阅读说明：本技术 靶向人Claudin18.2和NKG2DL的嵌合抗原受体细胞及其制备方法和应用 (Chimeric antigen receptor cell targeting human Claudin18.2 and NKG2DL, and preparation method and application thereof ) 是由 代红久 徐慧 朱靓婧 于 2021-10-08 设计创作，主要内容包括：本发明属于嵌合抗原受体细胞领域,一种靶向人Claudin18.2和NKG2DL的嵌合抗原受体细胞及其制备方法和应用,该嵌合抗原受体细胞含有靶向结合人Claudin18.2和靶向结合人NKG2DL的氨基酸序列。本发明的靶向Claudin18.2和NKG2DL的双特异性嵌合抗原受体修饰的免疫应答细胞能增强与肿瘤细胞的结合,明显提高抗肿瘤活性。(The invention belongs to the field of chimeric antigen receptor cells, and relates to a chimeric antigen receptor cell targeting human Claudin18.2 and NKG2DL, a preparation method and application thereof, wherein the chimeric antigen receptor cell contains an amino acid sequence targeting and combining human Claudin18.2 and human NKG2 DL. The immune response cell modified by the bispecific chimeric antigen receptor targeting Claudin18.2 and NKG2DL can enhance the combination with tumor cells and obviously improve the anti-tumor activity.)

1. Chimeric antigen receptors that target human claudin18.2 and NKG2DL bispecific, characterized in that:

the chimeric antigen receptor comprises:

targeting the extracellular recognition domain amino acid sequence of the human claudin18.2 protein: the amino acid sequence of the Claudin18.2 protein is targeted and combined as shown in SEQ ID No.2 or SEQ ID No. 3; or a variant modified by an amino acid having 80 to 99% homology with the amino acid sequence;

amino acid sequence of extracellular recognition domain targeting human NKG2DL protein: the amino acid sequence of human NKG2D shown in SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 and targeted to combine with human NKG2DL protein; or a variant modified by an amino acid having 80 to 99% homology with the amino acid sequence;

the chimeric antigen receptor according to claim 1, wherein: the amino acid sequence of the chimeric antigen receptor is as follows:

the amino acid sequence of a guide sequence, the amino acid sequence of an extracellular recognition domain targeted to be combined with human Claudin18.2 protein, the amino acid sequence of an extracellular recognition domain targeted to be combined with human NKG2DL protein, the amino acid sequence of a hinge region, the amino acid sequence of a transmembrane domain and the amino acid sequence of an intracellular signal domain which are sequentially connected from an amino terminal to a carboxyl terminal;

or the amino acid sequence of a guide sequence, the amino acid sequence of an extracellular recognition domain targeted to be combined with human NKG2DL protein, the amino acid sequence of an extracellular recognition domain targeted to be combined with human Claudin18.2, the amino acid sequence of a hinge region, the amino acid sequence of a transmembrane domain and the amino acid sequence of an intracellular signal domain which are sequentially connected from an amino terminal to a carboxyl terminal;

the transmembrane domain comprises a CD8 transmembrane domain, or a CD28 transmembrane domain, or a 4-1BB transmembrane domain, or an OX40 transmembrane domain, or an ICOS transmembrane domain;

wherein the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;

the immunoreceptor tyrosine activation motif comprises an intracellular signaling domain of the CD3 zeta chain or an fcsri gamma intracellular signaling structure;

the costimulatory signaling domain comprises a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX40 intracellular signaling domain, or an ICOS intracellular signaling domain;

the costimulatory signaling domain comprises a CD28 and 4-1BB intracellular signaling domain, or a 4-1BB and OX40 intracellular signaling domain, or a CD28 and OX40 intracellular signaling domain.

2. The chimeric antigen receptor according to claim 2, characterized in that the amino acid sequence of said chimeric antigen receptor is:

the polypeptide is formed by sequentially and serially connecting a leader peptide amino acid sequence shown as SEQ ID No.1, an antibody scFv amino acid sequence of Claudin18.2 shown as SEQ ID No.2 or SEQ ID No.3, an amino acid sequence of human NKG2D shown as SEQ ID No.5, an amino acid sequence of a human CD8 hinge region shown as SEQ ID No.10, an amino acid sequence of a human CD8 transmembrane region shown as SEQ ID No.11, an amino acid sequence of a human 4-1BB intracellular structural domain shown as SEQ ID No.13 and an amino acid sequence of a human CD3 zeta structural domain shown as SEQ ID No. 16;

or

The polypeptide is formed by sequentially and serially connecting an amino acid sequence of a leader peptide shown as SEQ ID No.1, an amino acid sequence of human NKG2D shown as SEQ ID No.5, an amino acid sequence of an antibody scFv of Claudin18.2 shown as SEQ ID No.2 or SEQ ID No.3, an amino acid sequence of a hinge region of human CD8 shown as SEQ ID No.10, an amino acid sequence of a transmembrane region of human CD8 shown as SEQ ID No.11, an amino acid sequence of an intracellular domain of human 4-1BB shown as SEQ ID No.13 and an amino acid sequence of a zeta domain of human CD3 shown as SEQ ID No. 16.

3. A nucleic acid molecule encoding the chimeric antigen receptor of claim 2 or 3, wherein:

the nucleotide sequence which is connected in series from 5 'to 3' and used for coding the guide sequence, the nucleotide sequence which is used for coding an antibody scFv targeting Claudin18.2, the nucleotide sequence which is used for coding human NKG2DL, the nucleotide sequence which is used for coding a hinge region, the nucleotide sequence which is used for coding the transmembrane domain and the nucleotide sequence which is used for coding the intracellular signal domain are included;

or comprises a nucleotide sequence which is connected in series from 5 'to 3' and is used for coding the guide sequence, a nucleotide sequence which is used for coding a target human NKG2DL, a nucleotide sequence which is used for coding an antibody scFv which is used for targeting Claudin18.2, a nucleotide sequence which is used for coding a hinge region, a nucleotide sequence which is used for coding the transmembrane domain and a nucleotide sequence which is used for coding the intracellular signal domain.

4. A recombinant vector comprising the nucleic acid molecule of claim 4.

5. A recombinant virus comprising the recombinant vector of claim 5 and a viral particle; the virus includes lentivirus, adenovirus, adeno-associated virus or retrovirus.

6. A functionalized immune-responsive cell obtained by infecting an immune effector cell with the recombinant virus of claim 6; the immune effector cells include cytotoxic T lymphocytes, NK cells, NKT cells, helper T cells, or macrophages.

7. A biological product characterized by comprising the amino acid sequence of claim 2 or 3; or comprising the nucleic acid molecule of claim 4; or a recombinant vector according to claim 5; or a recombinant virus according to claim 6; or a functionalized immunoresponsive cell of claim 7.

8. Use of a biological product according to claim 8 in the manufacture of a medicament for the treatment of cancer, autoimmune diseases or viral bacterial infections.

9. The use of claim 9, wherein the cancer is NKG2DL positive and/or claudin18.2 positive tumors, including gastric, pancreatic, liver, brain, prostate, lymphatic, leukemia, intestinal, lung, or breast cancer.

Technical Field

The invention belongs to the field of chimeric antigen receptor cells, and relates to an amino acid coding sequence of a bispecific chimeric antigen receptor for bispecific targeting human Claudin18.2(CLDN18.2) and NKG2DL, an immune response cell modified by the amino acid coding sequence, a preparation method of the amino acid coding sequence and the immune response cell, and application of the amino acid coding sequence and the immune response cell in medicine preparation.

Background

With the rapid development of biotechnology, immune cell therapy has become the fourth largest therapy in the field of cancer treatment.

Cancer immunotherapy mainly comprises adoptive cell therapy, immunomodulators, tumor vaccines, immune binding site blocking therapy and the like. Among them, in the field of cell therapy, CAR-T therapy has undoubtedly become a star in the dispute of research institutions and pharmaceutical companies as "pursuit".

The principle of the immunotherapy represented by CAR-T (Chimeric Antigen Receptor T-Cell) is mainly that Chimeric Antigen Receptor modification is carried out on T cells extracted from patients by genetic engineering means to form CAR-T cells, and the T cells can specifically recognize tumor surface associated antigens (tumor Cell markers) so as to kill tumors in a targeted manner.

Recent studies have shown that the expression of the NKG2DL protein is an indicator of "stress" in cells, and is rarely or only transiently expressed in healthy tissues, while it is usually expressed at higher levels on the surface of various tumor cells from different sources. The receptor of the NKG2DL protein is NKG2D, and research shows that the NKG2D-NKG2DL system plays an important role in the anti-tumor immunity of the organism, and NKG2D transmits an activation signal through recognizing NKG2DL generated on the surface of a tumor cell and activates the immune system, thereby playing a killing role on the tumor cell. In addition, the research finds that serum of patients with autoimmune diseases contains soluble NKG2DL, and the NKG2D-NKG2DL system plays a certain role in treating autoimmune diseases, resisting inflammation, resisting aging and the like (see Legroup L et al, Frontiers in Immunology, (2019)). Therefore, the expression of NKG2DL is used as a specific change on tumor cells when an organism generates tumors, provides a more accurate target point for the immunotherapy of the tumors, and provides a hint for the development of related new therapies and medicaments.

In addition, it has been shown that Claudin is a tetraspanin protein, the NH2 terminal and the COOH terminal are located intracellularly, having two extracellular loops, and a total of 27 family members have been found so far. Claudin18.2 is a tetraspanin protein expressed at the epithelial tight junction in the Claudin family, an important molecule of cellular tight junctions, which constitutes a paracellular barrier and controls the flow of molecules between cells. Claudin18.2 is a highly specific cell surface molecule, which is expressed only on differentiated gastric mucosal epithelial cells in normal tissues, so that the development of therapeutic antibodies against Claudin18.2 has greater anticancer potential, lower toxicity and larger optimal dosage space. The Claudin18.2 protein is highly expressed in solid tumors such as gastric Cancer, pancreatic Cancer, ovarian Cancer, bile duct Cancer and lung adenocarcinoma (see Sahin et al, Clinical Cancer Research (2008)). In addition, experts have disclosed that tumors with high Claudin18.2 expression often do not express PD-L1 and are not sensitive to PD-L1 targeted immunosuppressive drugs, and thus are a highly unmet field of clinical need. The unique expression profile of Claudin18.2 in normal tissues, as well as abnormal expression profiles in a variety of tumors, make it a very attractive target for anti-cancer therapy. While Claudin18.2 acts as a cell membrane surface protein, the exposed extracellular structure allows binding of antibodies, and these characteristics suggest that Claudin18.2 is an ideal therapeutic target.

In conclusion, we constructed novel highly specific killing immunoresponsive cells based on the activating targets claudin18.2 and NKG2DL of the CLDN18.2-NKG2DL system and their mutant peptides as bispecific chimeric antigen receptor modifications targeting tumor regions for tumor treatment.

Disclosure of Invention

In view of the above problems and/or other problems of the related art, it is an object of the present invention to overcome the problems of poor specificity and low killing efficiency of effector cells in the tumor environment encountered in the existing tumor clinical technologies, and to provide a bispecific targeting binding polypeptide domain targeting human claudin18.2 and NKG2DL or a functional variant thereof, a bispecific chimeric antigen receptor targeting human claudin18.2 and NKG2DL or a functional variant thereof, a coding nucleotide sequence thereof and an expression vector thereof, an engineered bispecific chimeric antigen receptor modified immune response cell targeting human claudin18.2 and NKG2DL, and applications thereof. The immune response cell modified by the engineered bispecific chimeric antigen receptor targeting human Claudin18.2 and NKG2DL can improve the specific killing efficiency of tumor cells, avoid the problem of treatment toxicity safety caused by off-target, and enhance the combination with the tumor cells, thereby providing a new means for tumor treatment with application prospect.

Technical scheme

An immune cell modified by a chimeric antigen receptor for bispecific targeting of human Claudin18.2 and NKG2DL, which is characterized in that the immune cell contains the chimeric antigen receptor, and the amino acid sequence of the chimeric antigen receptor is as follows:

amino acid sequences of a leader sequence, an extracellular recognition domain amino acid sequence, a hinge region amino acid sequence, a transmembrane domain amino acid sequence and an intracellular signal domain which are sequentially connected from an amino terminal to a carboxyl terminal, and target-binding human Claudin18.2 and NKG2 DL;

or a leader sequence, a hinge region amino acid sequence, a transmembrane domain amino acid sequence and an intracellular signal domain amino acid sequence which are sequentially connected from an amino terminal to a carboxyl terminal, and an extracellular recognition domain amino acid sequence, a hinge region amino acid sequence, a NKG2DL targeted to be combined with human Claudin18.2 and NKG2 DL;

the extracellular recognition structural domain amino acid sequence of the targeted binding human Claudin18.2 is as follows: an amino acid sequence of SEQ ID No.2 or SEQ ID No.3 that binds to CLDN 18.2; or a variant which is produced by amino acid modification and has 80-99% homology with the amino acid sequence shown in SEQ ID No.2 or SEQ ID No. 3.

The amino acid sequence of the extracellular recognition domain targeted to bind human NKG2DL is as follows: the amino acid sequence of human NKG2D shown in SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 and targeted to combine with human NKG2DL protein; or a variant which is produced by amino acid modification and has 80-99% homology with the amino acid sequence shown in SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No. 9.

Wherein the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;

the immune cell is characterized in that:

the nucleic acid molecule for coding the bispecific chimeric antigen receptor targeting human Claudin18.2 and NKG2DL comprises a nucleotide sequence for coding the guide sequence, a nucleotide sequence for coding the human NKG2D protein receptor targeting human NKG2DL, a nucleotide sequence for coding the extracellular recognition domain targeting human Claudin18.2, a nucleotide sequence for coding a hinge region, a nucleotide sequence for coding the transmembrane domain and a nucleotide sequence for coding the intracellular signal domain which are connected in series from 5 'to 3';

or a nucleic acid molecule encoding the bispecific chimeric antigen receptor targeting human Claudin18.2 and NKG2DL, comprising a nucleotide sequence encoding the leader sequence, a nucleotide sequence encoding an extracellular recognition domain targeted to bind human Claudin18.2, a nucleotide sequence encoding a human NKG2D protein receptor targeted to bind human NKG2DL, a nucleotide sequence encoding a hinge region, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signal domain, which are connected in series in sequence from 5 'to 3';

a recombinant vector or expression plasmid for bispecific targeting of chimeric antigen receptors of human Claudin18.2 and NKG2DL, characterized in that it comprises said nucleic acid molecule.

The recombinant vector or expression plasmid, characterized in that the recombinant vector or expression plasmid contains a promoter, wherein the promoter comprises the EF1 α long promoter, or the EFs short promoter.

A recombinant virus comprising the nucleotide sequence of said recombinant vector and a viral particle; the virus includes lentivirus, adenovirus, adeno-associated virus or retrovirus.

The immune cell is applied to preparing medicaments for resisting gastric cancer, pancreatic cancer, liver cancer, brain cancer, prostatic cancer, lymph cancer, leukemia, intestinal cancer, lung cancer or breast cancer.

Detailed description of the invention

In a first aspect, the present application provides a chimeric antigen receptor that targets human claudin18.2 and NKG2DL bispecific.

The amino acid sequence of the targeted binding human Claudin18.2 is as follows: an amino acid sequence shown as SEQ ID No.2 or SEQ ID No. 3; or a variant which is produced by amino acid modification and has 80-99% homology with the amino acid sequence shown in SEQ ID No.2 or SEQ ID No. 3.

The human NKG2D protein receptor that targets binding to human NKG2DL or a functional variant (analog) thereof comprising a sequence selected from the group consisting of: an amino acid sequence shown as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9, or a functional variant resulting from one or more amino acid modifications; wherein the functional variant modified by amino acid is polypeptide with 80-99% homology with amino acid sequence shown in SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No. 9.

The inventor continuously performs amino acid sequence design and sequence permutation, combination and screening through creative work, performs random screening test and targeting function verification (for example, tests of constructing a virus vector, further infecting T cells, obtaining modified T cells, detecting in vitro activity of the obtained modified T cells and the like) on sequences of hundreds of CAR molecules, then performs sequence adjustment according to comparison of a plurality of random combination results, and finally screens out sequences with best effect, thereby obtaining the amino acid sequence of the human NKG2D protein receptor which is efficiently targeted and combined with human NKG2DL and functional variants thereof.

The inventor continuously designs amino acid sequences, arranges and combines the sequences and screens through creative work, analyzes the biological characteristics of the sequences by using software, and selects the high-titer CLDN18.2 amino acid sequence with good stability and high binding force and a functional variant thereof.

In certain non-limiting embodiments, chimeric antigen receptors that target human Claudin18.2 and NKG2DL bispecific can further comprise a linker amino acid sequence represented by the formula (GGGGS) n, wherein 3. ltoreq. n.ltoreq.8.

In a second aspect, the present application provides a chimeric antigen receptor targeted to human claudin18.2 and NKG2DL bispecific, comprising the amino acid sequence of a leader sequence, the amino acid sequence of a human NKG2D protein receptor targeted to bind to human claudin18.2 and to bind to human NKG2DL, the amino acid sequence of the hinge region, the amino acid sequence of the transmembrane domain, the amino acid sequence of the intracellular signaling domain, sequentially linked from amino terminus to carboxy terminus. The amino acid sequence targeted to bind to the extracellular recognition domain of human NKG2DL comprises the human NKG2D protein receptor targeted to bind to human NKG2DL or a functional variant thereof as described in the first aspect of the present application.

The extracellular recognition domain (also referred to as the extracellular domain or simply consisting of the recognition element it contains) comprises a recognition element that specifically binds to a molecule present on the cell surface of a target cell.

In some non-limiting examples, the leader sequence is covalently linked to the 5' end of the extracellular antigen-binding domain.

In some embodiments, the chimeric antigen receptor that bispecific targets human claudin18.2 and NKG2DL comprises a hinge region.

In some embodiments, the transmembrane domain comprises a transmembrane region.

In some embodiments, the amino acid sequence of the human CD8 polypeptide of the hinge region is selected from the group consisting of the polypeptide shown in SEQ ID No.10 or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90 to 99% homology with the amino acid sequence shown in SEQ ID No. 10.

In some embodiments, the amino acid sequence of human CD8 of the transmembrane region is selected from the group consisting of the polypeptide of SEQ ID No.11 or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90 to 99% homology with the amino acid sequence of SEQ ID No. 11.

In some embodiments, the amino acid sequence of human CD28 of the transmembrane region is selected from the group consisting of the polypeptide of SEQ ID No.12 or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90 to 99% homology with the amino acid sequence of SEQ ID No. 12.

In some embodiments, the human 4-1BB intracellular domain is selected from: a polypeptide having an amino acid sequence as shown in SEQ ID No. 13; or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 13.

In some embodiments, the human CD28 intracellular domain is selected from the group consisting of: a polypeptide having an amino acid sequence as shown in SEQ ID No. 14; or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 14.

In some embodiments, the human OX40 intracellular domain is selected from the group consisting of: a polypeptide having an amino acid sequence as shown in SEQ ID No. 15; or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 15.

In some embodiments, the CD3 ζ intracellular domain is selected from: a polypeptide having an amino acid sequence as shown in SEQ ID No. 16; or a functional variant with amino acid modifications. Wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 16.

In some non-limiting embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;

in some non-limiting embodiments, the chimeric antigen receptor that bispecific targets human claudin18.2 and NKG2DL is recombinantly expressed or expressed from a vector.

In certain non-limiting embodiments, the intracellular domain of the chimeric antigen receptor of the present bispecific targeting human claudin18.2 and NKG2DL further comprises at least one costimulatory signaling region comprising at least one costimulatory ligand molecule that provides optimal lymphocyte activation.

In certain non-limiting embodiments, the chimeric antigen receptor that targets human claudin18.2 and NKG2DL bispecific may further comprise a spacer (spacer) that links the antigen binding domain to the transmembrane domain. The spacer may be sufficiently flexible to allow the antigen binding domain to be oriented in different directions to facilitate antigen recognition. The spacer may be a hinge region from IgG1, or part of the CH2CH3 region and CD3 of an immunoglobulin.

In certain non-limiting embodiments, the intracellular domain of the chimeric antigen receptor that bispecific targets human claudin18.2 and NKG2DL can comprise a human CD3 ζ polypeptide that can activate or stimulate cells (e.g., T cells of lymphoid lineage).

In certain non-limiting embodiments, the intracellular domain of a Chimeric Antigen Receptor (CAR) that bispecific targets human claudin18.2 and NKG2DL further comprises at least one costimulatory signaling region comprising at least one costimulatory molecule that provides optimal lymphocyte activation. As used herein, "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes to an antigen. The at least one co-stimulatory signaling region may comprise a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide (not based on a protein associated with an immune response), or a combination thereof.

In some embodiments, the costimulatory signaling region of the intracellular domain of the CAR comprises two costimulatory molecules: CD28 and 4-1BB, 4-1BB and OX40 or CD28 and OX 40.

In a third aspect, the present application provides a nucleic acid molecule encoding the bispecific human claudin18.2 and NKG2DL targeting chimeric antigen receptor according to the second aspect, said nucleic acid molecule comprising a nucleotide sequence encoding a leader sequence, a nucleotide sequence encoding a scFv targeted to bind human claudin18.2 and a human NKG2D protein targeted to bind human NKG2DL, a nucleotide sequence encoding a transmembrane domain, a nucleotide sequence encoding an intracellular signaling domain, connected in series in order from 5 'to 3'.

In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a hinge region. In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;

polynucleotides encoding extracellular recognition domains that target binding to human NKG2DL can be modified by codon optimization. Codon optimization can alter naturally occurring and recombinant gene sequences to achieve the highest possible level of productivity in any given expression system.

In a fourth aspect, the present application provides a recombinant vector or expression plasmid comprising the chimeric antigen receptor of the second aspect of the present application or the nucleic acid of the third aspect of the present application.

Genetic modification of immune-responsive cells (e.g., T cells, CTL cells, NK cells) can be achieved by transducing substantially homologous cellular compositions with recombinant DNA or RNA constructs. In one embodiment, the vector is a retroviral vector (e.g., a gammaretrovirus or lentivirus) that can introduce a DNA or RNA construct into the genome of a host cell. For example, polynucleotides that target the chimeric antigen receptors of human claudin18.2 and NKG2DL bispecific can be cloned into retroviral vectors and expression can be driven from their endogenous promoters, retroviral long terminal repeats, or from alternative internal promoters.

Non-viral vectors or RNA may also be used. Random chromosomal integration or targeted integration can be used (e.g., using nucleases, transcription activator-like effector nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and/or regularly clustered interspaced short palindromic repeats (CRISPRs) or transgene expression (e.g., using natural or chemically modified RNAs)).

In some embodiments, the vector is selected from the group consisting of a gamma-retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector.

In an exemplary embodiment, the vector is a gamma-retroviral vector.

In a fifth aspect, the present application provides a recombinant virus capable of expressing a chimeric antigen receptor that targets human claudin18.2 and NKG2DL bispecific according to the second aspect of the invention and is capable of infecting immunoresponsive cells.

In some embodiments, the immunoresponsive cell is a cytotoxic T lymphocyte, an NK cell, an NKT cell, a helper T cell, or a macrophage.

In an exemplary embodiment, the immunoresponsive cell is a cytotoxic T lymphocyte.

In some embodiments, the virus is a lentivirus, adenovirus, adeno-associated virus, retrovirus, or the like.

In an exemplary embodiment, the virus is a lentivirus.

In an exemplary embodiment, the virus is a retrovirus.

In a sixth aspect, the present application provides an isolated modified immunoresponsive cell comprising a chimeric antigen receptor according to the second aspect of the present application transformed with a recombinant vector or expression plasmid according to the third aspect of the present application.

For initial genetic modification of cells to provide the described bispecific human claudin18.2 and NKG2DL targeting chimeric antigen receptor modified immunoresponsive cells, typically retroviral vectors are used for transduction, however any other suitable viral vector or non-viral delivery system may be used. Retroviral gene transfer (transduction) has also proven effective for subsequent genetic modification of cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands. Combinations of retroviral vectors and suitable assembly lines are also suitable, wherein the capsid proteins are functional for infecting human cells.

In some embodiments, the immunoresponsive cell further comprises at least one exogenous co-stimulatory ligand.

Possible transduction methods also include direct co-culture of the cells with the producer cells. The transduced viral vectors can be used to express a co-stimulatory ligand (e.g., 4-1BBL) in immune-responsive cells. Preferably, the selected vector exhibits high infection efficiency and stable integration and expression.

In some embodiments, preferably, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, and combinations thereof, or more preferably, the co-stimulatory ligand is 4-1 BBL.

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

Multiple T cell subsets isolated from patients can be transduced with vectors for CAR expression.

In an exemplary embodiment, wherein the modified immunoresponsive cell is a CAR-T cell.

The bispecific chimeric antigen receptors targeting human claudin18.2 and NKG2DL can be used to generate genetically modified central memory T cells, which are then cryopreserved.

Seventh aspect the present application provides a method of making an isolated chimeric antigen receptor modified immunoresponsive cell of the sixth aspect of the application, comprising the steps of:

firstly, the nucleic acid molecule of the third aspect is connected into an expression vector by a molecular cloning mode to obtain the expression vector of the bispecific chimeric antigen receptor targeting human Claudin18.2 and NKG2 DL;

then, the obtained bispecific chimeric antigen receptor expression vector targeting human Claudin18.2 and NKG2DL is transfected into 293T cells to obtain virus solution;

finally, the virus liquid is used for infecting immune response cells, and the immune response cells expressing the bispecific targeted chimeric antigen receptor modification of human Claudin18.2 and NKG2DL are obtained from the infected cells.

In some non-limiting embodiments, the modified immunoresponsive cells of the invention can be cells of lymphoid lineage. The cells of the lymphoid lineage are selected from B, T and Natural Killer (NK) cells, and provide functions such as antibody production, regulation of cellular immune system, detection of foreign substances in blood, detection of foreign cells in a host, and the like. Non-limiting examples of cells of lymphoid lineage include T cells, Natural Killer (NK) cells, Cytotoxic T Lymphocytes (CTLs), regulatory T cells, macrophages, embryonic stem cells, and pluripotent stem cells (e.g., pluripotent stem cells that can differentiate into lymphoid cells).

In some embodiments, the immunoresponsive cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a Cytotoxic T Lymphocyte (CTL), a regulatory T cell, a macrophage, a human embryonic stem cell, and a pluripotent stem cell that can differentiate into lymphoid cells, 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 adaptive 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 (or stem-like memory T cells), and two types of effector memory T cells (e.g., TEM cells and TEMRA cells), regulatory T cells (also referred to as suppressor T cells), natural killer T cells, mucosa-associated constant T cells, and γ δ T cells.

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

In a preferred embodiment, the isolated modified immunoresponsive cell is a T cell.

In a preferred embodiment, the isolated modified immunoresponsive cell is a Natural Killer (NK) cell.

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

In an eighth aspect, the present application provides a pharmaceutical composition comprising an effective amount of the isolated modified immunoresponsive cell of the sixth aspect of the invention and a pharmaceutically acceptable excipient.

The present application discloses pharmaceutical compositions comprising isolated modified immunoresponsive cells expressing said bispecific human Claudin18.2 and NKG2 DL-targeting chimeric antigen receptor and a pharmaceutically acceptable carrier.

Administration of the pharmaceutical composition may be autologous or non-autologous. For example, immunoresponsive cells expressing said chimeric antigen receptors bispecific targeting human claudin18.2 and NKG2DL and compositions comprising the same can be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood-derived T cells of the presently disclosed subject matter or progeny thereof (e.g., in vivo, ex vivo, or derived in vitro) can be administered by including catheter administration, intravenous injection, or parenteral administration. When a pharmaceutical composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising said immunoresponsive cells bispecific targeting chimeric antigen receptors of human claudin18.2 and NKG2 DL) is administered, it is typically formulated in a unit dose injectable form (solution, suspension, emulsion).

The compositions of the present application may be formulations. The immunoresponsive cells expressing the Chimeric Antigen Receptors (CARs) for bispecific targeting of human claudin18.2 and NKG2DL disclosed herein 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, liquid compositions are more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition can comprise a carrier, which can be a solvent or dispersion medium comprising, for example, water, physiological saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

Various additives may be added that enhance the stability and sterility of the composition, including 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 Chimeric Antigen Receptors (CARs) bispecific targeting human claudin18.2 and NKG2DL of the presently disclosed subject matter.

If desired, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. The selection of suitable carriers 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-fill form).

In a ninth aspect, the present application provides a kit for the treatment or prevention of a disease comprising an immunoresponsive cell of the sixth aspect of the invention or a nucleic acid of the third aspect of the invention.

In a tenth aspect, the present application provides the use of a human NKG2D protein receptor or functional variant thereof targeted to bind to human claudin18.2 and human NKG2DL, a chimeric antigen receptor of bispecific targeting human claudin18.2 and NKG2DL, a recombinant vector or expression plasmid of the fourth aspect, a recombinant virus of the sixth aspect, an isolated modified immunoresponsive cell of the seventh aspect, and a kit of the ninth aspect, in a product for the treatment, or prevention of a disease, disorder or health disorder.

In some embodiments, the disease treated or prevented includes anti-tumor, anti-aging, autoimmune, anti-bacterial, and the like.

Principle of action

The bispecific targeting human Claudin18.2 and NKG2DL chimeric antigen receptor modified immune response cell of the invention, the engineered cell transmits activation signal and activates immune system by recognizing tumor cell surface antigen Claudin18.2 and NKG2DL, thereby exerting killing effect on tumor cells (as shown in figure 1); the immune response cell modified by the bispecific chimeric antigen receptor targeting Claudin18.2 and NKG2DL can improve the specific killing efficiency on tumor cells, avoid the problem of safe treatment toxicity caused by off-target, and enhance the combination with the tumor cells, thereby providing a new means for tumor treatment with application prospect.

Advantageous effects

The invention utilizes the chimeric antigen receptor modified T cell technology to prepare the bispecific chimeric antigen receptor modified engineering immunocyte which targets human Claudin18.2 and NKG2DL, the preparation method has simple steps, the obtained novel engineering immunocyte can specifically identify the tumor cell, can more effectively target and attack the tumor cell, has high killing rate to the tumor, and can be used for preparing anti-tumor products, in particular for preparing and treating the tumors with positive CLDN18A2 and NKG2 DL; the specifically targeted human Claudin18.2 and NKG2DL chimeric antigen receptor modified engineering immune cell enhances the combination with tumor cells, thereby obviously improving the killing efficiency of the engineering immune cells to the tumor cells. The invention can be hopefully used for preparing anti-tumor products, in particular to medicaments for resisting gastric cancer, pancreatic cancer, liver cancer, brain cancer, prostatic cancer, lymph cancer, leukemia, intestinal cancer, lung cancer or breast cancer, and has good industrial application prospect.

Drawings

FIG. 1 shows a schematic representation of the working pattern of CAR of KD-496 engineered cells of the present invention, wherein A is CLDN18.2-NKG2D-CAR and B is NKG2D-CLDN 18.2-CAR.

FIG. 2 is a schematic diagram showing the connection sequence of the parts of the chimeric antigen receptor in example 1, wherein A is KD-182, B is KD-025, and C is KD-496.

FIG. 3 shows the secondary structure diagram of the protein receptor human NKG2D protein targeting human NKG2DL in the chimeric antigen receptor of the present invention, wherein A-F are extracellular regions of the amino acid sequence of human NKG2D, respectively, and the characteristics of the protein receptor human NKG2D protein are analyzed by software, that is, based on the crystal structure of the complex formed by NKG2D and its ligand NKG2DL, the amino acids of the key sites affecting affinity are first obtained by alanine scanning, and then the saturation mutation of single-point mutation is performed, and according to the result of the saturation mutation, the secondary structure diagram of 6 sequences with good stability and high ligand binding force is selected by performing the calculation of multi-point mutation.

FIG. 4 shows the results of flow cytometry for T cell purity in example 3.

FIG. 5 shows the results of the KD-496CAR-T cell in vitro expression assay in example 4.

FIG. 6 shows the results of expression assay of KD-496 virus infected 293T cells in example 5. Wherein A is the expression of NKG2D and B is the expression of CLDN18.2 antibody scFv.

FIG. 7 is the results of the KD-496-1CAR-T cell killing experiment in vitro in example 6, wherein A is the killing of KD-496-1CAR-T cells on NUGC4 cells, B is the killing of KD-496-1CAR-T cells on AGS-18.2 cells, and C is the killing of KD-496-1CAR-T cells on MKN28-18.2 cells.

FIG. 8 shows the results of the in vitro cytokine IFN-. gamma.release assay for KD-496-1CAR-T cells in example 7, wherein A is a standard curve for ELISA assay and B is a histogram of cytokine IFN-. gamma.release.

FIG. 9 shows the results of mouse transplantation model (PDX) experiment of KD-496-1CAR-T cell therapy for human tumor in example 8, wherein A is the antigen expression of human tumor tissue in PDX model and B is the experiment of drug effect in KD-496CAR-T cell mouse.

FIG. 10 is the safety test of KD-496-1CAR-T in example 9 in mice, wherein A is the picture of HE staining of mouse visceral organs, and B is the survival rate of mice.

Detailed Description

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 modification of the parent structure, and refers to a variant of the structure that has the same or similar biological function and properties as the parent, e.g., the same targeted binding function as the parent. By way of non-limiting example, a "functional variant" may be obtained by making one or more conservative substitutions in the parent. Functional variants in the present application are modifications of the resulting structure binding to the human NKG2DL target on the basis of the receptor for human NKG2DL (human NKG2D amino acid sequence) and modifications of the resulting structure binding to the human claudin18.2 target on the basis of the claudin18.2 amino acid sequence.

The term "analog" refers to a structurally related polypeptide that has the function of the reference polypeptide molecule. In the application, the polypeptide refers to a polypeptide which is related to the amino acid sequence structure of human NKG2D and has a polyamino acid structure combined with human NKG2DL in a targeted way; and a polyamino acid structure related to the amino acid sequence structure of Claudin18.2 and having targeted binding with human Claudin18.2.

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 comprising an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.

The term "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid within the same group.

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

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

CARs can both bind antigen and transduce functions of T cell activation that 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 "recognition" 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 substantially recognize other molecules in a sample, e.g., other molecules in a biological sample that naturally includes a polypeptide of the invention.

The term "specific binding" refers to the association between two molecules (e.g., a ligand and a receptor) characterized by the ability of one molecule (ligand) to bind to another specific molecule (receptor), even in the presence of many other different molecules, i.e., the ability to show preferential binding of one molecule to another in a heterogeneous mixture of molecules. Specific binding of the ligand to the receptor was also demonstrated as follows: in the presence of excess unlabeled ligand, the detectably labeled ligand has reduced binding to the receptor (i.e., a binding competition assay).

The term "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes 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 a cell. 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, which contains the desired coding sequence and 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 a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers and terminators, as well as polyadenylation signals.

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

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

The term "modulate" as used herein refers to a change, either 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 not present at a level sufficient to achieve the functional effect achieved 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 described with reference to 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.

Example 1 expression plasmid for bispecific targeting of chimeric antigen receptors of human Claudin18.2 and NKG2DL

The overall design is as follows:

1. determination of the amino acid sequence of chimeric antigen receptors that target Claudin18.2 and NKG2DL bispecific

First, the full-length amino acid sequence of human NKG2D (NP _031386.2) was searched from the Genbank database of the national library of medicine (NCBI), the human amino acid sequence number of claudin18.2 is: NP _ 001002026.1. Immunizing a mouse with human Claudin18.2 protein, purifying to obtain an antibody fragment thereof, and carrying out humanization to obtain a humanized anti-human Claudin18.2 single-chain antibody (scFv); a single-chain antibody is composed of a heavy chain and a light chain, and the connecting segment between the heavy chain and the light chain is (G)₄S)₃Finally, finally4 scFv antibody fragments with high affinity with a target antigen Claudin18.2 are obtained by effect screening, wherein 2 amino acid sequences are shown as SEQ ID No.2 or SEQ ID No. 3.

Secondly, a chimeric antigen receptor which targets human Claudin18.2 and NKG2DL in a dual specificity mode is constructed, namely the amino acid sequence of the chimeric antigen receptor molecule is determined:

from amino terminal to carboxyl terminal, the amino acid sequence of guide peptide (shown as SEQ ID No. 1), the amino acid sequence of scFv of antibody targeting human Claudin18.2 (shown as SEQ ID No.2 or SEQ ID No.3), the amino acid sequence of human NKG2D (shown as SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No. 9), the amino acid sequence of hinge region of human CD8 (shown as SEQ ID No. 10), the amino acid sequence of the human CD8 transmembrane region (shown as SEQ ID No. 11) or the amino acid sequence of the human CD28 transmembrane region (shown as SEQ ID No. 12), the amino acid sequence of the human 4-1BB intracellular domain (shown as SEQ ID No. 13) or the amino acid sequence of the human CD28 intracellular domain (shown as SEQ ID No. 14) or the amino acid sequence of the human OX40 intracellular domain (shown as SEQ ID No. 15) or the combination of the two, and the amino acid sequence of the human CD3 zeta domain (shown as SEQ ID No. 16) are connected in series in sequence (shown as FIG. 2);

A-F shown in FIG. 3 are secondary structure diagrams of 6 sequences which are selected by the inventor through calculation of multi-point mutation and have good stability and high ligand binding force according to the result of saturation mutation, wherein the characteristics of the amino acids are analyzed by using software according to the extracellular region of the amino acid sequence of human NKG2D, namely based on the crystal structure (PDB number: 4S0U) of a compound consisting of NKG2D and ligand NKG2DL, the amino acids of key sites influencing affinity are firstly obtained through alanine scanning, then the saturation mutation of single-point mutation is carried out, and the calculation of the multi-point mutation is carried out according to the result of the saturation mutation;

the codon optimized sequences include: nucleotide sequence coding for a leader sequence, nucleotide sequence coding for Claudin18.2, nucleotide sequence coding for a human NKG2D sequence, nucleotide sequence coding for a human CD8 hinge region, nucleotide sequence coding for a human CD8 or CD28 transmembrane region, nucleotide sequence coding for a human 4-1BB or CD28 or OX40 intracellular domain, nucleotide sequence coding for a CD3 zeta domain.

Table 1 shows the expression of CAR in different combinations of the amino acid sequence targeting human Claudin18.2 and the amino acid sequence of NKG2D (SEQ ID No.5, see the most effective NKG2D sequence in patent CN 112142854B), the vectors constructed according to the following method, the viruses obtained, the corresponding CAR-T cells obtained, and the CAR detected according to the following method; the results show that: when the scFv amino acid sequence (shown as SEQ ID No.2 or SEQ ID No.3) of the human Claudin18.2 antibody is combined with the NKG2D (shown as SEQ ID No. 5), the CAR expression is optimal, and the human Claudin18.2 antibody can be used as a subsequent activity verification experiment.

TABLE 1 expression of CAR for different sequence combinations

2. Construction of chimeric antigen receptor molecule plasmids expressing bispecific targeting human Claudin18.2 and NKG2DL

The invention selects a CAR sequence in the step 1, which is named as KD-496-1 (the amino acid sequence is shown as SEQ ID No.17, and the nucleotide is shown as SEQ ID No. 19) and KD-496-2 (the amino acid sequence is shown as SEQ ID No.18, and the nucleotide is shown as SEQ ID No. 20) to complete the efficacy verification test of the subsequent embodiment, and the specific sequences are as follows:

the polypeptide is formed by sequentially connecting an amino acid sequence of a leader peptide (shown as SEQ ID No. 1), an antibody scFv amino acid sequence of Claudin18.2 (shown as SEQ ID No.2 or SEQ ID No.3), an amino acid sequence of human NKG2D (shown as SEQ ID No. 5), an amino acid sequence of a human CD8 hinge region (shown as SEQ ID No. 10), an amino acid sequence of a human CD8 transmembrane region (shown as SEQ ID No. 11), an amino acid sequence of a human 4-1BB intracellular domain (shown as SEQ ID No. 13) and an amino acid sequence of a human CD3 zeta domain (shown as SEQ ID No. 16) in series;

or

The polypeptide is formed by sequentially connecting an amino acid sequence of a leader peptide (shown as SEQ ID No. 1), an amino acid sequence of human NKG2D (shown as SEQ ID No. 5), an antibody scFv amino acid sequence of Claudin18.2 (shown as SEQ ID No.2 or SEQ ID No.3), an amino acid sequence of a human CD8 hinge region (shown as SEQ ID No. 10), an amino acid sequence of a human CD8 transmembrane region (shown as SEQ ID No. 11), an amino acid sequence of a human 4-1BB intracellular domain (shown as SEQ ID No. 13) and an amino acid sequence of a human CD3 zeta domain (shown as SEQ ID No. 16) in series;

the nucleotide sequence (Nanjing-one biosynthesis) of the chimeric antigen receptor molecule of the full-gene synthesis bispecific targeting human Claudin18.2 and NKG2DL is shown in SEQ ID No.19 or SEQ ID No.20, and is connected to a lentivirus vector lentiGuide-Puro (Addgene, USA) in a molecular cloning manner to construct a full-length CAR sequence expression frame with a single coding frame, and the full-length CAR sequence expression frame is expressed by using an EF1alpha promoter or an EFS promoter.

The specific operation steps are as follows:

the nucleotide sequence of the chimeric antigen receptor molecule of bispecific targeting human Claudin18.2 and NKG2DL is synthesized by whole gene synthesis, the CAR molecule sequence is artificially synthesized by PCR amplification, recovered by Axygen gel recovery kit (Hangzhou Zengheng), and is subjected to homologous recombination and connection with a vector lentiGuide-Puro (Addgene, USA) digested by restriction enzymes SmaI and MluI to form KD-496-1 and KD-496-2 expression vectors.

The specific recombination and ligation reaction system and conditions are as follows:

recombination and connection system:

PCR product 5. mu.l recovered from the gel, SmaI and MluI enzyme digestion lentiGuide-Puro plasmid (Addgene, USA) 3. mu.l recovered from the gel; 4X 1402 quick cloning Kit (Nanjing Kinuomei) 5. mu.l; 7 mu l of deionized water; the volume of the ligation reaction system is 20 μ l;

recombinant ligation conditions: and (3) placing the reaction system in a water bath at 50 ℃, reacting for 15min, and then placing on ice for 1 min.

10ul of the recombinant ligation product was transformed with competent Stbl3, using the following procedure.

Mu.l of the ligation product was added to 50. mu.l of competent cells (Stbl3, purchased from Invitrogen, USA) and ice-cooled for 30min at 42 ℃ for 45s for 2min, and then 500. mu.l of non-resistant LB liquid medium was added and shake-cultured at 37 ℃ and 200rpm for 40min, spread on ampicillin-resistant LB solid plates, and left overnight in a 37 ℃ incubator. After single colonies appeared, 5 colonies with the proper size were picked, plasmids were extracted and sent to a commercial sequencer for sequencing, and the sequencing results were compared with the fitted nucleotides (i.e., the nucleotides of the chimeric antigen receptors of bispecific targeting human Claudin18.2 and NKG2 DL) to confirm that the sequences were completely correct, thus confirming that the plasmids (KD-496-1 and KD-496-2 expression plasmids) of the chimeric antigen receptors of bispecific targeting human Claudin18.2 and NKG2DL were obtained.

Extraction and purification of chimeric antigen receptor expression plasmids that target human Claudin18.2 and NKG2DL bispecific.

The Stbl3 strain containing the KD-496 expression Plasmid was cultured in LB medium in large quantities and subjected to high-purity endotoxin-free extraction using Qiagen Plasmid Midi Kit (Qiagen, Germany) for infection. (see Qiagen plasmid extraction kit instructions for the specific detection procedures).

Example 2: preparation of viral solution of lentivirus vector

The recombinant plasmid (KD-496 expression plasmid) for bispecific targeting of chimeric antigen receptors of human claudin18.2 and NKG2DL obtained in example 1 and packaging vectors pol/gag, Rev and VSVG were used according to 12: 10: 5: 6 ratio of Lipofectamine^TM6000 transfection reagent (purchased from Biyuntian, product model is C0526) co-transfects 293T cells (the specific transfection operation process is shown in the transfection instruction), a complete culture medium (purchased from hyclone, product model is SH30243.01) is replaced 6 hours after transfection, cell supernatants rich in lentiviral particles are collected after culturing for 48 hours and 72 hours respectively, and virus supernatants are concentrated by ultracentrifugation to obtain virus solutions (hereinafter abbreviated as KD-496-1 and KD-496-2 virus solutions) of the lentiviral vectors carrying the chimeric antigen receptors of bispecific targeting human Claudin18.2 and NKG2 DL.

Example 3: isolated culture of T cells

Taking fresh peripheral blood of a healthy donor, and centrifugally separating the fresh peripheral blood mononuclear cells by density gradient; then, paramagnetic beads (purchased from Invitrogen, USA, and the product information is shown in the specification) coupled with anti-CD3 antibody and anti-CD 28 antibody are usedHuman T-Activator CD3/CD28) to enrich CD3+ T cells, specifically, peripheral blood mononuclear cells are diluted to a concentration of (10-30). times.10⁶The individual cells/ml were mixed with Magnetic beads at a ratio of 3:1, incubated at room temperature for 2-3 hours, and CD3+ T cells were enriched using a Magnetic particle collector (MPC, available from Invitrogen, USA). The enriched CD3+ T cells were finally resuspended in culture medium (purchased from Life Technologies, USA, under the product information OpTsizer^TMT-Cell Expansion SFM), adjusted to a Cell concentration of 1X 10⁶One/ml, finally 5% CO at 37 ℃₂The culture was carried out in an incubator for 2 days, and the results of the measurement are shown in FIG. 4.

Example 4: preparation of chimeric antigen receptor T cells (KD-496-1 and KD-496-2CAR-T) for bispecific targeting of human Claudin18.2 and NKG2DL

First, the CD3+ T cells obtained in example 3 were seeded in a 24-well plate at a seeding concentration of 1 to 10X 10⁵Cell/ml at 37 ℃ with 5% CO₂The culture is carried out in the environment for about 24 hours (the culture time depends on the specific practice, and generally, the cell confluence rate is ensured to be between 50 and 70 percent when the virus liquid is infected). Then, the KD-496 virus solutions collected in example 2 were taken, and added to a cell culture flask together according to an MOI of 10 to 40, and the flask was sealed, placed in a flat angle centrifuge, centrifuged at a low speed (500g to 1000g/min) for 1 hour, and then placed in an incubator to be cultured at 37 ℃. A chimeric antigen receptor T cell (KD-496CAR-T) which can target human Claudin18.2 and NKG2DL in a bispecific manner is obtained 48 hours after infection, and a next functional experiment can be carried out, as shown in figure 1, a working schematic diagram of the engineered cell is shown, namely the engineered cell transmits an activation signal and activates an immune system by recognizing Claudin18.2 and NKG2DL generated on the surface of a tumor cell, so that the tumor cell can be killed.

Detection of CAR protein expression using flow cytometry analysis:

the cells were centrifuged, washed twice with PBS and resuspended in FACS fluid (PBS with 0.1% sodium azide and 0.4% BSA); Anti-CD314 Antibody (APC-Anti-human CD314(NKG2D), biolegend, 320808) was added to the cell suspension, incubated at 4 ℃ for 1h, and Isotype control group (APC Mouse IgG1, kappa Isotype Ctrl Antibody, biolegend, 400120) was set; after washing the cells twice, 200. mu.L of FACS solution was added to resuspend the cells; BD facscan II was used to obtain stained cells and FlowJo was used to analyze the results. As shown in figure 5, the control group was T cells infected with empty viral fluid, and expression of the CAR molecule was barely detectable; the experimental group shows that the expression rates of the Claudin18.2 antibody scFv of the KD-496-1 and the KD-496-2 are 87.6 percent and 70.4 percent respectively and the expression rate of the NKG2D is 60.8 percent and 33.7 percent respectively for the T cells infected with KD-496-1 and KD-496-2 virus liquid.

Example 5: KD-496 binding assays to MICA, ULBP2/5/6 and CLDN18.2 proteins

The specific operation steps are as follows:

first, 293T cells (ATCC, USA) were inoculated into a 24-well plate at a concentration of 1-10X 10⁵Cell/ml at 37 ℃ with 5% CO₂The culture is carried out in the environment for about 24 hours (the culture time depends on the specific practice, and generally, the cell confluence rate is ensured to be between 50 and 70 percent when the virus liquid is infected). Thereafter, the KD-496 virus solution collected in example 2 was collected and used to infect 293T cells at an MOI of 10 to 40.

Culturing for 48h, collecting cells, and centrifuging at 4 deg.C for 5min at 300 g; washed twice with PBS and resuspended in FACS fluid (PBS with 0.1% sodium azide and 0.4% BSA); Anti-CD314 antibodies (APC-Anti-human CD314(NKG2D), biolegend, 320808) were added to the cell suspension, incubated at 4 ℃ for 1h, and Isotype controls (APC Mouse IgG1, kappa Isotype Ctrl Antibody, biolegend, 400120) were set. Adding primary anti-MICA (Mammalia, C-6His, C489), Recombinant Human NKG2DL2(C-6His, C508) and Claudin18.2 His (CR53) into the cell suspension, incubating for 4h at 4 ℃, and setting a control group; after washing the cells twice, 200. mu.L of FACS solution was added to resuspend the cells; adding the Anti-HIS-PE into the cell suspension, and incubating for 1h at 4 ℃; after washing the cells twice, 200. mu.L of FACS solution was added to resuspend the cells; BD facscan II was used to obtain stained cells and FlowJo was used to analyze the results. As shown in FIG. 6A, the positive rates of NKG2D, and CLDN18.2 antibody scFv of 293T cells of KD-496-1 and KD-496-2 virus solutions are respectively 98.8% and 98.6%, and 94.3% and 89.5%; the control group, shown in FIG. 6B, was 293T cells not infected with viral fluid, and had little binding to MICA, ULBP2/5/6, CLDN 18.2; the experimental group consisted of 293T cells infected with KD-496-1 and KD-496-2 virus fluids, with 95% and 93.9% binding to MICA, 64% and 58% binding to ULBP2/5/6, and 48.1% and 40.4% binding to CLDN 18.2.

Example 6: KD-496-1CAR-T cell in vitro killing experiment

An experimental group and 3 control groups are correspondingly arranged for each target cell line, wherein the experimental group is added with the cell suspension of the CAR-T cells specifically targeting human Claudin18.2 and NKG2DL obtained in example 4; the blank control group was supplemented with T cells not infected with virus (i.e., CD3+ T cells obtained in example 3); the KD-019 control group is added with CAR-T cells targeting CD19 (the preparation method is referred to CN 109803983B); KD-025 control group CAR-T cells targeting human NKG2DL were added (preparation method refer to CN 109803983B); the KD-182 control group was supplemented with CAR-T cells targeting human Claudin18.2 (preparation method referred to CN).

First, KD-019 CAR-T, KD-025 CAR-T, KD-182CAR-T, KD-496-1CAR-T cells were prepared by infection as described in example 4, and after further culturing for 72 hours after infection, the cells were subjected to killing inoculation, and after each target cell line was stained with CFSE fluorescence, the cells were counted under a fluorescence microscope, and the cell density was adjusted to about 2X 10⁶cells/ml, 20. mu.l/well, target cells were seeded in 96-well culture plates; adding T, KD-019 CAR-T, KD-025 CAR-T, KD-182CAR-T, KD-496-1CAR-T cells to effector cells at an effective to target ratio of 0.25:1, 1:1, 4: 1; then placing the cells in an incubator at 37 ℃ for culturing for 24 h; finally, the killing of the target cell line by KD-496-1CAR-T cells was evaluated using a 7-AAD/CFSE cytotoxicity test kit (purchased from Biovision, Inc., cat # K315-100) according to the instructions for the kit. As shown in FIG. 7, FIGS. 7A and 7B are the killing results of NUGC4 and AGS-18.2 cells, respectively, FIG. 7C is the killing result of MKN28-18.2 cells, and KD-025 CAR-T and KD-182CAR-T have some killing effect on tumor cells; KD-496-1CAR-T vs NUGC4, AGS-18.2 and MKN28-18.2 cells kill more than KD-025 CAR-T, and kill with KD-182CAR-T is equivalent, and then in vivo efficacy is further verified.

Example 7: KD-496-1CAR-T cell in vitro cytokine release assay

Experiment set up one experimental group and 2 control groups, wherein, the experimental group adds the cell suspension of the CAR-T cell specifically targeting human claudin18.2 and NKG2DL obtained in example 4; the blank control group was supplemented with T cells not infected with virus (i.e., CD3+ T cells obtained in example 3); the KD-019 control group was supplemented with unrelated CAR-T cells targeting CD 19.

First, KD-019 CAR-T, KD-496-1CAR-T cells were prepared by infection as described in example 4, and after further culturing for 72 hours after infection, killer inoculation was carried out with the target cell line adjusted to a cell density of about 1.5X 10⁶cells/ml, 20. mu.l/well, target cells were seeded in 96-well culture plates; according to the effective target ratio of 5:1 adding effector cells T, KD-019 CAR-T, KD-496-1CAR-T cells; then placing the cells in an incubator at 37 ℃ for culturing for 24 h; finally, the Human IFN-. gamma.ELISA Kit II Kit (purchased from BD Co., Cat.: 550612) was used to evaluate the release of killer cytokines from KD-496-1CAR-T cells on the above-mentioned target cell line according to the instructions of the Kit. As shown in FIG. 8, after the KD-496-1CAR-T cell and the tumor cell NUGC4 and the KD-496-1CAR-T cell were co-cultured with the tumor cell, INF-gamma was significantly increased compared with the control group, indicating that the KD-496-1CAR-T cell has a good anti-tumor effect.

Example 8: KD-496-1CAR-T mouse in vivo efficacy experiment

Human gastric cancer tissues removed from surgery were placed into 15mL centrifuge tubes containing 10mL of tissue preservation solution (L15 medium + 1% double antibody) and returned to the laboratory with a thermal bucket and ice bag. (PDX vaccination within 24h after tissue ex vivo); after the sample is sent to the laboratory, the tissue treatment is carried out in a biological safety cabinet, and the steps are as follows: the tissue in the centrifuge tube was removed to a small dish (L15 was added to the dish in advance) and photographed; the samples were placed under a stereomicroscope for tissue edge trimming: (removing fat, connective tissue, etc. on the edge of tumor tissue; placing 2X 2mm2 black outside the dish bottomColor card, then the tumor tissue after treatment is cut into uniform blocks with the size: 2X 2mm3, placed in a new dish containing L15 medium and placed on an ice box; tissue samples were inoculated in immunodeficient mice (B-NDG) with tissue block sizes of: 3 mm at 2X 2 mm/block, left locus inoculated with 2 blocks; an inoculation mode comprises the following steps: subcutaneous inoculation. As shown in fig. 9A, immunohistochemical IHC results showed high expression of gastric cancer tissues claudin18.2 and NKG2DL (MICA and ULBP2/5/6) antigens. When the tumor grows to 100mm³Left and right, random grouping was performed, each group was n-5, CAR-T cells were prepared by infection according to the method of example 4, and CAR-T cells were injected into tail vein, 1 × 10⁷cells/100 mu L, tumor size of the mice is measured for 2 times/week, and the result is shown in figure 9B, and compared with a control group, KD-025, KD-182 and KD-496-1 have obvious tumor inhibition effect.

Example 9: safety experiment of KD-496-1CAR-T in mice

First, KD-496-1CAR-T cells were prepared by infection as in example 4, and the effect of KD-496-1CAR-T cells on the major organs and survival cycle of mice is shown in FIG. 10. As can be seen from FIG. 10, the KD-496-1CAR-T cell does not cause inflammation, edema and necrosis of the major organs such as heart, liver, lung and kidney of the mice (FIG. 10A), and has no negative effect on the survival cycle of the mice (FIG. 10B).

In conclusion, the bispecific human Claudin18.2 and NKG2DL chimeric antigen receptor targeted virus vector constructed by the invention and the modified engineering immune cell thereof can be applied to the treatment of various tumors, including gastric cancer, pancreatic cancer, liver cancer, brain cancer, prostate cancer, lymph cancer, leukemia, intestinal cancer, lung cancer or breast cancer.

Sequence listing

<110> Nanjing Kaidi medical technology Co., Ltd

<120> chimeric antigen receptor cell targeting human Claudin18.2 and NKG2DL, and preparation method and application thereof

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> PRT

<213> leader sequence (amino acid sequence 2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 1

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> 2

<211> 246

<212> PRT

<213> Claudin18.2 Single chain antibody sequence 1(2 Ambystoma latex x Ambystoma jeffersonia)

<400> 2

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Tyr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Thr Phe Ser Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val

180 185 190

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

195 200 205

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

210 215 220

Ala Lys Leu Tyr Tyr Gly Asn Ser Met Asp Ser Trp Ser Gln Gly Leu

225 230 235 240

Ser Val Thr Val Ser Ser

245

<210> 3

<211> 246

<212> PRT

<213> Claudin18.2 Single chain antibody sequence 2(2 Ambystoma latex x Ambystoma jeffersonia)

<400> 3

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

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Leu Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn

85 90 95

Ala Tyr Tyr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Thr Phe Ser Ser Gly Gly Asp Tyr Thr Phe Tyr Pro Asp Ser Val

180 185 190

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

195 200 205

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

210 215 220

Ala Lys Leu Tyr Tyr Gly Asn Ser Met Asp Ser Trp Ser Gln Gly Leu

225 230 235 240

Ser Val Thr Val Ser Ser

245

<210> 4

<211> 136

<212> PRT

<213> NKG2D sequence 1(2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 4

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

1 5 10 15

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

20 25 30

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 5

<211> 136

<212> PRT

<213> NKG2D sequence 2(2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 5

Ser Leu Phe Asn Lys Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

1 5 10 15

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

20 25 30

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 6

<211> 136

<212> PRT

<213> NKG2D sequence 3(2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 6

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

1 5 10 15

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

20 25 30

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Lys Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Lys Arg Thr Val

130 135

<210> 7

<211> 136

<212> PRT

<213> NKG2D sequence 4(2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 7

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

1 5 10 15

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Lys

20 25 30

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 8

<211> 136

<212> PRT

<213> NKG2D sequence 5(2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 8

Ser Leu Phe Asn Lys Glu Val Lys Ile Pro Leu Thr Glu Ser Tyr Cys

1 5 10 15

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

20 25 30

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 9

<211> 136

<212> PRT

<213> NKG2D sequence 6(2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 9

Ser Leu Phe Asn Lys Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

1 5 10 15

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

20 25 30

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

35 40 45

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

50 55 60

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

65 70 75 80

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

85 90 95

Asn Leu Leu Thr Ile Ile Glu Met Lys Lys Gly Asp Cys Ala Leu Tyr

100 105 110

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

115 120 125

Tyr Ile Cys Met Gln Arg Thr Val

130 135

<210> 10

<211> 45

<212> PRT

<213> CD8 hinge region (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 10

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> 11

<211> 24

<212> PRT

<213> CD8 transmembrane region (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 11

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> 12

<211> 27

<212> PRT

<213> CD28 transmembrane region (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 12

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 13

<211> 42

<212> PRT

<213> 4-1BB intracellular Domain (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 13

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> 14

<211> 41

<212> PRT

<213> CD28 Domain (2 Ambystoma latex x Ambystoma jeffersonia)

<400> 14

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 15

<211> 36

<212> PRT

<213> OX40 Domain (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400> 15

Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly

1 5 10 15

Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr

20 25 30

Leu Ala Lys Ile

35

<210> 16

<211> 112

<212> PRT

<213> CD3 zeta Domain (2 Ambystoma laterale x Ambystoma jeffersonia)

<400> 16

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 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> 17

<211> 654

<212> PRT

<213> KD-496CAR molecule amino acid sequence 1(2 Ambystoma laterale x Ambystoma jeffersonanium)

<400> 17

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 Asp Ile Val Ile Thr Gln Ser Pro Asp Ser Leu

20 25 30

Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln

35 40 45

Ser Leu Leu Asn Ser Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln

50 55 60

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr

65 70 75 80

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

85 90 95

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val

100 105 110

Tyr Tyr Cys Gln Asn Ala Tyr Tyr Tyr Pro Phe Thr Phe Gly Gly Gly

115 120 125

Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

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

145 150 155 160

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

165 170 175

Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys

180 185 190

Arg Leu Glu Trp Val Ala Thr Phe Ser Ser Gly Gly Asp Tyr Thr Phe

195 200 205

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

210 215 220

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

225 230 235 240

Ala Val Tyr Tyr Cys Ala Lys Leu Tyr Tyr Gly Asn Ser Met Asp Ser

245 250 255

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

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

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

290 295 300

Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn

305 310 315 320

Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln

325 330 335

Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys

340 345 350

Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly

355 360 365

Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser

370 375 380

Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp

385 390 395 400

Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser

405 410 415

Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Ala Ala Thr

420 425 430

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

435 440 445

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

450 455 460

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

465 470 475 480

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

485 490 495

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

500 505 510

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

515 520 525

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

530 535 540

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

545 550 555 560

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650

<210> 18

<211> 654

<212> PRT

<213> KD-496CAR molecule amino acid sequence 2(2 Ambystoma laterale x Ambystoma jeffersonanium)

<400> 18

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 Asp Ile Val Ile Thr Gln Ser Pro Leu Ser Leu

20 25 30

Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln

35 40 45

Ser Leu Leu Asn Ser Gly Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Leu

50 55 60

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr

65 70 75 80

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

85 90 95

Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val

100 105 110

Tyr Tyr Cys Gln Asn Ala Tyr Tyr Tyr Pro Phe Thr Phe Gly Gly Gly

115 120 125

Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

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

145 150 155 160

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

165 170 175

Thr Phe Ser Lys Phe Gly Met Ser Trp Val Arg Gln Ala Pro Asp Lys

180 185 190

Arg Leu Glu Trp Val Ala Thr Phe Ser Ser Gly Gly Asp Tyr Thr Phe

195 200 205

Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

210 215 220

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

225 230 235 240

Ala Ile Tyr Tyr Cys Ala Lys Leu Tyr Tyr Gly Asn Ser Met Asp Ser

245 250 255

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

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

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

290 295 300

Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn

305 310 315 320

Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln

325 330 335

Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys

340 345 350

Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly

355 360 365

Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser

370 375 380

Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp

385 390 395 400

Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser

405 410 415

Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ala Ala Ala Thr

420 425 430

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

435 440 445

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

450 455 460

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

465 470 475 480

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

485 490 495

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

500 505 510

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

515 520 525

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

530 535 540

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

545 550 555 560

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650

<210> 19

<211> 1962

<212> DNA

<213> KD-496CAR molecule nucleotide sequence 1(2 Ambystoma laterale x Ambystoma jeffersonanium)

<400> 19

atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60

ccggatatcg ttataaccca atctcccgac tcattggcag tcagtttggg cgaacgggcg 120

actattaact gtaaatcatc acagtctttg ctcaactcag gcaatcagag aaattacctg 180

acttggtatc aacagaagcc cgggcaaccg ccaaaactgt tgttttattg ggcttcaaca 240

cgggaaagtg gggtgcctga tcggtttaca gggagcggat ccggcaccga ttttactttg 300

acaatctctt cactgcaagc cgaggacgta gcggtttact attgtcaaaa tgcctattat 360

tacccattta ccttcggagg cgggacaaaa cttgaaataa agggtggcgg aggctctggc 420

ggcgggggct cagggggtgg tggttctgag gttcaacttg tagagagtgg aggaggagtt 480

gtacaaccgg ggggcagtct tagactttct tgcgctgcat ctgggtttac attctctaag 540

tttggaatga gttgggtgag acaagcgccc ggtaagcgcc ttgagtgggt agcaactttt 600

agctcaggtg gtgactacac cttctaccca gactccgtga agggtcggtt caccattagc 660

cgagataact caaaaaacac gctttacctg caaatgaaca gtctgcgagc tgaggatacg 720

gcggtatatt actgtgcaaa gttgtattat ggaaatagca tggattcctg gagccagggt 780

ctctctgtga cagtttctag tggaggaggg ggttccgggg gaggcggctc tggcggcgga 840

gggagtggcg gggggggttc agggggtgga ggaagctctc tgttcaacca agaggtgcag 900

ataccactta ccgaatcata ttgtggcccc tgcccaaaga actggatatg ttacaaaaat 960

aattgctacc agtttttcga cgagtccaag aattggtatg aatcacaagc cagctgcatg 1020

tcccaaaatg cgtcattgtt gaaggtatat tctaaggagg accaagattt gttgaagttg 1080

gttaaatcct atcattggat ggggttggtc catataccta caaatggttc atggcagtgg 1140

gaagatggat ctatactgag cccaaatctt ctgacaataa ttgaaatgca aaaaggcgat 1200

tgtgcccttt acgctagtag cttcaaaggt tatattgaga actgtagcac accgaacact 1260

tatatctgta tgcagagaac ggttgccgct gcaaccacga cgccagcgcc gcgaccacca 1320

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 1380

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 1440

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1500

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1560

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1620

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1680

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1740

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1800

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1860

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1920

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 1962

<210> 20

<211> 1962

<212> DNA

<213> KD-496CAR molecule nucleotide sequence 2(2 Ambystoma laterale x Ambystoma jeffersonanium)

<400> 20

atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60

ccggacatag tcataacaca atctccgctt agcttgccgg tcactcctgg cgaaccagcc 120

tctatcagtt gtaaaagctc acaatcactg ctcaatagcg gaaaccagcg gaactatttg 180

acatggtacc tccaaaaacc tggtcaacct ccaaagctgc tgttttactg ggcctcaacg 240

cgggagtcag gggttcctga tcggtttact ggttcaggca gcggtacaga ttttacgctg 300

aaaataagca gggttgaggc agaagatgtc ggtgtctatt actgtcagaa cgcatattac 360

tacccgttta cctttggtgg cggtacaaag ctggaaatca aaggcggggg cggaagcgga 420

ggtggtggtt caggtggagg cggttccgaa gtgcagcttc ttgaaagtgg tggtgggttg 480

gtacaaccag gaggcagtct cagactgtcc tgtgccgctt ccggcttcac gttctctaag 540

tttggaatgt catgggtacg acaggcaccc gataagcgcc tcgaatgggt cgcaactttt 600

tccagcggtg gtgattacac gttctatcca gattcagtca aaggccggtt tacgatctcc 660

cgagataaca gtaagaatac actgtatctt caaatgaatt cacttcgggc agaagatacc 720

gcgatttatt attgcgctaa actttactac gggaactcta tggattcctg gagtcaaggc 780

ttgagtgtta ctgtatcaag tggaggaggg ggttccgggg gaggcggctc tggcggcgga 840

gggagtggcg gggggggttc agggggtgga ggaagctctc tgttcaacca agaggtgcag 900

ataccactta ccgaatcata ttgtggcccc tgcccaaaga actggatatg ttacaaaaat 960

aattgctacc agtttttcga cgagtccaag aattggtatg aatcacaagc cagctgcatg 1020

tcccaaaatg cgtcattgtt gaaggtatat tctaaggagg accaagattt gttgaagttg 1080

gttaaatcct atcattggat ggggttggtc catataccta caaatggttc atggcagtgg 1140

gaagatggat ctatactgag cccaaatctt ctgacaataa ttgaaatgca aaaaggcgat 1200

tgtgcccttt acgctagtag cttcaaaggt tatattgaga actgtagcac accgaacact 1260

tatatctgta tgcagagaac ggttgccgct gcaaccacga cgccagcgcc gcgaccacca 1320

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 1380

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 1440

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1500

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1560

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1620

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1680

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1740

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1800

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1860

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1920

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 1962