Antibody binding to steap1 or antigen-binding moiety and use thereof

Abstract

Provided are an antibody binding to STEAP1 or an antigen-binding moiety thereof and a use thereof. The antibody can bind to STEAP1 of a human, a cynomolgus monkey or a mouse, and the antibody has good affinity and an internalization effect, and has the potential to prepare an anti-tumor ADC drug.

Description

An antibody that binds to STEAP1 or its antigen-binding moiety and its applications

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 2024110544640, filed on August 1, 2024, entitled "An antibody binding to STEAP1 or its antigen-binding portion and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the fields of tumor immunotherapy and molecular immunology, specifically relating to an antibody that specifically binds to STEAP1 and its applications. Background Technology

[0004] Prostate cancer (PCa) is a major health concern, with approximately 1.3 million new cases diagnosed globally each year. Currently, about 10 million men are diagnosed with PCa, of whom approximately 700,000 have metastatic disease. Metastatic PCa causes more than 400,000 deaths annually, and this mortality rate is projected to more than double by 2040. Although the 5-year survival rate for locally transmitted PCa is greater than 99%, metastatic or recurrent PCa is generally considered incurable. Therefore, there is an urgent need for effective new drugs or combinations to treat this incurable disease.

[0005] In recent years, with the deepening research on immunotherapy, various related antigens have been discovered in prostate cancer, which have a significant impact on the occurrence, development, and prognosis of prostate cancer. Prostatic six-transmembrane epithelial antigen 1 (STEAP1) is a membrane protein containing 339 amino acids (39.9 kDa) and six transmembrane α-helices. STEAP1 is mainly expressed in prostate tissue, located in the epithelial cell membrane at intercellular junctions. In prostate cancer (PCa), STEAP1 is a highly enriched cell surface antigen.

[0006] STEAP1 acts as an ion channel or transporter at the intercellular junctions of the prostate and participates in intercellular communication. Knockout of the STEAP1 gene promotes apoptosis mediated by either exogenous or endogenous pathways. Furthermore, some studies have revealed the role of STEAP1 in cancer cells, showing that its overexpression inhibits apoptosis, enhances cell proliferation and invasion, and induces the transformation of epithelial cells into mesenchymal cells, ultimately promoting tumor progression and invasiveness. In short, STEAP1 is involved in molecular transport, cell growth, immune responses, and various biological processes.

[0007] Therefore, STEAP1 is considered a very promising target for PCa immunotherapy, and also an effective prognostic biomarker.

[0008] Among the existing treatment options for prostate cancer, the two most advanced antibody therapies are: (1) Bispecific antibody therapy: The most representative bispecific antibody molecule is Xaluritamig, a product of Amgen. This bispecific antibody targets both STEAP1 and CD3, and preclinical studies have demonstrated its efficacy against PCa cells and its safety in cynomolgus monkeys; (2) Antibody-drug conjugate (ADC) therapy: The most representative ADC molecule is Vandortuzumab vedotin, a product of Genentech. The phase 1 clinical results of this drug showed preliminary antitumor activity and tolerability in patients with non-metastatic castration-resistant prostate cancer (mPRPC). In summary, there is still a great deal of room for clinical research on the STEAP1 target for relapsed or refractory PCa. Immunotherapy antibodies and their derivatives (TCE or ADC) targeting STEAP1 are a potential high-quality therapy. Specifically, there are currently very few clinical studies targeting STEAP1 for the treatment of PCa, so it is necessary to develop STEAP1 antibodies with high efficacy and good safety. Summary of the Invention

[0009] This application provides, on the one hand, an isolated monoclonal antibody or its antigen-binding portion, which is capable of specifically binding to STEAP1, comprising i) a heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3, and ii) a light chain variable region comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:2, 3, and 4, or comprise amino acid sequences having 1-3 amino acid substitutions in each CDR compared to the above amino acid sequences; VL CDR1, VL CDR2, and VL CDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:6, 7, and 8, or comprise amino acid sequences having 1-3 amino acid substitutions in each CDR compared to the above amino acid sequences, for example, 1, 2, or 3 amino acid substitutions.

[0010] The heavy chain variable region of the isolated monoclonal antibody or its antigen-binding portion described in this application contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:1.

[0011] The light chain variable region of the isolated monoclonal antibody or its antigen-binding portion described in this application contains an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:5.

[0012] The isolated monoclonal antibody or its antigen-binding portion described in this application comprises, respectively, an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences shown in SEQ ID NOs:1 and 5.

[0013] The antibody or its antigen-binding portion thereof in this application is mouse-derived, chimeric, humanized, or fully human.

[0014] The second aspect of this application provides a humanized monoclonal antibody or its antigen-binding portion, which is capable of specifically binding to STEAP1, comprising i) a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, and ii) a light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein the sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 are as described in the first aspect of this application; and the heavy chain variable region and the light chain variable region respectively comprise (1) SEQ ID NO: 9 and 12; (2) SEQ ID NO: 9 and 13; (3) SEQ ID NO: 10 and 12; (4) SEQ ID NO: 10 and 13; (5) SEQ ID NO: 11 and 12; or (6) SEQ ID NO: 9 and 12. The sequences shown in NO:11 and 13 have amino acid sequences with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0015] In some embodiments, the antibody or its antigen-binding portion described in this application further comprises a heavy chain constant region and / or a light chain constant region, wherein the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region, and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region. The heavy chain constant region may be an IgG, IgD, IgA, IgM, or IgE heavy chain constant region, preferably a heavy chain constant region naturally or modified to have Fc receptor and / or complement system protein binding capacity, or a functional fragment thereof, such as a fragment containing the hinge region, CH2, and CH3 of the heavy chain constant region. In one embodiment, the heavy chain constant region may be an IgG1 heavy chain constant region, such as the human IgG1 heavy chain constant region. In some embodiments, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO:18. The light chain constant region may be a κ or λ light chain constant region, such as the human κ light chain constant region. In some embodiments, the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO:19.

[0016] In some embodiments, the antibody of this application may comprise two heavy chains and two light chains, or be composed of two heavy chains and two light chains, wherein each heavy chain comprises the aforementioned heavy chain constant region sequence, heavy chain variable region sequence, and / or CDR sequence, and each light chain comprises the aforementioned light chain constant region sequence, light chain variable region sequence, and / or CDR sequence. In some embodiments, the antibody of this application or its antigen-binding portion may be a Fab, F(ab')2 fragment, Fv, scFv, or (scFv)2, etc.

[0017] A third aspect of this application provides a nucleic acid molecule that encodes the antibody or its antigen-binding portion described in the first and / or second aspects.

[0018] A fourth aspect of this application provides a host cell comprising an antibody or antigen-binding portion thereof as described in the first and / or second aspects and / or a nucleic acid molecule as described in the third aspect.

[0019] In some embodiments, the host cell described in this application is a bacterial cell, a mammalian cell, a hybridoma cell, or a cell line.

[0020] The fifth aspect of this application provides a pharmaceutical composition comprising an antibody or antigen-binding portion thereof as described in the first or second aspect, a nucleic acid molecule as described in the third aspect, a host cell as described in the fourth aspect, and a pharmaceutically acceptable carrier.

[0021] In another aspect, this application provides a method for treating or alleviating STEAP1-related disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of this application to the subject.

[0022] In some embodiments, the use is the use of the antibody or antigen-binding fragment in the preparation of a medicament for diagnosing and / or treating STEAP1-expressing cancers.

[0023] In some implementations, the cancers for which STEAP1 is expressed are selected from the group consisting of: prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, stomach cancer, uterine cancer, and ovarian cancer.

[0024] In some implementations, the cancer expressed by STEAP1 is prostate cancer.

[0025] In some preferred embodiments, the prostate cancer is castration-resistant prostate cancer.

[0026] The sixth aspect of the invention also provides the use of a pharmaceutical composition for manufacturing a medicament for treating STEAP1-expressing cancers.

[0027] This invention provides an anti-STEAP1 antibody. The human-mouse chimeric antibody 91F9A9 exhibits superior binding affinity to the positive reference AMG509mAb (the anti-STEAP1 antibody portion of Xaluritamig, human IgG1 structure) on STEAP1-overexpressing cell lines. At the endocytic level, this chimeric antibody is superior to the positive reference 120.v24mab (the anti-STEAP1 antibody portion of Vandortuzumab vedotin, human IgG1 structure). Humanized mutants of 91F9A9 show significantly enhanced binding affinity to tumor cells and overexpressing cells while reducing immunogenicity. The antibody molecule 91F9A9 provided by this invention has a higher affinity than the existing Yangshen molecule AMG509mAb monoclonal antibody portion. Furthermore, the humanized variants 91F9A9-VH1+VL1, 91F9A9-VH1+VL2, 91F9A9-VH2+VL1, 91F9A9-VH2+VL2, 91F9A9-VH3+VL1, and 91F9A9-VH3+VL2 all showed superior affinity to AMG509mAb and 120.v24mAb on STEAP1-overexpressing cells and LNCaP tumor cells. At the same time, 91F9A9 exhibits stronger endocytosis levels on tumor cells than the Yangshen molecule 120.v24mAb, and may have better therapeutic effects as lead molecules for ADC and TCE drugs. Attached Figure Description

[0028] Figure 1: FACS binding results of chimeric antibody 91F9A9 and CHO-K1 / human STEAP1 overexpressing cells.

[0029] Figure 2: FACS binding results of chimeric antibody 91F9A9 and human prostate cancer cells LNCaP.

[0030] Figure 3: FACS binding results of chimeric antibody 91F9A9 and CHO-K1 / cyno STEAP1.

[0031] Figure 4: Epitope competition results between chimeric antibody 91F9A9 and monoclonal antibody AMG509mAb.

[0032] Figure 5: Epitope competition results between chimeric antibody 91F9A9 and monoclonal antibody 120.v24mAb.

[0033] Figure 6: Internalization level of chimeric antibody 91F9A9 in prostate cancer cells LNCaP.

[0034] Figure 7: FACS binding results of anti-STEAP1 humanized antibody with CHO-K1 / human STEAP1 cells.

[0035] Figure 8: FACS binding results of anti-STEAP1 humanized antibody with LNCaP cells. Detailed Implementation

[0036] Unless otherwise stated, the technical and scientific terms used in this invention have the meanings commonly understood by a person skilled in the art to which this invention pertains.

[0037] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form of the object referred to, unless the context clearly specifies otherwise.

[0038] The term "or" refers to a single element among the listed selectable elements, unless the context explicitly indicates otherwise.

[0039] The terms “comprising” or “including” mean that the stated elements, integers, or steps are included, but do not exclude the inclusion of any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps.

[0040] The term "antibody" in this article is intended to include full-length antibodies of IgG, IgA, IgD, IgE, and IgM, as well as any antigen-binding fragments (i.e., antigen-binding portions). A full-length antibody is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH or VL) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can also be divided into hypervariable regions called complementarity-determining regions (CDRs), separated by more conserved backbone regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. Methods and techniques for identifying CDRs within the amino acid sequences of the heavy and light chain variable regions are well known in the art and can be used to identify CDRs within the specific heavy and / or light chain variable region amino acid sequences disclosed herein. Exemplary specifications that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, MD, (1991); Al-Lazikani et al., *Journal of Molecular Biology*; 273:927-948 (1997); and Martin et al., *Proceedings of the National Academy of Sciences* (Proc. Natl. Acad. Sci. USA), 86:9268-9272, (1989). Public databases can also be used to identify CDR sequences within antibodies. This application adopts the Kabat definition.

[0041] The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including binding to various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system. The "functional fragment" of the antibody constant region refers to a segment within the constant region that retains certain desired functions; for example, fragments in the heavy chain constant region that retain FcR / complement system component binding activity, such as the Fc fragment.

[0042] In this article, the term "antigen-binding moiety" (or simply antibody moiety) of an antibody refers to one or more segments of an antibody that retain the ability to specifically bind to an antigen (e.g., the STEAP1 protein). It has been demonstrated that the antigen-binding function of an antibody can be exercised by segments of a full-length antibody. Examples of binding segments contained in the "antigen-binding moiety" of an antibody include (i) Fab segments, monovalent segments consisting of VL, VH, CL, and CH1; (ii) F(ab')2 segments, bivalent segments containing two Fab segments connected by a disulfide bridge in the hinge region; (iii) Fd segments consisting of VH and CH1; (iv) Fv segments consisting of the antibody's single arm VL and VH; (v) dAb segments consisting of VH (Ward et al., (1989) Nature 341:544-546); (vi) separated complementarity-determining regions (CDRs); and (vii) dAb-VL, a segment containing a single variable domain and a heavy chain constant domain. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked via a synthetic linker that makes them single-chain proteins through recombination, where the VL and VH regions pair to form a monovalent molecule. These single-chain antibodies are also intended to be included in the terminology. These antibody fragments can be obtained using techniques commonly known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0043] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to the STEAP1 protein is substantially free of antibodies that specifically bind to antigens other than STEAP1. However, an isolated antibody that specifically binds to human STEAP1 may have cross-binding to other antigens, such as STEAP1 proteins from other species. Furthermore, isolated antibodies are substantially free of other cellular material and / or chemicals.

[0044] The terms "monoclonal antibody," "monoclonal antibody," or "monoclonal antibody composition" refer to antibody molecules that consist of a single molecule. A monoclonal antibody composition exhibits specific binding specificity and affinity for a particular epitope.

[0045] Prostatic six-transmembrane epithelial antigen 1 (STEAP1) is a membrane protein containing 339 amino acids (39.9 kDa) and six transmembrane α-helices. STEAP1 is mainly expressed in prostatic tissue and is located in the epithelial cell membrane at intercellular junctions.

[0046] The term "pharmaceutically acceptable" means that when the molecular bulk and composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions. Specific examples of substances that can serve as pharmaceutically acceptable carriers or components of other substances include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc.

[0047] The anti-STEAP1 antibody of the present invention can also be used to detect and / or measure STEAP1 or STEAP1-expressing cells in a sample, for example, for diagnostic purposes. For example, the anti-STEAP1 antibody or a fragment thereof can be used to diagnose conditions or diseases characterized by abnormal expression of STEAP1 (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for STEAP1 may include, for example, contacting a sample obtained from a patient with the anti-STEAP1 antibody of the present invention, wherein the anti-STEAP1 antibody is labeled with a detectable marker or reporter molecule. Alternatively, an unlabeled anti-STEAP1 antibody may be combined with a second antibody that is itself detectably labeled for diagnostic applications. The detectable marker or reporter molecule may be a radioisotope such as 3H, 14C, 32P, 35S, or 125I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-STEAP1 antibody of the present invention comprises 89Zr-labeled antibodies, such as those labeled with 89Zr-deferroamine, for noninvasive identification and tracking of tumor cells in a subject (e.g., positron emission tomography (PET) imaging). (See, for example, Tavare, R et al., Cancer Research, January 1, 2016; 76(1):73-82; and Azad, BB et al., Oncotarget., March 15, 2016; 7(11):12344-58). Specific exemplary assays that can be used to detect or measure STEAP2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

[0048] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased from the market. Those skilled in the art will understand that the methods and materials described below are merely exemplary and should not be considered as limiting the scope of the present invention.

[0049] Example 1. Preparation of anti-STEAP1 monoclonal antibody

[0050] 1) Immunization

[0051] In accordance with current animal welfare regulations, human STEAP1 protein (protein sequence reference NCBI: AAF17479.1) was used to design mRNA lipid nanoparticles (Nanjing Chengshi), STEAP1-DNA (Nanjing Genscript), and to construct STEAP1 overexpressing cell lines as immunogens. The cells were then immunized in three groups.

[0052] 2) Screening of hybridomas secreting anti-STEAP1 antibodies

[0053] Based on the results of immunized serum testing, fusion animals were selected. Three to four days after the final immunization, spleen cells from selected mice were extracted in a sterile environment according to the standard hybridoma production protocol and fused with Sp2 / 0 cells. The fusion cells were cultured for 6–7 days in DMEM medium containing 1×HAT (hypoxanthine, aminopterin, and thymidine) and 10% fetal bovine serum. A total of 120 96-well plate hybridomas were screened. The binding ability of all hybridoma supernatants to STEAP1-overexpressing cells was analyzed by FACS, and positive clones against STEAP1 were selected. After four rounds of fusion, a total of 1179 parental hybridoma clones were selected (the ratio of the average fluorescence intensity of the supernatant binding to STEAP1-overexpressing cells in FACS was >2.5). These clones were screened and confirmed. Hybridoma parent clones were selected based on a FACS mean fluorescence intensity ratio greater than 5 for supernatant binding to STEAP1-overexpressing cells. Subcloning was then performed using limiting dilution, and the cells were cultured in DMEM medium containing 1×HT (hypoxanthine and thymidine) and 10% fetal bovine serum. FACS analysis was performed on the cell culture supernatant from the subcloned cells, yielding dozens of positive single clones. The FACS results of the 91F9A9 subclone supernatant are shown in Table 1. The results indicate that, compared to the isotype control Mouse IgG and the blank control PBS, the 91F9A9 subclone supernatant showed a FACS mean fluorescence intensity ratio greater than 100 for binding to STEAP1-overexpressing cells, and a FACS mean fluorescence intensity ratio greater than 10 for binding to tumor cells LNCaP, preliminarily demonstrating the specific binding ability of this clone to STEAP1.

[0054] Table 1: FACS binding detection of supernatant from hybridoma clone 91F9A9 subclone.

[0055] 3) Sequencing of antibodies and construction of chimeric antibodies

[0056] The hybridoma-positive subclone 91F9A9 antibody sequence was sequenced and chimeric antibody recombinant expression was validated. Its variable region and complementarity-determining region (CDR) sequences are shown in Table 2. The chimeric antibody 91F9A9 uses the following backbone sequence: human IgG1 heavy chain constant region (SEQ ID NO:18) and κ light chain constant region (SEQ ID NO:19). The expression method is as follows: an expression plasmid was constructed using the mammalian system expression vector pTT5, and the plasmid was transiently transfected into ExpiCHO-S cells for recombinant expression. The chimeric antibody obtained from recombinant expression was purified using Protein A. Meanwhile, based on the variable region sequences of the STEAP1 monoclonal antibody portions of Xaluritamig and Vandortuzumab vedotin, we recombined these two drugs in Expi293-F cells to form antibodies (named AMG509mAb and 120.v24mAb, respectively) with the human IgG1 heavy chain constant region (SEQ ID NO:18) and κ light chain constant region (SEQ ID NO:19) as the backbone, as positive control antibodies.

[0057] Table 2: Variable region and CDR sequence of 91F9A9 antibody and their SEQ ID NOs.

[0058] Example 2. Binding of CHO-K1 / human STEAP1 cells to anti-STEAP1 chimeric antibody 91F9A9

[0059] To test the binding ability of the chimeric antibody 91F9A9 obtained in Example 1 to STEAP1, FACS binding and half-maximal effector concentration (EC50) were performed using CHO-K1 / human STEAP1 overexpression cells (constructed from a robust strain; the nucleotide sequence of STEAP1 has NCBI index number NM_012449.3). The method was as follows: Each antibody was serially diluted 3-fold in PBS containing 0.1% BSA, with an initial concentration of 300 nM. 50 μL of antibody was taken and mixed with approximately 1 × 10⁻⁶ BSA in 50 μL of PBS. 5CHO-K1 / human STEAP1 cells were incubated at 4°C for 40 minutes. After washing the cells three times with PBS, 100 μL of 1 μg / ml goat anti-human IgG, Fc fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, catalog number: 109-605-098) was added, and the cells were incubated at 4°C for 30 minutes. Fluorescence signal was then measured using BD Canto. The FACS binding and EC50 values ​​of the chimeric antibody 91F9A9 to CHO-K1 / human STEAP1 overexpressing cells are shown in Table 3 and Figure 1. The results indicate that the purified chimeric antibody 91F9A9 showed FACS-specific binding ability in the STEAP1 overexpressing cell line, and the binding level was superior to the positive reference AMG509 mAb. However, compared to the positive reference 120.v24 mAb, the chimeric antibody 91F9A9 had a lower EC50.

[0060] Table 3: FACS EC50 of chimeric antibody 91F9A9 and CHO-K1 / human STEAP1 overexpressing cells

[0061] Example 3. Binding of anti-STEAP1 chimeric antibody 91F9A9 to human prostate cancer cells LNCap

[0062] To test the binding affinity of the chimeric antibody 91F9A9 obtained in Example 1 to STEAP1, the binding affinity of the chimeric antibody of this application to human prostate cancer cells LNCaP (manufacturer: China Center for Type Culture Collection, catalog number: GDC0284) was tested by FACS. The method is as follows: Each antibody was serially diluted 3-fold in PBS containing 0.1% BSA, with an initial concentration of 300 nM. 50 μL of antibody was taken and mixed with approximately 1 × 10⁻⁶ BSA in 50 μL of PBS. 5 LNCaP cells were incubated at 4°C for 40 minutes. Afterwards, the cells were washed three times with PBS, and then 100 μl of 1 μg / ml goat anti-human IgG, Fc fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, catalog number: 109-605-098) was added and incubated at 4°C for 30 minutes. Fluorescence signal was then measured using BD Canto. The FACS binding of the chimeric antibody to LNCaP cells is shown in Figure 2. The results showed that the purified chimeric antibody 91F9A9 exhibited FACS-specific binding ability on the tumor cell line LNCaP, and Figure 2 shows that the binding level of this chimeric antibody to tumor cells was comparable to that of the positive references AMG509 mAb and 120.v24 mAb.

[0063] Example 4. Binding of anti-STEAP1 chimeric antibody 91F9A9 to cynomolgus monkey STEAP1-expressing cells.

[0064] To test the species cross-binding reactivity of the chimeric antibody 91F9A9 in Examples 2 and 3 with cynomolgus monkeys, the binding affinity of the chimeric antibody to CHO-K1 / cyno STEAP1 primary cells transiently transfected with the cynomolgus monkey STEAP1 gene was tested using FACS. The method was as follows: Each antibody was serially diluted 3-fold in PBS containing 0.1% BSA, with an initial concentration of 300 nM. 50 μL of antibody was taken and mixed with approximately 1 × 10⁻⁶ BSA in 50 μL of PBS. 5 CHO-K1 / cyno STEAP1 cells were incubated at 4°C for 40 minutes. After washing the cells three times with PBS, 100 μl of 1 μg / mL goat anti-human IgG, Fc fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, catalog number: 109-605-098) was added, and the cells were incubated at 4°C for 30 minutes. Fluorescence signal was then measured using BD Canto. The FACS binding and EC50 values ​​of the chimeric antibody 91F9A9 to cynomolgus monkey STEAP1-expressing cells are shown in Table 4 and Figure 3. The results show that the chimeric antibody 91F9A9 can bind to cynomolgus monkey STEAP1-expressing cells in a concentration gradient, indicating that this chimeric antibody possesses human-cynomolgus monkey cross-reactivity.

[0065] Table 4: FACS EC50 of chimeric antibodies 91F9A9 and CHO-K1 / cyno STEAP1

[0066] Example 5. Epitope competition between anti-STEAP1 chimeric antibody 91F9A9 and monoclonal antibodies AMG509mAb and 120.v24mAb

[0067] To test whether antibody 91F9A9 in this application binds to the same epitope as the positive control comparison products AMG509mAb and 120.v24mAb, CHO-K1 / human STEAP cells were used, and FACS testing was performed to determine whether each antibody in this application competes with the epitope of the positive control product.

[0068] The AMG509 mAb and 120.v24 mAb monoclonal antibodies were biotinylated using the biotinylation agent N-[6-(biotinamino)hexanoyl]-6-aminohexanoic acid N-succinimide ester (manufacturer: Aladdin, catalog number: B122220-25mg). In short, the AMG509 mAb, 120.v24 mAb monoclonal antibodies, and the biotinylation agent were mixed at a molar ratio of 10:1 and incubated at room temperature for 2 hours. The biotinylated antibody was then dialyzed against PBS at pH 7.4 at 4°C. The biotinylated AMG509 mAb and 120.v24 mAb were then tested for binding EC50 to CHO-K1 / human STEAP1 cells using the method described in Example 2 via FACS, and a concentration of 0.01 μg / mL (0.067 nM) was selected for the following epitope competition assay.

[0069] Each antibody in this application was serially diluted 3-fold in PBS, starting at a concentration of 300 nM, resulting in a total of 12 concentrations. 50 μl of antibody was taken and mixed with approximately 1 × 10⁻⁶ ppm of PBS. 5 CHO-K1 / human STEAP1 cells were incubated at 4°C for 40 minutes. After washing the cells three times with PBS, 100 μL of biotin-conjugated AMG509 mAb and 120.v24 mAb (final concentration 0.01 μg / mL) were added, and the cells were incubated at 4°C for 40 minutes. Then, SA fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, catalog number: 016-600-084) was added, and the cells were incubated at 4°C for 30 minutes. Fluorescence signal was then measured in a BD Canto instrument. The epitope competition between the chimeric antibody 91F9A9 and the monoclonal antibodies AMG509 mAb and 120.v24 mAb is shown in Figures 4-5. The results showed that the chimeric antibody 91F9A9 exhibited concentration-dependent epitope competition with both the positive references AMG509mAb and 120.v24mAb, indicating that the binding epitope of the chimeric antibody on the STEAP1 antigen was consistent with or similar to that of the positive references.

[0070] Example 6. Internalization of anti-STEAP1 chimeric antibody 91F9A9 in prostate cancer cells LNCaP

[0071] Antibodies, as an important component of ADC drugs, possess targeting capabilities. They can specifically recognize tumor cell surface antigens, bind to these antigens, undergo endocytosis, and deliver cytotoxins to tumor cells to exert toxic effects. Therefore, antibody internalization ability is an important indicator for early screening of ADC drugs. Referring to Examples 2-5, to evaluate the ADC drug development potential of the chimeric antibody 91F9A9, we tested the internalization efficiency of the chimeric antibody 91F9A9 on LNCaP cells and compared it with the naked antibody portion 120.v24mAb of the clinical-stage ADC drug Vandortuzumab vedotin. The specific procedures are as follows.

[0072] Digest, centrifuge to collect target cells, and resuspend the target cells (LNCaP) in experimental buffer (appropriate complete culture medium) to adjust the target cell density (2×10⁻⁶). 5 Transfer the cell suspension (50 μL / well) to a 96-well plate. Incubate the 96-well plate overnight in a cell culture incubator (37°C / 5% CO2). Prepare the test / control working solution and the labeled reagent working solution (4×) (manufacturer: Sartorius, catalog number: 4649) using experimental buffer. Mix the test / control working solution and the labeled reagent working solution at a 1:3 molar ratio and a 1:1 volume ratio, and incubate in a cell culture incubator for 15 minutes to ensure complete coupling. Transfer the coupling mixture to the corresponding wells of the 96-well plate.

[0073] The experimental plate was incubated in a cell culture incubator (37℃ / 5% CO2) for a certain period of time, and a detection wavelength compatible with the specified wavelength was used. The instrument (Germany) takes pictures and images at specified time points. Use The Live-Cell Analysis System software calculates the total integrated red fluorescence intensity within the cell, which is used to represent the antibody internalization rate.

[0074] Figure 6 shows the internalization level of the chimeric antibody 91F9A9 after 48 hours of incubation. The results indicate that the chimeric antibody 91F9A9 exhibits concentration-dependent internalization in the LNCaP tumor cell line, with the highest internalization level at high concentrations, and the overall internalization level is higher than that of the positive reference standard 120.v24mAb. Therefore, this chimeric antibody has potential value as a high-quality ADC drug.

[0075] Example 7. Humanization design and recombinant production of anti-STEAP1 antibody molecule 91F9A9

[0076] The parental antibody structure was modeled using computer-aided homology modeling (MOE) software. A human natural germline sequence with high homology to the parental sequence was selected. CDRs of the positive monoclonal antibody were grafted into the human natural germline sequence using CDR grafting technology to obtain a humanized chimeric antibody derived from the parental antibody. Different amino acid residues of the chimeric antibody and the parental antibody were compared. Based on classical residues, interaction loop regions, core regions, and mutation hotspots, key points affecting subsequent affinity were identified, and appropriate sites were selected for combination design to obtain the variable region amino acid sequence of the humanized antibody. The amino acid sequence of the humanized antibody was codon-optimized, and the heavy and light chain variable region DNA fragments were inserted into the heavy chain plasmid Human IgG1-CH-pcDNA3.4 and the light chain plasmid Human Igkappa-CL-pcDNA3.4 expression vectors (constructed by Pengbo), respectively, to form expression plasmids.

[0077] The above-mentioned heavy and light chain plasmids were mixed 1:1 and transfected into CHO cells, which were then cultured in shake flasks at 37°C. The supernatant was collected for antibody purification. The filtered cell culture supernatant was loaded onto an affinity purification column at an appropriate flow rate. After washing and eluting with a suitable buffer, the product buffer was replaced with PBS (pH 7.2) to remove any elution buffer, and the sample was concentrated. After concentration, the antibody was quantified by OD280 nm. The molecular weight and purity of the purified antibody were determined by SDS-PAGE and SEC-HPLC.

[0078] The mutant sequences of the variable region of the humanized antibody are shown in Table 5.

[0079] Table 5. Sequences of humanized antibody variable region mutants and their corresponding sequence numbers

[0080] Example 8. Binding of anti-STEAP1 humanized antibody to CHO-K1 / STEAP1 cells

[0081] To test the binding ability of the humanized antibody obtained in Example 7 to STEAP1, FACS detection was performed using CHO-K1 cells (Burn-built, the nucleotide sequence of STEAP1 has NCBI index number NM_012449.3) overexpressing human STEAP1.

[0082] Each antibody was serially diluted 3-fold in PBS containing 0.1% BSA, starting at a concentration of 300 nM. Take 50 μl of antibody and mix it with approximately 1 × 10⁻⁶ BSA in 50 μl of PBS. 5CHO-K1 / STEAP1 cells were incubated at 4°C for 40 minutes. Afterwards, the cells were washed three times with PBS, and 100 μL of 1 μg / ml goat anti-human IgG fluorescent secondary antibody (manufacturer: Jackson ImmunoResearch, catalog number: 109-605-098) was added and incubated at 4°C for 30 minutes. Fluorescence signal was then measured using BD Canto.

[0083] Chimeric antibody 91F9A9 was used as an isotype control for humanized antibodies, and human IgG was used as a negative control.

[0084] The average fluorescence intensity and half-maximal effect concentration (EC50) of each antibody are shown in Table 6 and Figure 7. The results show that the humanized antibodies 91F9A9-VH1+VL1, 91F9A9-VH1+VL2, 91F9A9-VH2+VL1, 91F9A9-VH2+VL2, 91F9A9-VH3+VL1, and 91F9A9-VH3+VL2 exhibited superior affinity to the parental chimeric antibodies in STEAP1 overexpressing cell lines, and their EC50 values ​​were significantly reduced. This indicates that humanization of these six antibodies did not affect their affinity; on the contrary, it enhanced the affinity of the chimeric antibodies.

[0085] Table 6. EC50 of anti-STEAP1 humanized antibody binding to CHO-K1 / human STEAP1 cells

[0086] Example 9. Binding of anti-STEAP1 humanized antibody to LNCaP cells

[0087] The binding ability of the humanized antibody of this application to human prostate cancer cells LNCaP (manufacturer: China Center for Type Culture Collection, catalog number: GDC0284) was tested.

[0088] Each antibody was serially diluted 3-fold in PBS containing 0.1% BSA, starting at a concentration of 300 nM. Take 50 μL of antibody and mix it with approximately 1 × 10⁻⁶ BSA in 50 μL of PBS. 5 LNCaP cells were incubated at 4°C for 40 minutes. After washing the cells three times with PBS, 100 μL of 1 μg / ml goat anti-human IgG fluorescent secondary antibody (manufacturer: Jackson Immuno Research, catalog number: 109-605-098) was added, and the cells were incubated at 4°C for 30 minutes. The fluorescence signal was then measured in BD Canto.

[0089] Chimeric antibody 91F9A9 was used as an isotype control for humanized antibodies, and human IgG was used as a negative control.

[0090] The average fluorescence intensity of each antibody is shown in Figure 8. The results show that the humanized antibodies 91F9A9-VH1+VL1, 91F9A9-VH1+VL2, 91F9A9-VH2+VL1, 91F9A9-VH2+VL2, and 91F9A9-VH3+VL1 of this application all showed higher average fluorescence intensity than the parental chimeric antibody 91F9A9 at different concentration points on LNCaP cells, further demonstrating that the humanization process reduced immunogenicity while improving the affinity of the chimeric antibody.

[0091] The partial amino acid sequence of this application is shown below:

[0092] AMG509mAb - Heavy Chain Variable Region

[0093] AMG509 mAb - Light Chain Variable Region

[0094] 120.v24 mAb - Heavy Chain Variable Region

[0095] 120.v24 mAb - Light Chain Variable Region

[0096] Human IgG1 heavy chain constant region

[0097] Human κ chain constant region

[0098] The embodiments of the present invention are not limited to those described above. Without departing from the spirit and scope of the present invention, those skilled in the art can make various changes and improvements to the present invention in form and detail, and these are all considered to fall within the protection scope of the present invention.

Claims

1. An isolated monoclonal antibody, or an antigen binding portion thereof, capable of specific binding to STEAP1, comprising i) a heavy chain variable region comprising a VH CDR1, a VH CDR2, and a VH CDR3, and ii) a light chain variable region comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, or comprise an amino acid sequence having 1-3 amino acid substitutions in each CDR compared to the above-mentioned amino acid sequences; the VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, or comprise an amino acid sequence having 1-3 amino acid substitutions in each CDR compared to the above-mentioned amino acid sequences.

2. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

1.

3. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

5.

4. The isolated monoclonal antibody, or an antigen binding portion thereof, of claim 1, wherein the heavy chain variable region and the light chain variable region comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequences set forth in SEQ ID NOs: 1 and 5, respectively.

5. The antibody, or antigen binding fragment thereof, of any one of claims 1-4, which is mouse-derived, chimeric, humanized, or fully human.

6. A humanized monoclonal antibody or an antigen-binding portion thereof capable of specifically binding to STEAP1, comprising i) a heavy chain variable region comprising a VH CDR1, a VH CDR2, and a VH CDR3, and ii) a light chain variable region comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the sequences of the VH CDR1, the VH CDR2, the VH CDR3, the VL CDR1, the VL CDR2, and the VL CDR3 are as set forth in claim 1; and, the heavy chain variable region and the light chain variable region each comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in (1) SEQ ID NOs: 9 and 12; (2) SEQ ID NOs: 9 and 13; (3) SEQ ID NOs: 10 and 12; (4) SEQ ID NOs: 10 and 13; (5) SEQ ID NOs: 11 and 12; or (6) SEQ ID NOs: 11 and 13.

7. The antibody or antigen-binding portion thereof of any one of claims 1-6, further comprising a heavy chain constant region and / or a light chain constant region.

8. The antibody or antigen-binding portion thereof of claim 7, the heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:

18.

9. The antibody or antigen-binding portion thereof of claim 7, the light chain constant region comprising the amino acid sequence set forth in SEQ ID NO:

19.

10. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-9.

11. A host cell comprising the antibody or antigen-binding portion thereof of any one of claims 1-9 and / or comprising the nucleic acid molecule of claim 10.

12. The host cell of claim 11, wherein, The cell is a bacterial cell, a mammalian cell, a hybridoma cell, or a cell line.

13. A pharmaceutical composition comprising the antibody or antibody fragment of any one of claims 1-9, the nucleic acid molecule of claim 10, the host cell of claim 11 or 12, and a pharmaceutically acceptable carrier.

14. Use of the antibody or antigen-binding fragment of any one of claims 1-9 for the manufacture of a medicament for the diagnosis and / or treatment of a cancer expressing STEAP1.

15. The use of claim 14, wherein the cancer expressing STEAP1 is selected from the group consisting of prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, gastric cancer, uterine cancer, and ovarian cancer.

16. The use of claim 15, wherein the cancer expressing STEAP1 is prostate cancer.

17. The use of claim 16, wherein the prostate cancer is castration-resistant prostate cancer.

18. Use of the pharmaceutical composition of claim 13 for the manufacture of a medicament for the treatment of a cancer expressing STEAP1.

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