WO2018121679A1 - Lymphocyte modified with chimeric antigen receptor expressing cxcr4, preparation method, and use - Google Patents

Abstract

The present invention belongs to the field of cell therapy. Particularly, the present invention provides a lymphocyte modified with a chimeric antigen receptor expressing a chemokine receptor, CXCR4. The present invention also provides a method for preparing a specific lymphocyte, comprising modifying a non-specific lymphocyte by means of genetic engineering to the specific lymphocyte capable of recognizing a tumor-associated surface antigen and expressing a chemokine receptor, CXCR4, thereby being enriched in a specific site, and targeting and killing a specific cell. The present invention also provides a use of the same in malignant tumor treatment.

Description

Chimeric antigen receptor-modified lymphocyte expressing CXCR4, preparation method and use thereof Technical field

The invention relates to the field of cells, in particular to genetically engineering non-specific lymphocytes to be specifically transformed into a tumor surface-associated antigen and expressing a chemokine receptor CXCR4, thereby enriching and killing cells at specific sites. Preparation method of sex lymphocytes and its use in the treatment of malignant tumors.

Background technique

Immunotherapy is considered to be the most promising treatment for cancer. Among them, immunological checkpoint inhibitors (such as CTLA-4, PD-1, etc.) have shown superior effects over existing therapies in various tumor clinical trials, and are approved for malignancy such as non-small cell lung cancer and bladder cancer. Treatment of tumors. Unlike CTLA-4 and PD-1 antibodies, which exert anti-tumor effects by re-activating the body's immune response, chimeric antigen receptor-modified immune cell therapy does not depend on the immune status of the body, and the chimeric antigen receptor is genetically engineered. It is expressed on the surface of immune cells, recognizes tumor antigens, activates immune effector cells to produce direct anti-tumor effects. At present, chimeric antigen receptor-modified lymphocytes targeting CD19 have shown good results in clinical trials for the treatment of acute lymphoblastic leukemia and non-Hodgkin's lymphoma, with an effective rate of up to 90%, showing this The enormous therapeutic potential of therapies. Currently, more than 300 clinical trials related to chimeric antigen receptor-modified lymphocyte therapy are underway.

Although chimeric antigen receptor-modified lymphocytes have attractive prospects in tumor immunotherapy, there are still several problems: 1. Tumor-specific antigen deficiency. Currently, there are few tumor-specific surface antigens, and most tumors lack specific antigens, and most of them are tumor-associated antigens that are highly expressed in tumors but also expressed in normal tissues, thereby limiting their lymphocyte therapy as a chimeric antigen receptor. Target. For example, in the clinical study of chimeric antigen receptor-modified lymphocytes targeting HER2, since human lung tissue also expresses HER2, a non-specific cytotoxic killing reaction is caused, resulting in death of the patient. 2. Enrichment of chimeric antigen receptor-modified lymphocytes at the tumor site. At present, the progress of lymphocyte treatment for chimeric antigen receptor modification occurs in hematological tumors, and solid tumors have not been reported. One of the important reasons is that the chimeric antigen receptor-modified lymphocytes after in vitro expansion cannot be large and effective. Entering the solid tumor, the anti-tumor effect is not obvious. 3. Inhibition of tumor microenvironment on chimeric antigen receptor-modified lymphocytes. Relative to hematological tumors, there is a complex tumor immune microenvironment in solid tumors, which makes the chimeric antigen receptor-modified lymphocytes unable to function, and the latest literature suggests that necrotic tumor cells can also release lymphocytes by releasing K ⁺ Depletion. Therefore, there is still a need to strengthen research in the field with a view to further improving the efficacy of chimeric antigen receptor-modified lymphocytes.

The migration of lymphocytes to tumors is a complex process that relies on chemokines and involves the interaction between circulating lymphocytes and endothelial cells. Studies have shown that CXCL12/SDF-1α is an important chemotactic regulator of T cells and myeloid monocytes, and by binding to its receptor CXCR4, drives cells to reach relevant sites along the concentration gradient. After radiotherapy, the expression of CXCL12/SDF-1α was elevated at the irradiation site, and the myeloid mononuclear cells were recruited to migrate to the irradiation site. This phenomenon was also found in some glial tumors that highly express CXCL12. Studies have confirmed that HIV infection can lead to downregulation of CXCR4 expression in T cells, and the decline is more pronounced over time. We found that after T cell-mediated gene modification of T cells, the expression of chemokine receptor CXCR4 was significantly decreased, and chemotactic activity was limited.

Based on the above phenomenon, we hypothesized that CXCR4 can be expressed in CXCL12/SDF-1α-expressing gliomas or multiple myeloma by expressing CXCR4 on chimeric antigen receptor-modified lymphocytes, thereby enhancing anti-tumor effects. The expression of CXCL12/SDF-1α in the tumor site can be increased by radiotherapy and the like, and the chimeric antigen-modified lymphocytes can be induced to produce a homing effect, thereby producing a better anti-tumor effect.

Summary of the invention

The technical problem to be solved by the present invention is to provide a new and effective means for the treatment of malignant tumors.

The technical solution to solve the technical problem of the present invention is to provide a chimeric antigen receptor comprising an extracellular antigen binding region, a transmembrane region and an intracellular signal region.

Wherein, in the chimeric antigen receptor, the extracellular antigen-binding region is a single-chain variable region, receptor or ligand of a monoclonal antibody that binds to a tumor antigen.

Further, the tumor antigen includes but not limited to: (EGFR), epithelial cell adhesion molecule (EpCAM), mesothelin, interleukin 13 receptor α2 (IL-13Rα2), epidermal growth factor receptor 2 (ERBB2), epidermal growth factor receptor 3 (ERBB3), epidermal growth factor receptor 4 (ERBB4), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), ganglioside Antigen G _D2 (GD2), folate receptor 1 (FOLR1), prostate specific membrane antigen (PSMA), melanin precursor protein (gp100), mucin 1 (MUC1), mucin 16 (MUC16), carbonic anhydrase 9 (CA9), L1 cell adhesion molecule (CD171), tumor antigen 125 (CA125), tumor antigen 15-3 (CA15-3), tumor antigen 19-9 (CA19-9), NY-ESO-1, MART-1 , MAGE4, B cell mature antigen (BCMA), leukocyte differentiation antigen 19 (CD19), leukocyte differentiation antigen 20 (CD20), leukocyte differentiation antigen 22 (CD22), leukocyte differentiation antigen 30 (CD30), leukocyte differentiation antigen 33 (CD33) Carcinoembryonic antigen (CEA), leukocyte differentiation antigen 38 (CD38), leukocyte differentiation antigen 133 (CD133), leukocyte differentiation antigen 138 (CD138), leukocyte differentiation antigen 12 3 (CD123), EPH receptor A2 (EPHA2), EPH receptor A3 (EPHA3).

Wherein, in the chimeric antigen receptor, the transmembrane region is a leukocyte differentiation antigen 8 (CD8) transmembrane region or a leukocyte differentiation antigen 28 (CD28) transmembrane region.

Wherein, in the chimeric antigen receptor, the intracellular signal region is leukocyte differentiation antigen 28 (CD28), tumor necrosis factor receptor superfamily member 4 (TNFRSF9/CD134/OX40), tumor necrosis factor receptor superfamily Member 9 (TNFRSF9/CD137/4-1BB), lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell costimulatory molecule (ICOS), CD40, CD27 or DAP10 (DNAX activator protein 10) Or a variety of intracellular domains.

Wherein, in the chimeric antigen receptor, the monoclonal antibody single-chain variable region is a scFv single-chain antibody variable region.

In the chimeric antigen receptor, the amino acid sequence of the variable region of the scFv single-chain antibody is represented by SEQ ID No. 1.

SEQ ID No.1 Amino acid sequence of the variable region of scFv single-chain antibody

Wherein, in the chimeric antigen receptor, the coding nucleotide sequence of the variable region of the scFv single-chain antibody is represented by SEQ ID No. 2.

SEQ ID No. 2 Encoding nucleotide sequence of the variable region of scFv single-chain antibody

More specifically, the above chimeric antigen receptor is obtained by sequentially linking a signal peptide, an scFv single chain antibody, a CD8α transmembrane region, a CD28 intracellular segment, a 4-1BB intracellular segment, and a CD3 ζ chain through a nitrogen terminal to a carbon terminal.

The amino acid sequence of the signal peptide is shown in SEQ ID No. 3.

MVLLVTSLLLCELPHPAFLLIP. SEQ ID No. 3

The coding nucleotide sequence of the signal peptide is shown in SEQ ID No. 4.

Wherein, the amino acid sequence of the CD8 transmembrane region is represented by SEQ ID No. 5.

The coding nucleotide sequence of the CD8 transmembrane region is shown in SEQ ID No. 6.

The amino acid sequence of the intracellular domain of CD28 is shown in SEQ ID No. 7.

The coding nucleotide sequence of the CD28 intracellular domain is shown in SEQ ID No. 8.

The amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID No. 9.

The coding nucleotide sequence of the 4-1BB intracellular domain is shown in SEQ ID No. 10.

The amino acid sequence of the CD3ζ domain is shown in SEQ ID No. 11.

The coding nucleotide sequence of the CD3ζ domain is shown in SEQ ID No. 12.

SEQ ID No. 12 The coding nucleotide sequence of the CD3 ζ domain

Further, in the chimeric antigen receptor, the chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 13;

Or: (2) the same or similar function as the protein of SEQ ID No. 13 obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the protein represented by SEQ ID No. protein.

SEQ ID No. 13 amino acid sequence of chimeric antigen receptor

Further, in the chimeric antigen receptor, the nucleotide sequence of the coding gene of the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 14 or a degenerate sequence thereof;

Or (2): a nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein.

SEQ ID NO: 14 Nucleotide sequence of gene encoding chimeric antigen receptor

Particularly, in the above chimeric antigen receptor, the ligand is an APRIL ligand.

Further, in the above chimeric antigen receptor, the APRIL amino acid sequence is represented by SEQ ID NO.

The coding nucleotide sequence of the APRIL is represented by SEQ ID NO.

Further, the chimeric antigen receptor is obtained by sequentially linking a signal peptide, an APRIL, a CD8α transmembrane region, an CD28 intracellular segment, a 4-1BB intracellular segment, and a CD3 ζ chain through a nitrogen terminal to a carbon terminal.

Particularly, in the chimeric antigen receptor, the chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 17;

Or: (2) the same or similar function as the protein of SEQ ID No. 17 obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the protein represented by SEQ ID No. 17. protein.

Amino acid sequence of the chimeric antigen receptor of SEQ ID No. 17

Further, in the chimeric antigen receptor, the nucleotide sequence of the coding gene of the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 18 or a degenerate sequence thereof;

Or (2): a nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein.

SEQ ID NO: 18 Nucleotide sequence of gene encoding chimeric antigen receptor

The present invention also provides a chimeric antigen receptor-modified lymphocyte, which coexpresses the chemokine CXCR4.

Further, the CXCR4 receptor is (1) a protein having the amino acid sequence of SEQ ID No. 19;

Or: (2) the same or similar function as the protein of SEQ ID No. 19 obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the protein represented by SEQ ID No. 19. protein.

SEQ ID No. 19 Amino acid sequence of CXCR4

Further, in the chimeric antigen receptor-modified lymphocyte, the nucleotide sequence of the coding gene of CXCR4 is (1): the nucleotide sequence shown in SEQ ID No: 20 or a degenerate sequence thereof;

Or (2): a nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO: 20. Or a similar protein.

SEQ ID No: 20 nucleotide sequence encoding the CXCR4 gene

Wherein, the CXCR4 is co-expressed with a chimeric antigen receptor by P2A, and the amino acid sequence of the P2A is represented by SEQ ID No. 21.

SEQ ID No. 21 Amino acid sequence of P2A

Further, the nucleotide sequence of the coding gene of P2A is represented by SEQ ID No. 22.

SEQ ID No. 22 coding nucleotide sequence of P2A

Further, the P2A may be replaced by T2A, and the amino acid sequence of the T2A is represented by SEQ ID No. 23.

SEQ ID No. 23 Amino acid sequence of T2A

Further, the nucleotide sequence of the coding gene of T2A is represented by SEQ ID No. 24.

SEQ ID No. 24 The coding nucleotide sequence of T2A

In the present invention, both P2A and T2A can be used for co-expression of genes, and the advantage is that the shear efficiency is relatively high.

Wherein the chimeric antigen receptor comprises: an extracellular antigen binding region, a transmembrane region and an intracellular signal region, wherein the extracellular antigen binding region is a ligand that binds to a tumor surface specific antigen or a related antigen, and a transmembrane region In the CD8 transmembrane region or the CD28 transmembrane region, the intracellular signal region is an intracellular domain of one or more of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, CD40, CD27 or DAP10.

Wherein, the tumor surface specific antigen or related antigen includes, but not limited to, EGFR, EpCAM, mesothelin, IL-13Rα2, ERBB2, ERBB3, ERBB4, VEGFR1, VEGFR2, GD2, FR, PSMA, gp100, MUC1, MUC16, CA9, CD171, CD125, CD15-3, CD19-9, NY-ESO-1, MART-1, MAGE4, CD19, CD20, CD22, CD30, CD33, CEA, CD38, CD138, CD123, EphA2.

Further, in the chimeric antigen receptor, the chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 13;

Or: (2) the same or similar function as the protein of SEQ ID No. 13 obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the protein represented by SEQ ID No. protein.

Wherein, in the chimeric antigen receptor, the nucleotide sequence of the coding gene of the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 14 or a degenerate sequence thereof;

Or (2): a nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein.

Alternatively, in the chimeric antigen receptor, the chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 17;

Or: (2) the same or similar function as the protein of SEQ ID No. 17 obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the protein represented by SEQ ID No. 17. protein.

Wherein the nucleotide sequence of the coding gene of the chimeric antigen receptor in the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 18 or a degenerate sequence thereof;

Or (2): a nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein.

Among them, the above chimeric antigen receptor and CXCR4 are co-expressed by one vector.

The invention also provides an expression vector.

The expression vector is an expression vector for simultaneously expressing the chimeric antigen receptor of SEQ ID NO: 13 and CXCR4 of SEQ ID NO: 19. or

The expression vector is an expression vector for simultaneously expressing the chimeric antigen receptor of SEQ ID NO: 17 and CXCR4 of SEQ ID NO: 19.

Further, the expression vector is a viral vector. Further, the expression vector is a lentiviral vector.

The invention also provides a virus. The virus comprises the expression vector described above.

The invention also provides a host cell comprising the expression vector or virus described above.

Further, the above host cell is a lymphocyte. Further, the lymphocytes are T cells or monocytes, and the T cells are αβT cells, NKT cells or γδT cells.

The invention also provides the use of a chimeric antigen receptor, chimeric antigen receptor modified lymphocyte, as described above, alone or in combination with radiation therapy for the treatment of a malignant tumor. It is mainly used to prepare drugs for treating malignant tumors or to inhibit tumors in combination with radiation therapy. The malignant tumor is lung cancer, liver cancer, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer, Multiple myeloma or prostate cancer.

More specifically, the present invention includes three aspects, which are described as follows:

A first aspect of the invention relates to a chimeric antigen receptor that coexpresses CXCR4. The chimeric antigen receptor is composed of a ligand capable of binding to a tumor-specific or related antigen, a transmembrane region and an intracellular signal region, and is linked to the CXCR4 expression gene by a P2A sequence or a T2A sequence. Co-expressed on the same carrier. (for example, the structure shown in Figure 1).

A second aspect of the invention relates to obtaining a chimeric antigen receptor modified lymphocyte expressing CXCR4. The human peripheral lymphocytes are transfected with a vector which co-expresses CXCR4 and a chimeric antigen receptor provided by the first aspect of the present invention, thereby obtaining a chimeric antigen receptor-modified lymphocyte expressing CXCR4. Generally, through the lentiviral packaging system, a high titer of lentivirus is generated and transfected into human peripheral lymphocytes.

A third aspect of the invention relates to the use of a chimeric antigen receptor which coexpresses CXCR4 according to any one of the first aspects of the invention, or a lymphocyte according to the second aspect, alone or in combination with radiation therapy for the treatment of a malignant tumor.

In the present invention, a gene sequence similar to the expression "the nucleotide sequence in SEQ ID NO: 14 is substituted, deleted or added with at least one nucleotide-derived sequence" generally means that the encoding is encoded by SEQ ID NO: 14. The nucleotide sequence of the protein-active polypeptide and its degenerate sequence. The degenerate sequence refers to a sequence produced by the substitution of one or more codons in the sequence by degenerate codons encoding the same amino acid. Due to the degeneracy of the codon, a degenerate sequence having a homology of less than about 89% to SEQ ID NO: 14 can also encode the sequence set forth in SEQ ID NO: 14. In addition, the meaning of "substituting, deleting or adding at least one nucleotide-derived sequence in the nucleotide sequence of SEQ ID NO: 14" also includes being able to be under moderately stringent conditions, more preferably under highly stringent conditions. The nucleotide sequence of the nucleotide sequence of SEQ ID NO: 14 hybridization. The term also encompasses nucleotide sequences having at least 80%, preferably at least 89%, more preferably at least 90%, and optimally at least 95% homology to the nucleotide sequence of SEQ ID NO: 14.

The term "the nucleotide sequence in SEQ ID NO: 14 is substituted, deleted or added with at least one nucleotide-derived sequence" also includes SEQ ID encoding a protein having the same function as the protein encoded by SEQ ID NO: 14. NO: The variant form of the open reading frame sequence in 14. These variants include, but are not limited to, a plurality of (usually 1 to 90, preferably 1 to 60, more preferably 1 to 20, optimally 1 to 10) nucleotide deletions. , inserts and/or substitutions, and adding a few at 5' and / or 3' ends (usually within 60, preferably within 30, more preferably within 10, optimally within 5) ) nucleotides.

In the present invention, the expression "the amino acid sequence obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence" includes but is not limited to several (usually 1-50, compared Preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several at the C-terminus and/or N-terminus (usually Within 20, preferably less than 10, more preferably less than 5 amino acids. For example, in the protein, when substituted with amino acids of similar or similar properties, the function of the protein is usually not altered. As another example, the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein. The term also encompasses active fragments and active derivatives of the protein.

The expression "the amino acid sequence obtained by substituting, deleting or adding at least one amino acid in the amino acid sequence" also includes, but is not limited to, having up to 10 (ie one or several), preferably up to 8, More preferably, at most 5 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide, ie a conservative variant polypeptide. These conservative variant polypeptides are preferably produced by substitution according to Table 1.

Table 1 amino acid substitution table

Initial residue Representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln;His;Lys;Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu;Val;Met;Ala;Phe Leu Leu(L) Ile;Val;Met;Ala;Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu;Phe;Ile Leu Phe(F) Leu;Val;Ile;Ala;Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr;Phe Tyr Tyr(Y) Trp;Phe;Thr;Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

The beneficial effects of the invention are:

The present invention produces a lentivirus and transduces lymphocytes by constructing a viral expression vector which co-expresses a chimeric antigen receptor and a chemokine receptor CXCR4, thereby obtaining a chimeric antigen receptor T lymphocyte which coexpresses CXCR4, which can be reversed. The expression of CXCR4 in T cells decreased by virus modification, enhanced the chemotactic activity of chimeric antigen receptor T cells, and promoted the aggregation of lymphocytes to high concentration areas of SDF-1 (CXCL12), and the chemotaxis increased from 3% to 9%. It is beneficial to enhance the anti-tumor effect of chimeric antigen receptor T lymphocytes. When used in combination with radiation therapy, it also induces a homing effect on chimeric antigen receptor T lymphocytes, resulting in better anti-tumor effects.

DRAWINGS

Figure 1 is a schematic diagram showing the chimeric antigen receptor that coexpresses CXCR4; A indicates that the extracellular antigen binding region is a chimeric antigen receptor of scFv, B indicates that the extracellular antigen binding region is a chimeric antigen receptor of APRIL, and C indicates The extracellular antigen binding region is a chimeric antigen receptor of the receptor; wherein SP is a GM-CFSR signal peptide, scFv represents an antigen binding region, and APRIL represents a ligand binding region. CD8αhinge indicates the CD8α transmembrane region, CD28 and 4-1BB indicate the costimulatory signal domain, CD3ζ indicates the T cell activation domain, P2A is the ligated fragment, and CXCR4 is the chemokine receptor;

Figure 2 shows the expression of CXCR4 in T cells after lentivirus infection;

Figure 3 shows the chemotactic activity of T cells after lentivirus infection;

Figure 4 shows CXCR4 expression in CAR-T cells after coexpression of CXCR4;

Figure 5 shows the chemotactic activity of CAR-T cells after coexpression of CXCR4; A indicates the CAR-T chemotaxis activity induced by RPMI-8226 cell culture supernatant, and B indicates the CAR-T trend induced by SDF-1 (ie CXCL12). Activity

Figure 6 shows CAR expression of CAR-T and CAR/CXCR4-T cells;

Figure 7 shows the killing activity of CAR-T and CAR/CXCR4-T cells.

detailed description

The invention will be described in detail below with reference to the accompanying drawings. In the following examples, where no specific experimental conditions are indicated, they are in accordance with conventional conditions well known to those skilled in the art, such as Sambrook J, Russell DW, 2001, Molecular Cloning: A laboratory manual (3 ^rd ed), Spring Harbor. The conditions described in the Laboratory Press, or in accordance with the conditions recommended by the manufacturer.

Example 1 The full-length gene of CAR, CAR/CXCR4 was obtained, and the construction of recombinant plasmid vector was completed.

I. Acquisition of full-length scFv-CAR and scFv-CAR/CXCR4 genes

The scFv-CAR, scFv-CAR-P2A-CXCR4 gene fragment was obtained by whole gene synthesis.

At the same time, the following primers were designed to amplify the scFv-CAR fragment:

5' Primer: 5'AGGTTTAAACTACGGATGGTGCTGCTGGTGACCTCCC3'SEQ ID NO:25

3' Primer: 5'ATGACTAGTCCCGGGTTAGCGAGGGGGCAGGGCCTGC3'SEQ ID NO:26

The reaction conditions are as follows:

PCR reaction: denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 68 ° C for 1 minute. The reaction was carried out for 25 cycles. Then extend at 72 ° C for another 10 minutes.

The following primers were designed to amplify the scFv-CAR/CXCR4 fragment:

5' Primer: 5'AGGTTTAAACTACGGATGGTGCTGCTGGTGACCTCCC3'SEQ ID NO:27

3' Primer: 5'ATGACTAGTCCCGGGTCAGCTGGAGTGGAAGCTGGAG 3'SEQ ID NO:28

The reaction conditions are as follows:

PCR reaction: denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 68 ° C for 2 minutes. The reaction was carried out for 25 cycles. Then extend at 72 ° C for another 10 minutes.

2. Acquisition of full-length APRIL-CAR and APRIL-CAR/CXCR4 genes

The APRIL-CAR, APRIL-CAR-P2A-CXCR4 gene fragment was obtained by whole-genome synthesis.

At the same time, the following primers were designed to amplify the APRIL-CAR fragment:

5' Primer: 5'aggtttaaactacggATGGAGACCGACACCCTGCTGC3'SEQ ID NO:29

3' Primer: 5'atgactagtcccgggTTAGCGAGGGGGCAGGGCCTGC3'SEQ ID NO:30

The reaction conditions are as follows:

PCR reaction: denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 68 ° C for 1 minute. The reaction was carried out for 25 cycles. Then extend at 72 ° C for another 10 minutes.

The following primers were designed to amplify the APRIL-CAR/CXCR4 fragment:

5' Primer: 5'aggtttaaactacggATGGAGACCGACACCCTGCTGC3'SEQ ID NO:31

3' Primer: 5'atgactagtcccgggTCAGCTGGAGTGGAAGCTGGAG 3'SEQ ID NO:32

The reaction conditions are as follows:

PCR reaction: denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 68 ° C for 2 minutes. The reaction was carried out for 25 cycles. Then extend at 72 ° C for another 10 minutes.

Construction of lentiviral recombinant plasmid containing full-length CAR and CAR/CXCR4 genes

As described above, in the whole gene-synthesis fragment, there is no restriction site, and we used the method of homologous recombination to directly insert the fragment into the multiple cloning site EcoR I/BamH I of the eukaryotic expression vector pWPXLd (purchased from American addgene). In the middle, the restriction site will disappear after recombination. The construction process is as follows:

1. Construction of recombinant lentiviral plasmid pWPXLd-CAR

The lentiviral vector pWPXLd was linearized by restriction endonucleases EcoR1 and BamH1, and the full-length CAR gene amplified fragment was inserted into the EcoR I/ of pWPXLd using the Tiangen EasyGeno Rapid Recombination Cloning Kit (Tiangen Biochemical Technology Co., Ltd., VI201). At the BamH I site, the recombinant product pWPXLd-CAR was transformed into E. coli Stbl3, and 30 clones were randomly selected for sequencing. The sequencing results were consistent with the designed full-length CAR sequence.

2. Construction of recombinant lentiviral plasmid pWPXLd-CAR/CXCR4

The lentiviral vector pWPXLd was linearized by restriction endonucleases EcoR1 and BamH1, and the full-length CAR/CXCR4 gene amplified fragment was inserted into the ecoR of pWPXLd using the Tiangen EasyGeno Rapid Recombination Cloning Kit (Tiangen Biochemical Technology Co., Ltd., VI201). The I/BamH I site, the recombinant product pWPXLd-CAR/CXCR4 was transformed into E. coli Stbl3, and 30 clones were randomly selected for sequencing. The sequencing results were consistent with the designed full length CAR/CXCR4 sequence.

The schematic diagram of the constructed lentiviral recombinant vector is shown in Figure 1. Figure 1A, Figure 1B and Figure 1C show the recombinant vector structure when the extracellular antigen binding region of the chimeric antigen receptor is scFv, ligand and receptor binding region, respectively. .

Example 2 Lentiviral packaging and production of CAR-T cells

1, slow virus packaging

293T cells were cultured in DMEM + 10% fetal bovine serum (FBS) and used for virus packaging when grown to 70-90% density. The procedure was as follows: fresh pre-warmed DMEM medium (containing 10% FBS) 2 h before transfection The transfection system was prepared in a 15 ml centrifuge tube: the experimental conditions are shown in Tables 2 and 3.

Table 2 plasmid system

Table 3 Transfection system:

After mixing, the transfection solution was evenly added to the dish, and the cultivation was continued at 37 ° C in a 5% CO ₂ incubator. After 12h, it was replaced with DMEM+2% FBS medium. The virus supernatant was collected at 48 h and 72 h, respectively, and the virus solution was filtered through a 0.45 μm filter, centrifuged at 4 ° C, 160,000 g, ultracentrifuged for 90 min, resuspended in PBS, and stored in -80 °.

2, T cell infection

Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (sigma). The procedure was as follows: an equal volume of peripheral blood was slowly added to Ficoll-Hypaque, 1000 g, centrifuged at 18 ° for 30 minutes, and the middle layer of white membrane was taken, and erythrocyte lysis was performed. The solution was applied for 2 minutes, centrifuged at 300 g for 5 minutes, washed twice in 1640 medium, counted, and co-cultured with CD3/CD28 Dynabeads (life technologies 11131D) (cells: beads = 1:3). After 24h, the lentivirus infects the activated PBMC by the following steps: centrifugation of the cells, resuspending the cells with the medium containing the virus supernatant (infection multiplicity MOI = 1:10), 1 × 10 ⁶ /ml cells, adding polybrene (polybrene) ), the final concentration was 8 mg/ml, and the mixture was centrifuged at 1000 g for 90 minutes at 32 °, and then cultured in a 37 ° C 5% CO ₂ incubator.

Example 3 Detection of CXCR4 expression in T cells

The lentivirus-infected or uninfected T cells and peripheral blood mononuclear cells constructed in Example 1 were exchanged after 24 hours, and X-VIVO 15 (04-418Q, LONZA) complete medium + 10% human AB serum was used ( H4522, sigma) continued to culture for 5 days while adding recombinant human interleukin 2 (IL-2) cytokine 100 U/ml (C013, novaprotein). The cells were then collected, washed twice with PBS, stained with APC-labeled anti-human CXCR4 antibody (Cat. No. 306510, biolegend), and analyzed for cell membrane CXCR4 expression by flow cytometry. The results showed that after infection with a lentivirus containing the full-length gene of CAR, the periphery The expression of CXCR4 in blood mononuclear cells decreased significantly, and the fluorescence intensity (logarithm) decreased from 238 to 174 (Fig. 4). The expression of CXCR4 decreased more significantly after T cells infected with lentivirus, and the fluorescence intensity (logarithm) decreased from 239 to 108. , reduced by more than half (as shown in Figure 2).

Example 4 Detection of T cell chemotactic activity of lentivirus infection

After the decrease in CXCR4 expression was observed in the infected peripheral blood mononuclear cells and T cells in Example 3, in order to evaluate the effects thereof, the mediated chemotaxis activity was further examined. The specific steps are as follows:

Lymphocyte chemotactic activity assays were assessed by cell migration assays. The transwell polycarbonate membrane was soaked in PBS containing 20 μg/ml BSA at 4 ° C overnight at 5 μm pore size (Cat. No. 3421; Costar, Cambridge, MA) transwell, and placed in a 24-well plate to dry before use.

Pre-test, infected (CAR-T group) or uninfected (control group) lentiviral cell T cells and peripheral blood mononuclear cells in RPMI1640 complete medium (containing 1% BSA, 10 mM HEPES buffer, pH 6.9) serum Hunger for 4 hours.

At the time of the experiment, 500 ul of serum-free RPMI1640 complete medium containing 100 ng/ml SDF-1 was added to a 24-well plate; the dried transwell was placed in the well, and then 200 ul of Far red-labeled serum-starved cells (5 × 10 ⁵ ) were added. Transwell upper room. Incubate for 4 h at 37 ° C, 5% CO ₂ , collect the cells from the lower chamber, pellet the pellet, and resuspend in 300 ul of pre-cooled FACS buffer (containing 10% calf serum and 0.2% sodium azide), each Add 1 × 10 ⁵ quantity of 9 micron latex beads (BCR167, sigma) and 25 μl of 50 ug/ml Propidium Iodide/propidium iodide working solution (ST511, Biyuntian Biotechnology Co., Ltd.). The sample was detected by flow cytometry.

Since the number of latex beads in each group is the same, it is used as a reference. Statistical PI staining negative cells can be calculated

The analysis found that after infection with lentivirus, the migration rate of peripheral monocytes decreased from 16.7% to 9.2% and the migration rate of T cells decreased from 19.3% to 4.1% under SDF-1 chemotaxis (Fig. 3). As the expression of CXCR4 is decreased, the chemotactic activity of T cells and peripheral monocytes is also significantly decreased.

Example 5 Expression of CXCR4 enhances chimeric antigen receptor-modified T cell chemotactic activity

To reverse the chemotactic activity of T cells, we overexpressed CXCR4 in T cells to enhance the chemotactic activity of Lentivirus-infected CAR-T cells.

First, CXCR4 was co-expressed in CAR-T cells. Lentivirus-infected T cells containing full-length CAR (CAR-T group) or CAR/CXCR4 (CAR/CXCR4-T group), cells were collected 48 h later, washed twice with PBS, APC-labeled anti-human CXCR4 antibody (Cat. No. 306510, Biolegend staining and flow cytometry were used to detect the expression of CXCR4 in cell membrane. The results showed that compared with CAR-T group, the expression of CXCR4 in T cells of CAR/CXCR4 group was significantly increased, and the average fluorescence intensity (logarithm) increased from 108 to 229. (As shown in Figure 4).

Secondly, the chemotaxis activity was tested in the same manner as in Example 4: using a 5 μm pore size (Cat. No. 3421; Costar, Cambridge, MA) transwell, and the polycarbonate membrane in transwell was soaked overnight at 4 ° C in PBS containing 20 μg/ml BSA. Store in a 24-well plate before use.

Before the test, T cells expressing (CAR/CXCR4-T group) or not expressing (CAR-T group) CXCR4 were serum starved for 4 hours in RPMI1640 complete medium (containing 1% BSA, 10 mM HEPES buffer, pH 6.9). .

At the time of the experiment, 500 ul of serum-free RPMI1640 complete medium containing 100 ng/ml SDF-1 or RPMI-8226 cell culture supernatant was added to a 24-well plate; the dried transwell was placed in the well, and then 200 ul of Far red-labeled serum was starved. Cells (5 x 10 ⁵ ) were added to the transwell upper chamber. Incubate for 4 h at 37 ° C, 5% CO ₂ , collect the cells from the lower chamber, pellet the pellet, and resuspend in 300 ul of pre-cooled FACS buffer (containing 10% calf serum and 0.2% sodium azide), each Add 1 × 10 ⁵ quantity of 9 micron latex beads (BCR167, sigma) and 25 μl of 50 ug/ml Propidium Iodide/propidium iodide working solution (ST511, Biyuntian Biotechnology Co., Ltd.). The sample was detected by flow cytometry.

Since the number of latex beads in each group is the same, it is used as a reference. Statistical PI staining negative cells can be calculated

The results showed that culture supernatant of RPMI-8226 cells (highly expressed and secreted with SDF-1) promoted CAR/CXCR4-T cell migration from less than 2.9% to 4.15% (Fig. 5A); in chemokine SDF-1 (Fig. 5A) When the concentration of CXCL12) was 100 ng/ml, the chemotaxis efficiency of CAR-T cells co-expressing CXCR4 was significantly increased, which was increased from 3% to 9% in the CAR-T group, which was increased by three times (Fig. 5B); Co-expression of CXCR4 can enhance the chemotactic activity of CAR-T cells to migrate to high concentrations of SDF-1.

Example 6 CAR expression and activity assay

T cells were infected with lentivirus containing full-length CAR or CAR/CXCR4. After 5 days of culture, cells were harvested and stained with FITC-labeled recombinant human BCMA protein (novaprotein) to detect T cell CAR expression. The results showed that compared with the control group (less than 5%), the CAR expression was significantly increased (up to 40%) in the CAR group and the CAR/CXCR4 group, and the fluorescence peak shifted (Fig. 6).

其中阴性组未加T细胞)。 Different concentrations of the above T cells, CAR-T cells or CAR/CXCR4-T cells (ie, effector cells) were co-cultured with CSFE-labeled human myeloma cell line RPMI-8226 cells (target cells, BCMA high expression), 24 h later. A total of 1×10 ⁵ Far red-labeled K562 cells were added to each group, washed twice with PBS, and the ratio of fluorescent cells was detected by flow cytometry FITC and APC channels. Because the number of Far red-labeled K562 cells in each group is the same, K562 cells can be used as a reference.

Among them, the negative group did not add T cells).

The results showed that when the number of effector cells/number of target cells (E/T)=1:1, the killing efficiency of the control cells to RPMI-8226 cells was 23.7%, while the CAR-T cells and CAR/CXCR4-T cells were respectively. Up to 45.3% and 51%; when the number of effector cells/number of target cells (E/T)=5:1, the killing efficiency of the control cells to RPMI-8226 cells was about 44.7%, while CAR-T cells and CAR/ CXCR4-T cells reached 75.1% and 82.3%, respectively (Fig. 7). The results showed that chimeric antigen receptor (CAR) can enhance the killing activity of T cells against RPMI-8226, while coexpression of CXCR4 can enhance CAR-T cells. Killing activity.

In summary, the above assay shows that the present invention can eliminate the chemotaxis of T cells expressing CAR-T by constructing a co-expressing chimeric antigen receptor and a CXCR4 lentiviral expression vector and introducing into T cells. The defect provides a lymphocyte with improved killing activity and chemotactic activity, which is beneficial to enhance the antitumor effect of chimeric antigen receptor-modified lymphocytes.

Claims (33)

A chimeric antigen receptor-modified lymphocyte co-expressing the chemokine CXCR4. The chimeric antigen receptor-modified lymphocyte according to claim 1, wherein the CXCR4 receptor is (1) a protein having the amino acid sequence of SEQ ID No. 19; Or: (2) the same or similar function as the protein of SEQ ID No. 19 obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the protein represented by SEQ ID No. 19. protein. The chimeric antigen receptor-modified lymphocyte according to claim 2, wherein the nucleotide sequence encoding the CXCR4 is (1): the nucleotide sequence shown in SEQ ID No. 20 or Degenerate sequence thereof; Or (2): a nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein. The chimeric antigen receptor-modified lymphocyte according to claim 1, wherein said CXCR4 is co-expressed with a chimeric antigen receptor by P2A. The chimeric antigen receptor-modified lymphocyte according to claim 4, wherein the amino acid sequence of the P2A is represented by SEQ ID No. 21. The chimeric antigen receptor-modified lymphocyte according to claim 4, wherein the nucleotide sequence of the gene encoding the P2A is represented by SEQ ID No. 22. The chimeric antigen receptor-modified lymphocyte according to claim 1, wherein the chimeric antigen receptor comprises: an extracellular antigen binding region, a transmembrane region, and an intracellular signal region. The chimeric antigen receptor-modified lymphocyte according to claim 1, wherein: The extracellular antigen binding region is a single-chain variable region, receptor or ligand of a monoclonal antibody that binds to a tumor antigen; The transmembrane region is a CD8 transmembrane region or a CD28 transmembrane region; The intracellular signal region is an intracellular domain of one or more of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, CD40, CD27 or DAP10. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the tumor antigen comprises, but not limited to, EGFR, EpCAM, mesothelin, IL-13Rα2, ERBB2, ERBB3, ERBB4, VEGFR1, VEGFR2 , GD2, FR, PSMA, gp100, MUC1, MUC16, CA9, CD171, CD125, CD15-3, CD19-9, NY-ESO-1, MART-1, MAGE4, CD19, CD20, CD22, CD30, CD33, CEA , CD38, CD138, CD123, EphA2. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the monoclonal antibody single-chain variable region is a scFv single-chain antibody. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the amino acid sequence of the scFv single-chain antibody is represented by SEQ ID No. 1. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the coding nucleotide sequence of the scFv single-chain antibody is represented by SEQ ID No. 2. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the chimeric antigen receptor sequentially binds a signal peptide, a scFv single-chain antibody, and a CD8α transmembrane through a nitrogen terminal to a carbon terminal. Region, CD28 intracellular segment, 4-1BB intracellular segment, CD3 ζ chain obtained. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 13; Or: (2) the same or similar function as the protein of SEQ ID No. 13 obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the protein represented by SEQ ID No. protein. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein: The nucleotide sequence of the gene encoding the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 14 or a degenerate sequence thereof; Or (2): a nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the ligand is APRIL. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the amino acid sequence of the APRIL is represented by SEQ ID NO. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the nucleotide sequence encoding the APRIL is represented by SEQ ID NO. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein the chimeric antigen receptor sequentially binds a signal peptide, APRIL, CD8α transmembrane region, CD28 through a nitrogen terminal to a carbon terminal. Intracellular segment, 4-1BB intracellular segment, CD3 ζ chain obtained. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein: The chimeric antigen receptor is (1) a protein having the amino acid sequence of SEQ ID No. 17; Or: (2) the same or similar function as the protein of SEQ ID No. 17 obtained by substituting and/or deleting and/or adding at least one amino acid in the amino acid sequence of the protein represented by SEQ ID No. 17. protein. The chimeric antigen receptor-modified lymphocyte according to claim 8, wherein: The nucleotide sequence of the gene encoding the chimeric antigen receptor is (1): the nucleotide sequence shown in SEQ ID No. 18 or a degenerate sequence thereof; Or (2): a nucleotide sequence derived by substituting, deleting or adding at least one nucleotide in the nucleotide sequence defined by (1), and having the same function as the nucleotide sequence of SEQ ID NO. Or a similar protein. A chimeric antigen receptor according to any one of claims 1 to 21 which is co-expressed with CXCR4 by a vector. An expression vector comprising the expression vector encoding the nucleic acid of any one of claims 1 to 21. The expression vector according to claim 23, wherein the expression vector is a lentiviral vector. A virus system comprising the expression vector of claim 23 or 24 in the viral system. Use of an expression vector according to claim 23 or 24, characterized in that it is used for the preparation of chimeric antigen receptor-modified lymphocytes targeting tumor cells. A host cell comprising the expression vector or virus of any one of claims 23 to 24. The host cell according to claim 27, wherein the host cell is a T lymphocyte. The host cell according to claim 28, wherein the T lymphocytes are αβ T cells, NKT cells or γδ T cells. A method of producing the host cell of claim 28 or 29, comprising the step of expressing the chimeric antigen receptor of any of claims 1-21 on a lymphocyte. Use of the chimeric antigen receptor-modified lymphocytes of claims 1-21 for the preparation of a medicament for the treatment of malignancy. Use of the chimeric antigen receptor-modified lymphocytes of any of claims 1-21 for use in combination with radiation therapy to inhibit tumors. The use according to claim 32, characterized in that the malignant tumor is lung cancer, liver cancer, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, esophageal cancer, renal cancer, Glioma, melanoma, pancreatic cancer, multiple myeloma or prostate cancer.

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