CN117534767A - CLDN6 chimeric antigen receptor targeting macrophage and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of cellular immunotherapy, and particularly relates to a target CLDN6 chimeric antigen receptor macrophage, a preparation method and application thereof. The invention provides a chimeric antigen receptor of a specific target CLDN6, a gene and a recombinant expression vector thereof, an engineered immune response cell modified by the chimeric antigen receptor of the specific target CLDN6 and application thereof. The chimeric antigen receptor modified immune response cell of the specific target CLDN6 kills tumor cells with high specificity and has good safety, thereby providing a novel means for tumor treatment with application prospect.

Description

CLDN6 chimeric antigen receptor targeting macrophage and preparation method and application thereof

Technical Field

The invention belongs to the technical field of cellular immunotherapy, and particularly relates to a target CLDN6 chimeric antigen receptor macrophage, a preparation method and application thereof.

Background

The Claudins (CLDNs) protein family was cloned from chicken liver by furose Mikio et al and named as such in 1998 and is an important component of Tight Junctions (TJs). The tight junctions are mainly composed of occludin, CLDNs, tightly linked adhesion molecules. In mammals, 27 Claudins members have been found so far, and the Claudins family members will grow as the study proceeds. Numerous studies have demonstrated that the Claudins family is an essential scaffold protein for TJs, playing an important role in maintaining cell polarity, intercellular adhesion fixation, ion transport by cell bypass, etc. in epithelial and endothelial cells. Claudins members are widely distributed in different tissues and organs, and their expression has diverse properties. In recent years, a large amount of research data show that abnormal expression of Claudins is related to various tumors, and more Claudins family potential targets are emerging for clinical diagnosis and treatment of tumors. Plays an important role in cancer and is also closely related to other diseases.

CLDN6, one of the 27 CLDN family members, is an important molecule that makes up the intercellular tight junctions. CLDN6 is located on chromosome 16p3.3 and has a molecular weight of about 20-40kDa. The Claudins members have similar structures, with 4 transmembrane domains, 2 extracellular loops and 2 intracellular tail regions per Claudin molecule. CLDN6 has a PDZ binding site at the C-terminus, which allows the tight junction protein to interact with certain proteins in the cell, and thus plays an important role in cell attachment and epithelial tissue permeability. The study of the molecular structure of CLDN6 protein is a premise and a basis for further understanding of the properties and functions of CLDN6, and the study of the higher structure of CLDN6 is still in the primary stage. However, more and more researches are worth noting, which show that the abnormal expression of CLDN6 can be involved in the occurrence and development processes of various malignant tumors such as breast cancer, ovarian cancer, cervical cancer, gastric cancer, lung cancer and the like.

Because tumor cells are easy to invade and transfer, the structures among cells are disordered compared with the normal cell arrangement, so that the resistance of cell signaling factors or medicines is increased in the flowing process, the barriers among cells are difficult to break through to reach the inside of the tumor cells, and the antitumor medicines cannot play a role in high efficiency. Therefore, the method is used for exploring and opening the tight cell connection, is a transduction clearance barrier of anti-tumor drugs and normal signal paths, and becomes a new idea in tumor treatment. CLDN6, an important molecule for cytoskeletal formation and cell signaling pathway regulation, can affect the growth process of cells, which provides a new direction for tumor diagnosis and intervention. Currently, many drugs against CLDN6 are in clinical and preclinical stages for the treatment of a variety of tumors. CLDN6 and other family proteins play an important role in the formation of tight junctions between epithelial and endothelial cells. The CLDN family maintains a balance of intracellular environments through cellular barriers, cell bypass transport, and signal transduction. Abnormal expression of family proteins such as CLDN6 can cause impaired TJs function and reduced barrier function, thereby resulting in increased tissue permeability and finally in the occurrence of various diseases including hereditary and allergic diseases, infectious diseases of various systems and even tumors. Currently, studies on CLDN6 have focused mainly on tumors, especially ovarian, breast, cervical cancers.

The data shows that there are 10 more enterprises laying out CLDN6 development pipelines focusing on CLDN6 antibodies, ADCs, bispecific antibodies, CAR-T. In particular, the IMAB-027 antibody drug from the subflag of the japanese An Si taylor pharmaceutical (Astellas Pharma Global Development, inc.) directed against CLDN6, which is an inhibitor targeting CLDN6, has been in clinical stage two, and indications include germ cell and embryo tumors, testicular tumors, ovarian cancer, but no stage 2 clinical data for this project have been found. AMG794 bispecific antibody of incorporations, targeting CD3 x CLDN6, is in clinical stage 1, indicated for ovarian epithelial cancer, non-small cell lung cancer, and non-squamous non-small cell lung cancer. At 2022 AACR conference, the ann corporation disclosed that AMG794 had acceptable preclinical safety data for CLDN6 expressing non-small cell lung cancer and epithelial ovarian cancer cells, supporting entry of AMG794 into clinical development. There are three types of pipeline drugs under the flag of the company Bai Enteco, germany, in BNT211 (clinical stage 2), BNT-142 (clinical stage 1/2) and CARVac Claudin6 mRNA vaccine (clinical stage 1). In the AACR conference, germany byntake discloses clinical preliminary data of BNT211 CAR-t+camvac combined treatment of CLDN6 positive solid tumors, CLDN6 CAR-T treatment was well tolerated with disease control rates up to 86% and objective remission rates ORR of 43% in 16 treated patients. In addition, 4 bispecific antibody drugs were still in preclinical stages, from ContextTherapeutics LLC (CLDN 6 x CD3), novarock Biotherapeutics ltd (NBL-028;TNFRSF9 x CLDN6), astronomical organisms (TJ-C64B; TNFRSF9 x CLDN 6), integral Molecular, inc. Combination Context Therapeutics LLC (IM-171, CLDN6 x CD3), respectively. Preclinical data of NBL-028, where Novarock is an affiliated company with the stone drug group, showed that NBL-028 was effective in inhibiting tumor and inducing the formation of immune memory in the mouse model, and no signs of liver injury or systemic toxicity were detected.

With increasing research on the family of tight junctions proteins, the molecular mechanisms and drug studies of the CLDNs family associated with tumors are becoming a hotspot and focus. Following CLDN18.2, CLDN6 also serves as a key member of the compact junction protein, and abnormal expression of CLDN6 plays an important role in various malignant tumors such as ovarian cancer, in particular gynecological tumors. By utilizing the spatial structure and the regulation characteristics of the CLDN6, a target drug aiming at the CLDN6 is searched or designed to actively regulate and control the signal transduction of the CLDN6 and interfere the tumor progress, thus being a great difficulty in early diagnosis and accurate treatment of tumors such as lung adenocarcinoma and the like.

Disclosure of Invention

With the rapid development of biotechnology, immune cell therapy has become the fourth largest therapy in the field of cancer therapy. Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immunojunction blocking therapies. Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) macrophages refer to macrophages that express a chimeric receptor on their surface that recognizes a specific antigen and signals intracellular, the core being that the CAR molecule carries a single chain antibody (scFv) that specifically recognizes a cell surface antigen, thereby recognizing and killing cells expressing the specific antigen. At present, the CAR-M cell therapy is mainly applied to cancer treatment, shows a certain curative effect in partial solid tumors, and has few reports of applying the CAR-M to lung adenocarcinoma treatment. In conclusion, the development of an engineering immune response cell modified by a specific chimeric antigen receptor of a specific target CLDN6 has great application prospect. The engineering cell transmits an activation signal and activates an immune system through the CLDN6 generated on the surface of the high-specificity recognition tumor cell, and has a killing effect on the tumor cell and good safety, so that a good clinical treatment effect is achieved.

In view of the problems of the related art and/or other problems, the invention aims to overcome the problems of weak specificity and low safety of the effector cells tending to bind and kill tumor cells in the tumor microenvironment faced by the existing tumor clinical technology, and provides a chimeric antigen receptor specifically targeting CLDN6, a gene and recombinant expression vector thereof, an engineered immune response cell modified by the chimeric antigen receptor specifically targeting CLDN6 and application thereof. The chimeric antigen receptor modified immune response cell of the specific target CLDN6 kills tumor cells with high specificity and has good safety, thereby providing a novel means for tumor treatment with application prospect.

In order to achieve the aim, the invention discloses a target CLDN6 chimeric antigen receptor macrophage, a preparation method and application thereof, and adopts the following technical scheme.

The application provides a chimeric antigen receptor expressed on the surface of immune effector cells, which is characterized in that the chimeric antigen receptor comprises the following components connected in sequence: an extracellular binding region, a transmembrane region, and an intracellular signaling region, wherein the extracellular binding region comprises a protein that specifically recognizes CLDN 6.

More specifically, the immune effector cells are macrophages or peripheral blood mononuclear cells.

More specifically, the protein that specifically recognizes CLDN6 is an antibody; preferably, the antibody is a single chain antibody or a domain antibody.

More specifically, the amino acid sequence of the transmembrane region is a sequence comprising the transmembrane region and the hinge region of CD8 or CD 28.

More specifically, the intracellular signaling region is selected from the group consisting of: at least one of the intracellular signal region sequences of CD3 ζ, fceriγ, CD27, CD28, CD137, CD134, or a combination thereof.

Preferably, the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region and an intracellular signaling region, connected in that order: single chain antibodies, CD8 and CD3 ζ, specifically recognizing CLDN 6.

The chimeric antigen receptor has an amino acid sequence shown in SEQ ID NO. 5.

Nucleic acid encoding any of the chimeric antigen receptors.

The present application also provides an expression vector that expresses a chimeric antigen receptor that specifically recognizes CLDN6, comprising a nucleic acid encoding any of the chimeric antigen receptors.

The nucleic acid has a nucleotide sequence shown in SEQ ID NO. 10.

The present application also provides a virus comprising: an expression vector, wherein the expression vector comprises any of the nucleic acids, is an expression vector that expresses a protein chimeric antigen receptor that specifically recognizes CLDN 6.

More specifically, the expression vector is derived from a lentivirus, adenovirus or retrovirus. Preferably, the expression vector is derived from an adenovirus plasmid or a lentiviral plasmid.

Use of any of the chimeric antigen receptors described above, or the nucleic acid, or the expression vector, or the virus, for the preparation of a genetically modified immune effector cell targeting CLDN 6.

The application also provides a genetically modified specific targeted immune effector cell which expresses a chimeric antigen receptor of a protein specifically recognizing CLDN6, transduced with the nucleic acid, or the expression vector or the virus.

More specifically, the genetically modified specifically targeted immune effector cell expresses a chimeric antigen receptor having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, and a polypeptide sequence of the polypeptide.

More specifically, the application of the genetically modified immune effector cells is used for preparing medicines for inhibiting tumors, wherein the tumors are CLDN6 positive tumors.

The present application also provides a pharmaceutical composition, i.e. an immunocytodrug, characterized in that the pharmaceutical composition comprises: specifically targeting immune responsive cells and excipients.

The inventor continuously performs amino acid sequence design, sequence arrangement combination and screening, performs random screening test and targeting function verification (such as virus vector construction, macrophage further infection, macrophage cell modification, in-vitro killing activity detection and other tests) on sequences of hundreds of CAR molecules, then performs sequence adjustment according to the result comparison of a plurality of random combinations, and finally screens out 1 sequence with better effect, thereby obtaining 1 scFv amino acid sequence of the high-titer targeted CLDN6 and functional variants thereof.

The preparation method of the genetically modified immune effector cell comprises the following steps.

And step 1, connecting the nucleic acid molecules of the nucleic acid to an original expression slave vector in a molecular cloning mode to obtain the expression vector of the chimeric antigen receptor of the specific target CLDN 6.

More specifically, the original expression vector is obtained from a lentivirus, an adenovirus or a retrovirus.

And 2, transfecting 293T cells with the obtained chimeric antigen receptor expression vector of the specific target CLDN6 to obtain the virus liquid.

And 3, infecting immune response cells by using the virus liquid, and separating from the infected immune response cells to obtain the specific targeting immune response cells modified by the chimeric antigen receptor for expressing the specific targeting CLDN 6. Wherein the immunoresponsive cell is preferably a macrophage.

In some non-limiting embodiments, the modified immunoresponsive cell of the present invention may be a macrophage, peripheral blood mononuclear cell, or a cell of lymphoid lineage. The cells of lymphoid lineage are selected from B, T and Natural Killer (NK) cells, providing the functions of antibody production, regulation of cellular immune system, detection of foreign substances in blood, detection of host foreign cells, etc. Non-limiting examples of cells of the lymphoid lineage include T cells, natural Killer (NK) cells, cytotoxic T Lymphocytes (CTLs), regulatory T cells, embryonic stem cells, and pluripotent stem cells (e.g., pluripotent stem cells that can differentiate into lymphoid cells).

In some embodiments, the immune response cell is selected from the group consisting of a macrophage, a peripheral blood mononuclear cell or T cell, a Natural Killer (NK) cell, a Cytotoxic T Lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, and a pluripotent stem cell that can differentiate into a lymphoid cell, preferably a T cell or a Natural Killer (NK) cell.

In some exemplary embodiments, the T cells are lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the acquired immune system.

In some non-limiting embodiments, T cells include, but are not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells, stem-like memory T cells, and both types of effector memory T cells (e.g., TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated constant T cells, and γδ T cells, in some embodiments, CAR-expressing T cells express Foxp3 to achieve and maintain a T regulatory phenotype.

In some embodiments, the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, and combinations thereof. In one embodiment, the costimulatory ligand is 4-1BBL.

In a preferred embodiment, the isolated modified immune response cell is a macrophage.

In some non-limiting embodiments, the isolated modified immune response cells (e.g., macrophages or peripheral blood mononuclear cells or) may be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

A pharmaceutical composition comprising an effective amount of the isolated modified immune responsive cells and a pharmaceutically acceptable excipient.

The pharmaceutical compositions disclosed herein comprise isolated modified immune response cells expressing the chimeric antigen receptor specifically targeted to CLDN6 and a pharmaceutically acceptable carrier.

The administration of the pharmaceutical composition may be autologous or non-autologous. For example, immune response cells expressing the chimeric antigen receptor specifically targeted to CLDN6 and compositions comprising the same can be obtained from one subject and administered to the same subject or to different compatible subjects. The peripheral blood-derived T cells of the presently disclosed subject matter or their progeny (e.g., derived in vivo, ex vivo, or in vitro) may be administered by including catheter administration, intravenous injection, or parenteral administration. When administered, the immunocytodrugs of the presently disclosed subject matter (e.g., immunocytodrugs comprising said immunocytodrugs specifically targeting CLDN6 chimeric antigen receptor-responsive cells) are typically formulated in unit dose injectable forms (solutions, suspensions, emulsions).

The pharmaceutical compositions of the present application may be formulations. The disclosed CLDN 6-specific Chimeric Antigen Receptor (CAR) -expressing immune response cells and compositions comprising the same may be conveniently provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, the liquid composition is more convenient to administer, particularly by injection. On the other hand, the adhesive composition may be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition may comprise a carrier, which may be a solvent or dispersion medium comprising, for example, water, physiological saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

Various additives may be added to enhance the stability and sterility of the composition including, but not limited to, antimicrobial preservatives, antioxidants, chelating agents, and buffers.

According to the present application, any carrier, diluent or additive used must be compatible with the immunoresponsive cells expressing the CLDN6 specific Chimeric Antigen Receptor (CAR) of the presently disclosed subject matter.

If desired, the viscosity of the adhesive composition may be maintained at a selected level by the use of a pharmaceutically acceptable thickener. The choice of suitable delivery vehicles and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, such as a time-release form or a liquid-filled form).

A kit for treating or preventing a disease comprising said immunoresponsive cell or said nucleic acid.

The protein or functional variant thereof which targets and binds to the CLDN6, the chimeric antigen receptor which targets and targets the CLDN6 specifically, the recombinant vector or expression plasmid, the recombinant virus, the isolated modified immune response cell and the kit are applied to products for treating and preventing diseases, discomfort or health disorder.

In some embodiments, the disease treated or prevented comprises lung adenocarcinoma, gastric cancer, pancreatic cancer, liver cancer, brain cancer, prostate cancer, lymphoma, leukemia, bowel cancer, lung cancer, ovarian cancer, or breast cancer.

According to the invention, the chimeric antigen receptor of the specific target CLDN6 is used for modifying the immune response cell to obtain an engineering cell, and the engineering cell transmits an activation signal and activates an immune system by recognizing the CLDN6 generated on the surface of the tumor cell, so that the engineering cell can play a role in killing the tumor cell.

Compared with the prior art, the invention has the following beneficial effects.

The invention utilizes the chimeric antigen receptor modified macrophage technology to prepare the chimeric antigen receptor modified engineering immune cell of the specific target CLDN6, the preparation method has simple steps, the obtained novel engineering immune cell can specifically identify tumor cells, can more effectively target and attack the tumor cells, has high killing rate on tumors, and can be used for preparing anti-tumor products, in particular to preparing medicaments for treating CLDN6 positive tumors. The invention is expected to be used for preparing anti-tumor products, in particular to medicines for treating lung adenocarcinoma, gastric cancer, pancreatic cancer, liver cancer, brain cancer, prostate cancer, lymphoma, leukemia, intestinal cancer, lung cancer, ovarian cancer or breast cancer, and has good industrial application prospect.

Drawings

Fig. 1 is a schematic structural diagram of an anti-CLDN6 CAR.

FIG. 2 is a plasmid map of the CAR-CLDN6 adenovirus vector (pAd 5F 35-CAR-CLDN 6-mCherry-Puro).

Description of the embodiments

Unless defined otherwise, 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 belongs.

The term "functional variant" is a variant of a structure that has been modified from the parent structure to have the same or similar biological function and properties, such as the same targeted binding function as the parent. As a non-limiting example, a "functional variant" may be obtained by one or more conservative passages in the parent.

The term "amino acid modification" refers to a conservative amino acid modification that does not significantly affect or alter the binding characteristics of a CAR (e.g., extracellular recognition domain) of the present disclosure that comprises an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.

The term "homology" refers to a high proportion of matches of amino acids or nucleotides as revealed by comparison of a target amino or nucleotide sequence with a reference sequence. Homology herein can be determined using standard software such as BLAST or FASTA.

The term "scFv" refers to a single chain antibody (single chain antibody fragment variable, scFv) that is formed by the joining of the heavy and light chain variable regions of an antibody via a short peptide of 15 to 20 amino acids.

The term "Chimeric Antigen Receptor (CAR)" chimeric antigen receptor includes a leader peptide portion, an extracellular target recognition domain, a transmembrane domain, and an intracellular domain.

CARs can both bind antigen and transduce T cell activation functions, which are independent of MHC restriction. Thus, CARs are "universal" immune antigen receptors that can treat a population of patients with antigen positive tumors, regardless of their HLA genotype. Adoptive immunotherapy using T lymphocytes expressing tumor-specific CARs can be a powerful therapeutic strategy for treating cancer.

The term "recognize" refers to selective binding to a target. The term "specifically binds" or "specifically binds to" or "specifically targets" as used herein refers to a polypeptide or fragment thereof that recognizes and binds to a biological molecule of interest (e.g., a polypeptide), but which does not recognize other molecules in a binding sample, such as other molecules in a biological sample that naturally includes a polypeptide of the invention.

The term "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or ligand thereof that is required for an effective response of a lymphocyte to an antigen.

The term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with appropriate control elements and which can transfer gene sequences into cells. Thus, the term includes cloning and expression vectors, as well as viral vectors and plasmid vectors.

The term "expression vector" refers to a recombinant nucleic acid sequence, i.e., a recombinant DNA molecule, containing the desired coding sequence and the appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host organism. The nucleic acid sequences necessary for expression in prokaryotes typically include promoters, operators (optional) and ribosome binding sites, often accompanied by other sequences. Eukaryotic cells are known to utilize promoters, enhancers and terminators and polyadenylation signals.

The term "immunoresponsive cell" as used herein refers to a cell, or a progenitor cell, or progeny thereof, that plays a role in the immune response.

The term "isolated cell" refers to an immune cell that is separated from molecules and/or cellular components that naturally accompany the cell.

The term "modulating" as used herein refers to changing positively or negatively.

The term "exogenous" as used herein refers to a nucleic acid molecule or polypeptide that is not endogenously present in the cell or is present at a level insufficient to achieve the functional effect obtained upon overexpression. Thus, the term "exogenous" is intended to include any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as exogenous, heterologous, and overexpressed nucleic acid molecules and polypeptides.

The term "exogenous nucleic acid molecule or polypeptide" as used herein refers to a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or in a sample obtained from a cell. The nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.

The present invention will be further illustrated by the following examples, but the present invention is not limited to these specific embodiments. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

And constructing an anti-CLDN6 CAR virus vector.

In the embodiment, an anti-CLDN6 single-chain antibody is adopted as an antigen binding domain of a CAR molecule, is combined with a CD8 alpha hinge region, a CD8 alpha transmembrane region and CD3 zeta, an anti-CLDN6 CAR is designed, the amino acid sequence is shown as SEQ ID NO. 5, the nucleic acid sequence is shown as SEQ ID NO. 10, and the schematic diagram is shown as figure 1.

The nucleic acid sequence of the CD8 hinge region is shown in SEQ ID NO. 6.

The nucleic acid sequence of the anti-CLDN6 single-chain antibody is shown as SEQ ID NO. 7.

The nucleic acid sequence of the CD8 transmembrane region is shown in SEQ ID NO. 8.

The nucleic acid sequence of CD3 zeta is shown in SEQ ID NO. 9.

The anti-CLDN6 CAR coding gene is synthesized through the whole genes, the synthesized CAR molecule coding gene is cloned into an adenovirus vector through the steps of PCR, enzyme digestion, recombination and the like, and is imported into host bacteria and stored in glycerol. Taking 100 mu L of glycerinum in a 50mL centrifuge tube, adding 25mL of fresh LB culture medium, culturing for 16 hours in a shaking table at 37 ℃ and 220rpm, then extracting plasmids by using a plasmid small-extraction medium-quantity kit, taking a small quantity of plasmids for sequencing, checking whether the sequence of the plasmids is consistent with a constructed target map as shown in figure 2, and storing the rest plasmids at-20 ℃.

Packaging anti-CLDN6 CAR virus and detecting titer.

1. Packaging anti-CLDN6 CAR adenovirus and detecting titer.

anti-CLDN6 CAR-M cells were prepared.

1. Preparation of chimeric antigen receptor macrophages specifically targeted to CLDN 6.

2. Expression of the CAR protein was detected by flow cytometry analysis.

anti-CLDN6 CAR-M cell in vivo and in vitro anti-tumor effect evaluation experiment.

1. And (5) evaluating in vitro anti-tumor effect.

Construction of CLDN6 expression vector: the CLDN6 sequence is constructed on a pCDH-CMV-MCS-EF1-Puro framework vector, and the C end is added with two labels of V5 and HA.

(1) Phagocytosis of CLDN6 positive T cells.

2. And (5) evaluating in vivo anti-tumor effect.

According to another aspect of the present application, in order to provide a medicament for tumor treatment to a patient, the present application also discloses a pharmaceutical composition, more specifically an immunocytodrug, wherein the pharmaceutical composition comprises: specifically targeting immune responsive cells and excipients.

Still further, wherein the specifically targeted immune response cell is an immune response cell that specifically targets a chimeric antigen receptor of CLDN 6.

More specifically, wherein the excipient is a pharmaceutically acceptable delivery vehicle for mixing with the specific targeted immune responsive cells, the specific targeted cells disclosed herein are formulated into unit dose injectable forms (solutions, suspensions, emulsions).

Further, the pharmaceutical compositions of the present application may be formulated so as to provide the specific targeted cells as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, the liquid composition is more convenient to administer, in particular by injection into the human body. On the other hand, the adhesive composition may provide longer contact time with specific tissues. The liquid or viscous composition may comprise a carrier, which may be a solvent or dispersion medium comprising, for example, water, physiological saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

In another aspect, various additives that enhance the stability and sterility of the composition may be added to the pharmaceutical composition, including but not limited to antimicrobial preservatives, antioxidants, chelating agents, and buffers.

More specifically, any delivery vehicle, diluent or additive used must be compatible with the immunoresponsive cells expressing the CLDN 6-specific Chimeric Antigen Receptor (CAR) of the presently disclosed subject matter.

Alternatively, the viscosity of the adhesive composition may be maintained at a selected level by the use of a pharmaceutically acceptable thickener. The choice of suitable delivery vehicles and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, such as a time-release form or a liquid-filled form).

Further, the preparation method of the specific targeting immune response cell is as described above, and will not be described herein.

Based on the technical scheme, the application provides a pharmaceutical composition, more particularly an immune cell drug, which can more effectively target and attack tumor cells through the chimeric antigen receptor modified engineering immune cells of the specific target CLDN6, improves the killing rate of tumors and lays a foundation for preparing the drug for treating the CLDN6 positive tumors. In another aspect of the application, the invention is expected to be used for preparing various antitumor products, in particular medicines for treating lung adenocarcinoma, gastric cancer, pancreatic cancer, liver cancer, brain cancer, prostate cancer, lymphoma, leukemia, intestinal cancer, lung cancer, ovarian cancer or breast cancer, and has good industrial application prospect.

Claims (15)

1. A chimeric antigen receptor expressed on the surface of an immune effector cell, said chimeric antigen receptor comprising, in sequence: an extracellular binding region, a transmembrane region, and an intracellular signaling region, wherein the extracellular binding region comprises a protein that specifically recognizes CLDN 6.

2. The chimeric antigen receptor according to claim 1, wherein the immune effector cell is a macrophage or peripheral blood mononuclear cell.

3. The chimeric antigen receptor according to claim 1, wherein the protein that specifically recognizes CLDN6 is an antibody; the antibody is a single chain antibody or a structural domain antibody.

4. The chimeric antigen receptor according to claim 1, wherein the transmembrane region is a sequence comprising a transmembrane region and a hinge region of CD8 or CD 28.

5. The chimeric antigen receptor according to claim 1, wherein the intracellular signaling region is selected from the group consisting of: at least one of the intracellular signal region sequences of CD3 ζ, fceriγ, CD27, CD28, CD137, CD134, or a combination thereof.

6. The chimeric antigen receptor according to claim 1, wherein the extracellular binding region is a single chain antibody that specifically recognizes CLDN6, the transmembrane region is CD8, and the intracellular signaling region is cd3ζ.

7. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor has the amino acid sequence of SEQ ID No. 5.

8. An expression vector, wherein the expression vector is an expression vector that expresses a chimeric antigen receptor that specifically recognizes a CLDN6 protein, and wherein the expression vector comprises a nucleic acid encoding the chimeric antigen receptor of any one of claims 1-7.

9. The nucleic acid of claim 8, wherein said nucleic acid has the nucleotide sequence of SEQ ID NO. 10.

10. The expression vector of claim 8, wherein the expression vector is derived from an adenovirus plasmid or a lentiviral plasmid.

11. A virus comprising the expression vector of claims 8-10.

12. An immune effector cell, characterized in that it is a specifically targeted immune effector cell expressing a protein chimeric antigen receptor specifically recognizing CLDN6 transduced with the nucleic acid of claim 8 or 9, or the expression vector of claim 10 or 11 or the virus of claim 11.

13. The immune effector cell of claim 12, wherein the tumor is CLDN6 positive tumor, for use in the preparation of a medicament for inhibiting a tumor.

14. The use of an immune effector cell according to claim 12 or 13, wherein the tumor is lung adenocarcinoma.

15. A pharmaceutical composition comprising the specific targeted immune response cell of claim 12 and an excipient.