WO2018053885A1 - 一种加强型Slit2 CAR-T和CAR-NK细胞制备方法和应用 - Google Patents

一种加强型Slit2 CAR-T和CAR-NK细胞制备方法和应用 Download PDF

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WO2018053885A1
WO2018053885A1 PCT/CN2016/101751 CN2016101751W WO2018053885A1 WO 2018053885 A1 WO2018053885 A1 WO 2018053885A1 CN 2016101751 W CN2016101751 W CN 2016101751W WO 2018053885 A1 WO2018053885 A1 WO 2018053885A1
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car
cells
gene
seq
hac
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李华顺
王保垒
任宝永
方冬冬
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李华顺
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Definitions

  • 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.
  • PD-L1 full-length programmed death receptor-ligand 1 the English name programmed cell death-Ligand 1
  • PD-1 the full name of programmed death receptor 1, the English name for programmed death 1
  • PD-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).
  • APCs antigen-presenting cells
  • 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 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.
  • CAR-T 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.
  • CAR-T genetically modified chimeric antigen receptor T cell
  • the present invention provides a CAR chimeric antigen receptor, a potentiating CAR-immunoblast, and a preparation method thereof.
  • 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;
  • 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.
  • 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;
  • the intracellular immunostimulatory molecule is selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB And one or more of the OX40 intracellular domains;
  • the intracellular immunocostimulatory molecule comprises a 4-1BB and a CD3 sputum intracellular domain;
  • the above coding gene further comprises a signal peptide Signal peptide1 (SP1) encoding gene represented by SEQ ID No: 3;
  • SP1 signal peptide Signal peptide1
  • the above coding gene further comprises a nucleotide coding sequence as shown in SEQ ID No: 4;
  • the above coding gene further comprises a Linker coding gene having a nucleotide sequence as shown in SEQ ID No: 5;
  • the above coding gene further comprises a signal peptide Signal peptide 2 (SP2) encoding gene represented by SEQ ID No: 6;
  • SP2 signal peptide Signal peptide 2
  • the above coding gene comprises a SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 ⁇ fusion gene having a nucleotide sequence as shown in SEQ ID No: 7.
  • the above coding gene comprises a SP2-HAC-HSA fusion gene having a nucleotide sequence as shown in SEQ ID No: 8.
  • the gene encoding the CAR chimeric antigen receptor is SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 ⁇ -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;
  • 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;
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • the intracellular immunostimulatory molecule is selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB And one or more of the OX40 intracellular domains.
  • 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.
  • HAC high-affinity consensus
  • 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;
  • 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.
  • 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.
  • 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%.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID No: Medium selected from V39, N41, Y43, M45, S48, N49, Q50, T51, D52, K53
  • 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,
  • 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 G65
  • 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 ⁇
  • 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;
  • 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.
  • amino acid residue is changed to N41I or N41V.
  • the CAR chimeric antigen receptor comprises a SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3 ⁇ element having:
  • 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.
  • the CAR chimeric antigen receptor comprises an SP2-HAC-HSA element having:
  • 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.
  • the method for preparing the enhanced CAR-immune cell of the present invention comprises the following steps:
  • Construction of vector construction of a vector capable of expressing the above CAR receptor
  • packaging of virus transfected with immune cells packaging the virus using the vector constructed in step (1) to obtain a packaged virus
  • 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.;
  • 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.
  • HSV vector etc.
  • 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.
  • 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.
  • the method for preparing the enhanced CAR-T cell comprises the following steps:
  • step (3) 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-CD3 ⁇ -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.
  • the fusion of the HSA protein prolongs the half-life of the protein.
  • the enhanced CAR-immunized cells of the present invention 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.
  • Example 1 is a schematic diagram showing the construction of the Slit2D2-CAR gene provided in Example 1 of the present invention
  • FIG. 2 is a schematic diagram showing the construction of the Slit2D2-CD8TM-4-1BB-CD3 ⁇ -IRES-HAC-HSA gene provided in Example 1 of the present invention
  • FIG. 3 is a schematic diagram of a pRRSLIN-slit2D2 CAR&HAC-HSA lentiviral expression vector provided in Example 1 of the present invention
  • Example 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
  • the target cell in B is SMMC7721.
  • the second domain of Slit2, Slit2D2 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-CD3 ⁇ ) 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.
  • the gene sequence of Slit2D2-CD8-4-1BB-CD3 ⁇ -IRES-HAC-HSA was transformed into the PRRSLIN vector by double restriction enzyme ligation, and the upstream of the gene was the EP-1 ⁇ 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.
  • 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 ⁇ g pRRSLIN-Slit2D2-CAR-IRES-HAC-HSA (target plasmid); 39.5 ⁇ g pMD2.G (VSV-G envelop); 73 ⁇ g pCMVR8.74 (gag, pol, tat, Rev); 625 ⁇ L 2M calcium ion solution. Total volume of solution B: 6.25 mL.
  • 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 100 ⁇ L 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
  • PBMC peripheral blood mononuclear cells
  • 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 ⁇ L flow detection of the purity of T cells in PBMC.
  • FBS autologous AB
  • IL-2 interleukin-2
  • 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
  • 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.
  • 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.
  • 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.
  • the killing effect of CAR-T cells on tumor cells was detected by LDH release method, and LDH release was detected by ELISA.
  • target cells to a 96-well cell culture plate and add 100 ⁇ L 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 ⁇ L of the culture solution was added.
  • CAR-T cells effector cells
  • Killing rate experimental group LDH (OD) / maximum LDH release group (OD).
  • 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.

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Abstract

一种加强型Slit2 CAR-T和CAT-NK细胞制备方法和应用，所述加强型CAR-T和CAT-NK细胞分别为包含加强型CAR嵌合抗原受体的T细胞和NK细胞，其中加强型CAR嵌合抗原受体包括能够结合抗原的胞外结构域、跨膜结构域、胞内免疫共刺激分子、内部核糖体进入位点IRES和HAC-HSA串联；其中胞外结构域包括Slit2蛋白的D2结构域。所述的加强型CAR-T细胞和加强型CAR-NK细胞，可封闭PDL-1抑制性信号，增强CAR-免疫细胞杀伤活性，并激活肿瘤浸润的免疫细胞；加强型CAR-免疫细胞可作为细胞药物用于肿瘤类疾病的治疗，使改造后的免疫细胞能够特异性识别和杀伤肿瘤，且具备更高效的肿瘤杀伤活性。

Description

一种加强型Slit2 CAR-T和CAR-NK细胞制备方法和应用 技术领域
本发明涉及生物技术领域，尤其涉及一种针对抗肿瘤干细胞的嵌合抗原受体细胞，特别是加强型CAR-T细胞和加强型CAR-NK细胞。
发明背景
PD-L1全称程序性死亡受体-配体1，英文名字programmed cell death-Ligand 1，是大小为40kDa的第一型跨膜蛋白。PD-1全称程序性死亡受体1，英文名字为programmed death 1，是一种重要的免疫抑制分子，为CD28超家族成员。PD-1(programmed death-1)主要表达于T细胞及初级B细胞表面，PD-1的两个配体(PD-L1和PD-L2)，广泛表达于抗原提呈细胞(APCs)等。PD1与其受体的相互作用，在免疫应答的负性调控方面发挥着重要作用。在许多人类肿瘤组织中均可检测到PD-L1蛋白的表达，肿瘤部位的微环境可诱导肿瘤细胞上的PD-L1的表达，表达的PD-L1有利于肿瘤的发生和生长，诱导抗肿瘤T细胞的凋亡，进而逃避免疫***的攻击。抑制PD-1与其配体的结合，可以使肿瘤细胞暴露于免疫***的杀伤视野，进而达到杀伤肿瘤组织及治疗癌症的作用。
在2016AACR年会上，华盛顿大学医学院皮肤病学科教授Paul T.Nghiem，MD，PhD做出报道，pembrolizumab这一具有抗PD-1作用的单克隆抗体可诱导高级别默克尔细胞癌的高反应性，中位无进展生存期PFS是9个月，传统化疗的患者PFS是3个月。近日百时美PD-1抑制剂Opdivo在临床试验方面取得新进展，发布了两组数据，其中一组显示，对当前任何药物均无治疗反应的晚期黑色素瘤的患者在使用Opdivo治疗中取得了34％五年生存率的结果。值得注意的是，IV期黑色素瘤患者的五年生存率通常只有15％到20％。另一组数据表明Opdivo和Yervoy联用治疗晚期黑色素瘤患者中取得22％的总缓解率，并且达到了69％两年总体生存率。另一项研究表明难治复发性或转移性头颈部鳞状细胞癌(SCCHN)患者在使用Opdivo治疗后生存期和存活率大大提高。数据表明，与对照组相比Opdivo治疗组死亡风险显着降低30％，中位总生存期显着延长。Opdivo治疗组一年存活率为36％，对照组为16.6％。此外，该研究还通过口咽部肿瘤HPV状态及PD-L1表达状态评估了Opdivo相对于对照方案的疗效。近期，Keytruda获FDA治疗复发性或难治性(R/R)经典型霍奇金淋巴瘤(cHL)的突破性药物资格。
CAR-T(Chimeric Antigen Receptor T-Cell Immunotherapy)，即嵌合抗原受体T细胞免疫疗法。该疗法是一种出现了很多年但近几年才被改良使用到临床中的新型细胞疗法。在急性白血病和非霍奇金淋巴瘤的治疗上有着显著的疗效，被认为是最有前景的肿瘤治疗方式之一。嵌合抗原受体(chimeric antigen receptors,CAR)T细胞(CAR-T)是对病人自己的T细胞经过基因修饰而构建出的，在治疗血癌上取得成功，但是利用CAR-T治疗实体瘤(是由异质的细胞群体组成的，这些异质的细胞具有不同的表面分子)并未取得理想的治疗效果。
CN105505869A公开了一种针对肿瘤干细胞的嵌合受体T细胞，此T细胞嵌合了2～3个独立的抗原受体，每个嵌合的抗原受体分别由不同的肿瘤干细胞特异性标记物的抗体的抗原结合部与不同的功能蛋白结合组成。此嵌合受体T细胞仅能在2-3个嵌合抗原受体均识别抗原后，才激活T细胞抗肿瘤作用，此方法在一定程度上提高了特异性但对于隐藏抗原的肿瘤细胞的特异性较低。
Liza B.John报道的文献(Blockade of PD-1 immunosuppression boosts CAR T-cell therapy)中通过加入抗PD-1抗体阻碍了PD-1免疫抑制途径即PD-1与配体PD-L的结合，从而在一定程度上提高基因修饰的表达嵌合抗原受体T细胞(CAR-T)的抗肿瘤功效。
发明内容
为克服现有技术中的不足，本发明提供了一种CAR嵌合抗原受体、加强型CAR-免疫细胞及其制备方法。
本发明一方面提供一种编码CAR嵌合抗原受体的基因，包括能够结合抗原的胞外结构域、信号传导结构域、胞内免疫共刺激分子、内部核糖体进入位点IRES和HAC-HSA的编码基因。
所述胞外结构域包括Slit2蛋白的D2结构域；简称Slit2D2，其编码基因具有如SEQ ID No：1所示的核苷酸序列；
所述的HAC的编码基因具有如SEQ ID No：2所示的核苷酸序列；
优选的，所述信号传导结构域选自CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS、CD154的铰链区、跨膜区和胞内区结构域的一种或多种。
优选的，所述信号传导结构域选自CD8的Hinge区、跨膜区和胞内区，更优选的，所述CD8包括CD8的Hinge区和跨膜区；
优选的，所述胞内免疫共刺激分子选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40胞内结构域中的一种或多种；
优选的，所述胞内免疫共刺激分子包括4-1BB和CD3ζ胞内结构域；
优选的，上述编码基因还包括核苷酸序列如SEQ ID No：3所示的信号肽Signal peptide1(SP1)编码基因；
优选的，上述编码基因还包括核苷酸序列如SEQ ID No：4所示的Flag编码基因；
优选的，上述编码基因还包括核苷酸序列如SEQ ID No：5所示的Linker编码基因；
优选的，上述编码基因还包括核苷酸序列如SEQ ID No：6所示的信号肽Signal peptide2(SP2)编码基因；
优选的，上述编码基因包括核苷酸序列如SEQ ID No：7所示的SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ融合基因；
优选的，上述编码基因包括核苷酸序列如SEQ ID No：8所示的SP2-HAC-HSA融合基因；
在本发明的一个优选实施例中，上述编码CAR嵌合抗原受体的基因为SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ-IRES-SP2-HAC-HSA编码基因，IRES的核苷酸序列如SEQ ID No：9所示。
本发明另一方面提供一种装载有上述编码基因的重组载体、重组菌株；
优选的，所述的重组载体选自：慢病毒、逆转录病毒、腺病毒、腺相关病毒或质粒。
本发明另一方面提供一种上述编码基因、装载有上述编码基因的重组载体、重组菌株在修饰免疫细胞和制备抗肿瘤药物中的应用；
优选的，所述的免疫细胞选自：T细胞、NK细胞如NK92。
本发明另一方面提供一种加强型CAR-免疫细胞，所述的细胞中含有上述编码基因；
优选的，所述的加强型CAR-免疫细胞为加强型CAR-T细胞或加强型CAR-NK细胞如加强型CAR-NK92细胞。
本发明另一方面还提供一种上述加强型CAR-免疫细胞在制备抗肿瘤药物中的应用。
本发明另一方面提供了一种CAR嵌合抗原受体，由上述编码基因转录表达得到，包括：能够结合抗原的胞外结构域、跨膜结构域、胞内免疫共刺激分子、内部核糖体进入位点IRES和HAC-HSA。
所述胞外结构域包括Slit2蛋白的D2结构域。
优选的，所述信号传导结构域选自CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS、CD154中一种的铰链区、跨膜区和胞内区中的一种或多种。
优选的，所述信号传导结构域选自CD8的铰链区、跨膜区和胞内区，更优选的，所述CD8为CD8的Hinge区 和CD8TM跨膜结构域。
优选的，所述胞内免疫共刺激分子选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40胞内结构域中的一种或多种。
优选的，所述胞内免疫共刺激分子包括4-1BB和CD3ζ胞内结构域。
本发明所述HAC-HSA中HAC为具有高亲和力共识(High-affinity consensus,HAC)的PD-1蛋白，可在体内和体外的情况下阻碍野生型PD-1蛋白与PD-L配体的结合；
所述HAC相对于野生型PD-1蛋白缺少一个跨膜结构域，并且具有一个或几个氨基酸残基的改变，同时，提高了对PD-L1配体的亲和力；
所述氨基酸残基改变，可位于PD-1和PD-L1结合域中，和/或，
氨基酸残基改变可位于PD-1的免疫球蛋白结构域。
在一个具体实施例中，所述HAC包含一个氨基酸序列相对于野生型PD-1蛋白多肽同一性选自85％或更高，90％或更高，95％或更高，98％，99％或更高，99.2％或更高。
在另一具体实施例中，所述HAC包含一个氨基酸序列相对于野生型PD-1蛋白多肽的免疫球蛋白域的同一性为85％或更高，90％或更高，95％或更高，98％，99％或更高，99.2％或更高，99.8％或更高，99.9％或更高，或100％。
在另一具体实施例中，所述HAC包含一个氨基酸序列相对于如SEQ ID No：6所示的原始仿制PD-1多肽氨基酸序列同一性为85％或更高，90％或更高，95％或更高，98％，99％或更高，99.2％或更高，99.8％或更高，99.9％或更高，或100％。
优选的，所述HAC包含一个氨基酸的突变相对于野生型PD-1蛋白多肽，此氨基酸的突变提高了HAC对PD-L1的亲和性；
所述氨基酸的改变1个或多个，2个或多个，3个或多个，4个或多个，5个或多个，6个或多个，7个或多个，8个或多个，9个或多个，10个或多个，11个或多个，12个或多个，13个或多个，14个或多个，15个或多个，16个或多个，17个或多个，18个或多个，19个或多个，20个或多个等。
优选的，在一个具体实施例中，所述氨基酸残基改变，其位点，选自如SEQ ID No：6所示野生型PD-1片段中的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，A107中的一种或几种；或，其他野生型PD-1蛋白相应位置的氨基酸；所述氨基酸改变包括1个或多个氨基酸的改变；所述多个氨基酸改变为2个或多个，3个或多个，4个或多个，5个或多个，6个或多个，7个或多个，8个或多个，9个或多个，10个或多个，11个或多个，12个或多个，13个或多个，14个或多个，15个或多个，16个或多个，17个或多个，18个或多个。
优选的，在另一具体实施例中，所述氨基酸残基改变可位于PD-1和PD-L1结合域中，所述氨基酸改变位点位于如SEQ ID No：6所示的PD-1片段中，选自V39，N41，Y43，M45，S48，N49，Q50，T51，D52，K53，A56，Q63，G65，Q66，L97，S102，L103，A104，P105，K106，A107；或，其他野生型PD-1蛋白相应位置的氨基酸中的一种或几种；所述氨基酸改变包括1个或多个氨基酸的改变；所述多个氨基酸改变为2个或多个，3个或多个，4个或多个，5个或多个，6个或多个，7个或多个，8个或多个，9个或多个，10个或多个，11个或多个，12个或多个，13个或多个，14个或多个，15个或多个。
在另一具体实施例中，所述氨基酸残基改变，其位点位于如SEQ ID No：6所示的PD-1片段中，选自：(a)V39，N41，Y43，M45，S48，N49，Q50，K53，A56，Q63，G65，Q66，L97，A100，S102，L103，A104，K106 和A107；或，其他野生型PD-1蛋白相应位置的氨基酸；(b)V39，N41，Y43，M45，S48，Q50，T51，D52F，K53，A56，Q63，G65，Q66，L97，S102，L103，A104，K106和A107；或，其他野生型PD-1蛋白相应位置的氨基酸(c)V39，L40，N41，Y43，R44，M45，N49，K53，M83，L97，A100和A107；或，其他野生型PD-1蛋白相应位置的氨基酸；(d)V39，L40，N41，Y43，R44，M45，N49，Q66P，M83，L97和A107；或，其他野生型PD-1蛋白相应位置的氨基酸；(e)V39，L40，N41，Y43，M45，N49，K53，Q66P，H82，M83，L97，A100和A107；或，其他野生型PD-1蛋白相应位置的氨基酸；(f)V39，L40，N41，Y43，M45，N49，K53，M83，L97，A100，和A107；或，其他野生型PD-1蛋白相应位置的氨基酸；(g)V39，L40，N41，Y43，R44，M45，N49，K53，L97，A100和A107；(h)V39，L40，N41，Y43，M45，S48，N49，K53，L97，A100和A107；或，其他野生型PD-1蛋白相应位置的氨基酸。
在另一具体实施例中，所述氨基酸残基改变，其位点位于如SEQ ID No：6所示的PD-1片段中，选自：(1)V39H或V39R；(2)L40V或L40I；(3)N41I或N41V；(4)Y43F或Y43H；(5)R44Y或R44L；(6)M45Q，M45E，M45L或M45D；(7)S48D，S48L，S48N，S48G或S48V；(8)N49C，N49G，N49Y或N49S；(9)Q50K，Q50E或Q50H；(10)T51V，T51L或T51A；(11)D52F，D52R，D52Y或D52V；(12)K53T或K53L；(13)A56S或A56L；(14)Q63T；Q63I，Q63E，Q63L或Q63P；(15)G65N；G65R，G65I，G65L，G65F或G65V；(16)Q66P；(17)V72I；(18)H82Q；(19)M83L或M83F；(20)R90K；(21)Y96F；(22)L97Y，L97V或L97I；(23)A100I或A100V；(24)S102T或S102A；(25)L103I，L103Y或L103F；(26)A104S，A104H或A104D；(27)P105A；(28)K106G，K106E，K106I，K106V，K106R或K106T；(29)A107P；A107I或A107V；或，其他野生型PD-1蛋白相应位置的氨基酸。
在另一具体实施例中，所述氨基酸残基改变，其位点位于如SEQ ID No：6所示的PD-1片段中，选自：(a){V39H或V39R}，{N41I或N41V}，{Y43F，Y43H}，{M45Q，M45E，M45L或M45D}，{S48D，S48L，S48N，S48G或S48V}，{N49C，N49G，N49Y或N49S}，{Q50K，Q50E或Q50H}，{K53T或K53L}，{A56S或A56L}，{Q63T，Q63I，Q63E，Q63L或Q63P}，{G65N，G65R，G65I，G65L，G65F或G65V}，{L97Y，L97V或L97I}，{S102T或S102A}，{L103I，L103Y或L103F}，{S102T或S102A}，{L103I，L103Y或L103F}，{A104S，A104H或A104D}，{K106G，K106E，K106I，K106V，K106R或K106T}，{A107P，A107I或A107V}；或，其他野生型PD-1蛋白相应位置的氨基酸；
(b){V39H或V39R}，{N41I或N41V}，{Y43F或Y43H}，{M45Q，M45E，M45L或M45D}，{S48D，S48L，S48N，S48G或S48V}，{Q50K，Q50E或Q50H}，{T51V，T51L或T51A}，{D52F，D52R，D52Y或D52V}，{K53T或K53L}，{A56S或A56L}，{Q63T，Q63I，Q63E，Q63L或Q63P}，{G65N，G65R，G65I，G65L，G65F或G65V}，{Q66P}，{L97Y，L97V或L97I}，{S102T或S102A}，{L103I，L103Y或L103F}，{A104S，A104H或A104D}，{K106G，K106E，K106I，K106V，K106R或K106T}，{A107P，A107I或A107V}；或，其他野生型PD-1蛋白相应位置的氨基酸；
(c){V39H或V39R}，{L40V或L40I}，{N41I或N41V}，{Y43F或Y43H}，{R44Y或R44L}，{M45Q，M45E，M45L或M45D}，{N49C，N49G，N49Y或N49S}，{K53T或K53L}，{M83L或M83F}，{L97Y，L97V或L97I}，{A100I或A100V}，{A107P，A107I或A107V}；或，其他野生型PD-1蛋白相应位置的氨基酸；
(d){V39H或V39R}，{L40V或L40I}，{N41I或N41V}，{Y43F或Y43H}，{M45Q，M45E，M45L或M45D}，{N49C，N49G，N49Y或N49S}，{K53T或K53L}，{Q66P}，{M83L或M83F}，{L97Y，L97V或L97I}和{A107P， A107I或A107V}；或，其他野生型PD-1蛋白相应位置的氨基酸；
(e){V39H或V39R}，{L40V或L40I}，{N41I或N41V}，{Y43F或Y43H}，{M45Q，M45E，M45L或M45D}，{N49C，N49G，N49Y或N49S}，{K53T或K53L}，{Q66P}，{H82Q}，{M83L或M83F}，{L97Y，L97V或L97I}，{A100I或A100V}和{A107P，A107I或A107V}；或，其他野生型PD-1蛋白相应位置的氨基酸；
(f){V39H或V39R}，{L40V或L40I}，{N41I或N41V}，{Y43F或Y43H}，{M45Q，M45E，M45L或M45D}，{N49C，N49G，N49Y或N49S}，{K53T或K53L}，{M83L或M83F}，{L97Y，L97V或L97I}，{A100I或A100V}和{A107P，A107I或A107V}；或，其他野生型PD-1蛋白相应位置的氨基酸；
(g){V39H或V39R}，{L40V或L40I}，{N41I或N41V}，{Y43F或Y43H}，{R44Y或R44L}，{M45Q，M45E，M45L或M45D}，{N49C，N49G，N49Y或N49S}，{K53T或K53L}，{L97Y，L97V或L97I}，{A100I或A100V}和{A107P，A107I或A107V}；或，其他野生型PD-1蛋白相应位置的氨基酸；
(h){V39H或V39R}，{L40V或L40I}，{N41I或N41V}，{Y43F或Y43H}，{M45Q，M45E，M45L或M45D}，{N49C，N49G，N49Y或N49S}，{K53T或K53L}，{L97Y，L97V或L97I}，{A100I或A100V}和{A107P，A107I或A107V}；或，其他野生型PD-1蛋白相应位置的氨基酸；
在另一具体实施例中，所述氨基酸残基改变，其位点位于如SEQ ID No：6所示的PD-1片段中，选自：(a)V39R，N41V，Y43H，M45E，S48G，Q50E，K53T，A56S，Q63T，G65L，Q66P，L97V，S102A，L103F，A104H，K106V和A107I；或，其他野生型PD-1蛋白相应位置的氨基酸；
(b)V39R，N41V，Y43H，M45E，S48N，Q50H，T51A，D52V，K53T，A56S，Q63L，G65F，Q66P，L97I，S102T，L103F，A104D，K106R和A107I；或，其他野生型PD-1蛋白相应位置的氨基酸；
(c)V39H，L40V，N41V，Y43H，R44Y，M45E，N49G，K53T，M83L，L97V，A100I和A107I；或，其他野生型PD-1蛋白相应位置的氨基酸；
(d)V39H，L40V，N41V，Y43H，M45E，N49G，K53T，Q66P，M83L，L97V和A107I；或，其他野生型PD-1蛋白相应位置的氨基酸；
(e)V39H，L40V，N41V，Y43H，M45E，N49S，K53T，Q66P，H82Q，M83L，L97V，A100V和A107I；或，其他野生型PD-1蛋白相应位置的氨基酸；
(f)V39H，L40I，N41I，Y43H，M45E，N49G，K53T，M83L，L97V，A100V和A107I；或，其他野生型PD-1蛋白相应位置的氨基酸；
(g)V39H，L40I，N41I，Y43H，R44L，M45E，N49G，K53T，L97V，A100V和A107I；或，其他野生型PD-1蛋白相应位置的氨基酸；
(h)V39H，L40V，N41I，Y43H，M45E，N49G，K53T，L97V，A100V和A107I；或，其他野生型PD-1蛋白相应位置的氨基酸；
(i)V39H，L40V，N41V，Y43H，M45E，N49G，K53T，L97V，A100V和A107I；或，其他野生型PD-1蛋白相应位置的氨基酸；
所述具有高亲和力共识的PD-1蛋白HAC可为转录后修饰蛋白；所述修饰包括糖基化、PEG修饰等。
更优选的，所述氨基酸残基改变为N41I或N41V。
优选的，所述的CAR嵌合抗原受体包括SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ元件，其具有：
1)如SEQ ID No：10所示的氨基酸序列，或，
2)将1)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列。
优选的，所述CAR嵌合抗原受体包括SP2-HAC-HSA元件，其具有：
3)如SEQ ID No：11所示的氨基酸序列，或，
4)将3)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列。
优选的，所述的嵌合抗原受体中，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超10个氨基酸残基的取代和/或缺失和/或添加。
本发明所述的氨基酸序列的连接“-”为一片段的N末端与另一片段的C末端直接连接，中间没有任何连接肽，如，HAC-HSA，HAC结构域通过C末端与HSA结构域的N末端直接连接，或HAC结构域通过其N末端与的HSA结构域C末端直接连接，即HAC结构域与HSA结构域直接连接，中间没有任何连接肽。
本发明另一方面提供一种加强型CAR-免疫细胞的制备方法，本发明合成编码基因可通过常规方法，从指定CAR的氨基酸序列，容易地制备编码CAR的碱基序列，由氨基酸序列的NCBI Ref Seq ID或GenBenk检索号得到编码氨基酸序列的碱基序列，并且可使用标准分子生物学和/或化学程序制备本发明的核酸。
优选的，本发明所述加强型CAR-免疫细胞的制备方法，具体包括以下步骤：
(1)载体的构建：构建能够表达上述CAR受体的载体；
(2)转染免疫细胞的病毒的包装：采用步骤(1)中构建的载体包装病毒，获得经过包装的病毒；
(3)细胞分离与扩增培养：抽取患者自身血液，从中分离出免疫细胞并进行扩增培养；
(4)免疫细胞的转染与制备及检测：采用步骤(2)中经过包装的病毒对步骤(3)中培养所得T细胞进行转染并扩增培养，获得嵌合抗原受体T细胞简称CAR-免疫细胞。
所述嵌合抗原受体免疫细胞为在免疫细胞中引入了本发明所述的编码嵌合抗原受体的基因。
所述表达载体可使用缺乏复制能力、无法在转染细胞中自我复制的病毒载体例如逆转录病毒载体(包括致癌逆转录病毒载体、慢病毒载体和假型载体)、腺病毒载体、痘苗病毒载体或HSV载体等；
优选的，本发明中所述免疫细胞来源于人外周血的免疫细胞，更优选自：T细胞、NK细胞如NK92；更优选的，本发明中可根据逆转录病毒选择广泛市售的多种类的可用于包装逆转录病毒载体的包装质粒，也可使用具有高转染效率的293细胞或293T细胞制备逆转录病毒颗粒。
其中，步骤(1)中所述载体的构建包括对胞外结构域Slit2D2、跨膜结构域、胞内免疫共刺激分子、内部核糖体进入位点IRES和HAC-HSA基因的扩增、酶切连接和转化。
在本发明一个优选的实施方式中，加强型CAR-T细胞的制备方法，具体包括如下步骤：
(1)载体的构建：扩增连接Slit2D2-CD8TM-4-1BB-CD3ζ-IRES-HAC-HSA基因，将所述基因克隆至慢病毒表达载体上；
(2)转染T细胞的病毒的包装：利用慢病毒包装质粒和慢表达载体转染293T细胞，包装和制备慢病毒；
(3)细胞分离与扩增培养：抽取患者自身血液，从中分离人外周血T细胞，培养扩增，利用慢病毒转染T细胞，使T细胞表达Slit2D2-CD8TM-4-1BB-CD3ζ-IRES-HAC-HSA。
(4)T细胞的转染与制备及检测：采用步骤(2)中经过包装的慢病毒对步骤(3)中培养所得T细胞进行转染并扩增培养，使T细胞表达Slit2D2-CD8TM-4-1BB-CD3ζ-IRES-HAC-HSA。
本发明将CAR技术和PD-1抗体免疫检查点治疗方法融合，通过在常规CAR-免疫细胞表达载体上加入分泌性HAC-HSA融合基因，制备得到加强型CAR-免疫细胞，其中，HAC-HSA表达的PD-1蛋白与PDL-1具有较高的亲 和力，同时，融合的HSA蛋白延长了蛋白的半衰期。本发明中的加强型CAR-免疫细胞，特别是加强型CAR-T细胞、加强型CAR-NK细胞，具有传统CAR-免疫细胞靶向杀伤能力，同时能够分泌PD-1融合蛋白，封闭PDL-1抑制性信号，增强CAR-免疫杀伤活性，并激活肿瘤浸润的免疫细胞。
附图简要说明
图1所示为本发明实施例1提供的Slit2D2-CAR基因构建示意图；
图2所示为本发明实施例1提供的Slit2D2-CD8TM-4-1BB-CD3ζ-IRES-HAC-HSA基因构建示意图；
图3为本发明实施例1提供的pRRSLIN-slit2D2 CAR&HAC-HSA慢病毒表达载体示意图；
图4所示为本发明实施例4提供的CAR-T细胞的流式浸染效果图；
图5所示为本发明实施例4提供的CAR-NK92细胞流式浸染效果图；
图6所示为本发明实施例6提供的不同效靶比条件下加强型CAR-T(slit2D2 CAR&HAC-HSA)细胞和普通CAR-T(slit2D2 CAR)细胞体外杀伤实验结果；其中A图中的靶细胞为H1299，B图中的靶细胞为SMMC7721。
实施本发明的方式：
实施例1：慢病毒表达载体制备
1、根据已知的Slit2序列[GenBank:EAW92793.1]分析设计构建Slit2的第二个结构域Slit2D2(Hohenester2008)，其基因序列如SEQ ID NO：1所示，从GenBank数据库中搜寻已知的人CD8的Hinge区和CD8TM跨膜区基因序列、人4-1BB胞内区基因序列、CD3ζ胞内区、IRES内部核糖体进入位点，得到Slit2D2-CAR(SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ)基因如SEQ ID NO：7所示，其串联示意图如图1所示；合成HAC基因片段，其核苷酸序列如SEQ ID NO：2所示，同时在其C端引入HSA基因，得到HAC-HSA融合基因(SP2-HAC-HSA)如SEQ ID NO：8所示。
2、将上述HAC-HSA融合基因序列***到Slit2D2-CAR序列下游，形成完整的Slit2D2-CD8-4-1BB-CD3ζ-IRES-HAC-HSA，其构建示意图如图2所示。
3、将Slit2D2-CD8-4-1BB-CD3ζ-IRES-HAC-HSA的基因序列通过双酶切连接转化到PRRSLIN载体中，基因上游为EP-1α启动子。将载体转化到Stbl3大肠杆菌菌株后，转种到含有氨苄青霉素的固体培养基中进行繁殖，筛选，获得阳性克隆，提取质粒，酶切鉴定克隆，通过测序确认载体构建成功，获得pRRSLIN-Slit2D2慢病毒表达载体，慢病毒表达载体构建示意图参见附图3。
实施例2：慢病毒制备
1.转染前24小时，以每皿约8×106将293T细胞接种至15cm培养皿中。确保转染时细胞在80％左右的汇合度且均匀分布于培养皿中。
2.准备溶液A和溶液B
溶液A：6.25mL 2×HEPES buffer缓冲液(采用5个大皿一起包装的量，效果最好)。
溶液B：分别加入以下质粒的混合物：112.5μg pRRSLIN-Slit2D2-CAR-IRES-HAC-HSA(target plasmid)；39.5μgpMD2.G(VSV-G envelop)；73μg pCMVR8.74(gag，pol，tat，rev)；625μL 2M钙离子溶液。溶液B总体积：6.25mL。
3.充分混匀溶液B，轻轻涡旋溶液A的同时，逐滴加入溶液A，静置5-15分钟。轻轻涡旋上述A和B的混合溶液，逐滴加入含293T细胞的培养皿中，轻轻前后晃动培养皿使DNA与钙离子的混合物均匀分布。(不要旋转培养皿)放置于培养箱中培养16-18小时。
更换新鲜培养基，继续培养。
在转速500g，温度25℃下离心10min，使用PES膜(0.45μm)过滤；以70％乙醇消毒离心管(贝克曼库尔特ultra-clear SW28centrifuge tubes)，并置于紫外灯下消毒30min；将已过滤的含慢病毒的上清液转移至离心管中，在离心管底部小心铺上一层20％蔗糖(每8mL上清液加1mL蔗糖)，以PBS平衡离心管，在转速25,000rpm(82，700g)，温度4℃下离心2h；小心取出离心管，倒掉上清液，倒置离心管去掉残余液体；加入100μLPBS，密封离心管，在4℃放置2h，每20min轻轻涡旋一次，500g离心1min(25℃)，收集病毒上清；冰上冷却后，置于-80℃保存。
实施例3：CAR-T细胞制备
1.取0.5mL血进行快速的病原微生物检测，排除HBV、HCV、HDV和HEV、HIV-1/2、***及寄生虫等微生物感染；无菌条件下，用肝素瓶采血50mL(肝素抗凝)，立即(4℃，24小时内)送至细胞制备实验室，保证此过程无病原微生物污染。得到患者血液后，在GMP制备室，用酒精棉球擦拭肝素瓶表面进行消毒后放入生物安全柜。
2.预先打开2个50mL离心管，将血液转入两个50mL离心管中，旋紧；将上述装好血液的两个50mL离心管放入离心机离心，400g(2000rpm)离心10min，室温离心后收集上层血浆，留下沉淀层；收集的自体血浆经56℃，30min灭活，4℃放置15min后，900g，离心30min(4℃)，取上清备用。
3.将上述富集的血细胞用生理盐水稀释至30mL/管，打开2个新的50mL离心管，每个离心管分别加入15mL人淋巴细胞分离液，用移液管把稀释后的血细胞液缓缓加入到盛有人淋巴分离液的离心管中，旋紧。注意血液要加到淋巴分离液的上层，勿打破人淋巴分离液的界面。将加好的血细胞液放入离心机，调至最小的升降速率，400g(2000rpm)离心20min(常温)。收集两管的中层白细胞层于一支15mL无菌离心管中，加入5mL生理盐水，洗两次(400g，离心10min)，得外周血单核细胞(PBMC)。
4.配置完全生长培养基，V-VIVO15添加自体AB(FBS)浓度为5％，白细胞介素-2(IL-2)浓度为40ng/mL，将分离得到的PBMC用培养基稀释成2×106/mL，取50μL流式检测PBMC中T细胞的纯度。
5.Day 0，配置缓冲液(在PBS缓冲液中添加1％的胎牛血清(FBS))，选用微珠作为细胞培养载体，将微珠振荡30s或手动上下摇匀5min，按照微珠与T细胞的用量比为3：1取CD3/CD28微珠置于1.5mL EP管中，添加1mL缓冲液清洗微珠，之后使用磁铁从EP管向外吸微珠1min，弃洗液，重复两次，再使用培养基将微珠重悬到原体积，将细胞和微珠混合后按2×106PBMC/mL加到合适的培养瓶中。
6.Day 2将细胞密度调整至3-5×106/mL，按病毒与细胞的比例为1:5添加实施例2制备得到的pRRSLIN-Slit2D2-CAR-IRES HAC-HSA慢病毒，同时添加聚凝胺(polybrene)4μg/mL和40ng/mL IL-2。4h之后，补加新鲜的完全培养基将细胞密度调整至1×106/mL继续培养。将所有的细胞离心，加入新鲜的培养基，继续培养。
7.每隔2-3天进行半量换液，维持细胞密度在0.5-1×106/mL。
8.Day 10-12，细胞数量达到109级别，在400g下离心5min得到免疫细胞，再用预冷的PBS洗涤两遍(400g，5min)。
9.用血球计数板计数，流式细胞仪检测细胞类群，CART细胞比例。每天观察培养基的颜色变化、细胞密度、细胞形态并作相应记录。逐步扩大培养过程中，加入总体积所需的白细胞介素-2。
实施例4：CAR-NK92细胞制备
参考实施例3的实验步骤制备CAR-NK92细胞。
实施例5：CAR-T细胞和CAR-NK92细胞的流式分析
对实施例3制备的CAR-T细胞和实施例4制备的CAR-NK92细胞进行流式分析，其具体步骤如下：
1.取5×104细胞(包括T细胞、NK细胞、slit2D2 CAR-T细胞、slit2D2 CAR-NK细胞、slit2D2 &HAC-HSA CAR-T细胞、slit2D2 &HAC-HSA CAR-NK细胞)用于染色；
2.细胞与抗体(抗体可与FLAG标签识别结合，偶联APC荧光分子)共孵育45min，50μl，置于冰上；
3.PBS洗脱两次；
4.用120μl FACS试剂重悬细胞；
5.流式细胞仪器测量APC荧光信号，如果与对照T细胞或NK细胞对比，CAR细胞APC荧光信号增强，表面CAR细胞构建成功。
CAR-T细胞和CAR-NK92细胞的流式浸染效果分别如图4和图5所示。
图4中，A图和C图为对照组：不侵染病毒的T细胞；APC偶联检测CAR分子的抗体检测不到CAR分子表达；B图：转染slit2D2 CAR病毒的T细胞，经流式检测，有细胞成功转染slit2D2 CAR分子；D图：转染slit2D2&HAC-HSA CAR-病毒的T细胞，经流式检测，有细胞成功转染slit2D2&HAC-HSA CAR分子。B图和D图分别说明成功制备相应的CAR-T细胞。
图5中，A图和C图为对照组：不侵染病毒的NK细胞；APC偶联检测CAR分子的抗体检测不到CAR分子表达；B图：转染slit2D2 CAR病毒的NK细胞，经流式检测，有细胞成功转染slit2D2 CAR分子；D图转染slit2D2&HAC-HSA CAR-T病毒的NK细胞，经流式检测，有细胞成功转染slit2D2&HAC-HSA CAR分子。B图和D图分别说明成功制备相应的CAR-NK细胞。
实施例6：CAR-T细胞体外活性检测
采用LDH释放法检测CAR-T细胞对肿瘤细胞的杀伤效应，通过ELISA方法检测LDH释放。
1.用含5％小牛血清的RPMI-1640培养液将靶细胞调整到5×104/mL。
2.在96孔细胞培养板中加入靶细胞，每孔加100μL。取3个孔作为效应细胞(CAR-T细胞)自然释放对照孔，不加靶细胞，仅加100μL培养液。
3.向各孔加10μL效应细胞，效应细胞与靶细胞的比例10:1；5:1；1:1。自然释放孔不加效应细胞只加100μL培养液，效应细胞与靶细胞共孵育6小时，每个实验置三个复孔。
4.最大释放孔中(阳性对照)加10μL Lysis Solution(10×)，孵育45min-60min，每个实验置三个复孔。
5.取上述3和4中待测样品和对照样品各50μL，加入新鲜的96孔酶标板中，再加入反应液和底物，避光30min。
6.加入50μL终止液。
7.在酶联检测仪上测定各孔的光密度(OD值)，检测波长490nm或492nm，在1小时内测完。
8.特异性杀伤效率计算
杀伤率＝实验组LDH(OD)/最大LDH释放组(OD)。
计算公式：杀伤效率＝(实验组-效应自然释放-靶自然释放)/(靶最大释放-靶自然释放)×100％。
9.通过CBA试剂盒测定细胞因子分泌情况，同时计算CAR-T细胞各组中的增殖情况，并利用CD3和CD8抗体染色，确认增殖的T细胞中CD8阳性的T细胞的比例。
加强型CAR-T(slit2D2 CAR&HAC-HSA)细胞和普通CAR-T(slit2D2 CAR)细胞不同效靶比条件下体外杀伤实验结果见图6，由结果可知，由本实验制备的加强型CAR-T(slit2D2 CAR&HAC-HSA)细胞在接触肿瘤细胞后，可以特异性的发生活化和增殖，杀伤肿瘤细胞，同时slit2D2-HAC-HSA的加强型CAR-T的效果要优于slit2D2 CAR-T的效果。
Figure PCTCN2016101751-appb-000001
Figure PCTCN2016101751-appb-000002
Figure PCTCN2016101751-appb-000003
Figure PCTCN2016101751-appb-000004
Figure PCTCN2016101751-appb-000005
Figure PCTCN2016101751-appb-000006
Figure PCTCN2016101751-appb-000007

Claims (10)

  1. 一种编码CAR嵌合抗原受体的基因，包括能够结合抗原的胞外结构域、信号传导结构域、胞内免疫共刺激分子、内部核糖体进入位点IRES和HAC-HSA的编码基因。
  2. 如权利要求1所述的编码基因，其特征在于，所述胞外结构域为Slit2D2，其编码基因具有如SEQ ID No：1所示的核苷酸序列；和/或，
    所述的HAC的编码基因具有如SEQ ID No：2所示的核苷酸序列；和/或，
    所述信号传导结构域选自CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS、CD154的铰链区、跨膜区和胞内区的一种或多种；和/或，
    所述胞内免疫共刺激分子选自CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40胞内结构域中的一种或多种。
  3. 如权利要求2所述的编码基因，其特征在于，所述信号传导结构域为CD8的Hinge区、跨膜区；和/或，
    所述胞内免疫共刺激分子包括4-1BB和CD3ζ胞内结构域。
  4. 如权利要求1-3任一项所述的编码基因，其特征在于，所述编码基因还包括核苷酸序列如SEQ ID No：3所示的信号肽SP1编码基因；和/或，
    所述编码基因还包括核苷酸序列如SEQ ID No：4所示的Flag编码基因；和/或，
    所述编码基因还包括核苷酸序列如SEQ ID No：5所示的Linker编码基因；和/或，
    所述编码基因还包括核苷酸序列如SEQ ID No：6所示的信号肽SP2编码基因。
  5. 如权利要求1所述的编码基因，其特征在于，所述编码基因包括核苷酸序列如SEQ ID No：7所示的SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ融合基因；和/或，
    所述编码基因包括核苷酸序列如SEQ ID No：8所示的SP2-HAC-HSA融合基因。
  6. 如权利要求1所述的编码基因，其特征在于，所述编码CAR嵌合抗原受体的基因为SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ-IRES-SP2-HAC-HSA编码基因，IRES的核苷酸序列如SEQ ID No：9所示。
  7. 一种装载有如权利要求1-6任一项所述的编码基因的重组载体、重组菌株。
  8. 一种如权利要求1-6任一项所述的编码基因和如权利要求7所述的重组载体、重组菌株在修饰免疫细胞和制备抗肿瘤药物中的应用。
  9. 一种CAR嵌合抗原受体，其特征在于，所述CAR嵌合抗原受体由如权利要求1-6任一项所述的编码基因转录表达得到；优选的，所述CAR嵌合抗原受体包括SP1-Flag-Linker-Slit2D2-CD8-4-1BB-CD3ζ元件，其具有：
    1)如SEQ ID No：10所示的氨基酸序列，或，
    2)将1)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列；和/或，
    所述CAR嵌合抗原受体包括SP2-HAC-HSA元件，其具有：
    3)如SEQ ID No：11所示的氨基酸序列，或，
    4)将3)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的氨基酸序列。
  10. 一种加强型CAR-免疫细胞，所述的细胞中含有如权利要求1-6任一项所述的编码基因；优选的，所述的加强型CAR-免疫细胞为加强型CAR-T细胞或加强型CAR-NK细胞。
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US11299530B2 (en) 2017-01-05 2022-04-12 Kahr Medical Ltd. SIRP alpha-CD70 fusion protein and methods of use thereof
US11566060B2 (en) 2017-01-05 2023-01-31 Kahr Medical Ltd. PD1-CD70 fusion protein and methods of use thereof
US11702458B2 (en) 2017-01-05 2023-07-18 Kahr Medical Ltd. PD1-41BBL fusion protein and methods of use thereof
US11897937B2 (en) 2017-01-05 2024-02-13 Kahr Medical Ltd. SIRPalpha-41BBL fusion protein and methods of use thereof
CN113811604A (zh) * 2019-03-29 2021-12-17 得克萨斯大学体系董事会 Car-nk细胞的产生方法及其用途
CN114144430A (zh) * 2019-11-21 2022-03-04 博生吉医药科技(苏州)有限公司 Cd7-car-t细胞及其制备和应用
CN114144430B (zh) * 2019-11-21 2023-11-07 博生吉医药科技(苏州)有限公司 Cd7-car-t细胞及其制备和应用
CN117106727A (zh) * 2023-08-22 2023-11-24 翔鹏佑康(北京)科技有限公司 一种car-nk细胞制剂的制备方法

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