Hcar1 monoclonal antibodies and uses thereof

Abstract

The present disclosure provides HCAR1 monoclonal antibodies and uses thereof. The present disclosure also provides polynucleotides encoding the HCAR1 monoclonal antibodies, vectors comprising the polynucleotides, host cells comprising the vectors, methods of producing the antibodies, and compositions comprising the antibodies. The present disclosure further provides antibody drug conjugates comprising the HCAR1 monoclonal antibodies conjugated to a cytotoxic drug, and uses of the antibodies or the antibody drug conjugates in the manufacture of a medicament for treating cancer.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedical technology, specifically relating to monoclonal antibodies that specifically bind to human HCAR1 and antibody-drug conjugates thereof. This disclosure also relates to polynucleotides encoding antibodies, vectors containing polynucleotides, host cells, and the use of antibodies and ADCs in the preparation of medicaments for the treatment and diagnosis of cancer. Background Technology

[0002] Over the past 20 years, antibody-mediated cancer therapy has been established as a successful and important cancer treatment strategy. The importance of iterative approaches for antigen target selection and optimal antibody development has been demonstrated in clinical trials of antibodies for cancer patients. Although many antibody-based therapies have been developed and applied to lung cancer patients (Coleman N et al., 2023), there is still a need to focus on identifying novel targets.

[0003] A central factor in cancer cells and their metabolism is the lactate receptor HCAR1 (also known as GPR81). Hypoxia is a sentinel feature of solid tumors. In association with this hypoxia, lactate may act as a signaling molecule to regulate various cellular processes associated with cancer progression and other diseases (Adeva-Andany M et al., 2014; Yang L et al., 2023; Baltazar F et al., 2020). The lactate receptor HCAR1 is upregulated in cancer and promotes cancer cell proliferation and metastasis (Brown TP et al., 2020; Roland CL et al., 2014). Outside of cancer, HCAR1 is constitutively expressed only in adipocytes or cells that promote lipid storage. Database searches show that despite variable tissue RNA expression, there is little widespread protein expression. Elevated HCAR1 expression has been observed in cancer cells but not in control cells (Roland CL et al., 2014; Yang LB et al., 2025). The inventors and others have found that nuclear transport of HCAR1 promotes cancerous malignancies. Cancer cells with HCAR1 knockdown or blockade of HCAR1 subcellular transport exhibited inhibition of growth and metastasis (Yang LB et al., 2025). Therefore, HCAR1 is an attractive cancer-specific target (Kesireddy M et al., 2024; Filis P et al., 2023). Currently, HCAR1 has been found to have significantly upregulated protein and mRNA expression in various tumors, including diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, glioma, gastric adenocarcinoma, thymic carcinoma, and pancreatic ductal adenocarcinoma, and is directly associated with tumor malignancy and poorer prognosis.

[0004] Monoclonal antibodies can kill tumor cells through direct action, antibody-dependent cytotoxicity (ADCC), payload delivery, or specific action of antibodies on the tumor vascular system and matrix. Among these mechanisms, antibody-drug conjugates (ADCs) have emerged as a revolutionary therapeutic class, combining the precise targeting capabilities of monoclonal antibodies with the potent cytotoxic effects of chemotherapeutic drugs (Coleman N et al., 2023; Kesireddy M et al., 2024; Filis P et al., 2023). ADC therapy also shows promise in enhancing immune system responses through antibody-mediated cytotoxicity, tumor-specific immunity, and adaptive immune responses.

[0005] Monomethylaurestatin E (MMAE) is a potent toxic payload that has been used in many FDA-approved ADCs (Bordeau BM et al., 2023; Yaghoubi S et al., 2021; Yip V et al., 2024; Hingorani DV et al., 2022). The inventors developed a novel antibody targeting HCAR1 and conjugated it to MMAE, resulting in an ADC that was lethal to NSCLC cells in vitro. These data suggest that HCAR1 may be a potential therapeutic target for limiting the growth and metastasis of NSCLC lung cancer. Summary of the Invention

[0006] This disclosure provides an antibody that specifically binds to human HCAR1 or an antigen-binding fragment thereof, as well as an antibody-drug conjugate comprising the same, to meet the needs of the art.

[0007] On one hand, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human HCAR1, comprising: a heavy chain variable region (VH), the heavy chain variable region comprising CDR-H1 as shown in SEQ ID NO:1, CDR-H2 as shown in SEQ ID NO:2, and CDR-H3 as shown in SEQ ID NO:3; and a light chain variable region (VL), the light chain variable region comprising CDR-L1 as shown in SEQ ID NO:4, CDR-L2 as shown in SEQ ID NO:5, and CDR-L3 as shown in SEQ ID NO:6.

[0008] In one embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises: VH having at least 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:7, and VL having at least 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:8.

[0009] In a preferred embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises: VH as shown in SEQ ID NO:7 and VL as shown in SEQ ID NO:8.

[0010] In one embodiment, the antibody or antigen-binding fragment of the present disclosure comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3, or IgG4 subclasses. In one embodiment, the antibody or antigen-binding fragment of the present disclosure comprises a light chain constant region derived from human λ or κ light chains.

[0011] In one embodiment, the disclosed antibody or its antigen-binding fragment is a full-length antibody. In another embodiment, the disclosed antibody or its antigen-binding fragment is an antibody fragment selected from Fv, scFv, Fab, Fab', and F(ab')2.

[0012] In one embodiment, the antibody or its antigen-binding fragment disclosed herein has one or more of the following properties: (1) it can specifically bind to human HCAR1 protein; (2) it can specifically bind to cancer cells expressing human HCAR1; and (3) it can be internalized by cells expressing HCAR1.

[0013] On the other hand, this disclosure provides an antibody-drug conjugate comprising the antibody described herein or an antigen-binding fragment thereof, and a cytotoxic drug conjugated thereto. In a preferred embodiment, the cytotoxic drug is monomethylaurestatin E (MMAE).

[0014] On the other hand, this disclosure provides a polynucleotide that encodes an antibody or antigen-binding fragment thereof. In one embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO:9 and / or 10.

[0015] On the other hand, this disclosure provides a vector comprising the polynucleotides of this disclosure. On the other hand, this disclosure provides a host cell comprising the polynucleotides or vectors of this disclosure.

[0016] On the other hand, this disclosure provides a method for generating an antibody or an antigen-binding fragment thereof, comprising: (a) culturing a host cell of this disclosure under conditions suitable for expressing the antibody or the antigen-binding fragment thereof, and (b) optionally, recovering the antibody or the antigen-binding fragment thereof.

[0017] On the other hand, this disclosure provides a composition comprising the antibody or antigen-binding fragment thereof, an ADC, a polynucleotide, a vector, or a host cell.

[0018] On the other hand, this disclosure provides the use of the antibodies of this disclosure or their antigen-binding fragments or ADCs in the preparation of reagents for detecting the presence or level of HCAR1 in biological samples.

[0019] On the other hand, this disclosure provides the use of the antibody or its antigen-binding fragment or ADC disclosed herein in the preparation of a medicament for the prevention or treatment of cancer. Preferably, the cancer is an HCAR1-expressing cancer, such as non-small cell lung cancer, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, glioma, gastric adenocarcinoma, thymic carcinoma, and pancreatic ductal adenocarcinoma. Attached Figure Description

[0020] Figure 1 WB images of HCAR1 in different lung cancer cells provided for the examples.

[0021] Figure 2 The results of the monoclonal antibody 1A6 binding activity assay provided in the example are shown.

[0022] Figure 3 The affinity test results for the monoclonal antibody 1A6 provided in the example are shown.

[0023] Figure 4 The figure shows the statistical results of the killing effect of different HCAR1 antibodies on lung cancer cells provided in the examples.

[0024] Figure 5 The figure shows the statistical results of the binding activities of different HCAR1 antibodies and their ADCs against natural HCAR1 and recombinant HCAR1, respectively, provided in the examples.

[0025] Figure 6 The results of the ADC internalization experiment provided for the example are shown in the figure.

[0026] Figure 7 The figure shows the results of the in vitro cytotoxicity assay of the HCAR1 antibody and its ADC provided in the example.

[0027] Figure 8 The figure shows the time-dependent killing effect of different HCAR1ADCs on small cell lung cancer cells provided in the example.

[0028] Figure 9 The in vivo efficacy experiment results are shown in the example diagram. Detailed Implementation

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Otherwise, certain terms used herein have the meanings set forth in the specification.

[0030] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a / an”, “a / an”, and “” used herein and in the appended claims include plural indicators.

[0031] Unless otherwise stated, any numerical value (such as concentration or concentration range in this document) should be understood to be modified by the term "about" in all cases. Therefore, numerical values ​​typically include ±10% of the listed values.

[0032] As used herein, the terms “comprising,” “including,” “having,” “containing,” or any other variation thereof shall be understood to imply inclusion of the integers or groups of integers stated therein, but not to exclude any other integers or groups of integers.

[0033] As used in this article, “subject” or “individual” means any animal, preferably a mammal, and most preferably a human.

[0034] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" percentage refers to the fact that two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned to obtain the maximum correspondence, as measured by using sequence comparison algorithms or by visual inspection.

[0035] As used herein, the term "antibody" is used broadly and includes immunoglobulins or antibody molecules, including monoclonal or polyclonal human antibodies, humanized antibodies, chimeric antibodies, and antibody fragments. Antibody structures are well known. Based on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be classified into five major classes (i.e., IgA, IgD, IgE, IgG, and IgM). The antibodies of this disclosure can belong to any of the five major classes or corresponding subclasses, preferably IgG1, IgG2, IgG3, or IgG4. The antibody light chain can be classified into one of two types (i.e., κ and λ). The antibodies of this disclosure can contain either a κ or λ light chain constant domain.

[0036] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment containing at least one CDR of an immunoglobulin heavy chain and / or light chain that binds to a target antigen. The antigen-binding fragment of the antibody described herein may contain one, two, three, four, five, or all six CDRs of the VH and / or VL sequences described herein from an antibody binding to HCAR1.

[0037] As used in this article, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical, except for the possibility of naturally occurring mutations that may be present in small amounts.

[0038] As used herein, the term "antibody-drug conjugate" or "ADC" refers to a biological drug formed by conjugating an antibody to a cytotoxic drug via a linker. ADCs combine the targeting specificity of antibodies with the potent cell-killing ability of cytotoxic drugs.

[0039] As used herein, the term "epitope" includes any determinant capable of specifically binding to immunoglobulins or T-cell receptors, preferably polypeptide determinants.

[0040] The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer encoded information into a host cell.

[0041] The term “specific binding” used in this article to refer to antibodies that recognize a specific antigen but do not substantially recognize or bind to other molecules in the sample.

[0042] The structure and location of the immunoglobulin variable region (CDR) can be determined by referring to the following: the Kabat numbering scheme, the Chothia numbering scheme, or the IMGT numbering scheme. Unless otherwise specified, the location of the CDR is determined according to the Kabat or IMGT numbering scheme in this document.

[0043] "Human HCAR1" refers to the human hydroxycarboxylic acid receptor 1, also known as GPR81. Human HCAR1 is a G protein-coupled receptor composed of 346 amino acids.

[0044] This disclosure generally relates to isolated HCAR1 monoclonal antibodies, nucleic acids encoding the antibodies and expression vectors, recombinant cells containing the vectors, and compositions containing the antibodies. Methods for preparing the antibodies are also provided. The antibodies of this disclosure possess one or more desired functional properties, including but not limited to high affinity binding to HCAR1, high specificity for HCAR1, and the ability to be internalized by HCAR1-expressing cells. More specifically, the antigenic structural region bound by the HCAR1 monoclonal antibody is the HCAR1 membrane surface region.

[0045] In one general aspect, this disclosure relates to isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to HCAR1.

[0046] In one embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (VH) comprising CDR-H1 as shown in SEQ ID NO:1, CDR-H2 as shown in SEQ ID NO:2, and CDR-H3 as shown in SEQ ID NO:3; and a light chain variable region (VL) comprising CDR-L1 as shown in SEQ ID NO:4, CDR-L2 as shown in SEQ ID NO:5, and CDR-L3 as shown in SEQ ID NO:6.

[0047] In a preferred embodiment, the antibody or antigen-binding fragment thereof disclosed herein comprises VH as shown in SEQ ID NO:7 and VL as shown in SEQ ID NO:8.

[0048] In one embodiment, the antibody or its antigen-binding fragment disclosed herein further comprises a constant region. The heavy chain constant region may be derived from human IgG1, IgG2, IgG3, or IgG4 subclasses. The light chain constant region may be derived from human λ light chain or κ light chain.

[0049] The scope of antibodies disclosed herein also includes various forms, such as antibody fragments selected from Fv, scFv, Fab, Fab', and F(ab')2. The scope of antibodies disclosed herein also includes their derived forms, such as antibody-drug conjugates.

[0050] This disclosure also provides antibody-drug conjugates comprising the HCAR1 monoclonal antibody described herein or its antigen-binding fragment, and a cytotoxic drug conjugated thereto. The cytotoxic drug can be any cytotoxic drug known in the art, such as auristatins like MMAE or MMAF; maytansinoids like DM1 or DM4; camptothecin analogs like SN-38; or pyrrolobenzodiazepine (PBD) dimers, etc. In a preferred embodiment, the cytotoxic drug is MMAE.

[0051] Antibodies and cytotoxic drugs can be conjugated via linkers. Linkers can be cleavable (e.g., hydrazone bonds, disulfide bonds, or peptide bonds) or non-cleavable. Suitable linkers and conjugation techniques are well known in the art.

[0052] In one embodiment, the ADC of this disclosure has one or more of the following characteristics: (1) it can specifically bind to cancer cells expressing human HCAR1; (2) it can be internalized by cells expressing HCAR1; (3) it is cytotoxic to cancer cells expressing HCAR1 in vitro; and (4) it can inhibit the growth of tumors expressing HCAR1 in vivo.

[0053] This disclosure also provides polynucleotides encoding any of the antibodies disclosed herein. In some embodiments, isolated polynucleotides encoding antibodies or antibody fragments that bind to HCAR1 are disclosed herein, wherein the antibody or antibody fragment comprises three heavy chain CDRs having the amino acid sequences shown in SEQ ID NO:1-3; and three light chain CDRs having the amino acid sequences shown in SEQ ID NO:4-6. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding the VH shown in SEQ ID NO:7 and a nucleotide sequence encoding the VL shown in SEQ ID NO:8. In a preferred embodiment, the polynucleotide comprises sequences as shown in SEQ ID NO:9 and / or 10.

[0054] This disclosure also provides vectors comprising isolated nucleic acid molecules encoding monoclonal antibodies or antigen-binding fragments thereof of this disclosure. Any vector known to those skilled in the art can be used, such as plasmids, granules, phage vectors, or viral vectors. In some embodiments, the vector is a recombinant expression vector, such as a plasmid.

[0055] This disclosure also provides a host cell containing an isolated nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof of this disclosure. Any host cell known to those skilled in the art can be used for recombinant expression of the antibody or antigen-binding fragment thereof of this disclosure. In some embodiments, the host cell is a CHO cell, a 293F cell, or a HEK293 cell.

[0056] This disclosure also provides a method for generating a monoclonal antibody or an antigen-binding fragment thereof, the method comprising culturing a cell containing a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof under conditions for generating the monoclonal antibody or an antigen-binding fragment thereof, and recovering the antibody or an antigen-binding fragment thereof from the cell or cell culture.

[0057] This disclosure also provides pharmaceutical compositions comprising the isolated monoclonal antibody or antigen-binding fragment thereof or ADC of this disclosure, and a pharmaceutically acceptable carrier. The active ingredients of this disclosure and compositions comprising them can also be used to manufacture medicaments for the therapeutic applications mentioned herein.

[0058] HCAR1 signaling is an important pathway in the pathogenesis of cancer. In some embodiments, the antibodies or ADCs disclosed herein can be used to prepare drugs for treating cancer. The cancer to be treated is preferably an HCAR1-expressing cancer, examples of which include, but are not limited to, non-small cell lung cancer, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, glioma, gastric adenocarcinoma, thymic carcinoma, and pancreatic ductal adenocarcinoma.

[0059] As used in this article, “treatment” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0060] This disclosure is further described with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or conditions recommended by the manufacturer.

[0061] Example 1: Cloning, sequencing and expression of antibody heavy and light chain genes 1. Construction of recombinant expression vectors The relevant functional region amino acid sequences of the HCAR1 monoclonal antibody referred to in this disclosure are as follows: CDR-H1:EITVSSNYMN(SEQ ID NO:1) CDR-H2:VIYSGGTTYYADSVKG(SEQ ID NO:2) CDR-H3:DLMEVGGMDV(SEQ ID NO:3) CDR-L1:SGDNLGGNKYVS(SEQ ID NO:4) CDR-L2:RDTQRPS(SEQ ID NO:5) CDR-L3:QAWDSTTAV(SEQ ID NO:6) The full-length heavy chain variable region of the monoclonal antibody 1A6 contains the amino acid sequence shown in SEQ ID NO.7: EVQLLESGGGLVQPGGSLRLSCAAS PVIVEPDGN WVRQAPGKGLEWVS IYSMNYYSSVKG RFTISRDNSENTLYLQMNSLRAEDTAVYYCAR DRDEVSNMDVDV WGQGTTVTVSS; The full-length light chain variable region of the monoclonal antibody 1A6 comprises the amino acid sequence shown in SEQ ID NO. 8: QPVLTQPPSVSVSPGQTASIT CSGDSPVKYMVS WYEQKPGQSPVVVIY RGMTQDAPS GIPERYSGSNSGNTATLTISGTQALDEADYYC QAWDDEW VFGGGTKLTVL.

[0062] The nucleotide sequences encoding the heavy chain variable region (VH) and light chain variable region (VL) of the HCAR1 monoclonal antibody (1A6) referred to in this disclosure have been optimized to suit the CHO cell expression system.

[0063] Based on the heavy chain variable region amino acid sequence (SEQ ID NO: 7) and light chain variable region amino acid sequence (SEQ ID NO: 8) provided in the examples, reverse translation and sequence optimization were performed using human codon bias, and DNA sequences encoding VH and VL were designed and synthesized respectively.

[0064] Specifically, the heavy chain variable region coding gene of the monoclonal antibody 1A6 contains the nucleotide sequence shown in SEQ ID NO: 9, and the light chain variable region coding gene of the monoclonal antibody 1A6 contains the nucleotide sequence shown in SEQ ID NO: 10.

[0065] Heavy chain vector construction: A DNA fragment containing the sequence SEQ ID NO: 9 was directionally inserted into the commercial expression vector pcDNA3.4 via restriction enzyme sites. This vector contains a strong cytomegalovirus (CMV) promoter, a human IgG1 heavy chain constant region (CH) sequence, and a glutamine synthase (GS) selection marker.

[0066] Light chain construction: A DNA fragment containing the sequence SEQ ID NO: 10 was directionally inserted into the commercial expression vector pcDNA3.1(+) by enzyme digestion. This vector contains the CMV promoter, the human κ light chain constant region (Cκ) sequence, and the neomycin resistance gene.

[0067] After the recombinant plasmid was constructed, the sequence accuracy of the inserted fragment was verified by Sanger sequencing to ensure the absence of point mutations and frameshift mutations. The verified recombinant plasmid is then used as the recombinant expression vector for subsequent expression.

[0068] 2. Antibody Expression and Purification The recombinant heavy chain expression vector and recombinant light chain expression vector, verified by sequencing, were co-transfected into CHO-K1 cells in logarithmic growth phase at a 1:1 ratio. Cell culture supernatant was collected 48-72 hours post-transfection. The collected supernatant was sterilized by filtration through a 0.22 μm filter and then loaded onto a Protein A affinity chromatography column. The column was equilibrated with binding buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4), and unbound impurities were washed away. The target antibody was then eluted with a low-pH elution buffer (e.g., 0.1 M sodium citrate, pH 3.0). The elution peak was collected, and the pH was immediately adjusted with neutralization buffer. Subsequently, the eluent was replaced with PBS buffer (pH 7.4) for dialysis, and then sterilized by filtration through a 0.22 μm filter to obtain purified HCAR1 monoclonal antibody. SDS-PAGE electrophoresis and HPLC-SEC analysis showed that the antibody purity was above 95%.

[0069] Example 2: HCAR1 expression in lung cancer cell lines 1. Test Methods Western blotting was used to extract total protein from human large cell lung cancer cells NCI-H661, small cell lung cancer cell line NCI-H146, human lung adenocarcinoma cells A549 and NCI-H647, and human bronchial epithelial cells HBEC3-KT. Proteins were separated by SDS-PAGE electrophoresis, transferred to membranes, and immunohybridized with HCAR1 antibody and GAPDH antibody, respectively. Finally, the expression level of HCAR1 protein was quantitatively analyzed by chemiluminescence imaging, with GAPDH as an internal control protein.

[0070] 2. Test Results Figure 1 The results showed that the HCAR1 protein band had a significant signal intensity, and the GAPDH band remained uniform as an internal control. The relative expression level of HCAR1 protein in small cell lung cancer cells could be calculated by gray value quantification, proving that there is effective expression of HCAR1 protein in these cancer cells.

[0071] Significant HCAR1 protein expression was detected in small cell lung cancer cells, indicating that HCAR1 can serve as a targeted therapeutic target for this type of cancer, providing experimental evidence for target expression in the subsequent development of HCAR1 antibodies and ADCs.

[0072] Example 3: Detection of binding activity with antigen HCAR1 1. Test Methods Enzyme-linked immunosorbent assay (ELISA) was used. Recombinant HCAR1 protein was coated onto the microplate overnight at 4°C. After blocking with blocking buffer, serially diluted HCAR1 monoclonal antibody 1A6 was added and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human IgG secondary antibody was added and incubated at 37°C for 30 minutes. After washing again, TMB chromogenic buffer was added, and the reaction was terminated with 2M HCl after color development at room temperature. The absorbance of each well was read at 450 nm using a microplate reader. A binding curve was plotted with antibody concentration on the x-axis and absorbance on the y-axis, and the half-maximal effective concentration (EC50) was calculated.

[0073] The antibody affinity was determined using surface plasmon resonance (SPR) technology with a Biacore 8K instrument. The HCAR1 monoclonal antibody of this application was immobilized on the surface of a CM5 sensor chip, and then different concentrations of recombinant HCAR1 protein were injected into the chip surface. The changes in the sensor map were detected in real time. The sensor data were fitted using a 1:1 Langmuir binding model, and the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) were calculated.

[0074] 2. Experimental Data like Figure 2As shown, the HCAR1 monoclonal antibody 1A6 binds to the HCAR1 antigen in a concentration-dependent manner, and its EC50 value is calculated to be 0.047 nM through data fitting.

[0075] like Figure 3 As shown, the ka value of the HCAR1 monoclonal antibody is 1.21E+051 / Ms, the kd value is 1.37E-031 / s, and the KD value is 1.13E-08M.

[0076] This demonstrates that monoclonal antibody 1A6 can specifically and with high affinity bind to the HCAR1 antigen, and the binding ability increases with increasing antibody concentration, proving that the antibody has good antigen-binding activity. The HCAR1 monoclonal antibody of this application exhibits a fast binding rate and a slow dissociation rate with the HCAR1 antigen, with a KD value reaching 10. -8 The M grade indicates that the antibody has a high affinity binding property to the HCAR1 antigen.

[0077] Example 4: Comparison of killing function against small cell lung cancer cells 1. Test samples and materials Test sample: HCAR1 polyclonal antibody, OALA00401, Beijing Aovia Biotechnology; GPR81 / HCAR1 Polyclonal Antibody, AHR-011, AlomoneLabs; HCAR1 antibody, SAB1300089, Sigma-Aldrich (Merck); HCAR1 Monoclonal Antibody, BYMab-07408, NanjingBYabscienceTechnologyCo.,Ltd; HCAR1Antibody, F55103, NewSiteJointBiotechnologyCo.,Ltd); Multiple HCAR1 antibodies were used as test samples, and human-independent IgG (AMlg) was used as a negative control.

[0078] The small cell lung cancer cell line NCI-H146 was used. Normal lung epithelial cells BEAS-2B were used as a control cell line to evaluate specific toxicity.

[0079] 2. Test methods NCI-H69 cells and BEAS-2B cells were seeded into 96-well plates and divided into antibody treatment group, positive control group, negative control group, and specific control group.

[0080] Antibody treatment group: After NCI-H69 cells were cultured to adhere, the above-mentioned antibodies OALA00401, AHR-011, SAB1300089, BYMab-07408, F55103 and the antibody of this application were added to each cell well, with a final concentration of 5 μg / mL. Five parallel wells were set for each antibody.

[0081] Specific control group: After BEAS-2B cells adhered to the culture vessel, the above-mentioned antibodies OALA00401, AHR-011, SAB1300089, BYMab-07408, F55103 and the antibody of this application were added to each cell well, with a final concentration of 5 μg / mL. Five parallel wells were set for each antibody.

[0082] Positive control group: The same concentration of HCAR1 inhibitor, 3,5-Dihydroxybenzoic acid (DHBA), was added.

[0083] Negative control group: Add the same concentration of nonspecific IgG (NMlg).

[0084] After intervention, cells in each group were cultured for another 48 hours. Cell viability was detected by CCK-8 assay, and DH release assay was used to assess cell membrane integrity (a marker of cell death).

[0085] 2. Experimental Data like Figure 4 As shown, antibodies OALA00401, AHR-011, SAB1300089, BYMab-07408, and F55103 not only do not directly kill small cell lung cancer cells, but their binding to the HCAR1 receptor may activate the receptor signaling pathway (mimicking lactate binding), thereby promoting cancer cell growth or metabolic reprogramming. In contrast, the negative control NMlg showed no significant effect, while the antibody provided in this application exhibited a significant killing effect.

[0086] Example 5: Preparation and Functional Analysis of Antibody-Drug Conjugates 1. Preparation of antibody-drug conjugates The purified HCAR1 monoclonal antibody was chemically conjugated with MMAE via a cleavable linker to prepare an HCAR1 ADC. After the conjugation reaction was completed, the prepared ADC was detected by hydrophobic interaction chromatography (HIC) or mass spectrometry to determine the drug-antibody conjugation ratio (DAR) and ADC samples with the expected DAR were screened.

[0087] 2. ADC binding activity detection Referring to the ELISA detection method in Example 2, the binding activity of HCAR1ADC with recombinant HCAR1 protein and NCI-H146 cells (small cell lung cancer cells expressing HCAR1) was detected, with unconjugated HCAR1 naked antibody as a positive control. Binding curves were plotted and the EC50 values ​​of the two were compared to evaluate the binding activity of the ADC. After conjugation of HCAR1 antibody with MMAE, the drug-antibody conjugation ratio (DAR) of the resulting ADC was 2-4, which met the expected conjugation ratio requirements.

[0088] Example 6: ADC maintains binding activity to HCAR1 1. Test Methods Recombinant HCAR1 protein (RecGPR81) and native HCAR1 protein (NativeGPR81) were used as coating antigens. The antigens were diluted to 2 μg / mL with coating buffer, and 100 μL was added to each well of a 96-well microplate. The plates were coated overnight at 4°C. The coating solution was discarded the next day, and the microplates were washed three times with PBST buffer for 5 minutes each time. The plates were then patted dry and ready for use.

[0089] Add 200 μL of PBST blocking buffer containing 5% skim milk powder to each well, incubate at 37°C for 1 hour, discard the blocking buffer, wash 3 times with PBST, and pat dry for later use.

[0090] A negative control group (NMIgG1), a 1A6 group, and a 1A6-MMAE group were set up, with three replicates for each group. Each test sample was diluted to 1 μg / mL with blocking buffer, and 100 μL of the diluent was added to each well. The samples were incubated at 37°C for 1 hour. After incubation, the samples were washed five times with PBST and patted dry for later use.

[0091] Dilute HRP-labeled goat anti-mouse IgG secondary antibody with blocking buffer at a ratio of 1:5000. Add 100 μL of the diluted secondary antibody to each well and incubate at 37°C for 30 minutes. Discard the secondary antibody solution, wash 5 times with PBST, and pat dry for later use.

[0092] Add 100 μL TMB chromogenic solution to each well, incubate at room temperature in the dark for 10 minutes, and then add 50 μL 2M HCl to stop the chromogenic process. Use a microplate reader to measure the absorbance value (OD450nm) of each well at a wavelength of 450nm and record the experimental data.

[0093] The average OD450nm of the three replicates in each group was calculated to reflect the binding activity of each sample with HCAR1 protein. The higher the OD value, the stronger the binding activity.

[0094] 2. Experimental Data like Figure 5As shown, the negative control (NMIgG1) had an OD value of 0.08±0.02 when bound to recombinant HCAR1 and 0.07±0.01 when bound to natural HCAR1, both close to baseline, with no obvious binding signal.

[0095] Group 1A6: The OD value for binding with recombinant HCAR1 was 2.15±0.06, and the OD value for binding with natural HCAR1 was 2.08±0.05, indicating a significant binding signal.

[0096] The 1A6-MMAE group showed an OD value of 2.12±0.05 for binding to recombinant HCAR1 and 2.05±0.04 for binding to natural HCAR1, which was not significantly different from the 1A6 naked antibody group.

[0097] All positive samples showed no significant difference in binding OD values ​​between recombinant HCAR1 and natural HCAR1, indicating comparable binding activity.

[0098] HCAR1-ADC fully retains the antigen-binding activity of the parent antibody: the OD values ​​of 1A6-MMAE binding to recombinant HCAR1 and natural HCAR1 are basically consistent with the corresponding naked parent antibody 1A6, with no statistical difference. This indicates that after the antibody is coupled to MMAE through a linker, its specific binding structure against HCAR1 is not destroyed, and the ADC maintains its high-efficiency binding ability to the HCAR1 antigen.

[0099] Antibodies / ADCs can specifically recognize native conformation HCAR1 protein: 1A6 and its corresponding ADCs showed strong and comparable binding activity to both recombinant HCAR1 and native HCAR1, proving that this type of antibody can recognize native HCAR1 protein and can target and bind to native HCAR1 antigen on the surface of cancer cells. This lays the structural and functional foundation for the subsequent ADC to target and enter cancer cells and exert cytotoxic effects.

[0100] The negative control NMIgG1 showed a binding OD value close to 0 with both recombinant and natural HCAR1, indicating no obvious non-specific binding signal. This demonstrates that the binding of 1A6 and its ADC to HCAR1 protein is target-specific, eliminating the interference of non-specific binding on the experimental results.

[0101] Example 7: ADC Internalization Experiment 1. Test Methods HCAR1ADC (1A6-MMAE) was conjugated with a pH-sensitive fluorescent dye (pHrodo), with normal mouse IgG-MMAE (NMg-MMAE) as a negative control. The fluorescently labeled conjugate was co-cultured with small cell lung cancer NCI-H146 cells, and intracellular fluorescence signals were observed by fluorescence microscopy at two time points: 0.5 hours and 4 hours of culture. The pHrodo dye emitted green fluorescence in an acidic endosome / lysosomal environment, and the appearance of fluorescence indicated that the ADC was internalized by the cells.

[0102] 2. Experimental Data like Figure 6 As shown, after 0.5 hours of culture, only a weak fluorescence signal was observed in the cells; after 4 hours of culture, obvious green fluorescence appeared in the NCI-H146 cells of the 1A6-MMAE treatment group, while no obvious fluorescence signal was observed in the negative control NMg-MMAE treatment group at either time point, proving that the ADC was effectively internalized by cells expressing HCAR1.

[0103] 3. Test Results HCAR1ADC can be specifically internalized by small cell lung cancer cells expressing HCAR1, and the internalization process is time-dependent, proving that ADC can enter target cells through targeted binding, providing a prerequisite for the subsequent release of the cytotoxic drug MMAE and killing of target cells.

[0104] Example 8: Both naked antibody and ADC showed in vitro cytotoxicity. 1. Test Methods (1) Cell preparation Human large cell lung cancer cells NCI-H661 expressing HCAR1 were selected as target cells. Cells in the logarithmic growth phase were resuspended in RPMI-1640 medium containing 10% fetal bovine serum, and the cell concentration was adjusted to 5 × 10⁶ cells / year. 4 Cells per mL were seeded at 100 μL per well in a 96-well cell culture plate and incubated overnight at 37°C in a 5% CO2 incubator until the cells reached approximately 80% adherence and confluence.

[0105] (2) Drug administration A negative control group (NMIgG1), a naked antibody group (1A6), and an ADC group (1A6-MMAE) were set up, with three replicates for each group. Each treatment reagent was diluted to the working concentration with serum-free medium, and 100 μL of the diluted reagent was added to the corresponding well to ensure a consistent final concentration. The blank wells were filled with an equal volume of serum-free medium. The culture plates were then incubated at 37°C in a 5% CO2 incubator for 72 hours.

[0106] (3) Cell lysis rate detection The specific cell lysis rate was detected by the lactate dehydrogenase (LDH) release method: After incubation, the cell culture supernatant of each well was collected. The supernatant was mixed with the substrate working solution in proportion according to the LDH cytotoxicity assay kit instructions. The mixture was incubated at room temperature in the dark for 30 minutes. After adding the stop solution, the absorbance value (OD value) was measured at a wavelength of 490 nm using an ELISA reader.

[0107] (4) Data calculation The formula for calculating the specific cell lysis rate is: Specific cell lysis rate (%) = (OD value of experimental group - OD value of blank group) / (OD value of maximum lysis group - OD value of blank group) × 100%; where the maximum lysis group is the target cell well treated with 1% Triton X-100.

[0108] (5) Experimental data like Figure 7 As shown, the cell-specific lysis rate of the negative control (NMIgG1) was 9.5%±1.8%, the cell-specific lysis rate of the 1A6 group was 36.9%±3.2%, and the cell-specific lysis rate of the 1A6-MMAE group reached 65.4%±4.5%, which was the highest among all treatment groups.

[0109] After HCAR1 antibody was conjugated with MMAE to form an ADC, its cytotoxic activity against target cells was significantly enhanced, demonstrating that the cytotoxic effect of MMAE was efficiently exerted after targeted delivery of the ADC. The negative control NMIgG1 showed no significant cell lysis, proving that the killing effect of 1A6 and its ADC on small cell lung cancer cells was a specific effect targeting HCAR1, excluding interference from non-specific factors.

[0110] Example 9: Detection of the killing effect of different HCAR1ADCs on small cell lung cancer cells 1. Test Methods (1) Cell preparation Small cell lung cancer cell lines expressing HCAR1 (such as H661) were selected as target cells. Cells in the logarithmic growth phase were resuspended in RPMI-1640 medium containing 10% fetal bovine serum, and the cell concentration was adjusted to 5 × 10⁶ cells / year. 4 Cells per mL were seeded at 100 μL per well in a 96-well cell culture plate and incubated overnight at 37°C in a 5% CO2 incubator until the cells reached approximately 80% adherence and confluence.

[0111] (2) Drug administration A negative control group (NMig, normal mouse IgG), a naked antibody group (1A6), and an ADC group (1A6-MMAE) were set up, with three replicates for each group. Each treatment reagent was diluted to the working concentration with serum-free medium, and 100 μL of the diluted reagent was added to the corresponding well to ensure a consistent final concentration; blank wells were filled with only an equal volume of serum-free medium.

[0112] (3) Fluorescence intensity detection and time point setting Fluorescence detection based on cellular metabolic activity (such as CellTiter-Blue or similar methods) was used to detect the drug at 0, 4, 12, 24, 36, 48, 60, 72, 84, and 96 hours after drug administration. Add the fluorescent substrate working solution to each well and incubate at 37°C for 30 minutes; Use an ELISA reader to detect the fluorescence intensity of each well. Set the excitation / emission wavelength according to the kit instructions. Using the fluorescence intensity at 0 hours as a baseline, the fluorescence intensity at each time point was normalized to obtain the "normalized fluorescence intensity percentage".

[0113] Normalized fluorescence intensity percentage calculation formula: Normalized fluorescence intensity (%) = fluorescence intensity value at 0 hours and fluorescence intensity values ​​at each time point × 100%. The greater the decrease in fluorescence intensity, the more significant the reduction in cell viability / metabolic activity, that is, the stronger the drug killing effect.

[0114] (4) Experimental data like Figure 8 As shown, the negative control (NMig) exhibited a slow overall decrease with no significant cytotoxic effect. The 1A6 group showed a significantly faster decrease in fluorescence intensity than the NMig group, demonstrating a certain cytotoxic effect. The ADC1A6-MMAE group showed the fastest decrease in fluorescence intensity, with a significant decrease observed after 12 hours, and maintained at a low level of approximately 40% after 96 hours.

[0115] Normalized fluorescence intensity decreased gradually over time in all treatment groups, with the decrease being significantly greater in the 1A6 and 1A6-MMAE groups than in the NMig group, indicating that the killing / inhibitory effect of the drug on cancer cells cumulatively increases over time. The rate and magnitude of fluorescence intensity decrease in the 1A6-MMAE group were significantly better than in the 1A6 naked antibody group, showing a significant decrease in cell viability as early as 12 hours after administration, with only about 38% viability remaining after 96 hours; while the 1A6 group showed approximately 50% viability after 96 hours, and the NMig group maintained approximately 60%. This indicates that conjugation with MMAE significantly enhances the targeted cytotoxic effect of the ADC, resulting in faster onset and stronger efficacy. The fluorescence intensity decrease in the negative control NMig group was slow, with no significant killing effect, proving that the killing of small cell lung cancer cells by 1A6 and 1A8-MMAE is a specific effect targeting HCAR1, excluding interference from non-specific factors.

[0116] Example 10: In vivo efficacy experiment 1. Test Methods Logarithmically growing NCI-H146 cells were subcutaneously inoculated into the right back of nude mice. When the tumor volume grew to approximately 100-200 mm³, mice were divided into four groups: PBS control group, anti-HCAR1 nude antibody group, HCAR1 ADC group, and control ADC group (non-specific IgG-MMAE). The corresponding drugs were administered to each group of mice via tail vein injection. Appropriate dosage and administration period were set. The tumor volume and body weight of the mice were measured twice a week. Tumor growth curves were plotted with time on the x-axis and tumor volume on the y-axis to analyze the in vivo antitumor effect of different treatment groups. At the same time, the in vivo tolerance of the drugs was assessed by changes in body weight.

[0117] 2. Experimental Data Figure 9 As shown, the tumor volume in the negative control group mice continued to grow rapidly over time, while the tumor volume growth in the 1A6ADC treatment group stopped shortly after administration, and some mice even showed a decrease in tumor volume. In a small cell lung cancer xenograft mouse model, HCAR1ADC can effectively inhibit tumor growth in vivo, and there are significant differences in the in vivo killing / anti-tumor efficacy of different ADCs, with some ADCs showing faster and more significant in vivo anti-tumor effects.

[0118] It should be understood that after reading the teachings of this disclosure, those skilled in the art can make various modifications and changes to this disclosure, and these equivalent solutions also fall within the scope defined by the claims.

Claims

1. HCAR1 monoclonal antibody, which comprises: Heavy chain variable regions (VH) comprising CDR-H1 as shown in SEQ ID NO:1, CDR-H2 as shown in SEQ ID NO:2, and CDR-H3 as shown in SEQ ID NO:3; and The light chain variable region (VL) includes CDR-L1 as shown in SEQ ID NO:4, CDR-L2 as shown in SEQ ID NO:5, and CDR-L3 as shown in SEQ ID NO:

6.

2. An HCAR1 monoclonal antibody comprising: VH as shown in SEQ ID NO:7 and VL as shown in SEQ ID NO:

8.

3. The HCAR1 monoclonal antibody according to any one of claims 1-2, comprising a heavy chain constant region and a light chain constant region, preferably, the heavy chain constant region being derived from human IgG1, IgG2, IgG3 or IgG4 subclasses, and the light chain constant region being derived from human λ light chain or κ light chain.

4. The HCAR1 monoclonal antibody according to any one of claims 1-32, wherein the antibody fragment is selected from Fv, scFv, Fab, Fab', and F(ab')2.

5. An antibody-drug conjugate comprising an HCAR1 monoclonal antibody according to any one of claims 1-4, and a cytotoxic drug conjugated thereto.

6. The antibody-drug conjugate according to claim 5, wherein the cytotoxic drug is monomethylauratestatin E (MMAE).

7. A vector comprising a polynucleotide encoding an HCAR1 monoclonal antibody according to any one of claims 1-4, preferably comprising a sequence as shown in SEQ ID NO:9 and / or 10.

8. A host cell comprising the vector according to claim 7.

9. A composition comprising an HCAR1 monoclonal antibody according to any one of claims 1-4, an antibody-drug conjugate according to claim 5 or 6, a vector according to claim 7, or a host cell according to claim 8.

10. Use of an HCAR1 monoclonal antibody according to any one of claims 1-4 or an antibody-drug conjugate according to claim 6 or 7 in the preparation of a reagent for detecting the presence or level of HCAR1 in a biological sample, or for the prevention or treatment of cancer. Optionally, the cancer is a cancer expressing HCAR1, preferably selected from non-small cell lung cancer, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, glioma, gastric adenocarcinoma, thymic carcinoma and pancreatic ductal adenocarcinoma.

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