Antibodies targeting glyr1, methods of making the same, and uses in the prevention and treatment of tumors

Abstract

This application provides antibodies targeting GLYR1 (e.g., CL-ab-2), their preparation methods, and their applications in tumor prevention and treatment. Specifically, this application relates to an antibody targeting GLYR1 or its antigen-binding fragment, wherein the antibody comprises a heavy chain complementarity-determining region (VH CDR) and a light chain complementarity-determining region (VL CDR), wherein the VH CDR and VL CDR each have an amino acid sequence selected from SEQ ID NO: 13-18 or 21-26. This application also relates to nucleic acid molecules, vectors or host cells, products, and applications related to the antibody or its antigen-binding fragment.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, primarily targeting the field of tumor immunodiagnosis and treatment. Specifically, this invention relates to antibodies targeting GLYR1 (e.g., antibody CL-ab-2), their preparation methods, and their applications in the diagnosis and / or prevention of tumors (e.g., pancreatic cancer). Background Technology

[0002] Cancer has become a major public health problem worldwide, seriously affecting human life and health, national economy and social development, and imposing a heavy burden on families and society. For example, pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a high mortality rate, with a 5-year survival rate of only 9%, earning it the title of "King of Cancers" in the field of oncology. Currently, surgery remains the main treatment for PDAC, but most pancreatic cancer patients are no longer candidates for surgery at the time of diagnosis. In addition, due to short-term drug resistance and toxic side effects of conventional chemotherapy drugs, the overall treatment effect of PDAC has not yet been significantly improved. With the rapid development of genome sequencing technology, multiple molecules or pathways associated with the occurrence of PDAC have been discovered, and targeted therapy has brought new hope to pancreatic cancer patients. Drugs targeting mutated genes such as BRCA1 / 2, NRG1, KRAS, and TP53 have been approved for clinical use in tumor treatment, but the overall treatment effect is not good (J Hematol Oncol. 2020; 13:130). Therefore, there is an urgent need to find more effective treatment methods to break through the bottleneck in the treatment of pancreatic cancer.

[0003] Antibody immunotherapy has become a hot research topic in the field of tumor immunotherapy in recent years, especially in the clinical treatment of hematological diseases, where it has achieved remarkable efficacy. In recent years, the efficacy rate of PD-1 / PD-L1 targeting in PDAC patients with MSI-H / dMMR has been 18.2%, but since only 1% of PDAC patients present with MSI-H / dMMR, this greatly limits the clinical application of PD-1 / PD-L1 antibody drugs in pancreatic cancer (J Clin Oncol. 2020; 38(1):1–10). Molecularly targeted antibody drugs, such as antibody-drug conjugates (ADCs), bispecific antibodies, and antibody-immune factor fusion proteins, have also been applied to the clinical treatment of PDAC, but the progress has not been satisfactory. For example, a phase I clinical trial of an MMAE ADC drug targeting MUC16 and GC for PDAC failed (Ann Oncol. 2016; 27(11):2124-2130; Pharmacol Ther. 2017; 170:8-13); and a bispecific antibody targeting EpCAM / CD3 could not effectively inhibit the in vivo growth of pancreatic cancer cells (Oncoimmunology. 2018; 7(8):e1450710).

[0004] Given the limited selection of targets for tumors (such as pancreatic cancer), discovering new effective targets and developing novel antibody drugs are key issues that urgently need to be addressed in tumor antibody immunotherapy, especially PDAC antibody immunotherapy.

[0005] In summary, there is an urgent need in this field to develop new targets for the diagnosis and treatment of tumors (e.g., pancreatic cancer) and antibody-based targeted drugs. Summary of the Invention

[0006] This application provides antibodies targeting GLYR1 or their antigen-binding fragments, their encoding molecules, vectors, host cells, products, and their use in tumor diagnosis, prevention, and / or treatment.

[0007] In a first aspect of this application, an antibody or antigen-binding fragment thereof targeting GLYR1 is provided, wherein the antibody comprises a combination of a heavy chain complementarity-determining region (VH CDR) and a light chain complementarity-determining region (VL CDR), wherein the VH CDR and VL CDR each have an amino acid sequence selected from the group consisting of: VH CDR1: SEQ ID NO:13; VH CDR2: SEQ ID NO:14; VH CDR3: SEQ ID NO:15; VL CDR1: SEQ ID NO:16; VL CDR2: SEQ ID NO:17; and VL CDR3: SEQ ID NO:18.

[0008] In some embodiments, the amino acid sequences of the VH CDR and VL CDR in the antibody targeting GLYR1 or its antigen-binding fragment are as follows: VH CDR1: SEQ ID NO:13; VH CDR2: SEQ ID NO:14; VH CDR3: SEQ ID NO:15; VL CDR1: SEQ ID NO:16; VL CDR2: SEQ ID NO:17; and VL CDR3: SEQ ID NO:18.

[0009] In some embodiments, the antibody of this application comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL each have an amino acid sequence selected from the group consisting of: VH: (a) the sequence shown in SEQ ID NO: 11; or (b) a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO: 11; and VL: (a') the sequence shown in SEQ ID NO: 12; or (b') a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO: 12. In some embodiments, the difference between the antibody or its antigen-binding fragment and SEQ ID NO: 11 and / or 12 is not located in the CDR, wherein the antibody is capable of specifically binding to GLYR1, for example, the VH and / or VL contain substitutions of one or more amino acid residues in their non-CDR regions (e.g., humanization design and substitution).

[0010] In some embodiments, the antibody or its antigen-binding fragment in this application is selected from: monoclonal antibody, polyclonal antibody, Fab, Fab', F(ab')2, Fd, scFv, disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, small antibody, F(ab')3, tetraantibody, triantibody, bispecific antibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0011] In some embodiments, the antibody or its antigen-binding fragment is present in a form selected from the group consisting of: free; attached, embedded, or immobilized on a support (e.g., magnetic beads, agarose, gold surface, chip, microcarrier); forming part of a fusion protein; forming part of an antibody-drug conjugate (ADC); forming part of a multispecific antibody (e.g., a bispecific antibody); forming part of a chimeric antigen receptor (CAR) or a cell modified with it (e.g., CAR-T, CAR-NK, CAR-M).

[0012] In some embodiments, the antibody of this application is a chimeric antibody. In some embodiments, the antibody of this application comprises an IgG backbone. In some embodiments, the antibody of this application comprises a human IgG backbone chimeric with scFv.

[0013] In some embodiments, the antibody of this application has one or more features selected from the group consisting of:

[0014] (i) Its specific binding to GLYR1, for example, as determined by surface plasmon resonance (SPR), is its binding affinity K to GLYR1 (preferably film-type GLYR1). D Less than 1.5 x 10 -10 M, for example, is 0.3 x 10 -10 M~1.5x 10 -10 M; and

[0015] (ii) It can inhibit tumor growth (such as pancreatic cancer) and improve tumor prognosis.

[0016] In some aspects of this application, a nucleic acid molecule is provided that encodes the antibody or antigen-binding fragment thereof of this application. In some embodiments, the encoding nucleic acid molecule is codon-optimized.

[0017] In some aspects of this application, a vector or host cell containing the nucleic acid molecules of this application is provided.

[0018] In some aspects of this application, a product is provided that comprises the antibody of this application or an antigen-binding fragment thereof, a nucleic acid molecule, a vector, or a host cell.

[0019] In some embodiments, the product of this application may be a drug, a medicine box, a reagent kit, etc., depending on its application and form.

[0020] In some aspects of this application, the use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector or host cell of this application in the preparation of products is provided.

[0021] In some embodiments, the product of this application can be used in one or more applications selected from the group consisting of, for example:

[0022] (a) Diagnosis, prevention and / or treatment of membrane-type GLYR1 overexpressing tumors (e.g., pancreatic cancer (e.g., pancreatic ductal adenocarcinoma)); and / or

[0023] (b) Detect whether the cells in the sample are membrane-bound GLYR1 overexpressing cells; and / or

[0024] (c) Screening for therapeutic drugs or treatment regimens for tumors that overexpress membrane-type GLYR1 (e.g., pancreatic cancer).

[0025] In some aspects of this application, a method for treating tumors is provided, the method comprising administering to a patient in need a therapeutically effective amount of the antibody of this application or an antigen-binding fragment thereof, a nucleic acid molecule, and / or a vector or host cell, or a product thereof.

[0026] Those skilled in the art can combine the foregoing technical solutions and features in any way without departing from the inventive concept and protection scope of this invention. Other aspects of this invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0027] The present invention will be further described below with reference to the accompanying drawings. The figures are only for illustrating the embodiments of the present invention and are not intended to limit the scope of the present invention.

[0028] Figure 1 Results of relative expression of GLYR1 mRNA in pancreatic cancer and adjacent normal tissues.

[0029] Figure 2 Representative immunohistochemical staining (A) and subcellular GLYR1 expression scores (B) of GLYR1 in 177 pairs of PDAC specimens. Scale bar = 50 μm; data are expressed as mean ± standard deviation and analyzed using a two-tailed Student's t-test.

[0030] Figure 3 ELISA was used to detect the titer of antibodies secreted by GLYR1 hybridomas.

[0031] Figure 4 Western blotting was used to detect the efficiency of GLYR1 knockout.

[0032] Figure 5 Western blotting was used to detect the specificity of anti-GLYR1 antibody.

[0033] Figure 6 : Results of anti-GLYR1 antibody testing.

[0034] Figure 7 ELISA results of the binding of anti-GLYR1 antibody to human GLYR1 antigen.

[0035] Figure 8 Biacore assay results for anti-GLYR1 antibody affinity:

[0036] Figure 8 A: Biacore assay results for antibody CL-ab-3 affinity;

[0037] Figure 8 B: Biacore assay results for antibody CL-ab-2 affinity;

[0038] Figure 8 C: Biacore assay results for antibody CL-ab-1 affinity;

[0039] Figure 8 D: Biacore assay result for antibody CL-ab-4 affinity;

[0040] In each subplot, the curves from top to bottom represent the results at each decreasing concentration, as shown in the icon on the right.

[0041] Figure 9 SW1990-GLYR1 GPI cell immunofluorescence identification.

[0042] Figure 10 Analysis of the in vitro ADCC effect induced by anti-GLYR1 antibody:

[0043] Figure 10 A: Analysis results of BXPC3 cells that are positive for GLYR1 membrane expression;

[0044] Figure 10 B: Analysis results for PANC1-GLYR1 GPI cells.

[0045] Figure 11 Analysis of the in vivo ADCC effect induced by anti-GLYR1 antibody:

[0046] Figure 11 A: The inhibitory effect of isotype IgG control on the growth of pancreatic cancer cells in mice;

[0047] Figure 11 B: The inhibitory effect of CL-ab-3 antibody on the growth of pancreatic cancer cells in mice;

[0048] Figure 11 C: The inhibitory effect of CL-ab-2 antibody on the growth of pancreatic cancer cells in mice.

[0049] The significance shown in the figure is compared with WT. ns represents no significant difference, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. Detailed Implementation

[0050] Through in-depth preliminary research, our research team discovered that the GLYR1 molecule undergoes abnormal membrane localization in pancreatic cancer cells, and that membrane-type GLYR1 expression is significantly negatively correlated with the prognosis of pancreatic cancer. The histological staining level of GLYR1 can be used for the diagnosis and prognostic monitoring of pancreatic cancer. Furthermore, drugs targeting abnormally localized membrane GLYR1 may be used for the treatment of pancreatic cancer.

[0051] Specifically, the nuclear epigenetic enzyme GLYR1 (Glyoxylate reductase 1homolog), also known as NP60 and N-PAC, is a redox enzyme mainly located in the cytoplasm and nucleus. As an epigenetic decoder and nucleosome instability factor, GLYR1 plays a crucial role in chromatin modification and gene expression regulation through histone demethylation. Studies have found that GLYR1 mutants induce coronary heart disease by activating cardiac development genes (Cell. 2022; 185(5):794-814). Furthermore, GLYR1 has a high mutation frequency in microsatellite unstable colorectal cancer (MSI CRC) and is considered a novel tumor suppressor gene (J Exp Clin Cancer Res. 2020; 39(1):125).

[0052] Our previous immunohistochemical (IHC) analysis of 177 pairs of pancreatic dysplastic anemia (PDAC) cancer and adjacent normal tissues revealed that GLYR1 was abnormally expressed on the cancer cell membrane in PDAC cancer tissues, but not in adjacent normal cells, suggesting that membrane-type GLYR1 may be closely related to the occurrence and development of pancreatic cancer. Further preliminary studies have also found that silencing GLYR1 can effectively inhibit pancreatic cancer metastasis, prolong the survival of pancreatic cancer patients, and improve the chemosensitivity of pancreatic cancer. Therefore, we propose that GLYR1 is a novel potential target for tumor therapy, and antibody immunotherapy targeting GLYR1 has important application prospects and significance in the clinical treatment of solid tumors such as pancreatic cancer.

[0053] Building upon this foundation, the present invention further provides an anti-GLYR1 antibody or its antigen-binding fragment, which can specifically target cancer cells (e.g., pancreatic cancer cells) that highly express GLYR1 on their cell membranes and effectively inhibit the in vivo and in vitro growth of such cancer cells, providing a novel immunotherapy strategy for the clinical diagnosis, prevention, and / or treatment of tumors. Furthermore, this application also provides the application of the anti-GLYR1 antibody or its active fragment in the diagnosis, prevention, and / or treatment of tumors, particularly pancreatic cancer (e.g., PDAC).

[0054] This application demonstrates through examples that the antibody targeting GLYR1 can specifically bind to GLYR1, thereby enabling its use in the prevention and / or treatment of tumors. Therefore, the antibody of this application has broad application prospects in the prevention, treatment, and / or detection of tumors, especially those related to tumors overexpressing membrane-type GLYR1 (or membrane-localized GLYR1), such as pancreatic cancer, particularly pancreatic ductal adenocarcinoma.

[0055] All numerical ranges provided herein are intended to clearly include all values ​​falling between the endpoints of the range and the range of values ​​between them. Features mentioned in the invention or embodiments may be combined. All features disclosed in this specification may be used in any combination form, and each feature disclosed in the specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0056] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0057] GLYR1

[0058] As used herein, the term "GLYR1 gene" refers to a glyoxylate reductase 1 homolog. As is known in the art, the product of this gene is typically located in the nucleus and cytoplasm and possesses DNA-binding, methylated histone-binding, and nucleosome-binding activities. GLYR1 gene sequences are known in the art, for example, HGNC:24434; NCBIGene:84656, etc. In the embodiments of this application, the nucleotide sequence GLYR1 shown in SEQ ID NO:1 is used as an example.

[0059] The GLYR1 polypeptide sequence is known in the art, for example, the sequence shown in NP_115958. In the embodiments of this application, the GLYR1 polypeptide with the amino acid sequence shown in SEQ ID NO:2 is used as an example.

[0060] The term "membrane-type GLYR1" refers to the GLYR1 polypeptide and its corresponding coding sequence that are abnormally located on the cell membrane. In studies related to this invention, it has been found that abnormally located membrane-type GLYR1 exists on the surface of certain cancer cells (e.g., pancreatic cancer cells). The presence and level of this membrane-type molecule are associated with the presence, prognosis, metastasis, and treatment efficacy of these cancers (especially pancreatic cancer). Silencing or inhibiting this molecule can achieve the prevention and / or treatment of these cancers.

[0061] Therefore, the research and development of highly efficient inhibitory molecules that specifically target membrane-bound GLYR1 is of great clinical significance and value.

[0062] GLYR1-specific antibody and its preparation

[0063] This application provides a GLYR1-specific antibody. The antibody of this application exhibits high binding affinity for membrane-localized GLYR1 and can effectively mediate ADCC effects in vitro and in vivo.

[0064] As used herein, the terms "antibody" or "immunoglobulin" generally refer to a 150 kDa isotetraglycoprotein with similar structural characteristics, typically composed of two identical light chains (L) and two identical heavy chains (H), but can also be single-chain antibodies. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain may contain a heavy chain variable region (VH); each light chain may contain a light chain variable region (VL).

[0065] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ in sequence, which contributes to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments in the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are the framework regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases may form a partially β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0066] The antibody of this application comprises a heavy chain complementarity-determining region (VH CDR) and a light chain complementarity-determining region (VL CDR), wherein the VH CDR and VL CDR may be selected from the group consisting of: VH CDR1 having the amino acid sequence shown in SEQ ID NO:13; VH CDR2 having the amino acid sequence shown in SEQ ID NO:14; VH CDR3 having the amino acid sequence shown in SEQ ID NO:15; VL CDR1 having the amino acid sequence shown in SEQ ID NO:16; VL CDR2 having the amino acid sequence shown in SEQ ID NO:17; and VL CDR3 having the amino acid sequence shown in SEQ ID NO:18. In some embodiments, the antibody of this application has a combination of the six CDRs shown in SEQ ID NO:13 to 18. In some embodiments, the amino acid sequences of the six CDRs of the antibody of this application are respectively shown in SEQ ID NO:13 to 18.

[0067] In some embodiments, the antibody of this application comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL may each have a sequence selected from the group consisting of: a VH having an amino acid sequence as shown in SEQ ID NO:11 or having at least 80% sequence identity with SEQ ID NO:11 (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity); and a VL having an amino acid sequence as shown in SEQ ID NO:12 or having at least 80% sequence identity with SEQ ID NO:12 (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). Those skilled in the art will understand that, with the CDR unchanged, minor changes in amino acid residues in VH and / or VL can be tolerated without affecting or significantly affecting the overall performance of the antibody. For example, one or more amino acid residues can be substituted in the non-CDR regions of VH and / or VL to achieve humanization.

[0068] As used herein, the terms “sequence identity” or “identity %” refer to the percentage of identical residues (e.g., amino acids or nucleic acids) in a candidate sequence compared to a reference sequence after sequence alignment and (if necessary) introducing vacancies to obtain the maximum percentage of sequence identity. For example, as used herein, “at least 80% sequence identity” means that the sequence identity between the candidate sequence and the reference sequence is greater than 80%, such as 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any numerical point or range thereof.

[0069] The "light chain" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively referred to as α, δ, ε, γ, and μ. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art. In some embodiments of this application, the light chain of the antibody is κ or λ, preferably κ. In some embodiments of this application, the heavy chain of the antibody is of type α, δ, ε, γ, or μ, preferably γ. In some embodiments of this application, the antibody is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, preferably IgG1.

[0070] As used herein, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a substantially homogeneous population, meaning that the individual antibodies in this population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody formulations (which typically contain different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized through hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a substantially homogeneous population of antibodies, which should not be interpreted as requiring any special method to produce the antibody. The antibodies of this application are preferably monoclonal antibodies.

[0071] Monoclonal antibodies can be prepared using various methods well known to those skilled in the art. For example, methods for preparing monoclonal antibodies include, but are not limited to: hybridoma method (Kohler et al., Nature, 256:495(1975)); recombinant DNA method (US Patent No. 4,816,567); phage display technology (Clackson et al., Nature, 352:624-628(1991) and Marks et al., J. Mol. Biol., 222:581-597(1991)); and single B cell antibody preparation technology (Chi Xiangyang et al., Chinese Journal of Biotechnology, 2012, 28(6):651-660). In some embodiments of this application, single B cell antibody preparation technology is preferably used to prepare the antibody library. This technology retains the natural pairing of the light and heavy chain variable regions, and has advantages such as good gene diversity, high efficiency, fully human origin, and low cell requirement.

[0072] The antibodies or antigen-binding fragments thereof in this application may include, but are not limited to: monoclonal antibodies, polyclonal antibodies, scFv, Fab, Fab', F(ab')2, Fd, disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibodies, IgGΔCH2, small antibodies, F(ab')3, tetraantibodies, triantibodies, bispecific antibodies, single-domain antibodies, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0073] In some embodiments, the antibody of this application is a monoclonal antibody or a humanized antibody, such as selected from chimeric antibodies, CDR transplanted antibodies, homologous replacement antibodies, surface remodeling antibodies, compensatory change antibodies, and localized conserved humanized antibodies. In some embodiments, the constant region (C region) of the antibody of this application is a human or non-human mammal (e.g., mouse) constant region.

[0074] In some embodiments, the antibodies or antigen-binding fragments of the present application may exist in forms including but not limited to: free antibodies; attached, embedded or immobilized on a support (e.g., magnetic beads, agarose, gold surface, chip, microcarrier); forming part of a fusion protein; forming part of an antibody-drug conjugate (ADC); forming part of a multispecific antibody (e.g., bispecific antibody); forming part of a chimeric antigen receptor (CAR) or its modified cells (e.g., CAR-T, CAR-NK, CAR-M).

[0075] In some embodiments, the antibody of this application is a chimeric antibody. In some embodiments, the antibody of this application comprises an IgG backbone. In some embodiments, the antibody of this application comprises a human IgG backbone chimeric with scFv.

[0076] In some embodiments, the antibody of this application has one or more features selected from the group consisting of:

[0077] (i) Its specific binding to GLYR1, for example, as determined by surface plasmon resonance (SPR), is its binding affinity K to GLYR1 (preferably film-type GLYR1). D Less than 1.5 x 10 -10 M, for example, is 0.3 x 10 -10 M~1.5x 10 -10 M; and

[0078] (ii) It can inhibit tumor growth (such as pancreatic cancer), improve tumor prognosis, and / or enhance tumor chemosensitivity. The tumor is preferably one that highly expresses GLYR1 on its membrane surface, such as pancreatic cancer.

[0079] The encoding molecule of the antibody or its functional fragment, the expression vector containing the molecule, and the host cell.

[0080] This application also provides nucleic acid molecules encoding the anti-GLYR1 antibody or fragments thereof. The sequences of these nucleic acid molecules can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form a single-chain antibody.

[0081] In some embodiments, the nucleic acid molecule of this application may comprise: a nucleotide sequence encoding a VH CDR and / or a VL CDR. In some embodiments, the nucleic acid molecule of this application may comprise a nucleotide sequence encoding the sequences shown in SEQ ID NO:13, 14, 15, 16, 17 and / or 18. In some embodiments, the nucleic acid molecule of this application may comprise: a nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO:11 or a nucleotide sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) and encoding a functional heavy chain variable region; and / or a nucleotide sequence encoding the light chain variable region shown in SEQ ID NO:12 or a nucleotide sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) and encoding a functional light chain variable region. Those skilled in the art will understand that codon optimization and other operations can be performed on the coding sequence to introduce minor changes in nucleotide residues without affecting or significantly affecting the overall performance of the encoded antibody.

[0082] In some embodiments, this application also discloses expression vectors containing the above-described nucleotide sequences. In some embodiments, the expression vector may be selected from, but is not limited to: pFUSE-CHIg-hG1, pFUSE2ss-CLIg-hk, pFUSE-CHIg-mG1, pFUSEss-CHIg-hG1, pFUSEss-CHIg-mG1, pFUSE-CHIg-hk, pFUSE-CHIg-mk, pFUSEss-CHIg-hk, pFUSEss-CHIg-mk, pcDNA3.1, pPICZα, and pPIC9K.

[0083] In some embodiments, this application also discloses host cells comprising the encoded nucleic acid molecules described in this application, or host cells transformed by the expression vector. The host cells can be prokaryotic cells, such as bacterial cells, e.g., *Escherichia coli*, *Bacillus subtilis*, etc.; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. In some embodiments, the host cells can be selected from: monkey kidney cells (COS), Chinese hamster ovary cells (CHO), Vero cells, HeLa cells (HeLa), young hamster kidney cells (BHK), human liver cancer cells (such as HepG2), and many other cell lines. They provide post-translational modifications for protein molecules, including correct folding, correct disulfide bond formation, and glycosylation at the correct sites.

[0084] There are many methods for transforming host cells using expression vectors, and the transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene (1,5-dimethyl-1,5-diazadecylmethylene polymethyl bromide)-mediated transfection, protoplast fusion, electroporation, liposome-mediated transfection, and direct microinjection of DNA into the cell nucleus. In this invention, preferred methods include electroporation or liposome-mediated methods. For example, Invitrogen's liposome transfection kit can be used to transfect host cells such as COS and CHO. The transformed host cells are then cultured under conditions suitable for antibody expression. The antibodies of this application are then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography—conventional separation and purification methods well known to those skilled in the art.

[0085] Drugs or drug compositions

[0086] The present invention also provides a medicament or pharmaceutical composition comprising an effective amount of the antibody of the present application or its antigen-binding fragment, a nucleic acid molecule, a vector, or a host cell. As used herein, the terms “active substance” and “active substance of the present invention” are used interchangeably and refer to the anti-GLYR1 antibody of the present application or its antigen-binding fragment, the nucleic acid molecule encoded therein, a vector, or a host cell.

[0087] As used herein, the terms “containing” or “including” include “comprising,” “consistently composed of,” and “composed of.” As used herein, a “pharmaceutically acceptable” ingredient is a substance suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. As used herein, the term “effective amount” refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0088] As used herein, the term "pharmaceutically acceptable carrier" refers to a delivery vehicle used for therapeutic agents, including various excipients and diluents. This term refers to drug carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0089] Pharmaceutically acceptable carriers in the composition may contain liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffers, etc. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous transport medium, with a pH usually around 5-8, preferably around 6-8.

[0090] As used herein, the term "unit dosage form" refers to a dosage form in which the active substance of the present invention is prepared for a single application for ease of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release formulations.

[0091] It should be understood that the effective dose of the antibody or its encoding sequence or other active substance may vary depending on the severity of the patient being treated. The specific dosage depends on the individual patient's circumstances (e.g., weight, age, physical condition, and desired outcome), and is within the judgment of a skilled physician.

[0092] The active substances of this application can be used to treat tumors such as pancreatic cancer (e.g., pancreatic ductal adenocarcinoma). The pharmaceutical compositions of this invention may also contain other antitumor active substances, or may be used in combination with other antitumor active substances to obtain better therapeutic effects. Other antitumor active substances include, but are not limited to: gemcitabine, TNF-α, TGF-β, IFN-α, angiostatin, endostatin, glyphosate mustard, porphyrin, lycorine ammonium salt, croton oil, etoposide, dehydroeugenol, doxorubicin, tamoxifen, 5-fluorouracil, norcanthone, difuran fluorouracil, cucurbitacin, holoharbin, oridonin, iridoside, tragali glycopeptide, cytarabine, carboplatin, paclitaxel, lentinan, flutamide, ifosfamide, ubenmex, leuprolide acetate, deoxyfluorouridine, loboplatin, linnotkene, ledrozole, or teniposide, etc.

[0093] When two or more drugs are administered in combination, they generally have a better effect than when the two drugs are administered alone. Preferably, the drugs or other formulations administered in combination do not interfere with the therapeutic activity of the active substance of this application.

[0094] Reagent test kit

[0095] This application also provides a kit for detecting the presence, level, or activity of GLYR1, particularly membrane-localized GLYR1, comprising the antibody or its active fragment of this application.

[0096] In some embodiments, the kit of the present invention can be used to detect the presence or content of GLYR1 in biological samples. The detection method includes the steps of: (a) contacting the sample with an antibody or an active fragment thereof from the kit; and (b) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of GLYR1 in the sample, or quantitatively detecting the amount of the antigen-antibody complex formed to reflect the content of GLYR1 in the sample. The sample may be pretreated or unpretreated, for example, it may be extracted, purified, or concentrated.

[0097] Previous studies have shown that GLYR1 is abnormally localized on the cell membrane surface of pancreatic cancer cells, and this abnormal localization is closely related to the prognosis and metastasis of pancreatic cancer. Therefore, in some embodiments, the detection product of this application can be used to detect abnormally localized GLYR1 on the cell membrane surface of suspected samples or cells to diagnose the presence of pancreatic cancer. The detection product of this application can also be used to quantify and compare abnormally localized GLYR1 on the cell membrane surface of samples or cells to predict the prognosis and / or metastasis of pancreatic cancer. Furthermore, the detection product of this application can be used to detect abnormally localized GLYR1 on the cell membrane surface of samples or cells before and after treatment to determine the effectiveness of treatment and / or to select a treatment regimen.

[0098] The kit of this application may contain a container and the antibody or its antigen-binding fragment thereof contained within the container, or a detection plate containing the antibody or its antigen-binding fragment, and an instruction manual. The kit may also contain other reagents required for detection, such as buffer solutions, indicators, etc. Those skilled in the art can adjust the contents of the kit according to specific needs.

[0099] Example

[0100] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make appropriate modifications and variations to the present invention, and such modifications and variations are all within the scope of the present invention.

[0101] Experimental methods not specifically described in the following examples can be performed using conventional methods in the art, such as those described in *Molecular Cloning: A Laboratory Manual* (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the supplier. DNA sequencing methods are conventional in the art and can also be provided by commercial companies.

[0102] Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0103] Example 1: Detection of GLYR1 mRNA expression in pancreatic cancer tissue

[0104] (I) Experimental Materials

[0105] Tissue samples were obtained from fresh samples after surgical resection of pancreatic cancer (Shanghai Changhai Hospital).

[0106] (II) Experimental Methods

[0107] ① Sample processing: Pancreatic cancer tissue samples were obtained from the surgical resection of pancreatic cancer, and adjacent non-pancreatic cancer pancreatic tissue was collected as a control; the tissue samples were rapidly frozen in liquid nitrogen to maintain the integrity of the RNA.

[0108] ②RNA Extraction and cDNA Synthesis: Total RNA was extracted using the commercial RNA extraction reagent TRIzol; the concentration and purity of RNA were determined using a nanodrop spectrometer to ensure A 260 / A 280 The ratio was between 1.8 and 2.0. cDNA was synthesized using 1 μg of total RNA and a commercial reverse transcription kit (Invitrogen, SuperScriptIV).

[0109] ③ Real-time quantitative PCR: Primers specifically targeting GLYR1 mRNA were designed; primer pairs SEQ ID NO:6 and SEQ ID NO:8 were used. The qPCR reaction was set up, including cDNA template, SYBR Green or TaqMan probes, primers, and qPCR MasterMix. qPCR thermal cycling conditions were set to an initial denaturation step (95°C, 10 min), followed by 40 cycles, each cycle including denaturation (95°C, 15 sec) and annealing / extension (59°C, 45 sec). Threshold cycling (Ct value) acquisition: Ct values ​​were obtained for each sample using a qPCR instrument. Relative expression levels were calculated using the ΔΔCt method, and the internal reference gene (β-actin) was selected for normalization.

[0110] (III) Experimental Results and Analysis

[0111] Pancreatic cancer tissue samples were obtained from surgically removed pancreatic cancer tissue, and adjacent non-pancreatic cancer pancreatic tissue was collected as a control. Figure 1As shown, the relative expression level of GLYR1 mRNA in pancreatic cancer tissues was significantly upregulated compared to adjacent normal tissues. These results indicate that the expression level of GLYR1 mRNA in tissues is closely related to pancreatic cancer.

[0112] Example 2: Expression of membrane-type GLYR1 in pancreatic cancer and paired adjacent normal tissues

[0113] (I) Experimental Materials

[0114] Tissue samples were obtained from the Shanghai Changhai Hospital Sample Bank; the primary antibody was a rabbit monoclonal antibody purchased from Abcam (#ab167155); the secondary antibody was a biotin-labeled goat anti-mouse / rabbit antibody purchased from Zhongshan Jinqiao (#SAP-9100).

[0115] (II) Experimental Methods

[0116] ① Sample collection and preparation: Pancreatic cancer tissue and paired adjacent normal tissue samples were obtained from surgically removed pancreatic cancer patients; freshly removed tissue samples were immediately placed in a container containing 4% formaldehyde and fixed at room temperature for 24 hours, then dehydrated and embedded in paraffin; the embedded tissue was cut into 4-micrometer-thick continuous sections using a microtome and mounted on glass slides.

[0117] ② Slide preparation: The slides were treated twice in xylene for 15 minutes each time to remove paraffin, and then dehydrated by a series of ethanol solutions of decreasing concentrations (100%, 95%, 80%, 70%). The slides were then autoclaved in citrate buffer (pH 6.0) for 10 minutes for antigen retrieval.

[0118] ③ Immunostaining and Nuclear Staining: Incubate sections in PBS containing 5% bovine serum albumin for 30 minutes to block nonspecific antibody binding. Cover sections with pre-diluted primary antibody against GLYR1 (1:200) and incubate overnight at 4°C. Incubate sections with secondary antibody labeled with horseradish peroxidase for 1 hour, then wash sections three times with PBS; stain sections with DAB (3,3'-diaminobenzidine) chromogenic agent until appropriate staining intensity appears. Stain with Hermatolysin for 1-2 minutes to stain cell nuclei. Finally, mount the sections after washing with xylene.

[0119] ④ Evaluation of GLYR1 membrane type: Immunohistochemical image analysis was performed using Python. Image processing library OpenCV and image analysis library scikit-image were used to segment and analyze the immunohistochemical images. GLYR1 immunohistochemical scoring was determined based on the positive rate and staining intensity. The Immuno-Reactive Score (IRS) was used for comprehensive scoring, and the percentage of positive cells (PP) was calculated and divided into five levels: 0 (≤5%), 1 (6-10%), 2 (11-50%), 3 (51-75%), and 4 (>75%). Staining intensity (SI) was divided into: 0 (no staining), 1 (pale yellow), 2 (brownish-yellow), and 3 (brownish-brown). The GLYR1 immunohistochemical staining score IRS was calculated as follows:

[0120] GLYR1 Immunostaining Score (IRS) = Positive Percentage Score (PP) × Staining Intensity Score (SI).

[0121] (III) Experimental Results and Analysis

[0122] The results showed that the nuclear epigenetic enzyme GLYR1 exhibited abnormal cell membrane localization in pancreatic cancer cells. Figure 2 A); Tissue microarray (from Changhai Hospital sample bank) screening showed that nearly 60% of pancreatic cancer tissues were GLYR1 membrane positive. Figure 2 B). Cytoplasmic localization of GLYR1 indicates that the GLYR1 expressed inducible form is in an intermediate state of membrane translocation, suggesting that abnormal membrane localization of GLYR1 may continue to occur subsequently.

[0123] The above results indicate that membrane-type GLYR1 is highly expressed on the cell membrane of pancreatic cancer cells, thus it can serve as a biomarker for the diagnosis of pancreatic cancer or a target for treatment.

[0124] Example 3: Preparation and Identification of GLYR1-Targeted Hybridoma Cells

[0125] (I) Experimental Materials

[0126] C57 mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., Freund's adjuvant from Sigma-Aldrich, GLYR1 immunogen from Beijing Yiqiao Shenzhou Technology Co., Ltd., myeloma cells from ATCC, DPBS from Hyclone, and 0.4% trypan blue from Sangon Biotech Co., Ltd. Pierce TM Rapid antibody typing kit (with κ and λ) - mice were purchased from Thermo Fisher Scientific, SW1990 mice were purchased from ATCC, and PX459 plasmid was purchased from Yunzhou Biotechnology.

[0127] (II) Experimental Methods

[0128] 1. Immunization of mice: 6-8 week old female C57 mice were immunized by subcutaneous injection of 50 μg GLYR1 protein mixed with an equal volume of complete Freund's adjuvant. On day 14 and day 28, 25 μg of antigen mixed with an equal volume of incomplete Freund's adjuvant were immunized by subcutaneous injection. On day 63, 50 μg of antigen and incomplete Freund's adjuvant were immunized intravenously.

[0129] 2. Blood collection and testing: Blood was collected for the first time about 4 days before immunization, and then 7 days after each immunization. The serum of the immunized animals was tested by indirect ELISA to determine the level of immune response. If the immunized animals could reach the level of immune response against the immunogen (OD value > 1.0, titer 1:8000), cell fusion was performed.

[0130] 3. Cell Fusion: Spleen cells and myeloma cells were fused using an electrofusion method. All cells from each fusion round were seeded into 96-well plates for subsequent screening. Primary Screening: The supernatant of the fused cells was screened using an indirect ELISA method, selecting those that were positive for the target protein. Secondary Screening: All positive clones obtained in the primary screening stage were further screened using an indirect ELISA method, selecting all positive maternal clone cell supernatants.

[0131] 4. Subcloning: Positive maternal clones are subcloned using the limiting dilution method to ensure that these positive maternal clones are derived from a single maternal clone cell, with a maximum of 3 rounds of subcloning.

[0132] (1) ELISA method for detecting antibody titer

[0133] GLYR1 antigen protein (1 μg / ml, 100 μl per well) was coated onto the plate and incubated overnight at 4°C. The next day, the plate was washed three times with PBST (PBS + 0.05% Tween), and 200 μl of blocking buffer (PBS + 3% BSA) was added to each well for blocking at room temperature for one hour. The plate was washed three times with PBST. Hybridoma secretion supernatant (containing antibody) and negative control (without antibody) at different dilutions were added, and the plate was incubated at 37°C for 2 hours. The plate was washed three times with PBST. Diluted secondary antibody (100 μl per well) was added, and the plate was incubated at 37°C for 1 hour. The plate was washed three times with PBST. 100 μl of TMB chromogenic solution was added, and after color change, 100 μl of STOP Solution was added. The OD value was detected at 450 nm using a single wave.

[0134] (2) Specificity of antibodies detected by immunoblotting

[0135] The GLYR1-KO cell line (SW1990-GLYR1 KO) based on pancreatic cancer cell line SW1990 was prepared using the CRISPR / Cas9 system.

[0136] Basic procedure: gRNA (TGCGGCTCGGCGACTTGGTG, SEQ ID NO:10) was synthesized and integrated into the PX459 plasmid. The recombinant plasmid was transfected into SW1990 cells using jetPEI (Polyplus). Forty-eight hours after transfection, positive cells were selected using puromycin (5 μg / ml), and then single clones were selected for amplification in 96-well plates. The knockout efficiency was verified by Western blotting.

[0137] (III) Experimental Results and Analysis

[0138] Hybridoma cell secretion supernatant was serially diluted at 1:10, 1:30, 1:90, 1:270, 1:810, and 1:2430, and the antibody titer was detected by ELISA. Figure 3 The results showed that four hybridoma cell lines (clone numbers: 4D2F10, 8B7C3, 1D1E1, and 13E10C4) secreted antibody titers as high as 2430 or higher.

[0139] The SW1990-GLYR1 KO cell line was prepared using the CRISPR / Cas9 system, and Western blotting confirmed that GLYR1 had been successfully knocked out in this cell line. Figure 4 The specificity of the four antibodies was detected by immunoblotting, and it was found that all four antibodies had good specificity for GLYR1. Figure 5 ).

[0140] Example 4: Obtaining hybridoma cell antibody sequences and expressing, preparing, and identifying recombinant human antibodies.

[0141] (I) Experimental Materials

[0142] The heavy and light chain expression plasmids of the antibody were purchased from InvivoGen, the heavy and light chain genes of the antibody were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., the gel extraction kit was purchased from Takara, the homologous recombinase was purchased from Nanjing Novizan Biotechnology Co., Ltd., the expiCHO-S cell line and transfection reagent were purchased from Thermo Fisher Scientific, and the protein G column was purchased from GE.

[0143] (II) Experimental Methods

[0144] The four hybridoma cell-derived antibodies obtained in Example 3 were sequenced by Genewiz to obtain the scFv sequences of the heavy and light chains of the antibodies.

[0145] Construction of VH and VL sequence vectors for GLYR1 antibody: The antibody light chain and heavy chain scFv genes were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. Subcloning was performed using the synthesized plasmids as templates. The obtained PCR fragments were purified using a gel extraction kit and ligated into VH (pFUSEss-CHIg-hG1) and VL (pFUSE2ss-CLIg-hk) expression vectors via homologous recombination. The vectors were then transformed into DH5α competent cells, and positive clones were obtained by sequencing, yielding correctly paired antibody light and heavy chain expression plasmids.

[0146] CHO-S cell system expression antibody: Prepare reaction mixture A: 2 ml OptiPro-SFM + light chain and heavy chain expression plasmids (50 μg each) and mixture B: 1.84 ml OptiPro-SFM + 160 μl ExpiFectamine. TM After mixing CHO Reagent by vortexing and incubating at room temperature for 5 minutes, add mixture B to mixture A by vortexing and incubating at room temperature for 10-20 minutes. Slowly add 50 ml of CHO-S cell system. 18-22 hours after transfection, add 300 μl of ExpiCHO to the system. TM Enhancer, 12ml ExpiCHO TM Feed the cells and incubate them at 37°C. After 7 days, collect the cell culture supernatant.

[0147] Antibody purification: Antibody purification was performed using the AKTA protein purification system as follows: Cell culture supernatant was centrifuged or filtered to remove cell debris. The protein G column was washed with 1×PBS for 10 CV until the baseline stabilized. The cell supernatant containing antibodies was loaded onto the AKTA, and the column was equilibrated again with PBS to wash away unbound impurities. The antibody on the column was eluted with 0.1M glycine (pH 2.8) and collected in a tube containing neutralization buffer (1M Tris, pH 9.0) to neutralize the pH. The eluted antibody solution was centrifuged using an ultrafiltration tube to replace the PBS.

[0148] Antibody purity and molecular weight determination: The purity and molecular weight of the purified antibody were determined by 10% polyacrylamide gel electrophoresis.

[0149] (III) Experimental Results and Analysis

[0150] Hybridoma cells 4D2F10, 8B7C3, 1D1E1, and 13E10C4 were sequenced by Suzhou Genewiz Biotechnology Co., Ltd. to obtain the antibody light and heavy chains, as well as the scFv sequence. The antibody heavy and light chain gene sequences were synthesized and constructed into human VH and VL expression vectors via homologous recombination. The antibodies were expressed using the CHO-S expression system and purified using the AKTA protein purification system to obtain recombinant antibodies, named CL-ab-1, CL-ab-2, CL-ab-3, and CL-ab-4, respectively.

[0151] The purified antibody was analyzed for purity and molecular weight by 10% polyacrylamide gel electrophoresis. Under complete reduction conditions, the GLYR1 antibody showed two bands with molecular weights of approximately 50 kDa and 25 kDa, representing the heavy and light chain bands, respectively, with a purity of over 95%. Figure 6 The above results demonstrate that we have successfully prepared and expressed a high-purity GLYR1 antibody.

[0152] Example 5: Determination of anti-GLYR1 antibody affinity

[0153] (I) Experimental Materials

[0154] Antigen-coated 96-well plates were purchased from Thermo Fisher Scientific; GLYR1 protein (#526189) was purchased from Novopro; positive control (#A00186) was purchased from Genscript Biotech; IgG negative control (#ab12912), secondary antibody corresponding to GLYR1 antibody (#ab6759), TMB chromogenic solution and stop solution were purchased from Abcam; secondary antibodies corresponding to positive control and IgG negative control (#7076S and #7074S) were purchased from Cell Signaling Technology; S-series CM5 sensor chips were purchased from Cytiva.

[0155] (II) Experimental Methods

[0156] ELISA assay for antibody affinity: GLYR1 protein was diluted with PBS and coated into 96-well plates (100 ng / well). The plates were incubated overnight at 4°C. After washing four times with PBST, the plates were blocked with 3% BSA at room temperature for 1 h. After washing four times with PBST, 100 μl of sample (test sample and control sample, 8 dilution gradients, consecutive 5-fold dilutions, with the highest concentration being 1 μg / ml) was added. After incubation at room temperature for 1 h, the plates were washed four times with PBST. After incubation at room temperature for 1 h, the plates were incubated with 100 μl of secondary antibody. After washing four times with PBST, 100 μl of TMB chromogenic solution was added. After color change, 100 μl of stop solution was added. The OD value was detected at 450 nm using a single wave.

[0157] SPR method for antibody affinity detection: A chip was placed on a Biacore T200 (Cytiva) instrument. Using HBSEP buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20), the antigen protein GLYR1 was covalently linked to the experimental channel using an amino-coupled method. Serially diluted antibodies were used as analytes and flowed through the control and experimental channels at a rate of 30 μl / min. Binding time was 120 seconds, dissociation time was 400 seconds, and regeneration buffer was Glycine 2.0. Affinity (K0.05) was measured using Biacore T200 evaluation software 3.1 (Cytiva). D Analysis shows that a 1:1 combination of action modes is used.

[0158] (III) Experimental Results and Analysis

[0159] We first used ELISA to detect the binding of recombinant GLYR1 antibodies to the antigen, and found that the prepared and expressed CL-ab-1, CL-ab-2, CL-ab-3 and CL-ab-4 recombinant GLYR1 antibodies could all bind to human GLYR1 protein. Figure 7 Further analysis using Biacore to detect the affinity of GLYR1 antibodies showed that all four recombinant GLYR1 antibodies exhibited high affinity: antibodies CL-ab-1, K... D =3.51×10 -11 M( Figure 8 C); Antibody CL-ab-2, K D =7.58×10 -11 M( Figure 8 B); Antibody CL-ab-3, K D =3.81×10 -11 M( Figure 8 A) Antibody CL-ab-4, K D =1.43×10 -10 M( Figure 8 D).

[0160] Example 6: Detection of the in vitro ADCC effect of anti-GLYR1 antibody

[0161] (I) Experimental Materials

[0162] Human GLYR1 cDNA ORF clone (NM_032569.3) was purchased from SinoBiological (HG23492-UT); mouse GLYR1 cDNA ORF clone (NM_028720.2) was obtained from Creative-Biogene (CDCR261743); calcium phosphate kit was purchased from ViralTherapy; QuickTiter Lentivirus Quantitation Kit was purchased from Cell Biolabs; polybrene was purchased from Yeasen; anti-FLAG-APC antibody was purchased from Biolegend; Jurkat... FcγRIIIa / NFAT-luc cells were purchased from Aikon Biotechnology (Suzhou) Co., Ltd.; ADCC Reporter Bioassay (#G7010), low IgG serum (#G711A), and bioluciferase assay substrates (#G719A and #G720A) were purchased from Promega; pRRLSIN, psPAX2, and pMD2.G plasmids were purchased from Addgene.

[0163] (II) Experimental Methods

[0164] Preparation of GLYR1-GPI membrane-anchored PDAC cells

[0165] To construct an artificial membrane-anchored GLYR1 expression vector (GLYR1-GPI), the coding sequence of human GLYR1 was amplified by PCR (SEQ ID NO:1; amplification primers are shown in SEQ ID NO:6 and 8 or SEQ ID NO:7 and 9). A signal peptide was designed to be added to the N-terminus of the encoded GLYR1 polypeptide (SEQ ID NO:2) (as shown underlined in SEQ ID NO:4 in the sequence information attached to the instruction manual), and a 2×FLAG epitope was added after the signal peptide (as shown underlined in SEQ ID NO:4 in the sequence information attached to the instruction manual). A CD59 GPI sequence was added to the C-terminus (as shown underlined in SEQ ID NO:4 in the sequence information attached to the instruction manual). The coding sequence of this full-length artificial membrane-anchored GLYR1 is shown in SEQ ID NO:3. Human CD59 was used as a control (Ctrl-GPI).

[0166] A PDAC monoclonal cell line expressing GLYR1-GPI was constructed using lentiviral transfection. The GLYR1-GPI sequence was cloned into the pRRLSIN plasmid and then co-transfected into virus packaging cells with the psPAX2 and pMD2.G plasmids at a ratio of 4:3:1 using a calcium phosphate assay kit. At 48 and 72 hours post-transfection, the culture supernatant was concentrated by ultracentrifugation (82,700 g, 2.5 h). Lentiviral titers were assessed using the QuickTiter Lentivirus Quantitation Kit. Subsequently, PDAC cells (SW1990-GLYR1 KO / PANC1-GLYR1 KO) were incubated at 1 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 10 cells / well in 24-well plates, and 2 mg / mL polybrene was added to promote infection (MOI = 10). FLAG-positive cells were sorted by FACS, and cells were further sorted by labeling with anti-FLAG-APC antibody. Cells were then seeded individually into 96-well plates, and GLYR1-GPI membrane expression was detected by immunofluorescence.

[0167] In vitro ADCC effect detection of anti-GLYR1 antibody

[0168] The in vitro ADCC effect of anti-GLYR1 antibody was assessed using a reporter gene assay. Target cells were collected by digestion, washed three times with PBS, and incubated at 1000 rpm for 5 min. Target cells were resuspended in culture medium (containing 5% low IgG serum) and counted, adjusting the target cell density to 2 × 10⁶ cells / mL. 5 25 μl of cell suspension was added to each well of a white-based 96-well plate, resulting in 5000 target cells per well. GLYR1 antibody (10 dilutions, 3-fold dilution, highest concentration 300 μg / ml, lowest concentration 0 μg / ml) and control antibody IgG1 (9 dilutions, 3-fold dilution, highest concentration 100 μg / ml, lowest concentration 0 μg / ml) were serially diluted with culture medium. 25 μl of the serially diluted antibody was added to each well of the 96-well plate containing the target cells, with three replicates. Effector cells (Jurkat cells) were collected. FcγRIIIa / NFAT-luc, washed three times with PBS, 1000 rpm for 5 min, and the cell density was adjusted to 3 × 10⁶ cells / mL in culture medium. 6 25 μl of cell suspension per well was added to each well of a 96-well white-background plate containing target cells and antibody for co-incubation at an effector-to-target ratio of 15:1 (75,000 effector cells / well, 5,000 target cells / well), for a total volume of 75 μl. The plates were incubated at 37°C in a 5% CO2 incubator for 6 hours. Before detection, Bio-Glo... TMAfter the luciferase assay substrates equilibrate to ambient temperature, remove the 96-well plates from the 37°C incubator and allow them to equilibrate to room temperature for 15 minutes. Add 25 μl of assay substrate to each well of the 96-well plate. Incubate at room temperature for 5 minutes, then perform luciferase detection using the chemiluminescence module of a microplate reader. Calculate the EC50 of each antibody based on the fluorescence value. 50 .

[0169] (III) Experimental Results and Analysis

[0170] We successfully prepared SW1990-GLYR1 KO and SW1990-GLYR1 GPI cells (abbreviated as GLYR1-KO and GLYR1-GPI cells, respectively). Figure 9 ) and PANC1-GLYR1 GPI cells.

[0171] Using PANC1-GLYR1 GPI cells ( Figure 10 B) and BXPC3 cells that are positive for GLYR1 membrane expression ( Figure 10 A) As target cells, with Jurkat FcγRIIIa Using / NFAT-luc as effector cells, we examined antibody-mediated ADCC effects. We found that CL-ab-2 and CL-ab-3 mediated significant ADCC effects in both GLYR1-expressing cell types compared to the isotype control antibody IgG, and this effect was dose-dependent. Figure 10 (A and 10B).

[0172] Example 7: Detection of ADCC effect of anti-GLYR1 antibody in vivo

[0173] (I) Experimental Materials

[0174] hIgG1 isotype antibody (#BE0297) and mouse IgG1 isotype antibody (#BE0083) were purchased from Bioxcell, rIL2 (#GMP-CD66) was purchased from Suzhou Nearshore Protein Technology Co., Ltd., and NCG mice were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.

[0175] (II) Experimental Methods

[0176] Female NCG mice with severe immunodeficiency, aged 6-8 weeks, were selected and subcutaneously injected with 2×10⁻⁶ NCG mice. 6A mouse tumor model was constructed using tumor cells, and the cells were grouped as follows: SW1990 WT group, SW1990 Ctrl-KO group, SW1990 GLYR1-KO group, SW1990 KO+Ctrl-GPI group, and SW1990 KO+GLYR1-GPI group (n=4 for each group). On day 14 of tumor growth, 2 μg of control hIgG1 isotype or anti-GLYR1 antibody was injected via the tail vein. The antibodies were then resuspended in 1×10⁻⁶ cells. 7 NK92 cells (300 μL HBSS, containing 5 μg gIL2 and 5 μg mouse IgG1 isotype antibody). Antibody treatment and tumor size measurement were performed weekly for a total of 2 treatments, with tumor size measured after each treatment.

[0177] (III) Experimental Results and Analysis

[0178] We first constructed NCG tumor-bearing mouse models using the following strains: SW1990 WT, SW1990 Ctrl-KO, SW1990 GLYR1-KO, SW1990 KO+Ctrl-GPI, and SW1990 KO+GLYR1-GPI. The tumors were allowed to grow to 100 mm. 3 On the left and right sides, CL-ab-2 and CL-ab-3 antibodies and NK92 cells were adopted and reinfused, respectively, with IgG as a negative control, and the ADCC effect in vivo was observed.

[0179] The results showed that compared with the isotype IgG control ( Figure 11 Compared to A), CL-ab-2 antibody ( Figure 11 C) significantly inhibited the growth of pancreatic cancer cells in mice, while CL-ab-3 antibody did not mediate a significant ADCC effect in vivo. Figure 11 B).

[0180] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0181] Appendix: Sequence Information

[0182] SEQ ID NO:1: GLYR1 gene

[0183]

[0184] SEQ ID NO:2: GLYR1 peptide

[0185] MAAVSLRLGDLVWGKLGRYPPWPGKIVNPPKDLKKPRGKKCFFVKFFGTEDHAWIKVEQLKPYHAHKEEMIKINGKGKRFQQAVDAVEEFLRRAKGKDQTSSHNSSDDKNRRNSSEEERSRPNSGDEKRKLSLSEGKVKKNMGEGKKRVSSGSSERGSKSPLKRAQEQSPRKRGRPPKDEKDLTIPESSTVKGMMAGPMAAFKWQPTASEPVKDADPHFHFLLSQTEKPAVCYQAITKKLKICEEETGSTSIQAADSTAVNGSITPTDKKIGFLGLG LMGSGIVSNLLKMGHTVTVWNRTAEKCDLFIQEGARLGRTPAEVVSTCDITFACVSDPKAAKDLVLGPSGVLQGIRPGKCYVDMSTVDADTVTELAQVIVSRGGRFLEAPVSGNQQLSNDGMLVILAAGDRGLYEDCSSCFQAMGKTSFFLGEVGNAAKMMLIVNMVQGSFMATIAEGLTLAQVTGQSQQTLLDILNQGQLASIFLDQKCQNILQGNFKPDFYLKYIQKDLRLAIALGDAVNHPTPMAAAANEVYKRAKALDQSDNDMSAVYRAYIH

[0186] SEQ ID NO:3: Encoding sequence of artificial membrane-localized GLYR1

[0187]

[0188] SEQ ID NO:4: Artificial membrane-localized GLYR1 precursor peptide

[0189]

[0190] SEQ ID NO:5: Artificial membrane-localized GLYR1 mature peptide (de-signal peptide)

[0191] DYKDDDDKDYKDDDDKAAVSLRLGDLVWGKLGRYPPWPGKIVNPPKDLKKPRGKKCFFVKFFGTEDHAWIKVEQLKPYHAHKEEMIKINGKGKRFQQAVDAVEEFLRRAKGKDQTSSHNSSDDKNRRNSSEERSRPNSGDEKRKLSLSEGKVKKNMGEGKKRVSSGSSERGSKSPLKRAQEQSPRKRGRPPKDEKDLTIPESSTVKGMMAGPMAAFKWQPTASEPVKDADPHFHFLLSQTEKPAVCYQAITKKLKICEEETGSTSIQAADSTAVNGSITPTDKKIGFLGLGLMGSGI VSNLLKMGHTVTVWNRTAEKCDLFIQEGARLGRTPAEVVSTCDITFACVSDPKAAKDLVLGPSGVLQGIRPGKCYVDMSTVDADTVTELAQVIVSRGGRFLEAPVSGNQQLSNDGMLVILAAGDRGLYEDCSSCFQAMGKTSFFLGEVGNAAKMMLIVNMVQGSFMATIAEGLTLAQVTGQSQQTLLDILNQGQLASIFLDQKCQNILQGNFKPDFYLKYIQKDLRLAIALGDAVNHPTPMAAAANEVYKRAKALDQSDNDMSAVYRAYIHGGTSLSEKTVLLLVTPFLAAAWSLHP

[0192] SEQ ID NO:6: GLYR1 amplification upstream primer 1

[0193] GATGCTGATCGTGAACATGG

[0194] SEQ ID NO:7: GLYR1 amplification upstream primer 2

[0195] TGTTACCAGGCAATCACGAA

[0196] SEQ ID NO:8: GLYR1 amplification downstream primer 1

[0197] GGCTAAGCGGAGATCCTTC

[0198] SEQ ID NO:9: GLYR1 amplification downstream primer 2

[0199] TTGGAGACGATTCCACTTCC

[0200] SEQ ID NO:10: gRNA

[0201] TGCGGCTCGGCGACTTGGTG

[0202] SEQ ID NO:11: Antibody CL-ab-2-VH

[0203] QVQLQQSGPELVKPGASVKISCKASGYAFSSYWMNWVKQRPGKGLEWIGQIYPGDGDTYYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCADGAMDYWGQGTSVTVSS

[0204] SEQ ID NO:12: Antibody CL-ab-2-VL

[0205] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATNLRDGVPSRFSGSGSGTQFSLKINSLQSEDFGSYFCQHFWYTPYTFGGGTKLEIK

[0206] SEQ ID NO:13: Antibody CL-ab-2-VH CDR1

[0207] GYAFSSYW

[0208] SEQ ID NO:14: Antibody CL-ab-2-VH CDR2

[0209] IYPGDGDT

[0210] SEQ ID NO:15: Antibody CL-ab-2-VH CDR3

[0211] ADGAMDY

[0212] SEQ ID NO:16: Antibody CL-ab-2-VL CDR1

[0213] ENIYSN

[0214] SEQ ID NO:17: Antibody CL-ab-2-VL CDR2

[0215] AAT

[0216] SEQ ID NO:18: Antibody CL-ab-2-VL CDR3

[0217] QHFWYTPYT

[0218] SEQ ID NO:19: Antibody CL-ab-3-VH

[0219] QVQLQQSGAELVKPGASVKISCKASGYAFSRYWINWVKQRPGKGLEWIGQIYPGDGDTDYNGKFKGKATLTVDKTSSTAYMQLSSLTSEDSAVYFCADGAMDYWGQGTSVTVSS

[0220] SEQ ID NO:20: Antibody CL-ab-3-VL

[0221] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYRQKQGKSPQLLVYAATNLAEGVPSRFSGSGSGTQFSLKINSLQSEDFGSYYCQHFWITPYTFGGGTKLEIK

[0222] SEQ ID NO:21: Antibody CL-ab-3-VH CDR1

[0223] GYAFSRYW

[0224] SEQ ID NO:22: Antibody CL-ab-3-VH CDR2

[0225] IYPGDGDT

[0226] SEQ ID NO:23: Antibody CL-ab-3-VH CDR3

[0227] ADGAMDY

[0228] SEQ ID NO:24: Antibody CL-ab-3-VL CDR1

[0229] ENIYSN

[0230] SEQ ID NO:25: Antibody CL-ab-3-VL CDR2

[0231] AAT

[0232] SEQ ID NO:26: Antibody CL-ab-3-VL CDR3

[0233] QHFWITPYT

Claims

1. An antibody or antigen-binding fragment thereof targeting GLYR1, wherein, The antibody comprises a combination of a heavy chain complementarity-determining region (VHCDR) and a light chain complementarity-determining region (VL CDR), wherein the VHCDR and VL CDR are each composed of amino acid sequences selected from the group consisting of: VH CDR1: SEQ ID NO: 13; VH CDR2: SEQ ID NO: 14; VH CDR3: SEQ ID NO: 15; VL CDR1: SEQ ID NO: 16; VL CDR2: SEQ ID NO: 17; and VL CDR3: SEQ ID NO:

18.

2. The antibody or its antigen-binding fragment as described in claim 1, wherein, The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL each have an amino acid sequence selected from the group consisting of: VH: (a) the sequence shown in SEQ ID NO: 11; or (b) a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO: 11; and VL: (a') the sequence shown in SEQ ID NO: 12; or (b') a sequence that has at least 80% sequence identity with the sequence shown in SEQ ID NO:

12.

3. The antibody or its antigen-binding fragment as described in claim 1, wherein, The antibody has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 11 and / or the antibody has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 12, and the differences in amino acid residues are not located in each VH CDR and VL CDR, wherein the antibody is capable of specifically binding to GLYR1.

4. The antibody or its antigen-binding fragment as described in claim 3, wherein, The VH and / or VL contain substitutions of one or more amino acid residues in their non-CDR regions.

5. The antibody or its antigen-binding fragment as described in claim 4, wherein, The substitution of one or more amino acid residues is a humanized design and substitution.

6. The antibody or antigen-binding fragment thereof as described in claim 1, wherein, The antibody or its antigen-binding fragment is selected from: monoclonal antibodies and polyclonal antibodies; and / or The antibody or its antigen-binding fragment is selected from the group consisting of: free; attached, embedded, or immobilized on a support; and / or The antibody is a chimeric antibody containing a human IgG backbone.

7. The antibody or antigen-binding fragment thereof as described in claim 1, wherein, The antibody or its antigen-binding fragment is selected from: Fab, Fab', F(ab')2 and scFv.

8. The antibody or antigen-binding fragment thereof as described in claim 1, wherein, The antibody or its antigen-binding fragment is selected from: bispecific antibodies, triantibodies, tetraantibodies, F(ab')3 and (scFv)2.

9. The antibody or antigen-binding fragment thereof as described in claim 1, wherein, The antibody or its antigen-binding fragment is selected from: disulfide-linked Fv, IgGΔCH2, DVD-Ig, scFv-Fc and mAb2.

10. The antibody or antigen-binding fragment thereof as described in claim 6, wherein, The support material is selected from: magnetic beads, agarose, gold surface, chip and microcarrier.

11. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10, wherein, The antibody has one or more characteristics selected from the group consisting of: (i) which specifically binds to GLYR1 with a binding affinity K D less than 1.5 x 10 -10 M; and (ii) It can inhibit the growth of pancreatic cancer, improve the prognosis of pancreatic cancer, and increase the sensitivity of pancreatic cancer to chemotherapy.

12. The antibody or antigen-binding fragment thereof as described in claim 11, wherein, The antibody or its antigen-binding fragment specifically binds to membrane-bound GLYR1.

13. The antibody or antigen-binding fragment thereof as described in claim 11, wherein, The binding affinity K D is 0.3 x 10 -10 M~1.5 x 10 -10 M.

14. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 13.

15. The nucleic acid molecule of claim 14, wherein the codons have been optimized.

16. A vector or host cell comprising the nucleic acid molecule as described in claim 14 or 15.

17. A product comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 13, a nucleic acid molecule as described in claim 14 or 15, and / or a vector or host cell as described in claim 16.

18. The use of the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 13, the nucleic acid molecule as described in claim 14 or 15, the vector or host cell as described in claim 16, in the preparation of a product, wherein the product is used for: (a) Diagnosis, prevention, and / or treatment of pancreatic cancer with membranous GLYR1 overexpression; and / or (b) Detect whether the cells in the sample are membrane-bound GLYR1 overexpressing cells; and / or (c) Screening for therapeutic drugs or treatment regimens for pancreatic cancer with GLYR1 overexpression.

19. The application as described in claim 18, wherein, The pancreatic cancer mentioned is pancreatic ductal adenocarcinoma.

20. The application of claim 18, wherein the product is a drug, a medicine box, or a test kit.

Images