An antibody targeting membrane-bound hsp70, chimeric antigen receptor and use thereof

Abstract

This invention belongs to the fields of biomedicine and molecular biology, and discloses an antibody targeting membrane-bound Hsp70, a chimeric antigen receptor, and their applications. This invention successfully prepared a high-affinity antibody targeting the membrane-bound Hsp70 (mHsp70) molecule, and based on this high-affinity antibody, constructed CAR-T cells that specifically recognize mHsp70. In vitro and in vivo experiments demonstrated that these CAR-T cells have a therapeutic effect on Hsp70-overexpression-mediated diseases (such as small cell lung cancer and other neoplastic diseases).

Description

Technical Field

[0001] This invention relates to an antibody targeting membrane-bound Hsp70, a chimeric antigen receptor, and their applications. Specifically, it relates to an antibody specifically targeting membrane-bound Hsp70, a chimeric antigen receptor (CAR) containing the antibody and its modified T cells (CAR-T cells), and its application in the preparation of drugs or detection products for the prevention and / or treatment of diseases related to high Hsp70 expression. It belongs to the fields of biomedicine and molecular biology. Background Technology

[0002] Antibodies, as a core tool of modern medicine, have expanded their applications from preventative vaccines to targeted therapies, continuously driving clinical progress. Taking chimeric antigen receptor (CAR) T-cell therapy as an example, its core prerequisite for targeted killing relies entirely on antibodies capable of specifically recognizing tumor surface antigens. It is noteworthy that for the same tumor antigen, multiple monoclonal antibodies recognizing different epitopes can often be developed, leading to the construction of CAR-T products with diverse functions—this has become the mainstream R&D path in this field for overcoming tumor heterogeneity and optimizing efficacy. For instance, CAR-T cells based on FMC63 and SJ25C1 antibodies targeting CD19 in B-cell malignancies entered clinical application relatively early, achieving breakthrough efficacy in the treatment of B-cell lymphoma and leukemia, with several drugs approved for marketing. Similarly, CAR-T therapies developed based on different antibodies such as C11D5.3 and J22.9-xi targeting BCMA in multiple myeloma have also shown significant clinical potential, further confirming the feasibility of the "one target, multiple antibodies" strategy. However, compared to hematologic malignancies, the application of CAR-T therapy in solid tumors has progressed slowly due to obstacles such as high heterogeneity, lack of tumor-specific antigens, and an immunosuppressive microenvironment. In particular, the number of high-quality antibodies available for screening for the same solid tumor target is limited, and CAR-T cells constructed from different antibodies exhibit significant differences in affinity, epitope recognition, signal transduction, and antitumor activity. Therefore, systematically screening multiple candidate antibodies for the same target and selecting the optimal CAR-T construction regimen has become a key direction for advancing CAR-T therapy for solid tumors.

[0003] As a highly conserved heat shock protein, Hsp70 (Heat Shock Protein 70) is expressed at low levels under normal physiological conditions and is located intracellularly (cytoplasm, endoplasmic reticulum, mitochondria), participating in protein folding, anti-apoptosis, and stress protection. Hsp70 is highly expressed in most tumor samples and can serve as a molecular marker for early-stage prostate and liver cancer. It is also associated with treatment resistance and prognosis in various tumors, including breast cancer, endometrial cancer, and rectal cancer. Therefore, many drugs that directly or indirectly target Hsp70 and its partners have been developed for cancer treatment.

[0004] Previous studies have indicated that extracellular Hsp70 in the tumor microenvironment can activate immune effector cells such as natural killer (NK) cells, suggesting its potential as a target for immunotherapy (including CAR-T cell therapy) (see Hsp70: A Multifunctional Chaperone in Maintaining Proteostasis and Its Implications in Human Disease, Regulation of Heat Shock Protein Responses, 251-274). Further research has shown that, through stress induction, Hsp70 can be aberrantly translocated to the surface of tumor cell membranes (i.e., membrane-bound Hsp70, mHsp70), becoming a potential tumor-specific biomarker and therapeutic target. As a transmembrane protein, only a small C-terminal sequence of mHsp70 is located extracellularly. This C-terminal-derived TKD peptide (TKDNNLLGRFELSG) can be used to sensitize NK cells, enhancing their ability to recognize tumor cells and their cytotoxicity. Therefore, targeting and eliminating mHsp70-positive tumor cells has become a feasible and promising strategy for treating solid tumors or related diseases.

[0005] However, high-quality antibodies capable of specifically recognizing membrane-bound Hsp70 (mHsp70) are currently extremely limited. cmHsp70.1 is one of the few reported antibodies specifically targeting mHsp70, but its application in CAR-T cell construction is rarely reported, and systematic in vivo functional validation is lacking. In addition, patent US20180000914A1 discloses a chimeric antigen receptor targeting Hsp70, comprising an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signal transduction domain derived from an anti-Hsp70 monoclonal antibody. This receptor redirects engineered immune cells to Hsp70-positive cells to reduce graft-versus-host disease risk and improve therapeutic efficacy; however, this patented technology also lacks in vivo efficacy data supporting mHsp70-positive solid tumor models. Therefore, to fill the gaps in existing technologies, developing novel anti-mHsp70 antibodies with higher affinity and specificity, and corresponding chimeric antigen receptors, and systematically validating their functions through in vitro and in vivo experimental data, has significant clinical translational value. Summary of the Invention

[0006] The purpose of this invention is to provide an antibody targeting membrane-bound Hsp70, a chimeric antigen receptor, and their applications. Specifically, this invention successfully prepared a high-affinity antibody targeting the membrane-bound Hsp70 (mHsp70) molecule, and based on this high-affinity scFv, constructed CAR-T cells that specifically recognize mHsp70. Through in vitro and in vivo experiments, it was demonstrated that these CAR-T cells have a therapeutic effect on Hsp70 high-expression-mediated diseases (such as small cell lung cancer and other neoplastic diseases).

[0007] The present invention is achieved by the following technical solution: the antibody contains a complementarity-determining region, and the amino acid sequence of the complementarity-determining region is selected from any one of the amino acid sequences shown in SEQ ID NO.3 to SEQ ID NO.50.

[0008] Preferably, the complementarity determination region includes a heavy chain complementarity determination region and a light chain complementarity determination region. The heavy chain complementarity determination region includes: CDR1 of amino acid sequences as shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15 and / or SEQ ID NO.18; CDR2 of amino acid sequences as shown in SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.16 and / or SEQ ID NO.19; and CDR3 of amino acid sequences as shown in SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.14, SEQ ID NO.17 and / or SEQ ID NO.20. The light chain complementarity-determining region includes: CDR1 of amino acid sequences as shown in SEQ ID NO.21, SEQ ID NO.24, SEQ ID NO.27, SEQ ID NO.30, SEQ ID NO.33 and / or SEQ ID NO.36; CDR2 of amino acid sequences as shown in SEQ ID NO.22, SEQ ID NO.25, SEQ ID NO.28, SEQ ID NO.31, SEQ ID NO.34 and / or SEQ ID NO.37; and CDR3 of amino acid sequences as shown in SEQ ID NO.23, SEQ ID NO.26, SEQ ID NO.29, SEQ ID NO.32, SEQ ID NO.35 and / or SEQ ID NO.38.

[0009] Preferably, the antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence of the heavy chain antibody VH as shown in SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43 and / or SEQ ID NO.44; The light chain variable region comprises the amino acid sequence of the light chain antibody VL as shown in SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49 and / or SEQ ID NO.50.

[0010] Preferably, the heavy chain variable region is directly connected to the light chain variable region or connected via a linker peptide.

[0011] Preferably, the linker peptide is (G4S). n , where n is a positive integer.

[0012] Another technical solution of the present invention is to provide a chimeric antigen receptor that targets membrane-bound Hsp70, wherein the chimeric antigen receptor includes at least an antigen-binding domain, and the antigen-binding domain includes the antibody described above.

[0013] Preferably, the chimeric antigen receptor is composed of a signal peptide, an antigen-binding domain, a hinge region, a transmembrane region, a co-stimulatory signal transduction domain, and a signal transduction domain connected in series. The amino acid sequence of the signal peptide is shown in SEQ ID NO.51; The amino acid sequence of the hinge region is shown in SEQ ID NO.52; The amino acid sequence of the transmembrane region is shown in SEQ ID NO.53; The amino acid sequence of the co-stimulatory signal transduction domain is shown in SEQ ID NO.54; The amino acid sequence of the signal transduction domain is shown in SEQ ID NO.55.

[0014] Another technical solution of the present invention is to provide a nucleic acid molecule that encodes the above-mentioned antibody or the above-mentioned chimeric antigen receptor.

[0015] Another technical solution of the present invention is to provide a recombinant expression vector, wherein the recombinant expression vector comprises the above-mentioned nucleic acid molecules.

[0016] Another technical solution of the present invention is to provide a CAR-T cell, wherein the CAR-T cell contains the chimeric antigen receptor described above.

[0017] Another technical solution of the present invention is to provide a drug for preventing and / or treating diseases mediated by Hsp70 high expression, said drug comprising any one or more of the following: (a) The antibodies mentioned above; (b) The chimeric antigen receptors described above; (c) The aforementioned nucleic acid molecules; (d) The recombinant expression vectors described above; (e) The CAR-T cells mentioned above.

[0018] Preferably, the drug is used in combination with cisplatin to prevent and / or treat diseases mediated by Hsp70 overexpression.

[0019] Preferably, the diseases mediated by Hsp70 high expression include neoplastic diseases.

[0020] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0021] Another technical solution of the present invention is to provide a testing product, wherein the testing product comprises any one or more of the following: (a) The antibodies mentioned above; (b) The chimeric antigen receptors described above; (c) The aforementioned nucleic acid molecules; (d) The recombinant expression vectors described above; (e) The aforementioned CAR-T cells; (f) The aforementioned drugs.

[0022] The testing product is a test kit or a test device.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention targets membrane-bound Hsp70 (mHsp70) molecules. By immunizing mice with the extracellular fragment TKD peptide of the antigen and fusing them, hybridoma cell lines are obtained. After cloning the variable region of the antibody, a mouse antibody sequence with high affinity is obtained. High-affinity chimeric antigen receptor (CAR) and CAR-T cells targeting mHsp70 are successfully constructed, providing novel effector cells for the immunotherapy of solid tumors.

[0024] (2) In vitro experiments have shown that the CAR-T cells targeting mHsp70 constructed in this invention can efficiently recognize and eliminate tumor cells expressing mHsp70 on the membrane surface, including pancreatic cancer cells, small cell lung cancer cells and other solid tumor cell lines, showing broad-spectrum anti-tumor activity and good specificity.

[0025] (3) In a mouse model of pancreatic cancer, the mHsp70-targeting CAR-T cells constructed in this invention significantly inhibited tumor growth; in a small cell lung cancer model, the mHsp70-targeting CAR-T cells constructed in this invention, combined with the chemotherapeutic drug cisplatin, synergistically improved the survival of mice. This in vivo experiment demonstrates the in vivo effectiveness of this invention in the treatment of solid tumors and provides a new strategy and experimental basis for CAR-T immunotherapy combined with chemotherapy (such as cisplatin) to treat small cell lung cancer and even various mHsp70-positive solid tumors, which has important clinical translational value. Attached Figure Description

[0026] Figure 1 This diagram illustrates the strategy for obtaining supernatant containing numerous antibody sequences in immunized mice according to the present invention.

[0027] Figure 2 This invention is for screening mHsp70-specific antibodies.

[0028] Figure 3 This invention relates to the construction and screening of mHsp70 CAR-T cells.

[0029] Figure 4 The expression of mHsp70 in pancreatic cancer and small cell lung cancer cells.

[0030] Figure 5 This study aims to detect the killing effect of mHsp70 CAR-T on different tumor cell lines in vitro.

[0031] Figure 6 This invention relates to an in vivo experiment demonstrating the anti-pancreatic cancer effect of mHsp70 CAR-T cells in mice.

[0032] Figure 7 mHsp70 CAR T cells synergistically enhance the anti-small cell lung cancer effect with cisplatin in mice. Detailed Implementation

[0033] The invention's objective, technical solution, and beneficial effects will be further explained in detail below.

[0034] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the claimed invention. Unless otherwise stated, 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 invention pertains.

[0035] This invention aims to address the lack of high-quality antibodies that specifically recognize membrane-bound Hsp70 (mHsp70) in existing technologies, and the deficiency in systematic in vivo functional validation of existing cmHsp70.1 antibodies and anti-Hsp70 monoclonal antibodies in patent US20180000914A1 against mHsp70 in mHsp70-positive solid tumor models. To this end, this invention designs and obtains novel antibodies with high affinity and high specificity targeting the mHsp70 molecule, and successfully constructs chimeric antigen receptor (CAR) and CAR-T cells targeting mHsp70 based on these antibodies. In vitro experiments have demonstrated that these CAR-T cells can efficiently eliminate various mHsp70-positive solid tumor cells (including pancreatic cancer and small cell lung cancer), while having no significant killing effect on normal cells; in vivo pancreatic cancer tumor-bearing models show that they can significantly inhibit tumor growth; in small cell lung cancer tumor-bearing models, synergistic use with cisplatin can prolong the survival of mice without observing serious treatment-related side effects, thus possessing good clinical translational value.

[0036] The detailed technical solution of this invention can be summarized as follows: First, Balb / c mice were immunized with the extracellular C-terminal TKD peptide (TKDNNLLGRFELSG) of human mHsp70 as an antigen. After cell fusion, hybridoma screening and antibody variable region cloning, a series of mouse antibody sequences with high affinity were obtained. The amino acid sequence of the complementarity-determining region (CDR) was selected from any one of SEQ ID NO.3 to SEQ ID NO.50, specifically including multiple heavy chain / light chain CDR combinations and variable region sequences (SEQ ID NO.39 to SEQ ID NO.50).

[0037] Secondly, the aforementioned single-chain antibody, serving as the antigen-binding domain, is tandemly linked with a signal peptide, hinge region, transmembrane region, co-stimulatory signal transduction domain, and signal transduction domain to construct a chimeric antigen receptor (CAR) targeting mHsp70. Further, a nucleic acid molecule encoding the antibody or chimeric antigen receptor, and a recombinant expression vector containing the nucleic acid molecule, are provided. This vector is then integrated to obtain CAR-T cells expressing the chimeric antigen receptor.

[0038] Finally, a drug composition comprising any one or more of the above-mentioned antibodies, chimeric antigen receptors, nucleic acid molecules, recombinant expression vectors, or CAR-T cells (with the addition of pharmaceutically acceptable vectors) is provided for the prevention and / or treatment of Hsp70-mediated neoplastic diseases (such as small cell lung cancer, pancreatic cancer, etc.); at the same time, a detection product (such as a detection kit or detection device) containing the above-mentioned components is provided for the qualitative or quantitative detection of Hsp70 expression.

[0039] Further research has confirmed that the constructed mHsp70-targeting CAR-T cells can effectively eliminate various mHsp70+ tumor cells and effectively inhibit the growth of pancreatic cancer or small cell lung cancer in mice. Therefore, this invention is applicable to various tumor diseases mediated by high mHsp70 expression, including pancreatic cancer or small cell lung cancer.

[0040] In one alternative embodiment, the antibody may be a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment of these antibodies, thereby expanding its application scope, without specific limitation herein. Preferably, the antibody or its antigen-binding fragment includes a complete antibody, Fab, single-chain antibody (scFv), or nanobody. More preferably, the antibody or its antigen-binding fragment is a single-chain antibody (scFv).

[0041] In an optional embodiment, the amino acid sequence of the complementarity-determining region (CDR) may be selected from an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homologous or identical to the sequence shown in any one of SEQ ID NO. 3 to SEQ ID NO. 50.

[0042] In one optional implementation, the various heavy chain / light chain CDR combinations are as follows: (a) Heavy chain complementarity determination region: CDR1 of the amino acid sequence shown in SEQ ID NO.3, CDR2 of the amino acid sequence shown in SEQ ID NO.4, and CDR3 of the amino acid sequence shown in SEQ ID NO.5; Light chain complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO.21, CDR2 of the amino acid sequence shown in SEQ ID NO.22, and CDR3 of the amino acid sequence shown in SEQ ID NO.23; (b) Heavy chain complementarity determination regions: CDR1 of the amino acid sequence shown in SEQ ID NO.6, CDR2 of the amino acid sequence shown in SEQ ID NO.7, and CDR3 of the amino acid sequence shown in SEQ ID NO.8; Light chain complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO.24, CDR2 of the amino acid sequence shown in SEQ ID NO.25, and CDR3 of the amino acid sequence shown in SEQ ID NO.26.

[0043] (c) Heavy chain complementarity determination regions: CDR1 of the amino acid sequence shown in SEQ ID NO.9, CDR2 of the amino acid sequence shown in SEQ ID NO.10, and CDR3 of the amino acid sequence shown in SEQ ID NO.11; Light chain complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO.27, CDR2 of the amino acid sequence shown in SEQ ID NO.28, and CDR3 of the amino acid sequence shown in SEQ ID NO.29.

[0044] (d) Heavy chain complementarity determination regions: CDR1 of the amino acid sequence shown in SEQ ID NO.12, CDR2 of the amino acid sequence shown in SEQ ID NO.13, and CDR3 of the amino acid sequence shown in SEQ ID NO.14; Light chain complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO.30, CDR2 of the amino acid sequence shown in SEQ ID NO.31, and CDR3 of the amino acid sequence shown in SEQ ID NO.32.

[0045] (e) Heavy chain complementarity determination region: CDR1 of the amino acid sequence shown in SEQ ID NO.15, CDR2 of the amino acid sequence shown in SEQ ID NO.16, and CDR3 of the amino acid sequence shown in SEQ ID NO.17; Light chain complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO.33, CDR2 of the amino acid sequence shown in SEQ ID NO.34, and CDR3 of the amino acid sequence shown in SEQ ID NO.35.

[0046] (f) Heavy chain complementarity determination regions: CDR1 of the amino acid sequence shown in SEQ ID NO.18, CDR2 of the amino acid sequence shown in SEQ ID NO.19, and CDR3 of the amino acid sequence shown in SEQ ID NO.20; Light chain complementarity-determining regions: CDR1 of the amino acid sequence shown in SEQ ID NO.36, CDR2 of the amino acid sequence shown in SEQ ID NO.37, and CDR3 of the amino acid sequence shown in SEQ ID NO.38.

[0047] In an optional implementation, the heavy / light chain variable region sequence can be selected from: (a) The amino acid sequence of the heavy chain antibody VH as shown in SEQ ID NO. 39; (b) The amino acid sequence of the heavy chain antibody VH as shown in SEQ ID NO. 40; (c) The amino acid sequence of the heavy chain antibody VH as shown in SEQ ID NO. 41; (d) The amino acid sequence of the heavy chain antibody VH as shown in SEQ ID NO. 42; (e) The amino acid sequence of the heavy chain antibody VH as shown in SEQ ID NO. 43; (f) The amino acid sequence of the heavy chain antibody VH as shown in SEQ ID NO. 44; (g) The amino acid sequence of the light chain antibody VL as shown in SEQ ID NO.45; (h) The amino acid sequence of the light chain antibody VL as shown in SEQ ID NO.46; (i) The amino acid sequence of the light chain antibody VL as shown in SEQ ID NO.47; (j) The amino acid sequence of the light chain antibody VL as shown in SEQ ID NO.48; (k) The amino acid sequence of the light chain antibody VL as shown in SEQ ID NO.49; (l) The amino acid sequence of the light chain antibody VL as shown in SEQ ID NO.50.

[0048] In an optional implementation, the heavy chain variable region and the light chain variable region of the antibody can be directly linked or linked via a linker peptide. Preferably, the linker peptide is (G4S). n Where n is a positive integer, such as any positive integer from 1 to 6. More preferably, the linker peptide is a sequence as shown in SEQ ID NO.56, where n is 3, which is more conducive to the linking of heavy chain antibody VH and light chain antibody VL.

[0049] In an optional embodiment, the chimeric antigen receptor is composed of a light chain signal peptide (SP), an antigen-binding domain (SEQ ID NO.51), a CD8 hinge region (SEQ ID NO.52), a CD8 transmembrane region (SEQ ID NO.53), a 4-1BB co-stimulatory signal transduction domain (SEQ ID NO.54), and a CD3ζ signal transduction domain (SEQ ID NO.55) connected in series.

[0050] In one optional embodiment, the recombinant expression vector can be a viral vector, including retroviral vectors and lentiviral vectors. A lentiviral vector is preferred. More specifically, the recombinant expression vector is obtained by inserting a nucleic acid molecule encoding the aforementioned single-chain antibody or chimeric antigen receptor into a virus, thereby obtaining a recombinant viral expression vector expressing the aforementioned single-chain antibody or chimeric antigen receptor.

[0051] In one alternative implementation, CAR T cells can be obtained by infecting T cells with lentiviruses; wherein the lentivirus is obtained by transfecting lentivirus packaging cells with a recombinant lentivirus expression vector, followed by cell culture; the recombinant lentivirus expression vector is obtained by inserting the coding gene of the chimeric antigen receptor into a lentivirus expression vector. In fact, those skilled in the art can prepare the aforementioned CAR using existing known techniques. T cells are not specifically defined here.

[0052] In one alternative embodiment, the pharmaceutical composition is typically formulated in the form of oral, topical, suppository, or sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays, in accordance with conventional practices.

[0053] In an optional embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be a buffer, emulsifier, suspending agent, stabilizer, preservative, excipient, filler, coagulant and blending agent, surfactant, dispersant, or defoamer.

[0054] In an optional embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a microcapsule, liposome, nanoparticle, or polymer, or any combination thereof. The delivery carrier of the pharmaceutically acceptable carrier may be a liposome, a biocompatible polymer (including natural and synthetic polymers), a lipoprotein, a polypeptide, a polysaccharide, a lipopolysaccharide, an artificial viral envelope, inorganic (including metal) particles, and bacterial, bacteriophage, viscous, or plasmid carriers, thereby broadening its application scope.

[0055] In an optional embodiment, the pharmaceutical composition may also be used in combination with other drugs for the prevention and / or treatment of mHsp70 high expression-mediated diseases, and the other preventive and / or therapeutic compounds may be administered simultaneously with the main active ingredient or simultaneously in the same composition.

[0056] In an alternative embodiment, the pharmaceutical composition may be administered into the body in a known manner, such as via intravenous systemic delivery or local injection to the tissue of interest. Such administration may be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in this invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0057] As an example, the drug composition may be administered to humans or non-human mammals, including mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc., without specific limitations.

[0058] In one optional implementation, the detection product comprises at least one or more of the aforementioned antibodies, chimeric antigen receptors, nucleic acid molecules, recombinant expression vectors, CAR-T cells, or pharmaceutical compositions. As an example, the detection product can be used to qualitatively or quantitatively detect mHsp70 expression, and subsequently for basic research or practical clinical applications related to mHsp70-related physiological or pathological changes. Practical clinical applications include, but are not limited to, screening, (aiding) diagnosis, monitoring, or predicting the progression of diseases mediated by high mHsp70 expression, thereby broadening its application scope.

[0059] More specifically, the sequences involved in the embodiments of the present invention are shown in Table 1 below.

[0060] Table 1

[0061] [Terminology Definitions and Explanations] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0062] 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 invention pertains.

[0063] “Approximately”: When referring to a specific numerical value, the term “approximately” means that the value can vary by no more than 1% from the listed values. For example, “approximately 100” includes 99 and 101 and all values ​​in between.

[0064] "Treatment": refers to administering a therapeutic agent (including the antibodies, CAR-T cells, etc. of this invention) to a patient with symptoms of one or more diseases, said therapeutic agent having a therapeutic effect on these symptoms. It is usually administered in a therapeutically effective amount to effectively relieve one or more symptoms.

[0065] "Optional" or "optionally": This indicates that the event or situation described below may occur but is not required to occur. For example, "optionally includes linker peptides" means that linker peptides may or may not be present.

[0066] "Sequence identity": refers to the degree of identity between two nucleic acid or amino acid sequences under optimal alignment and comparison. In this invention, sequences with at least 85%, 90%, or 95% identity with the listed sequences are all included in the scope, preferably at least 95% (including any integer value between 85% and 100%).

[0067] "Antibody": Used in the broadest sense, it encompasses intact antibodies and their antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv), single-chain antibodies (scFv), VHH antibodies, bispecific antibodies, multispecific antibodies, fusion proteins, humanized antibodies, chimeric antibodies, glycosylated variants, and any immunoglobulin molecule containing a desired antigen recognition site. Intact antibodies typically consist of two heavy chains and two light chains; the heavy chains contain VH and CH1-CH3, and the light chains contain VL and CL (κ or λ). Antibody classes include IgA, IgD, IgE, IgG, IgM, and their subclasses (such as IgG1, IgG2, etc.).

[0068] "Antigen-binding fragment": refers to the molecular region that specifically binds to the antigen, including but not limited to Fab, Fab', F(ab')2, Fv, scFv, single-domain antibodies (VHH, VNAR, sdAb, nanobodies), etc.

[0069] "Variable region": refers to the variable region of the antibody light or heavy chain, which is composed of four frame regions (FR) and three complementarity-determining regions (CDR). The CDR determines the antigen binding specificity.

[0070] Monoclonal antibodies are antibodies derived from a basic homogeneous group of antibodies that are highly specific against a single antigen or epitope. They can be prepared using methods such as hybridoma assays, phage display, yeast display, recombinant DNA assays, or single-cell sequencing.

[0071] "Specific binding": refers to the binding equilibrium dissociation constant (KD) between the antibody and the antigen being at least 10. -6 M, preferred 10 -7 M to 10 -12 M.

[0072] "Conjugate" refers to a complex of an antibody or its antigen-binding fragment linked to a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, etc., via covalent bonds or non-covalent interactions (such as electrostatic forces). This linking can be achieved through a variety of connectors known in the art.

[0073] "Vector": refers to a tool used to carry exogenous nucleic acids into host cells. In this invention, vectors include plasmids, phage particles, and viral vectors (such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpesviruses). Lentiviral vectors are preferred due to their ability to transduce non-proliferating cells, achieve long-term stable integration, and have low immunogenicity. Expression vectors typically contain operable promoters (such as constitutive or inducible promoters like CMV, EF-1α, and SV40), enhancers, selection markers (such as antibiotic resistance genes), or reporter genes (such as luciferase, GFP, and mKate2). Methods for introducing nucleic acids into host cells include calcium phosphate precipitation, lipid transfection, electroporation, and viral transduction.

[0074] "Formulation": refers to a composition containing the chimeric antigen receptor, nucleic acid molecule, carrier, host cell, or engineered immune cell (such as CAR-T cell) of the present invention, and comprising a pharmaceutically acceptable carrier, diluent, or excipient. Liquid formulations are preferred, and injectable formulations are more preferred. The concentration of CAR-T cells is typically 1 × 10⁻⁶. 3 Up to 1×10 8 cells / mL, more preferably 1×10⁻⁶ cells / mL 4 Up to 1×10 7 Cells / mL. The formulation may contain buffer (such as neutral buffered saline), carbohydrates (such as glucose, sucrose, mannitol), proteins, amino acids, antioxidants, chelating agents (such as EDTA), adjuvants (such as aluminum hydroxide), and preservatives, preferably formulated for intravenous administration.

[0075] "Therapeutic Applications": This invention provides T cells transduced with a lentiviral vector encoding a chimeric antigen receptor (CAR-T cells) for therapeutic purposes. CAR-T cells can recognize mHsp70 on the surface of tumor cells, triggering a specific immune response and effectively killing tumor cells. Treatable cancers include hematologic malignancies (such as leukemia, lymphoma, and multiple myeloma) and solid tumors (such as pancreatic cancer, small cell lung cancer, breast cancer, colorectal cancer, and glioblastoma). CAR-T cells can be derived from the patient's own body (autologous) or a healthy donor (allogeneic), and are reinfused into the patient after in vitro genetic modification and expansion. Intravenous injection is preferred as the preferred method of administration, with a typical dose of 1 × 10⁻⁶. 6 Up to 1×10 10 One cell per treatment cycle. CAR-T cells can be used in combination with chemotherapy (such as cisplatin, cyclophosphamide, fludarabine), radiotherapy, immunosuppressants, or other immunotherapies. The frequency and dosage are determined by the physician based on the patient's age, weight, tumor burden, and individual differences in their condition.

[0076] The following examples illustrate specific embodiments of the present invention. However, the scope of protection of the present invention is not limited to these examples. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise specified, the reagents, consumables, etc., used in the following examples are commercially available.

[0077] Example 1: Antibody Development and Sequence Cloning (1) Mouse immunization Five Balb / c mice (Huaan Biotechnology) were immunized according to the following protocol: each mouse received three immunizations with human TKD peptide. A second immunization was administered 14 days after the first, with a 7-day interval between the second and third immunizations. On day 7 after the third immunization, a small serum sample was collected from the middle ear artery for testing. If the sample passed the test, a booster immunization was administered 7 days later. Whole blood samples were collected 7 days after the booster immunization (see [link to relevant documentation]). Figure 1 ).

[0078] (2) Serum titer detection Blood collection and testing: Seven days after immunization, mouse spleen B cells were hybridized with myeloma cells. Positive monoclonal antibody cell lines were obtained after screening. The antibody levels in the supernatant were detected by ELISA, and the optimal positive cell line was identified by immunofluorescence assay. Potency and other indicators were also measured (see [link to relevant documentation]). Figure 1 Serum titers were detected by ELISA.

[0079] Due to the large amount of data detected, only the mice corresponding to the effective acquisition of optimal cell lines and antibody results, along with their related data, are summarized and compiled. Other data are not included in this embodiment. Target mice with high titers meeting experimental requirements were selected for fusion after triple immunoassay serum testing.

[0080] In ELISA testing, when mouse serum is diluted 1:900, OD 450 The nm value should be greater than 2. When diluted at a ratio of 1:8100, the OD value should be... 450 The nm value should be greater than 1 to be considered as meeting the sorting criteria. The secondary antibody is goat anti-mouse IgG Fc secondary antibody (Invitrogen, 31437).

[0081] In this embodiment, one mouse (64#) was selected from five mice for fusion, and the blood test results are shown in Table 2.

[0082] Table 2

[0083] As shown in Table 1, all five mice, after three immunizations, produced titers when coated with TKD peptides. Mice #64 showed a qualified titer (the qualified standard is 1:8100, OD...). 450 Fusion can be performed if the nm value is >1.0 and the valence is >72900.

[0084] (3) Cell fusion and hybridoma preparation Cell fusion was performed using SP2 / 0 mouse myeloma cells and spleen B cells from selected mice (64#). After fusion, the cells were cultured, observed, tested, and subjected to positive and negative control experiments to obtain a batch of hybridoma cells that met the experimental requirements. These cells were then further cultured and selected.

[0085] (4) Antibody detection and strain identification After cloning and supernatant analysis using the fusion and limiting dilution method, 16 supernatant samples meeting the project requirements were obtained. The detection data are shown in Table 3. Further comparison and detection using Western blotting and cellular immunofluorescence were performed. Six hybridoma cell lines with high titers and superior affinity were amplified, cultured, and cryopreserved for later use.

[0086] From all evaluated candidate antibodies, HXHsp70.13 and HXHsp70.04, which exhibited high affinity for membranes, were purified and further affinity assessments were performed. Surface plasmon resonance (SPR) technology was used to evaluate their interaction with the target protein. Real-time SPR kinetic analysis showed that, compared with the negative control (mAb-G) and HXHsp70.13 (mAb-D), the HXHsp70.04 (mAb-F) antibody had a stronger binding affinity for recombinant human Hsp70 protein, exhibiting a faster binding rate and a slower dissociation rate. The detection data are shown in Table 4.

[0087] Among them, the immunoblotting detection of antibody recognition of Hsp70 in cell lysate in 16 supernatant samples is shown in [reference needed]. Figure 2 As shown in Figure A; representative images of antibody staining for mHsp70-positive tumor cells (AsPC-1, HCT-116) and mHsp70-negative tumor cells (PANC-1) in the six supernatant samples using immunofluorescence are shown in Figure A. Figure 2 As shown in B; the SPR kinetic analysis of the preferred antibody against recombinant human Hsp70 protein is shown in [reference]. Figure 2 As shown in C.

[0088] Table 3

[0089] Table 4

[0090] (5) Cloning of antibody scFv sequence RNA was extracted from 6 hybridoma cells and reverse transcribed into cDNA. The variable regions of the light and heavy chains were amplified by PCR using degenerate primers (see Table 5). After ligation with the pTomo vector to infect E. coli, positive clones were selected for sequencing. Plasmids were extracted from the bacterial culture of positive clones for bidirectional sequencing identification. The sequencing results confirmed the correct strains. After expansion culture, the plasmids were extracted and preserved.

[0091] PCR amplification: The specific operation was performed according to the instructions of the Thermo powerup™ SYBR Green Master Mix (A25742) kit. The program was as follows: 50℃, 2 min; 95℃, 2 min; 95℃, 15 s (40 cycles); 60℃, 1 min (40 cycles); 12℃, forever.

[0092] Table 5

[0093] Example 2: Construction and lethality testing of scFv CAR-T (1) CAR vector construction This embodiment employs a traditional second-generation CAR structure. The amino acid sequence of the anti-Hsp70 specific scFv is obtained from the heavy and light chain variable regions of the anti-Hsp70 mAb generated in Example 1. A second-generation CAR was constructed, consisting of the anti-Hsp70 scFv, a human CD8-derived hinge and transmembrane domain, and human 4-1BB co-stimulatory and CD3ζ signaling domains. The specific molecular sequence of the CAR is: SP-VL-(G4S)3-VH-CD8TM-41BB-CD3ζ. After the cloned vector was verified to be correct by enzyme digestion and sequencing, it was transformed into competent E. coli (Stbl3) and cultured. Then, it was extracted using an endotoxin-free extraction kit and identified by Hind III digestion.

[0094] (2) Acquisition of human T cells and preparation of CAR-T cells All blood samples were processed in accordance with prescribed ethical and safety procedures. hPBMCs from healthy donors were collected at West China Hospital of Sichuan University, following a protocol approved by the Human Research Ethics Committee (2022151). CD3 enrichment was performed using the EasySep Human T Cell Isolation Kit (STEMCELL Technologies, 17951) through negative selection. +T cells were activated with CD3 / CD28 activation beads, then cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U / mL), streptomycin (100 mg / mL), and recombinant human interleukin-2 (200 U / mL, PeproTech). Cells were cultured in a humidified incubator at 37°C and 5% CO2. To generate CAR-T cells, T cells were cultured for 72 h and then infected with lentiviral particles in the presence of LentiBOOST (Sirion Biotech). Ten hours later, the viral supernatant was replaced with fresh medium containing IL-2. T cells were further expanded by replacing half of the medium with fresh medium every two days. Transduction efficiency was determined by FCM, and CAR-T cells were adoptively transferred to mice or used in in vitro experiments. The lentiviruses used were packaged using standard maturation methods and their titers were measured.

[0095] To verify the cytotoxicity of the Hsp70 CAR-T cells in vitro, this embodiment generated luciferase-labeled cell lines by infecting them with pTomo-CMV-Luciferase-IRES-puro lentivirus (MOI≈10), and then screened them with puromycin (Life Technologies, 1 µg / ml) for 2 weeks to construct various tumor cell lines.

[0096] See Figure 3 , Figure 3 A shows the structure and components of the mHsp70 CAR. Figure 3 B was constructed from mHsp70 CAR-T cells, and the CAR-T cell positivity rate was detected by flow cytometry. Figure 3 C is the assay of mHsp70 CAR-T cells against AsPC-1 and HCT116 cytotoxicity using a luciferase activity assay at an effector-target ratio of 5:1.

[0097] The results show that this invention successfully constructed a second-generation CAR targeting the Hsp70 antigen (see [link to documentation]). Figure 3 (A) The CAR and mKate2 protein are expressed in parallel via the T2A sequence (see Table 6), and the CAR expression efficiency is shown by the mKate2 expression status. High-purity human T cells were infected with lentivirus to obtain CAR-T cells with a high positive rate (see Table 6). Figure 3 B). At the same effector-to-target ratio, CAR-T cells constructed based on HXHsp70.04, HXHsp70.13, and HXHsp70.29 scFv exhibited stronger killing effects, while the other clones showed relatively weaker effects. These three lines were subsequently selected as candidate CAR-T cells for further in-depth research (see [link]). Figure 3 C).

[0098] Table 6

[0099] Based on the above experiments and their results, the following conclusions can be drawn: Through molecular cloning experiments, a traditional second-generation CAR targeting the Hsp70 antigen was successfully constructed, and Hsp70 CAR-T cells were successfully constructed through expression detection. This lays the foundation for subsequent functional exploration of CAR-T cells and the smooth progress of in vitro and in vivo experiments.

[0100] Experimental Example 1: Detection of mHsp70 expression in pancreatic cancer and small cell lung cancer cells This experiment was used to evaluate the expression of mHsp70 in pancreatic cancer and small cell lung cancer cells.

[0101] Specific steps: 1) Place the cell culture medium and seed target cells into 24-well plates pre-coated with glass discs 24 hours in advance, at a density of 50,000-100,000 cells; 2) After the cells adhere and spread on the second / third day, wash twice with PBS and divide into control and experimental groups; 3) Add 4% paraformaldehyde to each well, incubate on a shaker at room temperature for 30 min, and then wash three times with PBS; 4) Add anti-Hsp70 Antibody [1H11] (SMC-249, StressMarq) antibody (diluted 1:200 with 2% FBS PBS) to the experimental group, and add 2% FBS PBS to the control group, and incubate on a shaker at room temperature for 1 h; 5) Centrifuge and discard the supernatant, and wash three times with 0.2% Tween 20 PBS; 6) Block each well with 10% goat serum in PBS, and incubate on a shaker at room temperature for 1 h; 7) Centrifuge and discard the supernatant, and wash three times with 0.2% Tween 20 PBS; 8) Add Cy3 to each well. Secondary antibody (diluted with PBS at 1:100), incubated on a shaker at room temperature for 1 h; 9) Centrifuge and discard the supernatant, add 0.2% Tween 20 PBS and wash three times; 10) Add DAPI (diluted with PBS at 1:1000) and stain for 10 min; 11) Mount the slide and examine under a confocal microscope.

[0102] See Figure 4 , Figure 4 Image A shows representative images of mHsp70 expression in pancreatic cancer cells (AsPC-1, BxPC-3, PANC-1), small cell lung cancer cells (NCI-H446, NCI-H1299, HCI-H82), and normal cells, as detected by immunofluorescence. Scale bar: 20 micrometers; Figure 4 B is a bar chart representing the immunofluorescence staining intensity of mHsp70, expressed as mean fluorescence intensity (MFI).

[0103] The results showed that pancreatic cancer cells (AsPC-1, BxPC-3, PANC-1) and small cell lung cancer cells (NCI-H446, NCI-H1299, HCI-H82) all expressed different levels of mHsp70, but mHsp70 protein expression was almost undetectable on the surface of two human normal cell lines, HEK-293T and EA.hy926 (see [link to study]). Figure 4 A and Figure 4 B).

[0104] Based on the above experiments and their results, the following conclusions can be drawn: mHsp70 is highly expressed in pancreatic cancer and small cell lung cancer cells, but is almost not expressed in normal human cells.

[0105] Experiment Example 2: In vitro experiment—the killing ability of mHsp70 CAR-T cells against tumor cells This experimental case was used to evaluate the killing activity of mHsp70 CAR-T cells against mHsp70 positive tumor cells and the release of cytokines in vitro.

[0106] Experimental method: Tumor cells carrying luciferase were digested, counted, and then the cell density was adjusted to 2 × 10⁻⁶. 4 / mL. 100 μL of tumor cells were seeded into 96-well plates, and the cell densities of mHsp70 CAR-T cells, CD19 CAR-T cells (target-negative control), and NTD cells were adjusted to 1×10⁶ cells / mL. 5 Target cells and T cells were seeded at an E:T ratio of 5:1 into black 96-well plates, with 100 μL seeded per well. The target cells and T cells were mixed and incubated for 24 hours. The cell supernatant was collected and frozen at -80°C to detect IFN-γ release. Cell killing was detected using a Promega fluorescence detection kit. Cells were first treated with 30 μL of 1×PLB lysis buffer for 20 minutes, and then 30 μL of substrate was added to each well before immediate detection using a BioTek microplate reader. After thawing the cell supernatant at -80°C, IFN-γ was detected using the IFN gamma Human ELISA Kit (Life Technology).

[0107] Cytotoxic cell killing percentage = (1 - Target cell fluorescence value with effector cells / Target cell fluorescence value without effector cells) × 100% See Figure 5 , Figure 5 A represents the CAR-T cell positivity rate detected by flow cytometry. Figure 5 B is the assay of mHsp70 CAR-T cells against pancreatic cancer and small cell lung cancer using luciferase activity assay at an effector-target ratio of 5:1. Figure 5C represents the detection of mHsp70 CAR by ELISA at an effector-to-target ratio of 5:1. The level of IFN-γ cytokines in the supernatant of T cells co-incubated with tumor cells; Figure 5 D is the assay of mHsp70 CAR-T cells against normal human cells using a luciferase activity assay at an effector-to-target ratio of 5:1.

[0108] The results showed that NTD and CD19 CAR-T cells had very low killing efficiency against target cells, while the mHsp70 CAR-T cell group had a relatively high target cell mortality rate (see [link to study]). Figure 5 C and Figure 5 D).

[0109] Based on the above experiments and their results, the following conclusions can be drawn: mHsp70 CAR-T cells exhibit strong killing ability against mHsp70+ cells in vitro, accompanied by the release of cytokines.

[0110] Experiment Example 3: In vivo experiment—mHsp70 CAR-T cells inhibit pancreatic cancer tumor growth This experimental case was used to evaluate the antitumor effect of mHsp70 CAR-T cells in an NCG mouse subcutaneous pancreatic cancer model.

[0111] Experimental methods: Severe immunodeficient mice were used to establish the model using NCG (purchased from Nanjing Jicui Pharmaceutical Co., Ltd.). After subcutaneous injection of human pancreatic cancer cells expressing mHsp70, mHsp70 CAR-T cells were reinfused.

[0112] See Figure 6 , Figure 6 A shows the mouse subcutaneous tumor modeling and mHsp70 CAR-T cell reinfusion strategy. Figure 6 B represents in vivo imaging monitoring of tumor burden in small animals at specific time points after CAR-T cell infusion; Figure 6 C represents the statistical change in mouse tumor cell volume; Figure 6 D and Figure 6 E represents the tumor images and volume statistics for each mouse at the ethical endpoint.

[0113] The results showed that mHsp70 CAR-T cells effectively inhibited tumor cell growth, and compared with mHsp70 CAR-T cells, the CD19 CAR-T cell group showed faster tumor growth in mice (see [link to study]). Figure 6 B and Figure 6 C).

[0114] Based on the above experiments and their results, the following conclusions can be drawn: mHsp70 CAR-T cells have strong anti-tumor function in vivo and significantly inhibit the growth of tumor cells, indicating that the mHsp70 scFv screened in this invention can effectively recognize and bind to the mHsp70 antigen in vivo, thereby killing tumor cells and inhibiting the growth of pancreatic cancer in mice.

[0115] Experiment Example 4: In vivo experiment—mHsp70 CAR-T cells inhibit small cell lung cancer and prolong survival This experiment was used to evaluate the antitumor effect and synergistic mechanism of mHsp70 CAR-T cells combined with the chemotherapy drug cisplatin in small cell lung cancer-bearing mice.

[0116] Experimental methods: Severe immunodeficient mice were used to establish a small cell lung cancer model using NCG. Each mouse was subcutaneously injected with human small cell lung cancer cells expressing mHsp70. When the tumor nodule was larger than 50 mm... 3 At day 0, mice were randomly divided into the following four groups carrying similar tumor burdens: CD19 CAR-T cell monotherapy group (control group): Mice were treated with an equal volume of physiological saline on day 0 (intraperitoneal injection once every 7 days), and simultaneously injected intravenously with 5×10⁻⁶ cells on day 5 of treatment. 6 One CD19 CAR-T cell.

[0117] CD19 CAR-T cell and cisplatin-treated group: Mice were treated with cisplatin on day 0 (3 mg / kg every 7 days, intraperitoneal injection), and simultaneously injected intravenously with 5 × 10⁻⁶ cisplatin on day 5 of treatment. 6 One CD19 CAR-T cell.

[0118] mHsp70 CAR-T cell monotherapy group: Mice were treated with an equal volume of physiological saline on day 0 (intraperitoneal injection every 7 days), and simultaneously injected intravenously with 5×10⁻⁶ mHsp70 CAR-T cells on day 5 of treatment. 6 70.04 HXHsp CAR-T cells.

[0119] mHsp70 CAR-T cell combined with cisplatin group: Mice were treated with cisplatin on day 0 (3 mg / kg every 7 days, intraperitoneal injection), and simultaneously injected intravenously with 5 × 10⁻⁶ cisplatin on day 5 of treatment. 6 70.04 HXHsp CAR-T cells.

[0120] See Figure 7 , Figure 7 A shows the mouse subcutaneous tumor modeling and mHsp70 CAR-T cell reinfusion strategy. Figure 7 B represents the statistical changes in mouse tumor cell volume, with tumor volume recorded every three days. Figure 7 C and Figure 7D shows images and volume statistics of tumors in each mouse on day 30 after treatment; Figure 7 E represents the survival curve of mice, indicating that the tumor volume in mice reached 2000 mmHg. 3 The mouse was declared dead.

[0121] The results showed that mHsp70 CAR-T cells effectively inhibited tumor cell growth. The tumor growth trend in the mHsp70 CAR-T cell group (HXHsp70.04 CAR-T) mice was similar to that of the CD19 CAR-T cell group and the cisplatin-treated group, but with a faster growth rate. Compared to the other three groups, tumor growth was significantly inhibited in the mHsp70 CAR-T cell combined with cisplatin group (Cispltin+HXHsp70.04 CAR-T) (see [link to relevant documentation]). Figure 7 B to Figure 7 D). Regarding mouse survival, the survival of mice treated with the combination of mHsp70 CAR-T cells and cisplatin (Cispltin + HXHsp70.04 CAR-T) was significantly prolonged, while the tumor growth trend of mice in the mHsp70 CAR-T cell group (HXHsp70.04 CAR-T) and the survival of mice treated with cisplatin alone was not significantly different. Figure 7 E).

[0122] Based on the above experiments and their results, the following conclusions can be drawn: mHsp70 CAR-T cells have strong anti-tumor function in vivo. Combined treatment with cisplatin significantly inhibited the growth of small cell lung cancer tumor cells and prolonged the survival of mice, indicating that the mHsp70 CAR-T cells we screened can synergistically enhance the anti-tumor effect of cisplatin in vivo.

[0123] In summary, this invention successfully developed a novel anti-mHsp70 single-chain antibody with high affinity and specificity, and constructed CAR-T cells targeting mHsp70 based on this antibody. These CAR-T cells efficiently and specifically eliminated mHsp70-positive pancreatic cancer and small cell lung cancer cells in vitro, significantly inhibited pancreatic cancer tumor growth in vivo, and synergistically inhibited small cell lung cancer growth and prolonged mouse survival when combined with the chemotherapeutic drug cisplatin, with promising preliminary safety. This invention not only fills the gap in the prior art regarding the lack of high-quality anti-mHsp70 antibodies and in vivo functional validation, but also provides new candidate drugs and combination therapy strategies for the immunotherapy of mHsp70-positive solid tumors (such as pancreatic cancer and small cell lung cancer), demonstrating significant clinical translational value.

[0124] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. An antibody targeting membrane-bound Hsp70, characterized in that: The antibody includes a complementarity-determining region, the amino acid sequence of which is selected from any one of the amino acid sequences shown in SEQ ID NO.3 to SEQ ID NO.

50.

2. The antibody of claim 1, wherein: The complementarity determination region includes a heavy chain complementarity determination region and a light chain complementarity determination region. The heavy chain complementarity-determining region includes: CDR1 of the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15 and / or SEQ ID NO.18; CDR2 of the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.16 and / or SEQ ID NO.19; and CDR3 of the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.14, SEQ ID NO.17 and / or SEQ ID NO.

20. The light chain complementarity-determining region includes: CDR1 of amino acid sequences as shown in SEQ ID NO.21, SEQ ID NO.24, SEQ ID NO.27, SEQ ID NO.30, SEQ ID NO.33 and / or SEQ ID NO.36; CDR2 of amino acid sequences as shown in SEQ ID NO.22, SEQ ID NO.25, SEQ ID NO.28, SEQ ID NO.31, SEQ ID NO.34 and / or SEQ ID NO.37; and CDR3 of amino acid sequences as shown in SEQ ID NO.23, SEQ ID NO.26, SEQ ID NO.29, SEQ ID NO.32, SEQ ID NO.35 and / or SEQ ID NO.

38.

3. The antibody according to claim 1, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence of the heavy chain antibody VH as shown in SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43 and / or SEQ ID NO.44; The light chain variable region comprises the amino acid sequence of the light chain antibody VL as shown in SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48, SEQ ID NO.49 and / or SEQ ID NO.

50.

4. The antibody according to claim 3, characterized in that: The heavy chain variable region is directly connected to the light chain variable region or connected through a linker peptide.

5. The antibody according to claim 4, characterized in that: the connecting peptide is (G4S) n n is a positive integer.

6. A chimeric antigen receptor targeting membrane-bound Hsp70, characterized in that: The chimeric antigen receptor includes at least an antigen-binding domain, wherein the antigen-binding domain comprises the antibody according to any one of claims 1 to 5.

7. The chimeric antigen receptor according to claim 6, characterized in that: The chimeric antigen receptor is composed of a signal peptide, an antigen-binding domain, a hinge region, a transmembrane region, a co-stimulatory signal transduction domain, and a signal transduction domain connected in series. The amino acid sequence of the signal peptide is shown in SEQ ID NO.51; The amino acid sequence of the hinge region is shown in SEQ ID NO.52; The amino acid sequence of the transmembrane region is shown in SEQ ID NO.53; The amino acid sequence of the co-stimulatory signal transduction domain is shown in SEQ ID NO.54; The amino acid sequence of the signal transduction domain is shown in SEQ ID NO.

55.

8. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody according to any one of claims 1 to 5, or encodes the chimeric antigen receptor according to claim 6.

9. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid molecule of claim 8.

10. A CAR-T cell, characterized in that: The CAR-T cells comprise the chimeric antigen receptor as described in claim 6.

11. A drug for preventing and / or treating diseases mediated by Hsp70 overexpression, characterized in that: The drug comprises any one or more of the following: (a) The antibody according to any one of claims 1 to 5; (b) The chimeric antigen receptor as described in claim 6; (c) The nucleic acid molecule of claim 8; (d) The recombinant expression vector of claim 9; (e) The CAR-T cells of claim 10.

12. The medicament according to claim 11, characterized in that: The drug is used in combination with cisplatin to prevent and / or treat diseases mediated by Hsp70 overexpression.

13. The medicament according to claim 11, characterized in that: The diseases mediated by high Hsp70 expression include neoplastic diseases.

14. The medicament according to claim 11, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

15. A testing product, characterized in that: The testing product includes any one or more of the following: (a) The antibody according to any one of claims 1 to 5; (b) The chimeric antigen receptor as described in claim 6; (c) The nucleic acid molecule of claim 8; (d) The recombinant expression vector of claim 9; (e) The CAR-T cells of claim 10; (f) The medicine according to claim 11.

16. The testing product according to claim 15, characterized in that, The testing product is a test kit or a test device.

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