CN116063575A - Chimeric Antigen Receptor (CAR) and application thereof in treating tumors - Google Patents

Abstract

The invention provides a Chimeric Antigen Receptor (CAR) and its use in the treatment of tumors. The antigen binding fragment with higher affinity with the target antigen CD19 is selected, so that the target antigen can be efficiently identified, the powerful anti-tumor effect is exerted, and the CD19 antigen binding fragment can be combined with various tumor cells, and has certain anti-cancer broad spectrum; CD27 and CD28 are used as double co-stimulators, so that the activation degree of T cells can be improved, the duration of CAR-T cells in vivo can be greatly prolonged, and the long-acting anti-tumor effect can be exerted; the nano liposome drug delivery system is constructed, and the liposome wraps the mammalian cell expression vector, so that the assembly of the CAR-T cells in the body is realized, the complicated process of manually preparing the CAR-T in the body is avoided, the production, transportation and storage costs are reduced, the over-expression of inflammation inhibition can be inhibited, and the safety of treatment is improved; the liposome wraps up the siRNA of the anti-TGF-beta, can inhibit the expression of TGF-beta effectively, relieve the immunosuppression to T cells, raise its anti-tumor activity; the nano liposome drug delivery system is externally modified by using an anti-CD 8 antibody, so that CD8+ T cells can be effectively captured, and then CAR-T cells with strong anti-tumor activity are generated, and the tumor treatment effect is improved.

Description

Chimeric Antigen Receptor (CAR) and application thereof in treating tumors

Technical field:

the invention belongs to the field of disease treatment, and particularly provides a Chimeric Antigen Receptor (CAR) and application thereof in treating tumors.

The background technology is as follows:

immunotherapy completely alters the traditional mode of cancer treatment, becoming the fourth largest tumor treatment after surgery, radiotherapy and chemotherapy, the most successful cases in tumor immunotherapy being immune checkpoint inhibitors and adoptive cell therapies (adoptive cell therapy, ACT) represented by chimeric antigen receptor (chimeric antigen receptor, CAR) T cells. Among metastatic cancers that cannot be cured using traditional therapies, CAR-T immunotherapy exhibits striking efficacy, some patients can achieve long-term remission and possibly cure, and Complete Remission Rates (CRR) of CD19 CAR-T treatment refractory acute B-lymphoblastic leukemia (B-ALL) and refractory B-cell non-hodgkin lymphoma (B-NHL) are reported to be 60% to 90% and 40% to 50%, respectively; b Cell Maturation Antigen (BCMA) CAR-T treatment the CRR of Multiple Myela (MM) refractory to treatment was 39% -80% (Feng Youqin, zhang Qiqi, hu Yongxian, yellow river, multi-target CAR-T treatment of hematological malignancy study progress, journal of clinical medicine 2022, 9, month 39, volume 9).

CARs generally consist of four domains: an extracellular antigen binding domain, a spacer or hinge region, a transmembrane domain, and an intracellular signaling domain. Antigen binding domains are typically formed from variable regions of antibody heavy (VH) and light (VL) chains joined by flexible linkers to form single chain fragment variable regions (scFv) and determine binding specificity; sometimes, instead of scFv, a native protein or peptide that binds to a receptor on its target cell can be used. Unlike TCRs that recognize antigens presented by MHC, CARs recognize and bind cell surface epitopes and determine target specificity independent of MHC. The hinge region is a spacer region on the surface of the exposed CAR-T cell that binds to the target antigen, and commonly used clinically is derived from CD8, CD28 or IgG, the length of the hinge region being determined empirically by the location of the target antigen, the antigen near the cell membrane requiring a longer hinge, while the antigen exposed on the cell surface requires a shorter hinge. The primary function of the transmembrane domain is to dock the CAR on the immune cell membrane, and some studies suggest that this region can affect CAR expression, stability, dimerization, and signal transduction. Intracellular signaling domains have been extensively studied in CAR engineering to generate CAR immune cells with the most active anti-tumor immunity, which, when bound to CARs, transduce signals to activate immune cells to spread out effective attacks.

First generation CAR-T cells contain only one cd3ζ signaling domain, and as optimal T cell activation and activation requires signaling from TCR-CD3 and CD28 signaling pathways, first generation CAR-T cell therapies are significantly inadequate in both therapeutic efficacy and persistence following adoptive transfer. To enhance anticancer activity, researchers designed second generation CAR-T cells that contained a costimulatory domain in addition to the cd3ζ signaling domain. The two most commonly used signal domains are the CD28 and 4-1BB (CD 137) intracellular domains. The CD28 domain is used by FDA approved axicabtagene ciloleucel and brexucabtagene autoleucel, both for CD19. The 4-1BB signaling domain was approved for Lisocabtagene maraleucel and tisagalecleucel targeting CD19 and idecabtagene vicleucel use for targeting B Cell Maturation Antigen (BCMA). Other intracellular co-stimulatory domains, such as OX40, CD27 and inducible T cell co-stimulators (ICOS), have also been tested in preclinical studies, with efficacy comparable to the CD28 and 4-1BB domains. Some of these co-stimulatory domains may have better beneficial effects on CAR-T cells, and one study showed that CD27 co-stimulation enhanced CAR-T cell persistence in vivo than CD28 domain (Song DG, ye Q, poussin M, harms GM, digini M, powell DJ jr.cd27costimulation augments the survival and antitumor activity of redirected human T cells in vivo.blood.2012;119 (3): 696-706). It has been found that intracellular CD28 and 4-1BB domains have different effects on CAR-T cell differentiation and metabolism, and the combination of 4-1BB and CD3 ζ induces central memory T cell differentiation and persistence, increases mitochondrial biosynthesis, enhances fatty acid oxidation and oxidative metabolism; on the other hand, CD28 and cd3ζ combination induce effector memory cell differentiation and glycolysis. To take advantage of the different properties of co-stimulatory domains and enhance the efficacy of CAR-T cells, researchers have designed third generation CAR-T therapies that combine two co-stimulatory domains in addition to cd3ζ, the combination of CD28 and 4-1BB can rescue CAR-T cells with low affinity for target antigens, enhance proliferative capacity, enhance central memory differentiation and enhance antitumor activity in vivo. When a library of CAR constructs comprising variable numbers of co-stimulatory domains was tested, it was found that CAR-T cells comprising two co-stimulatory domains DAP10 and CD27 achieved optimal antitumor activity in vivo (Huang R, li X, heY, zhu W, gao L, liu Y, et al, receptor additives in CAR-T cell engineering J Hematol Oncol.2020;13 (1): 86).

To date, five CAR-T cells have been approved by the us FDA, the first approved CAR-T being Kymriah, which was approved on day 1 of 2018, for the treatment of relapsed or refractory large B-cell lymphomas, including diffuse large B-cell lymphomas (DLBCL), high-grade B-cell lymphomas, and follicular lymphomas-induced DLBCL; the second approved CAR-T was yescanta, approved at 2017, 10, 18 for treatment of large B-cell lymphomas, including DLBCL, primary mediastinum large B-cell lymphomas, high grade B-cell lymphomas, and follicular lymphomas-induced DLBCL; the third CD-19 targeted CAR-T product Tecartus was FDA approved for recurrent or refractory mantle cell lymphoma at 24 of 7.2020. Breyanzi is a recently acquired batch of CD19 targeted CAR-T therapies against recurrent or refractory large B-cell lymphomas, undergoing two or more systemic treatments, including DLBCL/high grade B-cell lymphomas, primary mediastinum large B-cell lymphomas, and grade 3B follicular lymphomas. Abegma is the only FDA-approved CAR-T therapy not directed against CD19, FDA approved at day 26, 3, 2021, for relapsed or refractory multiple myeloma following four-line or more systemic treatment (including immunomodulators, proteasome inhibitors, and anti-CD 38 monoclonal antibodies).

Despite the better efficacy, immunotherapy has many limitations leading to CAR-T cell therapy failure, mainly manifested in:

ideal tumor antigens are difficult to obtain, currently approved CAR therapies are directed against B lineage markers such as CD19, BCMA, etc., while CD 19-targeted CAR-T treatment may lead to B cell dysgenesis, intravenous immunoglobulin supplements can easily compensate for most B cell functions. However, few such specific antigens in solid tumors do not affect normal function, making CAR-T therapy in solid tumors lag behind hematological malignancies.

The inefficiency of CAR-T cell transport and infiltration into the tumor, the aberrant vasculature with aberrant expression of adhesion molecules reduces CAR-T cell attachment, migration and infiltration into tumor sites; dense extracellular matrix, including cancer-associated fibroblasts, creates a physical barrier for CAR-T cells to enter the tumor site; and deregulated cytokine expression preferentially attracts inhibitory immune cells, also serving as a barrier to CAR-T cells.

Tumor microenvironments create adverse immune effects, and in some cancers, both cellular and stromal components create adverse microenvironments for cancer immunotherapy, and tumor-associated macrophages, regulatory T cells, myeloid-derived suppressor cells, and tumor-associated fibroblasts, among other cellular components, can directly suppress CAR-T function, VEGF, TGF-beta, IL-4, IL-10, and many others, also cause T cell dysfunction and suppress infiltration of immune cells, where TGF-beta is the primary inhibitor (Jordan Hartley, hinrich Abken, chimeric antigen receptors designed to overcome transforming growth factor-beta-mediated repression in the adoptive T-cell therapy of solid tumors, clin tranverse.immunology 22;8 (6): e 1064), TGF-beta directly inhibits T cell activity by binding to TGFBR1 and TGFBR 2; TGF-delta binding induces heterodimerization of the respective receptors and phosphorylation of the primary TGF-gamma signaling mediators SMAD2 and SMAD3, which induces inhibition of transcription programs, resulting in reduced cytokine production, cytotoxicity after antigen binding and T cell expansion; TGF-beta also promotes T cell differentiation into regulatory T cells, which in turn produce TGF-alpha, further promoting immunosuppression and tumor tolerance. For this reason, researchers have proposed the use of TGF- β inhibitors to eliminate or reduce the adverse effects of TGF- β on CAR-T cells.

After tumor cell escape, antigen loss and down-regulation are important mechanisms of treatment failure after CAR-T cells exert anticancer activity, it is counted that 7-25% of patients treated with CD 19-targeted CAR-T therapies will have malignant tumor recurrence due to reduced CD19 expression despite high initial response rates (Majzner RG, mackall cl. Turner Antigen Escape from CAR-T-cell therapy.2018; 8 (10): 1219-26).

Insufficient expansion and persistence of CAR-T cells in vivo, in addition to immune escape, is considered critical for long-term remission, particularly for those malignancies requiring long-term treatment, such as acute lymphoblastic leukemia (acute lymphoblastic leukemia, ALL). Lack of persistence of CAR-T cells may be related to host anti-transgenic immune responses to CAR-T cells, such as lymphodepletion with fludarabine, can reduce anti-transgenic immune responses, improve expansion and persistence of CAR-T cells, and enhance efficacy of CAR-T cells. Researchers believe that, in addition to the mode of administration, lack of CAR-T cell expansion and persistence is directly related to CAR-T cell own factors including CAR-T construction, parental T cell selection, T cell culture conditions, pharmacological manipulations, modification of CAR-T gene expression and metabolism, reversal of T cell failure, promotion of memory phenotype development, and the like. With respect to CAR-T structure, most clinical trials and FDA approved CAR-T cell products contain cd3ζ and one costimulatory domain, typically CD28 or 4-1BB, and some clinical studies have observed that the 4-1BB domain is longer in duration, up to 168 days, whereas CD 28-based CAR-T cells are about 30 days. Ex vivo culture, activation and expansion are not only prepared for transduction of CAR transgenic T cells, expansion to generate enough CAR-T cells for clinical use, but are also critical for maintaining CAR-T function and persistence after infusion. In vitro culture, which results in terminal differentiation of T cells and has a propensity to Activate Induced Cell Death (AICD) and failure, affects in vivo expansion and persistence, and optimization of culture conditions can improve the development and in vivo expansion and persistence of memory CAR-T cells.

In order to overcome the above difficulty, the present invention provides a novel chimeric antigen receptor and a delivery system thereof, wherein on one hand, the chimeric antigen receptor has a novel antigen binding domain targeting CD19, and uses CD27 and CD28 as dual co-stimulatory factors, so as to improve the persistence of CAR-T cells and promote cell proliferation; on the other hand, based on the delivery of liposome, the liposome internally wraps nucleic acid carrier carrying chimeric antigen receptor and siRNA against TGF-beta, and externally binds CD8 antibody, so that CD8+ T cells can be bound in vivo in a targeting manner, and autologous CAR-T cells are synthesized, so that the complicated process of preparing the CAR-T cells in vitro can be avoided, the time of the CAR-T cells can be prolonged, and the tumor treatment effect can be further improved.

Disclosure of Invention

In order to solve the technical problems, the invention provides a chimeric antigen receptor, which further comprises a signal peptide, a CD19 targeting antigen binding fragment, a hinge region, a transmembrane region, intracellular signal domains CD27, CD28 and CD3 zeta region, wherein the CD19 targeting antigen binding fragment has a light chain variable region and a heavy chain variable region, the chimeric antigen receptor further comprises a signal peptide, a CD19 targeting antigen binding fragment, a hinge region, a transmembrane region, intracellular signal domains CD27, CD28 and CD3 zeta region, the CD19 targeting antigen binding fragment has a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2.

The CD19 is a known tumor target expressed at a high level in various blood tumors, and the antigen binding fragment capable of effectively recognizing the CD19 is screened and obtained by taking the CD19 as a target antigen, and has higher binding activity with the target antigen, so that the off-target phenomenon can be prevented. In addition, costimulatory factors are one of the key factors in the stimulation and maintenance of CAR-T cell physiological activity, and studies have shown that CAR T cell inhibition by TGF- β can be overcome by CD28 costimulation provided by CD28 ζ CAR upon binding to cognate antigen, whereas 4-1BB-CD3 ζ CAR and the first generation CD3 ζ CAR-T cells are inhibited (golomba-Nagy V, kuehle J, holbach AA, abken H, CD28- ζ CAR T cells resist TGF- β repression through IL-2signaling,which can be mimicked by an engineered IL-7autocrine loop.Mol Ther 2018;26:2218-2230). Studies have also shown that in preclinical experiments, CARs containing CD28 generally have greater cytokine release and more aggressive killing of tumor cells than the 4-1BB co-stimulatory domain (Kathryn m.cappell, james n.kochenderefer, A comparison of chimeric antigen receptors containing CD ver 4-1BB costimulatory domains,Nature Reviews Clinical Oncology,2021,18:715-727). Thus, in order to reduce the adverse effects of TGF- β while increasing the anti-tumor effect, CD28 was selected as the co-stimulatory factor in the present invention; and to increase the duration of CAR-T cells, chimeric antigen receptors were constructed using both CD27 and CD28 co-stimulatory factors.

Furthermore, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 3.

A gene delivery drug is provided that employs liposomes to encapsulate a nucleic acid expression vector carrying a nucleotide sequence encoding the chimeric antigen receptor and an siRNA against TGF- β; the surface of the liposome is modified with a CD8 antigen binding fragment, the CD8 antigen binding fragment is a single domain antigen binding fragment, and the amino acid sequence of the fragment is shown as SEQ ID NO. 4; the siRNA of the anti-TGF-beta comprises siRNA-01 and siRNA-02, wherein the sense nucleotide sequence of the siRNA-01 is gagcaccauucuccuugaaaggacu, and the antisense nucleotide sequence is aguccuuucaaggagaauggugcuc; the sense nucleotide sequence of siRNA-02 was gcaacaacgccaucuaugatt and the antisense nucleotide sequence was ucauagauggcguuguugctt.

The nanometer delivery system can be used for delivering nucleic acid such as DNA or mRNA and the like to cells and promoting the expression of exogenous factors in vivo, and the technology is widely applied to the research of gene medicines and nucleic acid vaccines. In recent years, researchers have attempted to load CAR genes through nano-delivery systems and have made some research progress. For example, joel G Rurik et al (Joel G Rurik, istv n Tombacz et al CAR T cells produced in vivo to treat cardiac injury, science.2022Jan 7;375 (6576): 91-96) disclose a CAR-T therapy for treating cardiac fibrosis by delivering modified messenger RNA (mRNA) in T cell targeted lipid nanoparticles (lipid nanoparticles, LNPs), the modified mRNA encoding the CAR efficiently delivered cells to T lymphocytes, thereby generating transiently potent CAR-T cells in vivo, thereby reducing post-injury fibrosis and restoring cardiac function. This approach allows for rapid generation of CAR-T cells in vivo, but uses mRNA as the expression medium, a transient therapeutic approach. In tumor therapy, one would prefer to use long-acting therapeutic means in order to effectively inhibit tumor recurrence, and thus researchers have also attempted to construct a CD3 antibody modified lipid nanoparticle system using LNP-loaded DNA vectors, such as, for example, jing-e Zhou et al (Jing-e Zhou, lei Sun et al, lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6knockdown in vivo,Journal ofControlled Release,2022,35,298-307), and load plasmids containing interleukin 6 short hairpin RNA (IL-6 shRNA) and CD19-CAR (AntiCD 3 LNP/CAR19+shIL 6) binding genes, which target T cells via CD3 antibody mediation, stably transfect T cells, transform them into CAR-T cells with IL-6 knockout function, thereby killing CD19 high-expressing leukemia tumor cells and reducing IL-6-induced CRS.

The anti-tumor activity and persistence of CD4 and/or cd8+ T cells for CAR-T cells is more important than the prior art for CD3, CD5, etc. surface antigens used in LNP complexes, and several preclinical models demonstrate the advantage of different T cell subsets for effective CAR-T treatment: CD8 (+) CD45RA (+) CCR7 (+) CAR-T cells are closest to T memory stem cell phenotype cells, generate greater CAR-T anti-tumor activity (Xu Y, zhang M et al Closely related T-memory stem cells correlate with in vivo expansion of car.CD19-T cells and are preserved by IL-7and IL-15.blood.2014; 123:3750-3759), and therefore the LNP complex is linked to an anti-CD 8 antibody in the present invention so as to form CAR-T cells with stronger activity, thereby exerting good anti-tumor effect.

In addition, the LNP complex is also wrapped with siRNA for resisting TGF-beta, and can inhibit the expression of TGF-beta, so that the immunosuppression of T cells is relieved, and the anti-tumor effect is improved. The siRNA comprises two groups of siRNAs, is the interference RNA of a mammal TGF-beta conservation region such as a human, a mouse, a monkey and the like, which is obtained by screening in the prior art, and has certain universality, broad spectrum and high efficiency.

Further, the preparation method of the medicine comprises the following steps: introducing a nucleotide sequence encoding said chimeric antigen receptor into a mammalian expression vector, constructing a liposomal nanoparticle LNP with soybean lecithin, cholesterol, and DMG-PEG2000, such that said LNP encapsulates said mammalian expression vector and said siRNA against TGF- β; and then allowing the LNP surface to bind to the CD8 antigen binding fragment, thereby obtaining nano-delivery drug particles

Provides the application of the chimeric antigen receptor and/or gene delivery drug in preparing anti-tumor preparations

Further, the tumor is leukemia, lymphoma and/or myeloma.

Advantageous effects

The invention provides a chimeric antigen receptor and a nucleic acid delivery system thereof, and application thereof in treating tumors, and has the following advantages:

(1) The antigen binding fragment with higher affinity with the target antigen CD19 is selected, so that the target antigen can be identified efficiently, and the powerful anti-tumor effect is exerted;

(2) CD27 and CD28 are selected as dual co-stimulators, so that the activation degree of T cells is improved, the duration of CAR-T cells in vivo is greatly prolonged, and the long-acting anti-tumor effect can be exerted;

(3) Constructing a nano liposome drug delivery system, wherein the liposome wraps a mammalian cell expression vector, so that the assembly of the CAR-T cells in vivo is realized, the complicated process of manually preparing the CAR-T in vitro is avoided, and the production, transportation and storage costs are reduced;

(4) The liposome wraps up the siRNA of the anti-TGF-beta, can inhibit the expression of TGF-beta effectively, relieve the immunosuppression to T cells, raise its anti-tumor activity;

(5) The nano liposome drug delivery system is externally modified by using an anti-CD 8 antibody, so that CD8+ T cells can be effectively captured, and then CAR-T cells with strong anti-tumor activity are generated, and the tumor treatment effect is improved.

Drawings

Fig. 1: schematic representation of chimeric antigen receptor structure;

fig. 2: schematic diagram of nano liposome structure;

fig. 3: tumor cell killing rate;

fig. 4: survival of the experimental animal;

fig. 5: IL-2 expression level;

fig. 6: IFN-gamma expression levels;

fig. 7: CAR-T cell duration.

Detailed Description

The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. All techniques implemented based on the above description of the invention should be within the scope of the protection claimed in this application.

The experimental methods described in the following examples, unless otherwise specified, are all conventional; the reagent biological material and the detection kit can be obtained from commercial sources unless otherwise specified.

Example 1 design of CAR and CAR-T cell preparation

1.1 CAR structure design

According to the invention, a CAR structure is designed by taking CD19 as a target, the CD19 is a widely used target in blood tumors, various blood tumors can be effectively identified, and the corresponding antigen binding fragment is obtained by the inventor in earlier research, and has a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2.

The CAR structure of the present invention comprises a signal peptide, a CD 19-targeting antigen binding fragment, a hinge region, a transmembrane region, intracellular signal domains CD27, CD28 and CD3 zeta region, the structure of which is shown in figure 1. The CAR is obtained through total gene synthesis, and the amino acid sequence of the CAR is shown as SEQ ID NO. 3.

1.2 preparation of lentiviral vectors

In the invention, CAR-T cells are constructed by lentiviral vectors, and the preparation steps of the lentiviral vectors are as follows:

(1) Preparing a recombinant expression vector: introducing a nucleotide sequence encoding the CAR into a pWPXLd-2A lentiviral skeleton through enzyme digestion connection, and identifying positive clones through double enzyme digestion; introducing the positive cloning plasmid into DH5 alpha competent cells by an electrotransformation method, adding an LB liquid medium, and carrying out shake culture at 37 ℃ and 200rpm for 6 hours; competent cells were uniformly spread on LB plates with ampicillin resistance, and were placed in an incubator at 37℃overnight for culture; selecting positive clone strains, inoculating the positive clone strains into an ampicillin-resistant liquid LB culture medium, and carrying out shaking culture at 37 ℃ and 200rpm for overnight; the cells were collected by centrifugation, and plasmids, designated pWPXLd-2A-CAR, were extracted using a plasmid extraction kit (purchased from Shanghai JieRui bioengineering Co., ltd.), were sequence-identified, screened to obtain positive clone strains sequenced correctly, and stored.

(2) Packaging lentivirus, culturing 293T cells by using a DMEM culture medium containing 10% FBS, replacing a fresh serum-free DMEM culture medium when the cell fusion degree reaches more than 80%, and placing the culture medium into an incubator for standing for 2 hours; 4mL of 2 XHEPES buffer is used as solution A; adding helper plasmid pMD2.G plasmid, psPAX2 plasmid and pWPXLd-2A-CAR plasmid into 2.5M calcium ion solution according to the ratio of 1:1:2, and uniformly mixing to prepare solution B; mixing the solution A and the solution B in equal volume, and standing for 3min to obtain a mixed solution; gradually dripping the mixed solution into 293T cells, and standing for culturing for 10 hours; changing fresh DMEM medium containing 10% FBS, placing into an incubator, standing for culturing for 48h, centrifuging at 1500rpm for 5min, retaining supernatant, collecting supernatant containing lentiviral vector plasmid, and filtering the supernatant with 0.45 μm filter membrane to obtain filtrate containing lentiviral vector plasmid.

(3) Ultracentrifugation to obtain lentivirus: transferring the filtrate containing the lentiviral vector plasmid into an overspeed centrifuge tube, wherein 20% of sucrose is arranged at the bottom of the centrifuge tube; centrifuging at 25000rpm at 4deg.C for 2 hr, discarding supernatant, and dissolving the precipitate with sterile PBS to obtain concentrated lentiviral vector; the titer of the detected viruses was 2.4X10 ⁹ TU/mL。

1.3 Preparation of CAR-T cells

Collecting 20mL of peripheral blood of healthy volunteers, adding the peripheral blood into a 50mL centrifuge tube containing heparin, centrifuging at 2000rpm for 10min, and collecting cell sediment; resuspension the cells with sterile PBS, adding the mononuclear cell separating liquid preheated at 37 ℃, uniformly mixing, and centrifuging at 1000rpm for 20min; dividing into three layers after centrifugation, carefully sucking the middle buffy coat cells into a clean centrifuge tube, and washing the cells for 3 times by using sterile PBS; cells were resuspended and cultured using RPMI1640 medium with 10% fbs.

Adjusting the cell concentration to 1×10 ⁶ 1mL of each well was inoculated into a 6-well plate, and fresh medium containing 200IU/mL of IL-2 was added for culture at 37℃for 48 hours to activate T cells; according to viruses: the cells were 10:1 proportion is added into the slow virus prepared in section 1.2, and the slow virus is cultured in a culture incubator at 37 ℃; after 24h of transfection, the medium was changed and the growth of the cells was continuously observed for 10-12 days to give transfected CAR-T cells, designated CD27-CD28-CAR-T.

Antigen binding fragments with the same targeting to CD19 were prepared using a similar method, but only including CAR-T cells of the CD28 co-stimulatory factor, designated CD28-CAR-T.

Example 2 design and preparation of Liposome delivery systems

In this embodiment, as shown in fig. 2, the liposome is internally wrapped with siRNA carrying a plasmid expression vector encoding the CAR and anti-TGF- β, and externally linked with a CD8 capture antibody, so as to achieve in vivo assembly of the CAR-T cells.

The gene sequence of the CD27-CD28-CAR is introduced into a mammalian expression vector pcDNA3.1 (+) by enzyme digestion and ligation reaction, wherein the expression vector is pre-constructed with green fluorescent protein GFP gene and is named pcDNA3.1 (+) -CD27-CD28-CAR. Soybean lecithin, cholesterol and DMG-PEG2000 are taken as main components of liposome nano particles (lipid nanoparticles, LNP), the three components are mixed according to the proportion of 55:35:10, and pcDNA3.1 (+) -CD27-CD28-CAR plasmid and siRNA for resisting TGF-beta are slowly added; the siRNA of the anti-TGF-beta comprises siRNA-01 and siRNA-02 with the same proportion, wherein the sense nucleotide sequence of the siRNA-01 is gagcaccauucuccuugaaaggacu, and the antisense nucleotide sequence is aguccuuucaaggagaauggugcuc; the sense nucleotide sequence of siRNA-02 was gcaacaacgccaucuaugatt and the antisense nucleotide sequence was ucauagauggcguuguugctt. The ratio of plasmid, anti-TGF-beta siRNA to lipid material was 1:1:20, rapidly mixing by a nanoasssembrmixer, and encapsulating the plasmid in a liposome to form an LNP-pcDNA3.1 (+) -CD27-CD28-CAR-siRNA complex; the corresponding LNP complex product is obtained by washing, concentrating and changing the solution into the preparation solution.

The anti-CD 8 antibody is a single domain antigen binding fragment of a target CD8 antigen prepared and obtained by the inventor in advance, the amino acid sequence of the antibody is shown as SEQ ID NO. 4, the antibody not only can be combined with a target antigen in a high specificity manner, but also has a heavy chain variable region structure, so that the molecular mass of the antibody is smaller, the antibody is easy to combine with liposome, and the LNP complex is convenient to prepare.

Dissolving an anti-CD 8 antibody in PBS (phosphate buffer solution) with the pH of 7.4, slowly dripping the solution into 4mol/L DSPE-PEG2000 dimethyl sulfoxide (DMSO) solution, slowly stirring to uniformly mix the solution, standing for reaction for 6 hours, dialyzing and drying after the reaction is completed, and obtaining the anti-CD 8-DSPE-PEG2000. The anti-CD 8-DSPE-PEG2000 and LNP-pcDNA3.1 (+) -CD27-CD28-CAR-siRNA are mixed according to the proportion of 5:1, reacted for 4 hours at 37 ℃, washed and concentrated to obtain the anti-CD 8-LNP-CAR-siRNA complex.

Example 3 in vitro anti-tumor experiment

3.1 tumor cell culture

In this example, the human myelogenous leukemia cell line K562 and the lymphoma cell line Raji (purchased from the cell bank of the national academy of sciences) were used as target cells,the tumor-inhibiting ability of the CAR-T cells was studied. Resuscitating K562 and Raji cells maintained at-80deg.C, inoculating into RPMI1640 medium containing 10% FBS, 100U/ml penicillin, 100U/ml streptomycin, respectively, 37 deg.C, 5% CO ₂ Culturing in a saturated humidity incubator; when the cell fusion degree reaches more than 80%, subculturing is carried out; and co-transmitted for 3-5 passages to fully activate the cells.

3.2 in vitro anti-tumor experiments

T cells were prepared as described in section 1.3, and then the cell number was adjusted to 1X 10 ⁷ Inoculating 0.1mL of cell suspension into 6-well plate, placing at 37deg.C and 5% CO ₂ Culturing overnight in a saturated humidity incubator; 100 μg of the anti-CD 8-LNP-CAR complex prepared in example 2 was then added, incubated at 37℃for 48h, and CAR-T cells were collected by centrifugation and designated LNP-CD27-CD28-CAR-T.

The CD27-CD28-CAR-T, CD28-CAR-T and LNP-CD27-CD28-CAR-T provided by the invention are respectively mixed with K562 and Raji cells according to the proportion of 5:1, inoculated into a 96-well plate for co-culture (taking fresh culture medium as a control group), after being cultured for 48 hours, supernatant of each well is sucked, washed for 2 times by using sterile PBS, 20 mu L of CCK8 is added, incubated for 2 hours at 37 ℃, and absorbance of each well is detected by using an enzyme-labeling instrument according to the formula: killing rate= [ (control group 0D value-experimental group 0D value)/control group 0D value ], killing rate of tumor cells by each group was calculated, respectively.

As shown in fig. 3, all three CAR-T cells provided in the present invention were able to kill tumor cells effectively in vitro, with similar levels of action for CD27-CD28-CAR-T and CD28-CAR-T, but with significantly stronger action for LNP-CD27-CD28-CAR-T than the other two CAR-T cells, possibly due to inclusion of TGF- β inhibitors (siRNA) in the LNP complex, reducing or eliminating the immunosuppressive effects of TGF- β.

Example 4 in vivo anti-tumor experiment

4.1 animal model preparation

After Balb/c nude mice with 6-8 weeks of week age are adapted to feed for one week, K562 cells in logarithmic growth phase with good growth state are collected by centrifugation, cells are resuspended by using sterile physiological saline, and the cell concentration is regulated to be 1 multiplied by 10 ⁷ 0.5mL of the cell suspension was injected under the scapula on the right side of nude mice using a sterile syringe; then the tumor size was observed daily, and the long diameter (L) and the short diameter (W) of the tumor were recorded, and the tumor volume (V) was calculated as follows: v tumor= (major diameter L. Times. Minor diameter W/2), when tumor volume reaches 200-300mm ³ When the molding was successful for the subsequent experiments.

4.2 animal survival observations

The nude mice which are successfully molded are randomly divided into 4 groups, and 10 nude mice in each group are respectively: control group: 200. Mu.L of physiological saline was injected; CD28-CAR-T group: injection 1X 10 ⁶ A CD28-CAR-T cell; CD27-CD28-CAR-T group: injection 1X 10 ⁶ A CD27-CD28-CAR-T cell; LNP group: 200 μg of the anti CD8-LNP-CAR complex provided in the present invention was injected. The survival state of the experimental animals was observed and recorded daily. As shown in fig. 4, treatment with CAR-T can extend animal survival, but to a different extent, and LNP complexes are more potent than CD28-CAR-T and CD27-CD28-CAR-T, probably due to the LNP complex targeting cd8+ T cells in vivo to synthesize CAR-T cells, and the use of TGF- β inhibitors, relieving autoimmune inhibition, making them more potent antitumor.

4.3 inflammatory factor expression

Inflammatory factors in the tumor treatment process have double functions, on one hand, the expression of the inflammatory factors can mediate liquid immunity and cellular immunity, and the tumor killing effect is enhanced; on the other hand, a large amount of high-level immune factors possibly generate immune factor storm, which aggravates the immune burden born by the organism and clinically induces adverse reactions such as fever, respiratory distress and even death, so that the immune factor level of a patient needs to be monitored in the CAR-T treatment process so as to manage and intervene in time and ensure the safety of treatment.

In this example, in order to examine the expression of inflammatory factors in animals after treatment, blood of mice was taken after 10 days of treatment, placed in an anticoagulant tube and allowed to stand at room temperature for 1h, and centrifuged at 2000rmp for 20min to obtain serum of mice. The serum IL-2 and IFN-gamma content was measured using ELISA kit (from Abcam Inc. of America) and the results are shown in FIGS. 5 and 6. After the CAR-T treatment is adopted, the immune factor level in blood is greatly improved, and the CD28 stimulating factor is adopted in the CAR-T cells, so that the immune factor level in an animal body is greatly improved, and especially the IL-2 level is improved by more than about 4 times compared with the normal level, and the CAR-T cells can be helpful for killing tumor cells, but also can cause undesirable adverse reactions such as immune factor storm and the like; fortunately, there was no further enhancement of this trend with both CD27 and CD28 stimulators, and there was a decrease in the expression levels of IL-2 and IFN-gamma using the LNP group, presumably for the immunomodulatory effects of anti-TGF-beta siRNA on the one hand, and reduction of the graft-versus-host immune response by autologous T cell synthesis of CAR-T in vivo on the other hand.

Example 5 CAR-T cell persistence assay

To examine the in vivo duration of CAR-T cells, the present example injected CD28-CAR-T, CD27-CD28-CAR-T, antiCD-LNP-CAR complex into mice, extracted mouse peripheral blood on days 7, 14, 21, 35, 60 and 90, obtained T cells using mononuclear cell isolates (method referenced 1.3), examined the number of CAR-T cells expressing green fluorescent protein by flow cytometry, and calculated CAR-T cell duty cycle in T cells.

As shown in fig. 7, CD28-CAR-T cells started at 7 days of injection, and their levels had been shown to decrease, which was difficult to detect on day 35; in contrast, CD27-CD28-CAR-T is able to remain in vivo for longer, and the presence of some CAR-T cells is still detected on day 35, but this effect is also maintained only up to 60 days of the month; the anti-CD 8-LNP-CAR complex requires a certain process for preparing CAR-T cells in vivo, so that the CAR-T cell level is low in the early stage, but can reach a peak value before and after 21 days, then a high CAR-T cell proportion can be maintained for a long time, and the existence of a small amount of CAR-T cells can be detected by 90 days. This demonstrates that the addition of CD27 co-stimulators and the use of LNP nanomedicine delivery systems can effectively extend CAR-T cell duration in vivo, laying the foundation for long-acting antitumor effects.

Claims (6)

1. The chimeric antigen receptor is characterized by further comprising a signal peptide, a CD19 targeting antigen binding fragment, a hinge region, a transmembrane region, intracellular signal domains CD27, CD28 and CD3 zeta region, wherein the CD19 targeting antigen binding fragment has a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2.

2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 3.

3. A gene delivery drug, characterized in that the drug employs liposomes encapsulating a nucleic acid expression vector carrying a nucleotide sequence encoding the chimeric antigen receptor as claimed in claim 1 or 2 and an anti-TGF- β siRNA; the surface of the liposome is modified with a CD8 antigen binding fragment, the CD8 antigen binding fragment is a single domain antigen binding fragment, and the amino acid sequence of the fragment is shown as SEQ ID NO. 4; the siRNA of the anti-TGF-beta comprises siRNA-01 and siRNA-02, wherein the sense nucleotide sequence of the siRNA-01 is gagcaccauucuccuugaaaggacu, and the antisense nucleotide sequence is aguccuuucaaggagaauggugcuc; the sense nucleotide sequence of siRNA-02 was gcaacaacgccaucuaugatt and the antisense nucleotide sequence was ucauagauggcguuguugctt.

4. A gene delivery drug according to claim 3, wherein the method of preparing the drug comprises the steps of: introducing a nucleotide sequence encoding said chimeric antigen receptor into a mammalian expression vector, constructing a liposomal nanoparticle LNP with soybean lecithin, cholesterol, and DMG-PEG2000, such that said LNP encapsulates said mammalian expression vector and said siRNA against TGF- β; the LNP surface is then allowed to bind to the CD8 antigen binding fragment, thereby obtaining nano-delivery drug particles.

5. Use of the chimeric antigen receptor of any one of claims 1-2 and/or the gene delivery drug of any one of claims 3-4 for the preparation of an anti-tumor formulation.

6. The use according to claim 5, wherein the tumour is a leukemia, a lymphoma and/or a myeloma.

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