WO2021231434A1

WO2021231434A1 - Psma targeting tritacs and methods of use

Info

Publication number: WO2021231434A1
Application number: PCT/US2021/031790
Authority: WO
Inventors: Holger Wesche; Bryan D. LEMON; Richard J. Austin; Che-Leung Law
Original assignee: Harpoon Therapeutics, Inc.
Priority date: 2020-05-12
Filing date: 2021-05-11
Publication date: 2021-11-18

Abstract

Provided herein are prostate specific membrane antigen (PSMA) targeting trispecific molecules comprising a domain binding to CD3, a half-life extension domain, and a domain binding to PSMA. Also provided are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such PSMA targeting trispecific molecules. Also disclosed are methods of using the disclosed PSMA targeting trispecific proteins in the prevention, or treatment of diseases, conditions and disorders, particularly metastatic castration resistant prostate cancer. Dosages and administration regimen for treatment methods are further provided.

Description

PSMA TARGETING TriTACs AND METHODS OF USE

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/023,701 filed on May 12 ,2020, U.S. Provisional Application No. 63/024,749 filed on May 14, 2020, U.S. Provisional Application No. 63/056,200 filed on July 24, 2020 and U.S. Provisional Application No. 63/167,485 filed on March 29, 2021, each incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

[0002] The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged. One such method is by inducing an immune response against the tumor, to make immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumor cells. Metastatic, castration-resistant prostate cancer (mCRPC) kills 27,000 patients in the US each year. Once mCRPC has metastasized beyond regional lymph nodes, the 5-year survival rate is 30%. No curative treatment is available and new therapies are urgently needed. In normal tissues, PSMA expression outside the central nervous system is largely restricted to the prostate. PSMA is expressed in >90% of malignant lesions of mCRPC patients.

SUMMARY OF THE INVENTION

[0003] Provided herein is a method of treating prostate cancer, the method comprising administration of an effective amount of a prostate specific membrane antigen (PSMA) targeting trispecific protein to a subject, wherein said protein comprises (a) a first domain (A) which specifically binds to human CD3; (b) a second domain (B) which is a half-life extension domain; and (c) a third domain (C) which specifically binds to PSMA, wherein the domains are linked in the order H2N-(C)-(B)-(A)-COOH, or by linkers LI and L2, and wherein the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 10 mg/kg.

[0004] In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 10 pg/kg. In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 1000 ng/kg. In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 500 ng/kg. In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 200 ng/kg. In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 1.3 ng/kg to about 160 ng/kg.

[0005] In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 54 ng/kg. In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 72 ng/kg. In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 96 ng/kg. In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 120 ng/kg. In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 150 ng/kg. In some embodiments, the PSMA targeting trispecific protein is administered at a dosage of about 160 ng/kg.

[0006] In some embodiments, the PSMA targeting trispecific protein has an elimination half time of at least about 20 hours. In some embodiments, the PSMA targeting trispecific protein has an elimination half-time of at least about 50 hours. In some embodiments, the PSMA targeting trispecific protein has an elimination half-time of about 100 hours.

[0007] In some embodiments, the PSMA targeting trispecific protein is administered once a week. In some embodiments, wherein the PSMA targeting trispecific protein is administered twice per week. In some embodiments, the PSMA targeting trispecific protein is administered every other week. In some embodiments, the PSMA targeting trispecific protein is administered every three weeks.

[0008] In some embodiments, the subject's prostate surface antigen (PSA) level decreases from about 3.8% to about 76% compared to the baseline. In some embodiments, the subject's prostate surface antigen (PSA) level decreases over 50% compared to the baseline.

[0009] In some embodiments, the method further comprising administration of a dexamethasone (dex) premedication. In some embodiments, the dex premedication is administered prior to administration of the PSMA targeting trispecific protein. In some embodiments, the dex premedication is administered at a dosage of about 1 mg to about 20 mg.

[0010] In some embodiments, the third domain comprises a scFv, a VH domain, a VL domain, a non-Ig domain, a ligand, a knottin, or a small molecule entity that specifically binds to PSMA.

In some embodiments, the third domain comprises one or more sequences selected from the group consisting of SEQ ID NO: 113-140.

[0011] In some embodiments, the first domain comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3. In some embodiments, the first domain comprises one or more sequences selected from the group consisting of SEQ ID NO: 1-88. In some embodiments, the first domain is humanized or human. In some embodiments, the first domain has a KD of 150 nM or less for binding to CD3 on CD3 expressing cells.

[0012] In some embodiments, the second domain binds human serum albumin. In some embodiments, the second domain comprises a scFv, a variable heavy domain (VH), a variable light domain (VL), a peptide, a ligand, or a small molecule. In some embodiments, the second domain comprises one or more sequences selected from the group consisting of SEQ ID NOs: 89-112.

[0013] In some embodiments, linkers LI and L2 are each 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), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, linkers LI and L2 are each independently (GGGGS)4 (SEQ ID NO: 159) or (GGGGS)3 (SEQ ID NO: 160). In some embodiments, linkers LI and L2 are each independently GGGGSGGGS (SEQ ID NO: 170) In some embodiments, the domains are linked in the order EbN-(C)-Ll-(B)-L2-(A)-COOE[.

[0014] In some embodiments, the PSMA targeting trispecific protein is less than about 80 kDa. In some embodiments, the PSMA targeting trispecific protein is about 50 to about 75 kDa. In some embodiments, the PSMA targeting trispecific protein is less than about 60 kDa. In some embodiments, the PSMA targeting trispecific protein has increased tissue penetration as compared to an IgG to the same PSMA. In some embodiments, the PSMA targeting trispecific protein comprises a sequence selected from the group consisting of SEQ ID NO: 141-147. In some embodiments, the PSMA targeting trispecific protein comprises a sequence as set forth in SEQ ID NO: 147. In some embodiments, the PSMA targeting trispecific protein comprises a sequence selected from the group consisting of SEQ ID NO: 150-152.

[0015] In some embodiments, the prostate cancer is a metastatic prostate cancer. In some embodiments, the prostate cancer is a castration resistant prostate cancer.

INCORPORATION BY REFERENCE

[0016] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS [0017] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0018] Figure 1 is schematic representation of an exemplary PSMA targeting trispecific antigen-binding protein where the protein has an constant core element comprising an anti-CD3e single chain variable fragment (scFv) and an anti-HSA variable heavy chain region; and a PSMA binding domain that can be a VH, scFv, a non-Ig binder, or ligand. [0019] Figures 2A-2C compare the ability of exemplary PSMA targeting trispecific proteins (PSMA targeting TriTAC molecules) with different affinities for CD3 to induce T cells to kill human prostate cancer cells. Figure 2A shows killing by different PSMA targeting TriTAC molecules in prostate cancer model LNCaP. Figure 2B shows killing by different PSMA targeting TriTAC molecules in prostate cancer model 22Rvl. Figure 2C shows ECso values for PSMA targeting TriTAC in LNCaP and 22Rvl prostate cancer models.

[0020] Figure 3 shows the serum concentration of PSMA targeting TriTAC (SEQ ID NO: 144) in Cynomolgus monkeys after i.v. administration (100 pg/kg) over three weeks.

[0021] Figure 4 shows the serum concentration of PSMA targeting TriTAC molecules with different CD3 affinities in Cynomolgus monkeys after i.v. administration (100 pg/kg) over three weeks.

[0022] Figures 5A-5C show the ability of PSMA targeting TriTAC molecules with different affinities for PSMA to induce T cells to kill the human prostate cancer cell line LNCaP. Figure 5A shows the experiment performed in the absence of human serum albumin with a PSMA targeting BiTE as positive control. Figure 5B shows the experiment performed in the presence of human serum albumin with a PSMA targeting BiTE as positive control. Figure 5C shows EC50 values for PSMA targeting TriTAC in the presence or absence of HSA with a PSMA targeting BiTE as a positive control in LNCaP prostate cancer models.

[0023] Figure 6 demonstrates the ability of PSMA targeting TriTAC molecules to inhibit tumor growth of human prostate cancer cells in a mouse xenograft experiment.

[0024] Figures 7A-D illustrates the specificity of TriTAC molecules in cell killing assays with target cell lines that do or do not express the target protein. Figure 7A shows EGFR and PSMA expression in LNCaP, KMS12BM, and OVCAR8 cell lines. Figure 7B shows killing of LNCaP tumor cells by PSMA, EGFR, and negative control TriTACs. Figure 7C shows killing of KMS12BM tumor cells by PSMA, EGFR, and negative control TriTACs. Figure 7D shows killing of OVCAR8 cells by PSMA, EGFR, and negative control TriTACs.

[0025] Figures 8A-8D depict the impact of pre-incubation at 37°C and freeze/thaw cycles on TriTAC activity. Figure 8A shows PSMA TriTAC (SEQ ID NO: 144) activity after pre incubation at 37°C or freeze/thaw cycles. Figure 8B shows PSMA TriTAC (SEQ ID NO: 345) activity after pre-incubation at 37°C or freeze/thaw cycles. Figure 8C shows PSMA TriTAC (SEQ ID NO: 146) activity after pre-incubation at 37°C or freeze/thaw cycles. Figure 8D shows PSMA TriTAC (SEQ ID NO: 147) activity after pre-incubation at 37°C or freeze/thaw cycles. [0026] Figures 9A-9B depict the activity of a PSMA targeting TriTAC molecule of this disclosure in redirected T cell killing in T cell dependent cellular cytotoxicity assays (TDCC). Figure 9A shows the impact of the PSMA targeting TriTAC molecule in redirecting cynomolgus peripheral blood mononuclear cells (PBMCs), from cynomolgus monkey donor G322, in killing LNCaP cells. Figure 9B shows the impact of the PSMA targeting TriTAC molecule in redirecting cynomolgus PBMCs, from cynomolgus monkey donor D 173, to kill MDAPCa2b cells.

[0027] Figure 10 depicts the impact of a PSMA targeting TriTAC molecule of this disclosure on expression of T cell activation markers CD25 and CD69.

[0028] Figure 11 depicts the ability of a PSMA targeting TriTAC molecule of this disclosure to stimulate T cell proliferation in the presence of PSMA expressing target cells.

[0029] Figures 12A-12B depict redirected T cell killing of LnCaP cells by PSMA targeting TriTAC molecules. Figure 12A shows redirected T cell killing of LnCaP cells by PSMA PH1T TriTAC (SEQ ID No: 150) and PSMA PHI TriTAC (SEQ ID NO: 151) molecules. Figure 12B shows redirected T cell killing of LnCaP cells by PSMA Z2 TriTAC (SEQ ID NO: 152).

[0030] Figure 13 depicts an exemplary process for preparing a PSMA binding TriTAC molecule of this disclosure.

[0031] Figure 14 depicts an SDS-PAGE gel showing purity profile of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147). Molecular weight markers are labeled to the right of the gel. Lane 1 : PSMA HTS TriTAC under non-reducing conditions. Lane 2: empty. Lane 3: molecular weight standard. Lane 4: PSMA HTS TriTAC under reducing conditions.

[0032] Figure 15 depicts sedimentation velocity distribution of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147).

[0033] Figure 16 depicts stability profiled of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) determined using analytical size exclusion chromatography.

[0034] Figure 17 depicts stability profiled of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) determined using differential scanning calorimetry.

[0035] Figure 18 depicts binding of depicts binding of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) to human PSMA (left most panel), human albumin (center panel), and human CD3e (right most panel).

[0036] Figure 19 depicts binding of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) to MDAPCa2b cells (top panel), as compared to that of a control TriTAC molecule; and binding of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) to human T cells (bottom panel), as compared to that of a control TriTAC molecule. [0037] Figure 20 depicts that an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) was able to direct T cells from 4 health donors (donor 24; donor 8144; donor 72; and donor 41) to kill PSMA expressing cells LnCaP, whereas control GFP TriTAC molecule (SEQ ID NO: 169) was unable to direct T cell mediated killing of the VaCaP cells.

[0038] Figure 21 depicts that an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) was able to induce CD69 expression on T cells in presence of LNCaP cells with PSMA HTS TriTAC but not with a control GFP-TriTAC (SEQ ID NO: 169). [0039] Figure 22 depicts that an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) was able induce f TNF-a expression by T cells in presence of LNCaP cells but not PSMA-negative HCT116 cells.

[0040] Figure 23 depicts that an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) was able to inhibit tumor growth in mice injected with a mixture of human PBMC and 22Rvl prostate cancer cells at dosages of 2 pg/kg, 10 pg/kg, 50 pg/kg, and 250 pg/kg.

[0041] Figure 24 depicts pharmacokinetic profile of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147). Serum levels of PSMA HTS TriTAC, at various time points following injection into cynomolgus monkeys, at 0.1 mg/kg or 3 mg/kg, are shown in the plot.

[0042] Figure 25 depicts transient T lymphocyte activation after first dosing of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) at 0.1 mg/kg, 1 mg/kg, and 3 mg/kg.

[0043] Figure 26 depicts side scatter plots for CD69 activation following injection of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) at 3 mg/kg (bottom panel) or a vehicle control (top panel).

[0044] Figure 27 depicts transient cytokine increase after first dosing of an exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) at 0.1 mg/kg, 1 mg/kg, and 3 mg/kg or a vehicle control. The left panel shows transient increase of IL-6 and the right panel shows transient increase of IL10.

[0045] Figure 28 depicts PSMA trispecific antigen-binding protein Phase I trial design.

[0046] Figure 29 depicts patients time on treatment from clinical database.

[0047] Figure 30 depicts each patient’s PSA value on PSMA trispecific antigen-binding protein treatment.

[0048] Figure 31A depicts PSA value and disease assessment on treatment of one patient. Figure 31B depicts PSA value and disease assessment on treatment of one patient. [0049] Figure 32 depicts median concentration time profile for PSMA trispecific antigen binding protein at dose range of 1.3-96 ng/kg.

[0050] Figure 33 depicts mean cytokine levels at 5-hours post- 1st PSMA trispecific antigen binding protein exposure.

[0051] Figure 34 depicts post treatment changes in circulation tumor cell.

[0052] Figure 35 provides a cartoon diagram showing that TriTAC molecules have six possible configurations.

[0053] Figures 36A-36C depict results for various experiments carried out for biophysical characterization of an exemplary PSMA targeting TriTAC (SEQ ID NO: 147, PSMA HTS TriTACTriTAC). (Figure 36A) Overlay of electrophoretograms from SDS denatured capillary electrophoresis of the PSMA HTS TriTACTriTAC under reduced conditions, non-reduced conditions, reduced conditions after incubation at 37°C for two weeks, non-reduced conditions after incubation at 37°C for two weeks. An internal 10 kDa standard is indicated by an arrow above each electrophoretogram. (Figure 36B) Intrinsic fluorescence (barycentric mean plotted). TO: unstressed PSMA HTS TriTACTriTAC. 5x F/T: sample frozen and thawed five times. 72 h Shake: Sample incubated with shaking at room temperature for 72 hours. 37°C 2 wk: PSMA HTS TriTACTriTACstored at 37°C for two weeks. (Figure 36C) Static light scattering (SLS) counts made at 266 nm and 473 nm during a 0.5 °C/min thermal ramp of PSMA HTS TriTACTriTACsamples treated under stressed conditions TO: unstressed PSMA HTS TriTACTriTACat 266 nM and 473 nM. 5x F/T: sample frozen and thawed five times at 266 nM and 473 nM. 72 h Shake: Sample incubated with shaking at room temperature for 72 hours at 266 nM and 473 nM. 37°C 2 wk: PSMA HTS TriTACTriTACstored at 37°C for two weeks at 266 nM and 473 nM.

[0054] Figure 37 depicts the results of an assay for measuring PSMA HTS TriTACTriTACinduced activation of T cells in presence of PSMA-expressing cells and directs T cells to kill PSMA-expressing cells. A titration of PSMA HTS TriTACTriTAC was added to co-cultures of LNCaP cells and purified human T cells in the presence or absence of 15 mg/ml HSA. LNCaP cell viability was measured at 48 hours. Plotted are the mean viability values ± SEM (n=3).

[0055] Figure 38 depicts PSMA HTS TriTACTriTAC-induced expression of CD25 on T cells. Co-cultures of LNCaP cells and purified T cells from four different healthy donors were treated with a titration of PSMA HTS TriTACTriTACor an anti-GFP TriTAC. After a 48 incubation, flow cytometry analyses were performed. Plotted is the expression of CD25 expression on CD4+ and/or CD8+ T cells (± SEM, n=2). [0056] Figure 39 depicts the results of an assay demonstrating PSMA HTS TriTACTriTAC- induced expression of IFNy. Conditioned medium was collected from LNCaP or HCT116 cells co-cultured with purified human T cells from two different donors. Plotted are the mean relative IFNy levels in the conditioned medium measured with an AlphaLISA assay (± SEM, n=3). [0057] Figure 40 depicts the stability of PSMA HTS TriTACTriTACin cynomolgus monkey. Co-cultures of LNCaP cells and purified human T cells were treated with freshly diluted PSMA HTS TriTACTriTAC, freshly diluted anti-GFP TriTAC, or with serum sample collected from a cynomolgus monkey 168 h after being dosed with 0.3 mg/kg PSMA HTS TriTACTriTAC. TriTAC samples were diluted with pooled treatment naive cynomolgus monkey serum to achieve a final concentration of 17.7% serum across the entire assay. Plotted is the mean viability of the LNCaP cells after a 48-hour incubation (± SEM n=2).

[0058] Figures 41A-41B depict the effect of HSA on PSMA-Dependent PSMA HTS TriTACTriTAC-induced expression of CD69 and CD25 on T cells. Co-cultures of purified T cells and (Figure 41A) PSMA-expressing LNCaP cells or (Figure 41B) PSMA-negative NCI- H1563 cells were treated with 1 nM PSMA HTS TriTACTriTACor anti-GFP TriTACin the absence or presence of 15 mg/ml HSA. After a 48 incubation, flow cytometry analyses were performed. Plotted are the expression of CD69 (left y-axis) and CD25 expression (right y-axis) on CD4+ and/or CD8+ T cells (± SD, n=2).

[0059] Figure 42 depicts an updated PSMA trispecific antigen-binding protein Phase I trial design.

[0060] Figure 43 depicts an updated patients time on treatment.

[0061] Figure 44 depicts an updated each patient’s PSA value on PSMA trispecific antigen binding protein treatment.

[0062] Figure 45 shows patient 057’ s response level for the treatment. Figure 45A shows partial response values of patient 057 during the course of the treatment. Figure 45B shows patient 057’s scans at pre-treatment, week 18 treatment and week 36 treatment.

[0063] Figure 46 shows the patient time on treatment of AMG160.

[0064] Figure 47 depicts an updated patients time on treatment.

[0065] Figure 48 depicts an updated each patient’s PSA value on PSMA trispecific antigen binding protein treatment.

[0066] Figure 49 shows the circulating tumor cells (CTC) changes from baseline. Figure 49A shows the CTC best response, Figure 49B shows changes in CTCs at week 13 from baseline. [0067] Figure 50 shows patient 054’ s scans at pre-treatment and week 45 treatment. DETAILED DESCRIPTION OF THE INVENTION [0068] Described herein are trispecific proteins that target prostate specific membrane antigen (PSMA), pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such proteins thereof. Also provided are methods of using the disclosed PSMA targeting trispecific proteins in the prevention, and/or treatment of diseases, conditions and disorders. The PSMA targeting trispecific proteins are capable of specifically binding to PSMA as well as CD3 and have a half-life extension domain, such as a domain binding to human serum albumin (HSA). Figure 1 depicts one non-limiting example of a trispecific antigen-binding protein.

[0069] In one aspect, the PSMA targeting trispecific proteins comprise a domain (A) which specifically binds to CD3, a domain (B) which specifically binds to human serum albumin (HSA), and a domain (C) which specifically binds to PSMA. The three domains in PSMA targeting trispecific proteins are arranged in any order. Thus, it is contemplated that the domain order of the PSMA targeting trispecific proteins are:

H₂N-(A)-(B)-(C)-COOH,

H₂N-(A)-(C)-(B)-COOH,

H₂N-(B)-(A)-(C)-COOH,

H₂N-(B)-(C)-(A)-COOH,

H₂N-(C)-(B)-(A)-COOH, or H₂N-(C)-(A)-(B)-COOH.

[0070] In some embodiments, the PSMA targeting trispecific proteins have a domain order of H₂N-(A)-(B)-(C)-COOH. In some embodiments, the PSMA targeting trispecific proteins have a domain order of H₂N-(A)-(C)-(B)-COOH. In some embodiments, the PSMA targeting trispecific proteins have a domain order of H₂N-(B)-(A)-(C)-COOH. In some embodiments, the PSMA targeting trispecific proteins have a domain order of H₂N-(B)-(C)-(A)-COOH. In some embodiments, the PSMA targeting trispecific proteins have a domain order of H₂N-(C)-(B)-(A)- COOH. In some embodiments, the PSMA targeting trispecific proteins have a domain order of H₂N-(C)-(A)-(B)-COOH.

[0071] In some embodiments, the PSMA targeting trispecific proteins have the HSA binding domain as the middle domain, such that the domain order is H₂N-(A)-(B)-(C)-COOH or H₂N- (C)-(B)-(A)-COOH. It is contemplated that in such embodiments where the HSA binding domain as the middle domain, the CD3 and PSMA binding domains are afforded additional flexibility to bind to their respective targets.

[0072] In some embodiments, the PSMA targeting trispecific proteins described herein comprise a polypeptide having a sequence described in Table 10 (SEQ ID NO: 141-147 and 150-152) and subsequences thereof. In some embodiments, the trispecific antigen binding protein comprises a polypeptide having at least 70%-95% or more homology to a sequence described in Table 10 (SEQ ID NO: 141-147 and 150-152). In some embodiments, the trispecific antigen binding protein comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, or more homology to a sequence described in Table 10 (SEQ ID NO: 141-147 and 150-152). In some embodiments, the trispecific antigen binding protein has a sequence comprising at least a portion of a sequence described in Table 10 (SEQ ID NO: 141-147 and 150-152). In some embodiments, the PSMA trispecific antigen-binding protein comprises a polypeptide comprising one or more of the sequences described in Table 10 (SEQ ID NO: 141-147 and 150-152). In further embodiments, the PSMA trispecific antigen-binding protein comprises one or more CDRs as described in the sequences in Table 10 (SEQ ID NO: 141-147 and 150-152).

[0073] The PSMA targeting trispecific proteins described herein are designed to allow specific targeting of cells expressing PSMA by recruiting cytotoxic T cells. This improves efficacy compared to ADCC (antibody dependent cell-mediated cytotoxicity) , which is using full length antibodies directed to a sole antigen and is not capable of directly recruiting cytotoxic T cells.

In contrast, by engaging CD3 molecules expressed specifically on these cells, the PSMA targeting trispecific proteins can crosslink cytotoxic T cells with cells expressing PSMA in a highly specific fashion, thereby directing the cytotoxic potential of the T cell towards the target cell. The PSMA targeting trispecific proteins described herein engage cytotoxic T cells via binding to the surface-expressed CD3 proteins, which form part of the TCR. Simultaneous binding of several PSMA trispecific antigen-binding protein to CD3 and to PSMA expressed on the surface of particular cells causes T cell activation and mediates the subsequent lysis of the particular PSMA expressing cell. Thus, PSMA targeting trispecific proteins are contemplated to display strong, specific and efficient target cell killing. In some embodiments, the PSMA targeting trispecific proteins described herein stimulate target cell killing by cytotoxic T cells to eliminate pathogenic cells (e.g., tumor cells expressing PSMA). In some of such embodiments, cells are eliminated selectively, thereby reducing the potential for toxic side effects.

[0074] The PSMA targeting trispecific proteins described herein confer further therapeutic advantages over traditional monoclonal antibodies and other smaller bispecific molecules. Generally, the effectiveness of recombinant protein pharmaceuticals depends heavily on the intrinsic pharmacokinetics of the protein itself. One such benefit here is that the PSMA targeting trispecific proteins described herein have extended pharmacokinetic elimination half time due to having a half-life extension domain such as a domain specific to HSA. In this respect, the PSMA targeting trispecific proteins described herein have an extended serum elimination half-time of about two, three, about five, about seven, about 10, about 12, or about 14 days in some embodiments. This contrasts to other binding proteins such as BiTE or DART molecules which have relatively much shorter elimination half-times. For example, the BiTE CD19xCD3 bispecific scFv-scFv fusion molecule requires continuous intravenous infusion (i.v.) drug delivery due to its short elimination half-time. The longer intrinsic half-times of the PSMA targeting trispecific proteins solve this issue thereby allowing for increased therapeutic potential such as low-dose pharmaceutical formulations, decreased periodic administration and/or novel pharmaceutical compositions.

[0075] The PSMA targeting trispecific proteins described herein also have an optimal size for enhanced tissue penetration and tissue distribution. Larger sizes limit or prevent penetration or distribution of the protein in the target tissues. The PSMA targeting trispecific proteins described herein avoid this by having a small size that allows enhanced tissue penetration and distribution. Accordingly, the PSMA targeting trispecific proteins described herein, in some embodiments have a size of about 50 kD to about 80 kD, about 50 kD to about 75 kD, about 50 kD to about 70 kD, or about 50 kD to about 65 kD. Thus, the size of the PSMA targeting trispecific proteins is advantageous over IgG antibodies which are about 150 kD and the BiTE and DART diabody molecules which are about 55 kD but are not half-life extended and therefore cleared quickly through the kidney.

[0076] In further embodiments, the PSMA targeting trispecific proteins described herein have an optimal size for enhanced tissue penetration and distribution. In these embodiments, the PSMA targeting trispecific proteins are constructed to be as small as possible, while retaining specificity toward its targets. Accordingly, in these embodiments, the PSMA targeting trispecific proteins described herein have a size of about 20 kD to about 40 kD or about 25 kD to about 35 kD to about 40 kD, to about 45 kD, to about 50 kD, to about 55 kD, to about 60 kD, to about 65 kD. In some embodiments, the PSMA targeting trispecific proteins described herein have a size of about 50kD, 49, kD, 48 kD, 47 kD, 46 kD, 45 kD, 44 kD, 43 kD, 42 kD, 41 kD,

40 kD, about 39 kD, about 38 kD, about 37 kD, about 36 kD, about 35 kD, about 34 kD, about

33 kD, about 32 kD, about 31 kD, about 30 kD, about 29 kD, about 28 kD, about 27 kD, about

26 kD, about 25 kD, about 24 kD, about 23 kD, about 22 kD, about 21 kD, or about 20 kD. An exemplary approach to the small size is through the use of single domain antibody (sdAb) fragments for each of the domains. For example, a particular PSMA trispecific antigen-binding protein has an anti-CD3 sdAb, anti-HSA sdAb and an sdAb for PSMA. This reduces the size of the exemplary PSMA trispecific antigen-binding protein to under 60 kD. Thus in some embodiments, the domains of the PSMA targeting trispecific proteins are all single domain antibody (sdAb) fragments. In other embodiments, the PSMA targeting trispecific proteins described herein comprise small molecule entity (SME) binders for HSA and/or the PSMA. SME binders are small molecules averaging about 500 to 2000 Da in size and are attached to the PSMA targeting trispecific proteins by known methods, such as sortase ligation or conjugation. In these instances, one of the domains of PSMA tri specific antigen -binding protein is a sortase recognition sequence, e.g., LPETG (SEQ ID NO: 57). To attach a SME binder to PSMA trispecific antigen-binding protein with a sortase recognition sequence, the protein is incubated with a sortase and a SME binder whereby the sortase attaches the SME binder to the recognition sequence. Known SME binders include MIP-1072 and MIP-1095 which bind to prostate- specific membrane antigen (PSMA). In yet other embodiments, the domain which binds to PSMA of PSMA targeting trispecific proteins described herein comprise a knottin peptide for binding PSMA. Knottins are disufide-stabilized peptides with a cysteine knot scaffold and have average sizes about 3.5 kD. Knottins have been contemplated for binding to certain tumor molecules such as PSMA. In further embodiments, domain which binds to PSMA of PSMA targeting trispecific proteins described herein comprise a natural PSMA ligand.

[0077] Another feature of the PSMA targeting trispecific proteins described herein is that they are of a single-polypeptide design with flexible linkage of their domains. This allows for facile production and manufacturing of the PSMA targeting trispecific proteins as they can be encoded by single cDNA molecule to be easily incorporated into a vector. Further, because the PSMA targeting trispecific proteins described herein are a monomeric single polypeptide chain, there are no chain pairing issues or a requirement for dimerization. It is contemplated that the PSMA targeting trispecific proteins described herein have a reduced tendency to aggregate unlike other reported molecules such as bi specific proteins with Fc-gamma immunoglobulin domains.

[0078] In the PSMA targeting trispecific proteins described herein, the domains are linked by internal linkers LI and L2, where LI links the first and second domain of the PSMA targeting trispecific proteins and L2 links the second and third domains of the PSMA targeting trispecific proteins. Linkers LI and L2 have an optimized length and/or amino acid composition. In some embodiments, linkers LI and L2 are the same length and amino acid composition. In other embodiments, LI and L2 are different. In certain embodiments, internal linkers LI and/or L2 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, the internal linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the internal linker is a peptide bond. In certain embodiments, internal linkers LI and/or L2 are "long", i.e., consist of 15, 20 or 25 amino acid residues. In some embodiments, these internal linkers consist of about 3 to about 15, for example 8, 9 or 10 contiguous amino acid residues. Regarding the amino acid composition of the internal linkers LI and L2, peptides are selected with properties that confer flexibility to the PSMA targeting trispecific proteins, do not interfere with the binding domains as well as resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. Examples of internal linkers suitable for linking the domains in the PSMA targeting trispecific proteins include but are not limited to (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), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, internal linker LI and/or L2 is (GGGGS)4 (SEQ ID NO: 159) or (GGGGS)3 (SEQ ID NO: 160). CD3 Binding Domain

[0079] The specificity of the response of T cells is mediated by the recognition of antigen (displayed in context of a major histocompatibility complex, MHC) by the TCR. As part of the TCR, CD3 is a protein complex that includes a CD3y (gamma) chain, a CD35 (delta) chain, and two CD3e (epsilon) chains which are present on the cell surface. CD3 associates with the a (alpha) and b (beta) chains of the TCR as well as CD3 z (zeta) altogether to comprise the complete TCR. Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodies leads to T cell activation similar to the engagement of the T cell receptor but independent of its clone- typical specificity.

[0080] In one aspect, the PSMA targeting trispecific proteins described herein comprise a domain which specifically binds to CD3. In one aspect, the PSMA targeting trispecific proteins described herein comprise a domain which specifically binds to human CD3. In some embodiments, the PSMA targeting trispecific proteins described herein comprise a domain which specifically binds to CD3y. In some embodiments, the PSMA targeting trispecific proteins described herein comprise a domain which specifically binds to CD35. In some embodiments, the PSMA targeting trispecific proteins described herein comprise a domain which specifically binds to CD3e.

[0081] In further embodiments, the PSMA targeting trispecific proteins described herein comprise a domain which specifically binds to the TCR. In certain instances, the PSMA targeting trispecific proteins described herein comprise a domain which specifically binds the a chain of the TCR. In certain instances, the PSMA targeting trispecific proteins described herein comprise a domain which specifically binds the b chain of the TCR.

[0082] In certain embodiments, the CD3 binding domain of the PSMA targeting trispecific proteins described herein exhibit not only potent CD3 binding affinities with human CD3, but show also excellent crossreactivity with the respective cynomolgus monkey CD3 proteins. In some instances, the CD3 binding domain of the PSMA targeting trispecific proteins are cross reactive with CD3 from cynomolgus monkey. In certain instances, human: cynomolgous KD ratios for CD3 are between 5 and 0.2. [0083] In some embodiments, the CD3 binding domain of the PSMA tri specific antigen-binding protein can be any domain that binds to CD3 including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some instances, it is beneficial for the CD3 binding domain to be derived from the same species in which the PSMA trispecific antigen-binding protein will ultimately be used in. For example, for use in humans, it may be beneficial for the CD3 binding domain of the PSMA trispecific antigen-binding protein to comprise human or humanized residues from the antigen binding domain of an antibody or antibody fragment.

[0084] Thus, in one aspect, the antigen-binding domain comprises a humanized or human antibody or an antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-CD3 binding domain comprises one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti- CD3 binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-CD3 binding domain described herein, e.g., a humanized or human anti-CD3 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs.

[0085] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human light chain variable region specific to CD3 where the light chain variable region specific to CD3 comprises human or non-human light chain CDRs in a human light chain framework region. In certain instances, the light chain framework region is a l (lamda) light chain framework. In other instances, the light chain framework region is a k (kappa) light chain framework.

[0086] In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human heavy chain variable region specific to CD3 where the heavy chain variable region specific to CD3 comprises human or non-human heavy chain CDRs in a human heavy chain framework region.

[0087] In certain instances, the complementary determining regions of the heavy chain and/or the light chain are derived from known anti-CD3 antibodies, such as, for example, muromonab- CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, TR- 66 or X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, FI 11-409, 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. [0088] In one embodiment, the anti-CD3 binding domain is a single chain variable fragment (scFv) comprising a light chain and a heavy chain of an amino acid sequence provided herein.

As used herein, "single chain variable fragment" or "scFv" refers to an antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. In an embodiment, the anti-CD3 binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity with an amino acid sequence provided herein; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to an amino acid sequence provided herein. In one embodiment, the humanized or human anti-CD3 binding domain is a scFv, and a light chain variable region comprising an amino acid sequence described herein, is attached to a heavy chain variable region comprising an amino acid sequence described herein, via a scFv linker. The light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region- scFv linker-heavy chain variable region or heavy chain variable region- scFv linker-light chain variable region.

[0089] In some instances, scFvs which bind to CD3 are prepared according to known methods. For example, scFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a scFv linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. Accordingly, in some embodiments, the length of the scFv linker is such that the VH or VL domain can associate intermolecularly with the other variable domain to form the CD3 binding site. In certain embodiments, such scFv 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, the scFv linkers consist of about 12 or less amino acid residues. In the case of 0 amino acid residues, the scFv linker is a peptide bond. In some embodiments, these scFv linkers consist of about 3 to about 15, for example 8, 9 or 10 contiguous amino acid residues. Regarding the amino acid composition of the scFv linkers, peptides are selected that confer flexibility, do not interfere with the variable domains as well as allow inter-chain folding to bring the two variable domains together to form a functional CD3 binding site. For example, scFv linkers comprising glycine and serine residues generally provide protease resistance. In some embodiments, linkers in a scFv comprise glycine and serine residues. The amino acid sequence of the scFv linkers can be optimized, for example, by phage-display methods to improve the CD3 binding and production yield of the scFv. Examples of peptide scFv linkers suitable for linking a variable light chain domain and a variable heavy chain domain in a scFv include but are not limited to (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), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the scFv linker can be (GGGGS)4 (SEQ ID NO: 159) or (GGGGS)3 (SEQ ID NO: 160). Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.

[0090] In some embodiments, CD3 binding domain of PSMA trispecific antigen-binding protein has an affinity to CD3 on 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 PSMA trispecific antigen-binding protein has an affinity to CD3e, g, or d 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 further embodiments, CD3 binding domain of PSMA trispecific antigen-binding protein has low affinity to CD3, i.e., about 100 nM or greater.

[0091] The affinity to bind to 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 an assay plate; displayed on a microbial cell surface; 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, to a bead, substrate, cell, etc. Agents can be added in an appropriate buffer and the binding partners incubated for a period of time at a given temperature. After washes to remove unbound material, the bound protein can be released with, for example, SDS, buffers with a high pH, and the like and analyzed, for example, by Surface Plasmon Resonance (SPR).

[0092] In some embodiments, CD3 binding domains described herein comprise a polypeptide having a sequence described in Table 7 (SEQ ID NO: 1-88) and subsequences thereof. In some embodiments, the CD3 binding domain comprises a polypeptide having at least 70%-95% or more homology to a sequence described in Table 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 a sequence described in Table 7 (SEQ ID NO: 1-88). In some embodiments, the CD3 binding domain has a sequence comprising at least a portion of a sequence described in Table 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 7 (SEQ ID NO: 1-88).

[0093] In certain embodiments, CD3 binding domain comprises an scFv with a heavy chain CDR1 comprising SEQ ID NO: 16, and 22-33. In certain embodiments, CD3 binding domain comprises an scFv with a heavy chain CDR2 comprising SEQ ID NO: 17, and 34-43. In certain embodiments, CD3 binding domain comprises an scFv with a heavy chain CDR3 comprising SEQ ID NO: 18, and 44-53. In certain embodiments, CD3 binding domain comprises an scFv with a light chain CDR1 comprising SEQ ID NO: 19, and 54-66. In certain embodiments, CD3 binding domain comprises an scFv with a light chain CDR2 comprising SEQ ID NO: 20, and 67-79. In certain embodiments, CD3 binding domain comprises an scFv with a light chain CDR3 comprising SEQ ID NO: 21, and 80-86.

Half-Life Extension Domain

[0094] Contemplated herein are domains which extend the half-life of an antigen-binding domain. Such domains are contemplated to include but are not limited to HSA binding domains, Fc domains, small molecules, and other half-life extension domains known in the art.

[0095] Human serum albumin (HSA) (molecular mass ~67 kDa) is the most abundant protein in plasma, present at about 50 mg/ml (600 mM), and has a half-life of around 20 days in humans. HSA serves to maintain plasma pH, contributes to colloidal blood pressure, functions as carrier of many metabolites and fatty acids, and serves as a major drug transport protein in plasma. [0096] Noncovalent association with albumin extends the elimination half-time of short lived proteins. For example, a recombinant fusion of an albumin binding domain to a Fab fragment resulted in an in vivo clearance of 25- and 58-fold and a half-life extension of 26- and 37-fold when administered intravenously to mice and rabbits respectively as compared to the administration of the Fab fragment alone. In another example, when insulin is acylated with fatty acids to promote association with albumin, a protracted effect was observed when injected subcutaneously in rabbits or pigs. Together, these studies demonstrate a linkage between albumin binding and prolonged action.

[0097] In one aspect, the PSMA targeting trispecific proteins described herein comprise a half- life extension domain, for example a domain which specifically binds to HSA. In some embodiments, the HSA binding domain of PSMA trispecific antigen-binding protein can be any domain that binds to HSA including but not limited to domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the HSA binding domain is a single chain variable fragments (scFv), single domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived single domain antibody, peptide, ligand or small molecule entity 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 further embodiments, the HSA binding domain is a small molecule. It is contemplated that the HSA binding domain of PSMA trispecific antigen-binding protein is fairly small and no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD in some embodiments. In certain instances, the HSA binding is 5 kD or less if it is a peptide or small molecule entity.

[0098] The half-life extension domain of PSMA trispecific antigen-binding protein provides for altered pharmacodynamics and pharmacokinetics of the PSMA trispecific antigen-binding protein itself. As above, the half-life extension domain extends the elimination half-time. The half-life extension domain also alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the trispecific antigen-binding protein. In some embodiments, the half-life extension domain provides for improved tissue (including tumor) targeting, tissue distribution, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared with a protein without an half-life extension domain. In one embodiment, therapeutic methods effectively and efficiently utilize a reduced amount of the trispecific antigen-binding protein, resulting in reduced side effects, such as reduced non-tumor cell cytotoxicity.

[0099] In some embodiments, the elimination half-life of the PSMA targeting trispecific proteins of the present disclosure is about 20 hours to about 100 hours, about 50 hours to about 100 hours, or about 80 hours to about 100 hours. In some cases, the elimination half-life is over 20 hours, over 40 hours or over 50 hours.

[0100] Further, the binding affinity of the half-life extension domain can be selected so as to target a specific elimination half-time in a particular trispecific antigen-binding protein. Thus, in some embodiments, the half-life extension domain has a high binding affinity. In other embodiments, the half-life extension domain has a medium binding affinity. In yet other embodiments, the half-life extension domain has a low or marginal binding affinity. Exemplary binding affinities include KD concentrations at 10 nM or less (high), between 10 nM and 100 nM (medium), and greater than 100 nM (low). As above, binding affinities to HSA are determined by known methods such as Surface Plasmon Resonance (SPR).

[0101] In some embodiments, HSA binding domains described herein comprise a polypeptide having a sequence described in Table 8 (SEQ ID NO: 89-112) and subsequences thereof. In some embodiments, the HSA binding domain comprises a polypeptide having at least 70%-95% or more homology to a 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 a sequence described in Table 8 (SEQ ID NO: 89- 112). In some embodiments, the HSA binding domain has a sequence comprising at least a portion of a sequence described in Table 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 8 (SEQ ID NO: 89-112).

[0102] In some embodiments, HSA binding domains described herein comprise a single domain antibody with a CDR1 comprising SE ID NO: 96, and 99-101. In some embodiments, HSA binding domains described herein comprise a single domain antibody with a CDR1 comprising SE ID NO: 97, and 102-107. In some embodiments, HSA binding domains described herein comprise a single domain antibody with a CDR1 comprising SE ID NO: 98, 108 and 109. Prostate Specific Membrane Antigen (PSMA) Binding Domain

[0103] Prostate specific membrane antigen (PSMA) is a 100 kD Type II membrane glycoprotein expressed in prostate tissues having sequence identity with the transferrin receptor with NAALADase activity. PSMA is expressed in increased amounts in prostate cancer, and elevated levels of PSMA are also detectable in the sera of these patients. PSMA expression increases with disease progression, becoming highest in metastatic, hormone-refractory disease for which there is no present therapy.

[0104] In addition to the described CD3 and half-life extension domains, the PSMA targeting trispecific proteins described herein also comprise a domain that binds to PSMA. The design of the PSMA targeting trispecific proteins described herein allows the binding domain to PSMA to be flexible in that the binding domain to PSMA can be any type of binding domain, including but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the binding domain to PSMA is a single chain variable fragments (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived single domain antibody. In other embodiments, the binding domain to PSMA is a non-Ig binding domain, i.e., antibody mimetic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and monobodies. In further embodiments, the binding domain to PSMA is a ligand or peptide that binds to or associates with PSMA. In yet further embodiments, the binding domain to PSMA is a knottin. In yet further embodiments, the binding domain to PSMA is a small molecular entity. [0105] In some embodiments, the PSMA binding domain comprises the following formula: fl- rl-f2-r2-f3-r3-f4, wherein rl, r2, and r3 are complementarity determining regions CDR1, CDR2, and CDR3, respectively, and fl, f2, f3, and f4 are framework residues, and wherein rl comprises SEQ ID No. 114, SEQ ID No. 115, SEQ ID No. 116, or SEQ ID NOL 125, r2 comprises 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 comprises SEQ ID No. 124, or SEQ ID NO: 127.

[0106] In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163 (X3INPAX4X5TDYAEX6VKG), and(c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY). In some embodiments, the amino acid residues XI, X2, X3, X4, X5, X6, and X7 are independently selected from glutamic acid, proline, serine, histidine, threonine, aspartic acid, glycine, lysine, threonine, glutamine, and tyrosine. In some embodiments, XI is proline. In some embodiments, X2 is histidine. In some embodiments, X3 is aspartic acid. In some embodiments, X4 is lysine. In some embodiments, X5 is glutamine.

In some embodiments, X6 is tyrosine. In some embodiments, X7 is serine. The PSMA binding protein of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is glutamic acid, X2 is histidine, X3 is aspartic acid, X4 is glycine, X5 is threonine, X6 is serine, and X7 is serine.

[0107] In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1 YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163 (X3INPAX4X5TDYAEX6VKG), and (c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY), wherein XI is proline. In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1 YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163 (X3INPAX4X5TDYAEX6VKG), and(c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY), wherein X5 is glutamine. In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1 YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163

(X3INPAX4X5TDYAEX6VKG), and(c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY), wherein X6 is tyrosine. In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1 YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163 (X3INPAX4X5TDYAEX6VKG), and(c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY), wherein X4 is lysine, and X7 is serine. In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1 YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163 (X3INPAX4X5TDYAEX6VKG), and (c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY), wherein X2 is histidine, X3 is aspartic acid, X4 is lysine, and X7 is serine. In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163 (X3INPAX4X5TDYAEX6VKG), and(c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY), wherein XI is proline, X2 is histidine, X3 is aspartic acid, and X7 is serine. In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163 (X3INPAX4X5TDYAEX6VKG), and(c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY), wherein X2 is histidine, X3 is aspartic acid, X5 is glutamine, and X7 is serine. In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1 YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163 (X3INPAX4X5TDYAEX6VKG), and(c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY), wherein X2 is histidine, X3 is aspartic acid, X6 is tyrosine, and X7 is serine. In some embodiments, the PSMA binding domain comprises a CDR1, CDR2, and CDR3, wherein (a) the amino acid sequence of CDR1 is as set forth in SEQ ID No. 162 (RFMISX1YX2MH), (b) the amino acid sequence of CDR2 is as set forth in SEQ ID No. 163 (X3INPAX4X5TDYAEX6VKG), and(c) the amino acid sequence of CDR3 is as set forth in SEQ ID No. 164 (DX7YGY), wherein X2 is histidine, X3 is aspartic acid, and X7 is serine. [0108] The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is glutamic acid, X2 is histidine, X3 is threonine, X4 is glycine, X5 is threonine, X6 is serine, and X7 is serine. The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is glutamic acid, X2 is histidine, X3 is threonine, X4 is glycine, X5 is threonine, X6 is serine, and X7 is serine. The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is glutamic acid, X2 is serine, X3 is threonine, X4 is lysine, X5 is threonine, X6 is serine, and X7 is serine. The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is proline, X2 is serine, X3 is threonine, X4 is glycine, X5 is threonine, X6 is serine, and X7 is glycine. The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is glutamic acid, X2 is serine, X3 is threonine, X4 is glycine, X5 is glutamine, X6 is serine, and X7 is glycine. The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is glutamic acid, X2 is serine, X3 is threonine, X4 is glycine, X5 is threonine, X6 is tyrosine, and X7 is glycine. The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is glutamic acid, X2 is histidine, X3 is aspartic acid, X4 is lysine, X5 is threonine, X6 is serine, and X7 is serine. The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is proline, X2 is histidine, X3 is aspartic acid, X4 is glycine, X5 is threonine, X6 is serine, and X7 is serine. The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is glutamic acid, X2 is histidine, X3 is aspartic acid, X4 is glutamine, X5 is threonine, X6 is serine, and X7 is serine. The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein XI is glutamic acid, X2 is histidine, X3 is aspartic acid, X4 is glycine, X5 is threonine, X6 is tyrosine, and X7 is serine.

The PSMA binding domain of the present disclosure may in some embodiments comprise CDR1, CDR2, and CDR3 sequences wherein X2 is histidine, and X7 is serine. . Exemplary framework sequences are disclosed as SEQ ID NO: 165-168.

[0109] In some embodiments, PSMA binding domains described herein comprise a polypeptide having a sequence described in Table 9 (SEQ ID NO: 113-140) and subsequences thereof. In some embodiments, the HSA binding domain comprises a polypeptide having at least 70%-95% or more homology to a sequence described in Table 9 (SEQ ID NO: 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 a sequence described in Table 9 (SEQ ID NO: 113- 140). In some embodiments, the HSA binding domain has a sequence comprising at least a portion of a sequence described in Table 9 (SEQ ID NO: 113-140). In some embodiments, the HSA binding domain comprises a polypeptide comprising one or more of the sequences described in Table 9 (SEQ ID NO: 113-140).

[0110] In some embodiments, PSMA binding domains described herein comprise a single domain antibody with a CDR1 comprising SE ID NO: 114-116, and 125. In some embodiments, PSMA binding domains described herein comprise a single domain antibody with a CDR1 comprising SEQ ID NO: 117-123, and 126. In some embodiments, PSMA binding domains described herein comprise a single domain antibody with a CDR1 comprising SE ID NO: 124 and 127.

PSMA Trispecific Protein Modifications

[0111] The PSMA targeting trispecific proteins described herein encompass derivatives or analogs in which (i) an amino acid is substituted with an amino acid residue that is not one encoded by the genetic code, (ii) the mature polypeptide is fused with another compound such as polyethylene glycol, or (iii) additional amino acids are fused to the protein, such as a leader or secretory sequence or a sequence for purification of the protein.

[0112] Typical modifications include, but are not limited to, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0113] Modifications are made anywhere in PSMA targeting trispecific proteins described herein, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Certain common peptide modifications that are useful for modification of PSMA targeting trispecific proteins include glycosylation, lipid attachment, sulfation, gamma- carboxylation of glutamic acid residues, hydroxylation, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, and ADP-ribosylation.

Polynucleotides Encoding PSMA targeting trispecific proteins

[0114] Also provided, in some embodiments, are polynucleotide molecules encoding a PSMA trispecific antigen-binding protein described herein. 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.

[0115] The polynucleotide molecules are constructed by known methods such as by combining the genes encoding the 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 expressing it in bacteria or other appropriate expression system such as, for example CHO cells. In the embodiments where the PSMA binding domain is a small molecule, the polynucleotides contain genes encoding the CD3 binding domain and the half-life extension domain. In the embodiments where the half-life extension domain is a small molecule, the polynucleotides contain genes encoding the domains that bind to CD3 and PSMA. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. The promoter is selected such that it drives the expression of the polynucleotide in the respective host cell.

[0116] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. This recombinant vector can be constructed according to known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.

[0117] A variety of expression vector/host systems may be utilized to contain and express the polynucleotide encoding the polypeptide of the described trispecific antigen-binding protein. Examples of expression vectors for expression in E.coli are pSKK (Le Gall et ah, J Immunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells. [0118] Thus, the PSMA targeting trispecific proteins as described herein, in some embodiments, are produced by introducing a vector encoding the protein as described above into a host cell and culturing said host cell under conditions whereby the protein domains are expressed, may be isolated and, optionally, further purified.

Pharmaceutical Compositions

[0119] Also provided, in some embodiments, are pharmaceutical compositions comprising a PSMA trispecific antigen-binding protein described herein, a vector comprising the polynucleotide encoding the polypeptide of the PSMA targeting trispecific proteins or a host cell transformed by this vector and at least one pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents. [0120] In some embodiments of the pharmaceutical compositions, the PSMA targeting trispecific proteins described herein are encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposome, 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 instances, the PSMA trispecific antigen-binding protein are conjugated to the surface of liposomes. In some instances, the PSMA trispecific antigen-binding protein are encapsulated within the shell of a liposome. In some instances, the liposome is a cationic liposome.

[0121] The PSMA targeting trispecific proteins described herein are contemplated for use as a medicament. Administration is effected by different ways, e.g ., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. In some cases, the PSMA targeting trispecific proteins are administered through intravenous therapy.

[0122] The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently. An "effective dose" refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.

[0123] In some embodiments, the PSMA targeting trispecific proteins of this disclosure are administered at a dosage of up to 10 mg/kg at a frequency of once a week. In some cases, the dosage ranges from about 1 ng/kg to about 10 mg/kg, such as, from about 1 ng/kg to about 1 pg/kg. In some embodiments, the dose is from about 1 ng/kg to about 10 ng/kg, about 5 ng/kg to about 15 ng/kg, about 12 ng/kg to about 20 ng/kg, about 18 ng/kg to about 30 ng/kg, about 25 ng/kg to about 50 ng/kg, about 35 ng/kg to about 60 ng/kg, about 45 ng/kg to about 70 ng/kg, about 65 ng/kg to about 85 ng/kg, about 80 ng/kg to about 1 pg/kg, about 0.5 pg/kg to about 5 pg/kg, about 2 pg/kg to about 10 pg/kg, about 7 pg/kg to about 15 pg/kg, about 12 pg/kg to about 25 pg/kg, about 20 pg/kg to about 50 pg/kg, about 35 pg/kg to about 70 pg/kg, about 45 pg/kg to about 80 pg/kg, about 65 pg/kg to about 90 pg/kg, about 85 pg/kg to about 0.1 mg/kg, about 0.095 mg/kg to about 10 mg/kg. In some cases, the dosage is about 0.1 mg/kg to about 0.2 mg/kg; about 0.25 mg/kg to about 0.5 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.75 mg/kg to about 3 mg/kg, about 2.5 mg/kg to about 4 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 6 mg/kg, about 5.5 mg/kg to about 7 mg/kg, about 6.5 mg/kg to about 8 mg/kg, about 7.5 mg/kg to about 9 mg/kg, or about 8.5 mg/kg to about 10 mg/kg. The frequency of administration, in some embodiments, is about less than daily, every other day, less than once a day, twice a week, weekly, once in 7 days, once in two weeks, once in two weeks, once in three weeks, once in four weeks, or once a month. In some cases, the frequency of administration is weekly. In some cases, the frequency of administration is weekly and the dosage is up to 10 mg/kg. In some cases, duration of administration is from about 1 day to about 4 weeks or longer.

[0124] In some embodiments, the PSMA targeting trispecific proteins of this disclosure are administered at a dosage of about 1 ng/kg to about 10 ng/kg, about 1 ng/kg to about 20 ng/kg, about 1 ng/kg to about 50 ng/kg, about 1 ng/kg to about 80 ng//kg, about 1 ng/kg to about 100 ng/kg, about lng/kg to about 120 ng/kg, about 1 ng/kg to about 150 ng/kg, about 1 ng/kg to about 180 ng/kg, about 1 ng/kg to about 200 ng/kg, about 1 ng/kg to about 500 ng/kg, about lng/kg to about lpg/kg, about 1 ng/kg to about 10 pg/kg, about 1 ng/kg to about 100 pg/kg, about 1 ng/kg to about 1 mg/kg, about 1 ng/kg to about 10 mg/kg, about 10 ng/kg to about 20 ng/kg, about 10 ng/kg to about 50 ng/kg, about 10 ng/kg to about 80 ng//kg, about 10 ng/kg to about 100 ng/kg, about 10 ng/kg to about 120 ng/kg, about 10 ng/kg to about 150 ng/kg, about 10 ng/kg to about 180 ng/kg, about 10 ng/kg to about 200 ng/kg, about 10 ng/kg to about 500 ng/kg, about 10 ng/kg to about lpg/kg, about 10 ng/kg to about 10 pg/kg, about 10 ng/kg to about 100 pg/kg, about 10 ng/kg to about 1 mg/kg, about 10 ng/kg to about 10 mg/kg, about 20 ng/kg to about 50 ng/kg, about 20 ng/kg to about 80 ng//kg, about 20 ng/kg to about 100 ng/kg, about 20 ng/kg to about 120 ng/kg, about 20 ng/kg to about 150 ng/kg, about 20 ng/kg to about 180 ng/kg, about 20 ng/kg to about 200 ng/kg, about 20 ng/kg to about 500 ng/kg, about 20 ng/kg to about lpg/kg, about 20 ng/kg to about 10 pg/kg, about 20 ng/kg to about 100 pg/kg, about 20 ng/kg to about 1 mg/kg, about 20 ng/kg to about 10 mg/kg, about 50 ng/kg to about 80 ng//kg, about 50 ng/kg to about 100 ng/kg, about 50 ng/kg to about 120 ng/kg, about 50 ng/kg to about 150 ng/kg, about 50 ng/kg to about 180 ng/kg, about 50 ng/kg to about 200 ng/kg, about 50 ng/kg to about 500 ng/kg, about 50 ng/kg to about lpg/kg, about 50 ng/kg to about 10 pg/kg, about 50 ng/kg to about 100 pg/kg, about 50 ng/kg to about 1 mg/kg, about 50 ng/kg to about 10 mg/kg, about 80 ng/kg to about 100 ng/kg, about 80 ng/kg to about 120 ng/kg, about 80 ng/kg to about 150 ng/kg, about 80 ng/kg to about 180 ng/kg, about 80 ng/kg to about 200 ng/kg, about 80 ng/kg to about 500 ng/kg, about 80 ng/kg to about lpg/kg, about 80 ng/kg to about 10 pg/kg, about 80 ng/kg to about 100 pg/kg, about 80 ng/kg to about 1 mg/kg, about 80 ng/kg to about 10 mg/kg, about 100 ng/kg to about 120 ng/kg, about 100 ng/kg to about 150 ng/kg, about 100 ng/kg to about 180 ng/kg, about 100 ng/kg to about 200 ng/kg, about 100 ng/kg to about 500 ng/kg, about 100 ng/kg to about lpg/kg, about 100 ng/kg to about 10 pg/kg, about 100 ng/kg to about 100 gg/kg, about 100 ng/kg to about 1 mg/kg, about 100 ng/kg to about 10 mg/kg, about 120 ng/kg to about 150 ng/kg, about 120 ng/kg to about 180 ng/kg, about 120 ng/kg to about 200 ng/kg, about 120 ng/kg to about 500 ng/kg, about 120 ng/kg to about lgg/kg, about 120 ng/kg to about 10 gg/kg, about 120 ng/kg to about 100 gg/kg, about 120 ng/kg to about 1 mg/kg, about 120 ng/kg to about 10 mg/kg, about 150 ng/kg to about 180 ng/kg, about 150 ng/kg to about 200 ng/kg, about 150 ng/kg to about 500 ng/kg, about 150 ng/kg to about lgg/kg, about 150 ng/kg to about 10 gg/kg, about 150 ng/kg to about 100 gg/kg, about 150 ng/kg to about 1 mg/kg, about 150 ng/kg to about 10 mg/kg, about 180 ng/kg to about 200 ng/kg, about 180 ng/kg to about 500 ng/kg, about 180 ng/kg to about lgg/kg, about 180 ng/kg to about 10 gg/kg, about 180 ng/kg to about 100 gg/kg, about 180 ng/kg to about 1 mg/kg, about 180 ng/kg to about 10 mg/kg, about 200 ng/kg to about 500 ng/kg, about 200 ng/kg to about lgg/kg, about 200 ng/kg to about 10 gg/kg, about 200 ng/kg to about 100 gg/kg, about 200 ng/kg to about 1 mg/kg, about 200 ng/kg to about 10 mg/kg, about 500 ng/kg to about lgg/kg, about 500 ng/kg to about 10 gg/kg, about 500 ng/kg to about 100 gg/kg, about 500 ng/kg to about 1 mg/kg, about 500 ng/kg to about 10 mg/kg, about lgg/kg to about 10 gg/kg, about lgg/kg to about 100 gg/kg, about lgg/kg to about 1 mg/kg, about lgg/kg to about 10 mg/kg, about lOgg/kg to about 100 gg/kg, about lOgg/kg to about 1 mg/kg, about lOgg/kg to about 10 mg/kg, about lOOgg/kg to about 1 mg/kg, about lOOgg/kg to about 10 mg/kg, about 1 mg/kg to about 10 mg/kg.

[0125] In some embodiments, the PSMA targeting trispecific proteins of this disclosure are administered at a dosage of about 1.3 ng/kg, about 4 ng/kg, about 12 ng/kg, about 24 ng/kg, about 30 ng/kg, about 40 ng/kg, about 54 ng/kg, about 72 ng/kg, about 96 ng/kg, about 120 ng/kg, about 150 ng/kg, or about 160 ng/kg. In some cases, the dosage administered to a single patient is increased gradually from about 1.3 ng/kg to about 4 ng/kg, from about 1.3 ng/kg to about 12 ng/kg, from about 1.3 ng/kg to about 24 ng/kg, from about 4 ng/kg to about 12 ng/kg, from about 4 ng/kg to about 24 ng/kg, from about 4 ng/kg to about 30 ng/kg, from about 12 ng/kg to about 24 ng/kg, from about 12 ng/kg to about 30 ng/kg, from about 12 ng/kg to about 40 ng/kg, from about 12 ng/kg to about 54 ng/kg, from about 12 ng/kg to about 72 ng/kg, from about 12 ng/kg to about 96 ng/kg, from about 12 mg/kg to about 120 ng/kg, from about 12 mg/kg to about 150 ng/kg, from about 12 mg/kg to about 160 ng/kg, from about 24 ng/kg to about 30 ng/kg, from about 24 ng/kg to about 40 ng/kg, from about 24 ng/kg to about 54 ng/kg, from about 24 ng/kg to about 72ng/kg, from about 24 ng/kg to about 96 ng/kg, from about 24 mg/kg to about 120 ng/kg, from about 24 mg/kg to about 150 ng/kg, from about 24 mg/kg to about 160 ng/kg, from about 30 ng/kg to about 40 ng/kg, from about 30 ng/kg to about 54 ng/kg, from about 30 ng/kg to about 72 ng/kg, from about 30 ng/kg to about 96 ng/kg, from about 30 mg/kg to about 120 ng/kg, from about 30 mg/kg to about 150 ng/kg or from about 30 mg/kg to about 160 ng/kg. In some cases, the dosage administered to a single patient is decreased from about 24 ng/kg to about 12 ng/kg, from about 24 ng/kg to about 4 ng/kg, from about 24 ng/kg to about 1.3 ng/kg, from about 30 ng/kg to about 24 ng/kg, from about 30 ng/kg to about 12 ng/kg, from about 30 ng/kg to about 4 ng/kg, or from about 30 ng/kg to about 1.3 ng/kg. In some cases, the dosage administered to a single patient is increased first and then decreased, or is decreased first and then increased.

[0126] In some embodiments, dexamethasone is administered before, during, or after the administration of the PSMA targeting trispecific proteins to control adverse effects. In some cases, dexamethasone is administered as a premedication before the administration of the PSMA targeting trispecific proteins. In some embodiments, the frequency of administering the dexamethasone premedication is about daily, every other day, less than once a day, twice a week, weekly, once in 7 days, once in two weeks, once in two weeks, once in three weeks, once in four weeks, or once a month. In some cases, the frequency of administration is weekly. In some cases, the dexamethasone premedication is administered before every cycle of the PSMA targeting trispecific proteins administration. In some cases, the dexamethasone premedication is administered for less than every cycle of the PSMA targeting trispecific proteins administration. In some cases, the dexamethasone premedication is administered for 1 cycle, 2 cycles, 3 cycles,

4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 11 cycles, 12 cycles, 13 cycles, 14 cycles, 15 cycles, 16 cycles, 17 cycles, 18 cycles, 19 cycles, or 20 cycles.

[0127] In some embodiments, the dexamethasone premedication of this disclosure is administered at a dosage of up to 100 mg. In some cases, the dosage ranges from about 0.1 mg to about 100 mg. In some embodiments, the dose is from about 0.1 mg to about 0.5 mg, from about 0.1 mg to about 1 mg, from about 0.1 mg to about 5 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 100 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 5 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 50 mg, from about 0.5 mg to about 100 mg, from about 1 mg to about 5 mg, from about 1 mg to about 10 mg, from about 1 mg to about 50 mg, from about 1 mg to about 100 mg, from about 5 mg to about 10 mg, from about 5 mg to about 50 mg, from about 5 mg to about 100 mg, from about 10 mg to about 50 mg, from about 10 mg to about 100, or from about 50 mg to about 100 mg. In some cases, the dexamethasone premedication is administered at a dosage of about 20mg, about 10 mg, about 4 mg or about 2 mg.

Methods of treatment

[0128] Also provided herein, in some embodiments, are methods and uses for stimulating the immune system of an individual in need thereof comprising administration of a PSMA targeting trispecific protein described herein. In some instances, the administration of a PSMA targeting trispecific protein described herein induces and/or sustains cytotoxicity towards a cell expressing PSMA. In some instances, the cell expressing PSMA is a cancer cell.

[0129] Also provided herein are methods and uses for a treatment of a disease, disorder or condition associated with PSMA comprising administering to an individual in need thereof a PSMA targeting trispecific protein described herein. Diseases, disorders or conditions associated with PSMA include, but are not limited to, a proliferative disease or a tumorous 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 renal cancer.

[0130] In some embodiments, the prostate cancer is an advanced stage prostate cancer. In some embodiments, the prostate cancer is drug resistant. In some embodiments, the prostate cancer is anti-androgen drug resistant. In some embodiments, the prostate cancer is metastatic. In some embodiments, the prostate cancer is metastatic and drug resistant (e.g., anti -androgen 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 enzalutamide resistant. In some embodiments, the prostate cancer is enzalutamide and arbiraterone resistant. In some embodiments, the prostate cancer is enzalutamide, arbiraterone, and bicalutamide resistant. In some embodiments, the prostate cancer is docetaxel resistant. In some of these embodiments, the prostate cancer is enzalutamide, arbiraterone, bicalutamide, and docetaxel resistant.

[0131] In some embodiments, administering a PSMA targeting trispecific protein described herein inhibits prostate cancer cell growth; inhibits prostate cancer cell migration; inhibits prostate cancer cell invasion; ameliorates the symptoms of prostate cancer; reduces the size of a prostate cancer tumor; reduces the number of prostate cancer tumors; reduces the number of prostate cancer cells; induces prostate cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death; or enhances the therapeutic effects of a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and any combinations thereof.

[0132] In some embodiments, the method comprises inhibiting prostate cancer cell growth by administering a PSMA targeting trispecific protein described herein. In some embodiments, the method comprises inhibiting prostate cancer cell migration by administering a PSMA targeting trispecific protein described herein. In some embodiments, the method comprises inhibiting prostate cancer cell invasion by administering a PSMA targeting trispecific protein described herein. In some embodiments, the method comprises ameliorating the symptoms of prostate cancer by administering a PSMA targeting trispecific protein described herein. In some embodiments, the method comprises reducing the size of a prostate cancer tumor by administering a PSMA targeting trispecific protein described herein. In some embodiments, the method comprises reducing the number of prostate cancer tumors by administering a PSMA targeting trispecific protein described herein. In some embodiments, the method comprises reducing the number of prostate cancer cells by administering a PSMA targeting trispecific protein described herein. In some embodiments, the method comprises inducing prostate cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death by administering a PSMA targeting trispecific protein described herein.

[0133] As used herein, in some embodiments, “treatment” or “treating” or “treated” refers to therapeutic treatment wherein the object is to slow (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. In other embodiments, “treatment” or “treating” or “treated” refers to prophylactic measures, wherein the object is to delay onset of or reduce severity of an undesired physiological condition, disorder or disease, such as, for example is a person who is predisposed to a disease (e.g., an individual who carries a genetic marker for a disease such as prostate cancer).

[0134] In some embodiments of the methods described herein, the PSMA targeting trispecific proteins are administered in combination with an agent for treatment of the particular disease, disorder or condition. Agents include but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies (g-rays, X-rays, and/or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g., antisense, retroviral therapy and the like) and other immunotherapies. In some embodiments, the PSMA targeting trispecific proteins are administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics, opioids and/or non-steroidal anti-inflammatory agents. In some embodiments, the PSMA targeting trispecific proteins are administered before, during, or after surgery. Certain Definitions

[0135] As used herein, “elimination half-time” is used in its ordinary sense, as is described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass a quantitative measure of the time course of drug elimination. The elimination of most drugs is exponential (i.e., follows first-order kinetics), since drug concentrations usually do not approach those required for saturation of the elimination process. The rate of an exponential process may be expressed by its rate constant, k, which expresses the fractional change per unit of time, or by its half-time, tl/2 the time required for 50% completion of the process. The units of these two constants are time-1 and time, respectively. A first-order rate constant and the half time of the reaction are simply related (kxtl/2=0.693) and may be interchanged accordingly. Since first-order elimination kinetics dictates that a constant fraction of drug is lost per unit time, a plot of the log of drug concentration versus time is linear at all times following the initial distribution phase (i.e. after drug absorption and distribution are complete). The half-time for drug elimination can be accurately determined from such a graph.

[0136] As used herein, the phrase “prostate cancer” or “advanced stage prostate cancer” includes a class of prostate cancers that has progressed beyond early stages of the disease. Typically, advanced stage prostate cancers are associated with a poor prognosis. Types of advanced stage prostate cancers include, but are not limited to, metastatic prostate cancer, drug- resistant prostate cancer such as anti-androgen-resistant prostate cancer (e.g., enzalutamide- resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer, and the like), hormone refractory prostate cancer, castration-resistant prostate cancer, metastatic castration -resistant prostate cancer, docetaxel-resistant prostate cancer, androgen receptor splice variant-7 (AR-V7)-induced drug-resistant prostate cancer such as AR-V7- induced anti-androgen-resistant prostate cancer (e.g., AR-V7-induced enzalutamide-resistant prostate cancer), aldo-keto reductase family 1 member C3 (AKRlC3)-induced drug-resistant prostate cancer such as AKR1C3 -induced anti-androgen-resistant prostate cancer (e.g.,

AKR1C3 -induced enzalutamide-resistant prostate cancer), and combinations thereof. In some instances, the advanced stage prostate cancers do not generally respond, or are resistant, to treatment with one or more of the following conventional prostate cancer therapies: enzalutamide, arbiraterone, bicalutamide, and docetaxel. Compounds, compositions, and methods of the present disclosure are provided for treating prostate cancer, such as advanced stage prostate cancer, including any one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of the types of advanced stage prostate cancers disclosed herein. [0137] The terms “PSMA targeting trispecific proteins,” “PSMA binding trispecific proteins,” and “PSMA binding TriTAC molecules,” are used interchangeably herein.

EXAMPLES

Example 1 : Methods to assess binding and cytotoxic activities of trispecific antigen binding molecules

Protein Production

[0138] Sequences of trispecific molecules were cloned into mammalian expression vector pcDNA 3.4 (Invitrogen) preceded by a leader sequence and followed by a 6x Histidine Tag (SEQ ID NO: 161). Expi293F cells (Life Technologies A14527) were maintained in suspension in Optimum Growth Flasks (Thomson) between 0.2 to 8 x le6 cells/ml in Expi293 media. Purified plasmid DNA was transfected into Expi293 cells in accordance with Expi293 Expression System Kit (Life Technologies, A14635) protocols, and maintained for 4-6 days post transfection. Conditioned media was partially purified by affinity and desalting chromatography. Trispecific proteins were subsequently polished by ion exchange or, alternatively, concentrated with Amicon Ultra centrifugal filtration units (EMD Millipore), applied to Superdex 200 size exclusion media (GE Healthcare) and resolved in a neutral buffer containing excipients. Fraction pooling and final purity were assessed by SDS-PAGE and analytical SEC.

Affinity Measurements

[0139] The affinities of the all binding domains molecules were measured by biolayer inferometry using an Octet instrument.

[0140] PSMA affinities were measured by loading human PSMA-Fc protein (100 nM) onto anti-human IgG Fc biosensors for 120 seconds, followed by a 60 second baseline, after which associations were measured by incubating the sensor tip in a dilution series of the trispecific molecules for 180 seconds, followed by dissociation for 50 seconds. EGFR and CD3 affinities were measured by loading human EGFR-Fc protein or human CD3-Flag-Fc protein, respectively, (100 nM) onto anti-human IgGFc biosensors for 120 seconds, followed by a 60 second baseline, after which associations were measured by incubating the sensor tip in a dilution series of the trispecific molecules for 180 seconds, followed by dissociation for 300 seconds. Affinities to human serum albumin (HSA) were measured by loading biotinylated albumin onto streptavidin biosensors, then following the same kinetic parameters as for CD3 affinity measurements. All steps were performed at 30°C in 0.25% casein in phosphate-buffered saline. Cytotoxicity assays

[0141] A human T-cell dependent cellular cytotoxicity (TDCC) assay was used to measure the ability of T cell engagers, 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 target cancer cell line cells are mixed together at a 10:1 ratio in a 384 wells plate, and varying amounts of T cell engager are added. After 48 hours, the T cells are washed away leaving attached to the plate target cells that were not killed by the T cells. To quantitate the remaining viable cells, CellTiter-Glo® Luminescent Cell Viability Assay (Promega) is used. In some cases, the target cells are engineered to express luciferase. In these cases, viability of the target cells is assessed by performing a luminescent luciferase assay with STEAD YGLO® reagent (Promega), where viability is directly proportional to the amount of luciferase activity.

Stability assays

[0142] The stability of the trispecific binding proteins was assessed at low concentrations in the presence of non-human primate serum. TriTACs were diluted to 33 pg/ml in Cynomolgus serum (BioReclamationIVT) and either incubated for 2 d at 37°C or subjected to five freeze/thaw cycles. Following the treatment, the samples were assessed in cytotoxicity (TDCC) assays and their remaining activity was compared to untreated stock solutions.

Xenograft assays

[0143] The in vivo efficacy of trispecific binding proteins was assessed 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 5e622Rvl human prostate cancer cells and 5e6 resting, human T cells that were isolated from a healthy, human donor. The mice were randomized into three groups, and treated with vehicle, 0.5 mg/kg PSMA TriTAC (SEQ ID NO: 141) or 0.5 mg/kg PSMA BiTE. Treatments were administered daily for 10 days via i.v. bolus injection. Animals were checked daily for morbidity and mortality. Tumor volumes were determined twice weekly with a caliper. The study was terminated after 30 days.

PK assays

[0144] The purpose of this study was to evaluate the single dose pharmacokinetics of trispecific binding proteins following intravenous injection. 2 experimentally naive cynomolgus monkeys per group (1 male and 1 female) were given compound via a slow IV bolus injection administered over approximately 1 minute. Following dose administration, cage side observations were performed once daily and body weights were recorded weekly. Blood samples were collected and processed to serum for pharmacokinetic analysis through 21 days post dose administration. [0145] Concentrations of test articles were determined from monkey serum with an electroluminescent readout (Meso Scale Diagnostics, Rockville). 96 well plates with immobilized, recombinant CD3 were used to capture the analyte. Detection was performed with sulfo-tagged, recombinant PSMA on a MSD reader according to the manufacturer’s instructions. Example 2: Assessing the impact of CD3 affinity on the properties of trispecific molecules [0146] PSMA targeting trispecific molecules with distinct CD3 binding domains were studied to demonstrate the effects of altering CD3 affinity. An exemplary PSMA targeting trispecific molecule is illustrated in Figure 1. Table 1 lists the affinity of each molecule for the three binding partners (PSMA, CD3, HSA). Affinities were measured by biolayer interferometry using an Octet instrument (Pall Forte Bio). Reduced CD3 affinity leads to a loss in potency in terms of T cell mediated cellular toxicity (Figures 2A-2C). The pharmacokinetic properties of these trispecific molecules was assessed in cynomolgus monkeys. Molecules with high affinity for CD3 like a tool PSMA TriTAC (SEQ ID NO: 144) have a terminal half-life of approx. 90 h (Figure 3). Despite the altered ability to bind CD3 on T cells, the terminal half-life of two molecules with different CD3 affinities shown in Figure 4 is very similar. However, the reduced CD3 affinity appears to lead to a larger volume of distribution, which is consistent with reduced sequestration of trispecific molecule by T cells. There were no adverse clinical observations or body weight changes noted during the study period.

Table 1 : Binding Affinities for Human and Cynomolgus Antigens

Example 3: Assessing the impact of PSMA affinity on the properties of trispecific molecules [0147] PSMA targeting trispecific molecules with distinct PSMA binding domains were studied to demonstrate the effects of altering PSMA affinity. Table 2 lists the affinity of each molecule for the three binding partners (PSMA, CD3, HSA). Reduced PSMA affinity leads to a loss in potency in terms of T cell mediated cellular toxicity (Figures 5A-5C). Table 2: Binding Affinities for Human and Cynomolgus Antigens

Example 4: In vivo efficacy of PSMA targeting trispecific molecules

[0148] The PSMA targeting trispecific molecule (SEQ ID NO: 141) was assessed for its ability to inhibit the growth of tumors in mice. For this experiment, immunocompromised mice reconstituted with human T cells were subcutaneously inoculated with PSMA expressing human prostate tumor cells (22Rvl) and treated daily for 10 days with 0.5 mg/kg i.v. of either PSMA targeting BiTE or TriTAC molecules. Tumor growth was measured for 30. Over the course of the experiment, the trispecific molecule was able to inhibit tumor growth with an efficacy comparable to a BiTE molecule (Figure 6).

Example 5: Specificity of trispecific molecules

[0149] In order to assess the specificity of PSMA targeting TriTAC molecules, their ability to induce T cells to kill tumor cells was tested with tumor cells that are negative for PSMA (Figure 7A). An EGFR targeting TriTAC molecule served as positive control, a GFP targeting TriTAC molecule as negative control. All three TriTACs with distinct PSMA binding domains showed the expected activity against the PSMA positive cell line LNCaP (Figure 7B), but did not reach EC50s in the PSMA negative tumor cell lines KMS12BM and OVCAR8 (Figures 7C and 7D). The EC50s are summarized in Table 3. At very high TriTAC concentrations (> 1 nM), some limited off-target cell killing could be observed for TriTACs (such as p8, SEQ ID NO: 145 and HDS, SEQ ID NO: 146, while HTS, SEQ ID NO: 147 did not show significant cell killing under any of the tested conditions.

[0150] Table 3: Cell killing activity of TriTAC molecules in with antigen positive and negative tumor cell lines (EC50 [pM])

Example 6: Stress tests and protein stability

[0151] Four PSMA targeting trispecific molecules were either incubated for 48 h in Cynomolgus serum at low concentrations (33.3 pg/ml) or subjected to five freeze thaw cycles in Cynomolgus serum. After the treatment, the bio-activity of the TriTAC molecules was assessed in cell killing assays and compared to unstressed samples (“positive control”, Figure 8A-D). All molecules maintained the majority of their cell killing activity. TriTAC p8, SEQ ID NO: 145 was the most stress resistant and did not appear to lose any activity under the conditions tested here.

Example 7: Xenograft Tumor Model

[0152] The PSMA targeting trispecific proteins of the previous examples are evaluated in a xenograft model.

[0153] Male immune-deficient NCG mice are subcutaneously inoculated with 5 xlO⁶22Rvl cells into their the right dorsal flank. When tumors reach 100 to 200 mm³, animals are allocated into 3 treatment groups. Groups 2 and 3 (8 animals each) are intraperitoneally injected with 1.5xl0⁷ activated human T-cells. Three days later, animals from Group 3 are subsequently treated with a total of 9 intravenous doses of 50 pg PSMA trispecific antigen-binding protein of Example 1 (qdx9d). Groups 1 and 2 are only treated with vehicle. Body weight and tumor volume are determined for 30 days. It is expected that tumor growth in mice treated with the PSMA trispecific antigen-binding protein is significantly reduced in comparison to the tumor growth in respective vehicle-treated control group.

Example 8: Activity of an exemplary PSMA antigen-binding protein (PSMA targeting TriTAC molecule) in redirected T cell killing assays using a panel of PSMA expressing cell lines and T cells from different donors

[0154] This study was carried out to demonstrate that the activity of the exemplary PSMA trispecific antigen-binding protein is not limited to LNCaP cells or a single cell donor.

[0155] Redirected T cell killing assays were performed using T cells from four different donors and the human PSMA-expressing prostate cancer cell lines VCaP, LNCaP, MDAPCa2b, and 22Rvl. With one exception, the PSMA trispecific antigen-binding protein was able to direct killing of these cancer cell lines using T cells from all donors with ECso values of 0.2 to 1.5 pM, as shown in Table 4. With the prostate cancer cell line 22 Rvl and Donor 24, little to no killing was observed (data not shown). Donor 24 also only resulted approximately 50% killing of the MDAPCa2b cell line whereas T cells from the other 3 donors resulted in almost complete killing of this cell line (data not shown). Control assays demonstrated that killing by the PSMA trispecific antigen-binding protein was PSMA specific. No killing was observed when PSMA- expressing cells were treated with a control trispecific protein targeting green fluorescent protein (GFP) instead of PSMA (data not shown). Similarly, the PSMA trispecific antigen-binding protein was inactive with cell lines that lack PSMA expression, NCI- 1563 and HCT116, also shown in Table 4.

Table 4: ECso Values from TDCC Assays with Six Human Cancer Cell Lines and Four Different T Cell Donors

Example 9: Stimulation of cytokine expression in by an exemplary PSMA trispecific antigen binding protein (PSMA targeting TriTAC molecule) in redirected T cell killing assays [0156] This study was carried out to demonstrate activation of T cells by the exemplary PSMA trispecific antigen-binding protein during redirected T cell killing assays by measuring secretion of cytokine into the assay medium by activated T cells.

[0157] Conditioned media collected from redirected T cell killing assays, as described above in Example 9, were analyzed for expression of the cytokines TNFa and IFNy. Cytokines were measured using AlphaLISA assays (Perkin-Elmer). Adding a titration of the PSMA antigen binding protein to T cells from four different donors and four PSMA-expressing cell lines, LNCaP, VCaP, MDAPCa2b, and 22Rvl resulted in increased levels of TNFa. The results for TNFa expression and IFN g expression levels in the conditioned media are shown in Tables 5 and 6, respectively. The ECso values for the PSMA antigen-binding protein induced expression of these cytokines ranged from 3 to 15 pM. Increased cytokine levels were not observed with a control trispecific protein targeting GFP. Similarly, when assays were performed with two cell lines that lack PSMA expression, HCT116 and NCI-H1563, PSMA HTS TriTAC also did not increase TNFa or IFNy expression. Table 5: ECso Values for TNFa Expression in Media from PSMA Trispecific Antigen- Binding Protein TDCC Assays with Six Human Cancer Cell Lines and T Cells from Four Different Donors

Table 6: ECso Values for IFNy Expression in Media from PSMA Trispecific Antigen- Binding Protein TDCC Assays with Six Human Cancer Cell Lines and T Cells from Four Different Donors

Example 10: Activity of an exemplary PSMA trispecific antigen-binding protein (PSMA targeting TriTAC) in redirected T cell killing assay (TDCC) using T cells from cynomolgus monkeys

[0158] This study was carried out to test the ability of the exemplary PSMA trispecific antigen binding protein to direct T cells from cynomolgus monkeys to kill PSMA-expressing cell lines. [0159] TDCC assays were set up using peripheral blood mononuclear cells (PBMCs) from cynomolgus monkeys. Cyno PBMCs were added to LNCaP cells at a 10: 1 ratio. It was observed that the PSMA trispecific antigen-binding protein redirected killing of LNCaP by the cyno PBMCs with an ECso value of 11 pM. The result is shown in Figure 9A. To confirm these results, a second cell line was used, MDAPCa2b, and PBMCs from a second cynomolgus monkey donor were tested. Redirected killing of the target cells was observed with an ECso value of 2.2 pM. The result is shown in Figure 9B. Killing was specific to the anti-PSMA arm of the PSMA trispecific antigen-binding protein as killing was not observed with a negative control trispecific protein targeting GFP. These data demonstrate that the PSMA antigen- binding trispecific protein can direct cynomolgus T cells to kill target cells expressing human PSMA.

Example 11 : Expression of markers of T cell activation in redirect T cell killing assays with an exemplary PSMA trispecific antigen-binding protein (PSMA targeting TriTAC molecule)

[0160] This study was performed to assess whether T cells were activated when the exemplary PSMA trispecific antigen-binding protein directed the T cells to kill target cells.

[0161] The assays were set up using conditions for the redirected T cell killings assays described in the above example. T cell activation was assessed by measuring expression of CD25 and CD69 on the surface of the T cells using flow cytometry. The PSMA trispecific antigen-binding protein was added to a 10: 1 mixture of purified human T cells and the prostate cancer cell line VCaP. Upon addition of increasing amounts of the PSMA trispecific antigen binding protein, increased CD69 expression and CD25 expression was observed, as shown in Figure 10. ECso value was 0.3 pM for CD69 and 0.2 pM for CD25. A trispecific protein targeting GFP was included in these assays as negative control, and little to no increase in CD69 or CD25 expression is observed with the GFP targeting trispecific protein, also shown in Figure 10

Example 12: Stimulation of T cell proliferation by an exemplary PSMA trispecific antigen binding protein (PSMA targeting TriTAC molecule) in the presence of PSMA expressing target cells

[0162] This study was used as an additional method to demonstrate that the exemplary PSMA trispecific antigen-binding protein was able to activate T cells when it redirects them to kill target cells.

[0163] T cell proliferation assays were set up using the conditions of the T cell redirected killing assay using LNCaP target cells, as described above, and measuring the number of T cells present at 72 hours. The exemplary PSMA trispecific antigen-binding protein stimulated proliferation with an ECso value of 0.5 pM. As negative control, a trispecific protein targeting GFP was included in the assay, and no increased proliferation was observed with this protein. The results for the T cell proliferation assay are illustrated in Figure 11.

Example 13: Redirected T cell killing of LNCaP cells by three exemplary PSMA trispecific antigen-binding proteins (PSMA targeting TriTAC molecules PH I T. PH. and Z2)

[0164] This study was carried out to test the ability of three exemplary PSMA trispecific antigen-binding proteins, having the sequences as set forth in SEQ ID Nos: 150, 151, and 152, to redirect T cells to kill the LNCaP cell line.

[0165] In TDCC assays, set up as described in above examples, the PSMA PH1T TriTAC (SEQ ID No: 150) and PSMA PHI TriTAC (SEQ ID NO: 151) proteins directed killing with ECso values of 25 and 20 pM, respectively, as shown in Figure 12A; and the PSMA Z2 TriTAC (SEQ ID NO: 152) protein directed killing with an ECso value of 0.8 pM, as shown in Figure 12B. Example 14: PSMA HTS TriTAC. a half-life extended. PSMA/CD3-specific TriTAC for the treatment of metastatic prostate cancer

[0166] An exemplary PSMA targeting TriTAC molecule PSMA HTS TriTAC (SEQ ID NO: 147) was used for the studies described below.

[0167] Biophysical characterization : PSMA HTS TriTAC (SEQ ID NO: 147) was prepared out using a process outline in Figure 13, comprising following sequential steps: depth filtration, Protein A affinity chromatography, low pH viral inactivation, two steps of ion exchange, viral filtration, and ultrafiltration and diafiltration. Purity was determined by SDS-PAGE, as shown in Figure 14; analytical ultracentrifugation was also carried out with the purified protein (results shown below in Table 8 and sedimentation velocity distribution is shown in Figure 15).

Stability of the PSMA HTS TriTAC (SEQ ID NO: 147) protein was determined using analytical size exclusion chromatography, and the results are shown in Figure 16 and Table 7. Furthermore, stability was assessed using differential scanning calorimetry, results of which are shown in Figure 17 and Table 9.

Table 7: Biophysical characterization of PSMA HTS TriTAC (SEQ ID NO: 147) -Analytical Size Exclusion Chromatography

Table 8: Biophysical characterization of PSMA HTS TriTAC (SEQ ID NO: 147) -Analytical

Ultra Centrifugation

Table 9: Biophysical characterization of PSMA HTS TriTAC (SEQ ID NO: 147) -Differential

Scanning Calorimetry

[0168] In vitro binding : For this study, human and cynomolgus albumin, PSMA, CD3e were incubated with PSMA HTS TriTAC (SEQ ID NO: 147) and binding was measured using biolayer interferometry. The PSMA and CD3e bindings were measured in the presence of 15 mg/ml HSA. The results are shown in Figure 18 and Table 10. High affinity binding was observed between PSMA HTS TriTAC (SEQ ID NO: 147) and human PSMA, albumin (ALB), and CD3e. In case of cynomolgus proteins, high affinity binding was observed with ALB and CD3e, however the affinity towards cynomolgus PSMA was indeterminate.

Table 10: Binding of PSMA HTS TriTAC (SEQ ID NO: 147)

[0169] In another binding study, PSMA-expressing MDAPCa2b cancer cells were incubated with PSMA HTS TriTAC (SEQ ID NO: 147) or a control TriTAC molecule that does not target PSMA. Following incubation, the cells were washed to remove unbound PSMA HTS TriTAC (SEQ ID NO: 147) or control TriTAC molecules and further incubated with Protein A conjugated to FITC. Binding of PSMA HTS TriTAC (SEQ ID NO: 147) or that of the control TriTAC to the MDAPCa2b cells was measured by flow cytometry. Robust binding of PSMA HTS TriTAC (SEQ ID NO: 147) to the MDAPCa2b cells was observed, as seen in Figure 19 (left panel) but no binding was observed in case of the control TriTAC molecule, also shown in Figure 19 (left panel).

[0170] In a further binding study, human T cells were incubated with PSMA HTS TriTAC (SEQ ID NO: 147) or a control TriTAC molecule that does not target PSMA. Following incubation, the cells were washed to remove unbound PSMA HTS TriTAC (SEQ ID NO: 147) or control TriTAC molecules and further incubated with a secondary antibody, which is able to recognize the anti-albumin domain in the TriTAC molecules, conjugated to Alexa Fluor 647. Binding of PSMA HTS TriTAC (SEQ ID NO: 147) or that of the control TriTAC to the human T cells was measured by flow cytometry. Robust binding of PSMA HTS TriTAC (SEQ ID NO: 147) to the human T cells was observed, as seen in Figure 19 (right panel) but no binding was observed in case of the control TriTAC molecule, also shown in Figure 19 (right panel).

[0171] PSMA HTS TriTAC (SEQ ID NO: 147) potently directs T cell killing of PSMA expressing cells_^ The aim of this study was to assess if PSMA HTS TriTAC (SEQ ID NO: 147) was able to direct T cells to kill PSMA-expressing cell, VCaP. VCaP cells used in this study were engineered to express luciferase. T cells from a 4 healthy donors (donor 24; donor 8144; donor 72; donor 41) and VCaP cells were mixed and varying amounts of PSMA HTS TriTAC (SEQ ID NO: 147) was added to the mixture. The mixture was incubated for 48 hours at 37 °C. In control experiments, a trispecific molecule, GFP TriTAC (SEQ ID NO: 169), which targets GFP, was used in place of PSMA HTS TriTAC. After 48 hours, the remaining viable VCaP cells were quantified using a luminescence assay. It was observed that PSMA HTS TriTAC (SEQ ID NO: 147) was able to efficiently direct T cells from all 4 healthy donors to kill VCaP cells (as shown in Figure 20), whereas the control GFP TriTAC molecule was not able to do that (also shown in Figure 20). Further TDCC assays were carried out with PSMA HTS TriTAC (SEQ ID NO: 147) , with additional PSMA-expressing cells lines (LNCaP, MDAPCa2b, VCaP, 22Rvl) and PSMA non-expressing cell lines (NCI-1536 and HCT-116) and the ECso values are presented in Table 11. It was observed that PSMA HTS TriTAC (SEQ ID NO: 147) was not able to direct T cells to kill the non-PSMA expressing cells. The TDCC activity of the PSMA HTS TriTAC was also measured in the presence or absence of HSA, and it was observed that the ECso for LNCaP cell killing was 0.7 pM in absence of HSA versus 1 pM in the presence of HSA (Figure 37).

Table 11 : PSMA HTS TriTAC (SEQ ID NO: 147) directed killing of four prostate cancer cell lines with four T cell donors

[0172] PSMA-dependent activation ofT cells by PSMA HTS TriTAC (SEQ ID NO: 147): In this assay, T cells from 4 different healthy donors (donor 24; donor 41; donor 72; and donor 8144) and LNCaP cells were incubated with PSMA HTS TriTAC (SEQ ID NO: 147) for 48 hours at 37 °C. T cells from the same donors were also incubated for 48 hours at 37 °C with a control trispecific molecule, GFP TriTAC (SEQ ID NO: 169), which targets GFP, and LNCaP cells. After 48 hours, T cells were collected, and (i) CD69 expression on the T cells was measured by flow cytometry, (ii) TNF-a secretion from the T cells was measured.

[0173] CD69 expression was detected on T cells from all 4 healthy donors in presence of LNCaP cells and PSMA HTS TriTAC (SEQ ID NO: 147) but not in presence of the negative control GFP TriTAC and LNCaP cells, as shown in Figure 21. It was observed that TNF-a was secreted into the medium in presence of LNCaP cells and PSMA HTS TriTAC (SEQ ID NO: 147) but not in presence of LNCaP cells and the control GFP TriTAC molecule, as shown in Figure 22.

[0174] PSMA HTS TriTAC potently inhibits growth of 22Rvl xenograft : For this study, 5 x 10⁶ human PBMC (peripheral blood mononuclear cells) and 22Rvl prostate cancer cells were injected into mice, at day 0. After 5 days, mice were injected with a vehicle control or PSMA HTS TriTAC (SEQ ID NO: 147) , daily for 10 days (days 5-14) at doses of 2 pg/kg; 10 pg/kg; 50 pg/kg; and 250 pg/kg. Tumor volumes were measured every few days and the study was terminated at day 45. Significant inhibition of tumor growth was observed in the mice injected with PSMA HTS TriTAC (SEQ ID NO: 147) , at all doses, compared to those injected with the vehicle control, as shown in Figure 23.

[0175] PSMA HTS TriTAC has a half-life of ~ 3.3 days in cynomolgus monkeys: For this study, cynomolgus monkeys were injected with 0.1 mg/kg or 3 mg/kg dose of PSMA HTS TriTAC (SEQ ID NO: 147) , intravenously, and serum samples were collected at various time points after the injection. Two monkeys were injected for each dose. The amount of PSMA HTS TriTAC (SEQ ID NO: 147) in the serum was measured using anti-idiotype antibodies recognizing the PSMA HTS TriTAC (SEQ ID NO: 147) molecule, in an electrochemiluminescient assay. Figure 24 shows a plot for the serum PSMA HTS TriTAC (SEQ ID NO: 147) levels at various time points. The data was then used to calculate the pharmacokinetic properties of the PSMA HTS TriTAC (SEQ ID NO: 147) molecule, as provided in Table 12. The pharmacokinetic data suggested feasibility of once or twice weekly dosing in humans. To demonstrate the PSMA HTS TriTAC protein retains biological activity after being dosed in cynomolgus monkeys, a serum sample collected 168 hours (1 week) after dosing and a freshly thawed PSMA HTS TriTAC were tested in a TDCC assay. The serum sample and freshly thawed protein were serially diluted in cynomolgus serum to control for the impact of serum on TDCC activity. Both samples redirected T cells to lyse LNCaP cells with EC50 values of 2.6 and 4.8 pM, respectively (Figure 40), indicating that the PSMA HTS TriTAC protein retained its biological activity after circulating in cynomolgus monkeys for 1 week.

Table 12: Pharmacokinetics of PSMA HTS TriTAC (SEQ ID NO: 147)

[0176] PSMA HTS TriTAC is highly tolerated with repeat dosing in cynomolgus monkeys : In this study, safety and tolerability of repeat dosing of PSMA HTS TriTAC (SEQ ID NO: 147) was assessed. Cynomolgus monkeys were administered PSMA HTS TriTAC (SEQ ID NO: 147) once weekly for 4 weeks (qwx4), at a dose of 3 mg/kg, and no observed adverse effect level was observed. Similar results were observed with 1 mg/kg and 0.1 mg/kg qwx4 doses. CD3 and albumin binding domains of PSMA HTS TriTAC (SEQ ID NO: 147) were able to cross-react with cynomolgus targets but minimal binding of PSMA HTS TriTAC (SEQ ID NO: 147) to recombinant cynomolgus PSMA was observed. It was concluded that the pharmacodynamic effects of PSMA HTS TriTAC (SEQ ID NO: 147) were consistent with T cell engagement. Further studies indicated that PSMA HTS TriTAC (SEQ ID NO: 147) was able to lead to transient reduction in circulating T cells, NK cells, and monocytes; upregulation of activation markers (CD25 and CD69) in the remaining circulating T cells. It was further observed that a mild and transient increase in cytokines (IFNy, IL-6, IL-10) with 1^st dose, was changed after 4^th dose and was much less pronounced. No adverse histopathology findings were observed with the safety studies.

[0177] PSMA HTS TriTAC (SEQ ID NO: 147) induces transient T lymphocyte margination and activation. Following the 1^st dose of PSMA HTS TriTAC (SEQ ID NO: 147) , a rapid decline of circulating T cells within 8 hrs post dose was observed. However, much less lymphocyte margination was noted after 4th and final dose. This observation was consistent for all doses, 0.1 mg/kg, 1 mg/kg, and 3 mg/kg. Results are shown in Figure 25. CD69 activation was observed 8 hours after administering the PSMA HTS TriTAC (SEQ ID NO: 147) at 3 mg/kg not after administration of the vehicle, as shown in Figure 26.

[0178] PSMA HTS TriTAC induces limited cytokines, no evidence of cytokine release syndrome: Transient, dose-dependent increases in IL-6 and IL-10 as similarly reported with other bispecific T cell engagers were seen following administration of PSMA HTS TriTAC (SEQ ID NO: 147) but no observable trend of increase in IL-2, -4, -5, TNFa, and IFNy was noted. Results are shown in Figure 27. Here, again, the transient increase in cytokine levels was not seen after the 4^th and final dose, also shown in Figure 27.

[0179] Based on the above studies, it was concluded that: (a) PSMA HTS TriTAC (SEQ ID NO: 147) TriTAC is a stable, manufacturable, single chain molecule that binds with high affinity and specificity to PSMA, CD3 and albumin; (b) PSMA HTS TriTAC (SEQ ID NO: 147) potently activates and redirects T cells to kill PSMA expressing cells in both in vitro and in vivo prostate cancer models; (c) PSMA HTS TriTAC (SEQ ID NO: 147) has a long serum half-life and was very well tolerated, even at high doses, in cynomolgus monkeys. With its small size, PSMA HTS TriTAC (SEQ ID NO: 147) is, in certain cases, more able to penetrate solid tumors than antibodies. PSMA HTS TriTAC (SEQ ID NO: 147) is thus a safe, effective, and convenient treatment for patients with metastatic castration resistant prostate cancer.

Table 13: CD3 Binding Domain Sequences

Table 14: HSA Binding Domain Sequences

Table 15: PSMA Binding Domain Sequences

Table 16: PSMA Targeting Trispecific Protein Sequences

Table 17: PSMA Binding Domain CDR sequences

Table 18: Exemplary Framework Sequences

Table 19: Control TriTAC Molecule Sequence

Example 15: PSMA trispecific antigen-binding protein Phase dose escalation expansion

safety and pharmacokinetics study

[0180] This study was carried out to test the Phase I dose escalation, expansion, safety and pharmacokinetics of an exemplary PSMA trispecific antigen-binding protein in patients with mCRPC.

[0181] Target population is patients with: metastatic castrate-resistant prostate cancer (mCRPC); disease progression on the prior systemic regimen; at least two prior systemic therapies approved for mCRPC; prior PSMA-targeting therapy allowed; and prior chemotherapy allowed, but not required.

[0182] Trial Design: PSMA trispecific antigen-binding protein Phase I trial design is shown in Figure 28. Key objectives include characterization of safety, pharmacokinetics, and identification of dose for expansion phase. Tumor assessments are performed every 9 weeks and include conventional CT and bone scans and PSA. Additional assessments include cytokines, circulating tumor cells (CTC). [0183] Dosing & Administration: PSMA trispecific antigen-binding protein is administered once weekly, by one-hour IV infusion. One cycle is 3 weeks. Starting dose of 1.3 ng/kg is established by minimally anticipated biological effect level. 44 patients have been dosed across 11 cohorts (ranging from 1.3 ng/kg to 120 ng/kg and one step dosing cohort).

Table 20: Baseline characteristics and demographics of Phase I patients, n=44.

Median (Range) 1 (0 - 3)

[0184] Table 20 shows that a heterogeneous population are being treated in dose escalation. The target population patients have a median of 7 prior systemic therapies, and a median of 2 prior novel hormonal therapies. 73% of patients had prior chemotherapy in metastatic setting. Additional prior therapies include sipuleucel-T, radium-223, A2AR inhibitor, olaparib, rucaparib, pembrolizumab, nivolumab, durvalumab, ipilimumab, listeria vaccine, Lul77/Ac225- PSMA-617, other investigational agents.

[0185] Table 21 shows that adverse events occurred in more than 10% of patients. Grades for the adverse events are determined according to Criteria for Adverse Events (CTCAE v5.0). All cytokine release syndrome (CRS) events were resolved and patients were successfully re treated. Transaminitis was observed primarily in the setting of CRS; abnormalities were transient, no clinical sequelae. Short-term premedication with steroids was effective in limiting CRS and allowing long-term treatment. One dose-limiting toxicity (DLT) was observed at 96 ng/kg, as a Grade 3 lipase increase. Treatment related serious adverse events (SAEs) includes: cytokine release syndrome (n=4), Aspartate Aminotransferase (AST) increase (n=2), Alanine Aminotransferas (ALT) increase (n=l), myalgia (n=l), infusion related reaction (IRR) (n=l), pneumonitis (n=l), seizure (n=l).

Table 21: Adverse events (CTCAE v5.0) in >10% of patients by grade, regardless of relationship.

[0186] Figure 29 depicts patients time on treatment from clinical database. Eleven of 28 patients (39%) with more than 18 weeks follow-up remained on study beyond week 18. 8 of 26 (31%) patients remained on study with more than 24 weeks. Of the 8 patients on study with more than 24 weeks, 7 patients (88%) remained on PSMA trispecific antigen-binding protein treatment longer than the time on their most recent prior systemic regimen (data not shown). 11 patients remain active. Patients discontinued study due to progressive disease (PD) (63%), death due to PD (9%), death due to unrelated AE (6%), unrelated AE (3%) or other (18%).

[0187] Figure 30 depicts each patient’s prostate specific antigen (PSA) values on PSMA trispecific antigen-binding protein treatment. Eight patients had PSA decreases from baseline ranging from -3.8% to -76%, including 2 patients with PSA decline more than 50% from baseline. One patient had baseline and subsequent PSA values of 5000ng/mL and is not shown in Figure 30.

[0188] PSMA trispecific antigen-binding protein was initiated at 1.3 ng/kg with no dexamethasone (Dex) premedication. Dose-dependent, transient increases in serum cytokine and chemokines were observed in early cohorts ( See Figure 33). Two patients who received 24 ng/kg of PSMA trispecific antigen-binding protein with no Dex premedication experienced Grade 3 CRS; these patients were subsequently administered Dex premedication weekly. Dex taper was implemented at Cohort 5 based on the observation that peripheral cytokines attenuated with each successive dose. In a 6-Week taper, Dex taper is administered once weekly prior to PSMA trispecific antigen-binding protein infusion for 2 cycles. In a 3-Week taper, Dex taper is administered once weekly prior to PSMA trispecific antigen-binding protein infusion for 1 cycle.

Table 22: Dexamethasone (Dex) Premedication.

[0189] Patient Profile: Patient 003, ongoing, a 69-year old male, was diagnosed in March 2013. Table 23: Patient 003 baseline characteristics

[0190] Patient 003 initiated PSMA trispecific antigen-binding protein treatment at 12ng/kg and escalated twice to 40 then 72 ng/kg. Patient 003 demonstrated early rise in PSA followed by a steady decline starting Week 12, currently -9% PSA decline from baseline, as shown in Figure 31A. A drop in LDH from 2361 to 241 U/L was observed, coinciding with PSA decline. Patient 003 remains on study after 78 weeks of treatment.

[0191] Patient Profile: Patient 024, ongoing, a 76-year old male, was diagnosed in December 2009.

Table 24: Patient 024 Baseline Characteristics

[0192] Patient 024 initiated PSMA trispecific antigen-binding protein at 54ng/kg with a 6-week dexamethasone taper. Patient 024 demonstrated early rise in PSA followed by a slight decline starting Week 15, as shown in Figure 31B. Patient 024 remains on study after 36 weeks of treatment.

[0193] Measurable Disease: 18 patients of 44 (41%) had measurable disease at baseline, including 10 patients with more than 1 post-treatment protocol scheduled disease assessment. In those 10 evaluable patients, sum of target lesions in 6 patients remained stable (-30% < % change < 20%) and 4 patients had disease progression (% change > 20%) as best response. [0194] Pharmacokinetics (PK) and Immunogenicity of PSMA trispecific antigen-binding protein treatment.

[0195] PSMA tri specific antigen-binding protein demonstrated dose proportional increase in Cmax and AUC with a geometric mean T1/2 of 24.9 hours, as shown in Figure 32. Median clearance (CL) and volume of distribution (V_ss) for PSMA trispecific antigen-binding protein in the given dose range of 1.3-96 ng/kg appear to be dose independent as shown in Table 25, indicative of linear kinetics. Of the 27 patients measured for anti-drug antibodies (AD As), one patient was ADA positive at baseline (neutralizing activity detected at C7D1 and beyond), two other patients developed ADA post-treatment (one was non-neutralizing, second was neutralizing at C4D1 and beyond).

Table 25: Median PK parameters for PSMA trispecific antigen-binding protein in the given dose range of 1.3-96 ng/kg

[0196] Figure 33 shows dose-dependent, transient increases in peripheral cytokine and chemokine levels were observed, peaking at 5 hours post infusion and returning to baseline 24 hours post-administration. Maximal cytokine/chemokine release attenuated with each successive dose over first two cycles. Transient cytokine increases can be effectively managed with short term dexamethasone premedication.

[0197] Reduction in circulating tumor cells (CTCs) was seen in 12 of 27 patients with available CTC counts collected on C1D1 predose (baseline) and C1D15 predose (after 2 doses of PSMA trispecific antigen-binding protein), as shown in Figure 34. Transient lymphocyte margination was observed post-PSMA trispecific antigen-binding protein infusion across cohorts.

[0198] Summary: (1) PSMA trispecific antigen-binding protein represents a novel half-life extended PSMA-targeting T cell engager that can be safely administered once weekly. (2) Phase I dose escalation comprises a heterogeneous, heavily pretreated population. (3) Evidence of half- life extension supports once weekly PSMA trispecific antigen-binding protein administration.

(4) Cytokine increases indicate T-cell activation and CTC reductions in a subset of patients support target engagement. (5) Adverse events have been transient, manageable and consistent with expected mechanism of action. (6) Early clinical signals have been observed, including 8 patients on treatment more than 24 weeks and PSA reductions in multiple patients. (7) Dose escalation is ongoing to identify dose for expansion phase.

Example 16: Proof-of-Concept Clinical Trial Phase Protocol for Administration of the PSMA

trispecific antigen-binding protein of Example 1 to Prostate Cancer Patients [0199] A subsequent phase II section will be treated at the MTD with a goal of determining if therapy with PSMA targeting trispecific proteins of the previous examples results in at least a 20% response rate.

[0200] Primary Outcome for the Phase II — To determine if therapy of PSMA targeting trispecific proteins of the previous examples results in at least 20% of patients achieving a clinical response (blast response, minor response, partial response, or complete response)

[0201] Eligibility: Histologically confirmed newly diagnosed aggressive prostate cancer according to the current World Health Organisation Classification, from 2001 to 2007 Any stage of disease.

Treatment with docetaxel and prednisone (+/- surgery).

Age > 18 years

Karnofsky performance status > 50% or ECOG performance status 0-2 Life expectancy > 6 weeks

Example 17: Additional characterization of PSMA HTS TriTAC Development of PSMA HTS TriTAC (SEQ ID NO: 147)

[0202] Protein production: Sequences of TriTAC molecules, such as a TriTAC molecule comprising the sequence of SEQ ID NO: 147 (PSMA HTS TriTAC), were cloned into a suitable mammalian expression and transfected into host cells, in some cases stable pools were generated, and were cultured in production media prior to purification. Conditioned media from host cell was filtered and purified by protein A affinity and desalted or subjected to preparative size exclusion chromatography using an AKTA Pure chromatography system (GE Healthcare). Protein A purified TriTAC proteins were further polished by ion exchange and formulated in a buffered solution containing excipients. Final purity was assessed by SDS-PAGE.

[0203] Affinity measurements: Affinity of the PSMA HTS TriTAC analytes for albumin, CD3e and tumor target ligands was measured by biolayer interferometry using an Octet RED96 instrument with anti-human Fc or streptavidin tips (ForteBio / Pall). Experiments were performed in the absence or presence of 15 mg/ml HSA.

[0204] Stability assessment: Purified PSMA HTS TriTAC molecule, comprising the sequence of SEQ ID NO: 147 was aseptically transferred to Type I glass vials at a concentration of 1 mg/ml, sealed and stressed by several freeze-thaws cycled from -80 °C to room temperature, or by incubation at 37 °C for 2 weeks, or by shaking for 72 hours. Stressed samples were compared to the same analysis of control non-stressed samples. Each was evaluated for concentration and turbidity by UV spectrometry. Samples were further evaluated by SDS-PAGE, capillary electrophoresis, melting temperature and aggregation measurements, and analytical SEC as described above.

[0205] Cell killing and T cell activation assays: T cell killing assays were performed as described previously (Nazarian AA, Archibeque IL, Nguyen YH, Wang P, Sinclair AM, Powers DA. Characterization of bispecific T-cell engager (BiTE®) Antibodies with a high-capacity T- cell dependent cellular cytotoxicity (TDCC) assay. J Biomol Screen. 2015;20:519-27). Tumor cell viability was measured using CellTiterGlo or by labeling cells with luciferase and measuring luciferase activity. T cell activation assays were set up using the same conditions as the T cell killing assays. Cytokines were measured using AlphaLISA kits (Perkin Elmer). CD69 and CD25 expression on T cells was measured by flow cytometry using anti-CD25 and anti- CD69 antibodies.

[0206] Pharmacokinetic studies in cynomolgus monkeys: TriTAC molecules were administered intravenously by single slow bolus. Serum samples were stored frozen at -80 °C until serum TriTAC levels were measured using an electrochemiluminescent ELISA assay. Pharmacokinetic analyses were performed using Phoenix WinNonlin Version 7.0 software (Certara, Princeton, NJ).

[0207] PSMA HTS TriTAC (SEQ ID NO: 147) was developed as a PSMA-targeting TriTAC for treatment of mCRPC in the T:A:C configuration (Figure 35). This exemplary TriTAC contains optimized anti-PSMA, anti-HSA, and anti-CD3 binding domains.

Table 26. Binding affinities of the PSMA HTS TriTAC to target proteins. Binding affinities for PSMA, CD3e, and albumin (ALB) from different species were measured by biolayer interferometry.

[0208] The TriTAC was characterized using multiple analytical methods to demonstrate its purity and stability. Under denaturing conditions, capillary electrophoresis showed that 99.7% of the TriTAC was present in the intact main peak and 0.3% was in the form of lower molecular weight species (Figure 36A and Table 27). SDS-PAGE showed a prominent band of the TriTAC at its predicted molecular weight of 52.5 kDa (Figure 14). Analytical size exclusion chromatography confirmed that the TriTAC had 98.1 % monomer content and revealed 1.9% of a higher molecular weight species consistent with the size of a non-covalent dimer (Figure 16 and Table 28). To test the TriTAC’s stability, the protein was subjected to different stress conditions, including multiple freeze/thaw cycles, shaking for 72 hours at room temperature, and storage at 37°C for two weeks. Under all conditions, the TriTAC maintained greater than 97% monomer content with no detectable increase in higher molecular weight species as measured by denaturing capillary electrophoresis and native analytical size exclusion chromatography. Full spectrum fluorescence and static light scattering were used to measure the melting and aggregation temperatures of the TriTAC before and after exposure to stress conditions (Figures 36B and 36C and Table 29). For all samples tested, the primary melting transition was between 55.2 and 55.5 °C and the exponential aggregation temperature was between 83.3 and 84.3 °C. In summary, these data suggest that PSMA HTS TriTAC is a stable and stress-resistant monomer that is not prone to aggregation and non-specific T cell activation.

Table 27. Capillary electrophoresis analysis of the PSMA HTS TriTAC samples from an accelerated stability study were analyzed by capillary electrophoresis to measure monomer content under non-reduced and reduced denaturing conditions.

Table 28. Analytical size exclusion chromatography of the PSMA HTS TriTAC samples from an accelerated stability study were analyzed by size exclusion chromatography to measure monomer content under non-denaturing conditions.

Table 29. Melting and aggregation temperatures of the PSMA HTS TriTAC samples from an accelerated stability study were measured by full spectrum fluorescence and static light scattering at two wavelengths, 266 nm and 473 nm. Small aggregates initiated at the observed Tm of 55°C, but exponential particle growth occurred only above 83°C for all samples.

Condition T_m (°C) T_agg 266 (°C)*

TO 55.3 83.7

5 XFT 55.5 83.4 37°C 2W 55.5 84.3 72h Shake 55.2 83.3

[0209] The PSMA HTS TriTAC was confirmed to target T cells to kill PSMA-expressing cancer cells in a TDCC assay. Titrations of PSMA HTS TriTAC were added to purified, resting human T cells from four different donors were co-cultured with LNCaP prostate cancer cells, and viability of the LNCaP cells was measured 48 hours later. The PSMA HTS TriTAC directed highly efficient killing of the LNCaP cells by T cells from all four donors at ECso values of 0.22 to 1.5 pM, whereas no lysis was observed with a TriTAC molecule targeting green fluorescent protein (GFP) (Figure 20). In parallel experiments performed as described above, potent directed lysis was also observed with these same donors and three additional prostate cancer cell lines, VCaP, MDAPCa2b, and 22Rvl, with ECso values between 0.16 and 4.8 pM (Table 4).

The only exception was the combination of T cells from Donor 24 with 22Rvl target cells, which did not result in efficient cell killing under these assay conditions but the expectation is that more efficient killing would have been observed if viability of the 22Rvl cells had been measure 72 or 96 hours after the start of the assay. The TDCC activity of PSMA HTS TriTAC was also measured in the presence or absence of HSA, and it was observed that the ECso for LNCaP cell killing was 0.7 pM in absence of HSA versus 1 pM in the presence of HSA (Figure 37). [0210] To confirm that the PSMA HTS TriTAC activates T cells in the presence of target cells, T cells and conditioned media were collected from TDCC assays performed with LNCaP as target cells. Using flow cytometry, a TriTAC concentration-dependent increase in expression levels of the activation markers CD69 and CD25 was observed on T cells from four different donors. The ECso values for induction of CD69 and CD25 expression (Tables 30 and 31) were similar to those observed for cell killing (Table 4). Comparable results were observed with VCaP, MDAPCa2b, or 22Rvl as target cells (Tables 30 and 31), whereas a TriTAC molecule targeting GFP failed to activate CD69 expression (Figures 21 and 38). A separate study was performed to confirm that HSA binding to the TriTAC does not result in target independent activation of T cells as assessed by measurement of CD69 and CD25 expression. Co-cultures of T cells and PSMA-expressing LNCaP cells or PSMA-negative NCI-H1563 cells were incubated with 1 nM of the PSMA HTS TriTAC or a control GFP-targeting TriTAC, the results shown in Figures 41A and 41B.

[0211] In the presence of HSA, increased CD69 and CD25 expression was only observed with the PSMA HTS TriTAC and LNCaP cells and not with the NCI-H1563 cells or with the GFP- targeting TriTAC (Figures 21 and 38). Another hallmark of T cell activation is cytokine production. TNFa and IFNy levels were measured in conditioned medium collected from TDCC assays with PSMA-expressing LNCaP cells or PSMA-negative HCT116 cells using T cells from two different donors. With T cells from both donors, the PSMA HTS TriTAC-dependent secretion of TNFa and IFNy was observed with PSMA-expressing LNCaP cells but not with PSMA-negative HCT116 cells (Figures 22 and 39). ECso values for cytokine production were slightly higher than those for surface expression of CD69 and CD25 (compare Tables 30, 31, 5, and 6).

[0212] Interestingly, although T cells from donor 24 did not efficiently killing 22Rvl under these assay conditions, induction of TNFa and IFNy production and of CD69 and CD25 expression was observed on par with T cells from the other donors and with other PSMA- expressing cell lines (Tables 30, 31, 5, and 6, and data not shown). These results indicate that the PSMA HTS TriTAC actives T cells from donor 24 in the presence of PSMA-expression 22Rvl cells, but this activation was not sufficient for cell killing in a 48 hour assay. T cell activation was dependent on both the PSMA binding domain in the PSMA HTS TriTAC and the expression of PSMA on target cells. Even with TriTAC concentrations exceeding EC so values by 10,000-fold, no T cell activation was seen with PSMA-negative HCT116 or NCI-H1563 cells (Tables 11, 30, 31, 5 and 6).

Table 30. Induction of CD69 expression on T cells by the PSMA HTS TriTAC. ECso values for the PSMA HTS TriTAC -directed induction of CD69 expression on by T cells from four donors in the presence of PSMA-expressing cells. No induction of CD69 expression was observed in the presence of two cell lines lacking PSMA expression.

Table 31. Induction of CD25 expression on T cells by the PSMA HTS TriTAC. ECso values for the PSMA HTS TriTAC -directed induction of CD25 expression on T cells from four donors in the presence of PSMA-expressing cells. No induction of CD25 expression was observed in the presence of two cell lines lacking PSMA expression.

PSMA CD25 ECso (pM)

Cell Line Expression Donor 24 Donor 8144 Donor 72 Donor 41

LNCaP Positive 0.7 0.2 0.4 0.6

MDAPCa2b Positive 0.2 0.2 0.1 0.2

VCaP Positive 1.5 1.2 0.9 0.8

22Rvl Positive 0.9 0.3 0.5 0.6

HCT116 Negative >1000 >1000 >1000 >1000

NCI- 1563 Negative >1000 >1000 >1000 >1000

Example 18: PSMA trispecific antigen-binding protein Phase dose escalation expansion

safety and pharmacokinetics study

[0213] This study was carried out to test the Phase I dose escalation, expansion, safety and pharmacokinetics of an exemplary PSMA trispecific antigen-binding protein in patients with mCRPC. This Example contains updated clinical trial data from Example 15.

[0214] Target population is patients with: metastatic castrate-resistant prostate cancer (mCRPC); disease progression on the prior systemic regimen; at least two prior systemic therapies approved for mCRPC; prior PSMA-targeting therapy allowed, but not required.

[0215] Trial Design: PSMA trispecific antigen-binding protein Phase I trial design is shown in Figure 42. Key objectives include characterization of safety, pharmacokinetics, and identification of dose for expansion phase. Tumor assessments are performed every 9 weeks and include conventional CT and bone scans and PSA. Additional assessments include cytokines, circulating tumor cells (CTC).

[0216] Dosing, Administration and Exposure: PSMA trispecific antigen-binding protein is administered once weekly, by one-hour IV infusion. One cycle is 3 weeks. Starting dose of 1.3 ng/kg is established by minimally anticipated biological effect level. 65 patients have been dosed across 12 cohorts ranging from 1.3 to 160ng/kg fixed dose. Fourteen of 54 (26%) patients with treatment start at least 6 months ago have remained on treatment beyond 24 weeks. Twelve of 52 (23%) patients with >1 post-baseline PSA level had PSA reductions from baseline, including 3 PSA50, 2 PSA30 responses. Nine of 43 (21%) with >1 post-baseline CTC level had CTCO response. Figure 43 shows an updated patients time on treatment.

[0217] Figure 44 shows an updated figure of the kinetics of patients with PSA reductions during treatment. PSA reductions post-baseline were observed across cohorts, with no dose response. Durable, sustained PSA declines were seen in patients at low doses.

Table 32 shows the relevant adverse events. The Grade 3+ CRS observed are transient and manageable. Also observed transient elevation of liver enzymes, which majority are in the setting of CRS and no clinical sequelae.

[0218] Patient Profile - patient 057: 75-year old male, diagnosed Jan. 2002.

Table 33 shows baseline characteristics of patient 057.

[0219] Patient 057 was administered an exemplary PSMA trispecific antigen-binding protein at 160 ng/kg. The result remonstrated partial response (-32%) at 1^st post baseline scan at week 9, confirmed PR (-43%) at week 18 (Figure 45), response maintained at Week 36, it showed 10% PSA decrease from baseline and remains on study after 20 weeks of treatment and remains on study after 41 weeks of treatment. Figure 45A shows partial response values of patient 057 during the course of treatment. Figure 45B shows patient 057’ s scans at pre-treatment, week 18 treatment and week 36 treatment.

[0220] Comparison between the PSMA trispecific antigen-binding protein of the present application and another antitumor drug (i.e., AMG160). AMG160 is moving into dose expansion at 0.3mg dose level. Based on the 0.3mg dosing cohort: 9 patients were dosed, 3 patients had PSA responses (33%) (1 PSA70, 1 PSA50, 1 PSA30), and 1 patient had unconfirmed PR (11%). In dose escalation overall, AMG160 showed 34% PSA50 response, 69% of pts had PSA reductions and 20% objective response. Figure 46 shows the patient time on treatment of AMG160. Table 34 shows the comparison of adverse events between PSMA trispecific antigen-binding protein of the present application and AMG160.

Example 19: PSMA trispecific antigen-binding protein Phase dose escalation expansion

safety and pharmacokinetics study

[0221] This study was carried out to test the Phase I dose escalation, expansion, safety and pharmacokinetics of an exemplary PSMA trispecific antigen-binding protein in patients with mCRPC. This Example contains updated clinical trial data from Example 18.

[0222] Target population is patients with: metastatic castrate-resistant prostate cancer (mCRPC); disease progression on the prior systemic regimen; at least two prior systemic therapies approved for mCRPC; prior PSMA-targeting therapy allowed, but not required.

[0223] Trial Design: PSMA trispecific antigen-binding protein Phase I trial design is shown in Figure 42. Key objectives include characterization of safety, pharmacokinetics, and identification of dose for expansion phase. Tumor assessments are performed every 9 weeks and include conventional CT and bone scans and PSA. Additional assessments include cytokines, circulating tumor cells (CTC).

[0224] Dosing, Administration and Exposure: PSMA trispecific antigen-binding protein is administered once weekly, by one-hour IV infusion. One cycle is 3 weeks. Starting dose of 1.3 ng/kg is established by minimally anticipated biological effect level. Figure 47 shows an updated patients time on treatment.

[0225] Figure 48 shows an updated figure of the kinetics of patients with PSA reductions during treatment. PSA reductions post-baseline were observed across cohorts, with no dose response. Durable, sustained PSA declines were seen in patients at low doses.

[0226] Reduction in circulating tumor cells (CTCs) was seen in multiple patients as shown in

Figure 49A and 49B.

[0227] Patient Profile - patient 054: 66-year old male, diagnosed Aug. 2014. Table 35 shows baseline characteristics of patient 054.

[0228] Patient 054 was administered an exemplary PSMA trispecific antigen-binding protein at 96ng/kg and escalated to 120 ng/kg in cycle 4. The result demonstrated a steady PSA decline over course of treatment, currently -60% PSA decline from baseline and stable disease per RECIST with 18% reduction in target lesions from baseline to week 45 scan (Figure 50).

Patient 054 remains on study after 45 weeks of treatment.

[0229] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of treating prostate cancer, the method comprising administration of an effective amount of a prostate specific membrane antigen (PSMA) targeting trispecific protein to a subject, wherein said protein comprises

(a) a first domain (A) which specifically binds to human CD3;

(b) a second domain (B) which is a half-life extension domain; and

(c) a third domain (C) which specifically binds to PSMA, wherein the domains are linked in the order H2N-(C)-(B)-(A)-COOH, or by linkers LI and L2, and wherein the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 10 mg/kg.

2. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 10 pg/kg.

3. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 1000 ng/kg.

4. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 500 ng/kg.

5. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 1 ng/kg to about 200 ng/kg.

6. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 1.3 ng/kg to about 160 ng/kg.

7. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 54 ng/kg.

8. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 72 ng/kg.

9. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 96 ng/kg.

10. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 120 ng/kg.

11. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 150 ng/kg.

12. The method of claim 1, wherein the PSMA targeting trispecific protein is administered at a dosage of about 160 ng/kg.

13. The method of any one of claims 1-12, wherein the PSMA targeting trispecific protein has an elimination half-time of at least about 20 hours.

14. The method of any one of claims 1-12, wherein the PSMA targeting trispecific protein has an elimination half-time of at least about 50 hours.

15. The method of any one of claims 1-12, wherein the PSMA targeting trispecific protein has an elimination half-time of about 100 hours.

16. The method of any one of claims 1-12, wherein the PSMA targeting trispecific protein is administered once a week.

17. The method of any one of claims 1-12, wherein the PSMA targeting trispecific protein is administered twice per week.

18. The method of any one of claims 1-12, wherein the PSMA targeting trispecific protein is administered every other week.

19. The method of any one of claims 1-12, wherein the PSMA targeting trispecific protein is administered every three weeks.

20. The method of any one of claims 1-12, wherein the subject's prostate surface antigen (PSA) level decreases from about 3.8% to about 76% compared to the baseline.

21. The method of any one of claims 1-12, wherein the subject's prostate surface antigen (PSA) level decreases over 50% compared to the baseline.

22. The method of any one of claims 1-12, further comprising administration of a dexamethasone (dex) premedication.

23. The method of claim 22, wherein the dex premedication is administered prior to administration of the PSMA targeting trispecific protein.

24. The method of claim 23, wherein the dex premedication is administered at a dosage of about 1 mg to about 20 mg.

25. The method of any one of claims 1-24, wherein the third domain comprises a scFv, a VH domain, a VL domain, a non-Ig domain, a ligand, a knottin, or a small molecule entity that specifically binds to PSMA.

26. The method of any one of claims 1-25, wherein the third domain comprises one or more sequences selected from the group consisting of SEQ ID NO: 113-140.

27. The method of any one of claims 1-25, wherein the first domain comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.

28. The method of any one of claims 1-25, wherein the first domain comprises one or more sequences selected from the group consisting of SEQ ID NO: 1-88.

29. The method of any one of claims 1-25, wherein the first domain is humanized or human.

30. The method of any one of claims 1-25, wherein the first domain has a KD of 150 nM or less for binding to CD3 on CD3 expressing cells.

31. The method of any one of claims 1-25, wherein the second domain binds human serum albumin.

32. The method of any one of claims 1-24, wherein the second domain comprises a scFv, a variable heavy domain (VH), a variable light domain (VL), a peptide, a ligand, or a small molecule.

33. The method of any one of claims 1-25, wherein the second domain comprises one or more sequences selected from the group consisting of SEQ ID NOs: 89-112.

34. The method of any one of claims 1-25, wherein linkers LI and L2 are each 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), (GGGGS)n (SEQ ID NO: 158) or GGGGSGGGS (SEQ ID NO: 170), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10

35. The method of any one of claims 1-25, wherein linkers LI and L2 are each independently GGGGSGGGS (SEQ ID NO: 170).

36. The method of any one of claims 1-25, wherein the domains are linked in the order EbN- (C)-Ll-(B)-L2-(A)-COOH.

37. The method of any one of claims 1-25, wherein the PSMA targeting trispecific protein is less than about 80 kDa.

38. The method of any one of claims 1-25, wherein the PSMA targeting trispecific protein is about 50 to about 75 kDa.

39. The method of any one of claims 1-25, wherein the PSMA targeting trispecific protein is less than about 60 kDa.

40. The method of any one of claims 1-25, wherein the PSMA targeting trispecific protein has increased tissue penetration as compared to an IgG to the same PSMA.

41. The method of any one of claims 1-25, wherein the PSMA targeting trispecific protein comprises a sequence selected from the group consisting of SEQ ID NO: 141-147.

42. The method of any one of claims 1-25, wherein the PSMA targeting trispecific protein comprises a sequence as set forth in SEQ ID NO: 147.

43. The method of any one of claims 1-25, wherein the PSMA targeting trispecific protein comprises a sequence selected from the group consisting of SEQ ID NO: 150-152.

44. The method of any one of claims 1-43, wherein the prostate cancer is a metastatic prostate cancer.

45. The method of any one of claims 1-44, wherein the prostate cancer is a castration resistant prostate cancer.