Monospecific and bispecific antibodies and antibody-drug conjugates targeting nectin 2 (CD112) and PSMA.

Abstract

The present invention relates to an anti-nectin 2 antibody or its antigen-binding fragment, an anti-PSMA antibody or its antigen-binding fragment, a bispecific antibody construct that binds to nectin 2 and PSMA, and antibody-drug conjugates comprising such antibodies. The present invention further relates to antibody-drug conjugates for use in the treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to an anti-nectin 2 antibody or its antigen-binding fragment, an anti-PSMA antibody or its antigen-binding fragment, a bispecific antibody construct that binds to nectin 2 and PSMA, and antibody-drug conjugates comprising such antibodies. The present invention further relates to antibody-drug conjugates for use in the treatment of cancer. [Background technology]

[0002] Prostate cancer (PCa) remains a major cause of cancer-related mortality and morbidity in men, being the second most commonly diagnosed cancer and the sixth leading cause of cancer death worldwide. Predisposition to prostate cancer is high, with over 70% of all cases diagnosed in men over 65 years of age. The development of prostate cancer is a multi-stage process, beginning with benign prostatic hyperplasia (BPH), progressing to high-grade prostatic intraepithelial neoplasia (HGPIN), and eventually becoming invasive and metastatic.

[0003] Treatment typically involves chemotherapy and radiotherapy, with or without a castration-based strategy, but most treatments have limited duration of clinical and survival benefits due to primary and acquired resistance caused by the tumor's adaptive resistance (Sumanasuriya, S. & Bono, JDCold Spring Harb. Perspect. Med. 8, a030635 (2018)). Docetaxel and androgen deprivation therapy (ADT) are widely used to treat prostate cancer. However, almost all patients who undergo ADT usually progress to castration-resistant prostate cancer (CRPC), which can eventually evolve into metastatic castration-resistant prostate cancer (mCRPC) (Nakazawa, M., Paller, C. & Kyprianou, N. Curr. Oncol. Rep. 19, 13 (2017); Saad, F. & Fizazi, K. Urology 86, 852-861 (2015)).

[0004] Therefore, the goal of current research is to find new treatment options and overcome resistance mechanisms.

[0005] Furthermore, aging induction has been controversial in cancer because it benefits tumor growth and harms tumor growth regardless of whether it is caused by conventional therapies or genetic mutations (Collado, M. Future Oncol. 6, 687-689 (2010)).

[0006] While the therapeutic benefits of senescence induction are derived from blocking cell proliferation and activating anti-tumor immune responses, the accumulation of senescent cells can stimulate tumor growth and angiogenesis, leading to recurrence and treatment resistance (Hernandez-Segura, A. et al. Trends Cell Biol. 28, 436-453 (2018)). Removal of senescent cells has become an interesting strategy for improving the effectiveness of treatment. Senescent immune cells, particularly T cells, can also accumulate in the tumor microenvironment, a phenomenon known as immunosenescence, which may contribute to cancer development (Ye, J. et al. EMBO Mol. Med. 6, 1294-1311 (2014); Ye, J. et al. Blood 120, 2021-2031 (2012)). When cancer cells age, the amount of SASP components released affects the recruitment of immune cells, suggesting that the effect of SASP on immune cell populations is a double-edged sword. While SASP-mediated recruitment of T cells, macrophages, and NK cells can prevent tumor initiation and progression, SASP can also increase tumor infiltration by immunosuppressive myeloid cells. In the absence of tumor-derived factors, myeloid cells differentiate into dendritic cells, macrophages, or neutrophils that can contribute to immune surveillance. However, in the presence of tumor-derived factors, these myeloid cells lose their ability to differentiate and inhibit the function of other immune cells, creating an immune tolerance environment that allows tumor progression (Ohtani, N. Inflamm. Regen. 42, 11 (2022); Eggert, T. et al. Cancer Cell 30, 533-547 (2016)). Therefore, the idea of ​​a "one-two punch" approach has been proposed, in which a "senescence-accelerating" drug is used to establish a stable cessation of cell proliferation in cancer cells, and then subsequent treatment is carried out with a drug that can kill senescent cells (senescent cell scavenging agent) (Wang, C. et al. Nature 574, 268-272 (2019); Sieben C. et al., Trends in Cell Biology, 28, Issue 9, 723-737 (2018)).Cell senescence is a promising target for improving the outcomes of current therapies, and several senolytic agents have been identified and obtained from natural products and used in combination with senescence-accelerating drugs.

[0007] Therefore, eliminating senescent cells has emerged as a promising therapeutic strategy for preventing tumor recurrence and metastasis in various cancers. However, senescent tumor cells are difficult to kill because they upregulate several survival-promoting pathways, and current tools for effective senescent cell elimination only include a few compounds suitable for clinical trials (Zhu, Y. et al. Aging 9, 955 - 963 (2017); Zhu, Y. et al. Aging Cell 15, 428 - 435 (2016); Yousefzadeh MJ et al. EBioMedicine, 36, 18 - 28 (2018)).

[0008] The use of immune checkpoint inhibitors (ICIs) has transformed cancer treatment by reactivating the immune system against malignant cells. However, only a small number of patients show a persistent response to ICIs, and resistance to treatment is widespread. Resistance to ICIs may be influenced by both extrinsic and endogenous factors in tumor cells. Prostate cancer is known to have low neoantigen carrying, a highly immunosuppressive microenvironment, and a weak immune response. While a large number of tumor-infiltrating lymphocytes (TILs) is a positive prognostic indicator in many types of cancer, including melanoma and breast cancer, in prostate cancer, most T cells in the tumor are CD4+ regulatory T cells (Tregs). Currently, ongoing clinical trials are evaluating the use of ICIs alone or in combination therapy in metastatic hormone-sensitive and castration-resistant prostate cancer. Immune checkpoint inhibitors (ICIs) have revolutionized current approaches to treating cancer. In a healthy state, immune checkpoints prevent the activation of a strong immune response against normal cells in the body. This regulation is based on the specific binding of immune checkpoints expressed by immune cells to their partner binding proteins. In cancerous states, blocking this interaction via ICIs reactivates the immune system against malignant cells.

[0009] Currently, numerous antibodies and small molecules targeting immune checkpoints such as CTLA-4, PD-1, PD-L1, TIGIT, TIM3, and CD47 are in clinical development. To date, the most widely used ICIs are antibodies targeting CTLA-4 (e.g., ipilimumab), PD1 (pembrolizumab, nivolumab), and PD-L1 (e.g., atelolizumab). While the application of ICIs has improved patient outcomes across various tumor types, a lasting response is seen in only a few percent of patients. In fact, the development of resistance mechanisms to immune checkpoint inhibitors is a widespread clinical event. Even among patients with melanoma, which shows one of the highest positive responses to ICIs, more than 60% of them do not show an objective response to anti-PD-1 therapy (Ott, PA et al. J. Clin. Oncol. 37, 318-327 (2019)).

[0010] The development of ICI resistance depends on both exogenous and endogenous factors in tumor cells. Among the exogenous factors of the tumor, the microbiome, the expression level of PD-L1 on immune cells, and the composition of tumor and peripheral immune cells play important roles in determining treatment outcomes. On the other hand, epigenetic fluctuations, mutational burden, and the expression of nascent antigens are all endogenous characteristics of tumor cells and can indicate to patients whether or not they are responding well to ICI therapy (Bagchi, S., Yuan, R. & Engleman, EGAnnu. Rev. Pathol. 16, 223-249 (2021)).

[0011] Prostate cancer is considered an immunologically low-sensitivity disease, characterized by a weak immune response to it. In fact, it typically exhibits T cell exhaustion, low neoantigen carrying capacity, and a highly immunosuppressive microenvironment (de Bono, JSet al. Nat. Rev. Cancer 20, 455-469 (2020); Krueger, TE, Thorek, DLJ, Meeker, AK, Isaacs, JT & Brennen, WN The Prostate 79, 320-330 (2019)). The tumor microenvironment is primarily composed of Tregs, polarized M2 tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), all of which generally exhibit an immunosuppressive phenotype (Krueger, TE, Thorek, DLJ, Meeker, AK, Isaacs, JT & Brennen, WN The Prostate 79, 320-330 (2019)). In particular, MDSCs have been found to have potent immunosuppressive effects in castration-resistant prostate cancer (CRPC), and interleukin-23 (IL-23) produced by MDSCs can regulate castration resistance by inducing androgen receptor signaling (Calcinotto, A. et al. Nature 559, 363-369 (2018)). Within this framework of reference, several preclinical studies have already demonstrated the beneficial effects of anti-MDSC and ICI combination therapy in improving the effectiveness of immune checkpoint blockade in CRPC models (Calcinotto, A. et al. Nature 559, 363-369 (2018); Lu, X. et al. Nature 543, 728-732 (2017)).

[0012] As mentioned above, a higher number of tumor-infiltrating lymphocytes (TILs) is a prognostic indicator of a good outcome in many types of cancer, including melanoma and breast cancer. In PCa, in addition to a lower proportion of TILs, the majority of T cells in mCRPC tumors are CD4+ regulatory T cells (Tregs), and the low abundance of CD8+ T cells usually exhibits an exhausted phenotype, along with the expression of inhibitory receptors such as PD-1, LAG-3, and TIM-3 (Nava Rodrigues, D. et al. J. Clin. Invest. 128, 4441-4453 (2018); Brady, L. et al. Nat. Commun. 12, 1426 (2021); He, MX et al. Nat. Med. 27, 426-433 (2021)).

[0013] Currently, numerous ongoing clinical trials of ICIs in locally advanced or metastatic hormone-sensitive and castration-resistant prostate cancer are being conducted, in which ICIs are being used alone or in combination therapy (Rebuzzi, SE et al. Cancers 14, 1245 (2022)). Overall, consistent results regarding overall survival have not been observed. Patients who partially benefit from anti-PD1 therapies such as nivolumab and pembrolizumab have been found to have tumors with altered CDK12. Indeed, CDK12-mutated PCa are typically associated with a poor prognosis but show increased carrying of neogenic antigens and lymphocyte infiltration. Furthermore, numerous studies evaluating combination therapy with ICIs and standard therapies have clearly shown better responses in patients with specific pathway abnormalities (e.g., AR-V7 variants, HRD), CDK12-inactivated tumors, and high-frequency MSI tumors (Rebuzzi, SE et al. Cancers 14, 1245 (2022)). These clinical trials highlight the need to identify novel therapeutic targets and strategies to enhance the immune response in PCa patients based on the genetic signature of tumors.

[0014] Less than 10% of patients refractory to chemotherapy benefit from immune checkpoint blockade immunotherapy (Markowski, MC et al. The Prostate 80, 407-411 (2020)). Therefore, mCRPC represents a significant unmet medical need.

[0015] Antibody-drug conjugates (ADCs) are an alternative strategy to ICIs. ADCs consist of a monoclonal antibody covalently bound to a cytotoxic drug via a chemical linker (Fu et al., Signal Transduct Target Ther. 2022;7(1):9). Cytotoxic drugs can be delivered in a specific manner via the binding of allotumor-associated antigens (TAAs) on tumor cells. ADCs have already demonstrated clinical efficacy and are FDA-approved for the treatment of both hematological malignancies and solid tumors (Fu et al., Signal Transduct Target Ther. 2022;7(1):9). A major obstacle to the use of ADCs in solid tumors is the limited availability of tumor-specific targets. Most solid tumor TAAs are also expressed, albeit at lower levels, on non-malignant primary cells in important tissues. The main side effects include hematological toxicity, including neutropenia, thrombocytopenia, leukopenia, and anemia (Fu et al., Signal Transduct Target Ther. 2022;7(1):9). For example, these side effects can be mitigated by altering the Fc domain of the mAb (Oshima et al., 2018).

[0016] Currently, several preclinical trials (Boinapally et al., Eur J Nucl Med Mol Imaging, 2022, 49(13):4369-4381; Machulkin et al., Eur J Med Chem. 2022 Jan 5;227:113936) and clinical trials (Cho et al. Mol Cancer Ther. 2018;17(10):2176-2186; Milowsky et al. Urol Oncol, 2016;34(12):530.e15-530.e21; Petrylak et al., Prostate. 2020 Jan;80(1):99-108) are underway using PSMA-targeted ADCs for the treatment of mCRPC. In the preclinical trials, each ADC demonstrated potent and specific cytotoxicity. Unfortunately, in clinical trials, they demonstrate only limited antitumor activity, accompanied by common treatment-related side effects such as neutropenia and neuropathy. Therefore, the usefulness of this treatment strategy may be limited by several factors. Firstly, heterogeneous expression and / or downregulation of PSMA can lead to tumor escape, difficult pharmacokinetics of adjoining drugs, and commonly observed side effects.

[0017] Understanding adaptive immune responses in advanced prostate cancer (APC) has become crucial, especially after the failure of anti-CTLA-4 (ipilimumab) therapy in Phase III trials (Beer, TM et al. J. Clin. Oncol. Off. J. Am. Chem. Clin. Oncol. 35:40-47 (2017)). Furthermore, PD-L1, a major target of existing immunotherapies, is rarely expressed in prostate cancer (Haffner, MC et al. Am. J. Pathol. 188, 1478-1485 (2018)). Most recently, the largest clinical trial testing anti-CTLA-4 + anti-PD-1 in CRPC did not significantly improve patient clinical outcomes (Sharma, P. et al. Cancer Cell 38, 489-499.e3 (2020)). [Overview of the project]

[0018] The inventors hypothesized that further development of effective ADCs for CRPC should include the identification of robust tumor target antigens that are stably and homogeneously expressed, the incorporation of safety mechanisms to mitigate toxicity, and strategies to overcome the premature release of circulating cytotoxic payloads.

[0019] Therefore, the inventors aimed to identify novel immune checkpoints in prostate cancer and utilize them as new targets for developing immunotherapy strategies for prostate cancer. Through analysis of bulk RNA and single-cell data derived from human patients, we confirmed that the transmembrane protein nectin 2 is upregulated in epithelial prostate cancer cells. Nectin 2 has immunomodulatory functions and interacts with receptors expressed by T cells and NK cells. Nectin 2 upregulation was found to be even higher in in vitro and in vivo aging induction models of therapy-induced aging and PTEN deficiency-induced cellular senescence.

[0020] Based on these findings, nectin 2 is a viable target for removing senescent cancer cells from tumors and reactivating the immune response. Novel scFvs were isolated using a human phage display library for the extracellular domains of nectin 2 and PSMA, and candidates were selected based on their binding to human PCa cell lines expressing nectin 2 and / or PSMA. Their specificity was further confirmed using T cells modified to have chimeric antigen receptors (CARs) incorporating the selected anti-nectin 2 or anti-PSMA scFvs. Furthermore, we observed that the novel anti-nectin 2 scFvs did not bind to non-malignant nectin 2-expressing human cells but selectively bound to nectin 2 epitopes exposed to cancer (in vitro). Importantly, anti-nectin 2 scFv-Fc are internalized into human PCa cells upon staining. Therefore, using these validated scFvs, we develop bispecific ADCs for senescent cell removal targeting nectin 2 and PSMA.

[0021] Therefore, the present invention relates to (i) a heavy chain variable domain (V) comprising CDR1-H of the sequence of sequence number 11, CDR2-H of the sequence of sequence number 12, and CDR3-H of the sequence of sequence number 13. H ), and (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 14, CDR2-L of the sequence of sequence number 15, and CDR3-L of the sequence of sequence number 16. L This relates to an anti-nectin 2 antibody containing ) or its antigen-binding fragment.

[0022] In some embodiments, the anti-nectin 2 antibody or its antigen-binding fragment is scFv.

[0023] The present invention further relates to a polypeptide comprising the anti-nectin 2 scFv of the present invention.

[0024] Furthermore, the heavy chain variable domain (V) of the anti-nectin 2 antibody or its antigen-binding fragment. H ) and light chain variable domain (V L The system also provides pairs of isolated nucleic acids, each containing a sequence encoding ).

[0025] The present invention also provides isolated nucleic acids comprising a sequence encoding the anti-nectin 2 scFv, or a polypeptide containing the anti-nectin 2 scFv.

[0026] The present invention further relates to an antibody-drug conjugate (ADC) comprising (i) the anti-nectin 2 antibody of the present invention or an antigen-binding fragment thereof, (ii) a cytotoxic payload, and (iii) a linker that connects the anti-nectin 2 antibody or its antigen-binding fragment to the cytotoxic payload.

[0027] The present invention also relates to (i) a heavy chain variable domain (V) comprising CDR1-H of the sequence of SEQ ID NO: 20, CDR2-H of the sequence of SEQ ID NO: 21, and CDR3-H of the sequence of SEQ ID NO: 22. H ), and (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 23, CDR2-L of the sequence of sequence number 24, and CDR3-L of the sequence of sequence number 25. LThis relates to an anti-PSMA antibody containing ) or its antigen-binding fragment.

[0028] In some embodiments, the anti-PSMA antibody or its antigen-binding fragment is an scFv.

[0029] The present invention further relates to polypeptides comprising the anti-PSMA scFv of the present invention.

[0030] Furthermore, the heavy chain variable domain (V) of the anti-PSMA antibody or its antigen-binding fragment. H ) and light chain variable domain (V L The system also provides pairs of isolated nucleic acids, each containing a sequence encoding ).

[0031] The present invention also provides isolated nucleic acids comprising an anti-PSMA scFv or a sequence encoding a polypeptide containing the anti-PSMA scFv.

[0032] The present invention further relates to an antibody-drug conjugate (ADC) comprising (i) the anti-PSMA antibody of the present invention or an antigen-binding fragment thereof, (ii) a cytotoxic payload, and (iii) a linker that connects the anti-nectin 2 antibody or its antigen-binding fragment to the cytotoxic payload.

[0033] Furthermore, the present invention provides an ADC comprising a bispecific antibody construct that binds to at least nectin-2 and PSMA, the bispecific antibody, a cytotoxic payload, and a linker that connects the bispecific antibody construct and the cytotoxic payload.

[0034] The present invention further relates to the ADC of the present invention for use in the treatment of cancer, and to pharmaceutical compositions comprising such ADC. [Modes for carrying out the invention]

[0035] definition Prostate-specific membrane antigen (PSMA) is a type II membrane protein expressed in all forms of prostate tissue. The reference sequence for human PSMA is available in the Uniprot database under accession number Q04609 (entry version 222, May 3, 2023).

[0036] "Nectin 2" refers to a type I membrane glycoprotein possessing two Ig-like C2 domains and an Ig-like V domain, which is a plasma membrane component of adherent junctions. The reference sequence for human nectin 2 is available in the Uniprot database under accession number Q92692 (entry version 217, May 3, 2023). Nectin 2 is also known as poliovirus receptor-associated protein-2, poliovirus receptor-like 2, CD112, or PRR-2.

[0037] An antibody can be a natural or conventional immunoglobulin molecule in which two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. The light chain contains two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The specificity of an antibody depends on the structural complementarity between the antibody-binding site and the antigenic determinant. The antibody-binding site is mainly composed of residues in the hypervariable region or "complementarity-determining region" (CDR). Each light and heavy chain of an immunoglobulin has three CDRs, designated as CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and CDR-H3, respectively. Therefore, the antigen-binding site of a conventional antibody contains six CDRs, each consisting of a set of CDRs derived from the V region of the heavy chain and the light chain, respectively.

[0038] A "framework region" (FR) refers to an amino acid sequence inserted between CDRs, i.e., the relatively conserved portions of the light and heavy chain variable regions of immunoglobulins across different types of immunoglobulins. Each of the light and heavy chains of immunoglobulins has four FRs, designated as FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively.

[0039] In the context of the present invention, the definition of CDR / FR in immunoglobulin light chains or heavy chains is determined based on Chothia numbering (Chothia et al., J Mol Biol. 1987;196(4):901-17).

[0040] As used herein, the term “antibody” means a conventional antibody and its antigen-binding fragment, as well as a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0041] The terms “monoclonal antibody” or “mAb,” as used herein, refer to a single primary structure antibody molecule against a specific antigen and should not be interpreted as requiring antibody production by any particular method. Monoclonal antibodies can be produced by a single clone of a B cell or hybridoma, but they can also be produced by recombination, for example, by protein engineering.

[0042] A (typical) antibody "fragment" includes a portion of an intact antibody, particularly the antigen-binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and bispecific or multispecific antibodies formed from antibody fragments.

[0043] The antibody or antibody fragment may be an isotype or a subtype, preferably IgG, and more preferably IgG1.

[0044] The term "bispecific antibody" refers to an antibody having the ability to bind to two distinct epitopes, either on a single antigen or two different antigens. The bispecific antibodies of the present invention can be bivalent, trivalent, or tetravalent. As used herein, "valence", "valency", "multiple valencies", or other grammatical variations thereof mean the number of antigen-binding sites in an antibody molecule. These antigen recognition sites can recognize the same epitope or different epitopes.

[0045] As used herein, the percentage of identity is calculated after pairwise global sequence alignment using the Smith-Waterman algorithm, for example, using EMBOSS Needle with default settings (matrix BLOSUM62, gap open 10, gap extend 0.5, end gap penalty false, end gap open 10, end gap extend 0.5).

[0046] Throughout this application, the term "comprising" should be construed as including all of the specifically recited features, as well as any additional, unspecified features. As used herein, the use of the term "comprising" also discloses embodiments where no features other than the specifically recited features are present (i.e., "consisting of"). Further, the indefinite articles "a" or "an" do not exclude a plurality.

[0047] Anti-nectin 2 antibody The present invention provides a heavy chain variable domain (V H ) comprising CDR1-H of the sequence GYTFTSY (SEQ ID NO: 11), CDR2-H of the sequence SAYNGN (SEQ ID NO: 12), and CDR3-H of the sequence YGWKDAMDY (SEQ ID NO: 13), and a light chain variable domain (V LThis relates to an anti-nectin 2 antibody containing ) or an antibody-conjugated fragment thereof.

[0048] The anti-nectin 2 antibody or antibody fragment may be a recombinant antibody, monoclonal antibody, chimeric antibody, humanized antibody, or human antibody.

[0049] In some embodiments, the anti-nectin 2 antibody or its antibody-conjugated fragment is (a) V containing the sequence of sequence number 17 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto H , (b) V containing the sequence of sequence number 18 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto L , or (c) V containing the sequence of sequence number 17 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto H , and V containing the sequence of sequence number 18 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto. L Includes.

[0050] In some embodiments, the anti-nectin 2 antibody or its antigen-binding fragment binds to the cancer-exposed epitope of nectin 2. Thus, the anti-nectin 2 antibody or its antigen-binding fragment distinguishes between cancerous nectin 2-expressing human cells and non-malignant nectin 2-expressing human cells. In some embodiments, the anti-nectin 2 antibody or its antigen-binding fragment binds to an epitope containing or consisting of the sequence of SEQ ID NO: 28.

[0051] In some embodiments, the anti-nectin 2 antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv).

[0052] In some embodiments, anti-nectin 2 scFv includes (i) CDR1-H of the sequence of SEQ ID NO: 11, CDR2-H of the sequence of SEQ ID NO: 12, and CDR3-H of the sequence of SEQ ID NO: 13. H, and (ii) V containing CDR1-L of sequence number 14, CDR2-L of sequence number 15, and CDR3-L of sequence number 16 L , and (iii)V H and V L Includes a linker that connects them. In some embodiments, the V of anti-nectin 2 scFv H This includes the sequence of sequence number 17, or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0053] In some embodiments, the V of anti-nectin 2 scFv L This includes the sequence of sequence number 18, or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0054] In some embodiments, the V of anti-nectin 2 scFv H It contains the sequence of sequence number 17, or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto, and contains the V of anti-nectin 2 scFv L This includes the sequence of sequence number 18, or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0055] The linker is a chemical linker or a polypeptide linker. In some embodiments, the linker linking the VH and VL of anti-nectin 2 scFv includes the sequence GGGSGGGGSGGGGST (SEQ ID NO: 19), or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0056] Preferably, anti-nectin 2 scFv contains or consists of the sequence of sequence number 10.

[0057] Polypeptides containing anti-nectin 2 scFv also constitute the technology of the present invention.

[0058] In some embodiments, the polypeptide is not a chimeric antigen receptor (CAR), and in particular, is not a CAR that includes an antigen-binding domain that binds to nectin 2, or a CAR that includes an antigen-binding domain that binds to PSMA and an antigen-binding domain that binds to nectin 2.

[0059] The present invention further relates to the V of an anti-nectin 2 antibody or antibody fragment, as disclosed herein. H and V L The present invention relates to pairs of nucleic acids, each encoding a separate chain. The present invention further relates to nucleic acids comprising a sequence encoding anti-nectin 2 scFv, or a polypeptide containing anti-nectin 2 scFv.

[0060] In some embodiments, the nucleic acid includes the sequence of sequence number 9, or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0061] Anti-PSMA antibody The present invention relates to a heavy chain variable domain (V) comprising (i) CDR1-H of the sequence of GFTFSSY of SEQ ID NO: 20, CDR2-H of the sequence of SGSGGS of SEQ ID NO: 21, and CDR3-H of the sequence of APRSKMDY of SEQ ID NO: 22. H ), and (ii) a light chain variable domain (V) containing the CDR1-L of the sequence SGSSSNIGSNTVN of SEQ ID NO: 23, the CDR2-L of the sequence SNNQRPS of SEQ ID NO: 24, and the CDR3-L of the sequence AAWDIVGEQVV of SEQ ID NO: 25 L This relates to an anti-PSMA antibody containing ) or its antigen-binding fragment.

[0062] The anti-PSMA antibody or antibody fragment may be a recombinant antibody, monoclonal antibody, chimeric antibody, humanized antibody, or human antibody.

[0063] In some embodiments, the anti-PSMA antibody or its antigen-binding fragment is, (a) V containing the sequence of sequence number 26 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto H , (b) V containing the sequence of sequence number 27 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto L , or (c) V containing the sequence of sequence number 26 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto H , and V containing the sequence of sequence number 27 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto. L Includes.

[0064] In some embodiments, the anti-PSMA antibody or its antigen-binding fragment is a single-stranded variable fragment (scFv).

[0065] In some embodiments, the anti-PSMA scFv includes (i) CDR1-H of sequence 20, CDR2-H of sequence 21, and CDR3-H of sequence 22. H , and (ii) V containing CDR1-L of sequence 23, CDR2-L of sequence 24, and CDR3-L of sequence 25 L , and (iii)V H and V L Includes a linker that connects them. In some embodiments, the V of anti-PSMA scFv H This includes the sequence of sequence number 26, or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0066] In some embodiments, the V of anti-PSMA scFv L This includes the sequence of sequence number 27, or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0067] In some embodiments, the V of anti-PSMA scFv H This includes the sequence of sequence number 26 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto, and is anti-PSMA scFv V LThis includes the sequence of sequence number 27 or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0068] The linker is a chemical linker or a polypeptide linker. In some embodiments, the linker linking the VH and VL of anti-PSMA scFv includes the sequence GGGSGGGGSGGGGST (SEQ ID NO: 19), or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0069] Preferably, the anti-PSMA scFv contains or consists of the sequence of sequence number 8.

[0070] Polypeptides containing anti-PSMA scFv also constitute the technology of the present invention.

[0071] In some embodiments, the polypeptide is not a CAR, and in particular, does not include an antigen-binding domain that binds to PSMA, or is not a CAR that includes an antigen-binding domain that binds to PSMA and an antigen-binding domain that binds to nectin 2.

[0072] The present invention further relates to the V of an anti-PSMA antibody or antibody fragment, as disclosed herein. H and V L The present invention relates to pairs of nucleic acids, each encoding a separate strand. The present invention further relates to nucleic acids comprising sequences encoding anti-PSMA scFv, or polypeptides containing anti-PSMA scFv.

[0073] In some embodiments, the nucleic acid includes the sequence of SEQ ID NO: 7, or a sequence that is at least 80%, 85%, 90%, 95%, or 98% identical thereto.

[0074] Multispecific antibodies that bind to Nectin-2 and PSMA We also provide multispecific (e.g., bispecific) antibody constructs that bind to at least nectin 2 and PSMA. Such multispecific or bispecific antibody constructs typically comprise (a) a first antigen-binding moiety that specifically binds to nectin 2, and (b) a second antigen-binding moiety that specifically binds to PSMA, wherein the first and second antigen-binding moieties are linked directly or by a linker.

[0075] In some embodiments, the multispecific or bispecific antibody construct comprises an anti-nectin 2 antibody or its antigen-binding fragment, and an anti-PSMA antibody or its antigen-binding fragment, as disclosed herein. In some embodiments, the anti-nectin 2 antibody or its antigen-binding fragment and the anti-PSMA antibody or its antigen-binding fragment are linked by a linker.

[0076] In some embodiments, in multispecific or bispecific antibody constructs, [A] The first antigen-binding moiety that specifically binds to nectin 2 is a heavy chain variable domain (V) containing (i) CDR1-H of the sequence of SEQ ID NO: 11, CDR2-H of the sequence of SEQ ID NO: 12, and CDR3-H of the sequence of SEQ ID NO: 13. H ), and (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 14, CDR2-L of the sequence of sequence number 15, and CDR3-L of the sequence of sequence number 16. L ) including or [B] The second antigen-binding moiety that specifically binds to PSMA is a heavy chain variable domain (V) containing (i) CDR1-H of the sequence of SEQ ID NO: 20, CDR2-H of the sequence of SEQ ID NO: 21, and CDR3-H of the sequence of SEQ ID NO: 22. H ), and (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 23, CDR2-L of the sequence of sequence number 24, and CDR3-L of the sequence of sequence number 25. L ) including or [C] The first antigen-binding moiety that specifically binds to nectin 2 is a heavy chain variable domain (V) containing (i) CDR1-H of the sequence of SEQ ID NO: 11, CDR2-H of the sequence of SEQ ID NO: 12, and CDR3-H of the sequence of SEQ ID NO: 13. H), and (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 14, CDR2-L of the sequence of sequence number 15, and CDR3-L of the sequence of sequence number 16. L ) including and The second antigen-binding moiety that specifically binds to PSMA is a heavy chain variable domain (V) containing (i) CDR1-H of the sequence of SEQ ID NO: 20, CDR2-H of the sequence of SEQ ID NO: 21, and CDR3-H of the sequence of SEQ ID NO: 22. H ), and (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 23, CDR2-L of the sequence of sequence number 24, and CDR3-L of the sequence of sequence number 25. L ) including or [D] The first antigen-binding moiety that specifically binds to nectin 2 is a heavy chain variable domain (V) containing (i) CDR1-H of the sequence of SEQ ID NO: 11, CDR2-H of the sequence of SEQ ID NO: 12, and CDR3-H of the sequence of SEQ ID NO: 13. H ), and (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 14, CDR2-L of the sequence of sequence number 15, and CDR3-L of the sequence of sequence number 16. L ) including and The second antigen-binding moiety that specifically binds to PSMA contains the sequence of SEQ ID NO: 26 or a sequence that is at least 80% identical thereto. H , and / or V containing the sequence of sequence number 27 or a sequence that is at least 80% identical thereto L Includes, [E] V, which has a first antigen-binding moiety that specifically binds to nectin 2 and contains the sequence of SEQ ID NO: 17 or a sequence that is at least 80% identical thereto. H , and / or V containing the sequence of sequence number 18 or a sequence that is at least 80% identical thereto L including and The second antigen-binding moiety that specifically binds to PSMA is a heavy chain variable domain (V) containing (i) CDR1-H of the sequence of SEQ ID NO: 20, CDR2-H of the sequence of SEQ ID NO: 21, and CDR3-H of the sequence of SEQ ID NO: 22. H ), and (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 23, CDR2-L of the sequence of sequence number 24, and CDR3-L of the sequence of sequence number 25.L ) including or [F] V, which has a first antigen-binding moiety that specifically binds to nectin 2 and contains the sequence of SEQ ID NO: 17 or a sequence that is at least 80% identical thereto. H , and / or V containing the sequence of sequence number 18 or a sequence that is at least 80% identical thereto L including and The second antigen-binding moiety that specifically binds to PSMA contains the sequence of SEQ ID NO: 26 or a sequence that is at least 80% identical thereto. H , and / or V containing the sequence of sequence number 27 or a sequence that is at least 80% identical thereto L Includes or [G] V, which has a first antigen-binding moiety that specifically binds to nectin 2 and contains the sequence of SEQ ID NO: 17 or a sequence that is at least 80% identical thereto. H , and V containing the sequence of sequence number 18 or a sequence that is at least 80% identical thereto L including and The second antigen-binding moiety that specifically binds to PSMA contains the sequence of SEQ ID NO: 26 or a sequence that is at least 80% identical thereto. H , and V containing the sequence of sequence number 27 or a sequence that is at least 80% identical thereto L Includes.

[0077] In multispecific or bispecific antibody constructs, the linker is either a chemical linker or a polypeptide linker.

[0078] In some embodiments, the multispecific or bispecific antibody constructs include an anti-nectin 2 antibody and an anti-PSMA antibody, as disclosed herein. The IgG-based bispecific antibodies are structurally similar to native antibodies and all have an Fc region. Various formats of IgG-based bispecific antibodies are available, including knobs-into-holes format or CrossMab format.

[0079] In some embodiments, the multispecific or bispecific antibody construct comprises an anti-nectin 2 antibody fragment and an anti-PSMA antibody fragment, as disclosed herein.

[0080] In some embodiments, the multispecific or bispecific antibody construct comprises anti-nectin 2 scFv and anti-PSMA scFv, as disclosed herein.

[0081] Fragment-based bispecific antibodies consist of variable domains or Fab units of the light and heavy chains derived from two antibodies and lack an Fc region. These fragments are linked together by a linker (e.g., a disulfide bond or non-covalent interaction). Various fragment-based bispecific antibodies are available, including bispecific T cell engagers (BiTEs), TCR mimics, tandem diabodies (TandAbs), biaffinity retargeting (DARTs), or bi-nanobodies (Ma et al., Front Immunol. 2021;12:626616).

[0082] Antibody-drug conjugates The present invention further relates to an antibody-drug conjugate comprising an antibody or antibody fragment as defined herein.

[0083] An antibody-drug conjugate (ADC) typically comprises (i) an antibody or its antigen-binding fragment, (ii) a cytotoxic payload, and (iii) a linker that connects the antibody or its antigen-binding fragment to the cytotoxic payload.

[0084] In some embodiments, the ADC comprises (i) an anti-nectin 2 antibody or its antigen-binding fragment as disclosed herein, (ii) a cytotoxic payload, and (iii) a linker connecting the anti-nectin 2 antibody or its antigen-binding fragment to the cytotoxic payload.

[0085] In some embodiments, the ADC includes (i) an anti-PSMA antibody or its antigen-binding fragment as disclosed herein, (ii) a cytotoxic payload, and (iii) a linker connecting the anti-PSMA antibody or its antigen-binding fragment to the cytotoxic payload.

[0086] In some embodiments, the ADC comprises (i) a multispecific or bispecific antibody construct that binds to at least nectin 2 and PSMA, (ii) a cytotoxic payload, and (iii) a linker that connects the bispecific antibody construct to the cytotoxic payload. In some embodiments, the multispecific or bispecific antibody construct that binds to at least nectin 2 and PSMA comprises an anti-nectin 2 antibody or its antigen-binding fragment as disclosed herein, and an anti-PSMA antibody or its antigen-binding fragment as disclosed herein.

[0087] A suitable ADC linker includes both detachable and non-detachable linkers.

[0088] In some embodiments, the linker is a cleavable linker. Typical examples of such linkers include chemically cleavable linkers (e.g., hydrazone bonds and disulfide bonds) and enzymatically cleavable linkers (e.g., glucuronide bonds and peptide bonds).

[0089] In some embodiments, the linker is an uncleavable linker (e.g., a thioether or maleimidocaproyl group).

[0090] Non-limiting examples of cytotoxic payloads conventionally incorporated into ADCs include tubulin inhibitors (e.g., auristatin derivatives such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), DNA damaging agents (e.g., DNA double-strand break inducers such as calicheamicin, DNA alkylating agents such as duocalmycin, DNA intercalation agents such as topoisomerase I inhibitors, and DNA crosslinking agents such as pyrrolobenzodiazepines (PBD)), and immunomodulators (e.g., TLR agonists). Cytotoxic payloads also include radioligands such as actinium-225, lead-212, gallium-68, lutetium-177, radium-223, and zirconium-89.

[0091] Therapeutic uses and pharmaceutical compositions The ADC of the present invention is intended for use as a pharmaceutical, and in particular for use in treating prostate cancer, especially castration-resistant prostate cancer, or metastatic castration-resistant prostate cancer.

[0092] The present invention further relates to a method for treating cancer in a subject requiring such treatment, comprising administering an ADC disclosed herein to the subject.

[0093] A pharmaceutical composition comprising an ADC disclosed herein and a pharmaceutically acceptable carrier.

[0094] The subjects may be mammals such as primates (e.g., humans, monkeys), rodents (e.g., rats, mice), canids (e.g., dogs), or felines (e.g., cats). Preferably, the subject is humans.

[0095] In some embodiments, at least one prostate cancer treatment agent is used in combination with an ADC. The prostate cancer treatment agent may be selected from the group consisting of chemotherapeutic agents (e.g., docetaxel or enzalutamide) and immune checkpoint inhibitors (e.g., anti-PD1 or anti-TIGIT antibodies).

[0096] The present invention will be further described with reference to the following drawings and embodiments. [Brief explanation of the drawing]

[0097] [Figure 1-1] The nectin-2-PVRIG axis is upregulated in PCa. (A) Expression levels of mRNAs of various immune checkpoints expressed by epithelial and immune cells from bulk RNA data. (B) Schematic diagram of the interaction between nectin-2 and PVR with receptors expressed by T cells and NK cells. (C) Gene expression levels in human prostate cancer samples. Among nectin-like molecules, nectin-2 is the most abundant in epithelial cells. On the other hand, PVRIG is the most expressed receptor in immune cells. [Figure 1-2] The nectin-2-PVRIG axis is upregulated in PCa. (A) Expression levels of mRNAs of various immune checkpoints expressed by epithelial and immune cells from bulk RNA data. (B) Schematic diagram of the interaction between nectin-2 and PVR with receptors expressed by T cells and NK cells. (C) Gene expression levels in human prostate cancer samples. Among nectin-like molecules, nectin-2 is the most abundant in epithelial cells. On the other hand, PVRIG is the most expressed receptor in immune cells.

[0098] [Figure 2-1]Nectin 2 is upregulated in Pten-deficiency-induced cellular senescence (PICS). (A) Experimental design and volcano plot of differentially present proteins between FDG+ and FDG- epithelial cells. (B) Results of Western blot analysis in wild-type and pten- / - prostate samples, and quantification of fold changes compared to wild-type nectin 2 and p21. (C) Real-time PCR data of mRNA expression levels of nectin 2 and p16 in epithelial prostate tumor cells sorted by FDG+ and FDG-. (D) Expression of nectin 2 by RNA sequencing data in wild-type, pten- / - and pten- / -p53- / - mice. (E) PTEN null mice were treated with docetaxel 10 mg / kg once a week for 4 weeks. After 4 weeks, prostates were collected and senescence induction was quantified by β-galactosidase assay on OCT-embedded tissue. (F) The size of the anterior lobe was significantly reduced in treated mice compared to untreated mice, and (G) upregulation of nectin 2 was confirmed by real-time PCR of prostate samples. [Figure 2-2] Nectin 2 is upregulated in Pten-deficiency-induced cellular senescence (PICS). (A) Experimental design and volcano plot of differentially present proteins between FDG+ and FDG- epithelial cells. (B) Results of Western blot analysis in wild-type and pten- / - prostate samples, and quantification of fold changes compared to wild-type nectin 2 and p21. (C) Real-time PCR data of mRNA expression levels of nectin 2 and p16 in epithelial prostate tumor cells sorted by FDG+ and FDG-. (D) Expression of nectin 2 by RNA sequencing data in wild-type, pten- / - and pten- / -p53- / - mice. (E) PTEN null mice were treated with docetaxel 10 mg / kg once a week for 4 weeks. After 4 weeks, prostates were collected and senescence induction was quantified by β-galactosidase assay on OCT-embedded tissue. (F) The size of the anterior lobe was significantly reduced in treated mice compared to untreated mice, and (G) upregulation of nectin 2 was confirmed by real-time PCR of prostate samples. [Figure 2-3]Nectin 2 is upregulated in Pten-deficiency-induced cellular senescence (PICS). (A) Experimental design and volcano plot of differentially present proteins between FDG+ and FDG- epithelial cells. (B) Results of Western blot analysis in wild-type and pten- / - prostate samples, and quantification of fold changes compared to wild-type nectin 2 and p21. (C) Real-time PCR data of mRNA expression levels of nectin 2 and p16 in epithelial prostate tumor cells sorted by FDG+ and FDG-. (D) Expression of nectin 2 by RNA sequencing data in wild-type, pten- / - and pten- / -p53- / - mice. (E) PTEN null mice were treated with docetaxel 10 mg / kg once a week for 4 weeks. After 4 weeks, prostates were collected and senescence induction was quantified by β-galactosidase assay on OCT-embedded tissue. (F) The size of the anterior lobe was significantly reduced in treated mice compared to untreated mice, and (G) upregulation of nectin 2 was confirmed by real-time PCR of prostate samples. [Figure 2-4] Nectin 2 is upregulated in Pten-deficiency-induced cellular senescence (PICS). (A) Experimental design and volcano plot of differentially present proteins between FDG+ and FDG- epithelial cells. (B) Results of Western blot analysis in wild-type and pten- / - prostate samples, and quantification of fold changes compared to wild-type nectin 2 and p21. (C) Real-time PCR data of mRNA expression levels of nectin 2 and p16 in epithelial prostate tumor cells sorted by FDG+ and FDG-. (D) Expression of nectin 2 by RNA sequencing data in wild-type, pten- / - and pten- / -p53- / - mice. (E) PTEN null mice were treated with docetaxel 10 mg / kg once a week for 4 weeks. After 4 weeks, prostates were collected and senescence induction was quantified by β-galactosidase assay on OCT-embedded tissue. (F) The size of the anterior lobe was significantly reduced in treated mice compared to untreated mice, and (G) upregulation of nectin 2 was confirmed by real-time PCR of prostate samples.

[0099] [Figure 3-1] Treatment-induced aging (TIS) further enhances nectin 2 levels in PCa cells. (A) Senescence induction in PC3, 22RV1, and LNCaP human prostate cancer cell lines using palbociclib and docetaxel. Treatments were added to the culture medium for 3 days, then the medium was changed, and cells were seeded for beta-galactosidase assays after 3 days. (B) Real-time expression of nectin 2 in human prostate cancer cell lines after senescence induction using palbociclib or docetaxel, and (C) flow cytometry. (D) Senescence induction during treatment-induced aging, as measured by FDG staining in 22rv1 cells, and (E) mean fluorescence intensity (MFI) of nectin 2 in untreated and treated cells. (F) MFI of nectin 2 cells in FDG- and FDG+ populations, and percentage of FDG- and FDG+ LNCAP cells cultured in untreated or androgen-deficient (ADT). (G) Results of β-galactosidase assay in LNCAP during senescence induction using androgen deficiency, and (H) mRNA levels of nectin 2. [Figure 3-2] Treatment-induced aging (TIS) further enhances nectin 2 levels in PCa cells. (A) Senescence induction in PC3, 22RV1, and LNCaP human prostate cancer cell lines using palbociclib and docetaxel. Treatments were added to the culture medium for 3 days, then the medium was changed, and cells were seeded for beta-galactosidase assays after 3 days. (B) Real-time expression of nectin 2 in human prostate cancer cell lines after senescence induction using palbociclib or docetaxel, and (C) flow cytometry. (D) Senescence induction during treatment-induced aging, as measured by FDG staining in 22rv1 cells, and (E) mean fluorescence intensity (MFI) of nectin 2 in untreated and treated cells. (F) MFI of nectin 2 cells in FDG- and FDG+ populations, and percentage of FDG- and FDG+ LNCAP cells cultured in untreated or androgen-deficient (ADT). (G) Results of β-galactosidase assay in LNCAP during senescence induction using androgen deficiency, and (H) mRNA levels of nectin 2. [Figure 3-3]Treatment-induced aging (TIS) further enhances nectin 2 levels in PCa cells. (A) Senescence induction in PC3, 22RV1, and LNCaP human prostate cancer cell lines using palbociclib and docetaxel. Treatments were added to the culture medium for 3 days, then the medium was changed, and cells were seeded for beta-galactosidase assays after 3 days. (B) Real-time expression of nectin 2 in human prostate cancer cell lines after senescence induction using palbociclib or docetaxel, and (C) flow cytometry. (D) Senescence induction during treatment-induced aging, as measured by FDG staining in 22rv1 cells, and (E) mean fluorescence intensity (MFI) of nectin 2 in untreated and treated cells. (F) MFI of nectin 2 cells in FDG- and FDG+ populations, and percentage of FDG- and FDG+ LNCAP cells cultured in untreated or androgen-deficient (ADT). (G) Results of β-galactosidase assay in LNCAP during senescence induction using androgen deficiency, and (H) mRNA levels of nectin 2. [Figure 3-4] Treatment-induced aging (TIS) further enhances nectin 2 levels in PCa cells. (A) Senescence induction in PC3, 22RV1, and LNCaP human prostate cancer cell lines using palbociclib and docetaxel. Treatments were added to the culture medium for 3 days, then the medium was changed, and cells were seeded for beta-galactosidase assays after 3 days. (B) Real-time expression of nectin 2 in human prostate cancer cell lines after senescence induction using palbociclib or docetaxel, and (C) flow cytometry. (D) Senescence induction during treatment-induced aging, as measured by FDG staining in 22rv1 cells, and (E) mean fluorescence intensity (MFI) of nectin 2 in untreated and treated cells. (F) MFI of nectin 2 cells in FDG- and FDG+ populations, and percentage of FDG- and FDG+ LNCAP cells cultured in untreated or androgen-deficient (ADT). (G) Results of β-galactosidase assay in LNCAP during senescence induction using androgen deficiency, and (H) mRNA levels of nectin 2. [Figure 3-5]Treatment-induced aging (TIS) further enhances nectin 2 levels in PCa cells. (A) Senescence induction in PC3, 22RV1, and LNCaP human prostate cancer cell lines using palbociclib and docetaxel. Treatments were added to the culture medium for 3 days, then the medium was changed, and cells were seeded for beta-galactosidase assays after 3 days. (B) Real-time expression of nectin 2 in human prostate cancer cell lines after senescence induction using palbociclib or docetaxel, and (C) flow cytometry. (D) Senescence induction during treatment-induced aging, as measured by FDG staining in 22rv1 cells, and (E) mean fluorescence intensity (MFI) of nectin 2 in untreated and treated cells. (F) MFI of nectin 2 cells in FDG- and FDG+ populations, and percentage of FDG- and FDG+ LNCAP cells cultured in untreated or androgen-deficient (ADT). (G) Results of β-galactosidase assay in LNCAP during senescence induction using androgen deficiency, and (H) mRNA levels of nectin 2. [Figure 3-6] Treatment-induced aging (TIS) further enhances nectin 2 levels in PCa cells. (A) Senescence induction in PC3, 22RV1, and LNCaP human prostate cancer cell lines using palbociclib and docetaxel. Treatments were added to the culture medium for 3 days, then the medium was changed, and cells were seeded for beta-galactosidase assays after 3 days. (B) Real-time expression of nectin 2 in human prostate cancer cell lines after senescence induction using palbociclib or docetaxel, and (C) flow cytometry. (D) Senescence induction during treatment-induced aging, as measured by FDG staining in 22rv1 cells, and (E) mean fluorescence intensity (MFI) of nectin 2 in untreated and treated cells. (F) MFI of nectin 2 cells in FDG- and FDG+ populations, and percentage of FDG- and FDG+ LNCAP cells cultured in untreated or androgen-deficient (ADT). (G) Results of β-galactosidase assay in LNCAP during senescence induction using androgen deficiency, and (H) mRNA levels of nectin 2. [Figure 3-7]Treatment-induced aging (TIS) further enhances nectin 2 levels in PCa cells. (A) Senescence induction in PC3, 22RV1, and LNCaP human prostate cancer cell lines using palbociclib and docetaxel. Treatments were added to the culture medium for 3 days, then the medium was changed, and cells were seeded for beta-galactosidase assays after 3 days. (B) Real-time expression of nectin 2 in human prostate cancer cell lines after senescence induction using palbociclib or docetaxel, and (C) flow cytometry. (D) Senescence induction during treatment-induced aging, as measured by FDG staining in 22rv1 cells, and (E) mean fluorescence intensity (MFI) of nectin 2 in untreated and treated cells. (F) MFI of nectin 2 cells in FDG- and FDG+ populations, and percentage of FDG- and FDG+ LNCAP cells cultured in untreated or androgen-deficient (ADT). (G) Results of β-galactosidase assay in LNCAP during senescence induction using androgen deficiency, and (H) mRNA levels of nectin 2. [Figure 3-8] Treatment-induced aging (TIS) further enhances nectin 2 levels in PCa cells. (A) Senescence induction in PC3, 22RV1, and LNCaP human prostate cancer cell lines using palbociclib and docetaxel. Treatments were added to the culture medium for 3 days, then the medium was changed, and cells were seeded for beta-galactosidase assays after 3 days. (B) Real-time expression of nectin 2 in human prostate cancer cell lines after senescence induction using palbociclib or docetaxel, and (C) flow cytometry. (D) Senescence induction during treatment-induced aging, as measured by FDG staining in 22rv1 cells, and (E) mean fluorescence intensity (MFI) of nectin 2 in untreated and treated cells. (F) MFI of nectin 2 cells in FDG- and FDG+ populations, and percentage of FDG- and FDG+ LNCAP cells cultured in untreated or androgen-deficient (ADT). (G) Results of β-galactosidase assay in LNCAP during senescence induction using androgen deficiency, and (H) mRNA levels of nectin 2.

[0100] [Figure 4-1] Nectin 2 and PSMA expression are correlated in PCa. (A) Correlation matrix showing Pearson coefficients in human RNA sequencing samples. (B) Scatter plot showing the correlation between Nectin 2 and FOLH1, and between PVR and FOLH1. (C) Percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells. (D) Comparison of percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells from castration-sensitive and castration-resistant prostate cancer patients. (E) Western blot of patient-derived organoids shows a correlation between Nectin 2 and PSMA. (F) Western blot expression levels of PSMA protein in human prostate cancer cell lines at TIS. [Figure 4-2] Nectin 2 and PSMA expression are correlated in PCa. (A) Correlation matrix showing Pearson coefficients in human RNA sequencing samples. (B) Scatter plot showing the correlation between Nectin 2 and FOLH1, and between PVR and FOLH1. (C) Percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells. (D) Comparison of percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells from castration-sensitive and castration-resistant prostate cancer patients. (E) Western blot of patient-derived organoids shows a correlation between Nectin 2 and PSMA. (F) Western blot expression levels of PSMA protein in human prostate cancer cell lines at TIS. [Figure 4-3]Nectin 2 and PSMA expression are correlated in PCa. (A) Correlation matrix showing Pearson coefficients in human RNA sequencing samples. (B) Scatter plot showing the correlation between Nectin 2 and FOLH1, and between PVR and FOLH1. (C) Percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells. (D) Comparison of percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells from castration-sensitive and castration-resistant prostate cancer patients. (E) Western blot of patient-derived organoids shows a correlation between Nectin 2 and PSMA. (F) Western blot expression levels of PSMA protein in human prostate cancer cell lines at TIS. [Figure 4-4] Nectin 2 and PSMA expression are correlated in PCa. (A) Correlation matrix showing Pearson coefficients in human RNA sequencing samples. (B) Scatter plot showing the correlation between Nectin 2 and FOLH1, and between PVR and FOLH1. (C) Percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells. (D) Comparison of percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells from castration-sensitive and castration-resistant prostate cancer patients. (E) Western blot of patient-derived organoids shows a correlation between Nectin 2 and PSMA. (F) Western blot expression levels of PSMA protein in human prostate cancer cell lines at TIS. [Figure 4-5]Nectin 2 and PSMA expression are correlated in PCa. (A) Correlation matrix showing Pearson coefficients in human RNA sequencing samples. (B) Scatter plot showing the correlation between Nectin 2 and FOLH1, and between PVR and FOLH1. (C) Percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells. (D) Comparison of percentages of Nectin 2+PSMA-, Nectin 2+PSMA+, Nectin 2-PSMA+, and Nectin 2-PSMA- in tumor cells from castration-sensitive and castration-resistant prostate cancer patients. (E) Western blot of patient-derived organoids shows a correlation between Nectin 2 and PSMA. (F) Western blot expression levels of PSMA protein in human prostate cancer cell lines at TIS.

[0101] [Figure 5-1] Anti-PSMA and anti-nectin 2 combinatorial CAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cell lines. (A) Expression of target antigens on human PCa cell lines as assessed by flow cytometry. (B) Design of CARs for second-generation monospecific anti-PSMA and anti-nectin 2 CAR constructs. (C) Design of CARs for combinatorial CARs targeting PSMA and nectin 2. (D) Expression of CARs on non-transduction (NTD) and T cells as assessed by flow cytometry. (EF) CombiCAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cells in a 92-hour fluorescence-based lysis assay. (G) ELISA measurement of IFNg secretion by CAR-T cells in co-culture with tumor cells as described in (E-F). [Figure 5-2]Anti-PSMA and anti-nectin 2 combinatorial CAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cell lines. (A) Expression of target antigens on human PCa cell lines as assessed by flow cytometry. (B) Design of CARs for second-generation monospecific anti-PSMA and anti-nectin 2 CAR constructs. (C) Design of CARs for combinatorial CARs targeting PSMA and nectin 2. (D) Expression of CARs on non-transduction (NTD) and T cells as assessed by flow cytometry. (EF) CombiCAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cells in a 92-hour fluorescence-based lysis assay. (G) ELISA measurement of IFNg secretion by CAR-T cells in co-culture with tumor cells as described in (E-F). [Figure 5-3] Anti-PSMA and anti-nectin 2 combinatorial CAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cell lines. (A) Expression of target antigens on human PCa cell lines as assessed by flow cytometry. (B) Design of CARs for second-generation monospecific anti-PSMA and anti-nectin 2 CAR constructs. (C) Design of CARs for combinatorial CARs targeting PSMA and nectin 2. (D) Expression of CARs on non-transduction (NTD) and T cells as assessed by flow cytometry. (EF) CombiCAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cells in a 92-hour fluorescence-based lysis assay. (G) ELISA measurement of IFNg secretion by CAR-T cells in co-culture with tumor cells as described in (E-F). [Figure 5-4]Anti-PSMA and anti-nectin 2 combinatorial CAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cell lines. (A) Expression of target antigens on human PCa cell lines as assessed by flow cytometry. (B) Design of CARs for second-generation monospecific anti-PSMA and anti-nectin 2 CAR constructs. (C) Design of CARs for combinatorial CARs targeting PSMA and nectin 2. (D) Expression of CARs on non-transduction (NTD) and T cells as assessed by flow cytometry. (EF) CombiCAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cells in a 92-hour fluorescence-based lysis assay. (G) ELISA measurement of IFNg secretion by CAR-T cells in co-culture with tumor cells as described in (E-F). [Figure 5-5] Anti-PSMA and anti-nectin 2 combinatorial CAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cell lines. (A) Expression of target antigens on human PCa cell lines as assessed by flow cytometry. (B) Design of CARs for second-generation monospecific anti-PSMA and anti-nectin 2 CAR constructs. (C) Design of CARs for combinatorial CARs targeting PSMA and nectin 2. (D) Expression of CARs on non-transduction (NTD) and T cells as assessed by flow cytometry. (EF) CombiCAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cells in a 92-hour fluorescence-based lysis assay. (G) ELISA measurement of IFNg secretion by CAR-T cells in co-culture with tumor cells as described in (E-F). [Figure 5-6]Anti-PSMA and anti-nectin 2 combinatorial CAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cell lines. (A) Expression of target antigens on human PCa cell lines as assessed by flow cytometry. (B) Design of CARs for second-generation monospecific anti-PSMA and anti-nectin 2 CAR constructs. (C) Design of CARs for combinatorial CARs targeting PSMA and nectin 2. (D) Expression of CARs on non-transduction (NTD) and T cells as assessed by flow cytometry. (EF) CombiCAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cells in a 92-hour fluorescence-based lysis assay. (G) ELISA measurement of IFNg secretion by CAR-T cells in co-culture with tumor cells as described in (E-F). [Figure 5-7] Anti-PSMA and anti-nectin 2 combinatorial CAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cell lines. (A) Expression of target antigens on human PCa cell lines as assessed by flow cytometry. (B) Design of CARs for second-generation monospecific anti-PSMA and anti-nectin 2 CAR constructs. (C) Design of CARs for combinatorial CARs targeting PSMA and nectin 2. (D) Expression of CARs on non-transduction (NTD) and T cells as assessed by flow cytometry. (EF) CombiCAR-T cells specifically lyse PSMA+ / nectin 2+ and PSMA+ / nectin 2-human PCa cells in a 92-hour fluorescence-based lysis assay. (G) ELISA measurement of IFNg secretion by CAR-T cells in co-culture with tumor cells as described in (E-F).

[0102] [Figure 6-1]Identification of novel anti-nectin 2 and anti-PSMA scFv antibodies with high tumor specificity and internalization ability. (A) Quantification of immunofluorescence staining from human non-malignant cells (HEK293T, HUVEC, HK2, BPH, RWPE, MCF10A) and human prostate cancer cell lines (22rv1n LNCAP, PC3, PC3 nectin 2 KO, DU145) using commercially available anti-nectin 2 antibodies or anti-nectin 2 scFv antibodies, respectively. (B) Immunofluorescence staining of LNCaP cells using a secondary anti-human Fc antibody and quantification of detected fluorescence intensity. Cells were stained with anti-nectin 2 scFv-Fc at 37°C for 1 hour, washed, and permeabilized. Nuclei were counterstained with DAPI. [Figure 6-2] Identification of novel anti-nectin 2 and anti-PSMA scFv antibodies with high tumor specificity and internalization ability. (A) Quantification of immunofluorescence staining from human non-malignant cells (HEK293T, HUVEC, HK2, BPH, RWPE, MCF10A) and human prostate cancer cell lines (22rv1n LNCAP, PC3, PC3 nectin 2 KO, DU145) using commercially available anti-nectin 2 antibodies or anti-nectin 2 scFv antibodies, respectively. (B) Immunofluorescence staining of LNCaP cells using a secondary anti-human Fc antibody and quantification of detected fluorescence intensity. Cells were stained with anti-nectin 2 scFv-Fc at 37°C for 1 hour, washed, and permeabilized. Nuclei were counterstained with DAPI.

[0103] [Figure 7] Amino acid sequences of anti-nectin 2 scFv HRB725 showing the CDR of the VH and VL chains; the VH chain sequence is underlined, the (G4S)3 linker between the VH and VL chains is italicized, and the VL chain is shown in regular letters.

[0104] [Figure 8] Amino acid sequence of anti-PSMA scFv HRB730 showing the CDR of the VH and VL chains; the VH chain sequence is underlined, the (G4S)3 linker between the VH and VL chains is italicized, and the VL chain is shown in regular letters.

[0105] [Figure 9]Anti-nectin 2 scFv-Fc(725v5) binds to cancer-exposed epitopes of nectin 2. Anti-nectin 2 scFv-Fc(725v5) selectively binds to nectin 2 when expressed by tumor cells, in contrast to commercially available anti-nectin 2 monoclonal antibodies that bind indiscriminately to both normal and tumor cells expressing nectin 2. [Examples]

[0106] Example 1: Nectin-2 mRNA is highly expressed in prostate cancer cells. To identify novel targets in prostate cancer, the inventors analyzed the expression of the most well-known IC in publicly available datasets. The inventors compared mRNA expression levels of genes expressed by epithelial and immune cells using raw data from the TGCA database of the Human Protein Atlas (https: / / www.proteinatlas.org / (2022)). Interestingly, as shown in Figure 1A, the inventors observed significantly higher expression of nectin 2 in PCa compared to all other targets, including PD1 and CTLA4.

[0107] Nectin-2 is a Ca2+-independent cell-cell adhesion molecule widely expressed on antigen-presenting cells and tumor cells, and its expression is regulated in tumorigenesis. The role of nectin-2 as an immune checkpoint has already been studied in breast and ovarian cancer (Oshima, T. et al. Mol. Cancer 12, 60 (2013)). Overexpression of nectin-2 mRNA in prostate cancer tissue has also been reported (Oshima, T. et al. Mol. Cancer 12, 60 (2013)). Increased protein levels have been observed in various types of cancer, including acute myeloid leukemia, multiple myeloma, gallbladder cancer, and prostate cancer (Sanchez-Correa, B. et al. Immunol. Cell Biol. 90, 109-115 (2012); Casado, J. Get al. Cancer Immunol. Immunother. CII 58, 1517-1526 (2009); WO2020144697A1). While primarily a regulator of cell adhesion, motility, and proliferation, nectin-2 is also immunomodulatory (Takai, Y., et al. Nat. Rev. Mol. Cell Biol. 9, 603-615 (2008)). In fact, this membrane protein can co-stimulate T cells and NK cells by interacting with CD226, or suppress their responses by binding to the inhibitory receptors TIGIT and PVRIG (Zhu, Y. et al. J. Exp. Med. 213, 167-176 (2016)).

[0108] It should be emphasized that PVR, another member of the nectin family, is a ligand for the co-stimulatory receptor CD226 and the co-inhibitory receptor TIGIT (Figure 1B). On the other hand, due to the weakness of the nectin 2-TIGIT interaction, the PVR receptor is still considered a strong ligand for TIGIT in this ligand-receptor network (Yu, X. et al. Nat. Immunol. 10, 48-57 (2009)).

[0109] However, the nectin 2 / TIGIT / PVRIG axis in prostate cancer has not yet been studied.

[0110] Example 2: PVRIG is a major nectin-2 interacting agent in the microenvironment of prostate tumors. To better understand the role of nectin 2 in prostate cancer, the inventors then evaluated the expression of its ligand / interacting substances in bulk RNA sequencing data from human prostate cancer samples.

[0111] The inventors observed high expression levels of nectin-2 and low expression of TIGIT, suggesting that PVRIG is the major nectin-2 interacting agent in the prostate tumor microenvironment (Figure 1B). Interestingly, by analyzing single-cell RNA sequencing data from PCa biopsies, the inventors confirmed that nectin-2 is primarily expressed by epithelial and endothelial cells (Figure 1C). These data were obtained from 13 tissue samples derived from 12 primary tumors and one lymph node metastasis, and after standard data processing and quality control procedures, they obtained transcriptome profiles for 36,424 cells (Chen, S. et al. Nat. Cell Biol. 23, 87-98 (2021)). However, this analysis for primary prostate cancer samples is enhanced in epithelial cells. Therefore, the inventors verified PVRIG expression by single-cell data by confirming single-cell analysis of lethal PCs. The cohort consisted of 2,170 cells derived from 14 patients and 15 fresh biopsies from three common mCRPC metastatic sites (bone, lymph nodes, and liver). PVRIG has been confirmed to be expressed by CD4+ T cells, CD8+ T cells, and NK cells (Jordan, MA & Wilson, L. Nat. Rev. Cancer 4, 253-265 (2004)). Furthermore, the inventors confirmed the expression of nectin-2 protein in human prostate cancer samples by immunofluorescence (not shown).

[0112] Example 3: Nectin 2 upregulation correlates with aging. Next, in collaboration with the Proteomic Core Facility of Lausanne at Ecole Polytechnique Federale de Lausanne (EPFL), the inventors performed mass spectrometry and bioinformatics analysis to obtain Pten - / - We identified membrane proteins upregulated by an aging population in mice. Using this technique, we confirmed that nectin 2 is one of the most upregulated transmembrane proteins expressed by senescent cells (Figure 2A, right panel). Furthermore, bioinformatics analysis revealed that cells with the highest aging scores also had the highest levels of nectin 2.

[0113] Using our mouse model of Pten-deficiency-induced cellular senescence (PICS, Toso et al., Cell Reports 9, 75-89 (2014)), we then demonstrated a correlation between senescence and nectin 2 expression at both the transcriptional and translational levels. This correlation was confirmed at the translational level in the entire tumor by Western blotting (Figure 2B), and this data was also validated by qRT-PCR on selected C12-FDG+ and C12-FDG- PTEN null tumor cells (Figure 2C). In addition to nectin 2, we also identified several senescence markers, including p21 (at the translational level) and p16 (at the transcriptional level), which were upregulated in the non-proliferating cell fraction. Furthermore, results from RNA sequencing indicated that pten - / - ;p53 - / - Pten when compared to wild-type mice - / - Upregulation of nectin 2 was confirmed in mice (Figure 2D).

[0114] Next, the inventors in vivo investigated whether nectin 2 upregulation correlated with treatment-induced aging (TIS) in their genetically modified mouse model. Mice were treated with docetaxel 10 mg / kg once a week for 4 weeks, and aging induction was confirmed by beta-galactosidase staining at the end of the treatment (Figure 2E), which was consistent with a reduction in anterior lobe tumor size (Figure 2F). Real-time PCR data showed significant nectin 2 upregulation (Figure 2G).

[0115] The inventors verified the expression of their targets on human prostate cancer cell lines. To test tumor heterogeneity in terms of mutational load and genetic alteration, the inventors used human prostate cancer cell lines containing both wild-type and mutant forms of the PTEN and TP53 genes, in terms of their response to androgen receptor signaling.

[0116] To evaluate whether nectin 2 expression was associated with disease progression and resistance to treatment, we treated human PCa cell lines with two common chemotherapeutic agents used in the management of PCa (specifically, docetaxel and palbociclib) to induce cellular senescence. Docetaxel-based regimens have been shown to improve patient symptoms and overall survival in mCRPC patients (Mohler, J. Let al. J. Natl. Compr. Cancer Netw. JNCCN 17, 479-505 (2019)). Furthermore, palbociclib (a CDK4 / 6 inhibitor) is commonly used in combination with ADT to treat metastatic prostate cancer and has already been shown to induce senescence at subtoxic concentrations (ClinicalTrials.gov Identifier: NCT02905318).

[0117] Furthermore, docetaxel and palbociclib also induced senescence at levels below toxic concentrations, and it was observed that the onset of senescence led to resistance to treatment. Subsequently, the effectiveness of senescence induction was evaluated using a beta-galactosidase assay (Figure 3A), and nectin 2 mRNA levels were measured by real-time PCR in treated and untreated cells (Figure 3B). Palbociclib treatment induced a significant increase in nectin 2 mRNA levels in all cell lines tested. The induction of nectin 2 mRNA levels after docetaxel treatment was also considerable compared to the untreated PC3 population, but was not significant in 22rv1 and LNCAP cells.

[0118] Flow cytometry analysis confirmed the same results, clearly demonstrating nectin-2 upregulation at the translational level under all test conditions (Figure 3C).

[0119] Aging induction during treatment was also confirmed by FDG staining in 22rv1 (Figure 3D) and upregulation of nectin 2 in the treated fraction (Figure 3E).

[0120] Aging induction was also achieved by treatment with enzalutamide, mimicking androgen depletion therapy. For this purpose, LNCaP cells were used because they express androgen receptors and therefore are sensitive to treatment. As demonstrated by FDG staining (not shown) and β-galactosidase assay (Figure 3G), aging was induced by the treatment, and nectin 2 was upregulated at both the protein (Figure 3F) and mRNA levels (Figure 3H).

[0121] Example 4: In aging prostate cancer cells, PSMA expression is positively correlated with nectin 2 expression. While nectin 2 expression appears to be cell-specific in prostate TME, the inventors cannot rule out its expression in other organs.

[0122] Therefore, the inventors designed a specific and selective treatment strategy based on Nectin 2 by utilizing prostate-specific membrane antigen (PSMA). To design an effective drug delivery system and immunotherapy, the inventors first confirmed the correlation between Nectin 2 and all other known immune checkpoints and PSMA. To this end, the inventors used the TCGA database and applied correlation matrices. As shown in Figures 4A and 4B, the inventors found that FOLH1(PSMA) expression was positively correlated only with Nectin 2 expression in prostate cancer, and that correlation was twice as strong as that of PVR. Furthermore, human prostate samples were also analyzed by immunofluorescence. In this case, biopsies of prostate tumors from normal tissue and tumor tissue were stained with primary antibodies against PSMA and anti-Nectin 2. The results of the immunofluorescence analysis revealed a higher percentage of epithelial prostate tumor cells co-expressing Nectin 2 and PSMA (Figure 4C). Furthermore, when samples from castration-sensitive prostate cancer (CSPC) were compared to a model of castration-resistant prostate cancer (CRPC), the percentage of double-positive cells was significantly higher in CRPC patients (Figure 4D).

[0123] Furthermore, the inventors performed Western blot analysis on patient-derived human organoids (PDOs) and found that samples characterized by nectin 2 expression were also positive for PSMA (Figure 4E).

[0124] Despite its clinical applications, the regulation of PSMA expression in senescent cells has not been studied. Therefore, we identified PSMA protein levels in our aging model and evaluated the potential to target it in combination with nectin-2. Western blots of human cell lines treated with TIS showed increased PSMA expression in both LNCaP and 22RV1 cells, but no band was detected in PC3 cells, as they do not express PSMA (Figure 4F).

[0125] Example 5: Dual-target CAR-T cells targeting nectin 2 and prostate-specific membrane antigen (PSMA) to eliminate senescent cells. Today, targeted therapies for prostate cancer primarily target prostate-specific membrane antigen (PSMA). The FOLH1 gene encodes PSMA, a type II transmembrane glycoprotein with folate hydrolase and N-acetylated-α-linked-acid dipeptidase activity. PSMA is expressed in normal, benign, and malignant prostate tissue, including intraepithelial and metastatic neoplasms (Hupe, MC et al. Front. Oncol. 8, 623 (2018); Troyer, J. et al. Int. J. Cancer 62, 552-558 (1995)). However, PSMA expression has already been shown to be 100 to 1000 times higher in prostate cancer compared to benign prostate (Heston, WDW Urology 49, 104-112 (1997)). For this reason, many radiopharmaceuticals and diagnostic tools are designed to target PSMA.

[0126] To target PSMA and Nectin 2 and prevent on-target / off-tumor toxicity, we designed a combinatorial CAR-T cell approach, a design first described by Kloss et al. to enhance tumor specificity in the absence of tumor-specific antigens (Kloss, C. et al. Nat. Biotechnol. 31, 71-75 (2013)). Therefore, to design highly specific and efficient CAR-T cells, we screened phage display libraries against PSMA and Nectin 2 and generated scFv-Fc constructs with novel anti-PSMA and anti-Nectin 2 scFv candidates. To evaluate the specificity of their binding to PSMA or Nectin 2, we used a PC3 cell line that naturally lacks PSMA expression and also generated a Nectin 2-deficient human PCa cell line (PC3, LNCaP, 22Rv1) using CRISPR / Cas9 technology. The absence or presence of surface PSMA, as well as the knockout (KO) of Nectin 2, were confirmed by flow cytometry (Figure 5A). Anti-PSMA (HRB730) (SEQ ID NOs. 7-8, Figure 8) or anti-Nectin 2 (HRB725) scFv (SEQ ID NOs. 9-10, Figure 7)-Fc constructs were evaluated by flow cytometry staining of PCa cell lines using anti-PSMA or anti-Nectin 2 scFv-Fc constructs at various dilutions (1:1, 1:10, 1:500) and anti-human Fc-AF488 secondary antibody, confirming their specific binding to their target antigens in a dose-dependent manner.

[0127] Importantly, the anti-nectin 2(725v5)scFv-Fc construct was found to be selective for nectin 2 only when expressed on tumor cells (Figure 9). Nectin 2 scFv does not bind to non-malignant nectin 2-expressing human cells, but binds to cancer-exposed epitopes of nectin 2 (containing or consisting of the sequence of SEQ ID NO: 28, i.e., amino acids at positions 222-241 of the nectin 2 polypeptide available from Uniprot entry Q92692-1).

[0128] Next, the inventors designed and fabricated single-specific anti-PSMA (SEQ ID NOs: 1-2) or anti-nectin 2 (SEQ ID NOs: 3-4) CAR constructs (Figure 5B), as well as a novel CombiCAR construct (SEQ ID NOs: 5-6) (Figure 5C). Here, the scFv targeting PSMA is linked to a transmembrane domain (TMD), an intracellular CD3z signaling domain, and subsequently to a self-cleaving T2A peptide, while the scFv targeting anti-nectin 2 is linked to a different TMD and an intracellular 4-1BB costimulatory domain. Both intracellular CD3z signaling and costimulatory signaling are necessary for successful T cell activation. Therefore, this design ensures that both receptors must simultaneously bind to the target nectin 2+ cells in order to induce downstream signaling that activates T cells. After transduction of T cells and proliferation of CAR-T cells using each construct, CAR expression was evaluated by flow cytometry (Figure 5D). To measure their antitumor-killing ability, CAR-T cells were co-cultured with human PCa cell lines for 92 hours (Figures 5E-F). As shown in Figures 5E-F, PCa cell lines expressing PSMA were readily lysed by monospecific anti-PSMA CAR-T cells, while PSMA-deficient PCa cell lines, such as PC3 wild-type and its nectin 2 knockout counterpart, as well as the human glioblastoma cell line Ge904, remained intact. Similarly, monospecific anti-nectin 2 CAR-T cells specifically and efficiently target PCa cell lines expressing nectin 2. Interestingly, PCa cell lines co-expressing PSMA and nectin 2 were efficiently lysed by CombiCAR-T cells, while PSMA-deficient PC3 wild-type remained intact. PSMA+ / nectin 2-PCa cell lines were similarly lysed by CombiCAR-T cells, which is because PSMA-targeted CARs are first-generation CARs that can be stimulated in vitro with only the intracellular CD3z signaling domain. In vivo, first-generation CAR-T cells are insufficient for efficient induction and persistence of T cell responses, and their activity is limited (Brocker, T. & Karjalainen, KJ Exp. Med. 181, 1653-1659 (1995)). In vivo, the inventors primarily used PSMA + / Nectin2+ Targeting PSMA to a lesser extent + / Nectin2 - Targeting, PSMA - / Nectin2 + / - We expect that it will not bind to PCa cells. Furthermore, as an indicator of successful T cell activation and functioning, IFNg secretion was evaluated in the supernatant after co-culture using sandwich ELISA. As shown in Figure 5F, the IFNg secretion levels of monospecific anti-PSMA, monospecific anti-nectin 2, and CombiCAR-T cells are consistent with their lytic ability in the presence of their target antigens, as evaluated in Figure 5G.

Claims

1. An anti-nectin 2 antibody or its antigen-binding fragment, comprising (i) a heavy chain variable domain (V) containing CDR1-H of the sequence of SEQ ID NO: 11, CDR2-H of the sequence of SEQ ID NO: 12, and CDR3-H of the sequence of SEQ ID NO:

13. Ｈ (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 14, CDR2-L of the sequence of sequence number 15, and CDR3-L of the sequence of sequence number 16. Ｌ An anti-nectin 2 antibody or its antigen-binding fragment, containing )

2. V Ｈ V contains the sequence of sequence number 17 or a sequence that is at least 80% identical thereto, and / or V Ｌ The anti-nectin 2 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises the sequence of sequence number 18 or a sequence that is at least 80% identical thereto.

3. An anti-nectin 2 antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a single-stranded variable fragment (scFv).

4. The anti-nectin 2 antibody or antigen-binding fragment thereof according to claim 3, comprising or consisting of the sequence of SEQ ID NO:

10.

5. A polypeptide comprising anti-nectin 2 scFv as described in claim 3 or 4.

6. The heavy chain variable domain (V) of the anti-nectin 2 antibody or its antigen-binding fragment according to claim 1 or 2 Ｈ ) and light chain variable domain (V Ｌ A pair of isolated nucleic acids comprising sequences encoding ) respectively, or an isolated nucleic acid comprising a sequence encoding anti-nectin 2 scFv according to claim 3 or 4 or the polypeptide according to claim 5.

7. An antibody-drug conjugate (ADC) comprising (i) an anti-nectin 2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, (ii) a cytotoxic payload, and (iii) a linker that links the anti-nectin 2 antibody or its antigen-binding fragment with the cytotoxic payload.

8. An antibody-drug conjugate (ADC) as defined in claim 7, for use in the treatment of cancer.

9. The ADC for use according to claim 7, wherein the cancer is prostate cancer.

10. An anti-PSMA antibody or its antigen-binding fragment, comprising (i) a heavy chain variable domain (V) containing CDR1-H of the sequence of SEQ ID NO: 20, CDR2-H of the sequence of SEQ ID NO: 21, and CDR3-H of the sequence of SEQ ID NO: 22 Ｈ (ii) a light chain variable domain (V) containing CDR1-L of the sequence of sequence number 23, CDR2-L of the sequence of sequence number 24, and CDR3-L of the sequence of sequence number 25. Ｌ An anti-PSMA antibody or its antigen-binding fragment, which includes )

11. V Ｈ comprises the sequence of SEQ ID NO: 26 or a sequence that is at least 80% identical thereto, and / or V Ｌ comprises the sequence of SEQ ID NO: 27 or a sequence that is at least 80% identical thereto, the anti-PSMA antibody or antigen-binding fragment thereof according to claim 10.

12. The anti-PSMA antibody or antigen-binding fragment thereof according to claim 10 or 11, which is a single-stranded variable fragment (scFv).

13. An anti-PSMA antibody or antigen-binding fragment thereof according to claim 12, comprising or consisting of the sequence of SEQ ID NO:

8.

14. The heavy chain variable domain (V) of the anti-PSMA antibody or its antigen-binding fragment according to claim 10 or 11 Ｈ ) and light chain variable domain (V Ｌ A pair of isolated nucleic acids, each containing a sequence encoding ) respectively, or an isolated nucleic acid, each containing a sequence encoding anti-PSMA scFv according to claim 12 or 13.

15. An antibody-drug conjugate (ADC) comprising (i) an anti-PSMA antibody or antigen-binding fragment thereof according to any one of claims 10 to 13, (ii) a cytotoxic payload, and (iii) a linker connecting the anti-PSMA antibody or antigen-binding fragment thereof and the cytotoxic payload.

16. An antibody-drug conjugate (ADC) as defined in claim 15, for use in the treatment of cancer, preferably prostate cancer.

17. A bispecific antibody construct that binds to at least nectin-2 and PSMA.

18. The bispecific construct according to claim 17, comprising an anti-nectin 2 single-strand variable fragment (scFv) as defined in claim 3 or 4, and an anti-PSMA scFv as defined in claim 12 or 13.

19. An antibody-drug conjugate (ADC) comprising (i) a bispecific antibody construct bound to nectin-2 and PSMA as defined in claim 17 or 18, (ii) a cytotoxic payload, and (iii) a linker connecting the bispecific antibody construct and the cytotoxic payload.

20. An antibody-drug conjugate (ADC) as defined in claim 19, for use in the treatment of cancer, preferably prostate cancer.

21. A pharmaceutical composition comprising an antibody-drug conjugate as defined in any one of claims 7-9, 15-16, or 19, and a pharmaceutically acceptable carrier.

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