WO2018053885A1 - Preparation method for and use of enhanced slit2 car-t cell and car-nk cell - Google Patents

Abstract

A preparation method for and use of enhanced Slit2 CAR-T cells and CAR-NK cells. The enhanced CAR-T cells and CAR-NK cells are respectively T cells and NK cells containing an enhanced CAR chimeric antigen receptor. The enhanced CAR chimeric antigen receptor includes an extracellular domain capable of binding to an antigen, a transmembrane domain, an intracellular immune costimulatory molecule, an internal ribosome entry site IRES, and HAC-HAS series. The extracellular domain comprises D2 domain of Slit2 protein. The enhanced CAR-T cells and enhanced CAR-NK cells can block programmed cell death-ligand 1 (PDL-1) inhibitory signal, enhance the killing activity of CAR-immune cells, and activate tumor-infiltrating immune cells. The enhanced CAR-immune cells can be used as a cellular drug for the treatment of tumor diseases, so that the modified immune cells can specifically recognize and kill tumors and have an increased high tumor killing activity.

Description

Method and application for preparing enhanced Slit2 CAR-T and CAR-NK cells Technical field

The present invention relates to the field of biotechnology, and in particular to a chimeric antigen receptor cell against an anti-tumor stem cell, particularly a boosted CAR-T cell and a boosted CAR-NK cell.

Background of the invention

PD-L1 full-length programmed death receptor-ligand 1, the English name programmed cell death-Ligand 1, is a first type transmembrane protein with a size of 40 kDa. PD-1, the full name of programmed death receptor 1, the English name for programmed death 1, is an important immunosuppressive molecule and is a member of the CD28 superfamily. PD-1 (programmed death-1) is mainly expressed on the surface of T cells and primary B cells, and two ligands of PD-1 (PD-L1 and PD-L2) are widely expressed in antigen-presenting cells (APCs). The interaction of PD1 with its receptor plays an important role in the negative regulation of immune response. The expression of PD-L1 protein can be detected in many human tumor tissues. The microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells. The expression of PD-L1 is beneficial to the occurrence and growth of tumors, and induces anti-tumor. The apoptosis of T cells, in turn, evades the attack of the immune system. Inhibition of the binding of PD-1 to its ligand can expose tumor cells to the killing field of the immune system, thereby achieving the effect of killing tumor tissue and treating cancer.

At the 2016 AACR Annual Meeting, Paul T. Nghiem, MD, PhD, a professor of dermatology at the University of Washington School of Medicine, reported that pembrolizumab, a monoclonal antibody with anti-PD-1 effects, induces high levels of Merkel cell carcinoma. Reactivity, median progression-free survival PFS was 9 months, and patients with conventional chemotherapy had a PFS of 3 months. Recently, the Bristol-Myuis PD-1 inhibitor Opdivo has made new progress in clinical trials, and released two sets of data. One group showed that patients with advanced melanoma who have no treatment response to any current drug have achieved treatment with Opdivo. The result of a 34% five-year survival rate. It is worth noting that the five-year survival rate of patients with stage IV melanoma is usually only 15% to 20%. Another group of data showed that Opdivo and Yervoy had a 22% overall response rate in patients with advanced melanoma and achieved a two-year overall survival rate of 69%. Another study showed that patients with refractory recurrent or metastatic head and neck squamous cell carcinoma (SCCHN) had significantly improved survival and survival after treatment with Opdivo. The data showed a significant 30% reduction in the risk of death in the Opdivo-treated group compared with the control group, with a significant median overall survival. The annual survival rate was 36% in the Opdivo treatment group and 16.6% in the control group. In addition, the study evaluated the efficacy of Opdivo relative to the control regimen by HPV status of the oropharyngeal tumor and PD-L1 expression status. Recently, Keytruda was awarded the FDA's breakthrough drug qualification for the treatment of recurrent or refractory (R/R) classic Hodgkin's lymphoma (cHL).

CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy), chimeric antigen receptor T cell immunotherapy. The therapy is a new type of cell therapy that has been in clinical use for many years but has been improved in recent years. It has significant efficacy in the treatment of acute leukemia and non-Hodgkin's lymphoma and is considered to be one of the most promising treatments for cancer. Chimeric antigen receptors (CAR) T cells (CAR-T) are genetically modified for the patient's own T cells and are successful in treating blood cancer, but using CAR-T to treat solid tumors ( It is composed of heterogeneous cell populations with different surface molecules that do not achieve the desired therapeutic effect.

CN105505869A discloses a chimeric receptor T cell targeting tumor stem cells, which has two to three independent antigen receptors, each of which is composed of a different tumor stem cell specific marker. The antigen binding portion of the antibody is composed of a combination of different functional proteins. This chimeric receptor T cell activates T cell anti-tumor effects only after 2-3 chimeric antigen receptors recognize the antigen, and this method improves the specificity but the tumor cells that hide the antigen to some extent. Lower specificity.

In the literature reported by Liza B. John (Blockade of PD-1 immunosuppression boosts CAR T-cell therapy), the binding of PD-1 immunosuppressive pathway, PD-1, to ligand PD-L was blocked by the addition of anti-PD-1 antibody. Thereby, the anti-tumor efficacy of the genetically modified chimeric antigen receptor T cell (CAR-T) is improved to some extent.

Summary of the invention

To overcome the deficiencies in the prior art, the present invention provides a CAR chimeric antigen receptor, a potentiating CAR-immunoblast, and a preparation method thereof.

In one aspect, the invention provides a gene encoding a CAR chimeric antigen receptor, comprising an extracellular domain capable of binding an antigen, a signaling domain, an intracellular immunostimulatory molecule, an internal ribosome entry site IRES and a HAC-HSA The coding gene.

The extracellular domain comprises the D2 domain of the Slit2 protein; referred to as Slit2D2, the coding gene thereof has the nucleotide sequence shown in SEQ ID No: 1.

The coding gene of the HAC has a nucleotide sequence as shown in SEQ ID No: 2;

Preferably, the signaling domain is selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, CD154 hinge region, transmembrane region and intracellular region One or more of the zone domains.

Preferably, the signaling domain is selected from the Hinge region, the transmembrane region and the intracellular region of CD8, and more preferably, the CD8 comprises a Hinge region and a transmembrane region of CD8;

Preferably, the intracellular immunostimulatory molecule is selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB And one or more of the OX40 intracellular domains;

Preferably, the intracellular immunocostimulatory molecule comprises a 4-1BB and a CD3 sputum intracellular domain;

Preferably, the above coding gene further comprises a signal peptide Signal peptide1 (SP1) encoding gene represented by SEQ ID No: 3;

Preferably, the above coding gene further comprises a nucleotide coding sequence as shown in SEQ ID No: 4;

Preferably, the above coding gene further comprises a Linker coding gene having a nucleotide sequence as shown in SEQ ID No: 5;

Preferably, the above coding gene further comprises a signal peptide Signal peptide 2 (SP2) encoding gene represented by SEQ ID No: 6;

Preferably, the above coding gene comprises a SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ fusion gene having a nucleotide sequence as shown in SEQ ID No: 7.

Preferably, the above coding gene comprises a SP2-HAC-HSA fusion gene having a nucleotide sequence as shown in SEQ ID No: 8.

In a preferred embodiment of the invention, the gene encoding the CAR chimeric antigen receptor is SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ-IRES-SP2-HAC-HSA encoding gene, the core of IRES The nucleotide sequence is shown in SEQ ID No: 9.

Another aspect of the present invention provides a recombinant vector and a recombinant strain loaded with the above-mentioned coding gene;

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

Another aspect of the present invention provides a use of the above-described coding gene, a recombinant vector loaded with the above-mentioned coding gene, and a recombinant strain for modifying immune cells and preparing antitumor drugs;

Preferably, the immune cells are selected from the group consisting of: T cells, NK cells such as NK92.

Another aspect of the present invention provides a booster CAR-immunoblast comprising the above-described coding gene;

Preferably, the enhanced CAR-immunoblast is a booster CAR-T cell or a booster CAR-NK cell such as a booster CAR-NK92 cell.

Another aspect of the present invention also provides the use of the above-described enhanced CAR-immune cells for the preparation of an antitumor drug.

In another aspect, the present invention provides a CAR chimeric antigen receptor which is obtained by transcriptional expression of the above coding gene, comprising: an extracellular domain capable of binding antigen, a transmembrane domain, an intracellular immunostimulatory molecule, an internal ribosome Enter the sites IRES and HAC-HSA.

The extracellular domain comprises the D2 domain of the Slit2 protein.

Preferably, the signaling domain is selected from the group consisting of a hinge region and a transmembrane region of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, CD154 And one or more of the intracellular regions.

Preferably, the signal transduction domain is selected from CD8 hinge region, transmembrane region and intracellular region, more preferably, is the Hinge region CD8 CD8 and CD8 transmembrane domain ^(TM).

Preferably, the intracellular immunostimulatory molecule is selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB And one or more of the OX40 intracellular domains.

Preferably, the intracellular immune costimulatory molecule comprises a 4-1BB and a CD3 sputum intracellular domain.

The HAC in the HAC-HSA of the present invention is a PD-1 protein with high-affinity consensus (HAC), which can block the wild-type PD-1 protein and the PD-L ligand in vivo and in vitro. Combine

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 increasing the affinity for the PD-L1 ligand;

The amino acid residue is altered and can be located in the PD-1 and PD-L1 binding domains, and/or,

Amino acid residue changes can be located in the immunoglobulin domain of PD-1.

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

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

In another specific embodiment, the HAC comprises an amino acid sequence having an amino acid sequence identity of 85% or greater, 90% or greater, relative to the original mock PD-1 polypeptide as set forth in SEQ ID No: 6. % or higher, 98%, 99% or higher, 99.2% or higher, 99.8% or higher, 99.9% or higher, or 100%.

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

One or more of the amino acid changes, two or more, three or more, four or more, five or more, six or more, seven or more, eight or Multiple, 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 specific embodiment, the amino acid residue is altered, the site of which is selected from the group consisting of V39, L40, N41, Y43, R44, M45 in the wild type PD-1 fragment shown in SEQ ID No: 6. One of S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, V72, H82, M83, R90, Y96, L97, A100, S102, L103, A104, P105, K106, A107 or Several; or, amino acids at corresponding positions of other wild-type PD-1 proteins; the amino acid changes include one or more amino acid changes; the plurality of amino acids are changed to two or more, three 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 multiple, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more.

Preferably, in another specific embodiment, the amino acid residue alteration can be located in the PD-1 and PD-L1 binding domains, the amino acid alteration site being located in the PD-1 fragment as set forth in SEQ ID No: Medium, selected from V39, N41, Y43, M45, S48, N49, Q50, T51, D52, K53, A56, Q63, G65, Q66, L97, S102, L103, A104, P105, K106, A107; or other wild One or more of the amino acids at the corresponding positions of the PD-1 protein; the amino acid changes include one or more amino acid changes; the plurality of amino acids are changed to two or more, three 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 than 13 or more, 14 or more, 15 or more.

In another specific embodiment, the amino acid residue is altered, the site of which is located in the PD-1 fragment set forth in SEQ ID No: 6, selected from: (a) V39, N41, Y43, M45, S48, N49, Q50, K53, A56, Q63, G65, Q66, L97, A100, S102, L103, A104, K106 And A107; or other amino acid corresponding to the wild-type PD-1 protein; (b) V39, N41, Y43, M45, S48, Q50, T51, D52F, K53, A56, Q63, G65, Q66, L97, S102, L103, A104, K106 and A107; or, other wild type PD-1 protein corresponding amino acids (c) V39, L40, N41, Y43, R44, M45, N49, K53, M83, L97, A100 and A107; Amino acids at corresponding positions of other wild-type PD-1 proteins; (d) V39, L40, N41, Y43, R44, M45, N49, Q66P, M83, L97 and A107; or, amino acids at positions corresponding to other wild-type PD-1 proteins (e) V39, L40, N41, Y43, M45, N49, K53, Q66P, H82, M83, L97, A100 and A107; or, amino acid at the corresponding position of other wild-type PD-1 proteins; (f) V39, L40 , N41, Y43, M45, N49, K53, M83, L97, A100, and A107; or, other amino acids of the corresponding position of the wild-type PD-1 protein; (g) V39, L40, N41, Y43, R44, M45, N49 , K53, L97, A100 and A107; (h) V39, L40, N41, Y43, M45, S48, N49, K53, L97, A100 and A107; or, amino acids at corresponding positions of other wild-type PD-1 proteins.

In another specific embodiment, the amino acid residue is altered, the site of which is located in the PD-1 fragment set forth in SEQ ID No: 6, selected from: (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 A56L; (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 A100V; (24) S102T or S102A; (25) L103I, L103Y or L103F; (26) A104S, A104H or A104D (27) P105A; (28) K106G, K106E, K106I, K106V, K106R or K106T; (29) A107P; A107I or A107V; or, amino acids at corresponding positions of other wild-type PD-1 proteins.

In another specific embodiment, the amino acid residue is altered, the site of which is located in the PD-1 fragment set forth in SEQ ID No: 6, selected from: (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}, {L97Y, L97V or L97I}, {S102T or S102A}, {L103I, L103Y or L103F}, {S102T or S102A}, {L103I, L103Y or L103F}, {A104S, A104H or A104D}, {K106G, K106E, K106I, K106V, K106R or K106T}, {A107P, A107I or A107V }; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(b) {V39H or V39R}, {N41I or N41V}, {Y43F or Y43H}, {M45Q, M45E, M45L or M45D}, {S48D, S48L, S48N, S48G or S48V}, {Q50K, Q50E or Q50H} , {T51V, T51L or T51A}, {D52F, D52R, D52Y or D52V}, {K53T or K53L}, {A56S or A56L}, {Q63T, Q63I, Q63E, Q63L or Q63P}, {G65N, G65R, G65I, G65L, G65F or G65V}, {Q66P}, {L97Y, L97V or L97I}, {S102T or S102A}, {L103I, L103Y or L103F}, {A104S, A104H or A104D}, {K106G, K106E, K106I, K106V, K106R or K106T}, {A107P, A107I or A107V}; or, amino acids corresponding to 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, other wild type PD-1 protein corresponding amino acid position ;

(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 acid at the corresponding position 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, other wild-type PD-1 protein corresponding positions Amino acid

(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 corresponding to 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 corresponding to 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 corresponding to other wild-type PD-1 proteins;

In another specific embodiment, the amino acid residue is altered, the site of which is located in the PD-1 fragment set forth in SEQ ID No: 6, selected from: (a) V39R, N41V, Y43H, M45E, S48G, Q50E, K53T, A56S, Q63T, G65L, Q66P, L97V, S102A, L103F, A104H, K106V and A107I; or, amino acids corresponding to 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 A107I; or, other wild-type PD-1 proteins Amino acid at the corresponding position;

(c) V39H, L40V, N41V, Y43H, R44Y, M45E, N49G, K53T, M83L, L97V, A100I and A107I; or, amino acids at positions corresponding to other wild-type PD-1 proteins;

(d) V39H, L40V, N41V, Y43H, M45E, N49G, K53T, Q66P, M83L, L97V and A107I; 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 A107I; or, amino acids at positions corresponding to other wild-type PD-1 proteins;

(f) V39H, L40I, N41I, Y43H, M45E, N49G, K53T, M83L, L97V, A100V and A107I; 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 A107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(h) V39H, L40V, N41I, Y43H, M45E, N49G, K53T, L97V, A100V and A107I; or, amino acids at corresponding positions of other wild-type PD-1 proteins;

(i) V39H, L40V, N41V, Y43H, M45E, N49G, K53T, L97V, A100V and A107I; or, amino acids at positions corresponding to other wild-type PD-1 proteins;

The PD-1 protein HAC having a high affinity consensus may be a post-transcriptional modification protein; the 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-CD3ζ element having:

1) an amino acid sequence as shown in SEQ ID No: 10, or,

2) An amino acid sequence derived therefrom which is 1) substituted and/or deleted and/or added by one or several amino acid residues and having the same function.

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

3) the amino acid sequence shown as SEQ ID No: 11, or,

4) An amino acid sequence derived therefrom which is 3) substituted and/or deleted and/or added by one or several amino acid residues and having the same function.

Preferably, in the chimeric antigen receptor, the substitution and/or deletion and/or addition of the one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues. .

The "-" linkage of the amino acid sequence of the present invention is such that the N-terminus of one fragment is directly linked to the C-terminus of another fragment without any linker peptide in between, for example, HAC-HSA, and the HAC domain passes through the C-terminus and the HSA domain. The N-terminus is directly linked, or the HAC domain is directly linked by its N-terminus to the C-terminus of the HSA domain, ie, the HAC domain is directly linked to the HSA domain without any linker peptide in between.

Another aspect of the present invention provides a method for preparing a boosted CAR-immunized cell. The synthetic coding gene of the present invention can easily prepare a base sequence encoding CAR from a predetermined CAR amino acid sequence by a conventional method, and the NCBI of the amino acid sequence. The Ref Seq ID or GenBenk accession number gives the base sequence encoding the amino acid sequence, and the nucleic acid of the present invention can be prepared using standard molecular biology and/or chemical procedures.

Preferably, the method for preparing the enhanced CAR-immune cell of the present invention comprises the following steps:

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

(2) packaging of virus transfected with immune cells: packaging the virus using the vector constructed in step (1) to obtain a packaged virus;

(3) Cell separation and expansion culture: the patient's own blood is taken, and the immune cells are isolated and expanded and cultured;

(4) Transfection, preparation and detection of immune cells: the T cells cultured in the step (3) are transfected and expanded and cultured using the packaged virus in the step (2) to obtain a chimeric antigen receptor T cell abbreviation. CAR-immune cells.

The chimeric antigen receptor immune cell is a gene encoding a chimeric antigen receptor of the present invention introduced into an immune cell.

The expression vector may use a viral vector such as a retroviral vector (including an oncogenic retroviral vector, a lentiviral vector, and a pseudotype vector), an adenovirus vector, or a vaccinia virus vector that lacks replication ability and is unable to self-replicate in a transfected cell. Or HSV vector, etc.;

Preferably, the immune cells in the present invention are derived from immune cells of human peripheral blood, more preferably from: T cells, NK cells such as NK92; more preferably, various types of commercially available antibodies can be selected according to retrovirus in the present invention. The packaging plasmid can be used to package a retroviral vector, and retrovirus particles can also be prepared using 293 cells or 293T cells with high transfection efficiency.

Wherein, the construction of the vector described in the step (1) comprises amplification of the extracellular domain Slit2D2, a transmembrane domain, an intracellular immunostimulatory molecule, an internal ribosome entry site, an IRES and a HAC-HSA gene, and a restriction enzyme digestion. Connect and convert.

In a preferred embodiment of the present invention, the method for preparing the enhanced CAR-T cell comprises the following steps:

(1) Construction of vector: Amplification of the Slit2D2-CD8TM-4-1BB-CD3ζ-IRES-HAC-HSA gene, and the gene was cloned into a lentiviral expression vector;

(2) Packaging of virus transfected with T cells: 293T cells were transfected with a lentiviral packaging plasmid and a slow expression vector to package and prepare a lentivirus;

(3) Cell separation and expansion culture: the patient's own blood is taken, human peripheral blood T cells are isolated, cultured and expanded, and T cells are transfected with lentivirus, so that T cells express Slit2D2-CD8TM-4-1BB-CD3ζ-IRES -HAC-HSA.

(4) T cell transfection, preparation and detection: using the packaged lentivirus in step (2), the T cells cultured in step (3) are transfected and expanded to make T cells express Slit2D2-CD8TM- 4-1BB-CD3ζ-IRES-HAC-HSA.

The invention combines the CAR technology and the PD-1 antibody immunological checkpoint treatment method, and prepares the enhanced CAR-immunized cells by adding the secretory HAC-HSA fusion gene to the conventional CAR-immunized cell expression vector, wherein the HAC-HSA The expressed PD-1 protein has a higher affinity with PDL-1. At the same time, the fusion of the HSA protein prolongs the half-life of the protein. The enhanced CAR-immunized cells of the present invention, particularly the enhanced CAR-T cells and the enhanced CAR-NK cells, have the traditional CAR-immunized cell targeted killing ability, and can secrete PD-1 fusion protein and block PDL- 1 Inhibitory signal, enhances CAR-immuno killing activity, and activates tumor-infiltrating immune cells.

BRIEF DESCRIPTION OF THE DRAWINGS

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

2 is a schematic diagram showing the construction of the Slit2D2-CD8TM-4-1BB-CD3ζ-IRES-HAC-HSA gene provided in Example 1 of the present invention;

3 is a schematic diagram of a pRRSLIN-slit2D2 CAR&HAC-HSA lentiviral expression vector provided in Example 1 of the present invention;

4 is a flow chart showing the effect of flow infusion of CAR-T cells according to Example 4 of the present invention;

Figure 5 is a diagram showing the flow-through effect of CAR-NK92 cells provided in Example 4 of the present invention;

Figure 6 is a graph showing the results of in vitro killing experiments of enhanced CAR-T (slit2D2 CAR&HAC-HSA) cells and common CAR-T (slit2D2 CAR) cells under different target-target conditions according to Example 6 of the present invention; The target cell is H1299, and the target cell in B is SMMC7721.

Mode for carrying out the invention:

Example 1: Preparation of lentiviral expression vector

1. The second domain of Slit2, Slit2D2 (Hohenester2008), was constructed according to the known Slit2 sequence [GenBank: EAW92793.1]. The gene sequence is shown in SEQ ID NO: 1, and the known GenBank database is searched. Human CD8 Hinge region and CD8 ^TM transmembrane region gene sequence, human 4-1BB intracellular region gene sequence, CD3 intracellular region, IRES internal ribosome entry site, resulting in Slit2D2-CAR (SP1-Flag-Linker-Slit2D2- The CD8-4-1BB-CD3ζ) gene is shown in SEQ ID NO: 7, and its tandem schematic diagram is shown in Figure 1; the HAC gene fragment is synthesized, the nucleotide sequence of which is shown in SEQ ID NO: 2, and in its C The HSA gene was introduced at the end to obtain the HAC-HSA fusion gene (SP2-HAC-HSA) as shown in SEQ ID NO: 8.

2. Insert the above HAC-HSA fusion gene sequence downstream of the Slit2D2-CAR sequence to form a complete Slit2D2-CD8-4-1BB-CD3ζ-IRES-HAC-HSA. The schematic diagram of the construction is shown in Figure 2.

3. The gene sequence of Slit2D2-CD8-4-1BB-CD3ζ-IRES-HAC-HSA was transformed into the PRRSLIN vector by double restriction enzyme ligation, and the upstream of the gene was the EP-1α promoter. The vector was transformed into Stbl3 Escherichia coli strain, then transferred to a solid medium containing ampicillin for propagation, screened, positive clones were obtained, plasmids were extracted, and clones were identified by enzyme digestion. The vector was successfully constructed by sequencing, and pRRSLIN-Slit2D2 was obtained slowly. See Figure 3 for a schematic representation of the construction of the viral expression vector and lentiviral expression vector.

Example 2: Lentivirus preparation

1. 24 hours before transfection, 293T cells were seeded into a 15 cm culture dish at approximately 8 x 10 ⁶ per dish. Ensure that the cells are confluent at 80% and evenly distributed in the culture dish during transfection.

2. Prepare solution A and solution B

Solution A: 6.25 mL 2 x HEPES buffer buffer (the amount of packaging with 5 large dishes is the best).

Solution B: A mixture of the following plasmids was separately added: 112.5 μg pRRSLIN-Slit2D2-CAR-IRES-HAC-HSA (target plasmid); 39.5 μg pMD2.G (VSV-G envelop); 73 μg pCMVR8.74 (gag, pol, tat, Rev); 625 μL 2M calcium ion solution. Total volume of solution B: 6.25 mL.

3. Mix the solution B thoroughly, gently vortex the solution A, and add the solution A dropwise, and let stand for 5-15 minutes. Gently swirl the above mixed solution of A and B, add dropwise to the Petri dish containing 293T cells, and gently shake the Petri dish to spread the mixture of DNA and calcium ions evenly. (Do not rotate the culture dish) and place it in the incubator for 16-18 hours.

Replace the fresh medium and continue the culture.

Centrifuge at 500g, temperature 25 ° C for 10min, filter with PES membrane (0.45μm); disinfect the centrifuge tube (Beckman Coulter ultra-clear SW28centrifuge tubes) with 70% ethanol, and sterilize under UV lamp for 30min; The filtered lentivirus-containing supernatant was transferred to a centrifuge tube, carefully layered with 20% sucrose (8 mL of sucrose per 8 mL of supernatant) at the bottom of the tube, and the tube was equilibrated with PBS at 25,000 rpm ( 82,700g), centrifuge at 2 °C for 2h; carefully remove the centrifuge tube, pour off the supernatant, invert the centrifuge tube to remove residual liquid; add 100μL PBS, seal the centrifuge tube, place at 4 ° C for 2h, gently vortex every 20min The virus supernatant was collected by centrifugation at 500 g for 1 min (25 ° C); after cooling on ice, it was stored at -80 ° C.

Example 3: Preparation of CAR-T cells

1. Take 0.5mL of blood for rapid detection of pathogenic microorganisms, and exclude microbial infections such as HBV, HCV, HDV and HEV, HIV-1/2, Treponema pallidum and parasites; under sterile conditions, collect 50 mL of blood from heparin bottle (heparin antibiotic) Condensed), immediately (4 ° C, within 24 hours) to the cell preparation laboratory to ensure that this process is free of pathogenic microorganisms. After obtaining the patient's blood, in the GMP preparation room, the surface of the heparin bottle is wiped with an alcohol cotton ball for disinfection and then placed in a biological safety cabinet.

2. Open two 50mL centrifuge tubes in advance, transfer the blood into two 50mL centrifuge tubes, and tighten them. Place the two 50mL centrifuge tubes filled with blood into the centrifuge for centrifugation, centrifuge at 400g (2000rpm) for 10min, and centrifuge at room temperature. After the upper layer of plasma was collected, the precipitated layer was left; the collected autologous plasma was inactivated at 56 ° C for 30 min, placed at 4 ° C for 15 min, 900 g, centrifuged for 30 min (4 ° C), and the supernatant was taken for use.

3. Dilute the above enriched blood cells to 30 mL/tube with physiological saline, open two new 50 mL centrifuge tubes, add 15 mL of human lymphocyte separation solution to each centrifuge tube, and pipette the diluted blood cell solution with a pipette. Slowly add to the centrifuge tube containing the human lymphatic separation solution and tighten. Note that the blood should be added to the upper layer of the lymphatic separation solution, and the interface of the human lymphatic separation solution should not be broken. The added blood cell solution was placed in a centrifuge, adjusted to a minimum rate of lifting, and centrifuged at 400 g (2000 rpm) for 20 min (normal temperature). Two tubes of the middle white blood cell layer were collected in a 15 mL sterile centrifuge tube, and 5 mL of physiological saline was added thereto, and washed twice (400 g, centrifuged for 10 min) to obtain peripheral blood mononuclear cells (PBMC).

4. Configure complete growth medium, V-VIVO15 added autologous AB (FBS) concentration of 5%, interleukin-2 (IL-2) concentration of 40 ng / mL, and the isolated PBMC was diluted to 2 × with the culture medium. 10 ⁶ /mL, 50 μL flow detection of the purity of T cells in PBMC.

5.Day 0, configure buffer (add 1% fetal bovine serum (FBS) in PBS buffer), use microbeads as cell culture carrier, shake the beads for 30s or shake them up and down for 5min, according to the microbeads The ratio of T cells was 3:1, and the CD3/CD28 microbeads were placed in a 1.5 mL EP tube. The beads were washed with 1 mL of buffer, and then the beads were sucked from the EP tube for 1 min using a magnet, and the washing solution was discarded. Then, the beads were resuspended to the original volume using the medium, and the cells and the beads were mixed and added to a suitable flask at 2 × 10 ⁶ PBMC/mL.

6.Day 2 adjust the cell density to 3-5×10 ⁶ /mL, and add the pRRSLIN-Slit2D2-CAR-IRES HAC-HSA lentivirus prepared in Example 2 at a ratio of 1:5 to the virus, and add at the same time. Polybrene 4 μg/mL and 40 ng/mL IL-2. After 4 h, fresh complete medium was added to adjust the cell density to 1×10 ⁶ /mL to continue the culture. All cells were centrifuged, fresh medium was added, and the culture was continued.

7. Perform a half-change every 2-3 days to maintain a cell density of 0.5-1 × 10 ⁶ /mL.

8. Day 10-12, the number of cells reached 10 ⁹ grades, centrifuged for 5 min at 400 g to obtain immune cells, and washed twice with pre-cooled PBS (400 g, 5 min).

9. Count with a hemocytometer and measure the proportion of cell groups and CART cells by flow cytometry. The color change, cell density, and cell morphology of the medium were observed daily and recorded accordingly. Gradually expand the culture process and add the required interleukin-2 to the total volume.

Example 4: Preparation of CAR-NK92 cells

CAR-NK92 cells were prepared by referring to the experimental procedure of Example 3.

Example 5: Flow cytometry analysis of CAR-T cells and CAR-NK92 cells

The CAR-T cells prepared in Example 3 and the CAR-NK92 cells prepared in Example 4 were subjected to flow analysis, and the specific steps were as follows:

1. Take 5×10 ⁴ cells (including T cells, NK cells, slit2D2 CAR-T cells, slit2D2 CAR-NK cells, slit2D2 &HAC-HSA CAR-T cells, slit2D2 &HAC-HSA CAR-NK cells) for staining;

2. Cells and antibodies (antibodies can be combined with FLAG tag recognition, coupled with APC fluorescent molecules) for 45 min, 50 μl, placed on ice;

3. PBS is eluted twice;

4. Resuspend the cells with 120 μl of FACS reagent;

5. Flow cytometry measures APC fluorescence signal. If compared with control T cells or NK cells, the APC fluorescence signal of CAR cells is enhanced and the surface CAR cells are successfully constructed.

The flow infiltration effects of CAR-T cells and CAR-NK92 cells are shown in Figures 4 and 5, respectively.

In Fig. 4, A and C are the control group: T cells that do not infect the virus; APC coupled with the antibody detecting the CAR molecule does not detect the expression of the CAR molecule; B: T cells transfected with the slit2D2 CAR virus, the flow Detection, cells successfully transfected slit2D2 CAR molecules; D map: T cells transfected with slit2D2 & HAC-HSA CAR-virus, cells were successfully transfected into slit2D2 & HAC-HSA CAR molecules by flow cytometry. Panels B and D illustrate the successful preparation of the corresponding CAR-T cells, respectively.

In Fig. 5, A and C are the control group: NK cells that do not infect the virus; APC-conjugated antibodies for detecting CAR molecules do not detect CAR molecule expression; Panel B: NK cells transfected with slit2D2 CAR virus, flow through In the detection, cells successfully transfected with slit2D2 CAR molecules; D images were transfected into s cells of slit2D2 & HAC-HSA CAR-T virus, and cells were successfully transfected into slit2D2 & HAC-HSA CAR molecules by flow cytometry. Panels B and D illustrate the successful preparation of the corresponding CAR-NK cells, respectively.

Example 6: In vitro activity assay of CAR-T cells

The killing effect of CAR-T cells on tumor cells was detected by LDH release method, and LDH release was detected by ELISA.

1. Adjust the target cells to 5 × 10 ⁴ /mL with RPMI-1640 medium containing 5% calf serum.

2. Add target cells to a 96-well cell culture plate and add 100 μL per well. Three wells were used as effector cells (CAR-T cells) to naturally release control wells, and no target cells were added, and only 100 μL of the culture solution was added.

3. Add 10 μL of effector cells to each well, and the ratio of effector cells to target cells is 10:1; 5:1; 1:1. The natural release well was added with only 100 μL of the culture medium without effector cells, and the effector cells were incubated with the target cells for 6 hours, and three replicate wells were placed for each experiment.

4. In the largest release well (positive control) plus 10 μL Lysis Solution (10×), incubate for 45 min-60 min, and place three replicate wells for each experiment.

5. Take 50 μL of the sample to be tested and the control sample in the above 3 and 4, add to the fresh 96-well microtiter plate, and then add the reaction solution and the substrate, and protect from light for 30 min.

6. Add 50 μL of Stop Solution.

7. Determine the optical density (OD value) of each well on an enzyme-linked detector, and measure the wavelength at 490 nm or 492 nm, and measure within 1 hour.

8. Specific killing efficiency calculation

Killing rate = experimental group LDH (OD) / maximum LDH release group (OD).

Calculation formula: killing efficiency = (experimental group - effect natural release - target natural release) / (target maximum release - target natural release) × 100%.

9. The cytokine secretion was measured by CBA kit, and the proliferation of each group of CAR-T cells was calculated, and the ratio of CD8-positive T cells in the proliferating T cells was confirmed by staining with CD3 and CD8 antibodies.

The results of in vitro killing experiments of enhanced CAR-T (slit2D2 CAR&HAC-HSA) cells and common CAR-T (slit2D2 CAR) cells under different target-to-target ratios are shown in Fig. 6. The results show that the enhanced CAR-T prepared by this experiment ( The slit2D2 CAR&HAC-HSA) cells can specifically activate and proliferate after contact with tumor cells, killing tumor cells, and the effect of the enhanced CAR-T of slit2D2-HAC-HSA is better than that of slit2D2 CAR-T.

Claims (10)

A gene encoding a CAR chimeric antigen receptor, comprising a gene encoding an extracellular domain, a signaling domain, an intracellular immunostimulatory molecule, an internal ribosome entry site IRES, and HAC-HSA capable of binding an antigen. The coding gene according to claim 1, wherein the extracellular domain is Slit2D2, the coding gene thereof has the nucleotide sequence shown in SEQ ID No: 1; and/or The gene encoding the HAC has a nucleotide sequence as shown in SEQ ID No: 2; and/or, The signaling domain is selected from the group consisting of a hinge region, a transmembrane region, and an intracellular region of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, CD154 Kind or more; and/or, The intracellular immunostimulatory molecule is selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB and OX40 cells. One or more of the inner domains. The coding gene according to claim 2, wherein the signal transduction domain is a Hinge region, a transmembrane region of CD8; and/or The intracellular immune costimulatory molecules include the 4-1BB and CD3ζ intracellular domains. The coding gene according to any one of claims 1 to 3, wherein the coding gene further comprises a signal peptide SP1 encoding gene represented by SEQ ID No: 3; and/or The coding gene further comprises a nucleotide encoding sequence such as the Flag encoding gene set forth in SEQ ID No: 4; and/or, The coding gene further comprises a Linker encoding gene having a nucleotide sequence as set forth in SEQ ID No: 5; and/or, The coding gene also includes a signal peptide SP2 encoding gene having a nucleotide sequence as shown in SEQ ID No: 6. The coding gene according to claim 1, wherein the coding gene comprises a SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ fusion gene having a nucleotide sequence as shown in SEQ ID No: 7. and / or, The coding gene includes a SP2-HAC-HSA fusion gene having a nucleotide sequence as shown in SEQ ID No: 8. The coding gene according to claim 1, wherein the gene encoding the CAR chimeric antigen receptor is SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ-IRES-SP2-HAC-HSA coding. The nucleotide sequence of the gene, IRES is shown in SEQ ID No: 9. A recombinant vector or recombinant strain carrying the gene encoding according to any one of claims 1 to 6. Use of the coding gene according to any one of claims 1 to 6 and the recombinant vector of claim 7, a recombinant strain for modifying immune cells and preparing an antitumor drug. A CAR chimeric antigen receptor, characterized in that the CAR chimeric antigen receptor is obtained by transcriptional expression of the coding gene according to any one of claims 1 to 6; preferably, the CAR chimeric antigen is subjected to The body includes an SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 element, which has: 1) an amino acid sequence as shown in SEQ ID No: 10, or, 2) an amino acid sequence from which 1) is substituted and/or deleted and/or added by one or several amino acid residues and has the same function; and/or The CAR chimeric antigen receptor comprises an SP2-HAC-HSA element having: 3) the amino acid sequence shown as SEQ ID No: 11, or, 4) An amino acid sequence derived therefrom which is 3) substituted and/or deleted and/or added by one or several amino acid residues and having the same function. A reinforced CAR-immunoblast comprising the gene encoding according to any one of claims 1 to 6; preferably, the booster CAR-immunoblast is a booster CAR-T cell or Enhanced CAR-NK cells.

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