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

Abstract

The present invention relates to anti-adhesin 2 antibodies or antigen-binding fragments thereof, anti-PSMA antibodies or antigen-binding fragments thereof, bispecific antibody constructs that bind to adhesin 2 and PSMA, and antibody-drug conjugates comprising such antibodies. The invention also relates to the use of said antibody-drug conjugate for the treatment of cancer.

Description

[0001] This invention relates to anti-nectin 2 antibodies or antigen-binding fragments thereof, anti-PSMA antibodies or antigen-binding fragments thereof, bispecific antibody constructs binding to nectin 2 and PSMA, and antibody-drug conjugates comprising such antibodies. The invention also relates to the use of said antibody-drug conjugates in the treatment of cancer.

[0002] background

[0003] Prostate cancer (PCa) remains a leading cause of cancer-related mortality and morbidity in men, ranking as the second most diagnosed cancer and the sixth leading cause of cancer death worldwide. The incidence of prostate cancer is highly predisposing, with over 70% of all cases diagnosed in men over 65 years of age. The development of prostate cancer is a multi-step process, beginning with benign prostatic hyperplasia (BPH), progressing to high-grade prostatic intraepithelial neoplasia (HGPIN), and ultimately becoming aggressive and metastatic.

[0004] Treatment is typically based on chemotherapy and radiation therapy, with or without combination based on castration strategies, but most treatments show limited clinical and survival benefits for duration due to primary and acquired resistance caused by tumor adaptation (Sumanasuriya, S. & Bono, JD Cold Spring Harb. Perspect. Med. 8, a030635 (2018)). Docetaxel and androgen deprivation therapy (ADT) are widely used to treat prostate cancer. However, almost all patients receiving ADT typically progress to castration-resistant prostate cancer (CRPC), which may 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)).

[0005] Therefore, current research aims to find new treatment options and overcome drug resistance mechanisms.

[0006] Furthermore, the induction of aging (whether by conventional treatments or by gene mutations) plays a controversial role in cancer through both beneficial and harmful tumor growth (Collado, M. Future Oncol. 6, 687–689 (2010)).

[0007] Senescence-induced therapeutic benefits stem from the inhibition of cell proliferation and the activation of anti-tumor immune responses, while the accumulation of senescent cells can stimulate tumor growth and angiogenesis, leading to relapse and treatment resistance (Hernandez-Segura, A. et al. Trends Cell Biol. 28, 436–453 (2018)). Eliminating senescent cells has become an interesting strategy for improving therapeutic efficacy. Senescent immune cells, particularly T cells, can also accumulate in the tumor microenvironment, a phenomenon known as immunosenescence, which can promote 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 become senescent, the amount of SASP components released affects the recruitment of immune cells, and the effect of SASP on the immune cell population is considered a double-edged sword. SASP recruitment of T cells, macrophages, and NK cells can prevent tumor development and progression, but SASP can also increase the tumor infiltration of immunosuppressive myeloid cells. In the absence of tumor-derived factors, myeloid cells differentiate into dendritic cells, macrophages, or neutrophils, which contribute to immune surveillance. However, in the presence of tumor-derived factors, these myeloid cells lose their ability to differentiate and suppress the functions of other immune cells, thus creating an immune-tolerant environment that allows tumor progression (Ohtani, N. Inflamm. Regen. 42, 11 (2022); Eggert, T. et al. Cancer Cell 30, 533–547 (2016)). Therefore, the concept of a "one-two punch" approach has been proposed, in which "pro-senescence" drugs are first used to stabilize and halt cell proliferation in cancer cells, followed by subsequent treatment with agents that can kill senescent cells (senescent cell clearance drugs) (Wang, C. et al. Nature 574, 268–272 (2019); Sieben CJ et al., Trends in Cell Biology, 28, Vol. 9, 723-737 (2018)). Cellular senescence represents a promising target for improving current treatment outcomes, and several senescent cell-clearing drugs have been identified and obtained from natural products for use in combination with anti-aging drugs.

[0008] Therefore, eliminating senescent cells has become 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 pro-survival pathways, and the current armamentarium of effective senescent cell clearance drugs consists of only a small number of 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)).

[0009] The use of immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment by reactivating the immune system to fight malignant cells. However, only a small percentage of patients experience a durable response to ICIs, and resistance to treatment is common. Resistance to ICIs can be influenced by both extrinsic and intrinsic factors affecting tumor cells. Prostate cancer is known to have a weak immune response, with low neoantigen load and a highly immunosuppressive microenvironment. In many types of cancer, including melanoma and breast cancer, a high number of tumor-infiltrating lymphocytes (TILs) is a positive prognostic indicator, but in prostate cancer, the majority of T cells within the tumor are CD4+ regulatory T cells (Tregs). Currently, ongoing clinical trials are evaluating the use of ICIs, alone or in combination with other treatments, in metastatic hormone-sensitive and castration-resistant prostate cancer. Immune checkpoint inhibitors (ICIs) have revolutionized the way cancer is currently treated. 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 the cancer case, blocking this interaction with ICIs leads to the reactivation of the immune system against malignant cells.

[0010] Currently, many 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), PD-1 (pembrolizumab, nivolumab), and PD-L1 (e.g., atezolizumab). Although ICI administration has improved outcomes for patients with various tumor types, only a small percentage of patients achieve durable responses. In fact, the emergence of resistance mechanisms to immune checkpoint inhibitors is a common clinical event. Even among melanoma patients who show one of the highest positive responses to ICIs, over 60% do not show an objective response to anti-PD-1 therapy (Ott, PA et al. J. Clin. Oncol. 37, 318-327 (2019)).

[0011] The development of ICI resistance depends on both extrinsic and intrinsic factors of tumor cells. Among extrinsic factors, the microbiome, PD-L1 expression levels on immune cells, and the composition of tumor and peripheral immune cells play important roles in determining treatment outcomes. On the other hand, epigenetic variations, mutational burden, and neoantigen expression are intrinsic characteristics of tumor cells that can guide the effectiveness of patient responses to ICI therapy (Bagchi, S., Yuan, R. & Engleman, EGAnnu. Rev. Pathol. 16, 223–249 (2021)).

[0012] Prostate cancer is considered an immunologically cold disease, characterized by a weak immune response. In fact, it typically presents as a microenvironment characterized by T-cell depletion, low neoantigen load, and a highly immunosuppressive environment (de Bono, JS et 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, M2-polarized tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), all of which typically exhibit an immunosuppressive phenotype (Krueger, TE, Thorek, DLJ, Meeker, AK, Isaacs, JT & Brennen, WN The Prostate 79, 320–330 (2019)). Specifically, MDSCs have been found to have potent immunosuppressive effects in castration-resistant prostate cancer (CRPC), with interleukin-23 (IL-23) produced by MDSCs modulating castration resistance by triggering androgen receptor signaling (Calcinotto, A. et al., Nature 559, 363–369 (2018)). Within this framework, several preclinical studies have demonstrated the beneficial effects of combined anti-MDSC and ICI therapy in enhancing immune checkpoint blockade efficacy in CRPC models (Calcinotto, A. et al., Nature 559, 363–369 (2018); Lu, X. et al., Nature 543, 728–732 (2017)).

[0013] As previously mentioned, a higher number of tumor-infiltrating lymphocytes (TILs) is a prognostic indicator of better outcomes in many types of cancer, such as melanoma and breast cancer. In PCa, in addition to a low percentage of TILs, most of the T cells within the tumor in mCRPC also result in CD4+ regulatory T cells (Tregs), and low abundance of CD8+ T cells often presents an exhausted phenotype while expressing 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)).

[0014] Currently, numerous clinical trials of immunoglobulin inhibitors (ICIs) for locally advanced or metastatic hormone-sensitive and castration-resistant prostate cancer are underway, with ICIs used both alone and in combination therapy (Rebuzzi, SE et al., Cancers 14, 1245 (2022)). Overall, no consistent results have been found regarding overall survival. Patients found to partially benefit from anti-PD1 therapies (such as nivolumab and pembrolizumab) are characterized by CDK12-altered tumors. Indeed, CDK12-mutant prostate cancers (PCa) are generally associated with poor prognosis but exhibit increased neoantigen burden and lymphocyte infiltration. Furthermore, several studies evaluating ICIs in combination with standard therapy have highlighted better responses in patients with specific pathway abnormalities (e.g., AR-V7 variants, HRD), CDK12-inactivated tumors, and high MSI tumors (Rebuzzi, SE et al., Cancers 14, 1245 (2022)). These clinical trials underscore the need to identify novel therapeutic targets and strategies based on the genetic characteristics of tumors to enhance the immune response in PCa patients.

[0015] Less than 10% of chemotherapy-refractory patients benefit from immune checkpoint blockade immunotherapy (Markowski, MC et al., The Prostate 80, 407–411 (2020)). Therefore, mCRPC represents a crucial unmet medical need.

[0016] Antibody-drug conjugates (ADCs) represent an alternative strategy to ICI. ADCs consist of monoclonal antibodies covalently linked to cytotoxic drugs via chemical linkers (Fu et al., Signal Transduct Target Ther. 2022;7(1):9.). Cytotoxic drugs can be delivered in a specific manner through binding to homologous tumor-associated antigens (TAAs) on tumor cells. ADCs have shown clinical efficacy and have been approved by the FDA for the treatment of hematologic 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 poor accessibility of tumor-specific targets. Most solid tumor TAAs are also expressed on non-malignant primary cells in key tissues, although at lower levels. Major side effects include hematologic toxicities, 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 mAb (Oshima et al., 2018).

[0017] Currently, several preclinical studies (Boinapally et al., Eur J Nucl Med Mol Imaging, 2022, 49(13):4369-4381; Machulkin et al., Eur J Med Chem. Jan 5, 2022; 227:113936) and clinical trials (Cho et al., Mol Cancer Ther. 2018; 17(10):2176-2186, Milowsky et al., UrolOncol., 2016; 34(12):530.e15-530.e21; Petrylak et al., Prostate. Jan 2020; 80(1):99-108) are using PSMA-targeting ADCs to treat mCRPC. In preclinical studies, the corresponding ADCs have shown strong specific cytotoxicity. Unfortunately, in clinical trials, they have shown only limited antitumor activity, accompanied by common treatment-related side effects such as neutropenia and neuropathy. Therefore, the practicality of this treatment strategy may be limited by several factors. First, heterogeneous expression and / or downregulation of PSMA can lead to tumor escape, challenge the pharmacokinetics of adjacent drugs, and cause common side effects.

[0018] Understanding the adaptive immune response in advanced prostate cancer (APC) has become crucial, especially after failure of anti-CTLA4 (ipilimumab) therapy in phase III trials (Beer, TM et al., J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 35, 40–47 (2017)). Furthermore, PD-L1, a major target of existing immunotherapies, is poorly expressed in prostate cancer (Haffner, MC et al., Am. J. Pathol. 188, 1478–1485 (2018)). Recently, the largest clinical trial testing anti-CTLA-4 plus anti-PD-1 in CRPC did not significantly improve patient outcomes (Sharma, P. et al., Cancer Cell 38, 489–499.e3 (2020)). Invention Overview

[0020] The inventors hypothesize that further development of effective ADCs for CRPC should include the identification of robust tumor target antigens with stable and uniform expression, the integration of safety mechanisms to mitigate toxicity, and strategies to overcome the premature release of cytotoxic payloads in circulation.

[0021] Therefore, the inventors aimed to identify novel immune checkpoints in prostate cancer and to develop them as new targets for prostate cancer immunotherapy strategies. Through analysis of bulk RNA and single-cell data from human patients, the transmembrane protein adhesin 2 was identified as upregulated in epithelial prostate cancer cells. Adhesin 2 possesses immunomodulatory functions and interacts with receptors expressed on T cells and NK cells. In in vitro and in vivo models of treatment-induced senescence and PTEN loss-induced cellular senescence, the upregulation of adhesin 2 was found to be even higher during senescence induction.

[0022] Based on these findings, adhesin 2 represents a potential target for removing senescent cancer cells from tumors and reactivating the immune response. Novel scFvs were isolated using a human phage display library targeting the extracellular domains of adhesin 2 and PSMA, and candidates were selected for binding to human PCa cell lines expressing adhesin 2 and / or PSMA. Their specificity was further confirmed using T cells modified to contain a chimeric antigen receptor (CAR) integrating the selected anti-adhesin 2 or anti-PSMA scFv. Furthermore, we observed selective binding of the novel anti-adhesin 2 scFv to the cancer-exposed epitopes of adhesin 2 without affecting non-malignant human cells expressing adhesin 2 (in vitro). Importantly, the anti-adhesin 2 scFv-Fc was internalized into human PCa cells after staining. Therefore, using these validated scFvs, a bispecific ADC targeting adhesin 2 and PSMA for the clearance of senescent cells can be developed.

[0023] Therefore, this invention relates to anti-adhesion 2 antibodies or antigen-binding fragments thereof. ， It includes (i) a variable heavy chain structural domain (V H ), which includes the CDR1-H sequence of SEQ ID NO:11, the CDR2-H sequence of SEQ ID NO:12 and the CDR3-H sequence of SEQ ID NO:13, and (ii) a variable light chain domain (V L The sequence comprises CDR1-L with sequence SEQ ID NO:14, CDR2-L with sequence SEQ ID NO:15, and CDR3-L with sequence SEQ ID NO:16.

[0024] In some embodiments, the anti-adhesion 2 antibody or its antigen-binding fragment is scFV.

[0025] The present invention also relates to polypeptides comprising the antiadhesion 2 scFv of the present invention.

[0026] It also provides a pair of isolated nucleic acids containing variable heavy chain domains (V) encoding anti-adhesion 2 antibodies or their antigen-binding fragments, respectively. H ) and variable light chain structural domain (V L ) sequence.

[0027] Also provided are isolated nucleic acids (which contain a sequence encoding the antiadhesion 2 scFv of the present invention) or polypeptides containing said antiadhesion 2 scFv.

[0028] The present invention also relates to antibody-drug conjugates (ADCs) comprising (i) an anti-adhesion 2 antibody or an antigen-binding fragment thereof according to the present invention, (ii) a cytotoxic payload, and (iii) a linker connecting the anti-adhesion 2 antibody or the antigen-binding fragment thereof to the cytotoxic payload.

[0029] The present invention also relates to an anti-PSMA antibody or an antigen-binding fragment thereof, comprising (i) a variable heavy chain domain (V H ), which includes the CDR1-H sequence of SEQ ID NO:20, the CDR2-H sequence of SEQ ID NO:21 and the CDR3-H sequence of SEQ ID NO:22, and (ii) a variable light chain domain (V L The sequence comprises CDR2-L with sequence SEQ ID NO:23, CDR3-L with sequence SEQ ID NO:24, and CDR3-L with sequence SEQ ID NO:25.

[0030] In some embodiments, the anti-PSMA antibody or its antigen-binding fragment is scFV.

[0031] This invention also relates to peptides comprising the anti-PSMA scFv of this invention.

[0032] It also provides a pair of isolated nucleic acids containing variable heavy chain domains (V) encoding anti-PSMA antibodies. H ) and variable light chain structural domain (V L (or the sequence of its antigen-binding fragment)

[0033] Also provided are isolated nucleic acids (which contain a sequence encoding the anti-PSMA scFv of the present invention) or polypeptides containing said anti-PSMA scFv.

[0034] The present invention also relates to antibody-drug conjugates (ADCs) comprising (i) an anti-PSMA antibody or an antigen-binding fragment thereof according to the present invention, (ii) a cytotoxic payload, and (iii) a linker connecting an anti-adhesion 2 antibody or an antigen-binding fragment thereof to the cytotoxic payload.

[0035] Also provided are bispecific antibody constructs that bind to at least adhesin 2 and PSMA, and ADCs comprising the bispecific antibody, a cytotoxic payload, and a connector linking the bispecific antibody construct to the cytotoxic payload.

[0036] The present invention also relates to the use of the ADC according to the invention for the treatment of cancer, and to pharmaceutical compositions comprising such ADC.

[0037] Detailed description

[0038] definition

[0039] "Prostate-specific membrane antigen" or "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, accession number Q04609 (entry version 222, May 3, 2023).

[0040] "Adhesin 2" refers to a type I membrane glycoprotein with two Ig-like C2 domains and one Ig-like V domain, serving as a component of the plasma membrane for adhesion. The reference sequence for human adhesin 2 is available in the Uniprot database, accession number Q92692 (entry version 217, May 3, 2023). Adhesin 2 is also known as poliovirus receptor-associated protein-2, poliovirus receptor-like 2, CD112, or PRR-2.

[0041] Antibodies can be natural or conventional immunoglobulin molecules in which two heavy chains are linked together by disulfide bonds, and each heavy chain is connected to a light chain via disulfide bonds. The light chain comprises two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site consists primarily of residues from the hypervariable region, or "complementarity-determining region" (CDR). Both the light and heavy chains of immunoglobulins have three CDRs, named CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, respectively. Therefore, the antigen-binding site of a conventional antibody comprises six CDRs, including a set of CDRs from each of the V regions of the heavy and light chains.

[0042] The "frame region" (FR) refers to the amino acid sequence interspersed between CDRs, specifically those relatively conserved portions of the variable regions of the immunoglobulin light and heavy chains within a single species. Each immunoglobulin light and heavy chain has four FRs, named FR-L1, FR-L2, FR-L3, FR-L4 and FR-H1, FR-H2, FR-H3, FR-H4, respectively.

[0043] In the context of this invention, the CDR / FR definition in the immunoglobulin light or heavy chain will be determined based on the Chothia number (Chothia et al., J Mol Biol. 1987; 196(4):901-17).

[0044] As used in this article, the term "antibody" refers to conventional antibodies and their antigen-binding fragments, as well as chimeric, humanized, bispecific, or multispecific antibodies.

[0045] The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule with a single primary structure that targets a specific antigen, and should not be interpreted as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be produced by a single clone of B cells or hybridomas, but they can also be recombinant, i.e., produced through protein engineering.

[0046] A "fragment" of a (conventional) antibody contains a portion of the complete antibody, particularly the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, biantibodies, and bispecific and multispecific antibodies formed from antibody fragments.

[0047] The antibody or antibody fragment can be an isotype or a subtype; preferably IgG, more preferably IgG1.

[0048] The term "bispecific antibody" refers to an antibody that has the ability to bind to a single antigen or two different epitopes on two different antigens. The bispecific antibodies of this invention can be bivalent, trivalent, or tetravalent. As used herein, "valent," "valence," "valencies," or other grammatical variations refer to the number of antigen-binding sites in the antibody molecule. These antigen recognition sites can recognize the same epitope or different epitopes.

[0049] As used in this paper, after pairwise global sequence alignment using the Smith-Waterman algorithm, such as the EMBOSS Needle with default settings (matrix BLOSUM62, open 10 spaces, extended 0.5 spaces, false penalty for terminal spaces, open 10 spaces at the terminal, extended 0.5 spaces at the terminal), the percentage of identity is calculated.

[0050] Throughout this application, the term "comprising" should be interpreted as including all specifically mentioned features as well as optional, additional, or unspecified features. As used herein, the use of the term "comprising" also discloses embodiments in which no features other than those specifically mentioned (i.e., "composed of") are present. Furthermore, the indefinite articles "a" or "an" do not exclude plurality.

[0051] Anti-adhesion 2 antibody

[0052] This invention relates to an anti-adhesion 2 antibody or its antigen-binding fragment, comprising (i) a variable heavy chain domain (V H), which includes CDR1-H with the sequence GYTFTSY (SEQ ID NO:11), CDR2-H with the sequence SAYNGN (SEQ ID NO:12), and CDR3-H with the sequence YGWKDAMDY (SEQ ID NO:13), and (ii) a variable light chain domain (V L The CDR1-L contains the sequence SGSSSNIGNNYVS (SEQ ID NO:14), the CDR2-L contains the sequence DNNKRPS (SEQ ID NO:15), and the CDR3-L contains the sequence GTWDRPKQKVV (SEQ ID NO:16).

[0053] Anti-adhesion-2 antibodies or antibody fragments can be recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibodies.

[0054] In some embodiments, the anti-adhesion 2 antibody or its antigen-binding fragment comprises:

[0055] (a) A sequence containing SEQ ID NO:17 or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with it. H ,

[0056] (b) A sequence containing SEQ ID NO:18 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it. L ,or

[0057] (c) A sequence containing SEQ ID NO:17 or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with it. H and V containing the sequence SEQ ID NO:18 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it. L .

[0058] In some embodiments, the anti-adhesion 2 antibody or its antigen-binding fragment binds to cancer-exposed epitopes of adhesion 2. Therefore, the anti-adhesion 2 antibody or its antigen-binding fragment can distinguish between cancerous adhesion 2-expressing human cells and non-malignant adhesion 2-expressing human cells. In some embodiments, the anti-adhesion 2 antibody or its antigen-binding fragment can bind to epitopes containing or consisting of the sequence SEQ ID NO:28.

[0059] In some embodiments, the anti-adhesion 2 antibody or its antigen-binding fragment is a single-chain variable fragment (scFv).

[0060] In some embodiments, the antiadhesion 2 scFv comprises (i) V H It includes the sequence CDR1-H of SEQ ID NO:11, the sequence CDR2-H of SEQ ID NO:12, and the sequence CDR3-H of SEQ ID NO:13, and (ii)V L It includes CDR1-L with sequence SEQ ID NO:14, CDR2-L with sequence SEQ ID NO:15, and CDR3-L with sequence SEQ ID NO:16, and (iii) includes V H and V L The connector.

[0061] In some embodiments, the V of the anti-adhesion 2 scFv H It contains the sequence SEQ ID NO:17 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it.

[0062] In some embodiments, the V of the anti-adhesion 2 scFv L It contains the sequence SEQ ID NO:18 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it.

[0063] In some embodiments, the V of the anti-adhesion 2 scFv H Contains the sequence SEQ ID NO:17 or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with it, and the V of antiadhesin 2 scFv L It contains the sequence SEQ ID NO:18 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it.

[0064] The adapter is a chemical adapter or a peptide adapter. In some embodiments, the adapter connecting the VH and VL of antiadhesin 2 scFv comprises the sequence GGGSGGGGSGGGGST (SEQ ID NO:19) or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with it.

[0065] Preferably, the antiadhesion 2 scFv contains or consists of the sequence SEQ ID NO:10.

[0066] The polypeptide containing anti-adhesion 2 scFv is also part of this invention.

[0067] In some embodiments, the polypeptide is not a chimeric antigen receptor (CAR), especially not a CAR containing an antigen-binding domain that binds to adhesin 2, or a CAR containing an antigen-binding domain that binds to PSMA and an antigen-binding domain that binds to adhesin 2.

[0068] The present invention also relates to a pair of V antibodies that respectively encode the anti-adhesion 2 antibody or antibody fragment disclosed herein. H and V L The invention also relates to nucleic acids comprising a sequence encoding antiadhesin 2 scFv, or polypeptides comprising antiadhesin 2 scFv.

[0069] In some embodiments, the nucleic acid comprises the sequence SEQ ID NO:9 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it.

[0070] Anti-PSMA antibody

[0071] This invention relates to anti-PSMA antibodies or antigen-binding fragments thereof, said antibodies or antigen-binding fragments comprising (i) a variable heavy chain domain (V H ), which includes CDR1-H with the sequence GFTFSSY (SEQ ID NO:20), CDR2-H with the sequence SGSGGS (SEQ ID NO:21), and CDR3-H with the sequence APRSKMDY (SEQ ID NO:22), and (ii) a variable light chain domain (V L The CDR1-L contains the sequence SGSSSNIGSNTVN (SEQ ID NO:23), the CDR2-L contains the sequence SNNQRPS (SEQ ID NO:24), and the CDR3-L contains the sequence AAWDIVGEQVV (SEQ ID NO:25).

[0072] Anti-PSMA antibodies or antibody fragments can be recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or human antibodies.

[0073] In some embodiments, the anti-PSMA antibody or its antigen-binding fragment comprises:

[0074] (a) A sequence containing SEQ ID NO:26 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it. H ,

[0075] (b) A sequence containing SEQ ID NO:27 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it. L ,or

[0076] (c) A sequence containing SEQ ID NO:26 or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with it. H and V containing the sequence SEQ ID NO:27 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it. L .

[0077] In some embodiments, the anti-PSMA antibody or its antigen-binding fragment is a single-chain variable fragment (scFv).

[0078] In some implementations, the anti-PSMA scFv comprises: (i) V H It includes the sequence CDR1-H of SEQ ID NO:20, the sequence CDR2-H of SEQ ID NO:21, and the sequence CDR3-H of SEQ ID NO:22, and (ii) V L It comprises CDR1-L with sequence SEQ ID NO:23, CDR2-L with sequence SEQ ID NO:24, and CDR3-L with sequence SEQ ID NO:25, and (iii) connects the V H and the V L The connector.

[0079] In some implementations, the V of the anti-PSMA scFv H It contains the sequence SEQ ID NO:26 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it.

[0080] In some implementations, the V of the anti-PSMA scFv L It contains the sequence SEQ ID NO:27 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it.

[0081] In some implementations, the V of the anti-PSMA scFv H Contains the sequence SEQ ID NO:26 or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with it, and is anti-PSMA scFv V L It contains the sequence SEQ ID NO:27 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it.

[0082] The adapter is a chemical adapter or a peptide adapter. In some embodiments, the adapter connecting VH and VL of anti-PSMA scFv comprises the sequence GGGSGGGGSGGGGST (SEQ ID NO:19) or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with it.

[0083] Preferably, the anti-PSMA scFv contains or consists of the sequence SEQ ID NO:8.

[0084] The polypeptide containing the anti-PSMA scFv also constitutes part of this invention.

[0085] In some embodiments, the peptide is not a CAR, especially not a CAR containing an antigen-binding domain that binds PSMA, or a CAR containing an antigen-binding domain that binds PSMA and an antigen-binding domain that binds adhesin 2.

[0086] This invention also relates to a pair of V-bands that respectively encode the anti-PSMA antibody or antibody fragment disclosed herein. H and V L The invention also relates to nucleic acids comprising sequences encoding anti-PSMA scFv, or polypeptides comprising anti-PSMA scFv.

[0087] In some embodiments, the nucleic acid comprises the sequence SEQ ID NO:7 or a sequence having at least 80%, 85%, 90%, 95% or 98% identity with it.

[0088] Multispecific antibodies that bind to adhesin 2 and PSMA

[0089] Multispecific (e.g., bispecific) antibody constructs that bind at least to adhesin 2 and PSMA are also provided. Such multispecific or bispecific antibody constructs typically comprise (a) a first antigen-binding moiety that specifically binds to adhesin 2 and (b) a second antigen-binding moiety that specifically binds to PSMA, wherein the first and second antigen-binding moieties are directly connected or connected via a linker.

[0090] In some embodiments, the multispecific or bispecific antibody construct comprises an anti-adhesion 2 antibody or its antigen-binding fragment thereof and an anti-PSMA antibody or its antigen-binding fragment thereof, as disclosed herein. In some embodiments, the anti-adhesion 2 antibody or its antigen-binding fragment thereof and the anti-PSMA antibody or its antigen-binding fragment thereof are linked by a adapter.

[0091] In some implementations, in multispecific or bispecific antibody constructs:

[0092] [A] The first antigen-binding moiety that specifically binds to adhesin 2 includes (i) a variable heavy chain domain (V H ), which includes the sequence CDR1-H of SEQ ID NO:11, the sequence CDR2-H of SEQ ID NO:12, and the sequence CDR3-H of SEQ ID NO:13, and (ii) a variable light chain domain (V L ), which includes CDR1-L with sequence SEQ ID NO:14, CDR2-L with sequence SEQ ID NO:15, and CDR3-L with sequence SEQ ID NO:16; or

[0093] [B] The second antigen-binding moiety that specifically binds to PSMA contains (i) a variable heavy chain domain (V H ), which includes the sequence CDR1-H of SEQ ID NO:20, the sequence CDR2-H of SEQ ID NO:21, and the sequence CDR3-H of SEQ ID NO:22, and (ii) a variable light chain domain (V L ), which includes CDR1-L with sequence SEQ ID NO:23, CDR2-L with sequence SEQ ID NO:24, and CDR3-L with sequence SEQ ID NO:25; or

[0094] [C] The first antigen-binding moiety that specifically binds to adhesin 2 includes (i) a variable heavy chain domain (V H ), which includes the sequence CDR1-H of SEQ ID NO:11, the sequence CDR2-H of SEQ ID NO:12, and the sequence CDR3-H of SEQ ID NO:13, and (ii) a variable light chain domain (V L ), which includes CDR1-L with sequence SEQ ID NO:14, CDR2-L with sequence SEQ ID NO:15, and CDR3-L with sequence SEQ ID NO:16; and

[0095] The second antigen-binding moiety that specifically binds to PSMA includes (i) a variable heavy chain domain (V H ), which includes CDR1-H with sequence SEQ ID NO:20, CDR2-H with sequence SEQ ID NO:21 and CDR3-H with sequence SEQ ID NO:22, and (ii) a variable light chain domain (V L ), which includes CDR1-L with sequence SEQ ID NO:23, CDR2-L with sequence SEQ ID NO:24, and CDR3-L with sequence SEQ ID NO:25; or

[0096] [D] The first antigen-binding moiety that specifically binds to adhesin 2 includes (i) a variable heavy chain domain (V H ), which includes CDR1-H with sequence SEQ ID NO:11, CDR2-H with sequence SEQ ID NO:12 and CDR3-H with sequence SEQ ID NO:13, and (ii) a variable light chain domain (V L ), which includes CDR1-L with sequence SEQ ID NO:14, CDR2-L with sequence SEQ ID NO:15, and CDR3-L with sequence SEQ ID NO:16; and

[0097] The second antigen-binding moiety that specifically binds to PSMA contains V H and / or V L The V H The V contains the sequence SEQ ID NO:26 or a sequence having at least 80% identity with it. L Contains sequence SEQ ID NO:27 or a sequence having at least 80% identity with it;

[0098] [E] The first antigen-binding moiety of specifically binding adhesin 2 contains V H and / or V L The V H The V contains the sequence SEQ ID NO:17 or a sequence having at least 80% identity with it. L Contains the sequence SEQ ID NO:18 or a sequence having at least 80% identity with it; and

[0099] The second antigen-binding moiety that specifically binds to PSMA includes (i) a variable heavy chain domain (V H ), which includes CDR1-H with sequence SEQ ID NO:20, CDR2-H with sequence SEQ ID NO:21 and CDR3-H with sequence SEQ ID NO:22, and (ii) a variable light chain domain (V L ), which includes CDR1-L with sequence SEQ ID NO:23, CDR2-L with sequence SEQ ID NO:24, and CDR3-L with sequence SEQ ID NO:25; or

[0100] [F] The first antigen-binding moiety of specifically binding adhesin 2 contains V H and / or V L The V H The V contains the sequence SEQ ID NO:17 or a sequence having at least 80% identity with it. LContains the sequence SEQ ID NO:18 or a sequence having at least 80% identity with it; and

[0101] The second antigen-binding moiety that specifically binds to PSMA contains V H and / or V L The V H The V contains the sequence SEQ ID NO:26 or a sequence having at least 80% identity with it. L Contains the sequence SEQ ID NO:27 or a sequence having at least 80% identity with it; or

[0102] [G] The first antigen-binding moiety of specifically binding adhesin 2 contains V H and V L The V H The V contains the sequence SEQ ID NO:17 or a sequence having at least 80% identity with it. L Contains the sequence SEQ ID NO:18 or a sequence having at least 80% identity with it; and

[0103] The second antigen-binding moiety that specifically binds to PSMA contains V H and V L The V H The V contains the sequence SEQ ID NO:26 or a sequence having at least 80% identity with it. L A sequence containing the sequence SEQ ID NO:27 or a sequence having at least 80% identity with it.

[0104] In the multispecific or bispecific antibody construct, the linker is a chemical linker or a peptide linker.

[0105] In some embodiments, the multispecific or bispecific antibody construct comprises an anti-adhesionin 2 antibody and an anti-PSMA antibody, as disclosed herein. IgG-based bispecific antibodies are structurally similar to natural antibodies and all possess an Fc region. Different forms of IgG-based bispecific antibodies are available, including knob-in-hole format or CrossMab format.

[0106] In some embodiments, the multispecific or bispecific antibody construct comprises an anti-adhesion 2 antibody fragment and an anti-PSMA antibody fragment, as disclosed herein.

[0107] In some embodiments, the multispecific or bispecific antibody construct comprises anti-adhesionin 2 scFv and anti-PSMA scFv, as disclosed herein.

[0108] Fragment-based bispecific antibodies consist of variable light chain domains and variable heavy chain domains or Fab units from two different antibodies, and lack an Fc region. These fragments are linked together via linkers (e.g., disulfide bonds or non-covalent interactions). Various fragment-based bispecific antibodies are available, including bispecific T-cell adaptors (BiTEs), TCR mimics, tandem biantibodies (TandAbs), biaffinity-based targeted antibodies (DARTs), or bi-nanobodies (Ma et al., FrontImmunol. 2021; 12:626616).

[0109] Antibody drug conjugates

[0110] This invention also relates to antibody-drug conjugates comprising antibodies or antibody fragments as defined herein.

[0111] Antibody-drug conjugates (ADCs) typically comprise (i) an antibody or an antigen-binding fragment thereof, (ii) a cytotoxic payload, and (iii) a linker connecting the antibody or its antigen-binding fragment to the cytotoxic payload.

[0112] In some embodiments, the ADC includes (i) an anti-adhesion 2 antibody or an antigen-binding fragment thereof as disclosed herein, (ii) a cytotoxic payload, and (iii) a connector linking the anti-adhesion 2 antibody or an antigen-binding fragment thereof to the cytotoxic payload.

[0113] In some embodiments, the ADC includes (i) an anti-PSMA antibody or an antigen-binding fragment thereof as disclosed herein, (ii) a cytotoxic payload, and (iii) a connector linking the anti-PSMA antibody or its antigen-binding fragment and the cytotoxic payload.

[0114] In some embodiments, the ADC comprises (i) a multispecific or bispecific antibody construct that binds to at least adhesin 2 and PSMA, (ii) a cytotoxic payload, and (iii) a linker connecting the bispecific antibody construct and the cytotoxic payload. In some embodiments, the multispecific or bispecific antibody construct that binds to at least adhesin 2 and PSMA comprises an anti-adhesin 2 antibody or an antigen-binding fragment thereof as disclosed herein, and an anti-PSMA antibody or an antigen-binding fragment thereof as disclosed herein.

[0115] Suitable ADC connectors include both cuttable and non-cuttable connectors.

[0116] In some implementations, the adapter is a cleavable adapter. Such adapters typically include chemically cleavable adapters (e.g., hydrazone and disulfide bonds) and enzyme-cleavable adapters (e.g., glucuronide and peptide bonds).

[0117] In some implementations, the connector is an uncuttable connector (e.g., sulfide or maleimide hexanoyl).

[0118] Non-limiting examples of cytotoxic payloads commonly incorporated into ADCs include microtubule inhibitors (e.g., aureusstatin derivatives such as monomethylaureusstatin E (MMAE) and monomethylaureusstatin F (MMAF)), DNA damaging agents (e.g., inducing DNA double-strand breaks such as chaziomycin; DNA alkylation such as docalamycin; DNA intercalation such as topoisomerase I inhibitors; DNA cross-linking agents such as pyrrolobenzodiazepine (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.

[0119] Therapeutic Uses and Pharmaceutical Compositions

[0120] The ADC according to the invention is used as a drug, particularly for the treatment of prostate cancer, especially castration-resistant prostate cancer or metastatic castration-resistant prostate cancer.

[0121] The present invention also relates to a method for treating cancer in a subject in need, the method comprising administering the ADC disclosed herein to the subject.

[0122] The pharmaceutical composition comprises the ADC disclosed herein and a pharmaceutically acceptable carrier.

[0123] The subject can be a mammal, such as a primate (e.g., a human, a monkey), a rodent (e.g., a rat, a mouse), a canine (e.g., a dog), or a feline (e.g., a cat). Preferably, the subject is a human.

[0124] In some embodiments, at least one prostate cancer therapeutic agent is used in combination with the ADC. The prostate cancer therapeutic agent may be selected from chemotherapy agents (such as docetaxel or enzalutamide) and immune checkpoint inhibitors (such as anti-PD1 or anti-TIGIT antibodies).

[0125] The invention will be further described in conjunction with the following figures and embodiments. Attached Figure

[0126] Figure 1The adhesin 2-PVRIG axis is upregulated in PCa. (A) mRNA expression levels of different immune checkpoints expressed by epithelial cells and immune cells from bulk RNA data. (B) Schematic diagram of the interactions between adhesin 2 and PVR with receptors expressed by T cells and NK cells. (C) Gene expression levels in human prostate cancer samples. Among adhesin-like molecules, adhesin 2 is the most abundant in epithelial cells. On the other hand, PVRIG is the most expressed receptor in immune cells.

[0127] Figure 2 Adhesin 2 was upregulated in Pten deficiency-induced cellular senescence (PICS). (A) Experimental design and volcano plot of proteins that differed between FDG+ and FDG- epithelial cells. (B) Western blot analysis of wild-type and pten- / - prostate samples and quantification of fold changes in adhesin 2 and p21 compared to wild-type. (C) Real-time PCR data of adhesin 2 and p16 mRNA expression levels in FDG+ and FDG-sorted prostate epithelial tumor cells. (D) Adhesin 2 expression in wild-type, pten- / -, and pten- / -p53- / - mice based on RNA-seq data. (E) PTEN nonsense mice were treated with docetaxel 10 mg / kg weekly for four weeks. After 4 weeks, prostates were collected, and senescence induction was quantified by β-galactosidase assay of OCT-embedded tissue. (F) Compared with untreated mice, the anterior lobe size was significantly reduced in treated mice, and (G) Real-time PCR of prostate samples confirmed the upregulation of adhesin 2.

[0128] Figure 3 Treatment-induced senescence (TIS) further increased the level of adhesin 2 in PCa. (A) Senescence induced in PC3, 22RV1, and LNCaP human prostate cancer cell lines using palbociclib and docetaxel. Treatments were added to the culture medium for three days, followed by medium replacement, and cells were seeded three days later for β-galactosidase assay. (B) Real-time detection of adhesin 2 expression in human prostate cancer cell lines after senescence induced by palbociclib or docetaxel, and (C) Flow cytometry. (D) Senescence induction during treatment-induced senescence as measured by FDG staining in 22rv1 cells, and (E) Mean fluorescence intensity (MFI) of adhesin 2 in untreated and treated cells. (F) MFI of adhesin 2 cells in FDG- and FDG+ populations, and the percentage of untreated or androgen-deprived (ADT) FDG- and FDG+ LNCAP cells. (G) Results of β-galactosidase assay in LNCAP during androgen deprivation-induced aging and (H) mRNA levels of adhesin 2.

[0129] Figure 4The expression of adhesin 2 and PSMA in PCa is correlated. (A) Correlation matrix showing Pearson coefficients in human RNA seq samples. (B) Scatter plot showing the correlation between adhesin 2 and FOLH1 and between PVR and FOLH1. (C) Percentages of adhesin 2+PSMA-, adhesin 2+PSMA+, adhesin 2-PSMA+, and adhesin 2-PSMA- in tumor cells. (D) Comparison of the percentages of adhesin 2+PSMA-, adhesin 2+PSMA+, adhesin 2-PSMA+, and adhesin 2-PSMA- in tumor cells of castration-sensitive and castration-resistant prostate cancer patients. (E) Western blotting of patient-derived organoids showing the correlation between adhesin 2 and PSMA. (F) PSMA protein expression levels measured by Western blotting in human prostate cancer cell lines at TIS.

[0130] Figure 5 CombiCAR T cell-specific lysis of PSMA+ / adhesin 2+ and PSMA+ / adhesin 2- human PCa cell lines. (A) Target antigen expression on human PCa cell lines as assessed by flow cytometry. (B) CAR design of a second-generation monospecific anti-PSMA and anti-adhesin 2 CAR construct. (C) CAR design of a combination CAR targeting PSMA and adhesin 2. (D) CAR expression on T cells compared to non-transduced (NTD) cells as assessed by flow cytometry. (EF) CombiCAR T cells specifically lyse PSMA+ / adhesin 2+ and PSMA+ / adhesin 2- human PCa cells in a 92-hr fluorescence-based lysis assay. (G) ELISA assay of IFNg secreted by CAR T cells co-cultured with tumor cells, as described in (EF).

[0131] Figure 6 Identification of novel antiadhesin 2 and anti-PSMA scFv with high tumor specificity and internalization ability. (A) Immunofluorescence staining of human non-malignant cells (HEK293T, HUVEC, HK2, BPH, RWPE, MCF10A) and human prostate cancer cell lines (22rv1n LNCAP, PC3, PC3 Nectin2 KO, DU145) was performed using commercial antiadhesin 2 antibodies or the aforementioned antiadhesin 2 scFv antibodies, respectively. (B) Immunofluorescence staining of LNCaP cells was performed using a secondary anti-human Fc antibody, and the detected fluorescence intensity was quantified. Cells were stained with antiadhesin 2 scFv-Fc at 37°C for 1 h, washed, and permeabilized. Cell nuclei were counterstained with DAPI.

[0132] Figure 7The amino acid sequence of antiadhesion 2 scFv HRB725 shows V H and V L Chain CDR; V H The sequence of the chain is indicated by an underscore, V H With V L The (G4S)3 joint between chains is indicated in italics, V L The chain is displayed using regular characters.

[0133] Figure 8 The amino acid sequence of anti-PSMA scFv HRB730 shows V H and V L Chain CDR; V H The sequence of the chain is indicated by an underscore, V H With V L The (G4S)3 joint between chains is indicated in italics, V L The chain is displayed using regular characters.

[0134] Figure 9 Anti-adhesion 2 scFv-Fc (725v5) binds to the cancer-exposed epitopes of adhesion 2. Anti-adhesion 2 scFv-Fc (725v5) selectively binds to adhesion 2 expressed by tumor cells, in contrast to commercial anti-adhesion 2 monoclonal antibodies that bind indiscriminately to both normal and tumor cells expressing adhesion 2. Example

[0135] Example 1: Adhesin 2 mRNA is highly expressed in prostate cancer cells.

[0136] To identify novel targets for prostate cancer, we analyzed the expression of the most well-known ICs in publicly available datasets. We used raw data from the TGCA database of Human Protein Atlas (https: / / www.proteinatlas.org / (2022)) to compare the mRNA expression levels of genes expressed in epithelial cells and immune cells. Interestingly, such as Figure 1 As shown in Figure A, we observed that adhesin 2 was significantly more expressed in PCa compared to all other targets, including PD1 and CTLA4.

[0137] Adhesin 2 (A2) is a Ca2+-independent cell adhesion molecule widely expressed in antigen-presenting cells and tumor cells, and its expression is regulated in tumorigenesis. The role of A2 as an immune checkpoint has been investigated in breast and ovarian cancer (Oshima, T. et al. Mol. Cancer 12, 60 (2013)). Overexpression of A2 mRNA in prostate cancer tissues has also been reported (Oshima, T. et al. Mol. Cancer 12, 60 (2013)). Elevated protein levels have been observed in various types of cancer, such as acute myeloid leukemia, multiple myeloma, gallbladder cancer, and prostate cancer (Sanchez-Correa, B. et al. Immunol. Cell Biol. 90, 109–115 (2012); Casado, JG et al. Cancer Immunol. Immunother. CII58, 1517–1526 (2009); WO2020144697A1). Although adhesin 2 is primarily a regulator of cell adhesion, motility, and proliferation, it also has immunomodulatory effects (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 inhibit their responses by binding to the inhibitory receptors TIGIT and PVRIG (Zhu, Y. et al., J. Exp. Med. 213, 167–176 (2016)).

[0138] It must be emphasized that another member of the adhesin family, PVR, is a ligand for both the co-stimulatory receptor CD226 and the co-inhibitory receptor TIGIT. Figure 1 B). On the other hand, due to the weak interaction between adhesin 2 and TIGIT, the PVR receptor appears to remain the major ligand for TIGIT in this ligand-receptor network (Yu, X. et al. Nat. Immunol. 10, 48–57 (2009)).

[0139] However, the adhesin 2 / TIGIT / PVRIG axis in prostate cancer has not been studied.

[0140] Example 2: PVRIG is a major adhesin-2 interactor in the prostate tumor microenvironment.

[0141] To better understand the role of adhesin 2 in prostate cancer, we then evaluated the expression of its ligands / interacting factors in bulk RNA-seq data from human prostate cancer samples.

[0142] We observed high expression levels of adhesin 2 and low expression of TIGIT, thus indicating that PVRIG is a major adhesin 2 interacting factor in the prostate tumor microenvironment. Figure 1 B). Interestingly, by analyzing single-cell RNA-seq data from PCa biopsies, we confirmed that adhesin 2 is primarily expressed by epithelial and endothelial cells. Figure 1 (C). These data were initially obtained from 13 tissue samples from 12 primary tumors and 1 lymph node metastasis, yielding a transcriptomic profile of 36,424 cells after standard data processing and quality control procedures (Chen, S. et al. Nat. Cell Biol. 23, 87–98 (2021)). However, this analysis of primary prostate cancer samples was rich in epithelial cells. Therefore, we examined single-cell analysis of lethal PCs to validate PVRIG expression via single-cell data. This cohort included 2,170 cells from 14 patients and 15 fresh biopsies from three common mCRPC metastatic sites (bone, lymph nodes, and liver). PVRIG expression has been confirmed by CD4+ T cells, CD8+ T cells, and NK cells (Jordan, MA & Wilson, L. Nat. Rev. Cancer 4, 253–265 (2004)). Furthermore, we confirmed the expression of adhesin 2 protein in human prostate cancer samples by immunofluorescence (not shown).

[0143] Example 3: Upregulation of Adhesin 2 is associated with aging

[0144] Next, in collaboration with the Lausanne Proteomics Core Laboratory at the Ecole Polytechnique Fédérale de Lausanne (EPFL), we performed mass spectrometry and bioinformatics analyses to identify Pten. - / - Membrane proteins upregulated in aging mice. Using this technique, we demonstrated that adhesin 2 is one of the most upregulated transmembrane proteins expressed in aging cells. Figure 2 (A, right figure). Furthermore, bioinformatics analysis revealed that the cells with the highest senescence scores were also the cells with the highest adhesin 2 abundance.

[0145] Using our Pten-deficiency-induced cellular senescence mouse model (PICS, Toso et al., CellReports 9, 75–89 (2014)), we subsequently demonstrated a correlation between senescence and adhesin 2 expression at both transcriptional and translational levels. Western blot analysis confirmed this correlation at the translational level in total tumors. Figure 2B), and this data was also validated by qRT-PCR on sorted C12-FDG+ and C12-FDG-PTEN nonsense tumor cells. Figure 2 C). In addition to adhesin 2, we also detected several aging markers such as p21 (at the translational level) and p16 (at the transcriptional level), which were upregulated in non-proliferating cell fractions. Furthermore, RNA-seq results confirmed that, when compared with pten... - / - p53 - / - Compared to wild-type mice, pten - / - Adhesin 2 was upregulated in mice ( Figure 2 D).

[0146] We then validated in vivo in our genetically engineered mouse model whether the upregulation of adhesin 2 was associated with treatment-induced aging (TIS). Mice were treated with docetaxel 10 mg / kg weekly for four weeks, and at the end of treatment, we performed β-galactosidase staining to confirm aging-induced (TIS). Figure 2 E), consistent with the decrease in anterior lobe tumor size ( Figure 2 F). Real-time PCR data indicated a significant upregulation of adhesin 2 (F). Figure 2 G).

[0147] We validated the expression of our target in human prostate cancer cell lines. To test tumor heterogeneity in terms of mutational burden and genetic alterations, as well as in response to androgen receptor signaling, we used wild-type and mutant forms of human prostate cancer cell lines possessing both the PTEN and TP53 genes.

[0148] To assess whether adhesin 2 expression is associated with disease progression and treatment resistance, we treated human PCa cell lines with two commonly used chemotherapeutic agents for PCa management (specifically docetaxel and palbociclib) to induce cellular senescence. Docetaxel-based regimens have been shown to improve symptoms and overall survival in patients with mCRPC (Mohler, JL et al. J. Natl. Compr. Cancer Netw. JNCCN17, 479–505 (2019)). Furthermore, palbociclib (a CDK4 / 6 inhibitor) is commonly used in combination with ADT for the treatment of metastatic prostate cancer and has been shown to induce senescence at subtoxic concentrations (ClinicalTrials.gov identifier: NCT02905318).

[0149] Furthermore, within the subtoxic range, docetaxel and palbociclib also exhibited the same senescence-inducing effect, leading to treatment resistance. Subsequently, the efficacy of senescence-inducing therapy was assessed using a β-galactosidase assay. Figure 3A), and real-time PCR was performed on treated and untreated cells to measure adhesin 2 mRNA levels (A). Figure 3 B). Treatment with palbociclib induced a significant increase in adhesin 2 mRNA levels in all tested cell lines. Docetaxel treatment also significantly induced adhesin 2 mRNA levels compared to the untreated population in PC3, but not significantly in 22rv1 and LNCAP cells.

[0150] Flow cytometry analysis also confirmed the same results, clearly showing that adhesin 2 was upregulated at the translational level under all test conditions. Figure 3 C).

[0151] FDG staining in 22rv1 also confirmed the senescence-inducing effect after treatment. Figure 3 D), in the treatment fraction, adhesin 2 was upregulated ( Figure 3 E).

[0152] Senescence induction was also achieved by treating cells with enzalutamide to mimic androgen deprivation therapy. For this purpose, we used LNCaP cells because they express androgen receptors and are therefore sensitive to treatment. This was demonstrated by FDG staining (not shown) and β-galactosidase assay (…). Figure 3 As confirmed by G), this treatment induces aging and affects proteins ( Figure 3 F) and mRNA levels ( Figure 3 H) upregulated adhesin 2.

[0153] Example 4: PSMA expression and adhesin 2 expression are positively correlated in senescent prostate cancer cells.

[0154] Although the expression of adhesin 2 in the prostate TME shows cell specificity, we cannot rule out its expression in other organs.

[0155] Therefore, we designed a specific and selective therapeutic strategy based on adhesin 2 by utilizing prostate-specific membrane antigen (PSMA). To design an effective drug delivery system and immunotherapy, we first examined the correlation between adhesin 2 and all other known immune checkpoints and PSMA. We used the TCGA database and applied a correlation matrix to accomplish this. Figure 4 A and Figure 4 As shown in Figure B, we found that FOLH1 (PSMA) expression in prostate cancer was positively correlated only with adhesin 2 expression, and its intensity was twice that of PVR. Furthermore, human prostate samples were analyzed by immunofluorescence. In this case, prostate tumor biopsies from both normal and tumor tissues were stained using anti-PSMA and anti-adhesin 2 primary antibodies. The results of immunofluorescence analysis revealed a higher percentage of epithelial prostate tumor cells co-expressing adhesin 2 and PSMA. Figure 4 C). Furthermore, samples from castration-sensitive prostate cancer (CSPC) were compared with models of castration-resistant prostate cancer (CRPC), and the percentage of double-positive cells was significantly higher in CRPC samples. Figure 4 D).

[0156] In addition, we performed Western blot analysis on patient-derived human organoids (PDOs), and samples characterized by adhesin 2 expression were also PSMA-positive. Figure 4 E).

[0157] Although PSMA has been used clinically, its expression regulation in senescent cells has not been investigated. Therefore, we examined PSMA protein levels in our senescence model to assess the potential for targeting it in combination with adhesin 2. Western blot analysis of human cell lines treated with TIS showed increased PSMA expression in both LNCaP and 22RV1 cells, while no band was detected in PC3 cells because they do not express PSMA. Figure 4 F).

[0158] Example 5: Senescent cell-clearing dual CAR-T cells targeting adhesin 2 and prostate-specific membrane antigen (PSMA)

[0159] Currently, targeted therapy for prostate cancer primarily targets prostate-specific membrane antigen (PSMA). The FOLH1 gene encodes PSMA (a type II transmembrane glycoprotein with folate hydrolase and N-acetyl-α-linked acid dipeptidase activity). PSMA is expressed in normal, benign, and malignant prostate tissues, including intraepithelial and metastatic lesions (Hupe, MC et al. Front. Oncol. 8, 623 (2018); Troyer, J. et al. Int. J. Cancer 62, 552–558 (1995)). However, it has been shown that PSMA expression is 100 to 1000 times higher in prostate adenocarcinoma compared to benign prostate cancer (Heston, WDW Urology 49, 104–112 (1997)). For this reason, many radiopharmaceuticals and diagnostic tools have been engineered to target PSMA.

[0160] To prevent non-tumor-targeting toxicity targeting PSMA and adhesin 2, we designed a combined CAR T-cell approach, first described by Kloss et al., to enhance tumor specificity in the absence of tumor-restricting 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 for PSMA and adhesin 2 and generated scFv-Fc constructs with novel anti-PSMA and anti-adhesin 2 scFv candidates. To evaluate their binding specificity to PSMA or adhesin 2, we used the naturally PSMA-deficient PC3 cell line and generated adhesin 2-deficient human PCa cell lines (PC3, LNCaP, 22Rv1) using CRISPR / Cas9 technology. The absence or presence of surface PSMA and the knockout (KO) of adhesin 2 were confirmed by flow cytometry. Figure 5 A). Anti-PSMA (HRB730) (SEQ ID NO:7-8, Figure 8 ) or anti-adhesion 2 (HRB725) scFv (SEQ ID NO:9-10, Figure 7 The )-Fc construct was identified as specifically binding to its target antigen in a dose-dependent manner, as assessed by flow cytometry staining of PCa cell lines with different dilutions (1:1, 1:10, 1:500) of anti-PSMA or anti-adhesin 2 scFv-Fc construct and anti-human Fc-AF488 secondary antibody.

[0161] Importantly, it was found that the anti-adhesionin2 (725v5) scFv-Fc construct was selective only for adhesionin2 when expressed on tumor cells. Figure 9 The adhesin 2 scFv binds to the cancer-exposed epitopes of adhesin 2 (containing or consisting of the sequence SEQ ID NO: 28, amino acids at positions 222-241 of the adhesin 2 polypeptide obtained from Uniprot entry Q92692-1) without affecting non-malignant human cells expressing adhesin 2.

[0162] Subsequently, we designed and generated single-specific anti-PSMA (SEQ ID NO:1-2) or anti-adhesion 2 (SEQ ID NO:3-4) CAR constructs. Figure 5 B), and the novel CombiCAR construct (SEQ ID NO:5-6) Figure 5C). Here, the PSMA-targeting scFv is linked to a transmembrane domain (TMD) and an intracellular CD3z signaling domain, followed by a T2A peptide cleavage, while the anti-adhesion 2-targeting scFv is linked to a different TMD and an intracellular 4-1BB co-stimulatory domain. Successful T cell activation requires both intracellular CD3z signaling and co-stimulatory signals. Therefore, this design ensures that both receptors must simultaneously bind to the target adhesion 2+ cells to trigger downstream signaling, leading to T cell activation. After transducing T cells and expanding CAR T cells using their respective constructs, CAR expression was assessed by flow cytometry. Figure 5 D). To determine their antitumor killing ability, CAR T cells were co-cultured with human PCa cell line for 92 h ( Figure 5 E- Figure 5 F). For example Figure 5 E- Figure 5 As shown in F, PCa cell lines expressing PSMA were readily lysed by monospecific anti-PSMA CAR T cells, while the PSMA-deficient PCa cell line PC3 wt and its adhesin 2 KO counterpart, as well as the human glioblastoma cell line Ge904, were avoided. Similarly, monospecific anti-adhesin 2 CAR T cells specifically and efficiently targeted PCa cell lines expressing adhesin 2. Interestingly, PCa cell lines co-expressing PSMA and adhesin 2 were efficiently lysed by CombiCAR T cells, while the PSMA-deficient wild-type PC3 was avoided. PSMA+ / adhesin 2- PCa cell lines were also lysed by CombiCAR T cells because the PSMA-targeting CAR was a first-generation CAR, which can be activated alone in vitro by the intracellular CD3z signaling domain. In vivo, first-generation CAR T cells are insufficient to induce a high-efficiency T cell response and have limited persistence and activity (Brocker, T. & Karjalainen, KJ Exp.Med.181, 1653–1659 (1995)). In vivo, we hope to primarily target PPSMA. + / Adhesin 2 + Secondly, targeting PSMA + / Adhesin 2 - and avoid PSMA - / Adhesin 2 + / - PCa cells. Furthermore, as an indicator of successful T cell activation and function, IFNg secretion was assessed in the supernatant after co-culture using a sandwich ELISA. Figure 5 As shown in Figure F, the IFNg secretion levels of monospecific anti-PSMA, monospecific anti-adhesion 2, and CombiCAR T cells correspond to their cleavage capacity in the presence of their target antigens, such as... Figure 5 As assessed by G.

Claims

1. An anti-adhesion 2 antibody or an antigen-binding fragment thereof, comprising (i) a variable heavy chain domain (V H ), which includes the CDR1-H sequence of SEQ ID NO:11, the CDR2-H sequence of SEQ ID NO:12 and the CDR3-H sequence of SEQ ID NO:13, and (ii) a variable light chain domain (V L The sequence includes CDR1-L with sequence SEQ ID NO:14, CDR2-L with sequence SEQ ID NO:15, and CDR3-L with sequence SEQ ID NO:

16.

2. The anti-adhesion 2 antibody or its antigen-binding fragment according to claim 1, wherein the V H Contains sequence SEQ ID NO:17 or a sequence having at least 80% identity with it, and / or the V L It contains the sequence SEQ ID NO:18 or a sequence that has at least 80% identity with it.

3. The anti-adhesion 2 antibody or its antigen-binding fragment according to claim 1 or 2, wherein the adhesin 2 antibody is a single-chain variable fragment (scFv).

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

10.

5. A polypeptide comprising the antiadhesion 2 scFv according to claim 3 or 4.

6. A pair of isolated nucleic acids comprising variable heavy chain domains (V) encoding an anti-adhesion 2 antibody or an antigen-binding fragment thereof according to claim 1 or 2. H ) and variable light chain structural domain (V L The sequence of the antiadhesin 2 scFv as claimed in claim 3 or 4, or the isolated nucleic acid containing the sequence encoding the polypeptide as claimed in claim 5.

7. An antibody-drug conjugate (ADC) comprising (i) an anti-adhesion 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 connecting the anti-adhesion 2 antibody or the antigen-binding fragment thereof to the cytotoxic payload.

8. The antibody-drug conjugate (ADC) as defined in claim 7, for 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 an antigen-binding fragment thereof, comprising (i) a variable heavy chain domain (V H ), which includes CDR1-H with sequence SEQ ID NO:20, CDR2-H with sequence SEQ ID NO:21 and CDR3-H with sequence SEQ ID NO:22, and (ii) a variable light chain domain (V L The sequence includes CDR1-L with sequence SEQ ID NO:23, CDR2-L with sequence SEQ ID NO:24, and CDR3-L with sequence SEQ ID NO:

25.

11. The anti-PSMA antibody of claim 10 or its antigen-binding fragment, wherein the V H Contains sequence SEQ ID NO:26 or a sequence having at least 80% identity with it, and / or the V L It contains the sequence SEQ ID NO:27 or a sequence that has at least 80% identity with it.

12. The anti-PSMA antibody or its antigen-binding fragment according to claim 10 or 11, wherein the antigen-binding fragment is a single-chain variable fragment (scFv).

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

8.

14. A pair of isolated nucleic acids comprising variable heavy chain domains (V) encoding an anti-PSMA antibody or an antigen-binding fragment thereof according to claim 10 or 11. H ) and variable light chain structural domain (V L The sequence of ), or an isolated nucleic acid 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 an 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 the antigen-binding fragment thereof and the cytotoxic payload.

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

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

18. The bispecific antibody construct of claim 17, comprising an anti-adhesion 2 single-chain 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 that binds to adhesin 2 and PSMA as defined in claim 17 or 18, (ii) a cytotoxic payload, and (iii) a connector linking the bispecific antibody construct to the cytotoxic payload.

20. An antibody-drug conjugate (ADC) as defined in claim 19, for 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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