CN114805581B

CN114805581B - Antibodies targeting IL13RA2, chimeric antigen receptors and uses thereof

Info

Publication number: CN114805581B
Application number: CN202210743595.4A
Authority: CN
Inventors: 赵阳兵; 刘晓军; 訾振国; 朱庚振
Original assignee: Shanghai Youtijisheng Biomedical Co ltd
Current assignee: Shanghai Youtijisheng Biomedical Co ltd
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2022-10-14
Anticipated expiration: 2042-06-29
Also published as: CN114805581A

Abstract

The invention discloses an IL13RA2 targeted antibody, a chimeric antigen receptor and application thereof. The IL13RA 2-targeting antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3. The invention also discloses a preparation method of the antibody, a chimeric antigen receptor containing the antibody, nucleic acid for coding the chimeric antigen receptor, a recombinant expression vector, a transformant, an antibody drug conjugate, a pharmaceutical composition, a kit and a kit, and applications of the chimeric antigen receptor, the nucleic acid, the recombinant expression vector and the transformant. The anti-IL 13RA2 antibody provided by the invention only kills IL13RA2 high-expression cells, and has good specificity and safety.

Description

Antibodies targeting IL13RA2, chimeric antigen receptors and uses thereof

Technical Field

The present invention relates to the fields of molecular biology, cell biology and immunooncology. In particular, the invention provides anti-IL 13RA2 antibodies, chimeric Antigen Receptors (CARs) (IL 13RA2 CARs) comprising the anti-IL 13RA2 antibodies, genetically engineered immune effector cells expressing the IL13RA2 CARs, and their use in treating tumors or cancer.

Background

Malignant Gliomas (MGs), including glioblastoma multiforme and glioblastoma multiforme, present 20000 new cases in the united states each year. According to the statistics of the american brain tumor association, 140000 people in the united states had malignant brain tumors by 2010. Although MG is a rare disease, its malignancy and lethality rate are very high. The existing standard treatment means has very limited effect, and the five-year survival rate after surgical operation and radiotherapy is also very low. For patients who relapse after surgery, there are also very few new treatment options. Therefore, the development of new targets and new therapeutic means are urgently needed by patients.

Interleukin-13 Receptor a2 (Interleukin-13 Receptor subbunit alpha 2, IL13RA2) is a tumor specific marker that is specifically and highly expressed on the surface of malignant tumor cells such as human gliomas (Dehinski et al, (1995) clin. Cancer Res.1, 1253-1258). Human IL13RA2 has attracted attention from the FDA in the United states as a therapeutic target of human glioma as early as 1988, and the organization successively prepares drug IL-13-PE38 aiming at the therapeutic target of human IL13RA2 and single-chain antibody scFv-PE fusion molecule aiming at human IL13RA2. Although IL-13-PE38 has been shown to be effective in the treatment of malignant tumors such as glioma, head and neck tumor, ovarian cancer and renal cancer and approved by the FDA in the United states for clinical treatment, the IL-13-PE38 binds not only to human IL13RA2 specifically expressed on the surface of tumor cells but also to IL13RAl expressed on the surface of normal tissue cells during the treatment process, and thus damages normal tissues and cells. Further applications of IL-13-PE38 are limited due to the lack of strict targeting. Therefore, there is a need for further development of a safe therapeutic agent that specifically targets human IL13RA 2-highly expressing tumor cells.

Despite significant advances in understanding malignant gliomas, mortality has remained stable over the last decade and new innovative therapies are urgently needed. To date, chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) has become a promising cancer therapy, however, further and improved T-Cell Immunotherapy is still needed.

Disclosure of Invention

The technical problem to be solved by the invention is that the anti-IL 13RA2 antibody in the prior art has a non-specific killing problem on non-human IL13RA2 high-expression tumor cells, so that an anti-IL 13RA2 antibody with better specificity, chimeric Antigen Receptors (CARs) (IL 13RA2 CARs) containing the anti-IL 13RA2 antibody, genetically engineered immune effector cells expressing the IL13RA2 CARs and application of the antibodies in treating tumors or cancers are provided.

In order to solve the above technical problems, one of the technical solutions provided by the present invention is: an antibody or antigen-binding fragment thereof targeting IL13RA2, which comprises a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the amino acid sequence of HCDR1 is shown in SEQ ID NO. 14, the amino acid sequence of HCDR2 is shown in SEQ ID NO. 15, the amino acid sequence of HCDR3 is shown in SEQ ID NO. 16, the amino acid sequence of LCDR1 is shown in SEQ ID NO. 8, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 9, and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 10. The anti-IL 13RA2 antibody provided by the invention has killing effect on IL13RA2 high-expression cells, and has good specificity and safety.

The antibody or antigen-binding fragment thereof of any one of the above aspects, wherein the framework region of the heavy chain variable region is a human framework region; in a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 22; the framework region of the light chain variable region is a human framework region; in a preferred embodiment of the present invention, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO 21.

In the present invention, the amino acid sequences of the above-listed CDRs are all shown in accordance with the Kabat's rules of definition. However, it is well known to those skilled in the art that CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia (Chothia et al (1989) Nature 342: 877-883, al-Lazikani et al, "Standard constraints for the structural organization of immunology", journal of Molecular Biology, 273, 927-948 (1997)), kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. Demontent of Health and Human Services, national instruments of Health (1987)), abs (balance of balance), general connectivity, international Classification of Molecular Biology, and the use of the National standards for clustering of CDRs (correlation). It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are understood to encompass complementarity determining regions as defined by any of the above-described known schemes described by the present invention. Although the claimed scope of the present invention is shown based on the Kabat definition rules, the corresponding amino acid sequences according to the definition rules of other CDRs should also fall within the scope of the present invention, and the antibody CDR definition method is shown in Table 1.

TABLE 1 antibody CDR definition method

Wherein, laa-Lbb may refer to the amino acid sequence from aa to bb of the antibody light chain from the N-terminus; haa-Hbb may refer to the amino acid sequence from aa to bb, starting from the N-terminus of the heavy chain of the antibody. For example, L24-L34 can refer to the amino acid sequence from position 24 to 34 according to the Chothia coding rules, starting from the N-terminus of the antibody light chain; H26-H32 can refer to the amino acid sequence from position 26 to position 32, beginning at the N-terminus of the antibody heavy chain, according to the Chothia coding rules.

Thus, where reference is made to an antibody defined with a particular CDR sequence as defined herein, the scope of the antibody also encompasses an antibody whose variable region sequences comprise the particular CDR sequence but whose claimed CDR boundaries differ from the particular CDR boundaries as defined herein due to the application of different protocols (e.g., different assignment system rules or combinations).

The antibody or antigen binding fragment thereof according to one of the claims, wherein the antibody satisfies one or more of the following three conditions: (1) The antibody is full-length antibody, fab ', F (ab') ₂ Or an Fv, preferably an scFv, wherein when the antibody is an scFv, the amino acid sequence of the antibody is represented by SEQ ID NO: 23; (2) The antibody is a monospecific antibody, bispecific antibody or multispecific antibody; (3) The antibody is a monoclonal antibody or a polyclonal antibody prepared from the antibody.

The antibody or antigen binding fragment thereof according to any one of claims, wherein when the antibody is a full length antibody, it comprises a heavy chain constant region derived from a heavy chain of a human antibody or a variant thereof, and a light chain constant region derived from a kappa chain or a lambda chain of a human antibody or a variant thereof.

In the present invention, the term "full length antibody" is used interchangeably to refer to a glycoprotein comprising at least two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain is composed of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention). The light chain constant region consists of one domain CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as lambda or kappa. Immunoglobulins comprising alpha, delta, epsilon, gamma and mu heavy chains are classified as immunoglobulins (Ig) a, igD, igE, igG and IgM. The complete antibody formed a "Y" shape. The stem of Y consists of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have a constant region consisting of three tandem (in-line) Ig domains, and a hinge region for increased flexibility; heavy chains mu and epsilon have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain", respectively. Each arm of Y comprises the variable region and the first constant region of a single heavy chain joined to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

In the present invention, the "Fab fragment" consists of the CH1 and variable regions of one light chain and one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domains. A "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form F (ab ') ₂ A molecule. "F (ab') ₂ A fragment "contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus F (ab') ₂ The fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. The term "Fv" means an antibody fragment consisting of the VL and VH domains directed to a single arm of an antibody, but lacking a constant region.

In the present invention, the scFv (single chain antibody) can be a single chain antibody conventional in the art, and comprises a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids. Wherein the VL and VH domains pair to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain [ see, e.g., bird et al, science 242, 423-426 (1988) and Huston et al, proc. Natl. Acad. Sci. USA 85]. Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art joints are made of repeating G ₄ S amino acid sequence or a variant thereof. For example, a peptide having an amino acid sequence (G) ₄ S) ₄ Or (G) ₄ S) ₃ A linker, but variants thereof may also be used.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitopic specificity; an antibody having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; an antibody having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently, and the like.

The antibodies of the invention include monoclonal antibodies. The monoclonal antibody or mAb or Ab of the present invention refers to an antibody obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines.

In order to solve the above technical problems, the second technical solution provided by the present invention is: a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof targeting IL13RA2 according to one of the claims.

In a preferred embodiment of the invention, the chimeric antigen receptor further comprises a CD8 signal peptide, a hinge region, a transmembrane region, an intracellular signaling domain 4-1BB, and a CD3 zeta region.

In a more preferred embodiment of the present invention, the chimeric antigen receptor is, in order from N-terminus to C-terminus: a CD8 signal peptide, an antibody or antigen-binding fragment thereof targeting IL13RA2, a hinge region, a transmembrane region, an intracellular signaling region 4-1BB, and a CD3 zeta region; the transmembrane region is preferably a CD8 transmembrane region.

In a further more preferred embodiment of the present invention, the amino acid sequence of the CD8 signal peptide is shown as SEQ ID NO. 25, preferably the nucleotide sequence is shown as SEQ ID NO. 26; and/or the amino acid sequence of the hinge region is shown as SEQ ID NO. 27, and the preferable nucleotide sequence is shown as SEQ ID NO. 28; and/or the amino acid sequence of the membrane penetrating region is shown as SEQ ID NO. 29, and preferably the nucleotide sequence is shown as SEQ ID NO. 30; and/or the amino acid sequence of the intracellular signal domain 4-1BB is shown as SEQ ID NO. 31, and the preferable nucleotide sequence is shown as SEQ ID NO. 32; and/or the amino acid sequence of the CD3 zeta region is shown in SEQ ID NO. 33, and the preferable nucleotide sequence is shown in SEQ ID NO. 34.

In order to solve the technical problems, the third technical scheme provided by the invention is as follows: an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof targeting IL13RA2 according to claim one or a chimeric antigen receptor according to claim two.

In a preferred embodiment of the invention, the nucleotide sequence encoding the antibody or antigen-binding fragment thereof targeting IL13RA2 is shown in SEQ ID NO. 24.

As known in the art, "nucleic acid" in the context of the present invention refers to a chain of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into a strand by a DNA or RNA polymerase.

In order to solve the technical problems, the fourth technical scheme provided by the invention is as follows: a recombinant expression vector comprising an isolated nucleic acid as described in the third of the claims.

In a preferred embodiment of the invention, the recombinant expression vector is a plasmid, cosmid, phage, or viral vector, preferably a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

In a more preferred embodiment of the invention, the vector is a lentiviral vector.

As used herein, "vector" means a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

In order to solve the above technical problems, the fifth technical solution provided by the present invention is: a transformant comprising the recombinant expression vector according to the fourth aspect.

In a preferred embodiment of the present invention, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

In a more preferred embodiment of the invention, the eukaryotic cell is a yeast cell or a mammalian cell; wherein the mammalian cell is, for example, 293T cell or CHO cell.

Once an expression vector or DNA sequence has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are grown in culture and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and may be varied or optimized depending on the particular expression vector and mammalian host cell used based on the present specification and methods known in the art. Alternatively, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics), or heavy metal (e.g., copper) resistance, etc., to the auxotrophic host. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

In order to solve the technical problems, the sixth technical scheme provided by the invention is as follows: a genetically modified cell comprising a chimeric antigen receptor according to claim two.

In a preferred embodiment of the invention, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably immune cells, such as T cells.

The term "genetically modified cell" in the context of the present invention may include cells into which exogenous nucleic acid has been introduced, including progeny of such cells. It includes primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. The invention includes mutant progeny that have the same function or biological activity as the cell selected or selected for in the originally transformed cell.

In order to solve the above technical problems, the seventh technical solution provided by the present invention is: a method of making an antibody or antigen-binding fragment thereof targeting IL13RA2, comprising the steps of: culturing the transformant according to the fifth embodiment, and obtaining an antibody or an antigen-binding fragment thereof targeting IL13RA2 from the culture.

In order to solve the above technical problems, the eighth technical solution provided by the present invention is: an antibody drug conjugate comprising a cytotoxic agent or tag and an antibody or antigen binding fragment thereof targeting IL13RA2 according to one of the schemes.

In a preferred embodiment of the invention, the cytotoxic agent is MMAF or MMAE and the label is a fluorescent agent.

To solve the above technical problems, the ninth technical solution provided by the present invention is: a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof targeting IL13RA2 according to one of the schemes, a genetically modified cell according to the sixth of the schemes, and/or an antibody drug conjugate according to the eighth of the schemes, and a pharmaceutically acceptable carrier.

In a preferred embodiment of the invention, the pharmaceutical composition further comprises one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

In order to solve the above technical problems, the tenth technical solution provided by the present invention is: use of an antibody or antigen-binding fragment thereof targeting IL13RA2 according to claim one, a chimeric antigen receptor according to claim two, a genetically modified cell according to claim six, an antibody drug conjugate according to claim eight, and/or a pharmaceutical composition according to claim nine for the preparation of a medicament for the diagnosis, prevention and/or treatment of a tumor.

In a preferred embodiment of the invention, the tumor is an IL13RA2 positive tumor.

In a more preferred embodiment of the invention, the tumor is a glioma or a head and neck tumor.

In order to solve the above technical problems, the eleventh technical solution provided by the present invention is: a kit comprising an antibody or antigen-binding fragment thereof targeting IL13RA2 according to claim one, a chimeric antigen receptor according to claim two, a genetically modified cell according to claim six, or an antibody drug conjugate according to claim eight or a pharmaceutical composition according to claim nine.

In a preferred embodiment of the invention, the kit further comprises (i) a means for administering the antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.

To solve the above technical problems, the twelfth technical solution provided by the present invention is: a kit comprising kit a and kit B, wherein:

the kit a comprises an antibody or antigen-binding fragment thereof targeting IL13RA2 according to one of the claims, a chimeric antigen receptor according to the second of the claims, a genetically modified cell according to the sixth of the claims, an antibody drug conjugate according to the eighth of the claims and/or a pharmaceutical composition according to the ninth of the claims.

The kit B contains the other anti-tumor antibody or a pharmaceutical composition comprising the other anti-tumor antibody and/or one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a co-stimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

To solve the above technical problems, the thirteenth technical solution provided by the present invention is: a method of immunodetection or assay for IL13RA2 comprising using an antibody or antigen binding fragment thereof targeting IL13RA2 according to claim one, a chimeric antigen receptor according to claim two, an antibody drug conjugate according to claim eight or a pharmaceutical composition according to claim nine.

In a preferred embodiment of the invention, the assay is a non-diagnostic assay, suitable only for scientific purposes.

In order to solve the above technical problems, the fourteen technical solutions provided by the present invention are: a method of diagnosing, treating and/or preventing an IL13RA 2-mediated disease or disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof targeting IL13RA2 according to one of the claims, a chimeric antigen receptor according to the second of the claims, an antibody drug conjugate according to the eighth of the claims or a pharmaceutical composition according to the ninth of the claims, or treating a patient in need thereof using a kit according to the twelfth of the claims.

In a preferred embodiment of the invention, the disease or disorder is a tumor, which is an IL13RA2 positive tumor.

As used herein, the term "effective amount" means an amount of a drug or pharmaceutical agent that elicits the biological or pharmacological response in a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that causes improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a decrease in the rate of progression of a disease or condition, as compared to a corresponding subject not receiving that amount. The term also includes within its scope an amount effective to enhance normal physiological function.

To solve the above technical problems, the fifteenth technical solution provided by the present invention is: a combination therapy comprising separately administering to a patient in need thereof an antibody or antigen-binding fragment thereof targeting IL13RA2 according to one of the claims, a chimeric antigen receptor according to the second of the claims, an antibody drug conjugate according to the eighth of the claims or a pharmaceutical composition according to the ninth of the claims, and a second therapeutic agent; the second therapeutic agent preferably comprises an additional anti-tumor antibody or a pharmaceutical composition comprising the additional anti-tumor antibody, and/or one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a co-stimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

To solve the above technical problems, the sixteenth technical solution provided by the present invention is: the antibody or antigen-binding fragment thereof targeting IL13RA2 according to claim one, the chimeric antigen receptor according to claim two, the genetically modified cell according to claim six, the antibody drug conjugate according to claim eight, and/or the pharmaceutical composition according to claim nine, is used for the diagnosis, prevention and/or treatment of tumors.

In some embodiments, the pharmaceutical composition or pharmaceutical formulation of the invention comprises a suitable pharmaceutically acceptable carrier, e.g. a pharmaceutical excipient, such as a pharmaceutical carrier, pharmaceutical excipient, including buffer, as known in the art. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of excipients and their use, see also "Handbook of Pharmaceutical excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, pharmaceutical Press, london, chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin. A Pharmaceutical formulation or Pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention having the desired purity may be prepared by mixing the antibody or antigen-binding fragment thereof of the invention with one or more optional Pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Eds. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution. The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient as is required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators and the like. The active ingredients are suitably present in combination in an amount effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antigen-binding fragment thereof of the invention, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the anti-IL 13RA2 antibody, the Chimeric Antigen Receptors (CARs) (IL 13RA2 CARs) containing the anti-IL 13RA2 antibody and the genetic engineering immune effector cells expressing the IL13RA2 CARs provided by the invention only kill glioma cell lines with high IL13RA2 expression, and have no killing capacity on other cell lines, which indicates that the anti-IL 13RA2 antibody provided by the invention has obvious specific killing capacity and good safety.

Drawings

FIG. 1 is a schematic diagram of IL13RA2 CAR element.

FIG. 2 is a schematic diagram of the pDA vector.

FIG. 3 shows the binding ability of the selected scFv to IL13RA2 antigen.

FIG. 4 shows the expression of IL13RA2 in human glioma cell lines.

FIG. 5 is the killing ability of the selected scFv CAR-T cells on the U87 cell line.

Figure 6 is the killing ability of the screened scFv CAR-T cells against the U251 cell line.

FIG. 7 is the killing ability of the selected scFv CAR-T cells against the A549 cell line.

FIG. 8 shows IL13RA2 expression in various solid tumors.

FIG. 9 is the killing ability of the selected scFv CAR-T cells against IL13RA2 negative tumor cell lines.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1 preparation of human anti-IL 13RA2 antibody

The following steps were taken to prepare fully human anti-IL 13RA2 antibodies:

expression and purification of phage display libraries: log phase TG1 library cultures were infected with freshly thawed M13K07 helper phage with a multiple infection rate of 20:1 (phage to cell ratio) and induced overnight with IPTG; the phage library was purified by PEG/NaCl precipitation and then the phage titer was determined.

Selection of IL13RA 2-specific scFv-phages: in the first round of selection, the IL13RA2-6His protein was coated overnight at 4 ℃ with 20. Mu.g/ml on Maxisorp plates, washed with PBS, blocked with 5% milk +1% BSA in 1 XPBS, incubated in phage solution for 2 hours, and washed 10 times with PBST. The scFv phage bound to the antigen were eluted using acid elution buffer (ph 2.2), neutralized, inoculated in 15 ml log phase TG1 culture (OD 600= 0.5), incubated at 37 ℃ for 30 minutes and shaking for 30 minutes, inoculated onto 2xYT-GA agar plates, and incubated overnight at 30 ℃ for subsequent selection. In subsequent rounds of selection, more stringent selection conditions were used, including decreasing the coated protein concentration (2. Mu.g/ml in the second round and 0.5. Mu.g/ml in the third round) and increasing the wash cycle (20 in the second round and 30 in the third round).

And (4) mpELISA screening. After three rounds of selection, positive colonies were selected for monoclonal phage ELISA (mpaisa) screening. Phage supernatants from individual colony clones were incubated with pre-blocked Maxisorp plates coated with IL13RA2-6His protein at 2. Mu.g/mL. After three washes, 100 μ l/well of HRP-conjugated anti-His antibody diluted in blocking buffer (5% milk +1% bsa,1 × PBS) at a ratio of 1. After washing the plates 5 times with PBST, 100 μ l/well of TMB substrate solution was added and incubated for 10-30 min until blue color appeared. By adding 50. Mu.l/well of stop solution (2 NH) ₂ SO ₄ ) To terminate the reaction. Reading at 450nm in a microplate readerThe absorbance of (2). Among the positive colonies selected, the absorbance value of clone No. 5 was measured to be 1.61. Positive colonies with an absorbance of 450nm or more than or equal to 0.5 were identified as being capable of producing anti-IL 13RA2 antibodies that bind IL13RA2-6His protein.

Cloning and sequence analysis: positive clones were screened according to ELISA results and used as templates for PCR cloning of scFv sequences (forward primer sequence SEQ ID NO: 1:5 '-tgcagctggcacgacaggttttc-3', reverse primer sequence SEQ ID NO: 2. The PCR product was then sequenced by the sanger sequencing method (forward primer sequence SEQ ID NO: 3. The CDR regions of the scFv were analysed by the abysis website (http:// abysis. Org /), see tables 2 and 3, where H08 is the sequence of the scFv targeting IL13RA2 obtained from patent WO2019178078A 1.

TABLE 2 amino acid sequence of the scFv light chain CDR

TABLE 3 amino acid sequence of the CDR of the scFv heavy chain

The light and heavy chain amino acid sequences, as well as the full length amino acid and nucleotide sequences of the scFv are shown in table 4.

TABLE 4

Example 2 preparation of IL13RA2 CAR

Vectors for the production of mRNA targeting IL13RA2 CAR were constructed. First, the scFv sequence was amplified by PCR and connected to the CD8 signal peptide (amino acid sequence SEQ ID NO: 25, nucleotide sequence SEQ ID NO: 26), hinge region (amino acid sequence SEQ ID NO: 27, nucleotide sequence SEQ ID NO: 28) and transmembrane sequence (amino acid sequence SEQ ID NO: 29, nucleotide sequence SEQ ID NO: 30), intracellular signaling domain 4-1BB (amino acid sequence SEQ ID NO: 31, nucleotide sequence SEQ ID NO: 32) and CD3 zeta region (amino acid sequence SEQ ID NO: 33, nucleotide sequence SEQ ID NO: 34) in tandem to give a CAR element, as shown schematically in FIG. 1, which was then cloned into a pDA vector that can be transcribed in vitro (FIG. 2) for transcription of the mRNA. A targeted IL13RA2 CAR mRNA was prepared by In Vitro Transcription (IVT). The pDA-CAR plasmid was linearized by Spe1 cleavage and purified by PCR clean kit. After measuring the DNA concentration with nanodrop and checking by running agarose DNA gel, IVT was performed according to the manufacturer's instructions (Thermofisiher, cat No: AM 13455). The concentration of the RNA product was determined by nanodrop and checked by running a PAGE gel.

Example 3 preparation and characterization of transiently expressing IL13RA2 CARTs

Introduction of IL13RA2 CAR mRNA into T cells using electroporation transfection: t cells were harvested, washed twice with Opti-MEM medium and washed 1X 10 with Opti-MEM medium ⁷ Resuspending in/ml; uniformly mixing 10 mug of RNA and 100 mug of T cells, uniformly mixing, and performing electroporation, wherein the parameters are as follows (BTX machine): 500v,0.7ms; the cells were then transferred to pre-warmed medium and cultured at 37 ℃.

Binding of IL13RA2 CART cells to IL13RA2-Fc recombinant protein was measured by FACS staining. As shown in FIG. 3, T cells expressing a CAR with anti-IL 13RA2 scFv-5 were able to bind IL13RA2-Fc recombinant protein. NTD is a negative control T cell without a CAR molecule. H08 is a positive control T cell targeting IL13RA2.

And (3) detecting the expression level of IL13RA2 in the human glioma cell strains U87 and U251. Expression of IL13RA2 in tumor cells was measured by FACS staining of U87 and U251 cells with isotype or anti-IL 13RA2 antibody. As shown in fig. 4, both U87 and U251 highly expressed IL13RA2. While the human non-small cell lung cancer cell line a549 did not express IL13RA2.

The killing ability of IL13RA2 CART cells on tumor cells was measured in an in vitro cytotoxicity assay. Inoculating the EGFP-expressing U87, U251 and A549 tumor cells on a flat-bottom 96-well plate at 10000 cells/100 ul/well; CART cells were diluted to appropriate concentrations and seeded at an E/T ratio of 1 in 100 μ l/well tumor cells, then co-culture plates were placed in the IncuCyte S3 machine and scan parameters were set. After scanning for 3 days, the total green object integral intensity (GCU x mum/hole) is analyzed to calculate the killing efficiency.

The killing ability of mRNA-based anti-IL 13RA2 CART cells at an E/T ratio of 1 to U87-GFP tumor cells (fig. 5, where group #5 differs significantly from group H08, p < 0.05), U251-GFP tumor cells (fig. 6), and a549-GFP cells (fig. 7). As shown, CART cells expressing anti-IL 13RA2 scFv-C5 significantly prevented the growth of U87 and U251 cells, while having a lesser effect on the growth of a549, relative to the untransduced CAR-T cell (NTD) group. This indicates that the scFv-based CART cells have a strong killing ability against glioma cell lines with high IL13RA2 expression.

Example 4 CART cell safety assessment

And (3) detecting the expression level of the IL13RA2 in various human-derived different tissue tumor cell lines. Expression of IL13RA2 in tumor cells was detected by FACS staining of different tumor cell lines with isotype or anti-IL 13RA2 antibodies. As shown in FIG. 8, the renal cancer cell line 786-O, the ovarian cancer cell lines OVCAR3 and SKOV3, the pancreatic cancer cell line ASPC-1, and the lung cancer cell line H226 all expressed IL13RA2 at a very low level.

T cells of CAR against IL13RA2 scFv-5 were obtained by the following procedure using electroporation transfection of mRNA, and CAR-T cells targeting IL13RA 2H 08 scFv were obtained as positive controls using the same electroporation method: t cells were collected, washed with Opti-MEM medium, and washed with Opti-MEM medium at 1X 10 ⁷ Resuspending in/ml; 10 mug of RNA and 100 mug of T cells were equally divided, mixed evenly and electroporated, with the following parameters (BTX machine): 500v,0.7ms; the cells were then transferred to pre-warmed medium and cultured at 37 ℃.

The killing ability of CART cells targeting IL13RA2 against tumor cells was measured in an in vitro cytotoxicity assay. Various tumor cell lines expressing EGFP were seeded at 5000 cells/100 ul/well in flat-bottom 96-well plates; 100 μ l of the CART cells resuspended in R10 medium were seeded into tumor cell well plates at an E/T ratio of 3. After scanning for 3 days, the total green object integral intensity (GCU x μm/hole) is analyzed to calculate the killing efficiency.

The killing ability of mRNA-based anti-IL 13RA2 CART cells on different tissue-derived tumor cells at an E/T ratio of 3. As shown in fig. 9, relative to the H08 group, CART cells expressing anti-IL 13RA2 scFv-5 killed the IL13RA 2-highly expressed cell lines U87 and U251, while the H08 cell line killed or inhibited growth significantly and non-specifically on the ovarian cancer cell line SKOV3 and the lung cancer cell line H226 (fig. 9B, F); and #5 only kills glioma cell lines with high IL13RA2 expression and has no killing capacity to other cell lines (figure 9A-H), which shows that #5 has obvious specific killing capacity and good safety.

Example 5 preparation and characterization of stably expressing IL13RA2 CARTs

Lentivirus plasmid preparation: to verify the ability of targeting IL13RA2 CAR to eliminate tumors for long periods of time, #5 or H08 IL13RA2 CAR was cloned into a lentiviral vector (e.g., pLV or pTRPE), after sequencing was successful, the resulting plasmids were transformed into X-Blue strain, plated for selection, kanamycin positive monoclonals were picked up overnight, inoculated into 200 ml LB medium, shaken overnight and plasmids were extracted with QIAGEN plasmid macroextraction kit to yield lentiviral plasmids expressing #5 or H08 IL13RA2 CAR.

And (3) slow virus packaging: the lentivirus plasmid is taken as a main plasmid, the lentivirus plasmid and a virus packaging plasmid (pRRE, rev, pMD.2G) are respectively mixed in an opti-MEM culture medium, PEIpro is added according to a mass ratio of 1 to be mixed for 20 minutes, the mixed solution is added into 293T cells in a logarithmic growth phase, supernatants are collected after 24 hours and 48 hours, the lentivirus is obtained after ultracentrifugation, and the virus titer detection is carried out in an activated T cell line by a double dilution method.

Preparation and detection of stable transgenic CAR-T cells: the frozen T cells were thawed and resuspended in R10 (plus 100U/ml IL 2), anti-CD 3/CD28 magnetic beads added to the T cells, magnetic beads: t cell = 1, virus was added to the challenge after overnight activationLive T cells, virus: t cell = 3. Removing the magnetic beads with a magnetic frame after five days, continuously culturing the obtained CAR-T cells, adjusting the density to 5e5/ml, changing the liquid every other day, counting and recording the cell size until the cell size is reduced to 350 μm ³ Functional experiments were performed on the left and right (after about 13 days). CART cell expression was measured by FACS staining. The expression rate of H08 expression group (group H) CARs relative to untransfected T cells (group NTD) was about 63.5%; the expression rate of #5 expression group (# 5) CAR was approximately 62.3%.

SEQUENCE LISTING

<110> Shanghai eco-bio-pharmaceutical Co., ltd

<120> IL13RA 2-targeting antibody, chimeric antigen receptor and use thereof

<130> P22012032C

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Forward primer

<400> 1

tgcagctggc acgacaggtt tc 22

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer

<400> 2

cgtcagactg tagcacgtt 19

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Forward primer

<400> 3

aacaattgaa ttcaggagga 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> reverse primer

<400> 4

cctcctaaga agcgtagtc 19

<210> 5

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> H08 LCDR1

<400> 5

Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala

1 5 10

<210> 6

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> H08 LCDR2

<400> 6

Ser Ala Ser Tyr Arg Ser Thr

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> H08 LCDR3

<400> 7

Gln His His Tyr Ser Ala Pro Trp Thr

1 5

<210> 8

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> #5 LCDR1

<400> 8

Arg Ala Ser Gln Asp Ile Arg Ser Tyr Leu Ala

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> #5 LCDR2

<400> 9

Ala Ala Ser Thr Leu Gln Ser

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> #5 LCDR3

<400> 10

Gln Gln Leu Asn Ser Phe Pro Ala Thr

1 5

<210> 11

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> H08 HCDR1

<400> 11

Arg Asn Gly Met Ser

1 5

<210> 12

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> H08 HCDR2

<400> 12

Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 13

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> H08 HCDR3

<400> 13

Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val

1 5 10

<210> 14

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> #5 HCDR1

<400> 14

Ser Tyr Ser Met Asn

1 5

<210> 15

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> #5 HCDR2

<400> 15

Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 16

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> #5 HCDR3

<400> 16

Ala Gly Gly Ser Leu Gly Ala Phe Asp Tyr

1 5 10

<210> 17

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> H08 light chain (VL) amino acid sequence

<400> 17

Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

1 5 10 15

Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly

20 25 30

Thr Ala Val Ala Trp Tyr Gln Gln Ile Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Ser Ala Ser Tyr Arg Ser Thr Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Phe Ile Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ser Ala

85 90 95

Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 18

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> H08 heavy chain (VH) amino acid sequence

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Val Ser Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Gly Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Gly

115 120

<210> 19

<211> 248

<212> PRT

<213> Artificial Sequence

<220>

<223> full-Length amino acid sequence of H08 scFv

<400> 19

Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

1 5 10 15

Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly

20 25 30

Thr Ala Val Ala Trp Tyr Gln Gln Ile Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Ser Ala Ser Tyr Arg Ser Thr Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Phe Ile Ile Ser Ser Leu

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Ser Ala

85 90 95

Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

115 120 125

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

130 135 140

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Asn Gly Met Ser Trp

145 150 155 160

Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Ala Thr Val Ser

165 170 175

Ser Gly Gly Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Ser

195 200 205

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Gly

210 215 220

Thr Thr Ala Leu Ala Thr Arg Phe Phe Asp Val Trp Gly Gln Gly Thr

225 230 235 240

Leu Val Thr Val Ser Ser Ser Gly

245

<210> 20

<211> 744

<212> DNA

<213> Artificial Sequence

<220>

<223> full-length nucleotide sequence of H08 scFv

<400> 20

ggatccgaca tccaaatgac tcagagcccc tctagcctca gtgcaagcgt cggagaccgg 60

gtgaccatca cctgtaaagc gtcccaggat gttggaacgg cagtagcttg gtatcaacaa 120

atcccaggga aggctccaaa gctccttata tactctgcta gttacaggtc caccggggtg 180

cccgaccgat tctctggctc cgggagcggc actgactttt cattcatcat tagtagtctt 240

caacctgagg actttgccac ctattattgc cagcaccact actctgcgcc gtggactttc 300

ggaggaggca cgaaggttga aattaaaggt ggaggtgggt ctggcggagg tggaagtggt 360

ggaggcgggt ccgaggttca gttggtagag tcaggcggtg gtctggtgca gccaggtggg 420

tccctgcgcc tcagctgtgc agcttccggc tttactttct caaggaatgg tatgtcctgg 480

gtacggcaaa cgccggacaa acgccttgag tgggtagcta ccgtatcctc tgggggctct 540

tacatatact atgcagactc tgtgaaagga agatttacaa tttcacgcga caatgcaaaa 600

aatagtttgt acctccaaat gtctagtctt agggccgagg atactgccgt ctactactgt 660

gcacgccagg gaacgacggc tcttgctacc cgatttttcg acgtttgggg ccaaggaacg 720

ttggtgacag ttagcagttc cgga 744

<210> 21

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> #5 light chain (VL) amino acid sequence

<400> 21

Asp Ile Leu Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Phe Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 22

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> #5 heavy chain (VH) amino acid sequence

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Gly Gly Ser Leu Gly Ala Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 23

<211> 241

<212> PRT

<213> Artificial Sequence

<220>

<223> #5 scFv full length amino acid sequence

<400> 23

Asp Ile Leu Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Phe Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu

115 120 125

Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn Trp Val Arg

145 150 155 160

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Ser Ser

165 170 175

Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

180 185 190

Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu

195 200 205

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Gly Gly Ser

210 215 220

Leu Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ser

<210> 24

<211> 723

<212> DNA

<213> Artificial Sequence

<220>

<223> #5 scFv full length nucleotide sequence

<400> 24

gacatcctgt tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca ggacattaga agttatttag cctggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag cttaatagtt tccctgccac cttcggccaa 300

gggacacgac tggagattaa aggtggtggt ggttctggcg gcggcggctc cggaggtggt 360

ggatccgaag tgcagctggt ggagtcgggg ggaggcctgg tcaagcctgg ggggtccctg 420

agactctcct gtgcagcctc tggattcacc ttcagtagct atagcatgaa ctgggtccgc 480

caggctccag ggaaggggct ggagtgggtc tcatccatta gtagtagtag tagttacata 540

tactacgcag actcagtgaa gggccgattc accatctcca gagacaacgc caagaactca 600

ctgtatctgc aaatgaacag cctgagagcc gaggacacgg ctgtgtatta ctgtgcgaga 660

gcggggggat cgctcggggc ctttgactac tggggccagg gaaccctggt cactgtctcc 720

tca 723

<210> 25

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8 signal peptide amino acid sequence

<400> 25

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 26

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<223> CD8 signal peptide nucleotide sequence

<400> 26

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 27

<211> 45

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8 Hinge amino acid sequence

<400> 27

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 28

<211> 135

<212> DNA

<213> Artificial Sequence

<220>

<223> CD8 Hinge nucleotide sequence

<400> 28

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 29

<211> 24

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8 transmembrane domain amino acid sequence

<400> 29

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 30

<211> 72

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of CD8 transmembrane domain

<400> 30

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 31

<211> 42

<212> PRT

<213> Artificial Sequence

<220>

<223> 4-1BB diagnostic domain amino acid sequence

<400> 31

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 32

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<223> 4-1BB nesting domain nucleotide sequence

<400> 32

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 33

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3-zeta amino acid sequence

<400> 33

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 34

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> CD3-zeta nucleotide sequence

<400> 34

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgacg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

Claims

1. An antibody or antigen-binding fragment thereof targeting IL13RA2, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3,

the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 14, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 15, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 16, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 8, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 9, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 10.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the framework regions of the heavy chain variable region are human framework regions;

the framework region of the light chain variable region is a human framework region.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 22 and the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 21.

4. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody satisfies one or more of the following three:

(1) The antibody is full-length antibody, fab ', F (ab') ₂ Or Fv;

(2) The antibody is a monospecific antibody;

(3) The antibody is a monoclonal antibody.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the Fv is an scFv having the amino acid sequence set forth in SEQ ID NO. 23.

6. The antibody or antigen-binding fragment thereof of claim 4, wherein when the antibody is a full-length antibody, it comprises a heavy chain constant region derived from a heavy chain of a human antibody, and a light chain constant region derived from a kappa chain or a lambda chain of a human antibody.

7. A chimeric antigen receptor comprising the antibody or the antigen-binding fragment thereof targeting IL13RA2 according to any one of claims 1 to 3 or 5.

8. The chimeric antigen receptor of claim 7, further comprising a CD8 signal peptide, a hinge region, a transmembrane region, an intracellular signaling region 4-1BB and a CD3 zeta region.

9. The chimeric antigen receptor according to claim 8, which is, in order from N-terminus to C-terminus: a CD8 signal peptide, an antibody or antigen binding fragment thereof targeting IL13RA2, a hinge region, a transmembrane region, an intracellular signaling domain 4-1BB, and a CD3 zeta region.

10. The chimeric antigen receptor according to claim 9, wherein the transmembrane region is a CD8 transmembrane region.

11. The chimeric antigen receptor according to claim 9, wherein the amino acid sequence of the CD8 signal peptide is as set forth in SEQ ID No. 25; and/or the amino acid sequence of the hinge region is shown as SEQ ID NO. 27; and/or the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 29; and/or the amino acid sequence of the intracellular signal domain 4-1BB is shown as SEQ ID NO. 31; and/or the amino acid sequence of the CD3 zeta region is shown in SEQ ID NO. 33.

12. The chimeric antigen receptor according to claim 11, wherein the nucleotide sequence of the CD8 signal peptide is set forth in SEQ ID No. 26; and/or the nucleotide sequence of the hinge region is shown as SEQ ID NO. 28; and/or the nucleotide sequence of the membrane penetrating region is shown as SEQ ID NO. 30; and/or the nucleotide sequence of the intracellular signal domain 4-1BB is shown as SEQ ID NO. 32; and/or the nucleotide sequence of the CD3 zeta region is shown in SEQ ID NO 34.

13. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof targeting IL13RA2 according to any one of claims 1 to 6, or encoding the chimeric antigen receptor according to any one of claims 7 to 12.

14. The nucleic acid of claim 13, wherein the nucleotide sequence encoding the antibody or antigen-binding fragment thereof targeting IL13RA2 is set forth in SEQ ID No. 24.

15. A recombinant expression vector comprising the isolated nucleic acid of claim 13 or 14.

16. The recombinant expression vector of claim 15, wherein the recombinant expression vector is a plasmid, a phage, or a viral vector.

17. The recombinant expression vector of claim 16, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector, and the plasmid is a cosmid.

18. A transformant comprising the recombinant expression vector according to any one of claims 15 to 17.

19. The transformant according to claim 18, wherein the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

20. The transformant according to claim 19, wherein the eukaryotic cell is a yeast cell or a mammalian cell.

21. The transformant of claim 20, wherein the mammalian cell is a 293T cell or a CHO cell.

22. A genetically modified cell comprising a chimeric antigen receptor according to any one of claims 7 to 12.

23. The cell of claim 22, wherein the genetically modified cell is a eukaryotic cell.

24. The cell of claim 23, wherein the eukaryotic cell is an isolated human cell.

25. The cell of claim 24, wherein the cell is an immune cell.

26. The cell of claim 25, wherein the immune cell is a T cell.

27. A method of making an antibody or antigen-binding fragment thereof targeting IL13RA2, comprising the steps of: culturing the transformant according to any one of claims 18 to 21, and obtaining an antibody or an antigen-binding fragment thereof targeting IL13RA2 from the culture.

28. An antibody drug conjugate comprising a cytotoxic agent or a label, and the antibody targeting IL13RA2 or an antigen-binding fragment thereof according to any one of claims 1 to 6.

29. The antibody drug conjugate of claim 28, wherein the cytotoxic agent is MMAF or MMAE and the label is a fluorescent agent.

30. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof targeting IL13RA2 of any one of claims 1 to 6, the genetically modified cell of any one of claims 22 to 26, and/or the antibody drug conjugate of claim 28 or 29, and a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 30, further comprising one or more of the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.

32. Use of the antibody or antigen-binding fragment thereof targeting IL13RA2 according to any one of claims 1 to 6, the chimeric antigen receptor according to any one of claims 7 to 12, the genetically modified cell according to any one of claims 22 to 26, the antibody drug conjugate according to claim 28 or 29, and/or the pharmaceutical composition according to claim 30 or 31 in the preparation of a medicament for diagnosing, preventing and/or treating a tumor, wherein the tumor is glioma.

33. A kit comprising the antibody or antigen-binding fragment thereof targeting IL13RA2 according to any one of claims 1 to 6, the chimeric antigen receptor according to any one of claims 7 to 12, the genetically modified cell according to any one of claims 22 to 26, or the antibody drug conjugate according to claim 28 or 29 or the pharmaceutical composition according to claim 30 or 31.

34. The kit of claim 33, further comprising (i) a device for administering the antibody or antigen-binding fragment thereof or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.

35. A kit comprising kit a and kit B, wherein:

the kit A contains the antibody or the antigen-binding fragment thereof targeting IL13RA2 according to any one of claims 1 to 6, the chimeric antigen receptor according to any one of claims 7 to 12, the genetically modified cell according to any one of claims 22 to 26, the antibody drug conjugate according to claim 28 or 29, and/or the pharmaceutical composition according to claim 30 or 31;

36. A method for immunodetection or determination of IL13RA2, comprising using an antibody or antigen-binding fragment thereof targeting IL13RA2 according to any one of claims 1 to 6, a chimeric antigen receptor according to any one of claims 7 to 12, an antibody drug conjugate according to claim 28 or 29 or a pharmaceutical composition according to claim 30 or 31, said detection being a non-diagnostic detection.