Use of a bispecific antigen-binding molecule that binds PSMA and CD3 in combination with 4-1BB costimulation

Abstract

Provided herein are methods for treating cancer using bispecific antigen-binding molecules that bind to prostate-specific membrane antigen (PSMA) and CD3. According to certain embodiments, antibodies useful herein bind to human PSMA with high affinity and induce human T-cell proliferation by binding to CD3. According to certain embodiments, bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds to human CD3 and a second antigen-binding domain that specifically binds to human PSMA are particularly useful herein. In certain embodiments, bispecific antigen-binding molecules in combination with anti-4-1BB agonists can inhibit the growth of PSMA-expressing prostate tumors. Bispecific antigen-binding molecules in combination with anti-4-1BB agonists are useful for treating diseases and disorders in which an upregulated or induced targeted immune response is desirable and / or therapeutically beneficial, for example, in the treatment of various cancers.

Description

[Technical Field]

[0001] The present invention relates to a bispecific antigen-binding molecule that binds to prostate-specific membrane antigen (PSMA) and CD3 in combination with 4-1BB costimulation, and methods of use thereof.

[0002] Sequence Listing Reference An official copy of the Sequence Listing has been submitted electronically via EFS-Web as an ASCII formatted Sequence Listing contemporaneously with the present specification, with the filename "10595WO01_SEQ_LIST_ST25," a creation date of June 19, 2020, and a size of approximately 4,096 bytes. The Sequence Listing contained in this ASCII format document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Prostate-specific membrane antigen (PSMA), also known as folate hydrolase 1 (FOLH1), is an endogenous decidual glycoprotein highly expressed in prostate epithelial cells and a cell surface marker for prostate cancer. Its expression persists in castration-resistant prostate cancer, which has a poor prognosis and limited treatment options. Methods for treating prostate cancer by targeting PSMA are being investigated. For example, yttrium-90 capromab is a radiotherapy containing a monoclonal antibody directed against an intracellular epitope of PSMA; J591, a monoclonal antibody directed against an extracellular epitope of PSMA, is part of the radiotherapy lutetium-177 J591; and MLN2704, in which maytansinoid 1 (DM1, an antimicrotubule agent) is conjugated to J591. These therapies are associated with toxicity. PSMA is also expressed within the neovasculature of other tumors, such as bladder, kidney, stomach, and colorectal cancer.

[0004] CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from the dimeric association of two of four distinct chains: epsilon, zeta, delta, and gamma. CD3 dimer configurations include gamma / epsilon, delta / epsilon, and zeta / zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby triggering T cell activation in a manner similar to TCR engagement by peptide-loaded MHC molecules. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes, including T cell activation. Furthermore, bispecific antibodies capable of binding to CD3 and a target antigen have been proposed for therapeutic uses, including targeting T cell immune responses to tissues and cells expressing the target antigen.

[0005] In T cell activation, costimulation via the TNF receptor superfamily is key to survival, acquisition of effector function, and memory differentiation. 4-1BB (Tnfrsf9), also known as CD137, is a member of the TNF receptor superfamily. Receptor expression is induced by lymphocyte activation after TCR-mediated priming, and its levels can be enhanced by CD28 costimulation. CD8 + Exposure to ligands or agonistic monoclonal antibodies (mAbs) on T cells costimulates 4-1BB, which contributes to T cell clonal expansion, survival, and development, induces peripheral monocyte proliferation, activates NF-kappaB, enhances TCR / CD3-induced activation-induced T cell apoptosis, memory generation, and regulates CD28 costimulation to promote Th1 cell responses. Summary of the Invention [Means for solving the problem]

[0006] Provided herein are methods for treating cancer in a subject. In some aspects, the method comprises administering to the subject a pharmaceutical composition comprising an anti-PSMA / anti-CD3 bispecific antigen-binding molecule or an anti-PSMA antibody and a pharmaceutically acceptable carrier or diluent, and further administering to the subject an anti-4-1BB agonist. In some aspects, the method comprises administering to the subject a pharmaceutical composition comprising an anti-PSMA / anti-CD3 bispecific antigen-binding molecule or an anti-PSMA antibody, an anti-4-1BB agonist, and a pharmaceutically acceptable carrier or diluent. In some embodiments, the cancer is selected from the group consisting of prostate cancer, kidney cancer, bladder cancer, colorectal cancer, and gastric cancer. In some cases, the cancer is prostate cancer. In some cases, the prostate cancer is castration-resistant prostate cancer.

[0007] Further provided herein are methods for treating cancer or inhibiting tumor growth. In some embodiments, the methods comprise administering to a subject in need thereof a therapeutically effective amount of each of (a) an anti-PSMA antibody or antigen-binding fragment thereof, or an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, and (b) an anti-4-1BB agonist.

[0008] Also provided herein is a therapeutic method for targeting / killing tumor cells expressing PSMA. In some embodiments, the therapeutic method comprises administering a therapeutically effective amount of an anti-CD3 / anti-PSMA bispecific antigen-binding molecule or an anti-PSMA antibody and a therapeutically effective amount of an anti-4-1BB agonist to a subject in need thereof. In some embodiments, the anti-CD3 / anti-PSMA bispecific antigen-binding molecule or anti-PSMA antibody and the anti-4-1BB agonist are formulated separately. In some embodiments, the anti-CD3 / anti-PSMA bispecific antigen-binding molecule or anti-PSMA antibody and the anti-4-1BB agonist are formulated in the same pharmaceutical composition.

[0009] Also provided herein is the use of an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, or an anti-PSMA antibody, and an anti-4-1BB agonist in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by PSMA-expressing cells.

[0010] Administering an anti-PSMA antibody or antigen-binding fragment thereof, or an anti-PSMA / anti-CD3 bispecific antibody, in combination with an anti-4-1BB agonist to a subject in need thereof can reduce tumor volume compared to treatment in the absence of the anti-4-1BB agonist.

[0011] Administering an anti-PSMA antibody or antigen-binding fragment thereof, or an anti-PSMA / anti-CD3 bispecific antibody in combination with an anti-4-1BB agonist to a subject in need thereof can increase tumor-free survival compared to treatment in the absence of the anti-4-1BB agonist.

[0012] Administering an anti-PSMA antibody or antigen-binding fragment thereof, or an anti-PSMA / anti-CD3 bispecific antibody, in combination with an anti-4-1BB agonist to a subject in need thereof can increase TRAF1 expression in tumors by at least about four-fold compared to TRAF1 expression in tumors of subjects administered the anti-CD3 / anti-PSMA bispecific antigen-binding molecule in the absence of the anti-4-1BB agonist.

[0013] Administering an anti-PSMA antibody or antigen-binding fragment thereof, or an anti-PSMA / anti-CD3 bispecific antibody, in combination with an anti-4-1BB agonist to a subject in need thereof can increase Bcl2 expression in tumors by at least about two-fold compared to Bcl2 expression in tumors of subjects administered the anti-CD3 / anti-PSMA bispecific antigen-binding molecule in the absence of the anti-4-1BB agonist.

[0014] Administering an anti-PSMA antibody or antigen-binding fragment thereof, or an anti-PSMA / anti-CD3 bispecific antibody, in combination with an anti-4-1BB agonist to a subject in need thereof can increase BFL-1 expression in tumors by at least about three-fold compared to BFL-1 expression in tumors of subjects administered the anti-CD3 / anti-PSMA bispecific antigen-binding molecule in the absence of the anti-4-1BB agonist.

[0015] Administering an anti-PSMA antibody or antigen-binding fragment thereof, or an anti-PSMA / anti-CD3 bispecific antibody, in combination with an anti-4-1BB agonist to a subject in need thereof can enhance CD8+ T cell proliferation and / or increased survival of CD8+ T cells in the tumor compared to CD8+ T cells in the tumor of a subject administered the anti-CD3 / anti-PSMA bispecific antigen-binding molecule in the absence of the anti-4-1BB agonist.

[0016] The anti-4-1BB agonist may be a small molecule or biological agonist of 4-1BB, and in some embodiments is an antibody. Exemplary anti-4-1BB agonists include, for example, commercially available antibodies such as anti-mouse 4-1BB, and therapeutic antibodies such as urelumab and utomilumab.

[0017] According to the methods provided herein, anti-PSMA antibodies or their antigen-binding fragments, and bispecific antibodies and their antigen-binding fragments that bind to human PSMA and human CD3 are useful. Bispecific antibodies are particularly useful for targeting CD3-expressing T cells and stimulating T cell activation, for example, in situations where T cell-mediated killing of PSMA-expressing cells is beneficial or desirable. For example, bispecific antibodies can direct CD3-mediated T cell activation to specific PSMA-expressing cells, such as prostate tumor cells.

[0018] Anti-PSMA antibodies or antigen-binding fragments thereof that bind to PSMA are useful in combination with anti-4-1BB agonists to treat diseases and disorders associated with or caused by tumors that express PSMA, particularly larger and / or more difficult-to-treat tumors. Exemplary anti-PSMA antibodies and antigen-binding fragments thereof are described in detail in U.S. Patent No. 10,179,819. In some embodiments, the anti-PSMA antibody comprises the HCVR of SEQ ID NO: 66 and the common light chain of SEQ ID NO: 1386, as referred to in U.S. Patent No. 10,179,819. In some embodiments, the anti-PSMA antibody is the H1H11810P antibody referred to in U.S. Patent No. 10,179,819.

[0019] Bispecific antigen-binding molecules (e.g., antibodies) that bind to PSMA and CD3 are also referred to herein as "anti-PSMA / anti-CD3 bispecific molecules," "anti-CD3 / anti-PSMA bispecific molecules," "PSMAxCD3 bsAbs," or simply "PSMAxCD3." The anti-PSMA portion of the anti-PSMA / anti-CD3 bispecific molecule is useful for targeting cells (e.g., tumor cells) that express PSMA (e.g., prostate tumors), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. Simultaneous binding of PSMA on tumor cells and CD3 on T cells promotes directed killing (cytolysis) of the targeted tumor cells by activated T cells. Thus, the anti-PSMA / anti-CD3 bispecific molecules useful herein are particularly useful for treating diseases and disorders associated with or caused by PSMA-expressing tumors (e.g., prostate cancer). The anti-PSMA / anti-CD3 bispecific molecules are also useful in combination with anti-4-1BB agonists to treat diseases and disorders associated with or caused by PSMA-expressing tumors, particularly larger and / or more difficult to treat tumors.

[0020] The bispecific antigen-binding molecule comprises a first antigen-binding domain that specifically binds to human CD3 and a second antigen-binding domain that specifically binds to PSMA.

[0021] An example of a bispecific antibody useful according to the methods provided herein is an anti-CD3 / anti-PSMA bispecific molecule, in which the first antigen-binding domain that specifically binds to CD3 comprises any of the HCVR amino acid sequences, any of the LCVR amino acid sequences, any of the HCVR / LCVR amino acid sequence pairs, any of the heavy chain CDR1-CDR2-CDR3 amino acid sequences, or any of the light chain CDR1-CDR2-CDR3 amino acid sequences set forth in U.S. Publication No. 2014 / 0088295.

[0022] Useful according to the methods provided herein are anti-CD3 / anti-PSMA bispecific antigen-binding molecules, wherein a first antigen-binding domain that specifically binds to CD3 comprises any of the HCVR amino acid sequences and / or any of the LCVR amino acid sequences, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, as set forth in Tables 12, 14, 15, 18, and 20 of U.S. Patent No. 10,179,819. In some embodiments, the first antigen-binding domain that specifically binds to CD3 comprises the heavy chain variable region (HCVR-1) amino acid sequence of SEQ ID NO:2.

[0023] Useful according to the methods provided herein are anti-CD3 / anti-PSMA bispecific molecules, wherein the second antigen-binding domain that specifically binds to PSMA comprises any of the HCVR amino acid sequences and / or any of the LCVR amino acid sequences, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, as set forth in Table 1 of U.S. Patent No. 10,179,819. In some embodiments, the second antigen-binding domain that specifically binds to PSMA comprises the heavy chain variable region (HCVR-2) amino acid sequence of SEQ ID NO:1.

[0024] Useful according to the methods provided herein are anti-CD3 / anti-PSMA bispecific molecules, wherein a first antigen-binding domain that specifically binds CD3 comprises the HCVR-1 amino acid sequence of SEQ ID NO: 2, and a second antigen-binding domain that specifically binds PSMA comprises the HCVR-2 amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-CD3 / anti-PSMA bispecific molecule comprises a common light chain variable region (LCVR) amino acid sequence of SEQ ID NO: 3.

[0025] In one embodiment, provided herein is a pharmaceutical composition comprising an anti-PSMA antigen binding molecule or an anti-PSMA / anti-CD3 bispecific antigen binding molecule and a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition further comprises an anti-4-1BB agonist.

[0026] According to the method of the present disclosure, anti-PSMA antibodies and their antigen-binding fragments, as well as anti-CD3 / anti-PSMA bispecific antigen-binding molecules, with modified glycosylation patterns are useful. In some applications, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function, modifications to remove undesired glycosylation sites or antibodies lacking fucose moieties present on oligosaccharide chains may be useful (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cytotoxicity (CDC).

[0027] In one aspect, the present disclosure provides a pharmaceutical composition comprising an anti-PSMA antibody or antigen-binding fragment thereof or an anti-CD3 / anti-PSMA bispecific antigen-binding molecule disclosed herein, an anti-4-1BB agonist, and a pharmaceutically acceptable carrier. In a related aspect, the present disclosure features a composition that is a combination of an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, an anti-4-1BB agonist, and a third therapeutic agent. In one embodiment, the third therapeutic agent is any agent that can be advantageously combined with the anti-CD3 / anti-PSMA bispecific antigen-binding molecule. Exemplary agents that can be advantageously combined with the anti-CD3 / anti-PSMA bispecific antigen-binding molecule are described in detail elsewhere herein.

[0028] In another aspect, provided herein are radiolabeled anti-PSMA antibody conjugates and anti-CD3 / anti-PSMA bispecific antigen-binding molecule conjugates for use in immunoPET imaging. The conjugates comprise an anti-PSMA antibody or anti-CD3 / anti-PSMA bispecific antigen-binding molecule, a chelating moiety, and a positron emitter.

[0029] Provided herein are processes for synthesizing the conjugates and synthetic intermediates useful therefor.

[0030] Provided herein are methods for imaging PSMA-expressing tissues, the methods comprising administering to the tissue a radiolabeled anti-PSMA antibody conjugate or an anti-CD3 / anti-PSMA bispecific antigen-binding molecule conjugate described herein, and visualizing PSMA expression by positron emission tomography (PET) imaging.

[0031] Provided herein are methods for imaging tissues containing PSMA-expressing cells, the methods comprising administering to the tissue a radiolabeled anti-PSMA antibody conjugate or an anti-CD3 / anti-PSMA bispecific antigen-binding molecule conjugate described herein, and visualizing PSMA expression by PET imaging.

[0032] Provided herein is a method for detecting PSMA in a tissue, the method comprising administering a radiolabeled anti-PSMA antibody conjugate or anti-CD3 / anti-PSMA bispecific antigen-binding molecule conjugate described herein to the tissue and visualizing PSMA expression by PET imaging. In one embodiment, the tissue is present in a human subject. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject has a disease or disorder, such as cancer, an inflammatory disease, or an infection.

[0033] Provided herein are methods for detecting PSMA in tissue, the methods comprising contacting the tissue with an anti-PSMA antibody or anti-CD3 / anti-PSMA bispecific antigen-binding molecule conjugated to a fluorescent molecule as described herein, and visualizing PSMA expression by fluorescence imaging.

[0034] Provided herein is a method for identifying a subject suitable for anti-tumor therapy, the method comprising selecting a subject having a solid tumor, administering a radiolabeled anti-PSMA antibody conjugate or an anti-CD3 / anti-PSMA bispecific antigen-binding molecule conjugate described herein, and visualizing the administered radiolabeled antibody conjugate in the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor identifies the subject as suitable for anti-tumor therapy.

[0035] Provided herein are methods for treating tumors, including selecting a subject with a solid tumor, determining that the solid tumor is PSMA-positive, and administering anti-tumor therapy to the subject in need thereof. In certain embodiments, the anti-tumor therapy includes an inhibitor of the PD-1 / PD-L1 signaling axis (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody), an example of which is checkpoint inhibitor therapy. In certain embodiments, the subject is administered a radiolabeled anti-PSMA antibody conjugate or an anti-CD3 / anti-PSMA bispecific antigen-binding molecule conjugate described herein, and the localization of the radiolabeled antibody conjugate is imaged by positron emission tomography (PET) imaging to determine whether the tumor is PSMA-positive. In certain embodiments, the subject is further administered a radiolabeled anti-PD-1 antibody conjugate, and the localization of the radiolabeled antibody conjugate is imaged by positron emission tomography (PET) imaging to determine whether the tumor is PD-1-positive.

[0036] Provided herein is a method for monitoring the effectiveness of an anti-tumor therapy in a subject, the method comprising: selecting a subject having a solid tumor, wherein the subject is being treated with an anti-tumor therapy; administering to the subject a radiolabeled anti-PSMA antibody conjugate or an anti-CD3 / anti-PSMA bispecific antigen-binding molecule conjugate described herein; imaging the localization of the administered radiolabeled conjugate in the tumor by PET imaging; and determining tumor growth, wherein a decrease from baseline in uptake of the conjugate or radiolabeled signal indicates the effectiveness of the anti-tumor therapy.

[0037] In certain embodiments, the anti-tumor therapy includes a PD-1 inhibitor (e.g., REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab), a PD-1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, and those disclosed in Patent Publication No. US2015-0203580), a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor (e.g., ipilim ... PD-1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, and those disclosed in Patent Publication No. US2015-0203580), a PD-1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, and those disclosed in Patent Publication No. US2015-0203580), a PD-1 inhibitor (e.g., atezolizumab, a inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, GITR inhibitors, antagonists of another T cell co-inhibitor or ligand (e.g., antibodies to LAG3, CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., aflibercept or U.S. Patent No. 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab) or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesbacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guérin, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3xCD20 bispecific antibodies, or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, and antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC). ADC)

[0038] Provided herein is a method for increasing the proliferation of CD8+ T cells in tumor tissue.In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of each of (a) an anti-CD3 / anti-PSMA bispecific antigen binding molecule and (b) an anti-4-1BB agonist.

[0039] Provided herein is a method for inducing and / or enhancing a T cell response against a tumor. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of each of (a) an anti-CD3 / anti-PSMA bispecific antigen binding molecule and (b) an anti-4-1BB agonist.

[0040] In some embodiments, the ratio of CD8+ T cells to Tregs is increased in tumor tissue compared to the ratio of CD8+ T cells to Tregs in tumor tissue of a subject administered the anti-CD3 / anti-PSMA bispecific antigen binding molecule in the absence of an anti-4-1BB agonist. In some embodiments, subsequent exposure to tumor cells induces a memory response in subjects treated with the anti-CD3 / anti-PSMA bispecific antigen binding molecule in the presence of an anti-4-1BB agonist.

[0041] Other embodiments will be apparent from consideration of the detailed description that follows. [Brief explanation of the drawings]

[0042] [Figure 1-1] Figures 1A-1G show that the PSMAxCD3 bispecific antibody can bind to both low- and high-antigen-expressing cell lines, demonstrating that the PSMAxCD3 bispecific antibody induces target-dependent CD3-mediated T cell activation, resulting in killing of PSMA-expressing tumor cells. Data shown are from duplicate wells and are representative of three independent experiments. [Figure 1-2] Same as above.

[0043] [Figure 2]Figures 2A-2B show growth inhibition of human prostate cancer cells in a xenogeneic tumor model as a result of treatment with a PSMAxCD3 bispecific antibody. In Figure 2A, NSG mice were co-implanted subcutaneously with 22Rv1 cells and human PBMCs. Mice were administered 0.1 or 1 mg / kg of PSMAxCD3, or 1 mg / kg of a CD3-binding control on days 0, 3, and 7. In Figure 2B, NSG mice were co-implanted subcutaneously with C4-2 cells and human PBMCs. Mice were administered 0.01 or 0.1 mg / kg of PSMAxCD3, or 0.1 mg / kg of a CD3-binding control on days 0, 3, and 7. Mean tumor volumes are shown as SEM (n = 5, 3 replicates). ****P < 0.0001. Statistical significance compared to the CD3-binding control is determined by two-way ANOVA.

[0044] [Figure 3-1] Figures 3A-3D show PSMA expression and accumulation of PSMAxCD3 bispecific antibodies in PSMA-expressing tissues of HuT mice and drug clearance. Figure 3A shows relative PSMA expression in tissues of HuT mice by RT-PCR. Figures 3B and 3C show ex vivo tissue biodistribution measured on day 6, expressed as percent injected dose per gram of tissue (%ID / g) and tissue-to-blood ratio. Data are presented as mean + SD. Figure 3D shows PSMAxCD3 drug clearance over time measured in mice treated with 1 mg / kg PSMAxCD3. [Figure 3-2] Same as above.

[0045] [Figure 4]Figures 4A-4C show that PSMAxCD3 bispecific antibody treatment prevented or delayed tumor growth in HuT mice implanted with a murine prostate adenocarcinoma cell line expressing human PSMA in tumors less than 200 mm3. A short-term but transient antitumor response was observed in larger tumors. In Figure 4A, mice were treated with 5 mg / kg of CD3-binding control (circles) or PSMAxCD3 (squares) on days 0, 4, 7, and 11. Five of five mice were tumor-free. Mean tumor volumes are shown as SEM (n = 7, 3 replicates). ****P < 0.0001. In Figure 4B, 50 mm3 tumors were treated with 5 mg / kg of CD3-binding control (circles) or PSMAxCD3 (squares) on days 8, 12, 15, and 19. Two of five mice were tumor-free. Mean tumor volumes are shown as SEM (n = 5, 3 replicates). ****P<0.0001. In Figure 4C, 200 mm tumors were treated with 5 mg / kg of CD3-conjugated control (circles) or PSMAxCD3 (squares) on days 9, 12, 16, and 19. 0 / 5 mice were tumor-free. Mean tumor volumes are shown as SEM (n=5, 3 replicates). **P=0.0014.

[0046] [Figure 5-1]Figures 5A-5D show the results of PSMAxCD3 bispecific antibody treatment of HuT mice bearing two tumors of different sizes on opposite flanks. The data indicate that the bispecific antibody targets tumors regardless of size, but efficacy is limited to smaller tumors. Figure 5A shows the mean volume of small tumors, shown as SEM (n = 5, 3 replicates). ***P < 0.001. Figure 5B shows the mean volume of large tumors, shown as SEM (n = 5, 3 replicates). *P = 0.01. All statistical significance is determined by two-way ANOVA compared to the CD3-conjugated control. Figures 5C and 5D show the ex vivo tissue biodistribution measured 6 days after administration of 1 mg / kg of 89Zr-PSMAxCD3 or 89Zr-CD3-conjugated control to mice bearing small and large tumors, and are expressed as the percentage of injected dose per gram of tissue (%ID / g) and tissue-to-blood ratio. Data are shown as mean + SD. [Figure 5-2] Same as above.

[0047] [Figure 6-1]Figures 6A-6D show the antitumor efficacy of PSMAxCD3 bispecific antibodies with anti-4-1BB costimulation in large TRAMP-C2hPMSA tumors (200 mm). Figure 6A shows representative flow plots and MFI of 4-1BB expression on CD4 and CD8 T cells in tumors and spleens 48 hours after administration of 5 mg / kg of CD3-binding control or PSMAxCD3 (n=5). Figure 6B shows established 200 mm TRAMP-C2-hPMSA tumors were treated once on day 9 with 5 mg / kg CD3-binding control (open circles), 2.5 mg / kg anti-4-1BB (filled circles), 1 mg / kg PSMAxCD3 (open triangles), 5 mg / kg PSMAxCD3 (filled triangles), 1 mg / kg PSMA + 2.5 mg / kg anti-4-1BB (open squares), or 5 mg / kg PSMAxCD3 + 2.5 mg / kg anti-4-1BB (filled squares). Mean tumor volumes are shown as SEM (n = 10, 3 replicates). ****P < 0.0001. Statistical significance was determined by two-way ANOVA compared to the CD3-binding control. Figure 6C provides tumor-free survival curves showing euthanasia of mice bearing tumors greater than 2000 mm. Significance is determined by the Gehan-Breslow-Wilcoxon test compared to the CD3-binding control. ****P<0.0001. The number of tumor-free (TF) mice is 0 / 10 for the CD3-binding control, 0 / 10 for 5 mg / kg PSMAxCD3, 1 / 10 for 1 mg / kg PSMAxCD3, 2 / 10 for the anti-4-1BB control, 6 / 10 for 1 mg / kg PSMA + 2.5 mg / kg anti-4-1BB, and 5 / 10 for 5 mg / kg PSMAxCD3 + 2.5 mg / kg anti-4-1BB. Figure 6D provides the relative expression of 4-1BB pathway genes in tumors 72 hours after treatment administration. (n=6) ****P<0.0001, ***P<0.009, *P<0.05. Statistical significance is determined by one-way ANOVA. [Figure 6-2] Same as above.

[0048] [Figure 7]Figures 7A-7B show the expansion of CD8 T cells in tumors and immunological memory after combination therapy with PSMAxCD3 and anti-4-1BB. Figure 7B shows that mice with removed 50 mm3 tumors were protected from secondary tumors when rechallenged with TRAMP-C2-hPSMA tumor cells, demonstrating that tumor-specific immunological memory can be induced with a CD3 bispecific antibody. DETAILED DESCRIPTION OF THE INVENTION

[0049] Before the present invention is described, it is to be understood that this invention is not limited to the particular methods and experimental conditions described herein, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.As used herein, the term "about" when used in relation to a specific referenced numerical value means that the value can vary from the referenced value by 1% or less.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0051] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are now described. All patents, patent applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0052] As shown in the Examples, antitumor efficacy of PSMAxCD3 was observed against small tumors, but antitumor efficacy was significantly reduced against larger tumors, more realistically reflecting the challenges of treating solid tumors in the clinic.

[0053] As shown below, the PSMAxCD3 bispecific antibody resulted in CD8 T cell infiltration, activation, and proliferation, which was effective in smaller tumors but not in larger tumors. The inventors sought to enhance and prolong PSMAxCD3-induced T cell activity by providing a costimulatory signal using an anti-4-1BB agonist. The 4-1BB signaling pathway can enhance the magnitude and duration of T cell responses by promoting T cell survival, reversing T cell anergy, and subsequently generating memory T cells to promote potent antitumor activity.

[0054] As shown herein, combining a PSMAxCD3 bispecific antibody with anti-4-1BB costimulation enhances CD8 T cell infiltration, activation, and proliferation, resulting in significant antitumor efficacy in larger tumors after a single administration. This combination can also induce tumor-specific T cell memory.

[0055] The ability of anti-PSMA antibodies and PSMAxCD3 bispecific antibodies to activate intratumoral T cells and the ability of 4-1BB costimulation to enhance the magnitude and duration of T cell responses, resulting in significant anti-tumor efficacy, is demonstrated herein. Combining anti-PSMA antibodies and PSMAxCD3 bispecific antibodies with 4-1BB costimulation is useful in methods of treating established solid tumors to achieve improved overall survival.

[0056] Therapeutic Uses of Antigen-Binding Molecules The present disclosure includes methods comprising administering to a subject in need thereof an anti-PSMA antibody or antigen-binding fragment thereof, or a bispecific antigen-binding molecule that specifically binds CD3 and PSMA, together with an anti-4-1BB agonist. Therapeutic compositions useful according to the methods herein may comprise an anti-PSMA antibody or a PSMAxCD3 bispecific antigen-binding molecule and a pharmaceutically acceptable carrier or diluent. As used herein, the phrase "subject in need thereof" refers to a human or non-human animal that exhibits one or more symptoms or signs of cancer (e.g., a subject that develops a tumor or suffers from any of the cancers described below), or a subject that would otherwise benefit from inhibition or reduction of PSMA activity or depletion of PSMA+ cells (e.g., prostate cancer cells).

[0057] The antibodies and bispecific antigen-binding molecules disclosed herein (and therapeutic compositions comprising them) are particularly useful in combination with anti-4-1BB agonists to treat any disease or disorder in which stimulating, activating, and / or targeting the immune response is beneficial. In particular, anti-PSMA antibodies and anti-CD3 / anti-PSMA bispecific antigen-binding molecules, in combination with anti-4-1BB agonists, can be used to treat, prevent, and / or ameliorate any disease or disorder associated with or mediated by PSMA expression or activity or the proliferation of PSMA+ cells. The mechanisms of action achieved by the therapeutic methods disclosed herein include killing PSMA-expressing cells in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. PSMA-expressing cells that can be inhibited or killed using antibodies or bispecific antigen-binding molecules include, for example, prostate tumor cells. Furthermore, 4-1BB costimulation achieves therapeutic effects such as enhancing T cell clonal expansion, survival, and development, inducing proliferation in peripheral monocytes, activating NF-kappaB, enhancing T cell apoptosis induced by TCR / CD3-induced activation, and contributing to memory generation.

[0058] Antigen-binding molecules, including anti-PSMA antibodies and anti-PSMA / anti-CD3 bispecific antibodies, can be used in combination with anti-4-1BB agonists to treat primary and / or metastatic tumors arising, for example, in the gastrointestinal tract, prostate, kidney, and / or bladder. In certain embodiments, the antibodies or bispecific antigen-binding molecules are used to treat one or more of the following cancers: renal clear cell carcinoma, chromophobe renal cell carcinoma, (renal) oncocytoma, (renal) transitional cell carcinoma, prostate cancer, colorectal cancer, gastric cancer, urothelial carcinoma, (bladder) adenocarcinoma, or (bladder) small cell carcinoma. According to certain embodiments of the present disclosure, anti-PSMA antibodies and anti-PSMA / anti-CD3 bispecific antibodies, when combined with anti-4-1BB agonists, are useful for treating patients with castration-resistant prostate cancer. According to other related embodiments disclosed herein, there is provided a method comprising administering an anti-CD3 / anti-PSMA bispecific antigen binding molecule in combination with an anti-4-1BB agonist to a patient suffering from castration-resistant prostate cancer.

[0059] The present disclosure also includes methods for treating an established tumor in a subject, where established is defined as a measurable tumor, i.e., a tumor that can be measured by a method appropriate for the given cancer.

[0060] The present disclosure also includes methods for treating residual cancer in a subject. As used herein, the term "residual cancer" refers to the presence or persistence of one or more cancer cells in a subject after treatment with an anti-cancer therapy.

[0061] According to certain aspects, the present disclosure provides methods for treating a disease or disorder associated with PSMA expression (e.g., prostate cancer), comprising administering to a subject one or more bispecific antigen-binding molecules described elsewhere in combination with an anti-4-1BB agonist after the subject has been determined to have prostate cancer (e.g., castration-resistant prostate cancer). For example, the present disclosure includes methods for treating prostate cancer, comprising administering to a patient an anti-CD3 / anti-PSMA bispecific antigen-binding molecule 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the subject has received hormone therapy (e.g., antiandrogen therapy).

[0062] Definition of Terms As used herein, the term "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR) and consisting of a homodimer or heterodimer formed from the association of two of the four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment unless explicitly specified as being from a non-human species. Thus, the term "CD3" refers to human CD3 unless specified as being from a non-human species, e.g., "mouse CD3," "monkey CD3," etc.

[0063] As used herein, "antibodies that bind CD3" or "anti-CD3 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). Antibodies and antigen-binding fragments useful herein may bind to soluble CD3 and / or cell surface-expressed CD3. Soluble CD3 includes native CD3 protein as well as recombinant CD3 protein variants that lack the transmembrane domain or are otherwise not associated with the cell membrane, such as, for example, monomeric and dimeric CD3 constructs.

[0064] As used herein, the term "cell surface-expressed CD3" refers to one or more CD3 proteins expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the CD3 protein is exposed to the extracellular side of the cell membrane and accessible to the antigen-binding portion of an antibody. "Cell surface-expressed CD3" includes a CD3 protein contained in association with a functional T cell receptor on the cell membrane. The term "cell surface-expressed CD3" also includes a CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The term "cell surface-expressed CD3" also includes a CD3 chain expressed by itself, without other CD3 chain types, on the surface of a cell (e.g., CD3-epsilon, CD3-delta, or CD3-gamma). "Cell surface-expressed CD3" can include or consist of a CD3 protein expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, "cell surface-expressed CD3" can include or consist of CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0065] As used herein, the term "PSMA" refers to prostate-specific membrane antigen, also known as folate hydrolase 1 (FOLH1). PSMA is an endogenous non-decidual glycoprotein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. PSMA is an attractive cell surface target for late-stage malignant diseases. It is also expressed in the neovasculature of renal clear cell carcinoma, bladder cancer, colon cancer, and breast cancer.

[0066] As used herein, the term "4-1BB," also known as CD137, refers to a costimulatory molecule induced by activation. 4-1BB is a key regulator of immune responses and a member of the TNF receptor superfamily. The term "anti-4-1BB agonist" refers to any ligand that binds to 4-1BB and activates the receptor. Exemplary anti-4-1BB agonists include urelumab (BMS-663513) and utomilumab (PF-05082566), as well as commercially available anti-mouse 4-1BB antibodies. Furthermore, the term "4-1BB agonist" refers to any molecule that partially or fully promotes, induces, increases, and / or activates the biological activity of 4-1BB. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, such as bispecific antibodies that contain one arm that binds to 4-1BB on immune cells and another arm that binds to an antigen on, for example, a tumor target. The term also encompasses fragments or amino acid sequence variants of natural polypeptides, peptides, antisense oligonucleotides, small organic molecules, and the like. In some embodiments, activation in the presence of an agonist is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% higher than the signal measured in a negative control under comparable conditions. The effectiveness of an agonist can also be determined using functional assays, such as the ability of an agonist to activate or promote the function of a polypeptide. For example, a functional assay may involve contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. The potency of an agonist is typically measured by its EC 50The EC value is defined as the concentration required to activate 50% of the agonist response. 50 The lower the value, the more potent the agonist is, and the lower the concentration required to activate the maximum biological response.4-1BB agonists can also include molecules containing 4-1BB-ligand or fragments of 4-1BB-ligand, for example, bispecific molecules, including one arm containing 4-1BBL or its fragment, and the other arm binding to, for example, an antigen on a tumor.These fragments can also include Fc region.

[0067] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0068] The term "antibody," as used herein, refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., PSMA or CD3). The term "antibody" encompasses immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (heavy chain variable region, herein referred to as HCVR or VCR). H The heavy chain constant region comprises a C H 1. C H 2, and C H Each light chain comprises three domains: a light chain variable region (LCVR or VVR) and a light chain variable region (VVVV). L The light chain constant region comprises one domain (C L 1) V H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments disclosed herein, the FRs of an anti-PSMA antibody or anti-CD3 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0069] As used herein, the term "antibody" also includes antigen-binding fragments of a complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from complete antibody molecules using any suitable standard method, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0070] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0071] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally comprises at least one CDR adjacent to, or in frame with, one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, V H Domains and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region can be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0072] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody useful herein include: (i) a V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L、 (viii)V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies useful herein may be linked to each other and / or to one or more monomeric V H Or V L The domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association (e.g., via disulfide bonds).

[0073] Like intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in connection with the antigen-binding fragments of antibodies useful herein using routine techniques available in the art.

[0074] The antibodies useful herein may function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies disclosed herein in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, thereby resulting in lysis of the target cells. CDC and ADCC can be measured using assays known and available in the art. (See, e.g., U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656.) The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0075] In certain embodiments, the anti-PSMA / anti-CD3 bispecific antibody useful herein is a human antibody. The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences, for example, in the CDRs, particularly in CDR3 (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0076] Antibodies useful in the methods disclosed herein may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibody," as used herein, is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail below), antibodies isolated from a recombinant combinatorial human antibody library (described in more detail below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), thereby improving the V H Area and V L The amino acid sequence of the region is human germline V H Array and V L While derived from and related to sequences, they may not naturally occur in the human antibody germline repertoire in vivo.

[0077] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa, with the dimer held together by interchain heavy chain disulfide bonds. In the second form, the dimer is not linked via interchain disulfide bonds, and the approximately 75-80 kDa molecule is composed of covalently linked light and heavy chains (half antibodies). These forms have been very difficult to separate, even after affinity purification.

[0078] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present disclosure provides a method for determining the frequency of occurrence of the second form in various intact IgG isotypes. H 2nd area or C H It encompasses antibodies with one or more mutations in the three regions, which may be desirable, for example, to improve the yield of a desired antibody type in production.

[0079] An antibody useful herein may be an isolated antibody. As used herein, an "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody" for purposes of this disclosure. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0080] Anti-PSMA antibodies and anti-PSMA / anti-CD3 bispecific antibodies useful according to the methods disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody was derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody was derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are backmutated to residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies useful herein may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present disclosure.

[0081] Useful according to the methods provided herein are anti-PSMA / anti-CD3 antibodies containing variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein, with one or more conservative substitutions. For example, the present disclosure includes anti-PSMA / anti-CD3 antibodies having HCVR amino acid sequences, LCVR amino acid sequences, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR amino acid sequences or LCVR amino acid sequences set forth in Table 1 herein.

[0082] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. Epitopes may be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl moieties on the antigen.

[0083] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or Gap, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0084] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned using a program such as GAP or BESTFIT with a predetermined gap weight, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331 (incorporated herein by reference). Examples of groups of amino acids with side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0085] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences disclosed herein to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0086] Sequence variants Bispecific antibodies useful herein contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy chain variable domain compared to the corresponding germline sequence from which the antibody is derived.

[0087] Also useful herein are antibodies, and antigen-binding fragments thereof, derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"), and which exhibit weak or undetectable antigen binding.

[0088] Additionally, antibodies useful herein may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be tested for one or more desired properties, such as improved binding specificity, weaker or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner, given the guidance of the present disclosure, are encompassed by the present invention.

[0089] Useful according to the present disclosure are bispecific antibodies comprising variants of any of the HCVR or LCVR amino acid sequences provided herein with one or more conservative substitutions. The antibodies and bispecific antigen-binding molecules useful herein contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the HCVR and LCVR compared to the corresponding germline sequences from which the individual antigen-binding domains are derived, while maintaining or improving the desired antigen-binding properties. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Examples of groups of amino acids with side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0090] The present disclosure also includes antigen-binding molecules comprising antigen-binding domains having HCVR and / or CDR amino acid sequences that are substantially identical to any of the HCVR and / or CDR amino acid sequences disclosed herein while maintaining or improving the desired antigen affinity. The term "substantial identity" or "substantially identical" means that two amino acid sequences, when optimally aligned using, for example, the programs GAP or BESTFIT with predefined gap weights, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitutions. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.

[0091] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences disclosed herein to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410, and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0092] Once obtained, the antigen-binding domain containing one or more germline mutations was tested for reduced binding affinity using one or more in vitro assays. Antibodies that recognize a specific antigen are typically screened for this purpose by testing for high (i.e., strong) binding affinity to the antigen, but the antibodies useful herein exhibit weak or no detectable binding. Bispecific antigen-binding molecules containing one or more antigen-binding domains obtained in this general manner are also encompassed by the present disclosure and have been found to be advantageous as avidity-driven tumor therapy.

[0093] Unexpected benefits, such as improved pharmacokinetic properties and reduced toxicity to patients, may be realized from the methods described herein.

[0094] Antibody binding properties As used herein, in the context of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein binding to either a predetermined antigen, e.g., a cell surface protein or fragment thereof, the term "binding" typically refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0095] For example, binding affinities are typically about 10, as measured by surface plasmon resonance (SPR) techniques on, for example, a BIAcore 3000 instrument using an antigen as the ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand). -7 M or less, about 10 -8 M or less, about 10 -9 K such as M or less D Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlate well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0096] Thus, the antibodies or antigen-binding proteins disclosed herein have a K that is at most 1 / 10th their affinity for binding to a non-specific antigen (e.g., BSA, casein). D According to the present disclosure, a specific antigen or cell surface molecule (receptor) binds to the target antigen or cell surface molecule (receptor) with an affinity corresponding to a K value that is 10-fold or less than that of a non-specific antigen. DAlthough antibody affinities corresponding to the values ​​may be considered undetectable binding, such antibodies may be paired with a second antigen-binding arm to produce the bispecific antibodies disclosed herein.

[0097] "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. D There is an inverse relationship between the binding affinity and the K D The smaller the value, the higher the affinity, i.e., the stronger. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, and therefore a smaller K D Conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction and, therefore, a larger K D In some situations, the binding affinity (or K ) of a particular molecule (e.g., an antibody) to an interaction partner molecule (e.g., antigen X) is D ) is higher compared to the binding affinity of the molecule (e.g., an antibody) to another interacting partner molecule (e.g., antigen Y), a larger K D The smaller the K value (lower, or weaker, affinity), the D This may be expressed as a binding ratio determined by dividing by a value (higher, or stronger, affinity), and may be expressed as, for example, 5-fold or 10-fold higher binding affinity.

[0098] "k d The term "(sec -1 or 1 / s)" refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. This value is known as the k off Also called value.

[0099] "k a The term "(M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment.

[0100] "K A The term "(M-1 or 1 / M)" refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is k a k d It is obtained by dividing by

[0101] "EC50" or "EC 50 The term "half-maximal effective concentration" refers to the concentration of antibody that elicits a response halfway between baseline and maximum after a specified exposure time. 50 The EC essentially represents the concentration of an antibody at which 50% of its maximal effect is observed. In certain embodiments, the EC 50 The EC value is equal to the concentration of an antibody disclosed herein that confers half-maximal binding to cells expressing CD3 or a tumor-associated antigen, as determined, for example, by a FACS binding assay. 50 Alternatively, the greater the half-maximal effective concentration value, the lower or weaker binding is observed.

[0102] In one embodiment, the reduction in binding is determined by measuring the EC2 concentration that allows binding to half the maximum amount of target cells. 50 It can be defined as an increase in antibody concentration.

[0103] In another embodiment, EC 50 The values ​​represent the concentration of antibody that induces half-maximal depletion of target cells by the cytotoxic activity of T cells. Therefore, increased cytotoxic activity (e.g., T cell-mediated tumor cell killing) is associated with an increase in EC 50 , or a decrease in half-maximal effective concentration values ​​is observed.

[0104] Bispecific antigen binding molecules Antibodies useful herein may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for multiple target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. Anti-PSMA / anti-CD3 bispecific antibodies useful herein can be linked to or coexpressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical coupling, genetic fusion, or noncovalent association, or vice versa) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second or additional binding specificity.

[0105] The use of the phrase "anti-CD3 antibody" or "anti-PSMA antibody" herein is intended to include both monospecific anti-CD3 or anti-PSMA antibodies and bispecific antibodies comprising a CD3-binding arm and a PSMA-binding arm. Thus, the present disclosure includes monospecific antibodies that bind to PSMA, such as the anti-PSMA antibodies described in U.S. Pat. No. 10,179,819. Exemplary anti-PSMA antibodies include the H1H11810P antibody disclosed in U.S. Pat. No. 10,179,819 and antibodies comprising the CDRs within the H1H11810 antibody. Furthermore, the present disclosure includes bispecific antibodies in which one immunoglobulin arm binds to human CD3 and the other immunoglobulin arm is specific for human PSMA. Exemplary sequences of bispecific antibodies useful in the methods provided herein are shown in Table 1.

[0106] In certain embodiments, the CD3-binding arm binds to human CD3 and induces human T cell activation. In certain embodiments, the CD3-binding arm weakly binds to human CD3 and induces human T cell activation. In other embodiments, the CD3-binding arm weakly binds to human CD3 and, in the context of a bispecific or multispecific antibody, induces the killing of tumor-associated antigen-expressing cells. In other embodiments, the CD3-binding arm weakly binds to or associates with human and cynomolgus monkey (monkey) CD3, but the binding interaction is not detectable by in vitro assays known in the art.

[0107] According to certain exemplary embodiments, the present disclosure includes bispecific antigen-binding molecules that specifically bind to CD3 and PSMA. Such molecules may be referred to herein, for example, as "anti-CD3 / anti-PSMA," or "anti-CD3xPSMA," or "CD3xPSMA" bispecific molecules, or other similar terms (e.g., anti-PSMA / anti-CD3).

[0108] As used herein, the term "PSMA" refers to the human PSMA protein unless specified as being from a non-human species (e.g., "mouse PSMA," "monkey PSMA," etc.).

[0109] The aforementioned bispecific antigen-binding molecules that specifically bind to CD3 and PSMA have a K of greater than about 40 nM as measured by an in vitro affinity binding assay. D The anti-CD3 antigen-binding molecule may also include an anti-CD3 antigen-binding molecule that binds to CD3 with weak binding affinity, such as exhibiting

[0110] As used herein, the term "antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising or consisting of at least one complementarity-determining region (CDR), alone or in combination with one or more additional CDRs and / or framework regions (FRs), that specifically binds to a particular antigen. In certain embodiments, the antigen-binding molecule is an antibody or antibody fragment, as those terms are defined elsewhere herein.

[0111] As used herein, the term "bispecific antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain in a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In the context of the present disclosure, the first antigen-binding domain specifically binds to a first antigen (e.g., CD3), and the second antigen-binding domain specifically binds to a second, distinct antigen (e.g., PSMA).

[0112] In certain exemplary embodiments, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first and a second antigen-binding domain, the CDR of the first antigen-binding domain may be designated with the prefix "A1", and the CDR of the second antigen-binding domain may be designated with the prefix "A2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

[0113] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected to each other to form a bispecific antigen-binding molecule useful herein. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerizing domain. The association of one multimerizing domain with another multimerizing domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerizing domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or configuration. For example, a multimerizing domain can be connected to a second multimerizing domain of the same or similar structure or configuration. H The multimer-forming component may be a polypeptide comprising three domains. H 2~C H The Fc portion of an immunoglobulin (comprising three domains), for example, the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0114] Bispecific antigen-binding molecules useful herein will typically comprise two multimerization domains, e.g., two Fc domains, each of which is an individual portion of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0115] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that contain or consist of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0116] Any bispecific antibody format or technology may be used to generate bispecific antigen-binding molecules useful herein. For example, an antibody or fragment thereof having a first antigen-binding specificity can be operably linked (e.g., by chemical coupling, genetic fusion, or noncovalent association, or other methods) to one or more other molecular entities, such as another antibody or antibody fragment having a second binding specificity, to generate a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable region (DVD)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab. 2 and bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a discussion of such formats).

[0117] In the context of bispecific antigen-binding molecules useful herein, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a naturally occurring version of the wild-type Fc domain. For example, the present disclosure includes bispecific antigen-binding molecules containing one or more modifications in the Fc domain that result in a modified Fc domain with modified binding interactions (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H 2nd area or C H The FcRn-binding domain contains modifications in three regions that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes with a pH ranging from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification, a 428L, a 259I (e.g., V259I), and a 308F (e.g., V308F) modification, a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification, a 252, a 254, and a 256 (e.g., 252Y, 254T, and 256E) modification, a 250Q and a 428L modification (e.g., T250Q and M428L), and a 307 and / or a 308 modification (e.g., 308F and / or 308P).

[0118] The present disclosure also provides a first Ig C H 3 domain and second Ig C Ha bispecific antigen-binding molecule comprising three domains, a first and a second Ig C H In one embodiment, the first Ig C domain is a IgG1 domain, and the first Ig C domain is a IgG2 domain. H The 3 domain binds to protein A and the second Ig C H The C3 domain contains a mutation that reduces or eliminates Protein A binding, for example, the H95R (according to IMGT exon numbering; H435R in EU numbering) modification. H 3 may further include a Y96F modification (Y436F according to IMGT, in the EU). See, e.g., U.S. Patent No. 8,586,713. H Additional modifications that may be found in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT).

[0119] In certain embodiments, the Fc domain may be a chimera that combines Fc sequences from two or more immunoglobulin isotypes. For example, a chimeric Fc domain may be a chimeric Fc domain that combines Fc sequences from human IgG1, human IgG2, or human IgG4 C. H C derived from 2 regions H Part or all of the 2 sequence and C derived from human IgG1, human IgG2, or human IgG4 HThe chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any of the antigen-binding molecules presented herein is, from the N-terminus to the C-terminus, [IgG4 C H Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules presented herein comprises, from the N-terminus to the C-terminus, [IgG1 C H 1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules useful herein are described in U.S. Patent Application No. 2014 / 0243504, published August 28, 2014, and incorporated herein in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, may have altered Fc receptor binding, thereby affecting Fc effector function.

[0120] pH dependent binding The present disclosure includes anti-PSMA antibodies and anti-CD3 / anti-PSMA bispecific antigen-binding molecules with pH-dependent binding properties. For example, the anti-PSMA arm of a bispecific antigen-binding molecule useful herein may exhibit decreased binding to PSMA at acidic pH compared to neutral pH. Alternatively, an anti-CD3 / anti-PSMA bispecific antigen-binding molecule useful herein may exhibit enhanced binding to PSMA at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​below about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0121] In one particular example, "decreased binding to ... at acidic pH compared to neutral pH" refers to the K D value and the K of an antibody that binds to its antigen at neutral pH D For example, an antibody or antigen-binding fragment thereof may be used if the antibody or antigen-binding fragment thereof has an acidic / neutral K value of about 3.0 or greater. D For purposes of this disclosure, when a ratio is presented, it can be considered to indicate "decreased binding to PSMA at acidic pH compared to neutral pH." In certain exemplary embodiments, the acidic / neutral K ratio for an antibody or antigen-binding fragment D The ratio can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

[0122] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a collection of antibodies for decreased (or increased) binding to a specific antigen at acidic pH compared to neutral pH. In addition, modification of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent properties. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, an antibody can be obtained that has decreased antigen binding at acidic pH compared to neutral pH.

[0123] Antibodies containing Fc variants According to certain embodiments useful herein, there are provided anti-PSMA antibodies and anti-CD3 / anti-PSMA bispecific antigen binding molecules comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides H 2 or C HThe antibodies include those containing mutations in the FcRn region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes with a pH ranging from about 5.5 to about 6.0). Such mutations can increase the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification, a 428L, a 259I (e.g., V259I), and a 308F (e.g., V308F) modification, a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification, a 252, a 254, and a 256 (e.g., 252Y, 254T, and 256E) modification, a 250Q and a 428L modification (e.g., T250Q and M428L), and a 307 and / or a 308 modification (e.g., 308F and / or 308P).

[0124] For example, the present disclosure includes anti-PSMA antibodies and anti-CD3 / anti-PSMA bispecific antigen-binding molecules comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations in the antibody variable domains disclosed herein, are contemplated as being within the scope of the present disclosure.

[0125] Biological characteristics of antibodies and bispecific antigen-binding molecules Useful according to the present disclosure are antibodies that have high affinity (e.g., subnanomolar K) for CD3-expressing human T cells and / or human PSMA. D and antigen-binding fragments thereof. Such antibodies and their properties are disclosed in U.S. Patent No. 10,179,819, which is incorporated herein by reference. Such bispecific antibodies are particularly useful in combination with anti-4-1BB agonists in the treatment of tumors.

[0126] Useful herein are anti-PSMA antibodies and anti-CD3 / anti-PSMA bispecific antigen binding molecules that exhibit one or more characteristics selected from the group consisting of: (a) inhibiting tumor growth in immunodeficient mice bearing human prostate cancer xenografts, (b) inhibiting tumor growth in immunocompetent mice bearing human prostate cancer xenografts, (c) suppressing tumor growth in immunodeficient mice bearing human prostate cancer xenografts, and (d) reducing tumor growth of established tumors in immunocompetent mice bearing human prostate cancer xenografts (see, e.g., U.S. Pat. No. 10,179,819, Example 8).

[0127] Useful herein are antibodies and antigen-binding fragments thereof that bind to human CD3 with medium or low affinity, depending on the therapeutic situation and the desired specific targeting properties.For example, in the context of a bispecific antigen-binding molecule in which one arm binds to CD3 and the other arm binds to a target antigen (e.g., PSMA), it may be desirable for the target antigen-binding arm to bind to the target antigen with high affinity, while the anti-CD3 arm binds to CD3 with only medium or low affinity.In this way, the preferential targeting of the antigen-binding molecule to cells that express the target antigen can be achieved while avoiding general / non-target CD3 binding and the resulting adverse side effects associated therewith.

[0128] Bispecific antigen-binding molecules (e.g., bispecific antibodies) useful herein can simultaneously bind to human CD3 and human PSMA. The binding arm that interacts with cells expressing CD3 may exhibit weak or no detectable binding when measured in an appropriate in vitro binding assay. The degree to which a bispecific antigen-binding molecule binds to cells expressing CD3 and / or PSMA can be assessed by fluorescence-activated cell sorting (FACS), as shown in U.S. Patent No. 10,179,819, Example 5.

[0129] For example, useful herein are bispecific antibodies that specifically bind to human T cell lines (e.g., Jurkat) that express CD3 but do not express PSMA, primate T cells (e.g., cynomolgus monkey peripheral blood mononuclear cells [PBMCs]), and / or PSMA-expressing cells. Useful herein are bispecific antibodies that specifically bind to human T cell lines (e.g., Jurkat), primate T cells (e.g., cynomolgus monkey peripheral blood mononuclear cells [PBMCs]), and / or PSMA-expressing cells that express CD3 but do not express PSMA ... specifically bind to human T cell lines (e.g., Jurkat), primate T cells (e.g., cynomolgus monkey peripheral blood mononuclear cells [PBMCs]), and / or PSMA-expressing cells that specifically bind to human -8 (18nM) ~ approx. 2.1x10 -7 (210nM) or higher EC 50 value (i.e., weaker affinity) or undetectable EC 50 Also useful herein are bispecific antigen-binding molecules that bind to any of the aforementioned T cells and T cell lines at a binding affinity of 5.6 nM (5.6 x 10), as determined using the FACS binding assay described in U.S. Patent No. 10,179,819, Example 5, or a substantially similar assay. -9 ) below EC 50 It is a bispecific antibody that binds to PSMA-expressing cells and cell lines at low antibody levels.

[0130] In some aspects, the bispecific antibodies bind to human CD3 with weak (i.e., low) or even undetectable affinity. According to certain embodiments, the present disclosure provides a bispecific antibody that binds to human CD3 with a K of greater than about 11 nM as measured by surface plasmon resonance. D The present invention includes antibodies and antigen-binding fragments of antibodies that bind to human CD3 at RT (e.g., 37°C).

[0131] In some embodiments, the bispecific antibody binds to monkey (i.e., cynomolgus) CD3 with weak (i.e., low) or even undetectable affinity.

[0132] In some embodiments, the bispecific antibody binds to human CD3 and induces T cell activation. For example, certain anti-CD3 antibodies have an EC of less than about 113 pM as measured in an in vitro T cell activation assay. 50 Induces human T cell activation at high levels.

[0133] Bispecific antibodies useful herein can bind to human CD3 and induce T cell-mediated tumor antigen-expressing cell killing. For example, the present disclosure provides antibodies with an EC of less than about 1.3 nM as measured in an in vitro T cell-mediated tumor cell killing assay. 50 and bispecific antibodies that induce T cell-mediated tumor cell killing.

[0134] Bispecific antibodies useful herein have a dissociation half-life (t) of less than about 10 minutes as measured by surface plasmon resonance at 25°C or 37°C. 1 / 2 ) can bind to CD3.

[0135] Anti-CD3 / anti-PSMA bispecific antigen binding molecules useful herein may further exhibit one or more characteristics selected from the group consisting of: (a) inducing proliferation of PBMCs in vitro; (b) activating T cells in human whole blood via induction of IFN-gamma release and CD25 upregulation; and (c) inducing T cell-mediated cytotoxicity against anti-PSMA resistant cell lines.

[0136] The present disclosure includes anti-CD3 / anti-PSMA bispecific antigen-binding molecules that can deplete tumor antigen-expressing cells in a subject (see, e.g., U.S. Pat. No. 10,179,819, Example 8). For example, according to certain embodiments, anti-CD3 / anti-PSMA bispecific antigen-binding molecules are provided, wherein a single administration of 1 μg, or 10 μg, or 100 μg, or 1 mg, 3 mg, 5 mg, 10 mg, 30 mg, 50 mg, 100 mg, 300 mg, or 500 mg of the bispecific antigen-binding molecule per patient to a subject (e.g., at a dose of about 5 mg / kg, about 2.5 mg / kg, about 1 mg / kg, about 0.1 mg / kg, about 0.08 mg / kg, about 0.06 mg / kg, about 0.04 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, or less) reduces the number of PSMA-expressing cells in the subject (e.g., tumor growth is suppressed or inhibited in the subject) to below detectable levels. In certain embodiments, a single administration of an anti-CD3 / anti-PSMA bispecific antigen-binding molecule at a dose of about 0.4 mg / kg reduces tumor growth in a subject to below detectable levels by about day 7, about day 6, about day 5, about day 4, about day 3, about day 2, or about day 1 after administration of the bispecific antigen-binding molecule to the subject. According to certain embodiments, a single administration of an anti-CD3 / anti-PSMA bispecific antigen-binding molecule disclosed herein at a dose of at least about 0.01 mg / kg causes the number of PSMA-expressing tumor cells to remain below detectable levels for at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more days after administration. As used herein, the phrase "below detectable levels" means that tumor cells growing subcutaneously in a subject cannot be detected, either directly or indirectly, using standard caliper measurement methods, for example, as described in U.S. Pat. No. 10,179,819, Example 8, herein.

[0137] Also useful according to the methods provided herein are anti-CD3 / anti-PSMA bispecific antigen-binding molecules that exhibit one or more characteristics selected from the group consisting of: (a) inhibiting tumor growth in immunodeficient mice bearing human prostate cancer xenografts, (b) inhibiting tumor growth in immunocompetent mice bearing human prostate cancer xenografts, (c) suppressing tumor growth in immunodeficient mice bearing human prostate cancer xenografts, and (d) reducing tumor growth of established tumors in immunocompetent mice bearing human prostate cancer xenografts (see, e.g., U.S. Pat. No. 10,179,819, Example 8). Exemplary anti-CD3 / anti-PSMA bispecific antigen-binding molecules can further exhibit one or more characteristics selected from the group consisting of (a) inducing a transient, dose-dependent increase in circulating cytokines, and (b) inducing a transient decrease in circulating T cells.

[0138] Epitope mapping and related techniques The CD3 and / or PSMA epitopes bound by antigen-binding molecules useful herein may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids of the CD3 or PSMA protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of CD3 or PSMA. Antibodies useful according to the methods disclosed herein may interact with amino acids contained within a single CD3 chain (e.g., CD3-epsilon, CD3-delta, or CD3-gamma) or with amino acids on two or more different CD3 chains. As used herein, the term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have two or more epitopes. Therefore, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated by adjacent amino acid residues within a polypeptide chain. In certain circumstances, epitopes can include sugar, phosphoryl, or sulfonyl moieties on an antigen.

[0139] Various techniques known to those skilled in the art can be used to determine whether the antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, AntibodiesThese include conventional cross-blocking assays, such as those described by Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide truncation analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody's antigen-binding domain interacts is hydrogen / deuterium exchange, detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves labeling the target protein with deuterium and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis to reveal deuterium-labeled residues corresponding to specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography of the antigen / antibody complex can also be used for epitope mapping purposes.

[0140] Exemplary bispecific antigen-binding molecules useful herein can comprise a first antigen-binding domain that specifically binds to human CD3 and / or cynomolgus CD3 with low or detectable binding affinity, and a second antigen-binding domain that specifically binds to human PSMA, wherein the first antigen-binding domain binds to the same epitope on CD3 as any of the specific exemplary CD3-specific antigen-binding domains described herein, and / or the second antigen-binding domain binds to the same epitope on PSMA as any of the specific exemplary PSMA-specific antigen-binding domains described herein.

[0141] Similarly, a bispecific antigen-binding molecule useful herein can comprise a first antigen-binding domain that specifically binds human CD3 and a second antigen-binding domain that specifically binds human PSMA, wherein the first antigen-binding domain competes for binding to CD3 with any of the specific exemplary CD3-specific antigen-binding domains set forth in Table 1 herein, and / or the second antigen-binding domain competes for binding to PSMA with any of the specific exemplary PSMA-specific antigen-binding domains set forth in Table 1 herein.

[0142] Whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to the same epitope as a reference antigen-binding molecule of the present disclosure or competes for binding can be easily determined by routine methods known to those skilled in the art. For example, to determine whether a test antibody binds to the same epitope on PSMA (or CD3) as a reference bispecific antigen-binding molecule of the present disclosure, the reference bispecific molecule is first bound to the PSMA protein (or CD3 protein). The ability of the test antibody to bind to the PSMA (or CD3) molecule is then evaluated. If the test antibody can bind to PSMA (or CD3) after saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope of PSMA (or CD3) than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to the PSMA (or CD3) molecule after saturation binding with the reference bispecific antigen-binding molecule, the test antibody may bind to the same epitope of PSMA (or CD3) as the epitope bound by the reference bispecific antigen-binding molecule. Additional routine experiments (e.g., peptide mutations and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present disclosure, two antigen-binding proteins bind to the same (or overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antigen-binding protein inhibits binding of the other by at least 50%, but preferably by 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, two antigen-binding proteins are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other. Two antigen-binding proteins are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other.

[0143] To determine whether an antibody or its antigen-binding domain competes for binding with a reference anti-antigen binding molecule, the above-described binding technique is performed in two ways: In the first way, the reference antigen-binding molecule binds to the PSMA protein (or CD3 protein) under saturating conditions, and then the binding of the test antibody to the PSMA (or CD3) molecule can be evaluated. In the second way, the test antibody binds to the PSMA (or CD3) molecule under saturating conditions, and then the binding of the reference antigen-binding molecule to the PSMA (or CD3) molecule can be evaluated. In both ways, if only the first (saturating) antigen-binding molecule can bind to the PSMA (or CD3) molecule, it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to PSMA (or CD3). As will be understood by those skilled in the art, an antibody that competes for binding to a reference antigen-binding molecule does not necessarily bind to the same epitope as the reference antibody, and may sterically block the binding of the reference antibody by binding an overlapping or adjacent epitope.

[0144] Preparation of antigen-binding domains and construction of bispecific molecules Antigen-binding domains specific for a particular antigen can be prepared by any antibody generation technique known in the art. Once obtained, two different antigen-binding domains specific for two different antigens (e.g., CD3 and PSMA) can be appropriately positioned relative to one another to produce a bispecific antigen-binding molecule using routine methods. (A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the present disclosure is provided elsewhere herein.) In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecule are derived from chimeric, humanized, or fully human antibodies. Methods for producing such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules useful herein can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (see, e.g., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE® or any other human antibody generation technology), high-affinity chimeric antibodies to a particular antigen (e.g., CD3 or PSMA) with human variable regions and mouse constant regions are first isolated. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into bispecific antigen-binding molecules useful herein.

[0145] Genetically engineered animals can also be used to produce human bispecific antigen-binding molecules. For example, genetically engineered mice that are unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, and the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to mouse kappa constant genes at the endogenous mouse kappa locus. Such genetically engineered mice can be used to produce fully human bispecific antigen-binding molecules containing two different heavy chains associated with the same light chain, each containing a variable domain derived from one of two different human light chain variable region gene segments. (See, for example, U.S. Patent No. 10,143,186 for a detailed discussion of such engineered mice and their use to produce bispecific antigen-binding molecules.)

[0146] biological equivalent The methods of the present disclosure contemplate the use of antigen-binding molecules having amino acid sequences that differ from those of the exemplary molecules disclosed herein but that retain the ability to bind to CD3 and / or PSMA. Such variant molecules may contain one or more amino acid additions, deletions, or substitutions compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described bispecific antigen-binding molecules.

[0147] Useful herein are antigen-binding molecules that are biologically equivalent to any of the exemplary antigen-binding molecules listed in Table 1 herein. Two antigen-binding proteins, or antibodies, are considered to be bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not exhibit significant differences in the rate and extent of absorption when administered, for example, at the same molar dose under similar experimental conditions, either in single or multiple doses. Some antigen-binding proteins are considered equivalents or pharmaceutical substitutes if their extent of absorption is comparable but their absorption rates are not; furthermore, such differences in absorption rates may be considered bioequivalent because they are intentional, reflected in the labeling, are not essential for achieving effective body drug concentrations in chronic use, and are not considered medically significant for the particular pharmaceutical product being studied.

[0148] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, or efficacy.

[0149] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch one or more times between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continuous therapy without such switching.

[0150] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms of action, to the extent that such mechanisms are known, for one or more conditions of use.

[0151] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro studies that correlate with and reasonably predict in vivo bioavailability data in humans, (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding protein.

[0152] Biologically equivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antigen-binding proteins can include variants of the exemplary bispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation characteristics of the molecule, for example, mutations that eliminate or remove glycosylation.

[0153] Species selectivity and species cross-reactivity According to certain embodiments, bispecific antigen-binding molecules useful herein bind to human CD3 but not to CD3 from other species. Also useful herein are antigen-binding molecules that bind to human PSMA but not to PSMA from other species. The methods of the present disclosure also contemplate the use of bispecific antigen-binding molecules that bind to human CD3 and CD3 from one or more non-human species, and / or bispecific antigen-binding molecules that bind to human PSMA and PSMA from one or more non-human species.

[0154] According to certain exemplary embodiments, antigen-binding molecules useful herein bind to human CD3 and / or human PSMA, and may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee CD3 and / or PSMA. For example, certain exemplary embodiments of the present disclosure provide bispecific antigen-binding molecules comprising a first antigen-binding domain that binds to human CD3 and cynomolgus monkey CD3, and a second antigen-binding domain that specifically binds to human PSMA.

[0155] Radiolabeled immunoconjugates of anti-PSMA / anti-CD3 antigen-binding molecules for immunoPET imaging Provided herein are radiolabeled antigen binding proteins that bind to anti-PSMA antibodies or anti-PSMA / anti-CD3 antigen binding molecules. In some embodiments, the radiolabeled antigen binding protein comprises an antigen binding protein covalently bound to a positron emitter. In some embodiments, the radiolabeled antigen binding protein comprises an antigen binding protein covalently bound to one or more chelating moieties, where the chelating moiety is a chemical moiety capable of chelating a positron emitter.

[0156] Suitable radiolabeled antigen-binding proteins, e.g., radiolabeled antibodies, include those that do not impair or do not substantially impair T cell function upon exposure to the radiolabeled antigen-binding protein. In some embodiments, the radiolabeled antigen-binding protein that binds to the anti-PSMA / anti-CD3 antigen-binding molecule is a weak blocker of CD3 T cell function, i.e., does not impair or does not substantially impair T cell function upon exposure to the radiolabeled antibody. The use of a radiolabeled anti-CD3 binding protein with minimal effect on CD3-mediated T cell function according to the methods provided herein ensures that subjects treated with the molecule are not disadvantaged by their T cells being unable to clear infection.

[0157] In some embodiments, an anti-PSMA antibody or anti-PSMA / anti-CD3 antigen binding molecule, e.g., a bispecific antibody, is provided, wherein the antigen binding protein is covalently linked to one or more moieties having the following structure: -LM Z wherein L is a chelating moiety, M is a positron emitter, and z is independently 0 or 1 in each occurrence, and at least one of the z's is 1.

[0158] In some embodiments, the radiolabeled antigen binding protein is a compound of formula (I): MLA-[LM Z ] k (I) A is an anti-PSMA antibody or anti-PSMA / anti-CD3 antigen binding molecule, L is a chelating moiety, M is a positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1. In some embodiments, k is 2.

[0159] In certain embodiments, the radiolabeled antigen binding protein is a compound of formula (II): A-[LM] k (II) wherein A is an anti-PSMA antibody or anti-PSMA / anti-CD3 antigen binding molecule, L is a chelating moiety, M is a positron emitter, and k is an integer from 1 to 30.

[0160] In some embodiments, provided herein are compositions comprising a conjugate having the structure: AL k wherein A is an anti-PSMA antibody or anti-PSMA / anti-CD3 antigen binding molecule, L is a chelating moiety, and k is an integer between 1 and 30, and the conjugate is chelated with a positron emitter in an amount sufficient to provide a specific activity suitable for clinical PET imaging.

[0161] Suitable chelating moieties and positron emitters are provided below.

[0162] Positron emitters and chelating moieties Suitable positron emitters include, but are not limited to, those that form stable complexes with chelating moieties and have suitable physical half-lives for the purposes of immunoPET imaging. Exemplary positron emitters include: 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86 Suitable positron emitters also include, but are not limited to, Y. 76 Br and 124 I, and the like, as well as prosthetic groups, e.g., 18 Examples include those introduced via F.

[0163] The chelating moiety described herein is a chemical moiety covalently attached to an anti-PSMA / anti-CD3 antigen-binding molecule, and includes a moiety capable of chelating with a positron emitter, i.e., capable of reacting with a positron emitter to form a coordinate chelate complex. Suitable moieties include those that allow efficient loading of specific metals and form sufficiently stable metal chelator complexes for diagnostic use in vivo, for example, for immunoPET imaging. Exemplary chelating moieties include those that minimize positron emitter dissociation and accumulation in bone mineral, plasma proteins, and / or bone marrow deposits to an extent suitable for diagnostic use.

[0164] Examples of chelating moieties include positron emitters 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86Exemplary chelating moieties include, but are not limited to, those that form stable complexes with Y. Exemplary chelating moieties include those described in Nature Protocols, 5(4):739, 2010; Bioconjugate Chem., 26(12):2579 (2015); Chem Commun (Camb), 51(12):2301 (2015); Mol. Pharmaceutics, 12:2142 (2015); Mol. Imaging Biol., 18:344 (2015); Eur. J. Nucl. Med. Mol. Imaging, 37:250 (2010); Eur. J. Nucl. Med. Mol. Imaging (2016). doi:10.1007 / s00259-016-3499-x; Bioconjugate Chem., 26(12):2579(2015); WO2015 / 140212A1; and those described in U.S. Pat. No. 5,639,879.

[0165] Exemplary chelating moieties include desferrioxamine (DFO), 1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic) acid (DOTP), 1R,4R,7R,10R)-α'α''α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid (DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), H4 octapentaenoic acid, H6 phospa, H2 dedopa, H5 decapa, H2 azapa, HOPO, DO2A, 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1 ,4,7,10-Tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazacyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 1,4,7,10-tetraazacyclododecane (Cyclen), 1,4,8,11-tetraazacyclotetradecane (Cyclam), octadentate chelating agent, octadentate bifunctional chelating agent Chelating agents include, but are not limited to, DFO*, hexadentate chelating agents, phosphonate-based chelating agents, macrocyclic chelating agents, chelating agents containing macrocyclic terephthalamide ligands, bifunctional chelating agents, fusarinine C and fusarinine C derivative chelating agents, triacetylfusarinine C (TAFC), ferrioxamine E (FOXE), ferrioxamine B (FOXB), and ferrichrome A (FCHA).

[0166] In some embodiments, the chelating moiety is covalently attached to the anti-PSMA / anti-CD3 bispecific antigen-binding molecule via a linker moiety that covalently attaches the chelating portion of the chelating moiety to the binding protein. In some embodiments, these linker moieties are formed from the reaction between a reactive moiety on the bispecific antigen-binding molecule, e.g., a cysteine ​​or lysine on an antibody, and a reactive moiety attached to a chelator, e.g., a p-isothiocyanatophenyl group and the reactive moieties provided in the conjugation methods described below. In addition, such linker moieties optionally contain chemical groups used to adjust polarity, solubility, steric interactions, rigidity, and / or length between the chelating moiety and the anti-PSMA / anti-CD3 bispecific antigen-binding molecule.

[0167] Preparation of radiolabeled anti-PSMA / anti-CD3 bispecific antigen-binding molecule conjugates Radiolabeled anti-PSMA antibody conjugates and anti-PSMA / anti-CD3 bispecific antigen-binding molecule conjugates can be prepared by (1) reacting the antigen-binding molecule with a molecule containing a positron emitter chelator and a moiety reactive for the desired conjugation site on the bispecific binding protein, and (2) loading the desired positron emitter.

[0168] Suitable conjugation sites include, but are not limited to, lysine and cysteine, both of which can be naturally occurring or engineered, for example, present on the heavy or light chain of an antibody. Cysteine ​​conjugation sites include, but are not limited to, those obtained by mutation, insertion, or reduction of antibody disulfide bonds. Methods for producing cysteine-engineered antibodies include, but are not limited to, those disclosed in WO2011 / 056983. Site-specific conjugation methods can also be used to direct the conjugation reaction to specific sites on an antibody, to achieve a desired stoichiometry, and / or to achieve a desired chelator-to-antibody ratio. Such conjugation methods are known to those skilled in the art and include, but are not limited to, glutamine conjugation, Q295 conjugation, and transglutaminase-mediated conjugation, as well as cysteine ​​engineering and enzymatic and chemoenzymatic methods, including, but not limited to, those described in J. Clin. Immunol., 36:100 (2016), the entire contents of which are incorporated herein by reference. A suitable moiety reactive with the desired conjugation site generally enables efficient and facile coupling of the anti-PSMA / anti-CD3 bispecific antigen-binding molecule, e.g., a bispecific antibody, with a positron emitter chelator. Moieties reactive with lysine and cysteine ​​sites include electrophilic groups known to those skilled in the art. In certain embodiments, when the desired conjugation site is lysine, the reactive moiety is an isothiocyanate, e.g., a p-isothiocyanatophenyl group or a reactive ester. In certain embodiments, when the desired conjugation site is cysteine, the reactive moiety is a maleimide.

[0169] When the chelator is desferrioxamine (DFO), suitable reactive moieties include, but are not limited to, isothiocyanatobenzyl groups, n-hydroxysuccinimide esters, 2,3,5,6 tetrafluorophenol esters, n-succinimidyl-S-acetylthioacetate, and those described in BioMed Research International, Vol 2014, Article ID 203601, which is incorporated herein by reference in its entirety. In certain embodiments, the molecule comprising a positron emitter chelator and a moiety reactive with a conjugation site is p-isothiocyanatobenzyl-desferrioxamine (p-SCN-Bn-DFO). [ka]

[0170] Loading of the positron emitter is achieved by incubating the positron emitter with the anti-PSMA / anti-CD3 bispecific antigen binding molecule chelator conjugate for a time sufficient to allow coordination of the positron emitter to the chelator, for example, by performing the methods described in the Examples provided herein, or substantially similar methods.

[0171] Exemplary Embodiments of Conjugates Included in the present disclosure is a radiolabeled antibody conjugate comprising an anti-PSMA antibody or an anti-PSMA / anti-CD3 bispecific antigen-binding molecule and a positron emitter. Additionally, included in the present disclosure is a radiolabeled antibody conjugate comprising an anti-PSMA antibody or an anti-PSMA / anti-CD3 bispecific antigen-binding molecule, a chelating moiety, and a positron emitter.

[0172] In some embodiments, the chelating moiety is: 89 The compound includes a chelating agent capable of complexing with Zr. In certain embodiments, the chelating moiety includes desferrioxamine. In certain embodiments, the chelating moiety is p-isothiocyanatobenzyl-desferrioxamine.

[0173] In some embodiments, the positron emitter is 89 In some embodiments, less than 1.0% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter, less than 0.9% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter, less than 0.8% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter, less than 0.7% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter, and less than 0.6% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter. less than 0.5% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter; less than 0.4% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter; less than 0.3% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter; less than 0.2% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter; or less than 0.1% of the anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules are conjugated to a positron emitter.

[0174] In some embodiments, the chelating moiety-to-antibody ratio of the conjugate is 1.0 to 2.0. As used herein, "chelating moiety-to-antibody ratio" is the average chelating moiety-to-antibody ratio and is a measure of the chelating agent loading per antibody. This ratio is similar to the drug-to-antibody ratio used by those skilled in the art to measure the "DAR," i.e., the drug loading per antibody for antibody-drug conjugates (ADCs). For the conjugates described herein for iPET imaging, the chelating moiety-to-antibody ratio can be determined by the methods described herein and other methods known in the art for determining the DAR, e.g., Wang et al., Antibody-Drug Conjugates, The 21 st The antibody-to-chelating moiety ratio can be determined using the methods described in Century Magic Bullets for Cancer (2015). In some embodiments, the chelating moiety to antibody ratio is about 1.7. In some embodiments, the chelating moiety to antibody ratio is 1.0 to 2.0. In some embodiments, the chelating moiety to antibody ratio is about 1.7.

[0175] In certain embodiments, the chelating moiety is p-isothiocyanatobenzyl-desferarioxamine and the positron emitter is 89 In another particular embodiment, the chelating moiety is p-isothiocyanatobenzyl-desferarioxamine and the positron emitter is 89 Zr, and the chelating moiety to antibody ratio of the conjugate is 1-2.

[0176] In some embodiments, provided herein are anti-PSMA antibodies or anti-PSMA / anti-CD3 bispecific antigen-binding molecules, wherein the antigen-binding molecules are covalently linked to one or more moieties having the following structure: -LM Z wherein L is a chelating moiety, M is a positron emitter, and z is independently 0 or 1 in each occurrence, and at least one of the z's is 1. In certain embodiments, the radiolabeled antigen binding protein is a compound of formula (I): MLA-[LM Z ] k (I) A is an anti-PSMA antibody or an anti-PSMA / anti-CD3 bispecific antigen-binding molecule, L is a chelating moiety, M is a positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1. In some embodiments, k is 2.

[0177] In some embodiments, L is: [ka]

[0178] In some embodiments, M is 89 It is Zr.

[0179] In some embodiments, k is an integer from 1 to 2. In some embodiments, k is 1. In some embodiments, k is 2.

[0180] In some embodiments, -LM is: [ka]

[0181] Also included in the present disclosure are compounds of formula (III): [ka] of 89 A method for synthesizing a radiolabeled antibody conjugate, comprising contacting A with Zr, where A is an anti-PSMA antibody or an anti-PSMA / anti-CD3 bispecific antigen-binding molecule. In certain embodiments, the compound of formula (III) is synthesized by contacting the anti-PSMA antibody or anti-PSMA / anti-CD3 bispecific antigen-binding molecule with p-SCN-Bn-DFO.

[0182] a compound of formula (III) 89The product of the reaction between Zr is also provided herein.

[0183] Provided herein are compounds of formula (III): [ka] wherein A is an anti-PSMA / anti-CD3 bispecific antigen-binding molecule and k is an integer from 1 to 30. In some embodiments, k is 1 or 2.

[0184] Provided herein are antibody conjugates comprising (i) an anti-PSMA antibody or an anti-PSMA / anti-CD3 bispecific antigen-binding molecule, and (ii) one or more chelating moieties.

[0185] In some embodiments, the chelating moiety comprises: [ka] [ka] is a covalent bond to an antibody or antigen-binding fragment thereof.

[0186] In some embodiments, the antibody conjugate has a chelating moiety to antibody ratio of about 1.0 to about 2.0, hi some embodiments, the antibody conjugate has a chelating moiety to antibody ratio of about 1.7.

[0187] In some embodiments, provided herein are compositions comprising a conjugate having the structure: AL k wherein A is an anti-PSMA antibody or anti-PSMA / anti-CD3 bispecific antigen-binding molecule, L is a chelating moiety, and k is an integer between 1 and 30, and the conjugate is chelated with a positron emitter in an amount sufficient to provide a specific activity suitable for clinical PET imaging. In some embodiments, the amount of chelated positron emitter is sufficient to provide a specific activity of about 1 to about 50 mCi per 1 to 50 mg of anti-PSMA / anti-CD3 bispecific antigen-binding molecule.

[0188] In some embodiments, the amount of chelated positron emitter is sufficient to provide a specific activity in the range of up to 50 mCi, up to 45 mCi, up to 40 mCi, up to 35 mCi, up to 30 mCi, up to 25 mCi, or up to 10 mCi, e.g., about 5 to about 50 mCi, about 10 to about 40 mCi, about 15 to about 30 mCi, about 7 to about 25 mCi, about 20 to about 50 mCi, or about 5 to about 10 mCi, per 1 to 50 mg of anti-PSMA / anti-CD3 bispecific antigen binding molecule.

[0189] Methods using radiolabeled immunoconjugates In certain aspects, the present disclosure provides diagnostic and therapeutic methods using the radiolabeled antibody conjugates of the present disclosure.

[0190] According to one aspect, the present disclosure provides a method for detecting PSMA in a tissue, the method comprising administering a radiolabeled anti-PSMA antibody conjugate or an anti-PSMA / anti-CD3 bispecific antigen-binding molecule conjugate provided herein to the tissue and visualizing PSMA expression by positron emission tomography (PET) imaging. In certain embodiments, the tissue comprises a cell or cell line. In certain embodiments, the tissue is present in the body of a subject, and the subject is a mammal. In certain embodiments, the subject is a human subject. In certain embodiments, the subject has a disease or disorder selected from the group consisting of cancers that express the PSMA antigen, such as prostate cancer, kidney cancer, bladder cancer, colorectal cancer, and gastric cancer. In one embodiment, the subject has prostate cancer.

[0191] According to one aspect, the present disclosure provides a method for imaging PSMA-expressing tissue, the method comprising administering a radiolabeled anti-PSMA antibody conjugate or anti-PSMA / anti-CD3 bispecific antigen-binding molecule conjugate of the present disclosure to the tissue and visualizing PSMA expression by positron emission tomography (PET) imaging. In one embodiment, the tissue is contained within a tumor. In one embodiment, the tissue is contained within a tumor cell culture or tumor cell line. In one embodiment, the tissue is contained within a tumor lesion of a subject. In one embodiment, the tissue is an intratumoral lymphocyte within the tissue. In one embodiment, the tissue comprises PSMA-expressing cells.

[0192] According to one aspect, the present disclosure provides a method for determining whether a subject having a tumor is suitable for anti-tumor therapy, the method comprising administering a radiolabeled antibody conjugate of the present disclosure and localizing the administered radiolabeled antibody conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate within the tumor identifies the subject as suitable for anti-tumor therapy.

[0193] According to one aspect, the present disclosure provides a method for predicting the response of a subject having a solid tumor to an anti-tumor therapy, the method comprising determining whether the tumor is PSMA-positive, and if the tumor is PSMA-positive, predicting a positive response in the subject. In certain embodiments, the tumor is determined to be positive by administering a radiolabeled antibody conjugate of the present disclosure and localizing the radiolabeled antibody conjugate within the tumor by PET imaging, and the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is PSMA-positive.

[0194] According to one aspect, the present disclosure provides a method for detecting a PSMA-positive tumor in a subject. The method according to this aspect includes administering to the subject a radiolabeled antibody conjugate of the present disclosure and determining the localization of the radiolabeled antibody conjugate by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is PSMA-positive.

[0195] Provided herein is a method for predicting a positive response to anti-tumor therapy, comprising administering a radiolabeled anti-PSMA antibody conjugate or an anti-PSMA / anti-CD3 bispecific antigen-binding molecule conjugate to a subject and determining the presence of PSMA-positive cells in solid tumors. The presence of PSMA-positive cells predicts a positive response to anti-tumor therapy.

[0196] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal exhibiting one or more symptoms or signs of cancer and / or a human or non-human mammal diagnosed with cancer, including a solid tumor, and in need of treatment for cancer. In many embodiments, the term "subject" may be used interchangeably with the term "patient." For example, a human subject may have a primary tumor or a metastatic tumor and / or be diagnosed with one or more symptoms or signs, including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, abdominal distension, chest pain / tightness, enlarged spleen, and elevated levels of cancer-associated biomarkers (e.g., CA125). This phrase includes subjects with primary or established tumors. In certain embodiments, this phrase includes human subjects with and / or in need of treatment for solid tumors, such as colon cancer, breast cancer, lung cancer, prostate cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, cervical cancer, pancreatic cancer, head and neck cancer, and brain cancer. The term includes subjects with primary or metastatic tumors (advanced malignancies). In certain embodiments, the phrase "subject in need thereof" includes subjects with solid tumors that are resistant or refractory to previous therapies (e.g., treatment with anticancer agents) or not adequately controlled by previous therapies. For example, the phrase includes subjects who have been treated with one or more lines of previous therapy, such as chemotherapy (e.g., carboplatin or docetaxel). In certain embodiments, the phrase "subject in need thereof" includes subjects with solid tumors that have been treated with one or more lines of previous therapy but have subsequently recurred or metastasized. In certain embodiments, the methods of the present disclosure are used in subjects with solid tumors. The terms "tumor," "cancer," and "malignancy" are used interchangeably herein. As used herein, the term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign (not cancerous) or malignant (cancer). For purposes of this disclosure, the term "solid tumor" refers to a malignant solid tumor.The term includes different types of solid tumors, termed according to the cell types that form them: sarcomas, carcinomas, and lymphomas.

[0197] In certain embodiments, the cancer or tumor is selected from the group consisting of astrocytoma, anal cancer, bladder cancer, blood cancer, blood cancer, bone cancer, brain cancer, breast cancer, cervical cancer, renal clear cell carcinoma, colorectal cancer, microsatellite-intermediate colorectal cancer, cutaneous squamous cell carcinoma, diffuse large B-cell lymphoma, endometrial cancer, esophageal cancer, fibrosarcoma, gastric cancer, glioblastoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, leukemia, liver cancer, leiomyosarcoma, lung cancer, lymphoma, melanoma, mesothelioma, myeloma, nasopharyngeal carcinoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, primary and / or recurrent carcinoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small cell lung cancer, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thyroid cancer, triple-negative breast cancer, uterine cancer, and Wilms' tumor. In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic and / or recurrent cancer.

[0198] In certain embodiments, cancer or tumor is selected from PSMA-positive tumors, such as tumors derived from prostate epithelium, duodenal mucosa, proximal tubule or colonic crypt neuroendocrine cell.In some aspects, cancer is bladder cancer, kidney cancer, gastric cancer or colorectal cancer.In some aspects, cancer is prostate cancer.In some aspects, cancer is metastatic cancer derived from primary prostate tumor.

[0199] As used herein, the terms "treat," "treating," and the like mean alleviating symptoms, eliminating the cause of symptoms either temporarily or permanently, slowing or inhibiting tumor growth, reducing tumor cell burden or tumor mass, promoting tumor regression, causing tumor shrinkage, necrosis, and / or disappearance, preventing tumor recurrence, preventing or inhibiting metastasis, inhibiting metastatic tumor growth, and / or prolonging the survival of a subject.

[0200] In certain embodiments, the radiolabeled anti-PSMA antibody conjugate or anti-PSMA / anti-CD3 bispecific antigen-binding molecule conjugate is administered intravenously or subcutaneously to the subject. In certain embodiments, the radiolabeled antibody conjugate is administered intratumorally. Upon administration, the radiolabeled antibody conjugate is localized within the tumor. The localized radiolabeled antibody conjugate is imaged by PET imaging, and the uptake of the radiolabeled antibody conjugate by the tumor is measured by methods known in the art. In certain embodiments, imaging is performed 1, 2, 3, 4, 5, 6, or 7 days after administration of the radiolabeled conjugate. In certain embodiments, imaging is performed on the same day as administration of the radiolabeled antibody conjugate.

[0201] In certain embodiments, the radiolabeled anti-PSMA antibody conjugate or anti-PSMA / anti-CD3 bispecific antigen-binding molecule conjugate can be administered at a dose of about 0.1 mg / kg to about 100 mg / kg of the subject's body weight, e.g., about 0.1 mg / kg to about 50 mg / kg, or about 0.5 mg / kg to about 25 mg / kg, or about 0.1 mg / kg to about 1.0 mg / kg.

[0202] Therapeutic Formulation and Administration Pharmaceutical compositions comprising the antigen-binding molecules useful herein are useful according to the present disclosure. In some embodiments, the pharmaceutical composition further comprises an anti-4-1BB agonist. The pharmaceutical composition is formulated with suitable carriers, excipients, and other agents that improve transportation, delivery, tolerability, etc. Numerous suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0203] The dose of an antigen-binding molecule administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred doses are typically calculated based on body weight or body surface area. When a bispecific antigen-binding molecule is used for therapeutic purposes in an adult patient, it may be advantageous to administer the bispecific antigen-binding molecule intravenously at a single dose of typically about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. In some embodiments, it may be advantageous to administer the bispecific antigen-binding molecule intravenously at a single dose of typically about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for administering bispecific antigen-binding molecules can be determined empirically; for example, patient progress can be monitored by periodic evaluation, and dosages adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0204] The dose of an anti-4-1BB agonist administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. A preferred dose is typically calculated based on body weight or body surface area. When an anti-4-1BB agonist is used for therapeutic purposes in an adult patient, it may be beneficial to administer the agonist intravenously in a single dose of typically about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg, or about 2.5 mg / kg body weight.

[0205] Various delivery systems are known and can be used to administer the pharmaceutical compositions useful herein, such as liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0206] The pharmaceutical compositions useful herein can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are easily applicable to the delivery of the pharmaceutical compositions useful herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir inside the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0207] Numerous reusable pen and autoinjector delivery devices have application in the subcutaneous delivery of pharmaceutical compositions useful herein, including, but not limited to, the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN™, to name a few. STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices having application in subcutaneous delivery of pharmaceutical compositions useful herein include, but are not limited to, the SOLOSTAR™ Pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly & Co.), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ Pen (Abbott Labs, Abbott Park, Illinois).

[0208] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in the vicinity of the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0209] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, isotonic solutions containing saline, glucose, and other auxiliary agents, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solution prepared in this manner is preferably filled into appropriate ampoules.

[0210] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for adjusting the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 0.5 to about 500 mg per dosage form in unit dose. In particular, for injections, the antibody is preferably contained in an amount of about 5 to about 100 mg, and for other dosage forms, it is preferably contained in an amount of about 10 to about 250 mg.

[0211] Combination Therapies and Formulations The present disclosure provides methods comprising administering a pharmaceutical composition comprising any of the exemplary monospecific or bispecific antigen-binding molecules described herein in combination with an anti-4-1BB agonist and one or more additional therapeutic agents. Exemplary additional therapeutic agents that may be combined or administered in combination with the anti-4-1BB agonists and bispecific antigen-binding molecules useful herein include, for example, EGFR antagonists (e.g., anti-EGFR antibodies [e.g., cetuximab or panitumumab] or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), antagonists of another EGFR family member, such as Her2 / ErbB2, ErbB3, or ErbB4 (e.g., anti-ErbB2, anti-ErbB3, or anti-ErbB4). rbB4 antibodies, or small molecule inhibitors of ErbB2, ErbB3, or ErbB4 activity), EGFRvIII antagonists (e.g., antibodies that specifically bind to EGFRvIII), cMET agonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies), VEGF antagonists (e.g., VEGF-traps, e.g., see U.S. Pat. No. 7,087,411 (also referred to herein as "VEGF inhibitory fusion proteins"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)), DLL4 antagonists (e.g., the anti-DLL4 antibodies disclosed in U.S. Pat. No. 2009 / 0142354, such as REGN421), Ang2 antagonists (e.g., H1H6 85P and other antibodies disclosed in U.S. Patent No. 2011 / 0027286), FOLH1 (PSMA) antagonists, PRLR antagonists (e.g., anti-PRLR antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), uroplakin antagonists (e.g., anti-uroplakin antibodies), and the like.Other agents that may be beneficially administered in combination with the compositions provided herein include small molecule cytokine inhibitors, as well as cytokine inhibitors including antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, and IL-18, or their respective receptors. Pharmaceutical compositions useful herein (e.g., pharmaceutical compositions comprising the anti-CD3 / anti-PSMA bispecific antigen binding molecules disclosed herein) may be administered in combination with an anti-4-1BB agonist and one of the following compounds: "ICE": ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePeid®, VP-16); "DHAP": dexamethasone (e.g., Decadron®), cytarabine (e.g., Cytos®), It may also be administered as part of a treatment regimen that includes a combination of one or more therapies selected from: ar-U®, cytosine arabinoside, ara-C), cisplatin (e.g., Platinol®-AQ); and "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePeid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ).

[0212] The present disclosure also includes therapeutic combinations comprising any of the antigen-binding molecules described herein and one or more inhibitors of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementioned cytokines, where the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). The antigen-binding molecules disclosed herein may also be administered and / or co-formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, and / or NSAIDs. The antigen binding molecules disclosed herein may also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[0213] The additional therapeutically active ingredient may be administered immediately prior to, simultaneously with, or immediately following administration of an antigen-binding molecule useful herein; (for purposes of this disclosure, such administration regimens will be considered administration of the antigen-binding molecule "in combination" with the additional therapeutically active ingredient).

[0214] The present disclosure includes pharmaceutical compositions in which the antigen binding molecules useful herein are co-formulated with one or more of the additional therapeutically active ingredients described elsewhere herein.

[0215] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of an antigen-binding molecule (e.g., an anti-PSMA antibody or an anti-CD3 / anti-PSMA bispecific antigen-binding molecule) may be administered to a subject over a defined time course. Additionally, multiple doses of an anti-4-1BB agonist may be administered to a subject over a defined time course. The method according to this aspect comprises sequentially administering to a subject one or more doses of each therapeutic agent, i.e., one or more doses of an antigen-binding molecule and one or more doses of an anti-4-1BB agonist. As used herein, "sequentially administering" means that each dose of a therapeutic agent, e.g., an antigen-binding molecule, is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., several hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering to a patient a single initial dose of an antigen-binding molecule, referred to as a loading dose, followed by one or more secondary doses of the antigen-binding molecule, and, optionally, followed by one or more tertiary doses of the antigen-binding molecule. The present disclosure includes methods comprising sequentially administering to a patient a single initial dose of an anti-4-1BB agonist, referred to as a loading dose, followed by one or more secondary doses of the anti-4-1BB agonist, and optionally, thereafter one or more tertiary doses of the anti-4-1BB agonist.

[0216] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration of an antigen-binding molecule and / or anti-4-1BB agonist useful herein. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses all contain the same amount of antigen-binding molecule (or anti-4-1BB agonist), but generally may differ from each other in terms of administration frequency. However, in certain embodiments, the amount of antigen-binding molecule (or anti-4-1BB agonist) contained in the initial, secondary, and / or tertiary doses differs from each other (e.g., adjusted accordingly) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of a treatment regimen, with subsequent doses administered less frequently (e.g., "maintenance doses").

[0217] In one exemplary embodiment of the present disclosure, each secondary dose and / or tertiary dose is administered 1 to 26 weeks after the immediately preceding dose (e.g., 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, 8 weeks, 8.5 weeks, 9 weeks, 9.5 weeks, 10 weeks, 10.5 weeks, 11 weeks, 11.5 weeks, 12 weeks, 12.5 weeks, 13 weeks, After 13.5 weeks, 14 weeks, 14.5 weeks, 15 weeks, 15.5 weeks, 16 weeks, 16.5 weeks, 17 weeks, 17.5 weeks, 18 weeks, 18.5 weeks, 19 weeks, 19.5 weeks, 20 weeks, 20.5 weeks, 21 weeks, 21.5 weeks, 22 weeks, 22.5 weeks, 23 weeks, 23.5 weeks, 24 weeks, 24.5 weeks, 25 weeks, 25.5 weeks, 26 weeks, 26.5 weeks, or longer). As used herein, the phrase "immediately preceding dose" refers to the dose of an antigen-binding molecule (or anti-4-1BB agonist) administered to a patient in a multiple-dose sequence, without any intervening doses, prior to the administration of the immediately subsequent dose in the sequence.

[0218] The method according to this aspect of the disclosure may include administering any number of secondary and / or tertiary doses of an anti-4-1BB agonist, anti-PSMA antibody, or bispecific antigen-binding molecule that specifically binds to PSMA and CD3 to a patient. For example, in certain embodiments, only a single secondary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to a patient. Similarly, in certain embodiments, only a single tertiary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to a patient.

[0219] In embodiments including multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may be adjusted during the course of treatment by a physician depending on the individual patient's needs after clinical testing.

[0220] Diagnostic Uses of Antibodies The bispecific antibodies of the present disclosure can also be used to detect and / or measure PSMA or PSMA-expressing cells in a sample, e.g., for diagnostic purposes. For example, an anti-PSMA antibody or a fragment thereof may be used to diagnose a condition or disease characterized by abnormal expression (e.g., overexpression, underexpression, lack of expression, etc.) of PSMA. An exemplary diagnostic assay for PSMA can include, for example, contacting a sample obtained from a patient with an anti-PSMAxCD3 bispecific antibody, which is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-PSMAxCD3 bispecific antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be, for example, 3 H, 14 C. 32 P, 35 S, or 125 The antibody may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-PSMAxCD3 bispecific antibodies useful herein is for the non-invasive identification and tracking of tumor cells in a subject (e.g., positron emission tomography (PET) imaging). 89 Zr-desferrioxamine-labeled antibodies 89 and Zr-labeled antibodies. (See, e.g., Tavare, R. et al. Cancer Res. 2016 Jan 1;76(1):73-82; and Azad, B.B. et al. Oncotarget. 2016 Mar 15;7(11):12344-58.) Specific exemplary assays that can be used to detect or measure PSMA in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0221] The samples that can be used in the PSMA diagnostic assay according to the present disclosure include any tissue or fluid sample that can be obtained from a patient and that contains a detectable amount of PSMA protein or a fragment thereof under normal or pathological conditions. Generally, the PSMA level in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal PSMA levels or activity) is first measured to establish a reference or standard PSMA level. This reference PSMA level can then be compared with the PSMA level measured in a sample obtained from an individual suspected of having a PSMA-related disease (e.g., a tumor containing PSMA-expressing cells) or condition. [Example]

[0222] The following examples are provided so as to more fully disclose to those skilled in the art how to make and use the methods and compositions useful herein, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0223] Example 1: Generation of a bispecific antibody that binds prostate-specific membrane antigen (PSMA) and CD3 The present disclosure provides an anti-PSMA antibody useful according to the method disclosed herein. The antibody is produced according to the disclosure provided in U.S. Patent No. 10,179,819. Examples of antibodies useful herein include the H1H11810P antibody and the CDR, HCVR, and LCVR sequences contained in this antibody. Thus, an exemplary anti-PSMA antibody or its antigen-binding fragment comprises the HCVR of SEQ ID NO: 66 and the LCVR of SEQ ID NO: 1386 disclosed in U.S. Patent No. 10,179,819.

[0224] The present disclosure also provides bispecific antigen-binding molecules that bind to CD3 and prostate-specific membrane antigen (PSMA). Such bispecific antigen-binding molecules are also referred to herein as "anti-PSMA / anti-CD3 bispecific molecules." The anti-PSMA portion of the anti-PSMA / anti-CD3 bispecific molecule is useful for targeting tumor cells that express PSMA, and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. The simultaneous binding of PSMA on tumor cells and CD3 on T cells promotes directed killing (cytolysis) of the targeted tumor cells by the activated T cells.

[0225] Bispecific antibodies comprising an anti-PSMA-specific binding domain and an anti-CD3-specific binding domain were constructed using standard methodologies, with the anti-PSMA antigen-binding domain and the anti-CD3 antigen-binding domain each comprising a distinct HCVR paired with a common LCVR. In some examples, the bispecific antibodies were constructed using a heavy chain derived from an anti-CD3 antibody, a heavy chain derived from an anti-PSMA antibody, and a common light chain. In other examples, the bispecific antibodies were constructed using a heavy chain derived from an anti-CD3 antibody, a heavy chain derived from an anti-PSMA antibody, and a light chain derived from an anti-CD3 antibody. In some examples, the bispecific antibodies were constructed using a HCVR derived from an anti-CD3 antibody, a HCVR derived from an anti-PSMA antibody, and a common LCVR. In other examples, the bispecific antibodies were constructed using a HCVR derived from an anti-CD3 antibody, a HCVR derived from an anti-PSMA antibody, and a LCVR derived from an anti-CD3 antibody.

[0226] A summary of the component parts of exemplary anti-PSMAxCD3 bispecific antibody constructs is provided in Table 1. [Table 1]

[0227] Example 2: PSMA-targeted CD3 bispecifics induce anti-tumor responses that are enhanced by 4-1BB costimulation We evaluated the PSMAxCD3 bispecific antibody, which targets the prostate cancer tumor antigen PSMA, in several preclinical solid tumor models. Mice humanized for CD3 and PSMA were developed to examine antitumor efficacy in the presence of an intact immune system and PSMA expression in normal tissues. ImmunoPET imaging demonstrated that PSMAxCD3 accumulated in PSMA-expressing tissues and tumors, correlating with significant antitumor efficacy. However, PSMAxCD3 lost efficacy with increasing tumor burden. To enhance efficacy in mice with higher tumor burden, we combined PSMAxCD3 with anti-4-1BB (InVivoPlus anti-mouse 4-1bb, isotype rat IgG1, catalog no. BP0169) costimulation. This successfully stimulated T cell activation, cytokine production, proliferation, and memory, resulting in enhanced efficacy and a durable antitumor response. This example demonstrates that a CD3 bispecific antibody combined with anti-4-1BB costimulation is a viable therapeutic combination for solid tumors.

[0228] In all studies in this example, a CD3 bispecific with an irrelevant targeting arm (CD3 binding control) was used as a control. In vivo efficacy was evaluated in both xenogeneic and syngeneic mouse models. For the xenogeneic model, NSG mice were implanted with human PBMCs and C4-2 or 22Rv1 cells (sample size: 5 mice per group). For the syngeneic model, HuT mice were implanted with TRAMP-C2-hPSMA cells (sample size: 5–10 mice per group). All animal studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

[0229] PSMAxCD3 induces target-dependent T cell activation and tumor cell cytotoxicity The PSMAxCD3 bispecific antigen-binding molecule was generated by immunizing VelocImmune® mice with human PSMA and CD3. The resulting PSMAxCD3 bispecific antibody is a hinge-stabilized, effector-minimized IgG4 isotype.

[0230] Flow cytometry analysis was used to determine the binding of PSMAxCD3 to JURKAT and preactivated human T cells, followed by detection with PE-anti-human IgG antibody. Human T cells were preactivated with anti-CD3 / CD28 for 6 days. After activation, 2 × 10 5 Activated human T cells or JURKAT cells per well were incubated with 10 μg / ml PSMAxCD3 for 30 minutes at 4°C. After incubation, cells were washed twice with cold PBS (1% FBS). After washing, PE-anti-human secondary antibody was added to the cells and incubated for an additional 30 minutes. Wells containing no antibody or secondary antibody only served as controls. After incubation, cells were analyzed by flow cytometry on a BD FACS Canto II.

[0231] Flow cytometry analysis was also used to determine binding of PSMAxCD3 to PSMA-expressing cell lines: C4-2, 22Rv1, or TRAMP-C2-hPSMA cells (2 × 10 6 Cells (100 μg / well) were incubated with PSMAxCD3 (10 μg / ml) for 15 minutes at 4°C. After incubation, cells were washed twice with cold PBS (2% FBS) and APC-anti-human secondary antibody was added for an additional 20 minutes on ice. No staining or staining with secondary antibody only was included as controls. Samples were analyzed on a BD LSRFortessa cell analyzer.

[0232] Briefly, PSMA-expressing cell lines (22Rv1 and C4-2 cells) were labeled with 1 μM Violet Cell Tracker and seeded overnight at 37°C. Separately, human PBMCs were added at 1 × 10 6The cells were seeded in RPMI medium supplemented with 0.1% PBS-C1000 cells / mL and incubated overnight at 37°C to enrich for lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The following day, target cells were co-incubated with naive PBMCs (effector / target cells 4:1) depleted of adherent cells and serial dilutions of either PSMAxCD3 or a CD3-binding control for 48 hours at 37°C. Cells were removed from the culture plate using enzyme-free dissociation buffer and analyzed by flow cytometry. For FACS analysis, cells were stained with a dead / live far-red cell tracker (Invitrogen). To assess killing specificity, cells were gated on the violet cell tracker-labeled population. To calculate adjusted viability, the percentage of live target cells was reported as follows: adjusted viability = (R1 / R2) * 100, where R1 = the percentage of live target cells in the presence of antibody, and R2 = the percentage of live target cells in the absence of test antibody. T cell activation was assessed by directly incubating cells with conjugated antibodies against CD2, CD69, and CD25 and reporting the percentage of activated (CD69+) or (CD25+) T cells among total T cells (CD2+).

[0233] Flow cytometry analysis demonstrated that PSMAxCD3 specifically bound to CD3 on Jurkat T cells and human PBMCs (Figure 1A). Furthermore, PSMAxCD3 specifically bound to 22Rv1 and C4-2, human tumor cell lines expressing different levels of PSMA, indicating that PSMAxCD3 can bind to both low- and high-antigen-expressing cell lines (Figure 1B). To evaluate the cytotoxic potential of PSMAxCD3, an in vitro flow cytometry-based cell killing assay was performed. PSMAxCD3 specifically bound to CD3 on Jurkat T cells and human PBMCs (Figure 1A). Furthermore, PSMAxCD3 specifically bound to 22Rv1 (EC50 1.79x10). -11 ) cells and C4-2 (EC50 2.23x10 -11) induced cell killing, whereas the CD3-binding control did not (Figure 1C). In response to PSMAxCD3, the early activation marker CD69 (Figure 1D) and the late activation marker CD25 (Figure 1E) were elevated on T cells. PSMAxCD3 also induced cytokine release (IFNγ and TNFα) when T cells were incubated with C4-2 or 22Rv1 tumor cells (Figure 1F, G).

[0234] Collectively, these results demonstrate that PSMAxCD3 induces target-dependent CD3-mediated T cell activation, resulting in the killing of PSMA-expressing tumor cells.

[0235] PSMAxCD3 inhibits the growth of human prostate cancer cells in xenogeneic tumor models Two subcutaneous tumor xenograft mouse models were constructed using the 22Rv1 and C4-2 human tumor cell lines. Human PBMCs were delivered to NSG mice as a source of human CD3 T cells at the time of tumor implantation, and the mice were immediately treated with a CD3-binding control or PSMAxCD3. Mice implanted with 22Rv1 tumor cells exhibited tumor growth inhibition at 0.1 mg / kg and 1 mg / kg of PSMAxCD3 (Figure 2A), whereas mice implanted with C4-2 tumor cells exhibited significant tumor growth inhibition at as low as 0.01 mg / kg of PSMAxCD3 (Figure 2B).

[0236] Syngeneic tumor studies A syngeneic study was conducted in mice genetically modified to express human CD3 and portions of human PSMA using VelociGene® technology. Mice (5-7 per group, 8-16 weeks old) were administered 5x10 6TRAMP-C2-hPSMA cells were injected subcutaneously (SC). Mice received 5 mg / kg of PSMAxCD3 or a CD3-binding control twice weekly for a total of four treatments. Tumor growth was measured using calipers. Tumor volume based on caliper measurements was calculated by the formula: volume = (length x width) / 2. For studies using PSMAxCD3 + anti-4-1BB (LOB12.3, BioXcell), the CD3-binding control group was treated with a rat-IgG isotype control, and the anti-4-1BB group was treated with a CD3-binding control. For tumor memory studies, mice whose tumors had cleared in response to treatment were injected with 1 x 10 cells into the opposite flank 35 days after tumor injection. 7 The mice were re-challenged with TRAMP-C2-hPSMA cells.

[0237] Preparation of immunoconjugates and small animal PET PET / CT images were acquired using a pre-calibrated Sofie Biosciences G8 PET / CT system (Sofie Biosciences, Culver City, CA, and Perkin Elmer). The energy window ranged from 150 to 650 keV with a reconstructed resolution of 1.4 mm in the center of the field of view. Six days after administration, mice underwent induction anesthesia using isoflurane and were placed under continuous flow of isoflurane for 10 minutes of static PET imaging followed by CT imaging. Attenuation-corrected PET and CT data were processed into false-color co-registered PET-CT maximum intensity projections using VivoQuant software (Invicro Imaging Services) with a color scale calibrated to indicate a signal range of 0–15% of the injected dose per volume, expressed as %ID / g. For ex vivo biodistribution analysis, mice were euthanized after PET / CT imaging. Blood, normal tissues, and tumors were then collected and placed in counting tubes. The gamma-emitting radioactivity of all samples was then counted in an automatic gamma counter (AMG, Hidex), and results were reported in normalized counts per minute (cpm). The %ID for each sample was determined using the sample count relative to a dose standard count prepared from the original injection material. Individual %ID / g values ​​were then derived by dividing the %ID by the weight of the appropriate blood, tissue, or tumor sample.

[0238] ImmunoPET imaging demonstrates in vivo biodistribution of PSMAxCD3 in HuT mice Xenogeneic models use immunodeficient mice lacking mature B cells, T cells, and NK cells. To examine the efficacy of PSMAxCD3 in an immunocompetent mouse model, human-targeted mice (HuT) were genetically engineered to express human PSMA and CD3 by deleting the mouse sequence and replacing it with the orthologous regions of human CD3 and PSMA. Expression of human PSMA transcripts was detected in the spinal cord, brain, liver, kidney, testis, and salivary gland, but negligible expression was observed in the prostate (Figure 3A). Furthermore, PSMA protein expression was also confirmed by immunohistochemistry, showing a similar expression pattern (data not shown, Skokos et al., submitted). To determine the in vivo bioavailability of the PSMA antigen and the distribution of PSMAxCD3 in HuT mice, immunoPET (iPET) imaging was used to track antibody localization. HuT mice were then treated with iPET. 89 Zr-anti-PSMA (bivalent antibody used to generate PSMAxCD3), 89 Zr-PSMAxCD3 or 89 A Zr-CD3 conjugate control was injected to assess tissue distribution. 89 There was no specific targeting in mice injected with the Zr-CD3 conjugate control. 89 Mice injected with Zr-anti-PSMA showed specific uptake in the liver, kidney, epididymis, lacrimal gland, salivary gland, and draining lymph nodes. Notably, although the brain and testis were identified as PSMA-expressing tissues, iPET did not demonstrate targeting, likely due to the blood-brain barrier and inaccessibility of the antigen. 89 Mice injected with Zr-PSMAxCD3 showed no bivalent uptake except for decreased kidney uptake and increased spleen uptake. 89 The distribution profile of PSMAxCD3 was similar to that of Zr-anti-PSMA, indicating that the distribution of PSMAxCD3 was primarily due to the PSMA-binding arm (Figures 3B and 3C). To confirm this, we examined the serum drug clearance in mice humanized with CD3 alone or with PSMA. While serum drug concentrations in HuT(CD3) mice were similar to those in WT mice, HuT(PSMA and CD3) mice showed a faster serum drug clearance (Figure 3D).

[0239] Finally, humanization of these mice did not alter the polyclonal development of splenic CD8 and CD4 T cells, as determined by T cell receptor (TCR) Vβ usage. HuT mice also have similar total T cell numbers and relative proportions of CD4, CD8, and regulatory T cells (Tregs) compared to WT mice (data not shown; Crawford et al., Sci. Transl. Med. 11, eaau7534 (2019)).

[0240] Taken together, these data demonstrated that PSMAxCD3 distribution is driven by the PSMA-binding arm and localizes to select antigen-expressing tissues in HuT mice.

[0241] PSMAxCD3 is effective against small established tumors in HuT mice HuT mice were subcutaneously implanted with a mouse prostate adenocarcinoma cell line expressing human PSMA (TRAMP-C2-hPSMA). PSMAxCD3 treatment initiated on the day of tumor implantation completely prevented tumor growth compared with mice receiving the CD3-conjugated control (Figure 4A). Tumors grew approximately 50 mm. 3 PSMAxCD3 treatment initiated when tumors were approximately 200 mm (Figure 4B) also demonstrated significant antitumor efficacy. However, despite the significant efficacy induced by these treatment regimens, 3 Delaying treatment until 200 mm tumors resulted in reduced antitumor efficacy, indicating a short but transient antitumor response (Figure 4C). Flow cytometry confirmed that PSMA target expression was still maintained in TRAMP-C2-hPSMA tumors, indicating that the lack of efficacy was not due to the absence of target. Furthermore, a high dose of PSMAxCD3 at 20 mg / kg significantly reduced tumor growth, even when PSMA target expression was maintained at 200 mm tumors. 3 However, the efficacy of cyclosporine in the treatment of rheumatoid arthritis was still insufficient to control the tumors (data not shown).

[0242] PSMAxCD3 targets tumors regardless of size, but efficacy is limited to smaller tumors To determine whether antitumor efficacy was determined by the local tumor environment or the total tumor burden in mice, a bilateral tumor model was established, with each mouse bearing a large and small tumor on opposite flanks. HuT mice were inoculated with 1 × 10 7 (left flank) and 1.25 x 10 6 (Right flank) TRAMP-C2-hPSMA cells were injected subcutaneously (SC). Tumors were approximately 150 mm 3 (left flank) and 50 mm 3 On day 12, when the tumors reached size (right flank), mice were administered 5 mg / kg of PSMAxCD3 or a CD3-binding control twice weekly for a total of four treatments.

[0243] PSMAxCD3 was able to slow tumor progression in smaller tumors (Figure 5A), but had no effect on larger tumors in the opposite flank of the same animal (Figure 5B). These findings suggested that the efficacy of PSMAxCD3 was determined by tumor-intrinsic factors, rather than total tumor burden or systemic T cell dysfunction. Subsequently, to determine whether PSMAxCD3 could penetrate larger tumors, 89 Zr-PSMAxCD3 or 89 Zr-CD3 conjugate control was injected into HuT mice bearing bilateral tumors. Injected mice showed significant reductions in peripheral tissues and tumors. 89 In contrast, mice showed specific uptake of Zr-PSMAxCD3 in tumors or tissues. 89 The Zr-CD3 conjugated control showed no specific uptake. Furthermore, ex vivo biodistribution analysis confirmed similar uptake of PSMAxCD3 regardless of tumor size, demonstrating that the lack of response was not due to a lack of PSMAxCD3 targeting (Figures 5C and 5D).

[0244] Ex vivo flow cytometry: Flow cytometry was used to detect circulating T cells and to examine the activation status of intratumoral T cells 48 or 96 hours after treatment, or to examine PSMA target retention on tumor cells. Tumors were mechanically disrupted and digested for 9 minutes at 42°C in the presence of collagenase II (175 units / mL; Worthington), collagenase IV (200 units / mL; Gibco), and DNase 1 (400 units / mL; Sigma). The digested material was then passed through a cell strainer. To detect T cells, a combination of CD45 (30-F11, Biolegend), CD90.2 (30-H12, Biolegend), CD8 (53-6.7, BD Pharmingen), CD4 (GK1.5, BD Pharmingen), and FOXP3 (FJK-165, EBiosciences) was used. T cell activation was examined using antibodies against granzyme B (GB11, BD Pharmingen), Ki67 (16A8, Biolegend), and 4-1BB (IAH2, BD Pharmingen). Staining was performed using the Ebioscience FoxP3 staining buffer set. T cells were identified as CD45+, CD90.2+, CD8+, CD4+, or CD4+ FOXP3+.

[0245] PSMAxCD3 induces T cell infiltration and activation in both small and large tumors Tumors were analyzed by immunohistochemistry to assess the frequency and spatial distribution of intratumoral T cells. Five-micrometer paraffin sections of tissue or tumor were stained with either anti-PSMA (ERP6253, ABCAM), anti-CD3 (A045229, DAKO), anti-CD4 (Ab183685, ABCAM), anti-CD8 (4SM15, eBiosciences), or anti-FOXP3 (12653, Cell Signaling Technologies) for IHC using a Ventana Discovery XT (Ventana; Tucson, AZ). Immunohistochemistry was performed on the Discovery XT Automated IHC Staining System using the Ventana DAB Map detection kit. Slides were manually counterstained with hematoxylin (2 min), dehydrated, and coverslipped. Images were acquired with an Aperio AT 2 slide scanner (Leica Biosystems, Buffalo Grove, IL) and analyzed using Indica HALO software (Indica Labs, Corrales, NM). H&E staining was performed by Histoserv, Inc. (Germantown, MD, USA).

[0246] Both large and small tumors were infiltrated with CD4+ and CD8+ T cells at baseline without treatment. Tumors were then examined after treatment with PSMAxCD3 or a CD3-binding control. PSMAxCD3 treatment significantly reduced tumor size by 50 mm 3 and 200mm 3 The treatment promoted an increase in the frequency of CD8+ T cells in both tumors. In contrast, there was no significant effect on the frequency of CD4+ T cells. Furthermore, the frequency of FOXP3+ immunosuppressive Treg cells was similar across all groups (data not shown). Because T cells are present in both large and small tumors, we confirmed the activity of these T cells after administration of PSMAxCD3 or a CD3-binding control.

[0247] Flow cytometry analysis revealed that CD8+ and CD4+ T cells in both large and small tumors after PSMAxCD3 treatment upregulated the cytolytic marker granzyme B and the proliferation marker Ki67 (data not shown). Furthermore, serum cytokine concentrations of IFN-γ, IL-2, and TNF-α were examined after PSMAxCD3 administration to demonstrate T cell activation. Tumor-bearing HuT mice treated with PSMAxCD3 induced systemic cytokine production at 4 hours, but cytokine release returned to baseline levels by 72 hours, indicating a strong but transient T cell response (data not shown). In contrast, PSMAxCD3 combined with anti-4-1BB resulted in increased cytokine release 96 hours after treatment, suggesting a sustained T cell response. These results suggest that while the initial response may be sufficient to eliminate small tumors that have already shrunk in size by 48 hours, T cells are unable to overcome rapidly growing large tumors. Therefore, additional costimulation to promote the proliferation and expansion of tumor-specific T cells may be necessary for the anti-tumor response of large tumors.

[0248] Costimulation of PSMAxCD3 and 4-1BB is highly effective against larger tumors T cells derived from larger tumors were examined for 4-1BB expression. Flow cytometry analysis demonstrated that PSMAxCD3 induced activation-dependent 4-1BB surface expression that was restricted to intratumoral T cells, with no expression observed on splenic T cells (Figure 6A). Next, we determined whether costimulation of the 4-1BB pathway could enhance antitumor efficacy in mice bearing larger tumor burdens. While PSMAxCD3 or anti-4-1BB alone showed some delay in tumor growth, mice treated with a single dose of PSMAxCD3 in combination with anti-4-1BB achieved remarkable antitumor efficacy (Figure 6B), with 50–60% of tumors completely eliminated by day 60 (Figure 6C). Notably, mice receiving PSMAxCD3 in combination with anti-4-1BB experienced transient weight loss when receiving higher doses of PSMAxCD3 in combination with anti-4-1BB. This transient weight loss could be alleviated by reducing the dose of PSMAxCD3 in combination with anti-4-1BB from 5 mg / kg to 1 mg / kg without affecting overall antitumor efficacy (data not shown). Furthermore, mice treated with PSMAxCD3 in combination with anti-4-1BB showed elevated transcript expression of the TRAF1 adaptor protein, which is essential for the 4-1BB-induced activation pathway, as well as upregulation of the survival genes Bcl2, Bcl-XL (Bcl2l1), and BFL-1 (Bcl2a1a) (Figure 6D).

[0249] Costimulation of PSMAxCD3 and 4-1BB enhances CD8 T cell proliferation and prolongs survival We assessed serum cytokine release as an indicator of T cell activation. While mice treated with PSMAxCD3 alone returned to baseline levels, mice treated with PSMAxCD3 in combination with anti-4-1BB showed enhanced and sustained cytokine induction, even 96 hours after treatment (data not shown). Because survival genes are upregulated via the 4-1BB pathway, we examined tumor-infiltrating CD8 and CD4 T cells 96 hours after treatment. Indeed, we observed a significant expansion of the CD8 T cell compartment in mice treated with the combination therapy compared with CD3-binding controls, anti-4-1BB, or PSMAxCD3 alone (Figure 7A). Furthermore, the combination of PSMAxCD3 with anti-4-1BB increased the percentage and total number of granzyme B+ (data not shown) and Ki67+ (data not shown) CD8 T cells, suggesting that the combination therapy induces the expansion of tumor-infiltrating T cells capable of cytotoxic activity and continued proliferation. Although the total number of Treg cells was similar between treatment groups, the CD8+ T cell expansion in mice receiving the combination therapy significantly improved the CD8-to-Treg ratio (data not shown). Mice from which large tumors were removed were re-challenged with TRAMP-C2-hPSMA cells in the opposite flank. Mice receiving the combination therapy were able to control a secondary tumor challenge compared to naive mice, demonstrating the generation of tumor-specific immunological memory (Figure 7B and Table 2). [Table 2]

[0250] Overall, our data demonstrate that PSMAxCD3 can induce short-term T cell activation, cytokine production, and proliferation, while combination with anti-mouse 4-1BB can prolong and enhance these effects, achieving antitumor efficacy even in established tumors. Furthermore, mice treated with PSMAxCD3 or PSMAxCD3 plus anti-4-1BB were protected from secondary tumor challenge.

[0251] conclusion A CD3 bispecific antibody targeting the tumor antigen PSMA (PSMAxCD3) has demonstrated preclinical efficacy in multiple mouse models. Combining PSMAxCD3 with anti-4-1BB achieved durable antitumor activity and resulted in prolonged survival in mice, demonstrating that costimulation can enhance the efficacy of CD3 bispecific antibodies against advanced solid tumors.

[0252] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims. The present invention provides, for example, the following items. (Item 1) A method for treating cancer or inhibiting tumor growth, comprising administering to a subject in need thereof a therapeutically effective amount of each of (a) an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, and (b) an anti-4-1BB agonist. (Item 2) 2. The method of claim 1, wherein the cancer is selected from the group consisting of prostate cancer, kidney cancer, bladder cancer, colorectal cancer, and gastric cancer. (Item 3) 3. The method of item 2, wherein the cancer is prostate cancer. (Item 4) 4. The method of item 3, wherein the prostate cancer is castration-resistant prostate cancer. (Item 5) 2. The method of claim 1, wherein the anti-CD3 / anti-PSMA bispecific antibody and the anti-4-1BB agonist are administered separately. (Item 6) 2. The method of claim 1, wherein the anti-CD3 / anti-PSMA bispecific antibody and the anti-4-1BB agonist are administered simultaneously. (Item 7) 2. The method of claim 1, wherein the anti-CD3 / anti-PSMA bispecific antibody is administered before, simultaneously with, or after the anti-4-1BB agonist. (Item 8) 8. The method of claim 7, wherein the anti-CD3 / anti-PSMA bispecific antibody is administered before the anti-4-1BB agonist. (Item 9) 8. The method of claim 7, wherein the anti-CD3 / anti-PSMA bispecific antibody is administered on the same day as the anti-4-1BB agonist. (Item 10) 10. The method of any one of items 1 to 9, wherein the anti-CD3 / anti-PSMA bispecific antibody is administered in combination with the anti-4-1BB agonist. (Item 11) 2. The method of claim 1, wherein the anti-4-1BB agonist is selected from a small molecule or an antibody. (Item 12) Item 12. The method of item 11, wherein the anti-4-1BB agonist is an antibody selected from the group consisting of urelumab and utomilumab. (Item 13) 13. The method of any one of Items 1 to 12, wherein the bispecific antigen-binding molecule comprises a first antigen-binding domain that specifically binds to human CD3 and comprises the heavy chain variable region (HCVR-1) amino acid sequence of SEQ ID NO: 2. (Item 14) 14. The method of any one of Items 1 to 13, wherein the bispecific antigen-binding molecule comprises a second antigen-binding domain, wherein the second antigen-binding domain specifically binds to human PSMA and comprises the heavy chain variable region (HCVR-2) amino acid sequence of SEQ ID NO: 1. (Item 15) 15. The method of any one of items 13 and 14, wherein the bispecific antigen-binding molecule comprises a first antigen-binding domain that specifically binds to CD3 and comprises the HCVR-1 amino acid sequence of SEQ ID NO: 2, and a second antigen-binding domain that specifically binds to PSMA and comprises the HCVR-2 amino acid sequence of SEQ ID NO: 1. (Item 16) 16. The method of any one of items 1 to 15, wherein the bispecific antigen-binding molecule comprises a consensus LCVR of SEQ ID NO: 3. (Item 17) 2. The method of item 1, wherein the tumor volume is reduced compared to treatment in the absence of the anti-4-1BB agonist. (Item 18) 2. The method of item 1, wherein tumor-free survival is increased compared to treatment in the absence of the anti-4-1BB agonist. (Item 19) 2. The method of claim 1, wherein TRAF1 expression in the subject's tumor is increased by at least about 4-fold compared to TRAF1 expression in the subject's tumor administered the anti-CD3 / anti-PSMA bispecific antigen binding molecule in the absence of an anti-4-1BB agonist. (Item 20) 2. The method of claim 1, wherein Bcl2 expression in the subject's tumor is increased by at least about 2-fold compared to Bcl2 expression in the subject's tumor administered the anti-CD3 / anti-PSMA bispecific antigen binding molecule in the absence of an anti-4-1BB agonist. (Item 21) 2. The method of claim 1, wherein BFL-1 expression in the tumor of the subject is increased by at least about 3-fold compared to BFL-1 expression in the tumor of the subject administered the anti-CD3 / anti-PSMA bispecific antigen binding molecule in the absence of an anti-4-1BB agonist. (Item 22) 2. The method of claim 1, wherein CD8+ T cell proliferation and / or CD8+ T cell survival in the tumor of the subject is increased compared to CD8+ T cells in the tumor of a subject administered the anti-CD3 / anti-PSMA bispecific antigen binding molecule in the absence of an anti-4-1BB agonist. (Item 23) A method for increasing the proliferation of CD8+ T cells in tumor tissue, comprising administering to a subject in need thereof a therapeutically effective amount of each of (a) an anti-CD3 / anti-PSMA bispecific antigen-binding molecule and (b) an anti-4-1BB agonist. (Item 24) 24. The method of claim 23, wherein the ratio of CD8+ T cells to Tregs in the tumor tissue of a subject treated with the anti-CD3 / anti-PSMA bispecific antigen-binding molecule plus an anti-4-1BB agonist is increased compared to the ratio of CD8+ T cells to Tregs in the tumor tissue of a subject treated with the anti-CD3 / anti-PSMA bispecific antigen-binding molecule in the absence of the anti-4-1BB agonist. (Item 25) 2. The method of claim 1, wherein subsequent exposure to tumor cells induces a memory response in the subject treated with the anti-CD3 / anti-PSMA bispecific antigen binding molecule in the presence of an anti-4-1BB agonist. (Item 26) A method for inducing and / or enhancing a T cell response against a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of each of (a) an anti-CD3 / anti-PSMA bispecific antigen-binding molecule and (b) an anti-4-1BB agonist. (Item 27) 1. A pharmaceutical composition comprising: (a) a bispecific antigen-binding molecule comprising: (i) a first antigen-binding domain that specifically binds to human CD3 and comprises the HCVR-1 amino acid sequence of SEQ ID NO: 2; and (ii) a second antigen-binding domain that specifically binds to human PSMA and comprises the HCVR-2 amino acid sequence of SEQ ID NO: 1; (b) an anti-4-1BB agonist, and (c) A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent. (Item 28) 28. The pharmaceutical composition of item 27, wherein the bispecific antigen-binding molecule of part (a) comprises the consensus LCVR amino acid sequence of SEQ ID NO: 3. (Item 29) A radiolabeled bispecific antibody conjugate comprising a bispecific antigen-binding molecule that binds to PSMA and CD3, a chelating moiety, and a positron emitter. (Item 30) The bispecific antigen-binding molecule has the formula (A): -LM Z (A) is covalently bonded to said chelating moiety L, 30. The conjugate according to item 29, wherein M is the positron emitter, z is independently in each occurrence 0 or 1, and at least one of the z is 1. (Item 31) 31. The conjugate of claim 29 or 30, wherein the chelating moiety comprises desferrioxamine. (Item 32) The positron emitter is 89 32. The conjugate according to any one of items 29 to 31, wherein Zr. (Item 33) -LM,

change

Claims

1. 1. A combination for treating a PSMA-positive cancer or inhibiting the growth of a PSMA-positive tumor in a subject in need thereof, comprising: (a) an anti-CD3 / anti-PSMA bispecific antigen-binding molecule; and (b) a 4-1BB agonist, wherein the 4-1BB agonist is an antibody; and the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is: a first antigen-binding domain that specifically binds to human CD3 and comprises three heavy chain complementarity determining regions (HCDR1-1, HCDR2-1, and HCDR3-1) amino acid sequences within the heavy chain variable region (HCVR-1) amino acid sequence of SEQ ID NO: 2 and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; a second antigen-binding domain that specifically binds to human PSMA and includes three heavy chain complementarity determining regions (HCDR1-2, HCDR2-2, and HCDR3-2) amino acid sequences within the heavy chain variable region (HCVR-2) amino acid sequence of SEQ ID NO: 1 and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; and A combination comprising:

2. 2. The combination of claim 1, wherein the cancer is selected from the group consisting of prostate cancer, kidney cancer, bladder cancer, colorectal cancer, and gastric cancer.

3. The combination according to claim 2, wherein the cancer is prostate cancer.

4. The combination of claim 3, wherein the prostate cancer is castration-resistant prostate cancer.

5. The combination of claim 1, wherein the anti-CD3 / anti-PSMA bispecific antigen-binding molecule and the 4-1BB agonist are administered separately.

6. The combination of claim 1, wherein the anti-CD3 / anti-PSMA bispecific antigen-binding molecule and the 4-1BB agonist are administered simultaneously.

7. The combination of claim 1, wherein the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is administered before, simultaneously with, or after the 4-1BB agonist.

8. The combination of claim 7, wherein the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is administered before the 4-1BB agonist.

9. The combination of claim 7, wherein the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is administered on the same day as the 4-1BB agonist.

10. The combination of any one of claims 1 to 9, wherein the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is administered in combination with the 4-1BB agonist.

11. The combination of claim 1, wherein the 4-1BB agonist is selected from the group consisting of urelumab and utomilumab.

12. The combination according to any one of claims 1 to 11, wherein the first antigen-binding domain comprises the HCVR-1 amino acid sequence of SEQ ID NO:

2.

13. The combination according to any one of claims 1 to 12, wherein the second antigen-binding domain comprises the HCVR-2 amino acid sequence of SEQ ID NO:

1.

14. The combination of claim 12 or 13, wherein the first antigen-binding domain comprises the HCVR-1 amino acid sequence of SEQ ID NO: 2, and the second antigen-binding domain comprises the HCVR-2 amino acid sequence of SEQ ID NO:

1.

15. The combination according to any one of claims 1 to 14, wherein the bispecific antigen-binding molecule comprises the LCVR of SEQ ID NO:

3.

16. The combination of claim 1, wherein the volume of the tumor is reduced compared to treatment in the absence of a 4-1BB agonist.

17. The combination of claim 1, wherein tumor-free survival is increased compared to treatment in the absence of a 4-1BB agonist.

18. The combination of claim 1, wherein TRAF1 expression in the tumor of the subject is increased by at least about 4-fold compared to TRAF1 expression in the tumor of a subject administered the anti-CD3 / anti-PSMA bispecific antigen-binding molecule in the absence of a 4-1BB agonist.

19. 2. The combination of claim 1, wherein Bcl2 expression in the tumor of the subject is increased by at least about 2-fold compared to Bcl2 expression in the tumor of a subject administered the anti-CD3 / anti-PSMA bispecific antigen binding molecule in the absence of a 4-1BB agonist.

20. The combination of claim 1, wherein BFL-1 expression in the tumor of the subject is increased by at least about 3-fold compared to BFL-1 expression in the tumor of a subject administered the anti-CD3 / anti-PSMA bispecific antigen-binding molecule in the absence of a 4-1BB agonist.

21. 2. The combination of claim 1, wherein proliferation of CD8+ T cells in the tumor of a subject is increased and / or survival of CD8+ T cells is enhanced compared to CD8+ T cells in the tumor of a subject administered the anti-CD3 / anti-PSMA bispecific antigen-binding molecule in the absence of a 4-1BB agonist.

22. 1. A combination for increasing proliferation of CD8+ T cells in PSMA-positive tumor tissue in a subject in need thereof, comprising: (a) an anti-CD3 / anti-PSMA bispecific antigen-binding molecule; and (b) a 4-1BB agonist, wherein the 4-1BB agonist is an antibody, and the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is a first antigen-binding domain that specifically binds to human CD3 and comprises HCDR1-1, HCDR2-1, and HCDR3-1 amino acid sequences within the HCVR-1 amino acid sequence of SEQ ID NO: 2, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; a second antigen-binding domain that specifically binds to human PSMA and comprises HCDR1-2, HCDR2-2, and HCDR3-2 amino acid sequences within the HCVR-2 amino acid sequence of SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; and A combination comprising:

23. The combination of claim 22, wherein the ratio of CD8+ T cells to Tregs in the tumor tissue of a subject treated with the anti-CD3 / anti-PSMA bispecific antigen-binding molecule plus a 4-1BB agonist is increased compared to the ratio of CD8+ T cells to Tregs in the tumor tissue of a subject treated with the anti-CD3 / anti-PSMA bispecific antigen-binding molecule in the absence of a 4-1BB agonist.

24. 2. The combination of claim 1, wherein subsequent exposure to tumor cells induces a memory response in the subject treated with the anti-CD3 / anti-PSMA bispecific antigen binding molecule in the presence of a 4-1BB agonist.

25. 1. A combination for inducing and / or enhancing a T cell response against a PSMA-positive tumor in a subject in need thereof, the combination comprising: (a) an anti-CD3 / anti-PSMA bispecific antigen-binding molecule; and (b) a 4-1BB agonist, wherein the 4-1BB agonist is an antibody, and the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is a first antigen-binding domain that specifically binds to human CD3 and comprises HCDR1-1, HCDR2-1, and HCDR3-1 amino acid sequences within the HCVR-1 amino acid sequence of SEQ ID NO: 2, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; A combination comprising: a second antigen-binding domain that specifically binds to human PSMA and comprises the HCDR1-2, HCDR2-2, and HCDR3-2 amino acid sequences within the HCVR-2 amino acid sequence of SEQ ID NO: 1, and the three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO:

3.

26. A pharmaceutical composition for treating PSMA-positive cancer or inhibiting the growth of a PSMA-positive tumor in a subject in need thereof, comprising: (a) a bispecific antigen-binding molecule comprising: (i) a first antigen-binding domain that specifically binds to human CD3 and comprises the HCVR-1 amino acid sequence of SEQ ID NO: 2; and (ii) a second antigen-binding domain that specifically binds to human PSMA and comprises the HCVR-2 amino acid sequence of SEQ ID NO: 1; (b) a 4-1BB agonist that is an antibody, and (c) comprising a pharmaceutically acceptable carrier or diluent; 1. A pharmaceutical composition, wherein the first antigen-binding domain and the second antigen-binding domain of the bispecific antigen-binding molecule of part (a) each comprise the consensus LCVR amino acid sequence of SEQ ID NO:

3.

27. 1. A composition for treating PSMA-positive cancer or inhibiting the growth of PSMA-positive tumors, comprising an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, wherein the composition is administered to a subject in need of treatment of PSMA-positive cancer or inhibition of the growth of PSMA-positive tumors in combination with a 4-1BB agonist, wherein the 4-1BB agonist is an antibody, and the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is: a first antigen-binding domain that specifically binds to human CD3 and comprises HCDR1-1, HCDR2-1, and HCDR3-1 amino acid sequences within the HCVR-1 amino acid sequence of SEQ ID NO: 2, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; a second antigen-binding domain that specifically binds to human PSMA and comprises HCDR1-2, HCDR2-2, and HCDR3-2 amino acid sequences within the HCVR-2 amino acid sequence of SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; and A composition comprising:

28. 1. A composition for treating PSMA-positive cancer or inhibiting the growth of PSMA-positive tumors, comprising a 4-1BB agonist, wherein the composition is administered to a subject in need of treatment of PSMA-positive cancer or inhibition of the growth of PSMA-positive tumors in combination with an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, wherein the 4-1BB agonist is an antibody, and the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is: a first antigen-binding domain that specifically binds to human CD3 and comprises HCDR1-1, HCDR2-1, and HCDR3-1 amino acid sequences within the HCVR-1 amino acid sequence of SEQ ID NO: 2, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; a second antigen-binding domain that specifically binds to human PSMA and comprises HCDR1-2, HCDR2-2, and HCDR3-2 amino acid sequences within the HCVR-2 amino acid sequence of SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; and A composition comprising:

29. 1. A composition for increasing proliferation of CD8+ T cells in PSMA-positive tumor tissue, comprising an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, wherein the composition is administered to a subject in need thereof in combination with a 4-1BB agonist, wherein the 4-1BB agonist is an antibody, and the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is: a first antigen-binding domain that specifically binds to human CD3 and comprises HCDR1-1, HCDR2-1, and HCDR3-1 amino acid sequences within the HCVR-1 amino acid sequence of SEQ ID NO: 2, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; a second antigen-binding domain that specifically binds to human PSMA and comprises HCDR1-2, HCDR2-2, and HCDR3-2 amino acid sequences within the HCVR-2 amino acid sequence of SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; and A composition comprising:

30. 1. A composition for increasing proliferation of CD8+ T cells in PSMA-positive tumor tissue, comprising a 4-1BB agonist, wherein the composition is administered to a subject in need thereof in combination with an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, wherein the 4-1BB agonist is an antibody, and the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is: a first antigen-binding domain that specifically binds to human CD3 and comprises HCDR1-1, HCDR2-1, and HCDR3-1 amino acid sequences within the HCVR-1 amino acid sequence of SEQ ID NO: 2, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; a second antigen-binding domain that specifically binds to human PSMA and comprises HCDR1-2, HCDR2-2, and HCDR3-2 amino acid sequences within the HCVR-2 amino acid sequence of SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; and A composition comprising:

31. 1. A composition for inducing and / or enhancing a T cell response against a PSMA-positive tumor, comprising an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, wherein the composition is administered to a subject in need of inducing and / or enhancing a T cell response against a PSMA-positive tumor in combination with a 4-1BB agonist, wherein the 4-1BB agonist is an antibody, and the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is: a first antigen-binding domain that specifically binds to human CD3 and comprises HCDR1-1, HCDR2-1, and HCDR3-1 amino acid sequences within the HCVR-1 amino acid sequence of SEQ ID NO: 2, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; a second antigen-binding domain that specifically binds to human PSMA and comprises HCDR1-2, HCDR2-2, and HCDR3-2 amino acid sequences within the HCVR-2 amino acid sequence of SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; and A composition comprising:

32. 1. A composition for inducing and / or enhancing a T cell response against a PSMA-positive tumor, comprising a 4-1BB agonist, wherein the composition is administered to a subject in need of inducing and / or enhancing a T cell response against a PSMA-positive tumor in combination with an anti-CD3 / anti-PSMA bispecific antigen-binding molecule, wherein the 4-1BB agonist is an antibody, and the anti-CD3 / anti-PSMA bispecific antigen-binding molecule is: a first antigen-binding domain that specifically binds to human CD3 and comprises HCDR1-1, HCDR2-1, and HCDR3-1 amino acid sequences within the HCVR-1 amino acid sequence of SEQ ID NO: 2, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; a second antigen-binding domain that specifically binds to human PSMA and comprises HCDR1-2, HCDR2-2, and HCDR3-2 amino acid sequences within the HCVR-2 amino acid sequence of SEQ ID NO: 1, and LCDR1, LCDR2, and LCDR3 amino acid sequences within the LCVR amino acid sequence of SEQ ID NO: 3; and A composition comprising:

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