EA042513B1 - PSMA-TARGETED TRISPECIFIC PROTEINS AND METHODS FOR THEIR APPLICATION - Google Patents

Description

Sequence listing

This document contains a sequence listing that has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy, created on November 22, 2017, is named 47517-708_601_SL.txt and is 150,911 bytes in size.

Background of the Invention

Often, in a variety of clinical settings, it is desirable to selectively kill an individual cell or a specific cell type. For example, the main goal of cancer therapy is the specific destruction of tumor cells so that healthy cells and tissues remain intact and undamaged. One such method is to induce an immune response against the tumor to stimulate immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL) to attack and destroy the tumor cells.

Brief summary of the present invention

Provided herein is a trispecific antigen-binding protein, pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such trispecific antigen-binding proteins, and methods of use in the treatment of diseases, disorders, or conditions. In one aspect, trispecific proteins targeted to prostate specific membrane antigen (PSMA) are described herein, wherein said proteins comprise (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B), which is a domain that provides an increase in half-life; and (c) a third domain (C) that specifically binds to PSMA, the domains being joined in the order H ₂ N-(A)-(C)-(B)-COOH, H ₂ N-(B)-(A) -(C)-COOH, H ₂ N-(C)-(B)-(A)-COOH or via linkers L1 and L2. In some embodiments, the first domain comprises a variable light chain and a variable heavy chain, each of which is capable of specifically binding to human CD3. In some embodiments, the first domain contains one or more sequences selected from the group consisting of SEQ ID NOs: 1-88. In some embodiments, the first domain is humanized or is a human domain. In some embodiments, the first domain is characterized by CD3 binding KD on the surface of CD3-expressing cells

150 nM or less. In some embodiments, the second domain binds human serum albumin. In some embodiments, the second domain comprises an scFv, a heavy chain variable domain (VH), a light chain variable domain (VL), a peptide, a ligand, or a small molecule. In some embodiments, the second domain contains one or more sequences selected from the group consisting of SEQ ID NOs: 89-112. In some embodiments, the third domain comprises an scFv, a VH domain, a VL domain, a non-Ig type domain, a ligand, a nottin, or a small molecule that specifically binds to PSMA. In some embodiments, the third domain contains one or more sequences selected from the group consisting of SEQ ID NOs: 113-140.

In some embodiments, each of the L1 and L2 linkers is independently selected from (GS)n (SEQ ID NO: 153), (GGS)n (SEQ ID NO: 154), (GGGS)n (SEQ ID NO: 155), ( GGSG)n (SEQ ID NO: 156), (GGSGG)n (SEQ ID NO: 157) or (GGGGS)n (SEQ ID NO: 158), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each of the L1 and L2 linkers is independently (GGGGS) ₄ (SEQ ID NO: 159) or (GGGGS) ₃ (SEQ ID NO: 160). In some embodiments, the domains are connected in the order H ₂ N-(A)-(C)-(B)-COOH. In some embodiments, the domains are connected in the order H ₂ N-(B)-(C)-(A)-COOH.

In some embodiments, the protein is less than about 80 kDa in size. In some embodiments, the protein is about 50 to about 75 kDa in size. In some embodiments, the protein is less than about 60 kDa.

In some embodiments, the protein has a half-life of at least about 50 hours. In some embodiments, the protein has a half-life of at least about 100 hours. In some embodiments, the protein has increased tissue penetration compared to IgG to the same PSMA.

In some embodiments, the protein contains a sequence selected from the group consisting of SEQ ID NO: 140-152.

According to another aspect, provided herein is a pharmaceutical composition,

- 1 042513 containing (i) PSMA-targeted trispecific protein in accordance with any of the above embodiments and (ii) a pharmaceutically acceptable carrier.

Also provided herein are methods of treating an individual in need of cancer treatment, the method comprising administering an effective amount of a pharmaceutical composition or PSMA-targeted trispecific proteins according to any of the above embodiments. In some embodiments, the cancer is prostate cancer or kidney cancer.

One embodiment relates to a PSMA-targeted trispecific protein, wherein said protein comprises (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B), which is a domain that provides an increase in half-life; and (c) a third domain (C) that specifically binds to PSMA, the second domain containing one or more sequences selected from the group consisting of SEQ ID NOs: 113-140. In some embodiments, the domains are linked in the order H2N-(A)-(C)-(B)-COOH, H2N-(B)-(A)-(C)-COOH, H2N-(C)-(B)- (A)-COOH or via linkers L1 and L2. In some embodiments, the first domain contains one or more sequences selected from the group consisting of SEQ ID NOs: 1-88. In some embodiments, the second domain contains one or more sequences selected from the group consisting of SEQ ID NOs: 89-112.

One embodiment relates to a PSMA-targeted trispecific protein, wherein said protein contains a sequence selected from the group consisting of SEQ ID NOs: 140-152. In some embodiments, said protein contains a sequence selected from the group consisting of SEQ ID NOs: 150-152.

One embodiment relates to a prostate specific membrane antigen (PSMA) targeting trispecific protein, wherein said protein comprises (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B), which is a domain that provides an increase in half-life; and (c) a third domain (C) that specifically binds to PSMA, the domains being connected in the order H ₂ N-(C)-(B)-(A)-COOH or via linkers L1 and L2, and the third domain containing one or more sequences selected from the group consisting of SEQ ID NO: 113-140.

One embodiment relates to a PSMA-targeted trispecific protein, wherein said protein comprises (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B), which is a domain that provides an increase in half-life; and (c) a third domain (C) that specifically binds to PSMA, the domains being connected in the order H ₂ N-(C)-(B)-(A)-COOH or via linkers L1 and L2, and the first domain containing one or more sequences selected from the group consisting of SEQ ID NO: 1-88.

One embodiment relates to a method for treating prostate cancer, the method comprising administering an effective amount of a PSMA-targeted trispecific protein, said protein comprising (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B), which is a domain that provides an increase in half-life; and (c) a third domain (C) that specifically binds to PSMA, wherein the domains are connected in the order H2N-(C)-(B)-(A)-COOH or via linkers L1 and L2, and wherein the third domain contains one or more sequences selected from the group consisting of SEQ ID NO: 113-140.

One embodiment relates to a method for treating prostate cancer, the method comprising administering an effective amount of a PSMA-targeted trispecific protein, said protein comprising (a) a first domain (A) that specifically binds to human CD3; (b) a second domain (B), which is a domain that provides an increase in half-life; and (c) a third domain (C) that specifically binds to PSMA, wherein the domains are connected in the order H2N-(C)-(B)-(A)-COOH or via linkers L1 and L2, and wherein the first domain contains one or more sequences selected from the group consisting of SEQ ID NO: 1-88.

Inclusion by reference

All publications, patents, and patent applications referred to in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually designated for inclusion by reference.

Brief description of the drawings

New features of the present invention are detailed in the appended claims. A better understanding of the features and advantages of the present invention will be provided with reference to the following detailed description, which sets forth illustrative embodiments that use the principles of the present invention and the accompanying drawings.

Fig. 1 is a schematic representation of an exemplary PMSA-targeted trispecific antigen-binding protein, wherein the protein contains a constant core element providing a CD3ε-binding single-chain variable fragment (scFv) and an HSA-binding heavy chain variable region; and a PMSA binding domain, which may be a VH, scFv, non-Ig type binding domain, or ligand.

- 2 042513

In FIG. 2A-2C compare the ability of exemplary PSMA-targeted trispecific proteins (PSMA-targeted TriTAC molecules) with different CD3 affinities to induce T cells to kill human prostate cancer cells.

In FIG. 2A shows the results of killing due to different PMSA-targeted TriTAC molecules in the LNCaP prostate cancer model.

In FIG. 2B shows results on killing induced by various PMSA-targeted TriTAC molecules in the 22Rv1 prostate cancer model.

In FIG. 2C shows EC ₅₀ values for PMSA-targeted TriTACs in the LNCaP and 22Rv1 prostate cancer models.

In FIG. 3 shows the serum concentration of PSMA-targeted TriTAC C236 in cynomolgus monkeys after i.v. administration (100 mcg/kg) for three weeks.

In FIG. 4 shows the serum concentration of PSMA-targeted TriTAC molecules with different CD3 affinities in cynomolgus monkeys after i.v. administration (100 mcg/kg) for three weeks.

In FIG. 5A-5C show the ability of PSMA-targeted TriTAC molecules with different PSMA affinities to induce T cells to kill the human prostate cancer cell line LNCaP.

In FIG. 5A shows the results of an experiment performed in the absence of human serum albumin with PSMA-targeted BiTE as a positive control.

In FIG. 5B shows the results of an experiment performed in the presence of human serum albumin with PSMA-targeted BiTE as a positive control.

In FIG. 5C shows EC ₅₀ values for PMSA-targeted TriTAC in the presence or absence of HSA, with PSMA-targeted BiTE as a positive control, in LNCaP prostate cancer models.

In FIG. 6 demonstrates the ability of PSMA-targeted TriTAC molecules to inhibit tumor growth of human prostate cancer cells in a mouse xenotransplantation experiment.

In FIG. 7A-7D illustrate the specificity of TriTAC molecules in cell killing assays using target cell lines that express or do not express the target protein.

In FIG. 7A shows the results of EGFR and PSMA expression in LNCaP, KMS12BM and OVCAR8 cell lines.

In FIG. 7B shows the results of LNCaP tumor cell killing induced by PSMA-targeted, EGFR-targeted TriTAC and TriTAC as a negative control.

In FIG. 7C shows results of KMS12BM tumor cell killing induced by PSMA-targeted, EGFR-targeted TriTAC and TriTAC as a negative control.

In FIG. 7D shows the results of PSMA-targeted, EGFR-targeted TriTAC and TriTAC as a negative control in killing OVCAR8 cells.

In FIG. 8A-8D show the effect of pre-incubation at 37° C. and freeze-thaw cycles on TriTAC activity.

In FIG. 8A shows the activity of PSMA TriTAC C235 after pre-incubation at 37° C. or freeze-thaw cycles.

In FIG. 8B shows PSMA TriTAC C359 activity after pre-incubation at 37°C or freeze-thaw cycles.

In FIG. 8C shows PSMA TriTAC C360 activity after pre-incubation at 37°C or freeze-thaw cycles.

In FIG. 8D shows the activity of PSMA TriTAC C361 after pre-incubation at 37° C. or freeze-thaw cycles.

In FIG. 9A, 9B show the activity of the PSMA-targeted TriTAC molecule of the present disclosure in redirected T cell kill assays by T cell dependent cellular cytotoxicity (TDCC).

In FIG. 9A shows the effect of the PSMA-targeted TriTAC molecule on redirecting cynomolgus monkey peripheral blood mononuclear cells (PBMCs), from a G322 cynomolgus donor, to kill LNCaP cells.

In FIG. 9B shows the effect of a PSMA-targeted TriTAC molecule on redirecting cynomolgus monkey PBMC, from a donor cynomolgus monkey D173, to kill MDAPCa2b cells.

In FIG. 10 shows the effect of the PSMA-targeted TriTAC molecule of the present disclosure on the expression of CD25 and CD69 T cell activation markers.

In FIG. 11 shows the ability of the PSMA-targeted TriTAC molecule of the present disclosure to stimulate T cell proliferation in the presence of PSMA-expressing target cells.

In FIG. 12A, 12B show results of redirected T cells killing LnCaP cells mediated by PSMA-targeted TriTAC molecules.

- 3 042513

In FIG. 12A shows the results of redirected T cells killing LnCaP cells mediated by PSMA PH1T TriTAC (SEQ ID NO: 150) and PSMA PHI TriTAC (SEQ ID NO: 151).

In FIG. 12B shows results of redirected T cells killing LnCaP cells mediated by PSMA Z2 TriTAC molecules (SEQ ID NO: 152).

Detailed disclosure of the present invention

This document describes trispecific proteins targeting prostate specific membrane antigen (PSMA), pharmaceutical compositions based on them, as well as nucleic acids, recombinant expression vectors and host cells to obtain such proteins. Also provided are methods of using the disclosed PSMA-targeted trispecific proteins in the prevention and/or treatment of diseases, conditions, and disorders. PSMA-targeted trispecific proteins are capable of specific binding to PSMA as well as CD3 and have a half-life extension domain such as the human serum albumin (HSA) binding domain. In FIG. 1 shows one non-limiting example of a trispecific antigen binding protein.

In one aspect, the PSMA-targeted trispecific proteins comprise a domain (A) that specifically binds to CD3, a domain (B) that specifically binds to human serum albumin (HSA), and a domain (C) that specifically binds to PSMA. The three domains in PSMA-targeted trispecific proteins are in any order. Thus, it is envisaged that the order of domains of PSMA-targeted trispecific proteins is as follows:

H ₂ N-(A)-(B)-(C)-COOH H ₂ N-(A)-(C)-(B)-COOH, H ₂ N-(B)-(A)-(C) -COOH, H ₂ N-(B)-(C)-(A)-COOH, H ₂ N-(C)-(B)-(A)-COOH or H ₂ N-(C)-(A) -(B)-COOH.

In some embodiments, PSMA-targeted trispecific proteins have the order of H ₂ domains N-(A)-(B)-(C)-COOH. In some embodiments, PSMA-targeted trispecific proteins have the order of H ₂ domains N-(A)-(C)-(B)-COOH. In some embodiments, PSMA-targeted trispecific proteins have the order of H ₂ domains N-(B)-(A)-(C)-COOH. In some embodiments, PSMA-targeted trispecific proteins have the order of H ₂ domains N-(B)-(C)-(A)-COOH. In some embodiments, PSMA-targeted trispecific proteins have the order of H ₂ domains N-(C)-(B)-(A)-COOH. In some embodiments, PSMA-targeted trispecific proteins have the order of H ₂ domains N-(C)-(A)-(B)-COOH.

In some embodiments, the PSMA-targeted trispecific proteins contain the HSA binding domain as a middle domain, such that the order of the domains is H ₂ N-(A)-(B)-(C)-COOH or H ₂ N-(C) -(B)-(A)-COOH. It is envisaged that in such embodiments where the HSA binding domain is a medium domain, the CD3 and PSMA binding domains have additional flexibility to bind to their respective targets.

In some embodiments, the PSMA-targeted trispecific proteins described herein provide for a polypeptide with the sequence described in Table 1. 10 (SEQ ID NO: 140-152), and its subsequences. In some embodiments, the trispecific antigen-binding protein comprises a polypeptide having at least 70-95% or more homology to the sequence described in Table 1. 10 (SEQ ID NO: 140-152). In some embodiments, the trispecific antigen-binding protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or more homology to the sequence described in Table 1. 10 (SEQ ID NO: 140-152). In some embodiments, the trispecific antigen-binding protein has a sequence comprising at least a portion of the sequence described in Table 1. 10 (SEQ ID NO: 140-152). In some embodiments, the PSMA trispecific antigen binding protein comprises a polypeptide comprising one or more of the sequences described in Table 1. 10 (SEQ ID NO: 140-152). In still other embodiments, the PSMA trispecific antigen binding protein comprises one or more CDRs as described in the sequences in Table 1. 10 (SEQ ID NO: 140-152).

The PSMA-targeted trispecific proteins described herein are designed to specifically target PSMA-expressing cells by recruiting cytotoxic T cells. This improves efficiency compared to ADCC (Antibody Dependent Cell Mediated Cytotoxicity) which involves full length antibodies directed to a single antigen and which does not have the ability to directly recruit cytotoxic T cells. In contrast, by recruiting CD3 molecules specifically expressed on the surface of these cells, PSMA-targeted trispecific proteins can cross-link cytotoxic T cells with PSMA-expressing cells in a highly specific manner, thereby directing the T cell's cytotoxic potential towards the target cell. The PSMA-targeted trispecific proteins described herein provide for the recruitment of cytotoxic T cells by binding to surface-expressed CD3 proteins that form part of the TCR. The simultaneous binding of several PSMA trispecific antigen-binding proteins to CD3 and to PSMA expressed on the surface of specific cells causes T cell activation and mediates the subsequent lysis of the specific PSMA-expressing cell. Thus, PSMA-targeted trispecific proteins are contemplated to exhibit intense, specific, and efficient killing of the target cell. In some embodiments, the PSMA-targeted trispecific proteins described herein stimulate cytotoxic T cells to kill a target cell to eliminate pathogenic cells (eg, PSMA-expressing tumor cells). In some of these embodiments, cells are killed selectively, thereby reducing the likelihood of toxic side effects.

The PSMA-targeted trispecific proteins described herein offer additional therapeutic advantages over traditional monoclonal antibodies and other smaller bispecific molecules. In general, the efficacy of recombinant proteins as pharmaceuticals is highly dependent on the natural pharmacokinetic parameters of the protein itself. One such advantage is that the PSMA-targeted trispecific proteins described herein have an increased pharmacokinetic half-life due to the presence of an extended half-life domain, such as an HSA-specific domain. In this regard, the PSMA-targeted trispecific proteins described herein have an increased serum half-life of about 2, 3, about 5, about 7, about 10, about 12, or about 14 days, according to some embodiments. This distinguishes them from other binding proteins, such as BiTE or DART molecules, which have a relatively much shorter half-life. For example, in the case of an scFv-scFv fusion molecule in the form of an anti-CD19xCD3 bispecific antibody of the BiTE format, continuous intravenous infusion (i.v.) drug delivery is required due to its short half-life. The longer natural half-life of PSMA-targeted trispecific proteins overcomes this problem, thereby providing increased therapeutic potential, such as low dose pharmaceutical formulations, reduced periodic administration, and/or novel pharmaceutical compositions. The PSMA-targeted trispecific proteins described herein are also optimally sized for enhanced tissue penetration and tissue distribution. Larger sizes limit or impede the penetration or distribution of the protein in target tissues. The PSMA-targeted trispecific proteins described herein avoid this by virtue of their small size, which allows enhanced tissue penetration and distribution. Therefore, in some embodiments, the PSMA-targeted trispecific proteins described herein are about 50 to about 80 kDa, about 50 to about 75 kDa, about 50 to about 70 kDa, or about 50 to about 65 kDa . Thus, the size of PSMA-targeted trispecific proteins is advantageous compared to IgG antibodies, which are approximately 150 kDa, and BiTE and DART diabody molecules, which are approximately 55 kDa, but whose half-life is not increased, and, therefore, they are rapidly excreted by the kidney.

In still other embodiments, the PSMA-targeted trispecific proteins described herein are of optimal size for enhanced tissue penetration and distribution. In such embodiments, PSMA-targeted trispecific proteins are designed to be as small as possible while retaining specificity for their targets. Therefore, according to such embodiments, the PSMA-targeted trispecific proteins described herein are from about 20 to about 40 kDa, or from about 25 to about 35 kDa, to about 40 kDa, to about 45 kDa, to about 50 kDa, up to about 55 kDa, up to about 60 kDa, up to about 65 kDa. In some embodiments, the PSMA-targeted trispecific proteins described herein are about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, about 39, about 38, about 37, about 36, approximately 35, approximately 34, approximately 33, approximately 32, approximately 31, approximately 30, approximately 29, approximately 28, approximately 27, approximately 26, approximately 25, approximately 24, approximately 23,

- 5 042513 about 22, about 21 or about 20 kDa. An exemplary approach to providing a small size is to use single domain antibody (sdAb) fragments for each of the domains. For example, a particular PSMA trispecific antigen binding protein contains an anti-CD3 sdAb, an anti-HSA sdAb, and an anti-PSMA sdAb. This allows the exemplary PSMA trispecific antigen-binding protein to be reduced in size to 40 kDa. Thus, in some embodiments, all domains of the PSMA-targeted trispecific proteins are single domain antibody (sdAb) fragments. In other embodiments, the PSMA-targeted trispecific proteins described herein comprise small molecule entities (SMEs) that bind HSA and/or PSMA. SME binding structures are small molecules with an average size of about 500 to 2000 Da and are attached to PSMA-targeted trispecific proteins by known methods such as sortase ligation or conjugation. In such cases, one of the domains of the PSMA trispecific antigen binding protein is a sortase recognition sequence, eg LPETG (SEQ ID NO: 57). To attach the SME binding structure to the PSMA trispecific antigen binding protein with the sortase recognition sequence, the protein is incubated with the sortase and the SME binding structure, whereby the sortase allows the SME binding structure to be attached to the recognition sequence. Known SME binding structures include MIP-1072 and MIP-1095, which bind to prostate specific membrane antigen (PSMA). In still other embodiments, the domain of the PSMA-targeted trispecific proteins described herein that binds to PSMA comprises a nottin peptide for PSMA binding. Nottins are disulfide-stabilized peptides with a cysteine knot scaffold and their average size is approximately 3.5 kDa. Nottins are designed to bind to certain tumor-associated molecules such as PSMA. In still other embodiments, the domain of the PSMA-targeted trispecific proteins described herein that binds to PSMA contains a natural PSMA ligand.

Another feature of the PSMA-targeted trispecific proteins described herein is that they are designed as a single polypeptide with a flexible linkage between their domains. This makes it easy to obtain and produce PSMA-targeted trispecific proteins, since they can be encoded by a single cDNA molecule, easily introduced into the vector. In addition, since the PSMA-targeted trispecific proteins described herein are monomeric with a single polypeptide chain, there are no problems with chain pairing or the need for dimerization. The PSMA-targeted trispecific proteins described herein are contemplated to have a reduced tendency to aggregate, unlike other known molecules, such as bispecific proteins with Fc-gamma immunoglobulin domains.

In the PSMA-targeted trispecific proteins described herein, the domains are connected via internal linkers L1 and L2, where L1 connects the first and second domains of the PSMA-targeted trispecific proteins, and L2 connects the second and third domains of the PSMA-targeted trispecific proteins. Linkers L1 and L2 have an optimized length and/or amino acid composition. In some embodiments, the L1 and L2 linkers are the same length and amino acid composition. In other embodiments, L1 and L2 are different. In certain embodiments, the L1 and/or L2 internal linkers are short, i.e. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, internal linkers are composed of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, the L1 and/or L2 internal linkers are long, i. consist of 15, 20 or 25 amino acid residues. In some embodiments, such internal linkers consist of from about 3 to about 15, such as 8, 9, or 10, contiguous amino acid residues. With regard to the amino acid composition of the L1 and L2 internal linkers, peptides are chosen with properties that confer flexibility on PSMA-targeted trispecific proteins, do not affect binding domains, and are resistant to protease cleavage. For example, as a rule, glycine and serine residues provide resistance to proteases. Examples of internal linkers suitable for joining domains within PSMA-targeted trispecific proteins include, without limitation, (GS) _n (SEQ ID NO: 153), (GGS) n (SEQ ID NO: 154), (GGGS) n ( SEQ ID NO: 155), (GGSG)n (SEQ ID NO: 156), (GGSGG)n (SEQ ID NO: 157) or (GGGGS)n (SEQ ID NO: 158), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the L1 and/or L2 internal linker is (GGGGS)4 (SEQ ID NO: 159) or (GGGGS)3 (SEQ ID NO: 160).

CD3 binding domain.

The specificity of the T cell response is mediated by antigen recognition (presented in the context of the major histocompatibility complex, MHC) of the TCR. As part of the TCR, CD3 is a protein complex that includes a CD3y chain (gamma), a CD3δ chain (delta), and two CD3ε chains (epsilon) that are present on the cell surface. CD3 binds to the α (alpha) and β (beta) chains

TCR, as well as CD3 ζ (zeta), together forming a complete TCR. Clustering of CD3 on the surface of T cells, for example by immobilized anti-CD3 antibodies, results in T cell activation similar to T cell receptor involvement, but independent of its clonal specificity.

In one aspect, the PSMA-targeted trispecific proteins described herein contain a domain that specifically binds to CD3. In one aspect, the PSMA-targeted trispecific proteins described herein comprise a domain that specifically binds to human CD3. In some embodiments, the PSMA-targeted trispecific proteins described herein comprise a domain that specifically binds to CD3y. In some embodiments, the PSMA-targeted trispecific proteins described herein comprise a domain that specifically binds to CD3δ. In some embodiments, the PSMA-targeted trispecific proteins described herein comprise a domain that specifically binds to CD3ε.

In still other embodiments, the PSMA-targeted trispecific proteins described herein comprise a domain that specifically binds to a TCR. In certain instances, the PSMA-targeted trispecific proteins described herein contain a domain that specifically binds the TCR α chain. In certain instances, the PSMA-targeted trispecific proteins described herein contain a domain that specifically binds the TCR β chain.

In certain embodiments, the CD3 binding domain of the PSMA-targeted trispecific proteins described herein not only has a strong CD3 binding affinity for human CD3, but also exhibits excellent cross-reactivity with the corresponding cynomolgus monkey CD3 proteins. In some instances, the CD3 binding domain of PSMA-targeted trispecific proteins is cross-reactive with cynomolgus CD3. In certain instances, human KD:cynomolgus _KD ratios for CD3 range from 5 to 0.2.

In some embodiments, the CD3 binding domain of the PSMA trispecific antigen binding protein can be any domain that binds to CD3, including, but not limited to, monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody domains. In some cases, it is preferred that the CD3 binding domain be from the same species in which the PSMA trispecific antigen binding protein will ultimately be used. For example, for use in humans, it may be preferred that the CD3-binding domain of the PSMA trispecific antigen-binding protein contains human residues or humanized residues of the antigen-binding domain of an antibody or antibody fragment.

Thus, in one aspect, the antigen binding domain comprises a humanized antibody, or a human antibody, or an antibody fragment, or a mouse antibody, or an antibody fragment. In one embodiment, the humanized CD3 binding domain or human CD3 binding protein comprises one or more (e.g., all three) of light chain complementarity determining region 1 (CDR1 LC), light chain complementarity determining region 2 (CDR2 LC), and complementarity determining light chain region 3 (CDR3 LC) of the humanized CD3 binding domain or human CD3 binding domain described herein and/or one or more (e.g., all three) of the complementarity determining region 1 of the heavy chain (CDR1 HC) determining complementarity between heavy chain region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3) of the humanized CD3 binding domain or human CD3 binding domain described herein, e.g., humanized CD3 binding domain or human CD3 binding domain, containing one or more, such as all three, CDR LCs and one or more, such as all three, CDRs NS.

In some embodiments, the humanized CD3 binding domain or human CD3 binding domain comprises a humanized light chain variable region or a human CD3-specific light chain variable region, wherein the CD3-specific light chain variable region provides a human light chain CDR or non-human species in the framework region of the human light chain. In certain instances, the light chain framework is a λ-(lambda) light chain framework. In other cases, the framework region of the light chain is the framework region of the light to-(kappa)-chain.

In some embodiments, the humanized CD3 binding domain or human CD3 binding domain provides for a humanized CD3-specific heavy chain variable region or human heavy chain variable region specific for CD3, wherein the CD3-specific heavy chain variable region provides for a heavy chain CDR

- 7 042513 of a human or non-human species in the framework region of a human heavy chain.

In certain cases, the complementarity-determining regions of the heavy chain and/or light chain are derived from known anti-CD3 antibodies, such as e.g. -66 or X35-3, VIT3, VMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

In one embodiment, the CD3 binding domain is a single chain variable fragment (scFv) containing a light chain and a heavy chain with the amino acid sequence provided herein. As used herein, a single chain variable fragment or scFv refers to an antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are continuously connected via a short, flexible polypeptide linker, and capable of be expressed as a single polypeptide chain, while scFv retains the specificity of the intact antibody from which it is derived. In one embodiment, the CD3 binding domain comprises a light chain variable region comprising an amino acid sequence with at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions), the amino acid sequence of the variable region of the light chain provided herein, or a sequence characterized by 95-99% identity with the amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence with at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions), the amino acid sequence of the heavy chain variable region presented herein, or a sequence characterized by 95-99% identity with the amino acid sequence presented herein.

In one embodiment, the humanized CD3 binding domain or human CD3 binding domain is scFv, and a light chain variable region containing the amino acid sequence described herein is fused to a heavy chain variable region containing the amino acid sequence described herein, via scFv linker. The light chain variable region and the scFv heavy chain variable region may be in any of the following orientations, for example: light chain variable region-scFv-linker-heavy chain variable region or heavy chain variable region-scFv-linker-light chain variable region.

In some cases, scFvs that bind to CD3 are obtained according to known methods. For example, scFv molecules can be generated by joining the VH and VL regions together using flexible polypeptide linkers. The scFv molecules contain an scFv linker (eg, a Ser-Gly linker) with an optimized length and/or amino acid composition. Therefore, in some embodiments, the length of the scFv linker is such that the VH or VL domain is capable of intermolecular association with another variable domain to form a CD3 binding site. In certain embodiments, such scFv linkers are short, i. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in certain instances, scFv linkers are composed of approximately 12 or fewer amino acid residues. In the case of 0 amino acid residues, the scFv linker is a peptide bond. In some embodiments, such scFv linkers consist of approximately 3-15, eg 8, 9, or 10 contiguous amino acid residues. With regard to the amino acid composition of the scFv linkers, peptides are chosen that provide flexibility, do not affect the variable domains, and also provide interstrand folding to join two variable domains together to form a functional CD3 binding site. For example, scFv linkers containing glycine and serine residues typically confer protease resistance. In some embodiments, linkers in scFv contain glycine and serine residues. The amino acid sequence of scFv linkers can be optimized, for example, using phage display techniques, to improve CD3 binding and scFv product yield. Examples of peptidic scFv linkers suitable for joining a light chain variable domain and a heavy chain variable domain in scFv include, without limitation, (GS) _n (SEQ ID NO: 153), (GGS) _n (SEQ ID NO: 154), (GGGS)n (SEQ ID NO: 155), (GGSG)n (SEQ ID NO: 156), (GGSGG)n (SEQ ID NO: 157) or (GGGGS) _n (SEQ ID NO: 158), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the scFv linker may be (GGGGS) ₄ (SEQ ID NO: 159) or (GGGGS) ₃ (SEQ ID NO: 160). Changing the length of the linker can maintain or increase activity, resulting in superior performance in activity assays.

In some embodiments, the CD3 binding domain of the PSMA trispecific antigen binding protein has an affinity for CD3

- 8 042513 on the surface of CD3-expressing cells with a KD of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, the CD3 binding domain of the PSMA trispecific antigen binding protein has an affinity for CD3ε, γ, or δ with a KD of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In still other embodiments, the CD3 binding domain of the PSMA trispecific antigen binding protein has low affinity for CD3, i. about 100 nM or more.

Binding affinity for CD3 can be determined, for example, by the ability of the PSMA trispecific antigen-binding protein itself or its CD3-binding domain to bind to CD3 coated on the assay plate; presented on the cell surface of the microorganism; in solution, etc. The binding activity of the PSMA trispecific antigen binding protein itself or its CD3 binding domain of the present disclosure to CD3 can be assayed by immobilizing the ligand (e.g. CD3) or the PSMA trispecific antigen binding protein itself or its CD3 binding domain on the bead, substrate, cell surface and etc. Certain agents may be added to the appropriate buffer and binding partners may be incubated for a specified period of time at a given temperature. After washes to remove unbound material, the bound protein can be released using, for example, SDS, high pH buffers, etc. and subject to analysis, for example, using the method of surface plasmon resonance (SPR).

In some embodiments, the CD3 binding domains described herein comprise a polypeptide with the sequence described in Table 1. 7 (SEQ ID NO: 188), and its subsequences. In some embodiments, the CD3 binding domain comprises a polypeptide having at least 70-95% or more homology to the sequence described in Table 1. 7 (SEQ ID NO: 1-88). In some embodiments, the CD3 binding domain comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or more homology to the sequence described in Table 1. 7 (SEQ ID NO: 1-88). In some embodiments, the CD3 binding domain has a sequence comprising at least a portion of the sequence described in Table 1. 7 (SEQ ID NO: 1-88). In some embodiments, the CD3 binding domain comprises a polypeptide comprising one or more of the sequences described in Table 1. 7 (SEQ ID NO: 1-88).

In certain embodiments, the CD3 binding domain comprises an scFv with a heavy chain CDR1 comprising SEQ ID NOs: 16 and 22-33. In certain embodiments, the CD3 binding domain comprises an scFv with a heavy chain CDR2 comprising SEQ ID NOs: 17 and 34-43. In certain embodiments, the CD3 binding domain comprises an scFv with a heavy chain CDR3 comprising SEQ ID NOs: 18 and 44-53. In certain embodiments, the CD3 binding domain comprises an scFv with a light chain CDR1 comprising SEQ ID NOs: 19 and 54-66. In certain embodiments, the CD3 binding domain comprises an scFv with a light chain CDR2 comprising SEQ ID NOs: 20 and 67-79. In certain embodiments, the CD3 binding domain comprises an scFv with a light chain CDR3 comprising SEQ ID NOs: 21 and 80-86.

A domain that provides an increase in half-life.

Provided herein are domains that provide for an increase in the half-life of an antigen-binding domain. Such domains are contemplated to include, without limitation, HSA binding domains, Fc domains, small molecules, and other domains known in the art to provide increased half-life.

Human serum albumin (HSA) (molecular weight ~67 kDa) is the most abundant protein in plasma, present at approximately 50 mg/ml (600 μM), and in humans has a half-life of about 20 days. HSA serves to maintain plasma pH, affects oncotic blood pressure, acts as a carrier for many metabolites and fatty acids, and serves as the major protein drug transporter in plasma.

Non-covalent binding to albumin provides an increase in the half-life of short-lived proteins. For example, recombinant fusion of an albumin-binding domain to a Fab fragment results in a 25-fold and 58-fold reduction in clearance and a 26-fold and 37-fold increase in in vivo half-life when intravenously administered to mice and rabbits compared to administration of the Fab fragment alone. According to another example, when insulin was acylated with fatty acids to promote binding to albumin, a sustained effect was observed when injected subcutaneously in rabbits and pigs. Taken together, these studies demonstrate the relationship between albumin binding and sustained release.

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In one aspect, the PSMA-targeted trispecific proteins described herein contain a domain that provides an increase in half-life, for example a domain that specifically binds to HSA. In some embodiments, the HSA binding domain of the PSMA trispecific antigen binding protein can be any domain that binds to HSA, including, but not limited to, monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody domains. In some embodiments, the HSA binding domain is a single chain variable fragment (scFv), a single domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of a single domain antibody derived from representative of camelids, a peptide, ligand, or small molecule compound specific for HSA. In certain embodiments, the HSA binding domain is a single domain antibody. In other embodiments, the HSA binding domain is a peptide. In still other embodiments, the HSA binding domain is a small molecule. It is envisaged that the HSA-binding domain of the PSMA trispecific antigen-binding protein is relatively small and weighs no more than 25 kDa, no more than 20 kDa, no more than 15 kDa, or no more than 10 kDa according to some embodiments. In certain cases, the size of the HSA-binding domain is 5 kDa or less if it is a peptide or a small molecular weight compound.

The half-life extension domain of the PSMA trispecific antigen-binding protein provides altered pharmacodynamic and pharmacokinetic parameters of the PSMA trispecific antigen-binding protein itself. As mentioned above, a domain that provides an increase in half-life provides an increase in half-life. The half-life extension domain also provides a change in pharmacodynamic properties, including changes in the distribution, penetration and diffusion of the trispecific antigen-binding protein in tissues. In some embodiments, the half-life extension domain provides improved tissue (including tumor) targeting, tissue distribution, tissue penetration, tissue diffusion, and increased efficiency compared to a protein without a half-life extension domain. In one embodiment, the therapeutic methods successfully and effectively use a reduced amount of the trispecific antigen-binding protein, resulting in reduced side effects, such as reduced cytotoxicity to non-tumor cells.

In addition, the binding affinity of the half-life extension domain can be selected to control the specific half-life of a particular trispecific antigen-binding protein. Thus, in some embodiments, a domain that provides an increase in half-life has a high binding affinity. In other embodiments, a domain that provides an increase in half-life is characterized by an average binding affinity. In yet other embodiments, the half-life domain has low or minimal binding affinity. Exemplary binding affinities include Kd at concentrations of 10 nM or less (high), 10 to 100 nM (medium), and greater than 100 nM (low). As mentioned above, binding affinities for HSA are determined using known methods such as the surface plasmon resonance (SPR) method.

In some embodiments, the HSA binding domains described herein comprise a polypeptide with the sequence described in Table 1. 8 (SEQ ID NO: 89-112), and its subsequences. In some embodiments, the implementation of the HSA-binding domain provides a polypeptide characterized by at least 70-95% or more homology with the sequence described in table. 8 (SEQ ID NO: 89-112). In some embodiments, the HSA binding domain comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or more homology to the sequence described in Table 1. 8 (SEQ ID NO: 89-112). In some embodiments, the HSA binding domain has a sequence comprising at least a portion of the sequence described in Table 1. 8 (SEQ ID NO: 89-112). In some embodiments, the HSA binding domain comprises a polypeptide comprising one or more of the sequences described in Table 1. 8 (SEQ ID NO: 89-112).

In some embodiments, the HSA binding domains described herein provide for a single domain antibody with a CDR1 comprising SEQ ID NOs: 96 and 99-101. In some embodiments, the HSA binding domains described herein provide for a single domain antibody with a CDR1 comprising SEQ ID NOs: 97 and 102-107. In some embodiments, the HSA binding domains described herein provide for a single domain antibody with a CDR1 comprising SEQ ID NOS: 98, 108, and 109.

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Prostate specific membrane antigen (PSMA) binding domain.

Prostate Specific Membrane Antigen (PSMA) is a 100 kDa type II membrane glycoprotein expressed in prostate tissues that shares sequence identity with a transferrin receptor with NAALADase activity. PSMA is overexpressed in prostate cancer and elevated levels of PSMA can also be detected in the serum of these patients. PSMA expression levels increase with disease progression, being highest in metastatic hormone refractory disease, for which there is currently no therapy.

In addition to the CD3-binding and half-life extension domains described, the PSMA-targeted trispecific proteins described herein also contain a domain that binds to PSMA. The design of the PSMA-targeted trispecific proteins described herein provides flexibility to the PSMA-binding domain such that the PSMA-binding domain can be any type of binding domain, including but not limited to monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibodies. In some embodiments, the PSMA binding domain is a single chain variable fragment (scFv), a single domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of a single domain antibody derived from camelid representative. In other embodiments, the PSMA binding domain is a non-Ig type binding domain, ie. an antibody mimic, such as anticalins, affilins, affitel molecules, affimers, affitins, alphabodies, avimers, DARP-ins, finomers, Kunitz domain peptides and monobodies. In still other embodiments, the PSMA binding domain is a ligand or peptide that binds or associates with PSMA. In still other embodiments, the PSMA binding domain is nottin. In still other embodiments, the PSMA binding domain is a small molecule.

In some embodiments, the PSMA binding domain comprises the following formula: f1-r1-f2-r2-f3-r3-f4, wherein r1, r2, and r3 are the complementarity-determining regions of CDR1, CDR2, and CDR3, respectively, and f1, f2, f3, and f4 are framework residues, with r1 providing SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 125; r2 provides SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123 or SEQ ID NO: 126 and r3 provides SEQ ID NO: 124 or SEQ ID NO: 127.

In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX ₁ YX ₂ MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX4X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX7YGY). In some embodiments, the amino acid residues X _b X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X ₇ are independently selected from glutamic acid, proline, serine, histidine, threonine, aspartic acid, glycine, lysine, threonine, glutamine, and tyrosine. In some embodiments, X ₁ is proline. In some embodiments, X ₂ is histidine. In some embodiments, X3 is aspartic acid. In some embodiments, X4 is a lysine. In some embodiments, X ₅ is glutamine. In some embodiments, X ₆ is tyrosine. In some embodiments, X ₇ is serine. In some embodiments, the PSMA binding protein of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X1 is glutamic acid, X2 is histidine, _X3 is aspartic acid, _X4 is glycine, _X5 is threonine , X ₆ is serine and X ₇ is serine.

In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX ₁ YX ₂ MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX ₄ X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX7YGY), wherein X ₁ is proline.

In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX1YX2MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX ₄ X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX7YGY), wherein X ₅ is glutamine.

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In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX1YX ₂ MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX ₄ X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX7YGY), wherein X ₆ is tyrosine.

In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX1YX2MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX ₄ X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX7YGY), where X4 is lysine and X7 is serine. In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX1YX2MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX ₄ X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX7YGY), wherein X ₂ is histidine, X ₃ is aspartic acid, X ₄ is lysine, and X ₇ is serine.

In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX1YX2MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX ₄ X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX7YGY), wherein X1 is proline, X2 is histidine, X3 is aspartic acid, and X7 is serine.

In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX1YX2MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX ₄ X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX ₇ YGY), wherein X ₂ is histidine, X ₃ is aspartic acid, X ₅ is glutamine and X7 is serine.

In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX1YX2MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX ₄ X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX7YGY), where X2 is histidine, X ₃ is aspartic acid, X ₆ is tyrosine, and X ₇ is serine.

In some embodiments, the PSMA binding domain comprises CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as shown in SEQ ID NO: 162 (RFMISX1YX2MH); (b) the amino acid sequence of CDR2 is as shown in SEQ ID NO: 163 (X ₃ INPAX ₄ X ₅ TDYAEX ₆ VKG); and (c) the amino acid sequence of CDR3 is as shown in SEQ ID NO: 164 (DX7YGY), wherein X2 is histidine, X3 is aspartic acid, and X7 is serine.

In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X1 is glutamic acid, X2 is histidine, X3 is threonine, X4 is glycine, X ₅ is threonine, X ₆ is serine; and X7 is serine. In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X1 is glutamic acid, X ₂ is histidine, X ₃ is threonine, X ₄ is glycine, X ₅ is threonine , X ₆ is serine and X7 is serine.

In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X1 is glutamic acid, X2 is serine, X3 is threonine, X4 is lysine, X ₅ is threonine, X ₆ is serine; and X7 is serine.

In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X1 is proline, X ₂ is serine, X ₃ is threonine, X4 is glycine, X ₅ is threonine, X ₆ is serine; and X ₇ is glycine.

In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, where X1 is glutamic acid, _X2 is serine, _X3 is threonine, _X4 is glycine, X ₅ is glutamine, X ₆ is serine and X7 is glycine.

In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X1 is glutamic acid, X ₂ is serine, X ₃ is threonine, X ₄ is glycine, X ₅ is threonine , X ₆ is tyrosine and X7 is glycine. In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X ₁ is glutamic acid, X ₂ is histidine, X ₃ is aspartic acid, X ₄ is lysine, X ₅ is is threonine, X ₆ is serine and X7 is serine.

In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X1 is proline, X ₂ is histidine, X ₃ is aspartic acid, X ₄ is glycine, X ₅ is threonine , X ₆ is serine and X7 is serine.

In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X1 is glutamic acid, X ₂ is histidine, X ₃ is aspartic acid, X ₄ is glutamine, X ₅ is threonine, X ₆ is serine and X7 is serine.

In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, where X1 is glutamic acid, X ₂ is histidine, X ₃ is aspartic acid, X4 is glycine, X ₅ is threonine , X ₆ is tyrosine and X ₇ is serine.

In some embodiments, the PSMA binding domain of the present disclosure may comprise the sequences CDR1, CDR2, and CDR3, wherein X ₂ is histidine and X7 is serine. Illustrative framework sequences are disclosed as SEQ ID NOs: 165-168.

In some embodiments, the PSMA binding domains described herein comprise a polypeptide with the sequence described in Table 1. 9 (SEQ ID NO: 113-140), and its subsequences. In some embodiments, the implementation of the HSA-binding domain provides a polypeptide characterized by at least 70-95% or more homology with the sequence described in table. 9 (SEQ ID NOS: 113-140). In some embodiments, the HSA binding domain comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or more homology to the sequence described in Table 1. 9 (SEQ ID NOS: 113-140).

In some embodiments, the HSA binding domain has a sequence comprising at least a portion of the sequence described in Table 1. 9 (SEQ ID NOS: 113-140). In some embodiments, the HSA binding domain comprises a polypeptide comprising one or more of the sequences described in Table 1. 9 (SEQ ID NOS: 113-140).

In some embodiments, the PSMA binding domains described herein provide for a single domain antibody with a CDR1 comprising SEQ ID NOs: 114-116 and 125. In some embodiments, the PSMA binding domains described herein provide for a single domain antibody with a CDR1 comprising SEQ ID NOs: 117-123 and 126. In some embodiments, the PSMA binding domains described herein provide for a single domain antibody with a CDR1 comprising SEQ ID NOs: 124 and 127.

Modifications of PSMA trispecific proteins.

The PSMA-targeted trispecific proteins described herein encompass derivatives or analogs in which (i) an amino acid is replaced by an amino acid residue that is not encoded according to the genetic code, (ii) the mature polypeptide is fused to another compound such as polyethylene glycol, or (iii) ) additional amino acids are fused to the protein, such as a leader sequence, or a secretion signal sequence, or a protein purification sequence.

Representative modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, flavin covalent attachment, heme moiety covalent attachment, nucleotide or nucleotide derivative covalent attachment, lipid or lipid derivative covalent attachment, phosphatidylinositol covalent attachment, crosslinking, cyclization, disulfide formation. bonds, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gammacarboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer RNA mediated sulfation, addition of amino acids to proteins, such as arginylation, and ubiquitination.

Modifications are made to any part of the PSMA-targeted trispecific proteins described herein, including the peptide backbone, amino acid side chains, and amino or carboxy termini. Certain common peptide modifications that are useful for modifying PSMA-targeted trispecific proteins include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blocking of an amino or carboxyl group in a polypeptide, or both, via covalent modification, and ADP-ribosylation.

Polynucleotides encoding PSMA-targeted trispecific proteins.

Also in some embodiments, polynucleotide molecules encoding the PSMA trispecific antigen binding protein described herein are provided. In some embodiments, the polynucleotide molecules are provided as a DNA construct. In other embodiments, the polynucleotide molecules are provided as a messenger RNA transcript.

Polynucleotide molecules are constructed using known methods, for example, by combining genes encoding three binding domains, either separated by peptide linkers or, in other embodiments, directly linked by a peptide bond, into a single genetic construct operably linked to a suitable promoter, and optionally a suitable transcription terminator, and expression in bacterial cells or other appropriate expression system such as, for example, CHO cells. In embodiments where the PSMA binding domain is a small molecule, the polynucleotides comprise genes encoding a CD3 binding domain and a half-life extension domain. In embodiments where the half-life extension domain is a small molecule, the polynucleotides comprise genes encoding domains that bind to CD3 and PSMA. Depending on the vector system and host used, any number of suitable transcriptional and translational elements may be used, including constitutive and inducible promoters. The promoter is chosen to direct the expression of the polynucleotide in the appropriate host cell.

In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which is a further embodiment. Such a recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, viruses (eg, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.) and cosmids.

A number of expression vector/host systems can be used to contain and express a polynucleotide encoding a polypeptide of the described trispecific antigen binding protein. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J. Immunol. Methods. (2004), 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

Thus, PSMA-targeted trispecific proteins, as described herein, according to some embodiments, are obtained by introducing into a host cell a vector encoding a protein as described above, and culturing said host cell under conditions in which the protein domains are expressed, may be isolated and optionally further purified.

pharmaceutical compositions.

In some embodiments, pharmaceutical compositions are also provided comprising a PSMA trispecific antigen binding protein as described herein, a vector containing a polynucleotide encoding a polypeptide of PSMA-targeted trispecific proteins, or a host cell transformed with such a vector, and at least one pharmaceutically acceptable carrier. .

The term pharmaceutically acceptable carrier includes, without limitation, any carrier that does not adversely affect the efficacy of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers may be formulated using conventional methods and may be administered to the subject at a suitable dose. Preferably the compositions are sterile. Such compositions may also contain adjuvants such as preservatives, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents.

In some embodiments of the pharmaceutical compositions, the PSMA-targeted trispecific proteins described herein are encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposomes, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, the PSMA trispecific antigen binding protein is attached to liposomes. In some cases, the PSMA trispecific antigen binding protein is conjugated to the surface of the liposomes. In some cases, the PSMA trispecific antigen binding protein is encapsulated in a liposome envelope. In some cases, the liposome is a cationic liposome.

The PSMA-targeted trispecific proteins described herein are provided for use as a drug. The introduction is carried out in various ways, for example by intravenous, intraperitoneal, subcutaneous, intramuscular, local or intradermal administration. In some embodiments, the route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The schedule of application will be determined by the attending physician and taking into account other clinical factors. Dosages for any given patient depend on many factors including patient size, body surface area, age, sex, particular compound to be administered, duration and route of administration, type of therapy, general health, and other concomitantly administered drugs. An effective dose refers to amounts of the active ingredient that are sufficient to affect the course and severity of the disease, resulting in a reduction in the intensity or remission of such pathology, and they can be determined using known methods.

Methods of treatment.

In some embodiments, also provided herein are methods and uses for stimulating the immune system in an individual in need thereof, comprising administering a PSMA-targeted trispecific protein as described herein. In some cases, administration of a PSMA-targeted trispecific protein as described herein induces and/or maintains cytotoxicity against a PSMA-expressing cell. In some cases, the PSMA-expressing cell is a cancer cell.

Also provided herein are methods and uses for treating a disease, disorder, or condition associated with PSMA, comprising administering to an individual in need thereof the PSMA-targeted trispecific protein described herein. Diseases, disorders or conditions associated with PSMA include, without limitation, a proliferative disease or a neoplastic disease. In one embodiment, the disease, disorder, or condition associated with PSMA is prostate cancer. In another embodiment, the disease, disorder, or condition associated with PSMA is kidney cancer.

In some embodiments, the prostate cancer is advanced prostate cancer. In some embodiments, the prostate cancer is drug resistant. In some embodiments, the prostate cancer is resistant to antiandrogen drugs. In some embodiments, the prostate cancer is metastatic. In some embodiments, the prostate cancer is metastatic and drug resistant (eg, antiandrogen drug resistant). In some embodiments, the prostate cancer is castration resistant. In some embodiments, the prostate cancer is metastatic and castration resistant. In some embodiments, the prostate cancer is resistant to enzalutamide. In some embodiments, the prostate cancer is resistant to enzalutamide and abiraterone. In some embodiments, the prostate cancer is resistant to enzalutamide, abiraterone, and bicalutamide. In some embodiments, the prostate cancer is docetaxel resistant. In some of these embodiments, the prostate cancer is resistant to enzalutamide, abiraterone, bicalutamide, and docetaxel.

In some embodiments, administration of a PSMA-targeted trispecific protein as described herein results in inhibition of growth of prostate cancer cells; provides inhibition of migration of prostate cancer cells; provides inhibition of invasion of prostate cancer cells; provides a decrease in the intensity of symptoms of prostate cancer; provides a reduction in the size of prostate cancer; provides a reduction in the number of cancerous tumors of the prostate gland; provides a decrease in the number of prostate cancer cells; induces necrosis, pyroptosis, oncosis, apoptosis, autophagy or other mechanism of death of prostate cancer cells; or provides an enhancement of the therapeutic effects of a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and any combination thereof.

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In some embodiments, the method comprises inhibiting the growth of prostate cancer cells by administering a PSMA-targeted trispecific protein as described herein. In some embodiments, the method comprises inhibiting the migration of prostate cancer cells by administering a PSMA-targeted trispecific protein as described herein. In some embodiments, the method comprises inhibiting invasion of prostate cancer cells by administering a PSMA-targeted trispecific protein as described herein. In some embodiments, the method comprises ameliorating symptoms of prostate cancer by administering a PSMA-targeted trispecific protein as described herein. In some embodiments, the method comprises reducing the size of the prostate cancer by administering the PSMA-targeted trispecific protein described herein. In some embodiments, the method comprises reducing prostate cancers by administering a PSMA-targeted trispecific protein as described herein. In some embodiments, the method comprises reducing the number of prostate cancer cells by administering a PSMA-targeted trispecific protein as described herein. In some embodiments, the method comprises inducing necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other mechanism of death of prostate cancer cells by administering the PSMA-targeted trispecific protein described herein.

As used herein, in some embodiments, treatment or treatment or treated refers to therapeutic treatment in which the goal is to slow (alleviate) the course of an undesired physiological condition, disorder, or disease, or to produce beneficial or desired clinical results. For the purposes described herein, beneficial or desirable clinical outcomes include, without limitation, symptomatic relief; reducing the severity of the condition, disorder or disease; stabilization (ie, no worsening) of the course of the condition, disorder, or disease; delaying the manifestation or slowing the progression of the condition, disorder or disease; alleviation of the course of the condition, disorder or disease and remission (partial or complete), whether it is detectable or undetected, or an increase in positive scores or improvement in the course of the condition, disorder or disease. Treatment involves inducing a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival in the absence of treatment. In other embodiments, treating, or administering treatment, or being treated refers to prophylactic measures in which the goal is to delay the onset of, or reduce the severity of, an undesired physiological condition, disorder, or disease, for example, in a person with a predisposition to the disease (e.g., an individual who is carrier of a genetic marker for a disease, such as prostate cancer).

In some embodiments of the methods described herein, PSMA-targeted trispecific proteins are administered in combination with an agent to treat a particular disease, disorder, or condition. Means include, without limitation, types of therapy involving antibodies, small molecules (for example, chemotherapeutic drugs), hormones (steroid, peptide, etc.), types of radiation therapy (γ-radiation, x-rays and / or targeted delivery of radioisotopes, microwave radiation, UV radiation, etc.), gene therapies (eg, antisense, retroviral therapy, etc.), as well as immunotherapies. In some embodiments, the PSMA-targeted trispecific proteins are administered in combination with antidiarrheals, antiemetics, analgesics, opioids, and/or non-steroidal anti-inflammatory drugs. In some embodiments, PSMA-targeted trispecific proteins are administered before, during, or after surgery.

Some definitions.

For the purposes of this document, the term half-life is used in its usual sense as described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (ed. Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, 6th ed., 1980). Briefly, this term covers the quantitative measurement of the time of excretion of the drug. The elimination of most drugs is exponential (ie, first order kinetics) because drug concentrations typically do not reach those required to saturate the elimination process. The rate of an exponential process can be expressed by its rate constant k, which expresses the relative change per unit time, or by its half-cycle ti/ ₂ , the amount of time required for 50% completion of the process. The units of these two constants are time ^-1 and time, respectively. First order rate constant and half cycle

- 16 042513 tions are related by a simple ratio (kxt _1/2 =0.693) and can accordingly be interchanged. Since the first order elimination kinetics assume that a constant fraction of the drug is lost per unit time, the plot of the logarithm of the drug concentration versus time is linear at all times after the initial distribution phase (i.e., after drug absorption and distribution completion). The elimination half-life of the drug can be accurately determined from such a graph.

As used herein, the phrase prostate cancer or advanced prostate cancer includes a class of forms of prostate cancer that progresses beyond the early stages of the disease. As a rule, forms of prostate cancer at a late stage are associated with a poor prognosis. Types of advanced prostate cancer include, without limitation, metastatic prostate cancer, drug-resistant prostate cancer such as antiandrogen-resistant prostate cancer (e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer, etc.), hormone-refractory prostate cancer, castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, docetaxel-resistant prostate cancer induced by androgen receptor splice variant-7 (AR-V7) drug-resistant prostate cancer, such as AR-V7-induced antiandrogen-resistant prostate cancer (e.g., AR-V7-induced enzalutamide-resistant prostate cancer) induced by aldoke member 3 family 1 toreductase (AKR1C3) drug-resistant prostate cancer, such as AKR1C3-induced antiandrogen-resistant prostate cancer (eg, AKR1C3-induced enzalutamide-resistant prostate cancer), and combinations thereof. Some cases of advanced prostate cancer have not responded at all or have been refractory to treatment with one or more of the following traditional prostate cancer therapies: enzalutamide, abiraterone, bicalutamide, and docetaxel. Compounds, compositions, and methods of the present disclosure are provided for the treatment of prostate cancer, such as advanced prostate cancer, including any one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) types of advanced prostate cancer disclosed herein.

Examples

Example 1 Methods for Assessing the Binding and Cytotoxic Activity of Trispecific Antigen Binding Molecules.

Getting protein.

Trispecific molecule sequences preceded by a leader sequence and followed by a 6x histidine tag (SEQ ID NO: 161) were cloned into the pcDNA 3.4 expression vector (Invitrogen) for expression in mammalian cells. Expi293F cells (Life Technologies A14527) were maintained in suspension in Optimum Growth flasks (Thomson) at 0.2 to 8 x 1e6 cells/ml in Expi293 medium. Expi293 cells were transfected with purified plasmid DNA according to the protocols of the Expi293 Expression System kit (Life Technologies, A14635) and maintained for 4-6 days post transfection. The conditioned medium was partially purified by affinity and salting out chromatography. The trispecific proteins were then purified by ion exchange or alternatively concentrated using Amicon ultracentrifuge filters (EMD Millipore), loaded onto Superdex 200 size exclusion chromatography medium (GE Healthcare) and separated in neutral buffer containing excipients. Fractions were pooled and final purity assessed by SDS-PAGE and analytical SEC.

Affinity measurements.

Affinities for all binding domain molecules were measured by biolayer interferometry using an Octet instrument.

PSMA affinity was measured by loading the human PSMA-Fc protein (100 nM) onto anti-human IgG Fc biosensors for 120 s, followed by a 60 s baseline measurement, after which binding events were measured by incubating the sensor tip in dilution series trispecific molecules for 180 s, followed by dissociation for 50 s. Affinities for EGFR and CD3 were measured by loading human EGFR-Fc protein or human CD3-Flag-Fc protein, respectively (100 nM), on anti-human IgG Fc biosensors for 120 s, followed by a 60-s baseline measurement, after whereby binding events were measured by incubating the sensor tip in a dilution series of trispecific molecules for 180 s, followed by dissociation for 300 s. Affinities for human serum albumin (HSA) were measured by loading biotinylated albumin onto streptavidin biosensors, then the same kinetics were measured as for CD3 affinity measurements. All steps were performed at 30° C. in 0.25% casein in phosphate buffered saline.

Cytotoxicity assays.

Human T cell dependent cellular cytotoxicity (TDCC) assay was used to measure the ability of T cell recruiting activators, including trispecific molecules, to direct T cells to kill tumor cells (Nazarian et al. 2015. J. Biomol. Screen. 20:519 -27). In this assay, T cells and cancer cell line target cells were mixed together at a ratio of 10:1 in a 384-well plate, and various amounts of T-cell recruiting activator were added. After 48 hours, the T cells were washed out, leaving target cells attached to the plate that were not killed by the T cells. CellTiter-Glo® fluorescent cell viability assay (Promega) was used to quantify remaining living cells. In some cases, target cells have been engineered to express luciferase. In such cases, viability of target cells was assessed by performing a luminescent luciferase assay using the STEADYGLO® reagent (Promega), in which viability is directly proportional to the level of luciferase activity.

stability analyses.

The stability of trispecific binding proteins was evaluated at low concentrations in the presence of non-human primate blood serum. TriTAC was diluted to 33 μg/ml in cynomolgus monkey serum (BioReclamationIVT) and either incubated for 2 days at 37°C or subjected to five freeze-thaw cycles. After treatment, samples were evaluated in cytotoxicity assays (TDCC) and their residual activity compared to untreated stock solutions.

Xenograft analyzes.

The in vivo efficacy of trispecific binding proteins was evaluated in xenograft experiments (Crown Bioscience, Taicang). NOD/SCID mice deficient in the common gamma chain (NCG, Model Animal Research Center of Nanjing University) were inoculated on day 0 with a mixture of 5e6 human prostate cancer cells 22Rv1 and 5e6 resting T cells human, which were isolated from the body of a healthy human donor. Mice were randomized into three groups and treated with vehicle, 0.5 mg/kg PSMA TriTAC C324 or 0.5 mg/kg PSMA BiTE. The drugs were administered daily for 10 days via i.v. bolus injection. Animals were checked daily for morbidity and mortality. Tumor volume measurements were taken twice a week with a caliper. The study was terminated after 30 days.

PK analyses.

The aim of this study was to evaluate the pharmacokinetic parameters of a single dose of trispecific binding proteins after intravenous injection. Previously unexperimented two cynomolgus monkeys per group (1 male and 1 female) were given the compound by slow IV bolus injection given over about 1 minute. After dosing once daily, cell observations were made and body weights were recorded every week. Collected blood samples and processed them to obtain serum for pharmacokinetic analysis within 21 days after dosing.

Test drug concentrations were determined in monkey serum by electroluminescent reading (Meso Scale Diagnostics, Rockville). 96-well plates with immobilized recombinant CD3 were used to capture the analyte. Detection was performed using SULFO-TAG labeled recombinant PSMA on an MSD reader according to the manufacturer's instructions.

Example 2 Evaluation of the Effect of CD3 Affinity on the Properties of Trispecific Molecules PSMA-targeted trispecific molecules with different CD3 binding domains were studied to show the effects of altering CD3 affinity.

An exemplary PSMA-targeted trispecific molecule is shown in FIG. 1. In the table. 1 shows the affinity of each molecule for three binding partners (PSMA, CD3, HSA). Affinity values were measured by biolayer interferometry using an Octet instrument (Pall Forte Bio). Reduced affinity for CD3 results in a loss of efficacy in the context of T-cell mediated cellular cytotoxicity (FIGS. 2A-2C). The pharmacokinetic properties of these trispecific molecules were evaluated in cynomolgus monkeys. Molecules with high affinity for CD3, like TriTAC C236, had a terminal phase half-life of approximately 90 hours (FIG. 3). Despite the altered CD3 binding ability on the surface of T cells, the terminal phase half-life of two molecules with different CD3 affinities shown in FIG. 4 is very similar. However, reduced affinity for CD3 appears to result in a larger volume of distribution, consistent with reduced sequestration of the trispecific molecule by T cells. There were no adverse clinical observations or changes in body weight during the study period.

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Table 1

Binding affinities for human and cynomolgus monkey antigens.

KD value of anti-PSMA antibody (nM) KD value of anti-albumin antibody (nM) KD value of anti-CD3e antibody (nM) Human cynomolgus macaque ratio of y values pHSA CSA ratio of y values Human cynomolgus macaque ratio of y values

cynomolgus / human cynomolgus / human cynomolgus / human TriTAC, high aff., as instrument -C236 16.3 0 0 22.7 25.4 1.1 6.0 4.7 0.8 TriTAC CD3, high aff. - C324 17.9 0 0 9.8 9.7 1 7.4 5.8 0.8 TriTAC CD3, average aff. -C339 13.6 0 0 8.8 8.3 0.9 40.6 33.6 0.8 TriTAC CD3, low aff. - C325 15.3 0 0 10.1 9.7 1 217 160 0.7

Example 3 Evaluation of the effect of PSMA affinity on the properties of trispecific molecules.

PSMA-targeted trispecific molecules with different PSMA-binding domains were studied in order to show the effects of changing the affinity for PSMA. In table. 2 shows the affinity of each molecule for three binding partners (PSMA, CD3, HSA). Reduced affinity for PSMA results in a loss of efficacy in the context of T-cell mediated cellular cytotoxicity (FIGS. 5A-5C).

table 2

Binding affinities for human and cynomolgus monkey antigens

KD value of anti-PSMA antibody (nM) KD value of anti-albumin antibody (nM) KD value of anti-CD3e antibody (nM) Human cynomolgus macaque ratio of y values pHSA CSA ratio of y values Human cynomolgus macaque ratio of y values

cynomolgus / human cynomolgus / human cynomolgus / human PSMA- GpTAS (r8) - C362 22.0 0 n.a. 6.6 6.6 1.0 8.3 4.3 0.52 PSMA TriTAC (HDS) С363 3.7 540 146 7.6 8.4 1.1 8.0 5.2 0.65 PSMA TriTAC (HTS) - C364 0.15 663 4423 8.4 8.6 1.0 7.7 3.8 0.49

Example 4 Efficacy of PSMA-targeted trispecific molecules in vivo.

The PSMA-targeted trispecific C324 molecule was evaluated for its ability to inhibit tumor growth in mice. For this experiment, immunodeficient mice reconstructed with human T cells were subcutaneously inoculated with PSMA-expressing human prostate tumor cells (22Rv1) and treated daily for 10 days with 0.5 mg/kg i.v. either PSMA-targeted BiTE or TriTAC molecules. Tumor growth was measured over 30 days. Throughout the experiment, the trispecific molecule was able to inhibit tumor growth with an efficiency comparable to that of the BiTE molecule (FIG. 6).

Example 5 Specificity of Trispecific Molecules.

To evaluate the specificity of PSMA-targeted TriTAC molecules, their ability to induce T cells to kill tumor cells was tested using tumor cells that

- 19 042513 are negative for PSMA (Fig. 7A). An EGFR-targeted TriTAC molecule served as a positive control, a GFP-targeted TriTAC molecule served as a negative control. All three TriTACs with different PSMA-binding domains showed the expected activity against the PSMA-positive LNCaP cell line (Fig. 7B), but did not achieve EC50 in the PSMA-negative tumor cell lines KMS12BM and OVCAR8 (Fig. 7C and 7D). EC50 are given in table. 3. At very high TriTAC concentrations (>1 nM), some limited off-target cell killing could be observed for TriTAC C362 and C363, while no significant cell killing was observed for C364 under any of the conditions tested.

Table 3

Cell-killing activity of TriTAC molecules in antigen-positive and antigen-negative tumor cell lines (EC50 [pM])

TriTAC LNCaP KMS12BM OVCAR8 PSMAp8 TriTAC С362 13.0 >10000 >10000 PSMA HDS TriTAC C363 6.2 >10000 >10000 PSMA HTS TriTAC C364 0.8 >10000 >10000 EGFR TriTAC C131 9.4 >10000 6 GFP TriTAC C >10000 >10000 >10000

Example 6 Stress tests and protein stability.

The four PSMA-targeted trispecific molecules were either incubated for 48 hours in cynomolgus monkey serum at low concentrations (33.3 μg/ml) or subjected to five freeze-thaw cycles in cynomolgus monkey blood serum. After treatment, the biological activity of TriTAC molecules was assessed in cell killing assays and compared with non-stressed samples (positive control, FIGS. 8A-8D). All molecules retained most of their cell-killing activity. TriTAC C362 was the most stress tolerant and did not seem to lose activity at all under the conditions tested in this case.

Example 7 Tumor Xenograft Model The PSMA-targeted trispecific proteins from the previous examples were evaluated in a xenograft model.

Male immunodeficient NCG mice were subcutaneously inoculated with 5 x 10 ⁶ 22Rv1 cells in their dorsal right flank. If the size of the tumors reached 100-200 mm ³ , the animals were divided into 3 treatment groups. Groups 2 and 3 (8 animals each) received 1.5 x 10 ⁷ activated human T cells by intraperitoneal injection. Three days later, group 3 animals were treated with a total of 9 intravenous doses of 50 μg PSMA trispecific antigen-binding protein from Example 1 (qdx9d). Groups 1 and 2 were treated with vehicle medium only. Body weight and tumor volume were determined within 30 days.

It is expected that tumor growth in mice treated with PSMA-trispecific antigen-binding protein is significantly reduced compared to tumor growth in the corresponding control group treated with vehicle medium.

Example 8 Proof of Concept Clinical Study Protocol for Administering the PSMA Trispecific Antigen Binding Protein of Example 1 to Patients with Prostate Cancer.

This is a Phase I/II clinical trial to investigate the PSMA trispecific antigen-binding protein of Example 1 as a treatment for prostate cancer.

Research outcomes:

Primary: Maximum tolerated dose of PSMA-targeted trispecific proteins from previous examples

Secondary: Determination of whether the in vitro response to the PSMA-targeted trispecific proteins from the previous examples is associated with clinical response

Phase I

The maximum tolerated dose (MTD) will be determined in the Phase I study.

1.1. The maximum tolerated dose (MTD) will be determined in the Phase I study.

1.2. Patients who meet eligibility criteria will be included in the study of the PSMA-targeted trispecific proteins from the previous examples.

1.3. The aim is to identify the highest dose of PSMA-targeted trispecific proteins from the previous examples that can be administered safely without serious or uncontrolled side effects in participants. The dose administered will depend on the number of participants previously enrolled in the study and how well the dose was tolerated. Not all participants will receive the same dose.

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Phase II

2.1. In a subsequent second phase II, participants will receive treatment for MTD, with the aim being to determine whether therapy with the PSMA-targeted trispecific proteins from the previous examples provides at least a 20% response rate.

The primary outcome for phase II is to determine whether therapy with the PSMA-targeted trispecific proteins from the previous examples achieves a clinical response in at least 20% of patients (explosive response, minimal response, partial response, or complete response)

Eligibility to Participate:

Histologically confirmed newly diagnosed aggressive prostate cancer according to the current classification of the World Health Organization, from 2001 to 2007.

Any stage of the disease.

Treatment with docetaxel and prednisone (± surgery).

Age >18 years.

Patient's general condition index according to Karnofsky >50% or an indicator of the patient's general condition according to the ECOG scale 0-2.

Life expectancy >6 weeks.

Example 9 Activity of exemplary PSMA-targeted antigen-binding protein (PSMA-targeted TriTAC molecule) in redirected T cell kill assays using a panel of PSMA-expressing cell lines and T cells from different donors.

This study was carried out in order to demonstrate that the activity of the exemplary PSMA trispecific antigen binding protein is not limited to LNCaP cells or a single cell donor.

Redirected T cell kill assays were performed using T cells from four different donors and human PSMA-expressing prostate cancer cell lines VCaP, LNCaP, MDAPCa2b, and 22Rv1. With one exception, the PSMA trispecific antigen binding protein was able to direct the killing of these cancer cell lines involving T cells from all donors with EC ₅₀ values of 0.2 to 1.5 pM, as shown in Table 1. 4. In the case of the 22 Rv1 prostate cancer cell line and the 24 donor, little to no kill was observed (data not shown). Also, donor 24 provided only about 50% killing of the MDAPCa2b cell line, while T cells from the other 3 donors provided almost complete killing of this cell line (data not shown). In control assays, killing by PSMA trispecific antigen binding protein was demonstrated to be PSMA specific. When PSMA-expressing cells were treated with a control trispecific protein targeting green fluorescent protein (GFP) rather than PSMA, no killing was observed (data not shown). Similarly, the PSMA trispecific antigen binding protein was inactive in cell lines that lacked expression of PSMA, NCI-1563, and HCT116, also shown in Table 1. 4.

Table 4

EC ₅₀ values from TDCC analyzes with six human cancer cell lines and four different T cell donors

cell line ECso (M) values for TDCC Donor 24 Donor 8144 Donor 72 Donor 41 LNCaP 1.5E-12 2.2E-13 3.6E-13 4.3E-13 MDAPCa2b 4.8E-12 4DE-13 4.9E-13 6.5E-13 VCaP 6.4E-13 1.6E-13 2.0E-13 3.5E-13 22Rvl N.D. 7.2E-13 1.4E-12 1.3E-12 HCT116 >l,0E-8 >l,0E-8 >l,0E-8 >l,0E-8 NCI-1563 >l,0E-8 >l,0E-8 >l,0E-8 >l,0E-8

Example 10 Stimulation of Cytokine Expression by an Exemplary PSMA Trispecific Antigen Binding Protein (PSMA-Targeted TriTAC Molecule) in Redirected T Cell Kill Assays.

This assay was performed to demonstrate T cell activation by exemplary PSMA trispecific antigen binding protein in redirected T cell kill assays by measuring cytokine secretion into the activated T cell assay medium.

Conditioned medium collected after cell killing assays by redirected T cells as described in Example 9 above was analyzed for expression of the cytokines TNFa and

- 21 042513

IFNy. Cytokine levels were measured using AlphaLISA assays (Perkin-Elmer). Addition of a titer of PSMA-targeted antigen-binding protein to T cells from four different donors and four PSMA-expressing cell lines, LNCaP, VCaP, MDAPCa2b and 22Rv1, provided an increase in TNFa levels. The results on the levels of expression of TNFa and expression of IFNy in the conditioned medium are shown in table. 5 and 6, respectively. EC ₅₀ values for PSMA-targeted antigen-binding protein-induced expression of these cytokines ranged from 3 to 15 pM. In the case of the control trispecific GFP-targeted protein, elevated levels of cytokines were not observed. Similarly, when assays were performed using two cell lines that lacked PSMA expression, HCT116 and NCI-H1563, PSMA HTS TriTAC also did not increase TNFa or IFNy expression levels.

Table 5

EC ₅₀ values for TNFa expression in media from PSMA-trispecific antigen-binding protein-induced TDCC assays with six human cancer cell lines and T cells from four different donors

cell line Donor 24 Donor 8144 Donor 41 Donor 72 LNCaP 4.9E-12 2.8E-12 4.0E-12 3.2E-12 VCaP 3.2E-12 2.9E-12 2.9E-12 2.9E-12 MDAPCa2b 2, IE-11 4.0E-12 5.5E-12 3.6E-12 22Rvl 8.9E-12 2.5E-12 4.0E-12 3.3E-12 HCT116 >lE-8 >lE-8 >lE-8 >lE-8 NCI-H1563 >lE-8 >lE-8 >lE-8 >lE-8

Table 6

EC50 values for IFNy expression in media from PSMA-trispecific antigen-binding protein-induced TDCC assays with six human cancer cell lines and

T cells from four different donors

cell line Donor 24 Donor 8144 Donor 41 Donor 72 LNCaP 4.2E-12 4.2E-12 4.2E-12 2.8E-12 VCaP 5DE-12 1.5E-11 3.4E-12 4.9E-12 MDAPCa2b 1.5E-11 5.8E-12 9.7E-12 3.5E-12 22Rvl 7.8E-12 3.0E-12 9DE-12 3.0E-12 HCT116 >lE-8 >lE-8 >lE-8 >lE-8 NCI-H1563 >lE-8 >lE-8 >lE-8 >lE-8

Example 11 Activity of an exemplary PSMA trispecific antigen binding protein (PSMA-targeted TriTAC) in a redirected T cell kill (TDCC) assay using T cells from cynomolgus monkeys.

This study was performed to test the ability of exemplary PSMA trispecific antigen binding protein to direct T cells from cynomolgus monkeys to kill PSMA expressing cell lines.

TDCC analyzes were performed using peripheral blood mononuclear cells (PBMC) from cynomolgus monkeys. Cynomolgus monkey PBMCs were added to LNCaP cells at a ratio of 10:1. The PSMA trispecific antigen binding protein was shown to redirect the killing of LNCaP of cynomolgus monkey PBMC with an EC ₅₀ value of 11 pM. The result is shown in Fig. 9A. To confirm these results, a second cell line, MDAPCa2b, was used and PBMCs from a second cynomolgus monkey donor were tested. Redirected killing of target cells was observed with an EC ₅₀ value of 2.2 pM. The result is shown in Fig. 9B. Killing was specific to the PMSA-binding arm of the PSMA trispecific antigen-binding protein, as killing was not observed for the trispecific GFP-targeted protein as a negative control. From these data, it appears that PSMA-targeted antigen-binding trispecific protein can direct cynomolgus monkey T cells to kill target cells expressing human PSMA.

Example 12 Expression of T Cell Activation Markers in Redirected T Cell Kill Assays Using the Exemplary PSMA Trispecific Antigen Binding Protein (PSMA Targeted TriTAC Molecule).

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This assay was performed to evaluate whether T cells were activated when an exemplary PSMA trispecific antigen binding protein directed T cells to kill target cells.

The assays were performed using the conditions for cell kill assays by redirected T cells described in the example above. T cell activation was assessed by measuring the expression levels of CD25 and CD69 on the surface of T cells using flow cytometry. PSMA trispecific antigen binding protein was added to a 10:1 mixture of purified human T cells and VCaP prostate cancer cell line. After the addition of increasing amounts of PSMA trispecific antigen binding protein, increased levels of CD69 expression and CD25 expression were observed as shown in FIG. 10. The EC ₅₀ value was 0.3 pM for CD69 and 0.2 pM for CD25. A GFP-targeted trispecific protein was included as a negative control in these assays, and in the case of the GFP-targeted trispecific protein, an increase in CD69 or CD25 expression from negligible to no expression was observed, also shown in FIG. 10.

Example 13 Stimulation of T cell proliferation by an exemplary PSMA trispecific antigen binding protein (PSMA-targeted TriTAC molecule) in the presence of target cells expressing PSMA.

This assay was used as an additional method to demonstrate that the exemplary PSMA trispecific antigen binding protein was able to activate T cells when redirected to kill target cells.

T cell proliferation assays were performed using conditions for cell kill assay by redirected T cells using LNCaP target cells as described above and measuring the number of T cells present at 72 hours. an EC ₅₀ value of 0.5 pM. A trispecific GFP targeting protein was included in the assay as a negative control and no increase in proliferation was observed for this protein. The results of the T cell proliferation assay are shown in FIG. eleven.

Example 14 Redirected T cell killing of LNCaP cells mediated by three exemplary PSMA trispecific antigen binding proteins (PSMA-targeted TriTAC molecules PH1T, PH and Z2).

This study was performed to test the ability of three exemplary PSMA trispecific antigen-binding proteins with the sequences shown in SEQ ID NOs: 150, 151 and 152 to redirect T cells to kill the LNCaP cell line.

In the TDCC assays performed as described in the examples above, the PSMAPH1T TriTAC (SEQ ID NO: 150) and PSMAPH1 TriTAC (SEQ ID NO: 151) proteins provided kill redirection with EC ₅₀ values of 25 and 20 pM, respectively, as shown in FIG. 12A; and the PSMA Z2 TriTAC protein (SEQ ID NO: 152) provided kill redirection with an EC ₅₀ value of 0.8 pM as shown in FIG. 12V.

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Table 7

CD3 binding domain sequences

SEQ ID NO: Description Amino acid sequence 1 CD3-binding, clone 2B2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWV RQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISR DDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISY WAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQE PSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQ APRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCTLWYSNRWVFGGGTKLTVL 2 CD3-binding, clone 9F2 EVQLVESGGGLVQPGGSLKLSCAASGFEFNKYAMNW VRQAPGKGLEWVARIRSKYNKYAIYYADSVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSFGAVTSGNYPNWVQQKPGQ APRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYDNRWVFGGGTKLTVL 3 CD3-binding, clone 5A2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYAIYYADSVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSHIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSTGYVTSGNYPNWVQQKPG QAPRGLIGGTSFLAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCVLWYSNRWIFGGGTKLTVL 4 CD3-binding, clone 6A2 EVQLVESGGGLVQPGGSLKLSCAASGFMFNKYAMNW VRQAPGKGLEWVARIRSKSNNYAIYYADSVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYIS YWATWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSFGAVTSGNYPNWVQQKPGQ APRGLIGGTKLLAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCVLWYSNSWVFGGGTKLTVL CD3-binding, clone 2D2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNWV RQAPGKGLEWVARIRSKYNNYAIYYKDSVKDRFTISR DDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISY WAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQE PSLTVSPGGTVTLTCGSSTGAVVSGNYPNWVQQKPGQ APRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVL 6 CD3-binding, clone 3F2

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EVQLVESGGGLVQPGGSLKLSCAASGFTYNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADEVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSKGAVTSGNYPNWVQQKPG QAPRGLIGGTKELAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCTLWYSNRWVFGGGTKLTVL 7 CD3-binding, clone 1A2 EVQLVESGGGLVQPGGSLKLSCAASGNTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYEIYYADSVKDRFTIS RDD SKNTAYLQMNNLKTEDTAVYYCVRHTNFGNSYIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPG QAPRGLIGGIYFLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVLWYSNRWVFGGGTKLTVL 8 CD3-binding, clone 1С2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNNYAMNW VRQAPGKGLEWVARIRSKYNNYAIYYADAVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSQIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSTGAVTDGNYPNWVQQKPG QAPRGLIGGIKFLAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCVLWYSNRWVFGGGTKLTVL 9 CD3-binding, clone 2E4 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAVNWV RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR DDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISY WAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQE PSLTVSPGGTVTLTCGESTGAVTSGNYPNWVQQKPGQ APRGLIGGTKILAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVL 10 CD3-binding, clone 10E4 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYPMNWV RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR DDSKNTAYLQMNNLKNEDTAVYYCVRHGNFNNSYIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSTGAVTKGNYPNWVQQKPG QAPRGLIGGTKMLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCALWYSNRWVFGGGTKLTVL eleven CD3-binding, clone 2H2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNW VRQAPGKGLEWVARIRSKYNNYAIYYADEVKDRFTIS

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RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSTGAVVSGNYPNWVQQKPG QAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCVLWYSNRWVFGGGTKLTVL 12 CD3-binding, clone 2A4 EVQLVESGGGLVQPGGSLKLSCAASGNTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGDSYIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSTGAVTHGNYPNWVQQKPG QAPRGLIGGTKVLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVLWYSNRWVFGGGTKLTVL 13 CD3-binding, clone 10V2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNNYAMNW VRQAPGKGLEWVARIRSGYNNYATYYADSVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSYTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFNAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVLWYANRWVFGGGTKLTVL 14 CD3-binding, clone 1G4 EVQLVESGGGLVQPGGSLKLSCAASGFEFNKYAMNW VRQAPGKGLEWVARIRSKYNNYETYYADSVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSLIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSSGAVTSGNYPNWVQQKPGQ APRGLIGGTKFGAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCVLWYSNRWVFGGGTKLTVL 15 CD3-binding wt EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYIS YWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ EPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVLWYSNRWVFGGGTKLTVL 16 CD3-binding CDR1 HCwt GFTFNKYAMN

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17 CD3-binding CDR2 HCwt RIRSKYNNYATYYADSVK 18 CD3-binding CDR3 HCwt HGNFGNSYISYWAY 19 CD3-binding CDR1 LCwt GSSTGAVTSGNYPN 20 CD3-binding CDR2 LCwt GTKFLAP 21 CD3-binding CDR3 LC wt VLWYSNRWV 22 Option 1 CDR1 HC GNTFNKYAMN 23 Option 2 CDR1 HC GFEFNKYAMN 24 Option 3 CDR1 HC GFMFNKYAMN 25 Option 4 CDR1 HC GFTYNKYAMN 26 Option 5 CDR1 HC GFTFNNYAMN 27 Option 6 CDR1 HC GFTFNGYAMN 28 Option 7 CDR1 HC GFTFNIYAMN 29 Option 8 CDR1 HC GFTFNEYAMN thirty Option 9 CDR1 HC GFTFNKYPMN 31 Option 10 CDR1 HC GFTFNKYAVN 32 Option 11 CDR1 HC GFTFNKYAIN 33 Option 12 CDR1 HC GFTFNKYALN 34 Option 1 CDR2 NS RIRSGYNNYATYYADSVK 35 Option 2 CDR2 NS RIRSKSNNYATYYADSVK 36 Option 3 CDR2 NS RIRSKYNKYATYYADSVK 37 Option 4 CDR2 NS RIRSKYNNYETYYADSVK 38 Option 5 CDR2 NS RIRSKYNNYATEYADSVK 39 Option 6 CDR2 NS RIRSKYNNYATYYKDSVK 40 Option 7 CDR2 HC RIRSKYNNYATYYADEVK 41 Option 8 CDR2 NS RIRSKYNNYATYYADAVK 42 Option 9 CDR2 NS RIRSKYNNYATYYADQVK 43 Option 10 CDR2 NS RIRSKYNNYATYYADDVK 44 Option 1 CDR3 NS HANFGNSYISYWAY 45 Option 2 CDR3 NS HTNFGNSYISYWAY

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46 Option 3 CDR3 NS HGNFNNSYISYWAY 47 Option 4 CDR3 NS HGNFGDSYISYWAY 48 Option 5 CDR3 HC HGNFGNSHISYWAY 49 Option 6 CDR3 NA HGNFGNSPISYWAY 50 Option 7 CDR3 HC HGNFGNSQISYWAY 51 Option 8 CDR3 HC HGNFGNSLISYWAY 52 Option 9 CDR3 HC HGNFGNSGISYWAY 53 Option 10 CDR3 NS HGNFGNSYISYWAT 54 Option 1 CDR1 LC ASSTGAVTSGNYPN 55 Option 2 CDR1LC GESTGAVTSGNYPN 56 Variant 3 CDR1 LC GSYTGAVTSGNYPN 57 Variant 4 CDR1LC GS SFGAVTSGNYPN 58 Variant 5 CDR1 LC GSSKGAVTSGNYPN 59 Option 6 CDR1LC GSSSGAVTSGNYPN 60 Variant 7 CDR1 LC GSSTGYVTSGNYPN 61 Variant 8 CDR1 LC GSTGAVVSGNYPN 62 Variant 9 CDR1 LC GSSTGAVTDGNYPN 63 Variant 10 CDR1 LC GSSTGAVTKGNYPN 64 Variant 11 CDR1 LC GSSTGAVTHGNYPN 65 Variant 12 CDR1 LC GSSTGAVTVGNYPN 66 Variant 13 CDR1 LC GSSTGAVTSGYYPN 67 Option 1 CDR2 LC GIKFLAP 68 Option 2 CDR2LC GTEFLAP 69 Variant 3 CDR2 LC GIYFLAP 70 Option 4 CDR2LC GTSFLAP 71 Variant 5 CDR2 LC GTNFLAP 72 Option 6 CDR2 LC GTKLLAP 73 Variant 7 CDR2 LC GTKELAP 74 Variant 8 CDR2 LC GTKILAP 75 Variant 9 CDR2 LC GTKMLAP 76 Variant 10 CDR2 LC GTKVLAP 77 Option 11 CDR2 LC GTKFNAP 78 Variant 12 CDR2 LC GTKFGAP 79 Option 13 CDR2 LC GTKFLVP

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80 Option 1 CDR3LC TLWYSNRWV 81 Option 2 CDR3LC ALWYSNRWV 82 Variant 3 CDR3 LC VLWYDNRWV 83 Option 4 CDR3LC VLWYANRWV 84 Variant 5 CDR3 LC VLWYSNSWV 85 Option 6 CDR3LC VLWYSNRWI 86 Variant 7 CDR3 LC VLWYSNRWA 87 CD3-binding, clone 2G5 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYALNWV RQAPGKGLEWVARIRSKYNNYATEYADSVKDRFTISR DDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISY WAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQE PSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQ APRGLIGGTNFLAPGTPERFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWAFGGGTKLTVL 88 CD3-binding, clone 8A5 EVQLVESGGGLVQPGGSLKLSCAASGFTFNEYAMNWV RQAPGKGLEWVARIRSKYNNYATYYADDVKDRFTISR DDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSGISY WAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQE PSLTVSPGGTVTLTCGSSTGAVTVGNYPNWVQQKPGQ APRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVL

Table 8

HSA binding domain sequences.

SEQ ID NO: Description Amino acid sequence 89 sdAb to HSA, clone 6C EVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNA KTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTV SS 90 sdAb to HSA, clone 7A EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV RQAPGKGLEWVSSISGSGADTLYADSLKGRFTISRDNA KTTLYLQMNSLRPEDTAVYYCTIGGSLSKSSQGTLVTV SS

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91 sdAb to HSA, clone 7G EVQLVESGGGLVQPGNSLRLSCAASGFTYSSFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNA KTTLYLQMNSLRPEDTAVYYCTIGGSLSKSSQGTLVTV SS 92 sdAb to HSA, clone 8H EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV RQAPGKGLEWVSSISGSGTDTLYADSVKGRFTISRDNA KTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTV SS 93 sdAb to HSA, clone 9A EVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWV RQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNA KTTLYLQMNSLRPEDTAVYYCTIGGSLSKSSQGTLVTV SS 94 sdAb to HSA, clone 10G EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV RQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNA KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTV SS 95 HSA binder, wt S 96 HSA-binding CDR1 wt GFTFSSFGMS 97 HSA-binding CDR2 wt SISGSGSDTLYADSVK 98 HSACDR3 binding wt GGSLSSR 99 Option 1 CDR1 GFTFSRFGMS 100 Option 2 CDR1 GFTFSKFGMS 101 Variant 3 CDR1 GFIYSSFGMS 102 Option 1 CDR2 SISGSGADTLYADSLK 103 Option 2 CDR2 SISGSGTDTLYADSVK 104 Variant 3 CDR2 SISGSGRDTLYADSVK 105 Variant 4 CDR2 SISGSGSDTLYAESVK 106 Variant 5 CDR2 SISGSGTDTLYAESVK 107 Option 6 CDR2 SISGSGRDTLYAESVK 108 Option 1 CDR3 GGSLSK 109 Option 2 CDR3 GGSLSV BY sdAb to HSA, clone 6CE EVQLVESGGGLVQPGNSLRLSCAASGFTFSRFGMSWV RQAPGKGLEWVSSISGSGSDTLYAESVKGRFTISRDNA KTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTV SS 111 sdAb to HSA, clone 8HE EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV RQAPGKGLEWVSSISGSGTDTLYAESVKGRFTISRDNA KTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTV SS 112 sdAb to HSA, 10GE clone EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV RQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNA KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTV SS

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Table 9

PSMA binding domain sequences

SEQ ID NO: Description Amino acid sequence 113 PSMA binder, wt EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWV RQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAK NTLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVTVSS 114 Option 1 CDR1 RFMISEYHMH 115 Option 2 CDR1 RFMISPYSMH 116 Variant 3 CDR1 RFMISPYHMH 117 Option 1 CDR2 DINPAGTTDYAESVKG 118 Option 2 CDR2 TINPAKTTDYAESVKG 119 Variant 3 CDR2 TINPAGQTDYAESVKG 120 Variant 4 CDR2 TINPAGTTDYAEYVKG 121 Variant 5 CDR2 DINPAKTTDYAESVKG 122 Option 6 CDR2 DINPAGQTDYAESVKG 123 Variant 7 CDR2 DINPAGTTDYAEYVKG 124 Option 1 CDR3 DSYGY

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125 Variant 4 CDR1 RFMISEYSMH 126 Variant 8 CDR2 TINPAGTTDYAESVKG 127 Option 2 CDR3 DGYGY 128 PSMA binder, clone 1 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWV RQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAK NTLYLQMNSLRAEDTAVYYCDGYGYRGQGTLVTVSS 129 PSMA binder, clone 2 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWV RQAPGKGLEWVSDINPAGTTDYAESVKGRFTISRDNAK NTLYLQMNSLRAEDTAVYYCDSYGYRGQGTLVTVSS 130 PSMA binder, clone 3 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWV RQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAK NTLYLQMNSLRAEDTAVYYCDSYGYRGQGTLVTVSS 131 PSMA-binding, clone 4 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWV RQAPGKGLEWVSTINPAKTTDYAESVKGRFTISRDNAK NTLYLQMNSLRAEDTAVYYCDSYGYRGQGTLVTVSS 132 PSMA binder, clone 5 EVQLVESGGGLVQPGGSLRLSCAASRFMISPYSMHWV RQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAK NTLYLQMNSLRAEDTAVYYCDGYGYRGQGTLVTVSS 133 PSMA-binding, clone 6 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWV RQAPGKGLEWVSTINPAGQTDYAESVKGRFTISRDNAK NTLYLQMNSLRAEDTAVYYCDGYGYRGQGTLVTVSS 134 PSMA binder, clone 7 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWV RQAPGKGLEWVSTINPAGTTDYAEYVKGRFTISRDNA KNTLYLQMNSLRAEDTAVYYCDGYGYRGQGTLVTVS S 135 PSMA binder, clone 8 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWV RQAPGI<GLEWVSDINPAI<TTDYAESVI<GRFTISRDNAI< NTLYLQMNSLRAEDTAVYYCDSYGYRGQGTLVTVSS 136 PSMA binder, clone 9 EVQLVESGGGLVQPGGSLRLSCAASRFMISPYHMHWV RQAPGKGLEWVSDINPAGTTDYAESVKGRFTISRDNAK NTLYLQMNSLRAEDTAVYYCDSYGYRGQGTLVTVSS 137 PSMA binder, clone 10 EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWV RQAPGKGLEWVSDINPAGQTDYAESVKGRFTISRDNA KNTLYLQMNSLRAEDTAVYYCDSYGYRGQGTLVTVSS 138 PSMA binder, clone eleven EVQLVESGGGLVQPGGSLRLSCAASRFMISEYHMHWV RQAPGKGLEWVSDINPAGTTDYAEYVKGRFTISRDNA KNTLYLQMNSLRAEDTAVYYCDSYGYRGQGTLVTVSS 139 PSMA binder, clone 12 EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWV RQAPGKGLEWVSDINPAGTTDYAESVKGRFTISRDNAK NTLYLQMNSLKPEDTAVYYCDSYGYRGQGTQVTVSS 140 PSMA binder, clone 13 EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWV RQAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAK NTLYLQMNSLKPEDTAVYYCDSYGYRGQGTQVTVSS

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Table 10

PSMA-targeted trispecific protein sequences

SEQID NO: number Design Subsequence 141 С00324 PSMA TriTAC CD3, high aff. EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVR QAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKN TLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVTVSSGG GGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSK FGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQ GTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLS CAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNY ATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGG GGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTS GNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLL GGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKL TVLHHHHHH 142 С00339 PSMA TriTAC CD3, medium aff. EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVR QAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKN TLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVTVSSGG GGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSK FGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQ GTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLS CAASGFTFNNYAMNWVRQAPGKGLEWVARIRSGYNN YATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTA VYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSG GGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSYTGAV TSGNYPNWVQQKPGQAPRGLIGGTKFNAPGTPARFSGS LLGGKAALTLSGVQPEDEAEYYCVLWYANRWVFGGG TKLTVLHHHHHH

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143 C00325 PSMA TriTAC CD3, low aff. EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVR QAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKN TLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVTVSSGG GGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSK FGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQ GTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLS CAASGFEFNKYAMNWVRQAPGKGLEWVARIRSKYNN YETYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTA VYYCVRHGNFGNSLISYWAYWGQGTLVTVSSGGGGSG GGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSSGAV TSGNYPNWVQQKPGQAPRGLIGGTKFGAPGTPARFSGS LLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLHHHHHH 144 C00236 PSMA TriTAC as a tool EVQLVESGGGLVQPGGSLTLSCAASRFMISEYSMHWVR QAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKN TLYLQMNSLKPEDTAVYYCDGYGYRGQGTQVTVSSGG GGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSF GMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTI SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG TLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSC AASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNY ATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGG GGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTS GNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLL GGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKL TVLHHHHHH 145 C00362 PSMA p8 TriTAC EVQLVESGGGLVQPGGSLRLSCAASRFMISEYSMHWVR QAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKN TLYLQMNSLRAEDTAVYYCDGYGYRGQGTLVTVSSGG GGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSK FGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQ GTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLS CAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNY ATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGG GGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTS GNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLL GGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKL TVLHHHHHH 146 C00363 PSMA HDS TriTAC C363 EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVR QAPGKGLEWVSDINPAGTTDYAESVKGRFTISRDNAKN TLYLQMNSLKPEDTAVYYCDSYGYRGQGTQVTVSSGG GGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSK FGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQ GTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLS CAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNY ATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGG GGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTS GNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLL

- 34 042513

GGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKL TVLHHHHHH 147 C00364 PSMA HTS TriTAC C364 EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVR QAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKN TLYLQMNSLKPEDTAVYYCDSYGYRGQGTQVTVSSGG GGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSK FGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQ GTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLS CAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNY ATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGG GGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTS GNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLL GGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKL TVLHHHHHH 148 C00298 PSMA BiTE QVQLVESGGGLVKPGESLRLSCAASGFTFSDYYMYWV RQAPGKGLEWVAIISDGGYYTYYSDIIKGRFTISRDNAK NSLYLQMNSLKAEDTAVYYCARGFPLLRHGAMDYWG QGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCKASQNVDTNVAWYQQKPGQAPKSLIYSAS YRYSDVPSRFSGSASGTDFTLTISSVQSEDFATYYCQQY DSYPYTFGGGTKLEIKSGGGGSEVQLVESGGGLVQPGG SLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRS KYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKT EDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS SGG GGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSS TGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPAR FSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVLHHHHHH 149 C00131 EGFR TriTAC QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWF RQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNA KNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYD YWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGN SLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSG SDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAV YYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYL QMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGT LVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGT VTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKF LAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLW YSNRWVFGGGTKLTVLHHHHHH

- 35 042513

150 С00457 PSMA PH1T TriTAC QVQLVESGGGVVQAGRSLTLSCAYSGVTVNVYRMGW FRQAPGKEREFVANINWSGNNRDYADSVRGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCASEKPGRLGEYDYGS QGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRL SCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDT LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC TIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGL VQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWV ARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQM NNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVT VSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSN RWVFGGGTKLTVLHHHHHH 151 С00404 PSMA PHI TriTAC QVQLVESGGGVVQAGRSLRLSCAYSGVTVNVYRMGW FRQAPGKEREFVANINWSGNNRDYADSVRGRFTISRDN SKNTLYLQMNSLRAEDTAVYYCASEKPGRLGEYDYGS QGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRL SCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDT LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC TIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGL VQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWV ARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQM NNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVT VSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSN RWVFGGGTKLTVLHHHHHH 152 С00410 PSMA Z2 TriTAC EVQLVESGGGLVQPGGSLTLSCAASRFMISEYHMHWVR QAPGKGLEWVSTINPAGTTDYAESVKGRFTISRDNAKN TLYLQMNSLRAEDTAVYYCDSYGYRGQGTLVTVSSGG GGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSK FGMSWVRQAPGKGLEWVSSISGSGRDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQ GTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLS CAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNY ATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAV YYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGG GGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTS GNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLL GGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKL TVLHHHHHH

Table 11

CDR sequences of the PSMA binding domain.

SEQID No. Subsequence SEQIDNo. 162 RFMISXiYX ₂ MH SEQIDNo. 163 X3INPAX4X5TDYAEX6VKG SEQIDNo. 164 DX7YGY

Table 12

Illustrative framework sequences

SEQID NO: Description Subsequence 165 Frame (fl) EVQLVESGGGLVQPGGSLTLSCAAS 166 Frame (f2) WVRQAPGKGLEWVS 167 Framework (β) RFTISRDNAKNTLYLQMNSLRAEDTAVYYC 168 Frame (f4) DGYGYRGQGTLVTVS s

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that in the practice of the present invention, various alternatives to the embodiments of the present invention described herein may be used. It is intended that the following claims define the scope of the present invention and that methods and structures within the scope of this claim and their equivalents are also covered by it.

Claims (6)

1. Prostate specific membrane antigen (PSMA) targeted trispecific protein comprising the amino acid sequence of SEQ ID NO: 147. 2. The PSMA-targeted trispecific protein of claim 1, wherein the size of the PSMA-targeted trispecific protein is less than 80 kDa. 3. The PSMA-targeted trispecific protein according to claim 1, wherein the size of the PSMA-targeted trispecific protein is between 50 and 75 kDa. 4. The PSMA-targeted trispecific protein of claim 1, wherein the PSMA-targeted trispecific protein has a half-life of at least 50 hours. 5. A pharmaceutical composition comprising (i) a PSMA-targeted trispecific protein according to any one of claims 1-4 and (ii) a pharmaceutically acceptable carrier. 6. The use of a pharmaceutical composition according to claim 5 for the manufacture of a medicament for the treatment of cancer.