CN107868791B

CN107868791B - Preparation method and application of reinforced Slit2CAR-T and CAR-NK cells

Info

Publication number: CN107868791B
Application number: CN201610850567.7A
Authority: CN
Inventors: 李华顺; 王保垒; 任宝永; 方冬冬
Original assignee: Asclepius Suzhou Technology Company Group Co Ltd
Current assignee: Asco (suzhou) Biotechnology Co Ltd
Priority date: 2016-09-26
Filing date: 2016-09-26
Publication date: 2021-11-23
Anticipated expiration: 2036-09-26
Also published as: WO2018053885A1; CN107868791A

Abstract

The invention provides a preparation method and application of reinforced Slit2CAR-T and CAT-NK cells, wherein the reinforced CAR-T and CAT-NK cells are respectively T cells and NK cells containing reinforced CAR chimeric antigen receptors, and the reinforced CAR chimeric antigen receptors comprise extracellular domains capable of combining antigens, transmembrane domains, intracellular immune co-stimulatory molecules, internal ribosome entry sites IRES and HAC-HSA which are connected in series; wherein the extracellular domain comprises the D2 domain of Slit2 protein. The reinforced CAR-T cells and reinforced CAR-NK cells can seal PDL-1 inhibitory signals, enhance CAR-immune cell killing activity and activate tumor-infiltrated immune cells; the reinforced CAR-immune cell can be used as a cell drug for treating tumor diseases, so that the modified immune cell can specifically recognize and kill tumors, and has more efficient tumor killing activity.

Description

Preparation method and application of reinforced Slit2CAR-T and CAR-NK cells

Technical Field

The invention relates to the technical field of biology, in particular to a chimeric antigen receptor cell aiming at an anti-tumor stem cell, and particularly relates to a reinforced CAR-T cell and a reinforced CAR-NK cell.

Background

PD-L1 is known as programmed cell death receptor-Ligand 1, English name programmed cell death-Ligand 1, and is the first type transmembrane protein with the size of 40 kDa. PD-1 is known as programmed death receptor 1, and is called programmed death 1 in English, is an important immunosuppressive molecule and is a member of CD28 superfamily. PD-1(programmed death-1) is mainly expressed on the surfaces of T cells and primary B cells, and two ligands (PD-L1 and PD-L2) of PD-1 are widely expressed in Antigen Presenting Cells (APCs) and the like. The interaction of PD1 with its receptor plays an important role in the negative regulation of the immune response. The expression of PD-L1 protein can be detected in a plurality of human tumor tissues, the microenvironment of the tumor part can induce the expression of PD-L1 on tumor cells, and the expressed PD-L1 is beneficial to the generation and growth of tumors and induces the apoptosis of anti-tumor T cells so as to escape the attack of an immune system. The combination of PD-1 and its ligand is inhibited, so that the tumor cell is exposed to the killing visual field of immune system, and the tumor tissue and cancer are killed.

At the 2016AACR annual meeting, Prof. Paul T. Nghiem, MD, PhD, professor of the Dermatology of the medical school of Washington university, Pembrolizumab, a monoclonal antibody with anti-PD-1 action, induced high reactivity of high-grade merkel cell carcinoma, with a median progression-free survival PFS of 9 months, and a patient with traditional chemotherapy PFS of 3 months. Recent advances in clinical trials with the bevaet PD-1 inhibitor, Opdivo, released two sets of data, one of which showed that patients with advanced melanoma who did not respond to treatment with any of the current drugs achieved 34% of five-year survival in treatment with Opdivo. Notably, the five-year survival rate for stage IV melanoma patients is typically only 15% to 20%. Another set of data indicates that 22% total remission was achieved in patients with advanced melanoma treated with combinations of Opdivo and yrervoy, and that 69% overall survival was achieved for two years. Another study showed that refractory relapsed or metastatic Squamous Cell Carcinoma of Head and Neck (SCCHN) patients had greatly improved survival and survival rates following treatment with Opdivo. The data show a significant 30% reduction in the risk of death in the Opdivo-treated group compared to the control group and a significant prolongation of median overall survival. The one-year survival rate of the Opdivo treated group was 36%, and that of the control group was 16.6%. In addition, the study evaluated the efficacy of Opdivo relative to the control regimen by oropharyngeal tumor HPV status and PD-L1 expression status. Recently, Keytruda received a breakthrough drug qualification to FDA treatment of relapsed or refractory (R/R) classic Hodgkin lymphoma (cHL).

CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy), a Chimeric Antigen Receptor T-Cell Immunotherapy. This therapy is a new type of cell therapy that has been developed for many years but has only been improved into clinical use in recent years. Has significant efficacy in the treatment of acute leukemia and non-hodgkin's lymphoma, and is considered to be one of the most promising tumor treatment modalities. Chimeric Antigen Receptors (CAR) T cells (CAR-T) have been constructed by genetic modification of the patient's own T cells and have been successful in treating blood cancers, but the use of CAR-T to treat solid tumors, which consist of heterogeneous cell populations with different surface molecules, has not achieved the desired therapeutic effect.

CN105505869A discloses a chimeric receptor T cell for tumor stem cells, wherein the T cell is chimeric with 2-3 independent antigen receptors, and each chimeric antigen receptor is composed of an antigen binding part of an antibody of a different tumor stem cell specific marker and different functional proteins. The chimeric receptor T cell can activate the anti-tumor effect of the T cell only after 2-3 chimeric antigen receptors all recognize antigens, and the method improves the specificity to a certain extent but has lower specificity to tumor cells hiding the antigens.

The document reported by Liza B.John (Block of PD-1 immunosuppressive carbohydrates CAR T-cell therapy) discloses that the addition of anti-PD-1 antibodies blocks the PD-1 immunosuppressive pathway, i.e., the binding of PD-1 to the ligand PD-L, thereby increasing to some extent the anti-tumor efficacy of genetically modified chimeric antigen receptor expressing T-cells (CAR-T).

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a CAR chimeric antigen receptor, a reinforced CAR-immune cell and a preparation method thereof.

In one aspect, the invention provides a gene encoding a chimeric antigen receptor for a CAR, comprising a gene encoding an extracellular domain capable of binding an antigen, a signaling domain, an intracellular immune co-stimulatory molecule, an Internal Ribosome Entry Site (IRES), and HAC-HSA.

The extracellular domain comprises the D2 domain of Slit2 protein; slit2D2 for short, and the coding gene thereof has the nucleotide sequence shown in SEQ ID No: 1;

the HAC coding gene has the sequence shown in SEQ ID No: 2;

preferably, the signalling domain is selected from one or more of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, the hinge region, the transmembrane region and the intracellular region domain of CD 154.

Preferably, the signaling domain is selected from the group consisting of the Hinge region, transmembrane region and intracellular region of CD8, more preferably, the CD8 comprises the Hinge region and transmembrane region of CD 8;

preferably, the intracellular immune co-stimulatory molecule is selected from one or more of the intracellular domains of CD3 ζ, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB and OX 40;

preferably, the intracellular immune co-stimulatory molecule comprises the 4-1BB and CD3 ζ intracellular domains;

preferably, the coding gene also comprises a nucleotide sequence shown as SEQ ID No: 3 (SP1) encoding a Signal peptide 1;

preferably, the coding gene also comprises a nucleotide sequence shown as SEQ ID No: 4, a Flag-encoding gene;

preferably, the coding gene also comprises a nucleotide sequence shown as SEQ ID No: 5, a Linker coding gene;

preferably, the coding gene also comprises a nucleotide sequence shown as SEQ ID No: 6 (SP2) or a Signal peptide 2;

preferably, the coding gene comprises a nucleotide sequence shown as SEQ ID No: 7, SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 zeta fusion gene;

preferably, the coding gene comprises a nucleotide sequence shown as SEQ ID No: the SP2-HAC-HSA fusion gene shown in 8;

in a preferred embodiment of the invention, the gene encoding the CAR chimeric antigen receptor is a gene encoding SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 zeta-IRES-SP 2-HAC-HSA, and the nucleotide sequence of IRES is as shown in SEQ ID No: shown at 9.

In another aspect, the present invention provides a recombinant vector, a recombinant strain, and a recombinant vector containing the above-described coding gene;

preferably, the recombinant vector is selected from the group consisting of: lentivirus, retrovirus, adenovirus, adeno-associated virus or plasmid.

The invention also provides the application of the coding gene, the recombinant vector loaded with the coding gene and the recombinant strain in modifying immune cells and preparing antitumor drugs;

preferably, the immune cell is selected from the group consisting of: t cells, NK cells such as NK 92.

In another aspect, the invention provides a reinforced CAR-immune cell, wherein the cell contains the coding gene;

preferably, the enhanced CAR-immune cell is an enhanced CAR-T cell or an enhanced CAR-NK cell such as an enhanced CAR-NK92 cell.

The invention also provides application of the reinforced CAR-immune cell in preparation of antitumor drugs.

In another aspect, the present invention provides a CAR chimeric antigen receptor obtained by transcriptional expression of the above coding gene, comprising: an extracellular domain capable of binding antigen, a transmembrane domain, an intracellular immune co-stimulatory molecule, an Internal Ribosome Entry Site (IRES), and HAC-HSA.

The extracellular domain comprises the D2 domain of Slit2 protein.

Preferably, the signalling domain is selected from one or more of the hinge region, transmembrane region and intracellular region of one of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, CD 154.

Preferably, the signaling domain is selected from the group consisting of a Hinge region, a transmembrane region and an intracellular region of CD8, more preferably, the CD8 is the Hinge region of CD8 and CD8^TMA transmembrane domain.

Preferably, the intracellular immune co-stimulatory molecule is selected from one or more of the intracellular domains of CD3 ζ, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB and OX 40.

Preferably, the intracellular immune co-stimulatory molecule comprises the 4-1BB and CD3 zeta intracellular domains.

In the HAC-HSA, HAC is PD-1 protein with High-affinity consensus (HAC), and can block the combination of wild-type PD-1 protein and PD-L ligand in vivo and in vitro;

the HAC lacks a transmembrane domain relative to the wild-type PD-1 protein and has one or several amino acid residue changes, while at the same time, having an improved affinity for PD-L1 ligand;

the amino acid residue changes may be located in the PD-1 and PD-L1 binding domains, and/or,

the amino acid residue changes may be located in the immunoglobulin domain of PD-1.

In a specific embodiment, the HAC comprises an amino acid sequence selected from 85% or greater, 90% or greater, 95% or greater, 98%, 99% or greater, 99.2% or greater, relative to the wild-type PD-1 protein polypeptide identity.

In another specific embodiment, the HAC comprises an amino acid sequence that is 85% or greater, 90% or greater, 95% or greater, 98%, 99% or greater, 99.2% or greater, 99.8% or greater, 99.9% or greater, or 100% identical to the immunoglobulin domain of the wild-type PD-1 protein polypeptide.

In another embodiment, the HAC comprises an amino acid sequence as set forth in SEQ ID No: 6, is 85% or greater, 90% or greater, 95% or greater, 98%, 99% or greater, 99.2% or greater, 99.8% or greater, 99.9% or greater, or 100%.

Preferably, the HAC comprises an amino acid mutation that increases the affinity of the HAC for PD-L1 relative to a wild-type PD-1 protein polypeptide;

the amino acid change is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, etc.

Preferably, in a particular embodiment, said amino acid residue is altered at a position selected from the group consisting of amino acid residues as set forth in SEQ ID No: 6, one or more of V39, L40, N41, Y43, R44, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, V72, H82, M83, R90, Y96, L97, A100, S102, L103, A104, P105, K106 and A107 in the wild-type PD-1 fragment; or, amino acids at corresponding positions of other wild-type PD-1 proteins; the amino acid change comprises a change of 1 or more amino acids; the plurality of amino acid changes are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more.

Preferably, in another embodiment, the amino acid residue alteration may be in the PD-1 and PD-L1 binding domains, and the amino acid residue alteration site is located as set forth in SEQ ID No: 6, selected from the group consisting of V39, N41, Y43, M45, S48, N49, Q50, T51, D52, K53, a56, Q63, G65, Q66, L97, S102, L103, a104, P105, K106, a 107; or, one or more amino acids at corresponding positions of other wild type PD-1 proteins; the amino acid change comprises a change of 1 or more amino acids; the plurality of amino acid changes are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more.

In another embodiment, the amino acid residue is altered at a position as set forth in SEQ ID No: 6 selected from the group consisting of: (a) v39, N41, Y43, M45, S48, N49, Q50, K53, a56, Q63, G65, Q66, L97, a100, S102, L103, a104, K106 and a 107; or, amino acids at corresponding positions of other wild-type PD-1 proteins; (b) v39, N41, Y43, M45, S48, Q50, T51, D52F, K53, a56, Q63, G65, Q66, L97, S102, L103, a104, K106 and a 107; or, amino acids (c) V39, L40, N41, Y43, R44, M45, N49, K53, M83, L97, a100 and a107 at corresponding positions of other wild-type PD-1 proteins; or, amino acids at corresponding positions of other wild-type PD-1 proteins; (d) v39, L40, N41, Y43, R44, M45, N49, Q66P, M83, L97 and a 107; or, amino acids at corresponding positions of other wild-type PD-1 proteins; (e) v39, L40, N41, Y43, M45, N49, K53, Q66P, H82, M83, L97, a100 and a 107; or, amino acids at corresponding positions of other wild-type PD-1 proteins; (f) v39, L40, N41, Y43, M45, N49, K53, M83, L97, a100, and a 107; or, amino acids at corresponding positions of other wild-type PD-1 proteins; (g) v39, L40, N41, Y43, R44, M45, N49, K53, L97, a100 and a 107; (h) v39, L40, N41, Y43, M45, S48, N49, K53, L97, a100 and a 107; or, other wild-type PD-1 protein corresponding position amino acid.

In another embodiment, the amino acid residue is altered at a position as set forth in SEQ ID No: 6 selected from the group consisting of: (1) V39H or V39R; (2) L40V or L40I; (3) N41I or N41V; (4) Y43F or Y43H; (5) R44Y or R44L; (6) M45Q, M45E, M45L or M45D; (7) S48D, S48L, S48N, S48G or S48V; (8) N49C, N49G, N49Y or N49S; (9) Q50K, Q50E or Q50H; (10) T51V, T51L or T51A; (11) D52F, D52R, D52Y or D52V; (12) K53T or K53L; (13) a56S or a 56L; (14) Q63T; Q63I, Q63E, Q63L or Q63P; (15) G65N; G65R, G65I, G65L, G65F or G65V; (16) Q66P; (17) V72I; (18) H82Q; (19) M83L or M83F; (20) R90K; (21) Y96F; (22) L97Y, L97V or L97I; (23) a100I or a 100V; (24) S102T or S102A; (25) L103I, L103Y or L103F; (26) a104S, a104H or a 104D; (27) P105A; (28) K106G, K106E, K106I, K106V, K106R or K106T; (29) A107P; a107I or a 107V; or, other wild-type PD-1 protein corresponding position amino acid.

In another embodiment, the amino acid residue is altered at a position as set forth in SEQ ID No: 6 selected from the group consisting of: (a) { V39H or V39R }, { N41I or N41V }, { Y43F, Y43H }, { M45Q, M45E, M45L or M45D }, { S48D, S48L, S48N, S48G or S48V }, { N49C, N49G, N49Y or N49S }, { Q50K, Q50E or Q50H }, { K53T or K53L }, { a56S or a56L }, { Q63T, Q63I, Q63E, Q63L or Q63P }, { G65N, G65R, G65I, G65L, G65F or G65V, 97Y, L Y, Y a Y, Y K Y, Y K Y, Y K Y, Y K, Y K Y, Y K, Y K Y, Y K, Y; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(b) { V39 or V39 }, { N41 or N41 }, { Y43 or Y43 }, { M45, M45 or M45 }, { S48, S48 or S48 }, { Q50, Q50 or Q50 }, { T51, T51 or T51 }, { D52, D52 or D52 }, { K53 or K53 }, { a56 or a56 }, { Q63, Q63 or Q63 }, { G65, G65 or G65 }, { Q66 }, { L97, L97 or L97 }, { S102 or S102 }, { L103, L103 or L103 }, { a104, a104 or a104 }, { K106, K106 or K107, a107, or a107 }; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(c) { V39H or V39R }, { L40V or L40I }, { N41I or N41V }, { Y43F or Y43H }, { R44Y or R44L }, { M45Q, M45E, M45L or M45D }, { N49C, N49G, N49Y or N49S }, { K53T or K53L }, { M83L or M83F }, { L97Y, L97V or L97I }, { a100I or a100V }, { a107P, a107I or a107V }; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(d) { V39H or V39R }, { L40V or L40I }, { N41I or N41V }, { Y43F or Y43H }, { M45Q, M45E, M45L or M45D }, { N49C, N49G, N49Y or N49S }, { K53T or K53L }, { Q66P }, { M83L or M83F }, { L97Y, L97V or L97I } and { a107P, a107I or a107V }; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(e) { V39H or V39R }, { L40V or L40I }, { N41I or N41V }, { Y43F or Y43H }, { M45Q, M45E, M45L or M45D }, { N49C, N49G, N49Y or N49S }, { K53T or K53L }, { Q66P }, { H82Q }, { M83L or M83F }, { L97Y, L97V or L97I }, { a100I or a100V }, and { a107P, a107I or a107V }; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(f) { V39H or V39R }, { L40V or L40I }, { N41I or N41V }, { Y43F or Y43H }, { M45Q, M45E, M45L or M45D }, { N49C, N49G, N49Y or N49S }, { K53T or K53L }, { M83L or M83F }, { L97Y, L97V or L97I }, { a100I or a100V } and { a107P, a107I or a107V }; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(g) { V39H or V39R }, { L40V or L40I }, { N41I or N41V }, { Y43F or Y43H }, { R44Y or R44L }, { M45Q, M45E, M45L or M45D }, { N49C, N49G, N49Y or N49S }, { K53T or K53L }, { L97Y, L97V or L97I }, { a100I or a100V } and { a107P, a107I or a107V }; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(h) { V39H or V39R }, { L40V or L40I }, { N41I or N41V }, { Y43F or Y43H }, { M45Q, M45E, M45L or M45D }, { N49C, N49G, N49Y or N49S }, { K53T or K53L }, { L97Y, L97V or L97I }, { a100I or a100V } and { a107P, a107I or a107V }; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

in another embodiment, the amino acid residue is altered at a position as set forth in SEQ ID No: 6 selected from the group consisting of: (a) V39R, N41V, Y43H, M45E, S48G, Q50E, K53T, a56S, Q63T, G65L, Q66P, L97V, S102A, L103F, a104H, K106V and a 107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(b) V39R, N41V, Y43H, M45E, S48N, Q50H, T51A, D52V, K53T, a56S, Q63L, G65F, Q66P, L97I, S102T, L103F, a104D, K106R and a 107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(c) V39H, L40V, N41V, Y43H, R44Y, M45E, N49G, K53T, M83L, L97V, a100I and a 107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(d) V39H, L40V, N41V, Y43H, M45E, N49G, K53T, Q66P, M83L, L97V and a 107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(e) V39H, L40V, N41V, Y43H, M45E, N49S, K53T, Q66P, H82Q, M83L, L97V, a100V and a 107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(f) V39H, L40I, N41I, Y43H, M45E, N49G, K53T, M83L, L97V, a100V and a 107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(g) V39H, L40I, N41I, Y43H, R44L, M45E, N49G, K53T, L97V, a100V and a 107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(h) V39H, L40V, N41I, Y43H, M45E, N49G, K53T, L97V, a100V and a 107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(i) V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, a100V and a 107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

the PD-1 protein HAC with high affinity consensus can be a post-transcriptional modified protein; such modifications include glycosylation, PEG modification, and the like.

More preferably, the amino acid residue is changed to N41I or N41V.

Preferably, the CAR chimeric antigen receptor comprises a SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 zeta element having:

1) as shown in SEQ ID No: 10, or,

2) 1) amino acid sequences which are derived from the amino acid sequences and have the same functions through substitution and/or deletion and/or addition of one or more amino acid residues.

Preferably, the CAR chimeric antigen receptor comprises a SP2-HAC-HSA element having:

3) as shown in SEQ ID No: 11, or,

4) and 3) amino acid sequences which are derived from the amino acid sequences and have the same functions through substitution and/or deletion and/or addition of one or more amino acid residues.

Preferably, in the chimeric antigen receptor, the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The linkage "-" of the amino acid sequence according to the present invention is a direct connection of the N-terminus of one fragment to the C-terminus of another fragment without any connecting peptide in between, e.g., HAC-HSA, the HAC domain being directly connected via its C-terminus to the N-terminus of the HSA domain, or the HAC domain being directly connected via its N-terminus to the C-terminus of the HSA domain, i.e., the HAC domain being directly connected to the HSA domain without any connecting peptide in between.

In another aspect of the present invention, there is provided a method for preparing an enhanced CAR-immune cell, wherein the synthetic coding gene of the present invention can easily prepare a base sequence encoding a CAR from an amino acid sequence of a designated CAR by a conventional method, obtain a base sequence encoding an amino acid sequence from NCBI Ref Seq ID or genbank accession number of the amino acid sequence, and prepare the nucleic acid of the present invention using standard molecular biology and/or chemical procedures.

Preferably, the preparation method of the enhanced CAR-immune cell specifically comprises the following steps:

(1) construction of the vector: constructing a vector capable of expressing the CAR receptor;

(2) packaging of virus transfected immune cells: packaging viruses by adopting the vector constructed in the step (1) to obtain packaged viruses;

(3) cell separation and amplification culture: extracting the blood of a patient, separating immune cells from the blood, and performing amplification culture;

(4) transfection, preparation and detection of immune cells: and (3) transfecting the T cells obtained by culturing in the step (3) by using the packaged virus in the step (2), and performing amplification culture to obtain the chimeric antigen receptor T cells, namely CAR-immune cells.

The chimeric antigen receptor immune cell is characterized in that the gene for coding the chimeric antigen receptor is introduced into the immune cell.

The expression vector may use a viral vector which lacks replication ability and cannot replicate itself in transfected cells, such as a retroviral vector (including oncogenic retroviral vector, lentiviral vector and pseudotyped vector), an adenoviral vector, vaccinia viral vector or HSV vector, etc.;

preferably, the immune cells of the present invention are derived from human peripheral blood, more preferably from: t cells, NK cells such as NK 92; more preferably, in the present invention, a wide variety of commercially available packaging plasmids for packaging retroviral vectors can be selected depending on the retrovirus, and 293 cells or 293T cells having high transfection efficiency can also be used for preparing retroviral particles.

The construction of the vector in the step (1) comprises amplification, enzyme digestion connection and transformation of an extracellular domain Slit2D2, a transmembrane domain, an intracellular immune co-stimulatory molecule, an internal ribosome entry site IRES and a HAC-HSA gene.

In a preferred embodiment of the invention, the preparation method of the reinforced CAR-T cell specifically comprises the following steps:

(1) construction of the vector: amplifying and connecting a Slit2D2-CD8TM-4-1BB-CD3 zeta-IRES-HAC-HSA gene, and cloning the gene onto a lentivirus expression vector;

(2) packaging of virus transfected T cells: transfecting 293T cells by using a lentivirus packaging plasmid and a slow expression vector, packaging and preparing lentivirus;

(3) cell separation and amplification culture: the patient's own blood was drawn, human peripheral blood T cells were isolated therefrom, expanded in culture, and transfected with lentivirus to express Slit2D2-CD8TM-4-1BB-CD3 ζ -IRES-HAC-HSA on T cells.

(4) Transfection and preparation and detection of T cells: and (3) performing transfection and amplification culture on the T cells cultured in the step (3) by using the packaged lentivirus in the step (2), so that the T cells express the Slit2D2-CD8TM-4-1BB-CD3 zeta-IRES-HAC-HSA.

The CAR technology and the PD-1 antibody immune checkpoint treatment method are fused, and the secretory HAC-HSA fusion gene is added to a conventional CAR-immune cell expression vector to prepare the enhanced CAR-immune cell, wherein the PD-1 protein expressed by HAC-HSA has higher affinity with PDL-1, and the fused HSA protein prolongs the half-life of the protein. The reinforced CAR-immune cells, particularly reinforced CAR-T cells and reinforced CAR-NK cells, have the traditional CAR-immune cell targeted killing capability, and can secrete PD-1 fusion protein, block PDL-1 inhibitory signals, enhance CAR-immune killing activity and activate tumor-infiltrated immune cells.

Drawings

FIG. 1 is a schematic diagram showing the construction of Slit2D2-CAR gene provided in example 1 of the present invention;

FIG. 2 is a schematic diagram showing the construction of Slit2D2-CD8TM-4-1BB-CD3 ζ -IRES-HAC-HSA gene provided in example 1 of the present invention;

FIG. 3 is a schematic diagram of pRRSLIN-slit2D 2CAR & HAC-HSA lentiviral expression vector provided in example 1 of the present invention;

FIG. 4 is a graph showing the effect of flow-type transfection of CAR-T cells provided in example 4 of the present invention;

FIG. 5 is a graph showing the effect of CAR-NK92 cell flow-type transfection provided in example 4 of the present invention;

FIG. 6 shows the results of in vitro killing experiments of enhanced CAR-T (slit2D 2CAR & HAC-HSA) cells and normal CAR-T (slit2D2 CAR) cells under different effective target ratios provided in example 6 of the present invention; wherein the target cell in panel A is H1299, and the target cell in panel B is SMMC 7721.

The specific implementation mode is as follows:

example 1: lentiviral expression vector preparation

1. According to the known sequence of Slit2 [ GenBank: EAW92793.1 ]]The second structural domain Slit2D2 (Hohenister 2008) of the Slit2 is analyzed, designed and constructed, and the gene sequence of the Slit is shown as SEQ ID NO: 1, searching known human CD8 Hinge region and CD8 from GenBank database^TMTransmembrane region gene sequence, human 4-1BB intracellular region geneThe sequence, the intracellular region of CD3 zeta and the internal ribosome entry site of IRES are adopted to obtain the gene of the Slit2D2-CAR (SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 zeta) shown in SEQ ID NO: 7, the series schematic diagram is shown in FIG. 1; synthesizing HAC gene fragment, wherein the nucleotide sequence of the HAC gene fragment is shown as SEQ ID NO: 2, and introducing an HSA gene into the C end of the fusion gene to obtain an HAC-HSA fusion gene (SP2-HAC-HSA) shown as SEQ ID NO: shown in fig. 8.

2. The HAC-HSA fusion gene sequence is inserted into the downstream of the Slit2D2-CAR sequence to form a complete HAC-HSA fusion gene sequence

The construction scheme of the Slit2D2-CD8-4-1BB-CD3 zeta-IRES-HAC-HSA is shown in FIG. 2.

3. The gene sequence of Slit2D2-CD8-4-1BB-CD3 zeta-IRES-HAC-HSA is transformed into PRRSLIN vector by double digestion connection, and the upstream of the gene is EP-1 alpha promoter. Transforming the vector into a Stbl3 escherichia coli strain, transferring the strain into a solid culture medium containing ampicillin, breeding, screening to obtain positive clones, extracting plasmids, carrying out enzyme digestion identification cloning, confirming the success of vector construction through sequencing, and obtaining a pRRSLIN-Slit2D2 lentiviral expression vector, wherein the schematic diagram of the construction of the lentiviral expression vector is shown in the attached figure 3.

Example 2: lentiviral preparation

1. 24 hours before transfection, at about 8X 10 per dish⁶293T cells were seeded into 15cm dishes. Ensure that the cells are confluent at around 80% and evenly distributed in the culture dish during transfection.

2. Preparing solution A and solution B

Solution A: 6.25mL of 2 XHEPES buffer (5 large dishes were used together in the amount of best results).

Solution B: the following mixtures of plasmids were added separately: 112.5 μ g pRRSLIN-Slit2D2-CAR-IRES-HAC-HSA (target plasmid); 39.5 μ G pMD2.G (VSV-G envelop); 73. mu.g pCMVR8.74(gag, pol, tat, rev); 625 μ L of 2M calcium ion solution. Total volume of solution B: 6.25 mL.

3. And (3) fully mixing the solution B, adding the solution A dropwise while slightly swirling the solution A, and standing for 5-15 minutes. The mixed solution of A and B was vortexed gently, added dropwise to a culture dish containing 293T cells, and the dish was shaken gently back and forth to uniformly distribute the mixture of DNA and calcium ions. (without rotating the dish) was placed in an incubator for 16-18 hours.

Replacing the fresh culture medium and continuing the culture.

Centrifuging at 25 deg.C for 10min at 500g, and filtering with PES membrane (0.45 μm); sterilizing centrifuge tube (Beckmann Coultra-clear SW28centrifuge tubes) with 70% ethanol, and sterilizing under ultraviolet lamp for 30 min; the filtered lentivirus-containing supernatant was transferred to a centrifuge tube, carefully layered with a 20% sucrose (1 mL per 8mL supernatant) on the bottom of the tube, equilibrated with PBS and centrifuged at 25,000rpm (82, 700g) at 4 ℃ for 2 h; carefully taking out the centrifugal tube, pouring out the supernatant, and inverting the centrifugal tube to remove residual liquid; adding 100 μ LPBS, sealing the centrifuge tube, standing at 4 deg.C for 2h, gently vortexing once every 20min, centrifuging at 500g for 1min (25 deg.C), and collecting virus supernatant; cooling on ice, and storing at-80 deg.C.

Example 3: CAR-T cell preparation

1. 0.5mL of blood is taken for rapid detection of pathogenic microorganisms, and HBV, HCV, HDV and HEV, HIV-1/2, treponema pallidum, parasites and other microbial infections are eliminated; under the aseptic condition, 50mL (heparin anticoagulation) of blood is collected by a heparin bottle and immediately (at 4 ℃ within 24 hours) is sent to a cell preparation laboratory, so that the process is ensured to be free from the pollution of pathogenic microorganisms. After obtaining the blood of the patient, the surface of the heparin bottle is wiped by an alcohol cotton ball in a GMP preparation room for disinfection and then is put into a biological safety cabinet.

2. 2 50mL centrifuge tubes are opened in advance, blood is transferred into two 50mL centrifuge tubes, and the centrifuge tubes are screwed tightly; placing the two 50mL centrifuge tubes filled with the blood into a centrifuge for centrifugation, centrifuging for 10min at 400g (2000rpm), centrifuging at room temperature, collecting upper plasma, and leaving a precipitate layer; the collected autologous plasma is inactivated at 56 deg.C for 30min, placed at 4 deg.C for 15min, then 900g, centrifuged for 30min (4 deg.C), and the supernatant is taken for use.

3. Diluting the enriched blood cells to 30 mL/tube with normal saline, opening 2 new 50mL centrifuge tubes, adding 15mL human lymphocyte separation solution into each centrifuge tube, slowly adding the diluted blood cell solution into the centrifuge tube containing human lymphocyte separation solution with a pipette, and screwing. Note that blood is added to the upper layer of the lymph separation fluid without breaking the interface with the human lymph separation fluid. The added blood cell fluid is put into a centrifuge, the lifting speed is adjusted to the minimum, 400g (2000rpm) is centrifuged for 20min (normal temperature). The middle leukocyte layer of the two tubes was collected in a 15mL sterile centrifuge tube, and 5mL physiological saline was added thereto, and washed twice (400g, centrifugation for 10min) to obtain Peripheral Blood Mononuclear Cells (PBMC).

4. Preparing complete growth medium, adding autologous AB (FBS) to V-VIVO15 at a concentration of 5% and interleukin-2 (IL-2) at a concentration of 40ng/mL, and diluting the separated PBMC to 2X 10 with the medium⁶Per mL, 50. mu.L of the flow assay PBMC were used for purity of T cells.

5, Day 0, preparing a buffer solution (1% Fetal Bovine Serum (FBS) is added into a PBS buffer solution), selecting microbeads as cell culture carriers, shaking the microbeads for 30s or shaking up and down manually for 5min, and mixing the microbeads with the T cells according to the dosage ratio of 3: 1 placing CD3/CD28 microbeads into a 1.5mL EP tube, adding 1mL buffer solution to clean the microbeads, then sucking the microbeads outwards from the EP tube for 1min by using a magnet, discarding the washing solution, repeating the steps twice, then re-suspending the microbeads to the original volume by using a culture medium, mixing the cells and the microbeads, and then mixing the cells and the microbeads according to a ratio of 2 multiplied by 10⁶PBMC/mL were added to the appropriate flask.

Day 2 adjustment of cell density to 3-5X 10⁶Perml, pRRSLIN-Slit2D2-CAR-IRES HAC-HSA lentivirus prepared in example 2 was added at a virus-to-cell ratio of 1:5, and polybrene (polybrene) was added at 4. mu.g/mL and IL-2 at 40 ng/mL. After 4h, the cell density was adjusted to 1X 10 by adding fresh complete medium⁶The culture was continued at/mL. All cells were centrifuged, fresh medium was added and the culture was continued.

7. Half-amount of the liquid is changed every 2-3 days to maintain the cell density at 0.5-1 × 10⁶/mL。

Day 10-12, cell number up to 10⁹Grade, immune cells were centrifuged at 400g for 5min and washed twice with pre-chilled PBS (400g, 5 min).

9. Counting by using a blood counting chamber, and detecting the cell group and the ratio of CART cells by using a flow cytometer. The color change, cell density, cell morphology of the culture medium were observed daily and recorded accordingly. In the process of gradually expanding culture, interleukin-2 required by the total volume is added.

Example 4: CAR-NK92 cell preparation

CAR-NK92 cells were prepared by reference to the experimental procedure of example 3.

Example 5: flow analysis of CAR-T cells and CAR-NK92 cells

The CAR-T cells prepared in example 3 and CAR-NK92 cells prepared in example 4 were subjected to flow analysis, with the following specific steps:

1. take 5X 10⁴Cells (including T cells, NK cells, slit2D 2CAR-T cells, slit2D2 CAR-NK cells, slit2D2&HAC-HSA CAR-T cells, slit2D2&HAC-HSA CAR-NK cells) for staining;

2. incubating the cells and the antibody (the antibody can be identified and combined with a FLAG label and coupled with an APC fluorescent molecule) for 45min, and placing the cells and the antibody on ice in 50 mu l;

eluting with PBS twice;

4. resuspend cells with 120 μ l FACS reagent;

5. flow cytometry measures APC fluorescence signal, CAR cells APC fluorescence signal if compared to control T cells or NK cells

Number enhancement, surface CAR cell construction was successful.

The flow-staining effect of CAR-T cells and CAR-NK92 cells is shown in fig. 4 and fig. 5, respectively.

In FIG. 4, panels A and C are control groups: t cells that do not infect viruses; APC-coupled antibodies that detect the CAR molecule do not detect CAR molecule expression; and B, drawing: the T cells transfected with the slit2D 2CAR virus were subjected to flow detection, and some cells were successfully transfected with slit2D 2CAR molecules; and (D) diagram: the slit2D2& HAC-HSA CAR-virus transfected T cells were successfully transfected with slit2D2& HAC-HSA CAR molecules by flow assay. Panel B and D illustrate the successful production of corresponding CAR-T cells, respectively.

In FIG. 5, panels A and C are control groups: NK cells that are not virus-infecting; APC-coupled antibodies that detect the CAR molecule do not detect CAR molecule expression; and B, drawing: NK cells transfected with slit2D 2CAR virus are subjected to flow detection, and some cells are successfully transfected with slit2D 2CAR molecules; d picture transfection slit2D2& HAC-HSA CAR-T virus NK cells, flow detection shows that some cells successfully transfect slit2D2& HAC-HSA CAR molecules. Panel B and D illustrate the successful production of corresponding CAR-NK cells, respectively.

Example 6: CAR-T cell in vitro activity assay

And detecting the killing effect of the CAR-T cells on tumor cells by adopting an LDH release method, and detecting the LDH release by adopting an ELISA method.

1. The target cells were adjusted to 5X 10 with 5% calf serum in RPMI-1640 medium⁴/mL。

2. Target cells were added to 96 well cell culture plates at 100. mu.L per well. 3 wells were used as effector cell (CAR-T cells) spontaneous release control wells, and 100. mu.L of medium was added without target cells.

3. Add 10. mu.L of effector cells to each well, with a ratio of effector cells to target cells of 10: 1; 5: 1; 1:1. The natural release hole is not added with effector cells, only 100 mu L of culture solution is added, the effector cells and target cells are incubated for 6 hours, and three multiple holes are arranged in each experiment.

4. mu.L of lysine Solution (10X) was added to the maximum release well (positive control) and incubated for 45min-60min, with triplicate wells for each experiment.

5. And (3) adding 50 mu L of each sample to be detected and the control sample in the

samples

3 and 4 into a fresh 96-hole enzyme label plate, adding the reaction solution and the substrate, and keeping out of the sun for 30 min.

6. Add 50. mu.L of stop solution.

7. The optical density (OD value) of each well was measured on an enzyme-linked detector at a detection wavelength of 490nm or 492nm, and the measurement was completed within 1 hour.

8. Calculation of specific killing efficiency

Kill rate ═ experimental set LDH (OD)/maximum LDH release set (OD).

Calculating the formula: the killing efficiency ═ 100% (experimental group-effect natural release-target natural release)/(target maximum release-target natural release).

9. Cytokine secretion was measured by CBA kit, and proliferation in each group of CAR-T cells was calculated, and staining with CD3 and CD8 antibodies confirmed the proportion of CD 8-positive T cells in the proliferated T cells.

The results of in vitro killing experiments of reinforced CAR-T (slit2D 2CAR & HAC-HSA) cells and common CAR-T (slit2D2 CAR) cells under different effect-target ratio conditions are shown in FIG. 6, and the results show that the reinforced CAR-T (slit2D 2CAR & HAC-HSA) cells prepared by the experiments can be specifically activated and proliferated after contacting tumor cells to kill the tumor cells, and the effect of the reinforced CAR-T of slit2D2-HAC-HSA is better than that of slit2D2 CAR-T.

Claims

1. A gene encoding a CAR chimeric antigen receptor comprising a gene encoding an extracellular domain capable of binding an antigen, a signaling domain, an intracellular immune co-stimulatory molecule, an Internal Ribosome Entry Site (IRES), and HAC-HSA;

the extracellular domain is Slit2D2, and the coding gene is SEQ ID No: 1.

2. The gene of claim 1, wherein the gene encoding HAC is SEQ ID No: 2; and/or the presence of a gas in the gas,

the signaling domain is selected from the hinge, transmembrane and intracellular regions of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS or CD 154; and/or the presence of a gas in the gas,

the intracellular immune co-stimulatory molecule is selected from one or more of the intracellular domains of CD3 ζ, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB or OX 40.

3. The gene of claim 2 wherein said signaling domain is the hinde region, transmembrane region of CD 8; and/or the presence of a gas in the gas,

the intracellular immune co-stimulatory molecule is the intracellular domains of 4-1BB and CD3 zeta.

4. The gene of any one of claims 1 to 3, further comprising a nucleotide sequence as set forth in SEQ ID No: 3, a gene encoding a signal peptide SP 1; and/or the presence of a gas in the gas,

the gene also comprises a nucleotide sequence shown as SEQ ID No: 4, a Flag-encoding gene; and/or the presence of a gas in the gas,

the gene also comprises a nucleotide sequence shown as SEQ ID No: 5, a Linker coding gene; and/or the presence of a gas in the gas,

the gene also comprises a nucleotide sequence shown as SEQ ID No: 6, the signal peptide SP 2.

5. The gene of claim 1, comprising a nucleotide sequence as set forth in SEQ ID No: 7, the fusion gene of SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 zeta and the nucleotide sequence are shown as SEQ ID No: SP2-HAC-HSA fusion gene shown in 8.

6. The gene of claim 1, wherein the gene is a gene encoding SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 ζ -IRES-SP2-HAC-HSA, and wherein the nucleotide sequence of IRES is as set forth in SEQ ID No: shown at 9.

7. A recombinant vector, a recombinant strain, loaded with the gene of any one of claims 1-6.

8. Use of the gene according to any one of claims 1 to 6 and the recombinant vector and the recombinant strain according to claim 7 in the preparation of antitumor drugs.

9. A CAR chimeric antigen receptor, wherein said CAR chimeric antigen receptor is transcriptionally expressed from a gene according to any of claims 1-6.

10. The CAR chimeric antigen receptor of claim 9, wherein said CAR chimeric antigen receptor comprises an amino acid sequence as set forth in SEQ ID No: 10, the SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 zeta element and the amino acid sequence are shown as SEQ ID No: 11, SP2-HAC-HSA element.

11. A reinforced CAR-immune cell comprising the gene of any one of claims 1-6.

12. The CAR-immune cell of claim 11, wherein the reinforced CAR-immune cell is a reinforced CAR-T cell or a reinforced CAR-NK cell.