CD3-binding mAb arm CD3B217-F405L, R409K and the PSMA-binding mAb arm PSMB121-R409.

The PS3B4 bispecific antibody contains the CD3-binding mAb arm CD3B217-F405L, R409K, and the PSMA-binding mAb arm I3RB122-R409.

Bispecific antibody PS3B5 contains the CD3-binding mAb arm CD3B217-F405L, R409K and the PSMA-binding mAb arm PSMB123-R409.

Bispecific antibody PS3B7 contains the CD3-binding mAb arm CD3B217-F405L, R409K and the PSMA-binding mAb arm PSMB58-R409.

Bispecific antibody PS3B8 contains the CD3-binding mAb arm CD3B217-F405L, R409K and the PSMA-binding mAb arm PSMB126-R409.

Bispecific antibody PS3B9 contains the CD3-binding mAb arm CD3B217-F405L, R409K and the PSMA-binding mAb arm PSMB127-R409.

Bispecific antibody PS3B10 contains the CD3B217-F405L, R409K mAb arm that binds

- 106 046641 with CD3, and the mAb PSMB128-R409 arm that binds to PSMA.

Bispecific antibody PS3B11 contains the CD3-binding mAb arm CD3B217-F405L, R409K and the PSMA-binding mAb arm PSMB129-R409.

Bispecific antibody PS3B12 contains the CD3-binding mAb arm CD3B217-F405L, R409K and the PSMA-binding mAb arm PSMB130-R409.

Bispecific antibody PS3B19 contains the CD3-binding mAb arm CD3B219-F405L, R409K and the PSMA-binding mAb arm PSMB119-R409.

Bispecific antibody PS3B20 contains the CD3-binding mAb arm CD3B219-F405L, R409K and the PSMA-binding mAb arm PSMB120-R409.

Bispecific antibody PS3B21 contains the CD3-binding mAb arm CD3B219-F405L, R409K and the PSMA-binding mAb arm PSMB121-R409.

Bispecific antibody PS3B22 contains the CD3-binding mAb arm CD3B219-F405L, R409K and the PSMA-binding mAb arm PSMB122-R409.

Bispecific antibody PS3B23 contains the CD3-binding mAb arm CD3B219-F405L, R409K and the PSMA-binding mAb arm PSMB123-R409.

Bispecific antibody PS3B24 contains the CD3-binding mAb arm CD3B219-F405L, R409K and the PSMA-binding mAb arm PSMB124-R409.

Bispecific antibody PS3B25 contains the CD3-binding mAb arm CD3B219-F405L, R409K and the PSMA-binding mAb arm PSMB165-R409.

Bispecific antibody PS3B26 contains the CD3-binding mAb arm CD3B219-F405L, R409K and the PSMA-binding mAb arm PSMB126-R409.

The bispecific antibody PS3B27 contains the CD3-binding arm CD3B219-F405L, R409K, and the PSMA-binding arm of the mAb PSMB127-R409.

The bispecific antibody PS3B28 contains the CD3-binding arm CD3B219-F405L, R409K, and the PSMA-binding arm of the mAb PSMB128-R409.

The bispecific antibody PS3B29 contains the CD3-binding arm CD3B219-F405L, R409K, and the PSMA-binding arm of the mAb PSMB129-R409.

The bispecific antibody PS3B30 contains the CD3-binding arm CD3B219-F405L, R409K, and the PSMA-binding arm of the mAb PSMB130-R409.

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Table 11

Sequences of bispecific antibodies to PSMA x CD3

ID. bispec number Amino acid severe sequence chains: Leverage to PSMA Shoulder to CD3 SEQ Ш NO Amino acid light chain sequences: Leverage to PSMA Shoulder to CD3 SEQ ID NO PS3B2 (PSMB12 0 X CD3B217) EVQLVQSGAEVKKPGESLKI SCKGSGYSFTSYWIGWVRQ MPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSISTA YLQWSSLKASDTAMYYCA RGWAYDRGLDYWGQGTLV TVS S ASTKGPS VFPLAPC SRS TSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVP SSSL GTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEAA GGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG K 99 DIVMTQSPDSLAVSLGE RATINCKSSQSVLYSSN NKNYLAWYQQKPGQPP KLLIYWASTRESGVPDR FSGSGSGTDFTLTISSLQ AEDVAVYYCQQYYSTP LTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKV QWKVDNALQSGNSQES VTEQDSKDSTYSLSSTL TLS KADYEKHKVYACE VTHQGLSSPVTKSFNRG EU 100

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EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY YCVKHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLLSGAQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 109 PS3B3 (PSMB12 1 X CD3B217) QVQLVQSGAEVKKPGSSVK VSCKASGGTFSSYAISWVRQ APGQGLEWMGGIIPIFGTAN YAQKFQGRVTITADESTSTA YMELS SLRSEDTAVYYC AR ASRVWHASYGYLDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSV VTVP 97 EIVLTQSPATLSLSPGER ATLSCRASQSVSKWLA WYQQKPGQAPRLLIYD ASNRATGIPARF SGSGS GTDFTLTIS SLEPEDF AV YYCQQRFTAPWTFGQG TKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLL NNFYPREAKVQWKVD NALQSGNSQESVTEQDS 98

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SSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAP EAAGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVY TLPP SQEEMTKNQ CL V KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG K KDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGL SSPVTKSFNRGEC EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY YCVKHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKD YFPEP VT VS WNSGALT SGVHTFPAVLQSSGLYSLSS V VTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLLSGAQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 109

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PS3B4

CD3B217 (PSMB12 x

WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHEALHNHYT QKSLSLSLGK QVQLVQSGAEVKKPGSSVK VSCKASGGTFSSYAISWVRQ APGQGLEWMGWIIPYNGNA NYAQKFQGRVTITADESTST AYMELS SLRSEDTAVYYC A RVNSAALVWERLDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVP S S SLGTKT YTCNVDHKP SNT KVDKRVESKYGPPCPPCPAP EAAGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVY TLPP SQEEMTKNQ V SLTCL V KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG K 96 DIQMTQSPSSLSASVGD RVTITCRASQSIDRWLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSPRYPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKAD Y EKHKVYACEVTHQGLS SPVTKSFNRGEC 83 EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY YCVKHGNFGNSYVSWFAY 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGAQPE DEAEYYCALWYSNLW 109

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WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTK PREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTEC s PS3B5 (PSMB12 3 x CD3B217) EVQLVQSGAEVKKPGESLKI SCKGSGYSFTSYWIGWVRQ MPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSISTA YLQWSSLKASDTAMYYCA RGLPIWYLDYWGQGTLVTV SSASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSL GT KTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLN 94 EIVLTQSPATLSLSPGER ATLSCRASQSVASDLA WYQQKPGQAPRLLIYF ASNRATGIPARF SGSGS GTDFTLTIS SLEPEDF AV YYCQQSITWPFTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKH KVYACEVTHQGLS SPVTKSFNRGEC 95

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GKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY YCVKHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHEALHNHYT QKSLSLSLGK 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLLSGAQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 109 PS3B7 (PSMB87 X EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI 109

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CD3B217) YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY YCVKHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSV FLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHEALHNHYT QKSLSLSLGK GGTNKRAPGTPARFSGS LLGGKAALTLLSGAQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTEC s EVQLVQSGAEVKKPGESLKI SCKGSGYSFTSYWISWVRQ MPGKGLEWMGIIYPGDSYT RYSPSFQGQVTISADKSISTA YLQWSSLKASDTAMYYCA RD YEWELFD SRLD YWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVT VP SSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAP EAAGGPSVFLFPPKPKDTLM 125 DIQMTQSPSSLSASVGD RVTITCRASQSISSYLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSYSTPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKHKVYACEVTHQGLS SPVTKSFNRGEC 91

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ISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVY TLPP SQEEMTKNQ VSLTCL V KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHE ALHNHYTQKSLSLSLG K PS3B8 (PSMB 12 6 X CD3B217) QVQLVQSGAEVKKPGSSVK VSCKASGGTFDDYAISWVR QAPGQGLEWMGRIDPIEGT ANYAQKFQGRVTITADESTS TAYMELSSLRSEDTAVYYC ARDRYYYDGVYWYSDYFD YWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLY SLS S VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPS QEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQ EGNVF SC S VMHE ALHNHYT 90 DIQMTQSPSSLSASVGD RVTITCRASQSISSYLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTIS SLQPEDF AT YYCQQSYSTPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKAD Y EKHKVYACEVTHQGLS SPVTKSFNRGEC 91

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QKSLSLSSLGK EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY YCVKHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLLSGAQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 109 EVQLLESGGGLVQPGGSLR LSCAASGFTFKSDAMHWVR QAPGKGLEWVSEISGSGGY TNYADSVKGRFTISRDNSKN TLYLQMNSLRAEDTAVYYC ARDSYDSSLYVGDYFDYW GQGTLVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSG 88 EIVLTQSPATLSLSPGER ATLSCRASQSVSSYLA WYQQKPGQAPRLLIYD ASNRATGIPARF SGSGS GTDFTLTIS SLEPEDF AV YYCQQRSNWPLTFGQG TKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLL NNFYPREAKVQWKVD 89

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VHTFPAVLQ SSGL YSLS S VV T VP S S SLGTKT YTCNVDHKP SNTKVDKRVESKYGPPCPPC PAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLT VLHQDWLNGKEYKCKVSN KGLPS SIEKTI SKAKGQPREP QVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLS LSLGK NALQSGNSQESVTEQDS KDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGL SSPVTKSFNRGEC EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY YCVKHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLLSGAQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 109

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PS3B10 (PSMB12

CD3B217

NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHEALHNHYT QKSLSLSLGK EVQLLESGGGLVQPGGSLR LSCAASGFTFDSDWMSWVR QAPGKGLEWVSAISGNGGS TEYADSVKGRFTISRDNSKN TLYLQMNSLRAEDTAVYYC ARDPYYYYDGDSYYGMDV WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQ EGNVF SC S VMHEALHNHYT QKSLSLSLGK 86 DIQMTQSPSSLSASVGD RVTITCRASQSISSYLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSYSTPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKHKVYACEVTHQGLS SPVTKSFNRGEC 87 EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGAQPE 109

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YCVKHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQF NWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTEC s PS3B11 (PSMB12 9 x CD3B217) QVQLVQSGAEVKKPGSSVK VSCKASGGTFSSYAISWVRQ APGQGLEWMGWISPYNGN ANYAQKFQGRVTITADESTS TAYMELSSLRSEDTAVYYC ARVNSAALVWERLDYWGQ GTLVTVSSASTKGPSVFPLA PCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTV SL GTKT YTCNVDHKPSN TKVDKRVESKYGPPCPPCPA PEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKT 82 DIQMTQSPSSLSASVGD RVTITCRASQSIDRWLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSPRYPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKAD Y EKHKVYACEVTHQGLS SPVTKSFNRGEC 83

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KPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSL GK EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY YCVKHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCP APEAAGGP S VFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK N QVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLLSGAQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 109 QVQLVQSGAEVKKPGSSVK 84 EIVLTQSPGTLLSLSPGER 85

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(PSMB13 0 X CD3B217) VSCKASGGTFKSYDISWVR QAPGQGLEWMGGIIPIEGTA NYAQKFQGRVTITADESTST AYMELS SLRSEDTAVYYC A RDYPAGYGFDYWGQGTLV TVS S AS TKGPS VFPLAPC SRS TSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKV D KRVESKYGPPCPPCPAPEAA GGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPE NNY KTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG K ATLSCRASQSVSSSYLA WYQQKPGQAPRLLIYG AS SRATGIPDRF SGSGS GTDFTLTISRLEPEDFAV YYCQQYGSSPLTFGQG TKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLL NNFYPREAKVQWKVD NALQSGNSQESVTEQDS KDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGL SSPVTK SFNRGEC EVQLLESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVY YCVKHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD 108 QAVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGAQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL 109

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NQVSLTCLVKGFYPSDIAVE W ESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK SLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTEC s PS3B29 (PSMB12 9 x CD3B219) QVQLVQSGAEVKKPGSSVK VSCKASGGTFSSYAISWVRQ APGQGLEWMGWISPYNGN ANYAQKFQGRVTITADESTS TAYMELSSLRSEDTAVYYC ARVNSAALVWERLDYWGQ GTLVTVSSASTKGPSVFPLA PCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTV TKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPA PEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCL VK GFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLY 82 DIQMTQSPSSLSASVGD RVTITCRASQSIDRWLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSPRYPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKAD Y EKHKVYACEVTHQGLS SPVTKSFNRGEC... 83

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SRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSL GK EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 111 PS3B30 (PSMB13 0 X CD3B219) QVQLVQSGAEVKKPGSSVK VSCKASGGTFKSYDISWVR QAPGQGLEWMGGIIPIEGTA NYAQKFQGRVTITADESTST AYMELS SLRSEDTAVYYC A RDYPAGYGFDYWGQGTLV TVS S ASTKGPS VFPLAPC SRS 84 EIVLTQSPGTLLSLSPGER ATLSCRASQSVSSSYLA WYQQKPGQAPRLLIYG AS SRATGIPDRF SGSGS GTDFTLTISRLEPEDFAV YYCQQYGSSPLTFGQG TKVEIKRTVAAPSVFIFP 85

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TSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEAA GGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQ DWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPP SQEEMTKNQ VSLTCL V KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG K PSDEQLKSGTASVVCLL NNFYPREAKVQWKVD NALQSGNSQESVTEQDS KDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGL SSPVTKSFNRGEC EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 111

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CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVFSCSVMHEALHNHYT QKSLSLSLGK PS3B28 (PSMB12 8 x CD3B219) EVQLLESGGGLVQPGGSLR LSCAASGFTFDSDWMSWVR QAPGKGLEWVSAISGNGGS TEYADSVKGRFTISRDNSKN TLYLQMNSLRAEDTAVYYC ARDPYYYYDGDSYYGMDV WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKD YFPEP VT VS WNSGALT SGVHTFPAVLQSSGLYSLSS V VTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK 86 DIQMTQSPSSLSASVGD RVTITCRASQSISSYLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSYSTPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKHKVYACEVTHQGLS SPVTKSFNRGEC 87 EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI 111

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YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSV FLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHEALHNHYT QKSLSLSLGK GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTEC s PS3B27 (PSMB12 7 x CD3B219) EVQLLESGGGLVQPGGSLR LSCAASGFTFKSDAMHWVR QAPGKGLEWVSEISGSGGY TNYADSVKGRFTISRDNSKN TLYLQMNSLRAEDTAVYYC ARDSYDSSLYVGDYFDYW GQGTLVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSG VHTFPAVLQ SSGL YSLS S VV T VP S S SLGTKT YTCNVDHKP SNTKVDKRVESKYGPPCPPC PAPEAAGGPSVFLFPPKPKD 88 EIVLTQSPATLSLSPGER ATLSCRASQSVSSYLA WYQQKPGQAPRLLIYD ASNRATGIPARF SGSGS GTDFTLTIS SLEPEDF AV YYCQQRSNWPLTFGQG TKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLL NNFYPREAKVQWKVD NALQSGNSQESVTEQDS KDSTYSLSSTLTLSKAD YEKHK VYACEVTHQGL SSPVTKSFNRGEC 89

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S CSVMHEALHNHYTQKSLS LSLGK EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHEALHNHYT 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 111

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QKSLSLSSLGK PS3B26 (PSMB12 6 X CD3B219) QVQLVQSGAEVKKPGSSVK VSCKASGGTFDDYAISWVR QAPGQGLEWMGRIDPIEGT ANYAQKFQGRVTITADESTS TAYMELSSLRSEDTAVYYC ARDRYYYDGVYWYSDYFD YWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLY SLS S VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPS QEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK 90 DIQMTQSPSSLSASVGD RVTITCRASQSISSYLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSYSTPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKHKVYACEVTHQGLS SPVTKSFNRGEC 91 EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT 111

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SGVHTFPAVLQSSGLYSLSS VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKA K GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTEC s PS3B25 (PSMB87 X CD3B219 ) EVQLVQSGAEVKKPGESLKI SCKGSGYSFTSYWISWVRQ MPGKGLEWMGIIYPGDSYT RYSPSFQGQVTISADKSISTA YLQWSSLKASDTAMYYCA RD YEWELFD SRLD YWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVT VP SSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAP EAAGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVY TLPP SQEEMTKNQ CL V 125 DIQMTQSPSSLSASVGD RVTITCRASQSISSYLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSYSTPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKHKVYACEVTHQGLS SPVTKSFNRGEC 91

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KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG K EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHEALHNHYT QKSLSLSLGK 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 111 PS3B24 (PSMB12 4 x CD3B219 QVQLVQSGAEVKKPGSSVK VSCKASGGTFSSYAISWVRQ APGQGLEWMGWISPYNGN ANYAQKFQGRVTITADESTS TAYMELSSLRSEDTAVYYC 92 DIQMTQSPSSLSSASVGD RVTITCRASQSISGWLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT 93

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) ARD SDRS YNLDYWGQGTL YYCQQSYSTPLTFGQGT VTVSSASTKGPSVFPLAPCS RSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSS SLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPE AAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTK K KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKHKVYACEVTHQGLS SPVTKSFNRGEC EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFP AVLQS SGL YSLS S VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 111

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VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHEALHNHYT QKSLSLSLGK PS3B23 (PSMB12 3 x CD3B219) EVQLVQSGAEVKKPGESLKI SCKGSGYSFTSYWIGWVRQ MPGKGLEWMGIIYPGD SDT RYSPSFQGQVTISADKSISTA YLQWSSLKASDTAMYYCA RGLPIWYLDYWGQGTLVTV SSASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSS SLGT KTYTCNVDHKPSNTKVDKR VESKYGPPCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTC LVKGFYP SDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK 94 EIVLTQSPATLSLSPGER ATLSCRASQSVASDLA WYQQKPGQAPRLLIYF ASNRATGIPARF SGSGS GTDFTLTIS SLEPEDF AV YYCQQSITWPFTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKH KVYACEVTHQGLS SPVTKSFNRGEC 95 EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY 111

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QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVES KYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SD GSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTEC s PS3B22 (PSMB12 3 x CD3B219) QVQLVQSGAEVKKPGSSVK VSCKASGGTFSSYAISWVRQ APGQGLEWMGWIIPYNGNA NYAQKFQGRVTITADESTST AYMELS SLRSEDTAVYYC A RVNSAALVWERLDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVP SSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAP 96 DIQMTQSPSSLSASVGD RVTITCRASQSIDRWLN WYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFAT YYCQQSPRYPLTFGQGT KVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLN NFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKAD Y EKHKVYACEVTHQGLS 83

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EAAGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVY TLPP SQEEMTKNQ VSLTCL V KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RL TVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG TO SPVTKSFNRGEC EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 111

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EGNVF SC S VMHEALHNHYT QKSLSLSSLGK PS3B21 (PSMB12 ix, CD3B219) QVQLVQSGAEVKKPGSSVK VSCKASGGTFSSYAISWVRQ APGQGLEWMGGIIPIFGTAN YAQKFQGRVTITADESTSTA YMELS SLRSEDTAVYYC AR ASRVWHASYGYLDYWGQG TLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSV VTVP SSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAP EAAGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVY TLPP SQEEMTKNQ VS LTCL V KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG K 97 A KVYACEVTHQGL SSPVTKSFNRGEC 98 EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN 111

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VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHE ALHNHYT QKSLSLSLGK KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTEC s PS3B20 (PSMB12 0 X CD3B219) EVQLVQSGAEVKKPGESLKI SCKGSGYSFTSYWIGWVRQ MPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSISTA YLQWSSLKASDTAMYYCA RGWAYDRGLDYWGQGTLV TVS S ASTKGPS VFPLAPC SRS TSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVP SSSL GTKTYTCNVDHKPSNTKVD KRVESKYGPPPPCPPAPEAA GGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYT 99 DIVMTQSPDSLAVSLGE RATINCKSSQSVLYSSN NKNYLAWYQQKPGQPP KLLIYWASTRESGVPDR FSGSGSGTDFTLTISSLQ AEDVAVYYCQQYYSTP LTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKV QWKVDNALQSGNSQES VTEQDSKDSTYSLSSTL TLS KADYEKHKVYACE VTHQGLSSPVTKSFNRG EC 100

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LPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG K EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSS S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQ VSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLD SDGSFLLYSKLTVDKSRWQ EGNVF SC S VMHEALHNHYT QKSLSLSLGK 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 111 PS3B19 (PSMB11 9 x CD3B219 EVQLVQSGAEVKKPGESLKI SCKGSGYSFTSYWIGWVRQ MPGKGLEWMGIIYPGDSDT RYSPSFQGQVTISADKSISTA 101 DIVMTQSPDSLAVSLGE RATINCKSSQSVLYSSN NKNYLAWYQQKPGQPP KLLIYWASTRESGVPDR 100

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) YLQWSSLKASDTAMYYCA RAYHYSKGLDYWGQGTLV TVS S ASTKGPS VFPLAPC SRS TSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEAA GGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQED PEVQ FNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLG K FSGSGSGTDFTLTISSLQ AEDVAVYYCQQYYSTP LTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKV QWKVDNALQSGNSQES VTEQDSKDSTYSLSSTL TLSKADYEKHKVYACE VTHQGLSSPVTKSFNRG EC EVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVR QAPGKGLEWVARIRSKYNN YATYYAASVKGRFTISRDDS KNSLYLQMNSLKTEDTAVY YCARHGNFGNSYVSWFAY WGQGTLVTVSSASTKGPSV FPLAPCSRSTSESTAALGCL VKD YFPEP VT VS WNSGALT SGVHTFPAVLQSSGLYSLSS VVTVP S S SLGTKT YTCNVD HKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVD 110 QTVVTQEPSLTVSPGGT VTLTCRSSTGAVTTSNY ANWVQQKPGQAPRGLI GGTNKRAPGTPARFSGS LLGGKAALTLSGVQPE DEAEYYCALWYSNLW VFGGGTKLTVLGQPKA APSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSY SCQ VTHEGSTVEKTVAPTEC s 111

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VSQEDPEVQFNWYVDGVE

VHNAKTKPREEQFNSTYRV

VSVLTVLHQDWLNGKEYK

CKVSNKGLPSSIEKTISKAK

GQPREPQVYTLPPSQEEMTK

NQVSLTCLVKGFYPSDIAVE

WESNGQPENNYKTTPPVLD

SDGSFLLYSKLTVDKSRWQ

EGNVF SC S VMHE ALHNHYT

QKSLSLSSLGK

Table 12

VH/VL sequences of bispecific antibodies to PSMA x CD3

Bispecific antibody SHOULDER 1 SEQ Ш NO: SHOULDER 2 SEQ Ш NO: (VH;VL IgG4PAA F405L, R409K) VH VL VH VL PS3B2 PSMB120 (FAB PSMB25) 77 78 CD3B217 102 103 PS3B3 PSMB121 (FAB PSMB49) 75 76 CD3B217 102 103 PS3B4 PSMB122 (FAB PSMB51) 74 61 CD3B217 102 103 PS3B5 PSMB123 (FAB PSMB55) 75 76 CD3B217 102 103 PS3B7 PSMB87 (FAB PSMB58) 160 64 CD3B217 102 103 PS3B8 PSMB126 68 69 CD3B217 102 103

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(FAB PSMB80) PS3B9 PSMB127 (FAB PSMB83) 66 67 CD3B217 102 103 PS3B10 PSMB128 (FAB PSMB84) 64 65 CD3B217 102 103 PS3B11 PSMB129 (FAB PSMB109) 60 61 CD3B217 102 103 PS3B12 PSMB130 (FAB PSMB86) 62 63 CD3B217 102 103 PS3B19 PSMB119 (FAB PSMB18) 79 78 CD3B219 104 105 PS3B20 PSMB120 (FAB PSMB25) 77 178 CD3B219 104 105 PS3B21 PSMB121 (FAB PSMB49) 75 76 CD3B219 104 105 PS3B22 PSMB122 (FAB PSMB51) 74 61 CD3B219 104 105 PS3B23 PSMB123 (FAB PSMB55) 75 76 CD3B219 104 105 PS3B24 PSMB124 (FAB 70 71 CD3B219 104 105

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PSMB56) PS3B25 PSMB87 (FAB PSMB58) 160 65 CD3B219 104 105 PS3B26 PSMB126 (FAB PSMB80) 68 69 CD3B219 104 105 PS3B27 PSMB127 (FAB PSMB83) 66 67 CD3B219 104 105 PS3B28 PSMB128 (FAB PSMB84) 64 65 CD3B219 104 105 PS3B29 PSMB129 (FAB PSMB109) 60 61 CD3B219 104 105 PS3B30 PSMB130 (FAB PSMB86) 62 63 CD3B219 104 105

Example 7 Cell Binding Characterization.

PSMA x CD3 bispecific antibodies were tested for binding to PSMA-positive LNCAP cell lines, human PSMA-HEK, chimpanzee PSMA-HEK, and cynomolgus PSMA-HEK. A cell binding assay (described previously) was used to evaluate the binding capabilities of PSMA bispecific antibodies. Briefly, PSMA-expressing tumor cells are bound by bispecific antibodies at known concentrations, and bound antibody is detected using PE-conjugated human kappa light chain detection reagent (Invitrogen). Mean fluorescence intensity (MFI) is a measure of the bound bispecific antibody. CIF is converted to relative EC50. The EC50 is a widely used dose response curve where the half-maximal effective concentration or EC ₅₀ point is defined as the inflection point of the curve. EC ₅₀ was determined by measuring bispecific and known cell-bound concentrations. High concentrations resulted in maximum binding of the target antigen, i.e., complete saturation of binding. Dose-response curves were then diluted to baseline or until no bispecific binding occurred. The inflection point of the curve reflects the EU ₅₀ point. The calculated _EC50 is determined by taking the amount of bispecific antibody at the EC50 μg/ml point and converting it to a molarity value based on the molecular weight of the bispecific antibody. Bispecific antibodies were normalized to protein concentration and then incubated with the same number of cells expressing human or cynomolgus PSMA. The SIF for each concentration was obtained by flow cytometry and plotted as a function of concentration. Data were log10 transformed and then plotted. To determine EC50, binding curves were constructed using nonlinear regression. These relative values were used to rank PSMA binding to target cells. In table 12 shows the relative EC ₅₀ binding values for whole cell binding studies using cell lines expressing PSMA LNCaP, cynomolgus and chimpanzee.

In fig. 11 shows the binding of all bispecific antibodies obtained to LNCAP. The binding data indicates that 3 binding populations are observed: 1) strong binding, 2) moderate binding, and 3) weak/no binding. PSMA x null arm bispecific antibodies retained binding to LNCAP cells (Fig. 11 E), but no binding was observed for anti-CD3 x null arm bispecific antibodies (Fig. 11 F). Medium-affinity and high-affinity CD3 bispecific antibodies to the same PSMA arm bind in a similar manner. In the remaining assays to evaluate the binding activity of chimpanzee PSMA-HEK (Fig. 12), Java PSMA

- 141 046641 macaque (Fig. 13), human PSMA-HEK (Fig. 14) or the parent HEK293 (data not shown) used only bispecific antibodies that were positive binding to LNCaP. High- and medium-affinity CD3 binding was tested for each cell line. Human PSMA-HEK binding data indicate that there may be little difference between LNCaP and this cell line; however, the same general binding order is evident. PS3B19 produced by LNCaP panning appears to bind strongly to human PSMA-HEK cells. Bispecific antibodies exhibit a wide range of binding profiles to human PSMA-HEK. Interestingly, hits panned on the chimpanzee PSMA cell line have a stronger binding profile, whereas hits panned on the LNCaP line show weaker binding. No binding was observed in this experiment with parental HEK cells (data not shown).

Once recombined into bispecific antibodies, several clones consistently outcompete others and bind cross-species. These bispecific antibodies are PS3B21, PS3B22, PS3B26, PS3B27, PS3B28 and PS3B30, corresponding to the mAbs PSMB121, PSMB122, PSMB126, PSMB127, PSMB128 and PSMB130. Cellular binding of EC5o and calculated EC5o are shown in Table. 13.

Table 13

Cellular binding EC50

bsAb LNCaP Cynomolgus macaque Chimpanzee ec ₅₀ (µg/ml) Estimated EC ₅₀ (nM) EC ₅₀ (µg/ml) Estimated EC ₅₀ (nM) EC ₅₀ (µg/ml) Estimated EC ₅₀ (nM) PS3B19 too weak N/A 1.757 12.4 5.10 35.93 PS3B20 122.7 864.1 2,003 14.1 3.33 23.44 PS3B21 0.7368 5.2 0.8604 6.1 1.25 8.81 PS3B22 2,603 18.3 1.137 8 3.02 21.23 PS3B23 too weak N/A 3,744 26.4 0.88 6.18 PS3B24 too weak n/a 18.22 128.3 1,Yu 7.75 PS3B26 0.07147 0.5 0.3706 2.6 0.05 0.34 PS3B27 2.07 14.6 1.403 9.9 3.24 22.83 PS3B28 1.157 8.1 0.6879 4.8 1.01 7.08 PS3B29 7,766 54.7 2,539 17.9 4,I 28.93 PS3B30 1.203 8.5 5.37 37.8 3.29 23.19

All bispecific antibodies retained the ability to bind to PSMA-positive cell lines. Some of the antibodies bound well to chimpanzee cells and cynomolgus PSMA expression, but only weakly bound to LNCaP cells. LNCAP-binding EC50s ranged from 0.5 nM to 864 nM, while cynomolgus PSMA-expressing EC50s ranged from 0.9 to 128 nM, and chimpanzee HEK-binding PSMAs ranged from 36 to 0.3 nM ( table 12). Based on EC ₅₀ cell binding, several anti-PSMA bispecific antibodies met the criteria of 20 nM or stronger binding to human PSMA and 50 nM or stronger binding to cynomolgus PSMA.

Example 8. Affine characterization by Proteon and Biacore.

To further evaluate the antibodies, association and dissociation rates with chimpanzee PSMA were measured for hits that were obtained from cell binding assays. The interactions of PSMA x CD3 bispecific mAbs with the target (recombinant chimpanzee, PSMA) were examined by surface plasmon resonance (SPR) using the ProteOn XPR36 system (BioRad). The biosensor surface was prepared by binding anti-human IgG Fc antibody (Jackson

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ImmunoResearch Laboratory cat. No. 109-005-098) with an alginate polymer layer modified surface of the GLC chip (BioRad, Cat. No. 176-5011) using the manufacturer's instructions for performing the amine coupling chemistry. Approximately 4400 units (RU) of anti-human IgG Fc antibodies were immobilized. Kinetic experiments were performed at 25°C in a running buffer solution (DPBS + 0.03% polysorbate P20 + 100 μg/ml BSA). To perform kinetic experiments, 100 RU of bispecific antibodies were captured, followed by injections of analytes (recombinant chimpanzee PSMA ECD) at concentrations ranging from 3.7 nM to 300 nM (in 3-fold serial dilution). The association phase was monitored for 3 minutes at 50 μl/min followed by 15 minutes of buffer flow (dissociation phase). The chip surface was regenerated with two 18-second pulses of 100 mM phosphoric acid (H ₃ PO ₄ Sigma, cat. no. 7961) at 100 μl/min.

The obtained data were processed using ProteOn Manager software. First, the data was corrected in relation to the background using intermediate sections. Subsequently, double subtraction of the data reference was performed by injecting the buffer solution instead of injecting the analytes. Kinetic data analysis was performed using a 1:1 Langmuir binding model. The result for each bispecific antibody was recorded in the format k _a (association rate), kd (dissociation rate) and KD (equilibrium dissociation constant). The results are shown in table. 14-18.

Results.

Table 14. Summary of binding kinetics and affinity of PS3B25 and PS3B27 to recombinant human PSMA (3.7-300 nM). The parameters shown in this table were obtained from the 1:1 Langmuir binding model. Affinity, KD=kd/k _a .

Table 14

also called ID. bispecific protein number k _a (1/M*s) 10 ⁵ ka (1/s) 10' 03 K _D (nM) PSMB87 x CD3B219 PS3B25 1.88±0.13 1,ONO,05 5.38±0.55 PSMB127 x CD3B219 PS3B27 2.87±0.36 2.89±0.70 10.3±3.2

n=3 independent experiments with 2 replicates

Results are presented as mean ± standard deviation.

Table 15. Summary of binding kinetics and affinity of PS3B25 and PS3B27 to recombinant chimpanzee PSMA (3.7-300 nM). The parameters shown in this table were obtained from the 1:1 Langmuir binding model. Affinity, KD=kd/k _a .

Table 15

also called ID. protein number k _a (1/M*s) ka (1/s) 10 K _D (nM) bispecific antibodies 10 ⁵ 03 PSMB87 x CD3B219 PS3B25 2.81±0.08 0.99±0.04 3.54±0.25 PSMB127 x CD3B219 PS3B27 2.08±0.38 1.56±0.37 7.48±0.97

n=3 independent experiments with 2 replicates

Results are presented as mean ± standard deviation.

Table 16. Summary of binding kinetics and affinity of PS3B25 and PS3B27 to recombinant cynomolgus PSMA (3.7-300 nM). The parameters shown in this table were obtained from the 1:1 Langmuir binding model. Affinity, KD=kd/k _a .

Table 16

also called ID. bispecific Ab protein number k _a (1/M*s) 10 ⁵ k _d (1/s) Yu ⁰³ K _D (nM) PSMB87 x CD3B219 PS3B25 0.98±0.04 7.97±0.34 81.1=1=3.3 PSMB127 x CD3B219 PS3B27 1.59±0.12 1.10±0.04 7.00±0.68

n=3 independent experiments with 2 replicates

- 143 046641

Results are presented as mean ± standard deviation.

Table 17. Comparison of binding affinities of PS3B25 and PS3B27 in humans, chimpanzees and cynomolgus monkeys. Affinity, KD=kd/k _a .

Table 17

Sample K _D (nM) human K _D (nM) chimpanzee K _D (nM) cynomolgus macaque PS3B25 5.38±0.55 3.54±0.25 81.1=1=3.3 PS3B27 10.3±3.2 7.48±0.97 7.00±0.68

Table 18. Summary of data on the kinetics and binding affinity of bispecific mAbs to recombinant chimpanzee PSMA (3.7-300 nM). The parameters shown in this table were obtained from the 1:1 Langmuir binding model. Affinity, KD=kd/k _a .

Table 18

ID. bispecific Ab protein number ka (1/M*s) kd (1/s) KD (nM) PS3B19 1.26E+04 2.98E-04 23.6 PS3B20 1.76E+04 3.78E-04 21.4 PS3B21 6.70E+04 4.73E-04 7.1 PS3B22 6.51E+04 1.24E-03 19 PS3B23 9.89E+05 1.76E-03 1.8 PS3B24 6.26E+03 2.52E-04 40.3 PS3B25 1.95E+05 1.06E-03 5.5 PS3B26 No binding PS3B27 1.70E+05 1.76E-03 10.4 PS3B28 1.34E+05 2,ZZE-OZ 17.4 PS3B29 6.71E+04 6.80E-03 101 PS3B30 7.34E+04 1.65E-03 22.4 PS3B31 No binding PS3B32 No binding PS3B33 No binding PS3B34 No binding PSMA zero No binding

For the most part, protein binding parallels cell binding. However, one of the bispecific antibodies did not demonstrate binding to recombinant chimpanzee PSMA ECD, although it bound to chimpanzee PSMA expressed on the cell surface of HEK cells. This PS3B26 antibody is a cell-only binder and was discarded from subsequent binding experiments. One of the positive binders, PS3B23, exhibited biphasic binding and did not fit the 1:1 binding pattern. Ten bispecific antibodies were positive for binding to recombinant chimpanzee PSMA ECD using Proteon, and their affinities were further profiled using BIACORE.

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Binding to recombinant chimpanzee PSMA via Biacore. Surface plasmon resonance (SPR) affinity measurements were performed using a Biacore 3000 optical biosensor (Biacore-GE Healthcare). The biosensor surface was prepared by coupling anti-human IgG Fc antibody (Jackson cat. no. 109-005-098) to carboxymethylated dextran on the surface of a CM-5 chip (BioRad, cat. no. BR-1000-12) using the manufacturer's chemical reaction instructions amine combination. Approximately 16,000 units (RU) of anti-human IgG Fc antibodies were immobilized in each of four flow cells. Kinetic experiments were performed at +25°C in a running buffer solution (DPBS + 0.03% polysorbate P20 + 100 μg/ml BSA). Antigen dilutions were prepared in running buffer (recombinant chimpanzee PSMA, concentration 1.2300 nM or 3.7-900 nM in 3-fold serial dilution). Approximately 100 RU of PSMA x CD3 bispecific mAb were captured in flow cells 2 to 4 of the sensor chip. Flow cell 1 was used as a reference. After PSMA x CD3 capture, a 3-5 minute injection (association phase) of antigen (recombinant chimpanzee PSMA) was performed at a rate of 50 μl/min, followed by a 15 or 20 minute injection of flow buffer (dissociation phase). The chip surface was regenerated with two 18-second injections of 100 mM phosphoric acid (Sigma H3PO4, cat. no. 7961) at 50 μl/min.

The obtained data were processed using the BIAevaluation software (Biacore). First, double reference subtraction of the data was performed by subtracting the plots obtained from the buffer injections from the reference plots obtained from the analyte injections. Kinetic data analysis was then performed using a 1:1 exact fit Langmuir binding model. The result for each PSMAxCD3 bispecific mAb was recorded in the format k _a (association rate), k _d (dissociation rate) and K _D (equilibrium dissociation constant).

Table 19

Summary of binding kinetics and affinities of bispecific mAbs to recombinant chimpanzee PSMA ECD using Biacore.

Ka (1/Ms) kd (1/s) KD (nM) PS3B19 6.24E+03 4D4E-04 66.3 PS3B20 7.82E+03 4.61E-04 59.1 PS3B21 1D0E+04 4.89E-04 44.4 PS3B22 1.66E+04 1.00E-03 60.4 PS3B23 1.34E+05 1.22E-03 9.1 PS3B24 2.35E+04 9.07E-04 38.6 PS3B25 6.59E+04 1.08E-03 16.4 PS3B27 8.90E+04 2.00E-03 22.4 PS3B28 6.20E+04 1.91E-03 30.8 PS3B29 2.74E+04 1.59E-03 58.1 PS3B30 3.48E+04 1.82E-03 52.3

All bispecific antibodies that bind to chimpanzee PSMA ECD by Proteon also bind by Biacore. Binding affinities were slightly weaker with Biacore.

Example 9: Evaluation of PSMA x CD3 bispecific antibodies in a functional cell killing assay.

T cell-mediated cytotoxicity assays were used to functionally screen for bispecific antibody activity. The bispecific antibodies were tested for their ability to lyse HEK cells overexpressing human PSMA, as well as the human prostate cancer cell line LNCAP. In addition, bispecific antibodies were tested for their ability to kill cynomolgus HEK PSMA cells to confirm species cross-reactivity.

A chromium-51 release assay was used to measure the cytotoxicity of individual bispecific antibodies. Cytotoxicity is measured by the amount of chromium released into the culture medium as a result of cell lysis in the presence of activated T cells. The magnitude of release is compared with spontaneous release of chromium only in target cells and maximum release through general lysis of target cells with Triton-X.

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Human pan-T cells (CD3+) from multiple donors were preactivated overnight in OKT3-coated flasks (1 μg/ml) and IL-2 at a concentration of 20 U/ml. T cells were washed twice. Target cells were labeled for 1 h with chromium-51. T cells and target cells were cultured in a 5:1 ratio for 18-24 hours, after which the culture supernatant was collected and analyzed. All points were analyzed in triplicate and reported as the average cytotoxicity value for triplicates and SEM. Bispecific antibody dilutions from 10 μg/ml to 0.00001 μg/ml were used.

In fig. 15 shows T cell-mediated killing of all anti-PSMA x CD3 bispecific antibodies against human PSMA-HEK cells. This experiment tested medium- and high-affinity CD3 bispecific antibodies. Cell killing is evident for most molecules with high affinity CD3 arms, with cell killing occurring at the lowest concentration tested. PS3B9 was the only intermediate affinity bispecific antibody demonstrating significant cell killing at lower concentrations. In fig. 16 shows T cell-mediated killing of several pairs of bispecific antibodies. It is apparent from these data that high-affinity bispecific antibodies binding to CD3 generate the most cell killing, and this has been the subject of additional cell killing experiments.

In fig. 17 shows cell killing data for anti-PSMA x CD3 bispecific antibodies with a high affinity CD3 arm generated against LNCAP cells. PS3B25, PS3B27, PS3B28, PS3B30, PS3B23, and PSB22 all exhibited T cell redirection activity. PS3B29 showed activity against human PSMA-HEK cell at the highest concentrations, but not against LNCAPB, indicating that since it is a weak PSMA binder, it may not bind well at low levels of PSMA expression. In contrast, PS3B25, PS3B27, and PS3B28 showed the highest tumor cell lysis by LNCAP. The bispecific antibody PS3B38, consisting of PSMB125 and a null arm against the RSV protein, had no cytolytic activity, as expected, due to its inability to bind to T cells.

To confirm the redirection function of cynomolgus PSMA T cells, bispecific antibodies were tested to lyse cynomolgus PSMA-HEK cells as shown in FIG. 18. All bispecific antibodies tested were able to kill cells expressing cynomolgus PSMA, with PS3B27 and PS3B28 having the highest activity. From previous cell binding studies, these PSMA arms had higher affinity for cynomolgus PSMA compared to human PSMA, and this observation was also reflected in the cell killing of cynomolgus PSMA targets. PS3B27 and PS3B30 were assayed to kill the parental HEK cell line and did not kill the parental HEK cell line (data not shown).

Anti-PSMA antibodies PSMB127 and PSMB130, which produce bispecific anti-PSMA x CD3 antibodies PS3B27 and PS3B30, respectively, were selected for additional analysis. PS3B27 and PS3B30 killed both human and cynomolgus PSMA targets and had a high-affinity CD3 arm. PS3B27 bound to cell lines expressing human PSMA with essentially the same EC50 of ~14.6 nM and cynomolgus monkey ~9.9 nM. PS3B30 bound to human PSMA with approximately a 5-6 fold difference in affinity, with an _EC50 of ~8-8.5 nM and cynomolgus PSMA ~37.857 nM. Although the difference between human binding and cynomolgus binding may be more than 5-fold, PS3B27 showed functional killing of both human and cynomolgus targets.

The interactions of PS3B27 with recombinant chimpanzee PSMA ECD were repeated, and interactions with recombinant cynomolgus PSMA ECD and human PSMA ECD were studied by surface plasmon resonance (SPR) using the ProteOn XPR36 system (BioRad), as described previously for recombinant chimpanzee PSMA ECD. A summary of the binding kinetics and affinity to ECD of chimpanzee, cynomolgus and human PSMA is shown in Table 1. 20. This bispecific antibody binds to all targets with essentially the same affinity.

Table 20

Summary of binding kinetics and affinity of PS3B25 and PS3B27 to recombinant PSMA ECDs

target k _a (1/M*s) 10 ⁵ cd (1/s) NG ⁰³ K _D (nM) PS3B25 PS3B27 PS3B25 PS3B27 PS3B25 PS3B27 Chimpanzee 2.81±0.08 2.08±0.38 0.99±0.04 1.56±0.37 3.54±0.25 7.48±0.97 Cynomolgus macaque 0.98±0.04 1.59±0.12 7.97±0.34 1.10±0.04 81.1=1=3.3 7.00±0.68 Human 1.88±0.13 2.87±0.36 1,ONO,05 2.89±0.70 5.38±0.55 10.3±3.2

- 146 046641

Evaluation of the bispecific antibody PS3B27 in a caspase assay.

T cell-mediated killing of PS3B27 was measured using a second cell toxicity assay. The caspase cytotoxicity assay indirectly measures cell death through cleavage of a fluorescent substrate by active caspase 3/7. Cleavage of the substrate results in the formation of a fluorescent DNA dye with fluorescence limited to the cell nucleus. Repeated fluorescence measurements are taken in each well throughout the assay using a motorized 10× magnification objective capable of accurately visualizing the cell(s) at the same coordinates. Target cell populations are identified based on certain size restrictions and/or the use of a secondary label.

Frozen pan CD3+ T cells (purchased from Biological Specialty Corporation, Colmar, PA) were isolated by negative selection against normal healthy donors. PSMA-expressing prostate cancer cells (LNCaP, C42) were cultured in RPMI 1640 supplemented with 10% HI FBS+ supplements (purchased from Life Technologies).

T cells and target cells were combined at an effector-target (E:T) ratio of 3:1 in RPMI without phenol red + 10% FBS and supplement (Life Technologies) without selection reagents, and 0.6 μl was added to each ml of cells NucView caspase reagent (Essen Bioscience) according to the manufacturer's recommendations. The total volume of cells was 0.1 ml in the corresponding wells of a transparent 96-well flat-bottom plate (BD Falcon). Bispecific antibodies PS3B27 (CD3xPSMA), CD3B288 (CD3xNull) or PS3B46 (PSMAxNull) were prepared at 2X final concentration in RPMI without phenol red prepared as above, and 0.1 ml of compounds was added to each well. After 30 min of incubation at room temperature to minimize cell aggregation at the edge of the wells, the plates were transferred to a Zoom Incucyte instrument (Essen Bioscience). Incucyte Instrument is placed in a humidified incubator at 37°C, 5% CO2.

Cell processing definitions were developed for each cell line tested according to the manufacturer's instructions. Measurements were taken every six hours until a plateau in caspase signal was observed, followed by three or more consecutive decreases from the maximum signal in the well(s) containing the highest concentration of test compound(s).

After completion of the assay, each plate was analyzed using the appropriate treatment definition. Raw fluorescence data were exported from Incucyte Zoom software and inserted into GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). Caspase 3/7 activity was determined by calculating the area under the curve (AUC) for each well in GraphPad. AUC values were plotted against Log10 nM compound. The EC ₅₀ for each dose curve, in nanomoles (nM), was recorded after nonlinear regression analysis (parameter fit 4, least ordinary squares). Each assay contained a minimum of three biological replicates, and each cell line was tested using five healthy donors. Data were further analyzed using preclinical statistics using a nonlinear regression model. Examples of graphs are shown in Fig. 19. The calculation results are shown in table. 21.

Table 21

Summary of EC ₅₀ values for analysis dependent on

T cell cytotoxicity

Donor LNCaP FGc S-42V M2550 0.27 nM 0.11 nM M5524 0.09 nM 0.06 nM M5772 0.04 nM 0.03 nM M7259 0.05 nM 0.06 nM M7444 0.09 nM 0.05 nM

Example 10 T cell activation by PS3B27 in PSMA positive cell lines.

Purified pan CD3+ T cells were obtained from normal healthy donors by Biological Specialty Corporation through negative selection of leukoapheresis white blood cells and stored frozen at -80°C or in liquid nitrogen until ready for use. Intact non-activated T cells were combined with target cells and anti-CD3 x PSMA bispecific antibodies or null controls (CD3 x null or PSMA x null) at an effector:target ratio of 3:1. After incubation for 48 hours, the supernatant was analyzed for cytokine secretion using an enzyme-linked immunosorbent assay (ELISA) (Meso Scale Discovery). Expression Mar

- 147 046641 CD25 T cell activation kernel was measured by flow cytometry by staining T cells for CD45, CD8, CD25 and near-infrared staining. CD8+/CD25+ populations were determined by initially gating a large population of cells (FSC-A versus SSC-A) to exclude cell debris and cell aggregates. The summed cell subset was further narrowed down to cells considered alive, eliminating staining for live/dead cells. Live cells were then gated for CD45+/CD8+ cells. Finally, a CD8+/CD25+ positive subset was identified. The EC ₅₀ for PS3B27 or control was obtained by plotting the percentage of CD8+/CD25+ versus Log10 nM bispecific antibody or control followed by nonlinear regression (parameter fit 4, least squares) (FIG. 20). All data analysis was performed in GraphPad Prism.

Example 11. Antitumor efficacy in preventing tumorigenesis in HEK293-PSMA xenografts in PBMC-humanized NSG mice.

All in vivo experiments were performed in accordance with the Institutional Animal Care and Use Guidelines and approved by the Institutional Animal Care and Use Committee of Janssen R&D, Spring House, PA, USA.

The efficacy of PS3B27 (PSMA x CD3 bispecific antibody) was assessed for the prevention of tumorigenesis (preventative model) in human HEK293-PSMA xenografts using inoculated human donor peripheral blood mononuclear cells (PBMCs) in male NSG mice (NOD.Cg-Prkdc ^scid IL2rg ^tmlWjl /SzJ or NOD SCID Gamma, Jackson Laboratories, Bar Harbor, Maine, USA). Mice were injected intravenously (i.v.) into the lateral tail vein with 1 x ¹⁰⁷ human PBMCs 7 days before tumor cell implantation. Mice were then implanted subcutaneously (SC) with 1 x 10 ⁷ HEK293-PSMA cells in the posterior right iliac fossa. Starting on the day of tumor implantation, control PBS (phosphate-buffered saline), PS3B27, CD3B288 (CD3 x null), or PS3B46 (PSMA x null) were administered IV at a dose of 0.4 mg/kg once every 2 to once every 3 days, 5 doses total on days 0, 3, 5, 7 and 10.

Tumor volume was calculated using the formula: Tumor volume (mm ³ ) = (a x b /2); where a represents the length and b the width of the tumor as determined using a measuring instrument, and was monitored twice a week throughout the study. The percentage of tumor growth inhibition (TGI) was defined as the difference between the mean tumor volumes in the treatment group and the control group, calculated as TGI = [((TVc - TVt) / TVc) * 100], where TVc represents the mean tumor volume in a given control group , and TVt represents the mean tumor volume in the treatment group. As defined by NCI criteria, a TGI >60% is considered biologically significant (Johnson, et al (2001) Br J Cancer 84(10): 1424-31). Animals were excluded from studies when the maximum tumor volume reached 1500 ^mm3 .

Engraftment of human PBMCs ultimately caused graft-versus-host disease (GVHD) in mice, in which the implanted donor T cells were activated and invaded host tissues, leading to weight loss, organ failure, and inevitable death. To monitor the onset and severity of GVHD, body weight was recorded twice weekly and expressed in grams (g). The percentage change in body weight was calculated using the formula: Change in body weight = [((Β,Β _ο )/Β _ο )*1ΟΟ], where B _t represents the body weight on a given day of the study, and B ₀ represents the body weight at the beginning of treatment . Animals with a permanent loss of body weight of more than 20% of the initial body weight were considered dying and excluded from the study.

Statistical significance was determined by 1-way analysis of variance (ANOVA) with multiple comparisons using Dunnett's multiple comparison test using Graph Pad Prism software (version 6). Treatment with PS3B27 effectively slowed down HEK293-PSMA tumorigenesis and tumor growth (Figure 21). Small but palpable HEK293PSMA tumors were detected in seven of eight mice in the PBS treatment group on day 16 of the study (6 days after the last therapeutic treatment), while only one of eight mice in the PS3B27 treatment group had a tumor. Five of eight mice had palpable tumors in the CD3B288 treatment group, and two of eight mice had small tumors in the PS3B46 group. Tumor growth inhibition was assessed 27 days after cessation of treatment (day 37 after tumor implantation), when there were at least 7 animals in each group. Tumor growth in the PSMA x CD3 bispecific antibody (PS3B27) treatment group was inhibited by 90% compared to PBS control animals (n=8 per group, p < 0.001). PSMA x null bispecific antibody (PS3B46) also inhibited tumorigenesis and growth in a statistically significant manner (TGI = 42%, N = 7) compared to PBS control (p < 0.05), although it was not considered a biologically significant effect based on the criteria NCI [1].

The group of animals receiving PBMCs eventually developed a progressive GVHD response, but there was little weight loss in the present study. There was a significant difference between the mean body weights of animals treated with 0.4 mg/kg PS3B27 compared to PBS, as shown in FIG. 22 to day 37 after tumor implantation (p>0.05). Therefore, PS3B27-mediated

- 148 046641 T cell redirection did not further contribute to GVHD-related weight loss.

Despite minor weight loss in the present study, deaths associated with sporadic GVHD were observed. One mouse in the PSMA bispecific antibody x PS3B46 null group was euthanized due to excessive (>20%) body weight loss at day 30 after tumor implantation. By day 42 after tumor implantation, additional GVHD-related deaths were recorded in PBS (n=1), anti-PSMA bispecific antibody PS3B46 group x null (n=2), and several additional mice were removed from the study due to reaching the volume endpoint tumors 1500 mm ³ , at which point the entire study was stopped.

Example 12: Efficacy of PS3B27 in preventing tumorigenesis in a mixture of HEK293-PSMA/T cell xenografts in male CD1 nude mice.

The efficacy of PS3B27 was assessed in a xenotransplantation model in which human pan CD3+ T cells and tumor cells were co-injected into male CD1 nude mice (NU-Foxn1nu, Charles River Laboratories, Wilmington, MA, USA).

Human anti-PSMA x CD3 bispecific antibody PS3B27 or control bispecific antibodies were administered intravenously every 2 to 3 days (once every 2 days or once every 3 days) for a total of 5 doses as indicated. Throughout the studies, mice were monitored (body weight and tumor measurement with calipers) twice a week. Drug doses expressed in μg/animal were converted to mg/kg based on 25 g body weight (example: 10 μg/animal=0.4 mg/kg). Drug doses administered in mg/kg were administered at 10 ml/kg based on body weight (example: mouse 25 g=0.25 ml).

Tumor volume was calculated using the formula: Tumor volume (mm ³ ) = (a x b2/2); where a represents the length and b the width of the tumor as determined using a measuring instrument, and was monitored every week throughout the study. The percentage of tumor growth inhibition (TGI) was defined as the difference between the mean tumor volumes in the treatment group and the control group, calculated as TGI = [((TVc - TVt) / TVc) * 100], where TVc represents the mean tumor volume in a given control group , and TVt represents the mean tumor volume in the treatment group. According to NCI criteria, TGI >60% is considered biologically significant [1]. Animals were excluded from studies when the maximum tumor volume reached 1500 ^mm3 .

The tolerability of PS3B27 cannot be assessed for CD3 binding in host tissues due to the lack of cross-reactivity of the CD3 arm to the corresponding murine antigens. However, the T cell injected with tumor cells expresses human CD3 and can bind to PS3B27 and CD3X x null controls. The percentage change in body weight was calculated using the formula: Change in body weight = [((Bt B0)/B0)*100], where Bt is the body weight on a given study day and B0 is the body weight at the start of treatment.

Statistical significance was determined by 1-way analysis of variance (ANOVA) with multiple comparisons using Dunnett's multiple comparison test using Graph Pad Prism software (version 6).

The efficacy of PS3B27 was assessed by preventing tumorigenesis in xenograft mixtures containing HEK293-PSMA cells and activated and expanded OO3-positive pan T cells at an effector-to-target ratio of 1:5 in male CD1 nude mice (reference ELN: CD3PSMA-2013-00003). T cells were activated and expanded in vitro using the IL-2 T Cell Activation/Expansion Kit (Miltenyi Biotech, Auburn, CA, USA, cat. no. 130-091-441, 130-097 -743), within 12 days. Mice were implanted sc with a mixture of HEK293-PSMA cells in the amount of 5x10 ⁶ and activated 1x10 ⁶ and T cells were expanded per mouse in 50% Cultrex (Trevigen, Gaithersburg, MD, USA, cat. no. 3433-005-01) and 50% serum-free medium RPMI 1640 into the right rear side. Beginning on the same day as tumor implantation, PBS, PS3B27 at a dose of 0.005-0.5 mg/kg, CD3B288 (bispecific antibody to CD3 x null) at a dose of 0.5 mg/kg, or PS3B46 (bispecific antibody to PSMA x zero) at a dose of 0.5 mg/kg IV, by body weight once every 2 - once every 3 days, a total of 5 doses on days 0, 2, 4, 7 and 9. (n=10 on group). Treatment with PS3B27 was also evaluated by IP administration (data not shown). One animal was removed every 46 and 49 days in the PBS control group for excess tumor burden. Tumor volume data were plotted until day 64, after which half of the control animals were removed from the study due to excess tumor volume.

As shown in FIG. 23, tumorigenesis and growth were assessed for 55 days after cessation of treatment (up to day 64). PS3B27 treatment significantly inhibited tumorigenesis and slowed growth compared to PBS control at all doses (0.005, 0.05, or 0.5 mg/kg), resulting in TGI of 73%, 81%, and 82%, respectively (p < 0.001, p <0.0001, p<0.001, respectively) on day 64. Treatment with PS3B27 by IP administration was as effective as IV administration (data not shown). Animals treated with CD3B288 (anti-CD3 bispecific antibody x null) or PS3B46 (anti-PSMA bispecific antibody x null) showed some antitumor activity with TGI of 51% and 38%, respectively, at day 64 (p<0.05, p= not significant, respectively), however this is not considered biologically significant based on the NCI 60% TGI criteria, demonstrating a requirement for bispecific binding

- 149 046641 antibodies with both CD3 and PSMA to achieve effectiveness.

No body weight loss was observed during the study, but animals treated with PS3B27 at 0.5 and 0.005 mg/kg had significantly less weight gain compared to PBS (p<0.001, p<0.0001, respectively, Fig 24), however this may be due to a lower tumor burden in these animals.

Example 13. Crystal structure of human PSMA ECD bound to the anti-PSMA Fab arm from the bispecific antibody PS3B27.

PSMA is a homodimeric protein expressed on the cell surface. PSMA is a type II integral glycoprotein of 750 residues per monomer, consisting of a large ECD domain (705 residues) with peptidase activity, a single-pass TM domain, and a short intracellular domain of 19 residues. The crystal structure of the extracellular region (ECR) of human PSMA bound to the Fab arm of PSMA from the bispecific antibody PS3B27 was determined at 3.15 Å resolution to better characterize the binding site between PSMA and the antibody.

The extracellular region of human PSMA (residues 44-750) was expressed in High Five insect cells with an N-terminal gp67 signal peptide followed by a cleavable hexahistidine tag (SEQ ID NO: 158).

Secreted protein was purified from the supernatant using a three-step procedure involving an initial Ni ²⁺ -NTA affinity capture, TEV-mediated histidine tag cleavage followed by a reverse affinity chromatography step and a final size exclusion chromatography step. Purified PSMA-ECD was frozen in liquid nitrogen and stored at -80°C in 10 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl ₂ , 0.1 mM ZnCl ₂

Fab PSMB83, which is the parent anti-PSMA Fab arm of the bispecific antibody PS3B27, was expressed in HEK293 Expi cells with a hexahistidine tag (SEQ ID NO: 158) and purified by affinity (HisTrap, GE Healthcare) and gel filtration chromatography (SEC- 300, Phenomenex Yarra). Fab was stored at 4°C in 50 mM NaCl, 20 mM Tris, pH 7.4.

The human PSMA/Fab PSMB83 ECD complex was prepared using a three-step procedure. Fab was first replaced with 20 mM MES, pH 6.0, 150 mM NaCl. Fab and PSMA were then mixed (1.5 molar excess of Fab relative to PSMA monomer) and incubated overnight at 4°C with dialysis in 20 mM MES, pH 6.0. Finally, the complex was coupled to a monoS 5/50 column in 20 mM MES, pH 6.0, and eluted with a NaCl gradient.

Crystals suitable for X-ray diffraction analysis were obtained using the sessile drop vapor diffusion method at 20°C and a Mosquito LCP robot (TTP Labtech). Fab crystals of PSMB83 associated with human PSMA ECD were grown from 18% PEG 3 kDa, 0.2 M (NH ₄ ) ₂ SO ₄ , 0.1 M Tris, pH 8.5 with microseeds and the PSMA/Fab complex initially at a concentration 7.3 mg/ml. Crystals of unbound PSMB83 Fab were prepared from 25% PEG 3 kDa, 0.2 M LiCl, 0.1 M acetate pH 4.5, with Fab initially contained at a concentration of 8.8 mg/ml.

The structures were solved by molecular replacement (MR) using Phaser (Phaser Crystallographic Software, University of Cambridge). The MR search model for the Fab PSMB83 structure was PDB code 4M6O. The structure of the PSMA/Fab complex was determined using the crystal structures of PSMA (PDB code: 2C6G) and PSMB83 Fab (1.93 Å resolution structure; data not shown) as MR search models. Structures were refined using PHENIX (Adams, et al, 2004) and model adjustments were made using COOT (Emsley and Cowtan, 2004). All other crystallographic calculations were carried out using the CCP4 software package (Collaborative Computational Project Number 4, 1994). All molecular graphics were generated using PyMol (PyMOL Molecular Graphics System, version 1.4.1, Schrodinger, LLC) and complementarity determining regions (CDRs) were defined using the Kabat definition.

The PSMA/Fab structure includes residues 1-211 of the Fab light chain, residues 1-224 of the Fab heavy chain (except residues 138-146, which are disordered), and residues 56-750 of PSMA, corresponding to a protease (residues 56-116 and 352-590 ), apical (residues 117-351) and helical (residues 591-750) domains and seven of ten possible N-linked glycans (at Asn-76, -121, -140, -195, -459, -476 and -638 ) to the PSMA dimer subunit. The active site of cynomolgus PSMA is located at the interface between three domains, and it contains two zinc atoms coordinated by histidine (H377 and H553) and glutamate/aspartate residues (D387, catalytic E424, E425 and D453), and a water molecule. The crystalline asymmetric unit contains one PSMA dimer, with each subunit bound similarly to Fab PSMB83. The Fab/PSMA binding site is well defined by the electron density map, allowing reliable positioning of binding residues. The Fab and PSMA molecules are numbered sequentially in FIG. 25-30.

Epitope, paratope and interactions of PSMB83. PSMB83, which is the original Fab arm to PSMA in the bispecific antibody PS3B27, recognizes a conformational and discontinuous epitope in the apical domain of PSMA (Fig. 25). The PSMA surface area immersed in the Fab is approximately 700 A ² . Specifically, the PSMB83 epitope residues are I138, F235, P237, G238, D244, Y299, Y300, Q303, K304, E307, and K324-P326. Helix α7 (residues Y299-E307) is the predominant region

- 150 046641 epitope and binds through the CDRs of the heavy and light chains of Fab. At one end of the helix, Y299 and Y300 form an aromatic cluster with Fab residues Y57 ^H , W94L and PSMA residues F235 and P237, while E307 at the other end of the helix forms a salt bridge with R91 ^L and hydrogen bonds Y32 ^L. In fig. 26 and 27 show the major interactions of PSMA with the light and heavy chains of PSMB83. Residues of the PSMB83 epitope are conserved between human and cynomolgus monkeys (FIG. 28), and the bispecific antibody PS3B27 was demonstrated to bind with similar affinity to human and cynomolgus monkey PSMA. In contrast, human-to-mouse mutations of the G238A and especially Y300D epitope are expected to reduce the binding affinity of PSMB83 to mouse PSMA compared to human PSMA. The Y300D mutation disrupts the hydrogen bond contact with N59 ^H and the docking interaction with W94L. The PSMB83 paratope consists of residues from all CDRs except CDR-L2 and CDR-H1 (Fig. 29). Specifically, the paratope residues are S30 ^L , Y32 ^L , R91 ^L , S92 ^L , W94 ^L of the light chain and G56 ^H -N59 ^H , K65 ^H , G66 ^H , Y101 ^H , V107 ^H and D109 ^H of the heavy chain. In fig. 30 shows the interaction pins between PSMA and PSMB83. The distant location of the epitope promotes binding of the Fab arm to PSMB83 in the bispecific anti-PS3B27 antibody to membrane-bound PSMA, while the other Fab arm remains associated with CD3 in the T cell membrane. PSMB83 is not predicted to inhibit the enzymatic activity of PSMA because the antibody binds to the active site and does not induce any significant structural changes in PSMA that could affect enzymatic function, such as loop movements that occlude the active site or displacement of catalytic residues (RMSD 0.3 A for Ca superposition of PSMA molecules in bound and unbound Fab (Barinka et al, 2007) structures).

Example 14 Anti-PSMA Antibody Affinity Maturation.

Affinity maturation was performed on anti-PSMA phage Fab clones from two PSMA affinity maturation libraries to identify an antibody with increased binding affinity compared to the parent PSMB127 (fab ID=PSMB83). Two PSMB127 affinity maturation libraries were generated. In the first library, heavy chain CDR1 and CDR2 were randomized according to the design shown in Table. 22 (PH9H9L1). The H-CDR3 fragment was amplified by PCR from pDR000024032 and digested with SacII+XhoI. This fragment was cloned into the PH9H9L1/PH9L3 library. It was transformed into E. coli MC1061F' cells and a phage displaying this Fab library was obtained. In the second library, light chain CDRs were randomized according to the design in Table 1. 25 (PH9L3L3). The heavy chain from PSMB83 (PSMH360) was amplified by PCR and digested with NcoI+XhoI. This fragment was cloned into the DNA of the PH9L3L3 library (ELN: De Novo 2010 phage library SRI-021). It was used to transform E. coli MC1061F' cells, and a phage displaying this Fab library was obtained.

Table 22

Library design PH9H9L1

Position Initial a. To. A. k. libraries thirty S D, K, S 31 S D, N, S, T 32 Y A, D, S, Y 33 A A, D, G, S, W, Y 35 S Η, N, S 50 A A, E, L, N, R, T, W, Y 52 S A, D, L, N, R, S 54 S A, E, N, S, Y 57 S D, N, R, S, T, Y 59 Y E, G, N, Q, R, Y

- 151 046641

Table 23

Library design PH9L3L3

Position Initial a. To. A. k. libraries thirty S D, N, R, S 31 S N, S, T 32 Y D, N, R, S, Y 49 Y E, N, K, Y 50 D D, G, S, W, Y 53 N D, N, S, T, Y 91 R A, D, E, G, Η, N, R, S, W, Y 92 S A, D, E, G, Η, N, R, S, W, Y 93 N A, D, E, G, Η, N, R, S, W, Y 94 W A, D, E, G, Η, N, R, S, W, Y 96 L F, I, L, N, R, W, Y

Solution panning of PSMA affinity maturation Fab-pIX libraries was performed against biotinylated human PSMA ECD for three rounds. Phage-bound antigen was captured with neutravidin beads (GE Health Care Life Science, cat. no. 78152104011150) according to the manufacturer's protocol, followed by extensive washes with 1x PBST (0.05% Tween-20) and prolonged incubation with unlabeled PSMA ECD at 500× molar excess of biotinylated antigen. As a result of panning, the clones obtained were PSMXP46R3_59H09, PSMXP46R3_59H06, PSMXP46R3_59E03, PSMXP46R3_59C09, PSMXP46R3_59H01, PSMXP46R3_59F11, and PSMXP46R3_59F07.

To determine the level of expression of anti-PSMA fab clones, Maxisorb 96-well plates were coated overnight at 4°C with anti-human Fd IgG, washed and blocked with 3% milk-PBS0.05% Tween for 1 h. Phage supernatant samples were serially diluted 2-fold for 11 dilutions in blocking buffer with the last control well. 100 μl of these solutions were captured on coated plates for 1 h. The plates were washed and 100 μl of HRP-conjugated anti-F(ab')2 antibody was added for 1 h. The plates were washed and developed using 100 μl of peroxidase reagent, and the luminescence was read at Envision (Fig. 31).

To determine the binding of anti-PSMA Fab clones to human and cynomolgus recombinant protein, 96-well Maxisorb plates were coated with 100 μl of 5 μg/ml neutravidin overnight at 4°C. The plates were washed and blocked with 3% aqueous PBS-0.05% Tween for 1 hour. Recombinant biotinylated human and cynomolgus PSMA proteins were captured at a concentration of 2.5 μg/ml for 1 h at room temperature. The plates were washed and 100 μl of 2-fold serially diluted Fab supernatant was collected for 1 h at room temperature. The plate was washed and then incubated for 2.5 h with 200 μl of 0.3% milk in PBST to wash away some of the weak affinity Fabs. A further 30 min of incubation was then carried out with freshly prepared 200 µl of 0.3% milk in PBST to remove weaker affinity Fabs. The plates were washed and 100 μl of HRP-conjugated anti-F(ab')2 antibody was added for 1 hour. The plates were washed and developed using 100 μl of peroxidase reagent, and the luminescence was read on an Envision (Fig. 32 and Fig. 33). In fig. 31 shows that the protein expression of the original Fab and the affinity matured Fab was similar. Y-axis values represent the luminescence of the detection reagent, which corresponds to the fab protein content of the dilution curve; the higher the luminescence index, the more protein in the well, which decreases with successive twofold dilutions. Affinity mature fab wells had more protein, but the increase over baseline was no more than five times greater, as demonstrated by the EC ₅₀ values (which represent the protein concentration that produces half the maximum response). These data demonstrate that the difference in PSMA binding profiles in FIG. 32 and fig. 33 is not associated with differences in Fab concentration.

In fig. 32 shows improved affinity matured Fab binding to human recombinant antigen compared to the original PSMA Fab (PSMB83). Again, the y-axis of the graph represents the luminescence values. In this case, the higher the value, the more Fab binds to the human PSMA protein. It represents a measure of binding since increased Fab concentrations (along the x-axis) result in higher luminescence values. Under these conditions, negligible binding of the original Fab was observed, as evidenced by the absence of signal even at high concentrations (open circles along the X axis). Binding of Fab to Affinity Mature

- 152 046641 was observed for the tested concentrations, which corresponds to a stronger binding ability to the human PSMA protein. Given that the binding of the original Fab to the human PSMA protein was zero, it was not possible to obtain an EC50.

In fig. 33 shows improved affinity matured Fab binding to recombinant cynomolgus antigen compared to the original PSMA Fab (PSMB83). Again, the y-axis of the graph represents the luminescence values. In this case, the higher the value, the more Fab binds to the cynomolgus PSMA protein. It represents a measure of binding since increased Fab concentrations (along the x-axis) result in higher luminescence values. Under these conditions, negligible binding of the original Fab was observed, as evidenced by the absence of signal even at high concentrations (open circles along the X axis). Affinity-matured Fab binding was observed at the concentrations tested, consistent with stronger binding ability to the human PSMA protein. Given that binding of the original Fab to human PSMA protein was nil, EC ₅₀ could not be obtained for direct comparison.

Overall, the phage Fab binding profiles demonstrate improved binding to the recombinant human and cynomolgus antigen compared to the parent PSMA mAb (PSMB127). This improvement is not the result of differences in Fab expression profiles as shown in FIG. 34 and fig. 35 showing affinity matured Fab binding normalized to Fab expression levels. The top five Fab candidates identified by ELISA-based screening were generated in the IgG4 PAA monoclonal antibody format. In table 24 lists subsequent mAb identifiers, and table. 25 and 26 provide sequence information.

Table 24

Five Major Affinity Mature Antibodies Identified by ELISA

NS L.C. W cells SEQ ID SEQ ID mAb protein ID PSMXP46R3_59C09 PSMH859 PSML160 PSMB346 PSMXP46R3 59E03 PSMH859 PSML159 PSMB345 PSMXP46R3_59F07 PSMH862 PSML158 PSMB349 PSMXP46R3 59H01 PSMH860 PH9L3 PSMB347 PSMXP46R3 59H06 PSMH859 PH9L3 PSMB344

- 153 046641

Table 25

VH and VL sequences for the top five PSMA Fab candidates

ID mAb Amino acid VH sequence SEQ ID NO Amino acid subsequence VL SEQ ID NO PSMB3 44 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS 138 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRSNWPLTFGQGTK VEIL 67 PSMB3 45 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS 138 EIVLTQSPATLSLSPG ERATLSCRASQSVSN YLAWYQQKPGQAP RLLIHDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIL 142 PSMB3 46 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF 138 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASYRATGIP ARFSGSGSGTDFTLT 143

- 154 046641

DYWGQGTLVTVSS IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIL PSMB3 47 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKS RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS 139 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRSNWPLTFGQGTK VEIL 67 PSMB3 49 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSLKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS 140 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRGNWPLTFGQGTK VEIL 144

Table 26

Heavy and light chain sequences of the top five PSMA candidates in IgG4 PAA monoclonal antibody format

ID mAb Amino acid heavy chain sequence SEQ ID NO Amino acid subsequence light chain SEQ ID NO PSMB344 EVQLLESGGGLVQPGGSLR LSCAASGFTFKSDAMHWV RQAPGKGLEWVSEISGSGG YTNYADSMKGRFTISRDNS KNTLYLQMNSLRAEDTAV YYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGL YSLSSVVTVP SSSLGTKTY 145 EIVLTQSPATLSLSPGER ATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGT DFTLTIS SLEPEDF A VYY CQQRSNWPLTFGQGTK VEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLN NFYPREAKVQWKVDNA LQSGNSQESVTEQDSKD E 89

- 155 046641

TCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLP S SIEKTISKAKGQPREPQ VY TLPPSQEEMTKNQVSLTCL VKGF YPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLS LSLGK KHKVYACEVTHQGLSS PVTKSFNRGEC PSMB345 EVQLLESGGGLVQPGGSLR LSCAASGFTFKSDAMHWV RQAPGKGLEWVSEISGSGG YTNYADSMKGRFTISRDNS KNTLYLQMNSLRAEDTAV YYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGL YSLSSVVTVP SSSLGTKTY TCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLP S SIEKTISKAKGQPREPQ VY TLPPSQEEMTKNQVS LTCL VKGFYPSDIAVEWESNGQP 145 EIVLTQSPATLSLSPGER ATLSCRASQSVSNYLA WYQQKPGQAPRLLIHD ASNRATGIPARFSGSGS GTDFTLTIS SLEPEDF AV YYCQQRRNWPLTFGQG TKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLL NNFYPREAKVQWKVDN ALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADY EKHKVY ACEVTHQGLS SPVTKSFNRGEC 148

- 156 046641

ENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLS LSLGK PSMB346 EVQLLESGGGLVQPGGSLR LSCAASGFTFKSDAMHWV RQAPGKGLEWVSEISGSGG YTNYADSMKGRFTISRDNS KNTLYLQMNSLRAEDTAV YYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAAL GCLVKD YFPEP VTVSWNS GALTSGVHTFPAVLQSSGL YSLSSVVTVP SSSLGTKTY TCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLP S SIEKTISK AKGQPREPQ VY TLPPSQEEMTKNQV SLTCL VKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLS LSLGK 145 EIVLTQSPATLSLSPGER ATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDAS YRATGIPARFSGSGSGT DFTLTIS SLEPEDF A VYY CQQRRNWPLTFGQGTK VEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLN NFYPREAKVQWKVDNA LQSGNSQESVTEQDSKD E KHKVYACEVTHQGLSS PVTKSFNRGEC 149 PSMB347 EVQLLESGGGLVQPGGSLR LSCAASGFTFKSDAMHWV RQAPGKGLEWVSEISGSGG YTNYADSMKSRFTISRDNS KNTLYLQMNSLRAEDTAV YYCARDSYDSSLYVGDYF 146 EIVLTQSPATLSLSPGER ATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGT DFTLTIS SLEPEDF A VYY CQQRSNWPLTFGQGTK 89

- 157 046641

DYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTK PRE EQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLP S SIEKTISK AKGQPREPQ VY TLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLS LSLGK VEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLN NFYPREAKVQWKVDNA LQSGNSQESVTEQDSKD STYSLSSTLTLSKADYE KHKVYACEVTHQGLSS PVTKSFNRGEC PSMB349 EVQLLESGGGLVQPGGSLR LSCAASGFTFKSDAMHWV RQAPGKGLEWVSEISGSGG YTNYADSLKGRFTISRDNS KNTLYLQMNSLRAEDTAV YYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGL YSLSSVVTVP SSSLGTKTY TCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPRE 147 EIVLTQSPATLSLSPGER ATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGT DFTLTIS SLEPEDF A VYY CQQRGNWPLTFGQGTK VEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLN NFYPREAKVQWKVDNA LQSGNSQESVTEQDSKD STYSLSSTLTLSKADY E KHKVYACEVTHQGLSS PVTKSFNRGEC 150

- 158 046641

EQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLP

HS SIEKTISKAKGQPREPQ VY TLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLS LSLGK of different NSs and 4 different LCs were combined in a matrix format to increase the diversity of the selected options (Table 26). Considering that the methionine in CDR2 of PSMH860 is at risk for post-translational modification, a new sequence with M64L was created and designated PSMH865. PSMH865 was combined with PSML160 to create the mAb PSMB365. In table 27 and 28 provide sequence information.

Table 26

Matrix format of combined 3 heavy chains and 4 light chains

PSMH859 PSMH860 PSMH862 PH9L3 PSMB344 PSMB347 PSMB358 PSML158 - PSMB361 PSMB349 PSML159 PSMB345 PSMB362 PSMB359 PSML160 PSMB346 PSMB363 PSMB360

Table 27

VH and VL sequences of selected compounds to PSMA from the recombined matrix

ID mAb Amino acid VH sequence SEQ ID NO Amino acid subsequence VL SEQ ID NO PSMB3 58 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSLKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS 140 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASNRATGIP ARFSGSGSGTDFTLT ISSLEPEDFAVYYCQ QRSNWPLTFGQGTK VEIL 67 PSMB3 EVQLLESGGGLVQPGGSLRLSCA 140 EIVLTQSPATLSLSPG 142

- 159 046641

59 ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSLKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS ERATLSCRASQSVSN YLAWYQQKPGQAP RLLIHDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIL PSMB3 60 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSLKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS 140 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASYRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIL 143 PSMB3 61 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKS RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS 139 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRGNWPLTFGQGTK VEIL 144 PSMB3 62 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKS RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS 139 EIVLTQSPATLSLSPG ERATLSCRASQSVSN YLAWYQQKPGQAP RLLIHDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIL 142 PSMB3 63 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKS RFTISRDNSKNTLYLQMNSLRAE 139 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASYRATGIP 143

- 160 046641

DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSS ARFSGSGSGTDFTLT ISSLEPEDFAVYYCQ QRRNWPLTFGQGTK VEIL PSMB3 65 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSLKSR FTISRDNSKNTLYLQMNSLRAED TAVYYCARDSYDSSLYVGDYFD YWGQGTLVTVSS 141 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASYRATGIP ARFSGSGSGTDFTLT ISSLEPEDFAVYYCQ QRRNWPLTFGQGTK VEIL 143

Table 28

Sequences of the heavy and light chains of selected compounds to PSMA from the recombined matrix

ID mAb Amino acid sequence of heavy chains SEQ ID NO Light chain amino acid sequence SEQ ID NO PSMB3 58 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSLKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVP SSSLGT KTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVK GFYPSDIA VEWESNGQPENNYKTTPPVLDS 147 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRSNWPLTFGQGTK VEIKRTVAAPSVFIFP PSDEQLKSGTASVVC LLNNFYPREAKVQW KVDNALQSGNSQES VTEQDSKDSTYSLSS TLTLSKADYEK HKV YACEVTHQGLSSPV TKSFNRGEC 89

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DGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSL GK PSMB3 59 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSLKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVP SSSLGT KTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVK GFYPSDIA VEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSL GK 147 EIVLTQSPATLSLSPG ERATLSCRASQSVSN YLAWYQQKPGQAP RLLIHDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIKRTVAAPSVFIFP PSDEQLKSGTASVVC LLNNFYPREAKVQW KVDNALQSGNSQES VTEQDSKDSTYSLSS TLTLSKADYEK HKV YACEVTHQGLSSPV TKSFNRGEC 148 PSMB3 60 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSLKG RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVP SSSLGT KTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEAAGGPSVFLF 147 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASYRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIKRTVAAPSVFIFP PSDEQLKSGTASVVC LLNNFYPREAKVQW KVDNALQSGNSQES 149

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PPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQ EGNV FSCSVMHEALHNHYTQKSLSLSL GK VTEQDSKDSTYSLSS TLTLSKADYEKHKV YACEVTHQGLSSPV TKSFNRGEC PSMB3 61 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKS RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSS SLGT KTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGF YPSDIA VEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSL GK 146 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASNRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRGNWPLTFGQGTK VEIKRTVAAPSVFIFP PSDEQLKSGTASVVC LLNNFYPREAKVQW KVDNALQSGNSQES VTEQDSKDSTYSLSS TLTLSKADYEK HKV YACEVTHQGLSSPV TKSFNRGEC 150 PSMB3 62 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKS RFTISRDNSKNTLYLQMNSLRAE 146 EIVLTQSPATLSLSPG ERATLSCRASQSVSN YLAWYQQKPGQAP RLLIHDASNRATGIP 148

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DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDV SQEDPEVQF NWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSL GK ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIKRTVAAPSVFIFP PSDEQLKSGTASVVC LLNNFYPREAKVQW KVDNALQSGNSQES VTEQDSKDSTYSLSS TLTLSKADYEKHKV YACEVTHQGLSSPV TKSFNRGEC PSMB3 63 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSMKS RFTISRDNSKNTLYLQMNSLRAE DTAVYYCARDSYDSSLYVGDYF DYWGQGTLVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSS SLGT KTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGF YPSDIA VEWESNGQPENNYKTTPPVLDS 146 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASYRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIKRTVAAPSVFIFP PSDEQLKSGTASVVC LLNNFYPREAKVQW KVDNALQSGNSQES VTEQDSKDSTYSLSS TLTLSKADYEK HKV YACEVTHQGLSSPV TKSFNRGEC 149

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DGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSL GK PSMB3 65 EVQLLESGGGLVQPGGSLRLSCA ASGFTFKSDAMHWVRQAPGKG LEWVSEISGSGGYTNYADSLKSR FTISRDNSKNTLYLQMNSLRAED TAVYYCARDSYDSSLYVGDYFD YWGQGTLVTVSSASTKGPSVFPL APCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGT KT YTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFY PSDIAVE WESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVF SC SVMHEALHNHYTQKSLSLSLGK 151 EIVLTQSPATLSLSPG ERATLSCRASQSVSS YLAWYQQKPGQAP RLLIYDASYRATGIP ARFSGSGSGTDFTLT IS SLEPEDF AVYYCQ QRRNWPLTFGQGTK VEIKRTVAAPSVFIFP PSDEQLKSGTASVVC LLNNFYPREAKVQW KVDNALQSGNSQES VTEQDSKDSTYSLSS TLTLSKADYEK HKV YACEVTHQGLSSPV TKSFNRGEC 149

In table 29 shows the CDRs for all affinity-mature matches.

Table 29

CDR sequences of selected affinity matured PSMA compounds

ID mAb

CDR (SEQ ID NO: )

CDR1 CDR2 CDR3 PSMB3 44 H c SDAMH (25) EISGSGGYTNYADS MKG(130) DSYDSSLYVGDYFDY (27) L C RASQSVSSYLA (28) DASNRAT (29) QQRSNWPLT (30) PSMB3 45 H C SDAMH (25) EISGSGGYTNYADS MKG (130) DSYDSSLYVGDYFDY (27)

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L WITH RASQSVSNYLA (131) DASNRAT (29) QQRRNWPLT (132) PSMB3 46 Η With SDAMH (25) EISGSGGYTNYADS MKG(130) DSYDSSLYVGDYFDY (27) L WITH RASQSVSSYLA (28) DASYRAT (133) QQRRNWPLT (132) PSMB3 47 Η WITH SDAMH (25) EISGSGGYTNYADS MKS (134) DSYDSSLYVGDYFDY (27) L WITH RASQSVSSYLA (28) DASNRAT (29) QQRSNWPLT (thirty) PSMB3 49 Η WITH SDAMH (25) EISGSGGYTNYADS LKG (135) DSYDSSLYVGDYFDY (27) L WITH RASQSVSSYLA (28) DASNRAT (29) QQRGNWPLT (136) PSMB3 58 Η WITH SDAMH (25) EISGSGGYTNYADS L.K.G. (135) DSYDSSLYVGDYFDY (27) L WITH RASQSVSSYLA (28) DASNRAT (29) QQRSNWPLT (30) PSMB3 59 Η WITH SDAMH (25) EISGSGGYTNYADS LKG (135) DSYDSSLYVGDYFDY (27) L WITH RASQSVSNYLA (131) DASNRAT (29) QQRRNWPLT (132) PSMB3 60 Η WITH SDAMH (25) EISGSGGYTNYADS LKG (135) DSYDSSLYVGDYFDY (27) L WITH RASQSVSSYLA (28) DASYRAT (133) QQRRNWPLT (132) PSMB3 61 Η WITH SDAMH (25) EISGSGGYTNYADS MKS (134) DSYDSSLYVGDYFDY (27) L WITH RASQSVSSYLA (28) DASNRAT (29) QQRGNWPLT (136) PSMB3 62 Η WITH SDAMH (25) EISGSGGYTNYADS MKS (134) DSYDSSLYVGDYFDY (27)

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L WITH RASQSVSNYLA (131) DASNRAT (29) QQRRNWPLT (132) PSMB3 63 N WITH SDAMH (25) EISGSGGYTNYADS MKS (134) DSYDSSLYVGDYFDY (27) L WITH RASQSVSSYLA (28) DASYRAT (133) QQRRNWPLT (132) N SDAMH EISGSGGYTNYADS DSYDSSLYVGDYFDY PSMB3 WITH (25) LKS (137) (27) 65 L WITH RASQSVSSYLA (28) DASYRAT (133) QQRRNWPLT (132)

Example 15. Generation of bispecific antibodies with affinity matured to PSMA x CD3.

Two types of affinity-matured PSMA x CD3 bispecific antibodies were generated: one specific for the targeting arm (eg, affinity-matured anti-PSMA antibody) recombined with a high-affinity CD3 arm [CD3B219 (VH SEQ ID NO: 104, VL SEQ ID NO: 105; NS with SEQ ID NO: 110, LC SEQ ID NO: 111) ] or low affinity CD3 arm called CD3B376 [CD3B376 (VH SEQ ID NO: 152, VL SEQ ID NO: 153; NS with SEQ ID NO : 154, LC SEQ ID NO: 155)].

Table 30 Sequences for the low affinity arm to CD3 (CD3B376)

ID mAb Amino acid VH sequence SEQ ID NO Amino acid VL sequence SEQ ID NO CD3B37 6 QVQLQQSGPRLVRPSQTLSL TCAISGDSVFNNNAAWSWI RQSPSRGLEWLGRTYYRSK WLYDYAVSVKSRITVNPDT SRNQFTLQLNSVTPEDTALY YCARGYSSSFDYWGQGTLV TVSS 152 QSALTQPASVSGSPGQSI TISCTGTSSNIGTYKFVS WYQQHPDKAPKVLLYE VSKRPSGVSSRFSGSKSG NTASLTISGLQAEDQAD YHCVSYAGSGTLLFGGG TKLTVL 153 Amino acid heavy chain sequence Amino acid subsequence light chain MAWVWTLLFLLMAAAQSIQ AQVQLQQSGPRLVRPSQTLS LTCAISGDSVFNNNAAWSW 154 MARS ALLIL ALLLLGLFS PGAWGQSALTQPASVS GSPGQSITISCTGTSSNIG 155

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IRQSPSRGLEWLGRTYYRSK WL YD YAVS VK SRITVNPDT SRNQFTLQLNSVTPEDTALY YCARGYSSSFDYWGQGTLV TVS S ASTKGPS VFPLAPC SRS TSESTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDK RVESKYGP PCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNY KTTPPVLDSDGSFLLYSK LT VDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK

TYKFVSWYQQHPDKAP KVLLYEVSKRPSGVSSR FSGSKSGNTASLTISGLQ AEDQADYHCVSYAGSG TLLFGGGTKLTVLGQPK AAPSVTLFPPSSEELQAN KATLVCLISDFYPGAVT VAWKADSSPVKAGVET TTPSKQSNNKYAASSYL SLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS

These parent mAbs are in the GenMab format (Labrijn et al, 2013), in which the targeting parent mAb (PSMA) contains the 409R GenMab mutation (the native amino acid for IgG4) and the killing parent mAb (CD3) contains the F405L GenMab mutation and the R409K mutation. Monospecific anti-CD3 antibody was expressed as IgG4 having Fc substitutions S228P, F234A, L235A, F405L and R409K (arm to CD3) (EU numbering) in the Fc regions. The targeting parent mAb (PSMA) is in the human IgG4 format with substitutions in Fc S228P, F234A, L235A. Monospecific antibodies were expressed in HEK cell lines under the control of CMV promoters.

Stock antibodies to PSMA and CD3 were purified using a protein A column with an elution buffer containing 100 mM NaAc, pH 3.5, and a neutralization buffer of 2.5 M Tris, pH 7.2. Neutralized parent mAbs were used to generate bispecific antibodies to PSMAxCD3. Some of the original mAbs were additionally replaced with D-PBS buffer, a pH 7.2 buffer for analytical measurements and analyses.

After purification, controlled Fab arm exchange was performed to produce bispecific antibodies as described in Example 6.

The final bispecific antibodies obtained along with the parent mAbs (i.e. PSMA, CD3 or Null) used in the recombination reactions are listed in Table. 27 and 28.

Selected appropriate PSMA antibodies were also combined with the non-killing arm (Null) to generate negative controls for testing purposes. For the control bispecific antibodies B2M1, an anti-RSV antibody was generated, purified in IgG4 PAA format and combined either with the CD3 arm CD3B219-F405L, R409K to obtain CD3B288 (CD3 x null), or with the arms to PSMA, PSMB122, PSMB126, PSMB130 to obtain PS3B37 , PS3B39 and PS3B40, respectively (PSMA x zero). These PSMA-specific affinity matured mAbs were crossed (as in the methods above) with CD3B219 and CD3B376 to produce the bispecific antibodies shown in Table 1. 31.

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Table 31

Generation of affinity-matured anti-PSMA x CD3 bispecific antibodies derived from affinity-matured compounds PSMB127

NS L.C. NS L.C. ID. number Shoulder 1 SEQ ID: SEQ ID: Shoulder 2 SEQ ID: SEQ ID PS3B60 PSMB344 145 89 CD3B219 110 111 PS3B61 PSMB345 145 148 CD3B219 110 111 PS3B62 PSMB346 145 149 CD3B219 110 111 PS3B63 PSMB347 146 89 CD3B219 110 111 PS3B64 PSMB349 147 150 CD3B219 110 111 PS3B70 PSMB358 147 89 CD3B219 110 111 PS3B71 PSMB359 147 148 CD3B219 110 111 PS3B72 PSMB360 147 149 CD3B219 110 111 PS3B73 PSMB361 146 150 CD3B219 110 111 PS3B74 PSMB362 146 148 CD3B219 110 111 PS3B75 PSMB363 146 149 CD3B219 110 111 PS3B76 PSMB358 151 149 CD3B376 154 155 PS3B77 PSMB349 145 89 CD3B376 154 155 PS3B78 PSMB359 145 148 CD3B376 154 155 PS3B79 PSMB360 145 149 CD3B376 154 155 PS3B80 PSMB347 146 89 CD3B376 154 155

PS3B81 PSMB361 147 150 CD3B376 154 155 PS3B82 PSMB362 147 89 CD3B376 154 155 PS3B83 PSMB363 147 148 CD3B376 154 155 PS3B84 PSMB344 147 149 CD3B376 154 155 PS3B85 PSMB345 146 150 CD3B376 154 155 PS3B86 PSMB346 146 148 CD3B376 154 155 PS3B90 PSMB365 146 149 CD3B376 154 155 digitally e antibodies to PSMAxCD3 tested on । tying with PSMA line

positive cells, LNCAP, with a PSMA-negative cell line, PC3. A cell binding assay (described previously) was used to evaluate the binding capabilities of PSMA bispecific antibodies. Bispecific antibodies were normalized to protein concentration and then incubated with the same number of cells expressing human or cynomolgus PSMA. The SIF for each concentration was obtained by flow cytometry and plotted as a function of concentration. Data were log10 transformed and then plotted. To determine EC5o, binding curves were constructed using nonlinear regression.

These relative values were used to rank PSMA binding to target cells. In fig. 36-38 show the binding of all bispecific antibodies obtained to LNCAP. In fig. 38 Neither construct demonstrated binding to a PSMA‐negative cell line. In fig. 36 and Fig. 37 all selected affinity matured compounds showed increased binding affinity with left-shifted curves and increased cMax compared to the parent mAb, PS2B27.

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The interaction of affinity-matured bispecific antibodies with recombinant cynomolgus PSMA ECD and human PSMA ECD was studied by surface plasmon resonance (SPR) using the ProteOn XPR36 system (BioRad), as described previously for recombinant chimpanzee PSMA ECD. All bispecific antibodies bind to both targets with essentially the same affinity, with KD ranging from 0.05 nM to 0.27 nM for human PSMA ECD and from 0.05 nM to 0.23 nM for cynomolgus PSMA ECD.

Example 16: Evaluation of affinity matured bispecific antibodies to CD3 and PSMA in a functional cell killing assay.

Based on the above data, affinity and sequence identity measurements, three anti-PSMA antibodies, PSMB347, PSMB360 and PSMB365, as bispecific antibodies with CD3B219 or CD3B376, were further characterized for the ability to mediate PSMA-specific T cell redirected cytotoxicity. T cell-mediated killing was measured using a caspase cytotoxicity assay, which indirectly measures cell killing through cleavage of a fluorescent substrate by active caspase 3/7. Cleavage of the substrate results in the formation of a fluorescent DNA dye with fluorescence limited to the cell nucleus. Repeated fluorescence measurements are taken in each well throughout the assay using a motorized 10× magnification objective capable of accurately visualizing the cell(s) at the same coordinates. Target cell populations are identified based on certain size restrictions and/or the use of a secondary label. Frozen pan CD3+ T cells (purchased from Biological Specialty Corporation, Colmar, PA) were isolated by negative selection against normal healthy donors. PSMA-expressing prostate cancer cells (LNCaP, C42) were cultured in RPMI 1640 supplemented with 10% HI FBS+ supplements (purchased from Life Technologies).

T cells and target cells were combined at an effector-target (E:T) ratio of 3:1 in RPMI without phenol red + 10% FBS and supplement (Life Technologies) without selection reagents, and 0.6 μl was added to each ml of cells NucView caspase reagent (Essen Bioscience) according to the manufacturer's recommendations. The total volume of cells was 0.1 ml in the corresponding wells of a transparent 96-well flat-bottom plate (BD Falcon). Bispecific antibodies PS3B27 (CD3xPSMA), CD3B288 (CD3xNull) or PS3B46 (PSMAxNull) were prepared at 2X final concentration in RPMI without phenol red prepared as above, and 0.1 ml of compounds was added to each well. After 30 min of incubation at room temperature to minimize cell aggregation at the edge of the wells, the plates were transferred to a Zoom Incucyte instrument (Essen Bioscience). Incucyte Instrument is placed in a humidified incubator at 37°C, 5% CO2.

Cell processing definitions were developed for each cell line tested according to the manufacturer's instructions. Measurements were taken every six hours until a plateau in caspase signal was observed, followed by three or more consecutive decreases from the maximum signal in the well(s) containing the highest concentration of test compound(s). As shown in FIG. 39, the curves for PS3B80, pS3B79, PS3B89, PS3B90, PS3B63 and PS3B72 shift to the left, indicating increased activity relative to PS3B27. As expected, control null arm antibodies did not induce cell death.

Example 17 Antitumor efficacy in preventing tumor formation of LnCaP xenografts in humanized NSG mice.

The efficacy of PS3B79 and PS3B90 was assessed in established 3D LnCaP AR.TB human prostate cancer cell xenografts in male NOD.Cg-Prkdc ^scid Il2rgtm ^lWjl /SzJ (NSG) mice humanized intraperitoneally (i.p.) with human T cells. PS3B79 and PS3B90 at doses of 2.5 and 5 mg/kg or control antibody NullxCD3B376 were administered every 3–4 days on days 36, 39, 43, 47, 50, 53, 56, 60, and 63 for a total of 8 doses. On day 53 post tumor implantation, which was the last date of the study when nine (9) animals remained in the group, tumor growth inhibition (%TGI) was calculated. Statistically significant inhibition of tumor growth was observed for PS3B79 at a dose of 5 mg/kg with 42% TGI (two-way ANOVA with Bonferroni correction, *p<0.0001, Fig. 40) and for PS3B90 at a dose of 2.5 and 5 mg/kg with 53% and 33%, respectively, compared to the CD3 null control (two-way ANOVA with Bonferroni test, *p<0.001, Fig. 41). Thus, CD3B376 is capable of inducing T cell activation and cytotoxicity in vivo and is capable of leading to tumor growth inhibition in a bispecific format via the high-affinity PSMA-binding arms, PSMB360 and PSMB365.

Example 18: Efficacy of PSMA x CD3 in an established LuCaP 86.2 prostate xenograft model in T cell humanized NSG mice.

The antitumor efficacy of PS3B72 was assessed in an established patient-derived xenograft (PDX) LuCaP 86.2 prostate tumor model in male NOD.CgPrkdcscid Il2rg ^tmlwjl /SzJ (NSG) mice humanized intraperitoneally (ip) with human pan-T cells. PS3B72 at a dose of 0.5 or 5 mg/kg or null x CD3 control antibody was administered once every three days or once every four days on days 45, 49, 52, 56, 59, 63, 66, 70, 73 and 77 , after tumor implantation it is generally difficult

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Claims (14)

sti 10 doses. On day 83 after tumor implantation, which was the last day of the study when all ten animals remained in the group, tumor growth inhibition (%TGI) was calculated. Statistically significant tumor growth inhibition was observed for PSMA x CD3 at both 0.5 and 5 mg/kg doses with 108% and 101% A TGI (linear mixed effects analysis using FDR adjustment, * p < 0.0001, Fig 42), respectively, compared to the CD3 null control. By the end of the 5-week dosing period, 9 of 10 complete regressions (CRs) were observed in the PSMA x CD3 0.5 mg/kg group. These mice 9 remained tumor-free until the study was completed. At the end of the study, 4 of 10 CRs were observed in the PS3B72 5 mg/kg group. No antitumor efficacy was observed after administration of PS3B72 at a dose of 5 mg/kg to mice in the absence of T cell humanization. CLAIM 1. An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 with SEQ ID NO: 25, 26, 27, 28, 29 and 30, respectively. 2. An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region (VH) of SEQ ID NO: 66 and a light chain variable region (VL) of SEQ ID NO: 67. 3. An isolated recombinant anti-PSMA antibody or antigen-binding fragment thereof according to claim 2, comprising VH SEQ ID NO: 66 and VL SEQ ID NO: 67. 4. An isolated recombinant anti-PSMA antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, where the antibody is of the IgG4 isotype. 5. An isolated recombinant anti-PSMA antibody or its antigen-binding fragment according to claim 4, containing substitutions S228P, F234A and L235A, where the residue numbering corresponds to the EU numbering. 6. A bispecific antibody comprising a first domain that specifically binds PSMA and a second domain that specifically binds CD3, wherein the first domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 SEQ ID NO: 25, 26, 27, 28, 29 and 30, respectively. 7. Bispecific antibody according to claim 6, characterized in that the first domain contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 SEQ ID NO: 25, 26, 27, 28, 29 and 30, respectively, and VH is SEQ ID NO: 66, and VL is SEQ ID NO: 67. 8. The bispecific antibody according to claim 7, wherein the first domain contains VH SEQ ID NO: 66 and VL SEQ ID NO: 67, and the second domain contains VH SEQ ID NO: 104 and VL SEQ ID NO: 105. 9. The bispecific antibody of claim 8, comprising a first heavy chain (HC1), a first light chain (LC1), a second heavy chain (HC2) and a second light chain (LC2), wherein HC1 and LC1 contain the amino acid sequences of SEQ ID NO: 88 and 89 respectively. 10. Bispecific antibody according to claim 9, characterized in that HC2 and LC2 contain SEQ ID NO: 110 and 111, respectively. 11. The bispecific antibody of claim 10, comprising HC1, LCI, HC2 and LC2 selected from SEQ ID NO: 88, 89, 110, 111, respectively. 12. Bispecific antibody according to any one of claims 6-11, characterized in that the antibody belongs to the IgG4 isotype. 13. The bispecific antibody of claim 12, comprising at least one substitution in the CH3 constant domain of the antibody. 14. The bispecific antibody according to claim 13, characterized in that the substitution in the constant CH3 domain of the antibody is a 409R, F405L or F405L/R409K substitution, where the residue numbering corresponds to the EU numbering. - 171 -