CN114540311A - Immune cell and application thereof - Google Patents

Abstract

The invention discloses an immune cell, which expresses a fusion protein of an intracellular truncated TGF-beta II type receptor and an ANTI-PD1 blocking type antibody on an immune cell membrane and simultaneously expresses a chimeric antigen receptor. The immune cell simultaneously expressing the intracellular truncated TGF-beta II type receptor, the ANTI-PD1 blocking type antibody fusion protein and the chimeric antigen receptor can specifically recognize the targeted tumor cell surface antigen, so that the immune cell successfully secretes the fusion protein, thereby enhancing the proliferation capacity, the ANTI-apoptosis capacity and the killing capacity to the tumor of the immune cell, and adjusting the negative balance between the tumor and the T cell; meanwhile, the immune cell killing effect is accurate, the safety is higher, the recurrence is not easy, and the life quality of patients is improved.

Description

Immune cell and application thereof

Technical Field

The invention relates to the technical field of immune cells, in particular to an immune cell for expressing a chimeric antigen receptor, and specifically relates to an immune cell for simultaneously expressing a fusion protein of an intracellular truncated TGF-beta II type receptor and an ANTI-PD1 blocking type antibody and application thereof.

Background

Tumor (tumor) refers to a new organism (neograwth) formed by local tissue cell proliferation of the body under the action of various tumorigenic factors, because the new organism is mostly in the form of space-occupying block-shaped protrusion, also called neoplasms (neoplasms). Among them, malignant tumors are easy to be metastasized, and they are easy to recur after treatment and are very difficult to cure in some special microenvironments.

Transforming growth factor beta (TGF- β) is an important promoter of immune homeostasis and immune tolerance, inhibiting the expansion and function of various components of the immune system. Interfere with TGF-beta signal transduction and promote the generation of inflammatory diseases. TGF-beta receptors are widely expressed in cells of body tissues, and thus, TGF-beta can play a regulatory role in the growth and development of many cells. In cells in different environments or developmental stages of tumor growth, the effect of TGF- β on cells appears to be either stimulatory or inhibitory, depending mainly on the type of cell, the differentiation state of origin, and the growth conditions. In the process of resisting cancer, TGF-beta can inhibit the process of tumor by inhibiting cell proliferation, inducing apoptosis, inhibiting expression of inflammatory factors and cell growth inhibition. These effects can maintain homeostasis in normal tissues, preventing early stages of tumor formation.

The mechanism is mainly that the TGF-beta inhibits cell proliferation to play a role in blocking the cell cycle, and the expression level and the functional state of cyclin (cyclin), Cyclin Dependent Kinase (CDK) and c-myc are mainly regulated to stop the cell at the G1 stage. TGF- β inhibits cell proliferation by inducing expression of 4EBP1 and Cyclin Dependent Kinase (CDK) inhibitors (p15, p21 and p 57). 4EBP1 binds to eukaryotic initiation factor 4E (eIF4E) and inhibits protein translation, while p15, p21 and p57 prevent cell cycle progression by inhibiting the activity of the CDK-cyclin complex necessary for the G1/S transition.

In addition, TGF- β also inhibits expression of Cdc25a phosphatase, which is also required for CDK-cyclin activation, and negatively regulates expression of a number of other factors that drive cell cycle progression and cell proliferation, including the Id protein, E2F, and c-Myc. The proto-oncogene c-myc is a transcriptional activator that promotes cell progression from G1 to S phase, and TGF-. beta.also inhibits cell growth by down-regulating c-myc expression in most cells.

TGF-. beta.s also play an important role in immunosuppression in the tumor microenvironment. TGF-. beta.s regulate the production and function of many types of immune cells. It controls adaptive immunity by directly promoting expansion of Treg cells, inhibiting production and function of effector T cells and antigen presenting dendritic cells (DC cells).

Similarly, TGF- β controls the innate immune system by inhibiting natural killer cells (NK cells) and regulating the complex behavior of macrophages and neutrophils, thereby forming a negative immune regulatory import network.

The role of TGF- β in immune regulation is among the broader roles of this cytokine and other members of its family in development, homeostasis and tissue regeneration. While dysfunction of this pathway can lead to congenital defects, fibrous diseases, immune disorders and cancer. The effects of TGF- β on most adult mammalian cells are from cell proliferation, differentiation, adhesion, motility, metabolism, communication and death. In addition, TGF-. beta.acts as a potent tumor suppressor by inhibiting the proliferation and promoting apoptosis of malignant tumor precursor cells. Interfering with the TGF- β signaling pathway through cell mutations not only converts these cells into a mature malignant state, but also allows them to create an immunosuppressive Tumor Microenvironment (TME) using TGF- β and produce additional stromal modifiers that promote tumor progression and metastasis.

When tumors develop to an advanced stage, many tumor cells have already developed some tolerance to the effects of TGF- β. Most tumor cells produce TGF-beta via autocrine or paracrine, resulting in higher than physiological levels of TGF-beta in tumor tissues. At this time, TGF-beta can promote tumor cells to generate EMT, and infiltration and metastasis of tumors are promoted.

Disclosure of Invention

In view of the above, the present invention provides an immune cell that simultaneously expresses a fusion protein of an intracellular truncated TGF- β II receptor and an ANTI-PD1 blocking antibody and a chimeric antigen receptor; the fusion protein can improve the activity of immune cells and the killing effect on tumors; wherein, the ANTI-PD1 blocking antibody is also called ANTI-PD1 immune checkpoint antibody; truncated TGF-beta type II receptors are also known as loss-of-activity TGF-beta type II receptors.

The fusion protein is a protein obtained by removing the intracellular active region of a TGF-beta II type receptor and fusing the extracellular domain of the TGF-beta II type receptor with the scFv region of an ANTI-PD1 blocking type antibody.

The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain; the extracellular domain comprises a signal peptide CD8SP and an antigen targeting sequence scFv; the transmembrane domain includes CD8 hinder and CD8 TM; the intracellular domain comprises one or more of the activation elements 4-1BB, CD28, ICOS, and CD3 ζ.

In one embodiment, the fusion protein of the intracellular truncated TGF-beta II type receptor and the ANTI-PD1 blocking antibody is expressed in series according to the sequence of ANTI-PD 1scFv, G4S x 4Linker, the extracellular domain of the TGF-beta II type receptor, the transmembrane domain of the TGF-beta II type receptor and the TGF-beta II type receptor with 100-300 amino acids at the intracellular N terminal.

The fusion protein and the chimeric antigen receptor are expressed by constructing one or more expression frames, the expression frames are transferred into immune cells through a delivery vector, and the delivery mode of the vector during constructing the expression frames comprises any one of lentivirus, retrovirus, common plasmid, episome, nano delivery system, electric transduction or transposon; alternatively, the vector for transferring the gene of the immune cell into the chimeric antigen receptor may comprise: lentiviruses, retroviruses, common plasmids, episomes, nano-delivery systems, electrical transduction, transposons or other delivery systems, and the like.

Wherein when the split between the chimeric antigen receptor and the fusion protein is in the same expression frame, a protein cleavage functional element is employed between the chimeric antigen receptor and the fusion protein, and the protein cleavage functional element is any one of T2A, P2A, E2A, F2A, and IRES; when the split between the chimeric antigen receptor and the fusion protein is in multiple expression cassettes, there is no need for split between the chimeric antigen receptor and the fusion protein and expression or separate delivery is performed.

In one embodiment, the expression of the chimeric antigen receptor is a chimeric antigen receptor that targets one target or multiple targets.

In one embodiment, the target of the chimeric antigen receptor comprises one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA, and EBV; preferably, the chimeric antigen receptor is targeted to an anti-CLDN 18.2 antibody.

In one embodiment, the binding region of the chimeric antigen receptor to the target can be scFv, Fab, or a combination of scFv and Fab; wherein, the scFv region structure can be replaced by any single-chain antibody, single-chain variable fragment (scFv) or Fab fragment of any target point; alternatively, the binding region of the chimeric antigen receptor to the target is a bispecific antibody that binds to one target or both targets.

In one embodiment, the chimeric antigen receptor comprises a leader sequence, a binding region (scFv) that recognizes a tumor associated antigen, a hinge region and transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signal CD3 ζ; wherein the scFv of the binding region is a scFv of an anti-idiotype antibody; the hinge region and transmembrane domain is CD28, or the CD8hinge region and transmembrane domain; the intracellular co-stimulatory domain is CD28, CD137(4-1BB), or an ICOS intracellular co-stimulatory domain.

In one embodiment, the binding region between the chimeric antigen receptor and the target can be a single target, a bispecific antibody that binds to two targets, or two or more chimeric antigen receptors that are formed across membranes and that recognize different targets.

The immune fusion cell provided by the invention can be prepared into a biological preparation, and the biological preparation is a pharmaceutically acceptable carrier, diluent or excipient. Administration of the biological agent may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The biological agent can be applied to drugs for preventing and/or treating solid tumors.

Compared with the prior art, the invention has the following beneficial effects:

the immune cell simultaneously expressing the intracellular truncated TGF-beta II type receptor, the ANTI-PD1 blocking type antibody fusion protein and the chimeric antigen receptor can specifically recognize the targeted tumor cell surface antigen, so that the immune cell successfully secretes the fusion protein, thereby enhancing the proliferation capacity, the ANTI-apoptosis capacity and the killing capacity to tumors of the immune cell and adjusting the negative balance between the tumors and T cells; meanwhile, the immune cell killing effect is accurate, the safety is higher, the recurrence is not easy, and the life quality of patients is improved.

Drawings

FIG. 1 is a graph showing the results of a target cell phenotype flow assay; wherein HGC-27-CLDN18.2 cells correspond to FITC-CLDN18.2 and APC-PD-L1 flow detection maps; accordingly, HGC-27 cells also corresponded to FITC-CLDN18.2 and APC-PD-L1 flow charts;

FIGS. 2a and 2b are CAR structural plans; wherein, FIG. 2a is the CAR-only expression cassette structure, named CT 008; FIG. 2b is an expression cassette, designated CT008, for the addition of an intracellular truncated TGF-beta type II receptor and ANTI-PD1 fusion protein to a control CAR

FIGS. 3a, 3b are secretory CAR-T amplification profiles; wherein, FIG. 3a shows the proliferation rates of three T cells (CT008, CT009 and NT) without antigen stimulation; FIG. 3b shows the proliferation rates of three T cells (CT008, CT009 and NT) under antigen stimulation;

FIGS. 4a, 4b, 4c show CAR-T cell positivity; wherein, figure 4a is the NT control-like CAR-T cell positivity rate; FIG. 4b is CAR-T cell positivity for CT 008; FIG. 4c is CAR-T cell positivity at CT 009;

FIGS. 5a, 5b, 5c show the results of phenotypic flow assays; wherein, figure 5a is the results of flow assay of the NT control CAR-T cell phenotype; FIG. 5b is the CAR-T cell phenotype flow assay result of CT 008; FIG. 5c is the CAR-T cell phenotype flow assay result at CT 009;

FIGS. 6a, 6b and 6c show the results of phenotypic flow assay for cell proliferation potency; wherein, fig. 6a is the result of flow-testing the cell proliferation potency of the NT control-like CAR-T cell phenotype; FIG. 6b is the CAR-T cell phenotype flow assay cell proliferation potency results of CT 008; FIG. 6c is the result of flow-testing the proliferative capacity of cells for CAR-T cell phenotype at CT 009;

FIGS. 7a, 7b, 7c are CAR-T cell in vitro tumoricidal function evaluations; wherein FIG. 7a is a graph showing the results of a test for smad expression inhibitory ability of NT control-like CAR-T cells; FIG. 7b is a graph showing the results of the CAR-T cell smad expression inhibition assay of CT 008; FIG. 7c is a graph showing the results of the CAR-T cell smad expression inhibition assay at CT 009;

FIG. 8 is a graph showing the results of the measurement of cytokine release capacity in three T cells (CT008, CT009, and NT); wherein, the line I represents contrast CT008 cells, the line II represents NT cells, the line III represents contrast CT009 cells, the line II represents NT + HGC27-LV035 cells, the line IV represents CT008+ HGC27-LV035 cells, and the line VI represents CT009+ HGC27-LV035 cells;

FIGS. 9a and 9b show the secretion of IFN-. gamma.and IL-2 factor after 24h coculture of CAR-T cells and target cells; wherein FIG. 9a is a graph showing the results of IL-2 cytokine concentration release assay of CAR-T cells after coculture with Claudin18.2+ target cells; FIG. 9a is a graph showing the results of IFN- γ cytokine concentration measurements released by CAR-T cells after coculture with Claudin18.2+ target cells;

FIG. 10 is a graph of experimental survival of mouse animals.

Detailed Description

The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

PD-1 (programmed death receptor 1), also known as CD279 (cluster of differentiation 279), is an important immunosuppressive molecule that regulates the immune system and promotes self-tolerance by down-regulating its response to human cells, and by inhibiting T-cell inflammatory activity. This may prevent autoimmune diseases, but it may also prevent the immune system from killing cancer cells. PD-1 is 288 amino acids type I membrane protein, it is originally cloned from mouse T cell hybridoma 2B4.11 of apoptosis, take PD-1 as immune regulation of the target point to resist tumor, resist infection, resist autoimmune disease and organ transplantation survival etc. have important meanings. The ligand PD-L1 of PD-1 can also serve as a target, and corresponding antibodies can also serve the same function. PD-1 and PD-L1 bind to initiate programmed death of T cells, allowing tumor cells to gain immune escape. The anti-PD-1 therapy removes immunosuppressive effects, activates T cell functions, enhances the immune surveillance ability and killing ability of T cells to tumors and generates tumor immune responses by combining PD-1 and blocking the combination of PD-1 with PD-L1 and PD-L2.

The design of chimeric antigen receptors CARs goes through the following process:

the first generation CARs had only one intracellular signaling component, CD3 ζ or Fc γ RI molecule, and, because of the single activation domain in the cell, it caused only transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve good clinical efficacy.

The second generation CARs introduce a costimulatory molecule such as CD28, 4-1BB, OX40 and ICOS on the basis of the original structure, and compared with the first generation CARs, the function of the second generation CARs is greatly improved, and the persistence of CAR-T cells and the killing capability of the CAR-T cells on tumor cells are further enhanced. On the basis of the second generation CARs, a plurality of novel immune co-stimulatory molecules such as CD27 and CD134 are connected in series, the third generation CARs and the fourth generation CARs are developed, and a double CAR or a multi CAR which can target 2 targets or a plurality of targets is expressed on the same cell.

The invention provides an immune cell simultaneously expressing intracellular truncated TGF-beta II type receptor and ANTI-PD1 blocking type antibody fusion protein and a chimeric antigen receptor, wherein the intracellular truncated TGF-beta II type receptor and ANTI-PD1 blocking type antibody fusion protein can improve the activity of the immune cell and the killing effect on tumors. (ii) a Wherein the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain; the extracellular domain comprises an antigen binding domain; the intracellular domain comprises a costimulatory signaling region, an intracellular region, and a CD3 zeta chain moiety; the costimulatory signaling region refers to a portion of the intracellular domain that includes costimulatory molecules, cell surface molecules required for the effective response of lymphocytes to antigens.

The fusion protein of the intracellular truncated TGF-beta II type receptor and the ANTI-PD1 blocking type antibody is sequentially expressed in series according to the sequence of ANTI-PD 1scFv, G4S x 4Linker, the extracellular domain of the TGF-beta II type receptor, the transmembrane domain of the TGF-beta II type receptor and the TGF-beta II type receptor with 100-300 amino acids at the intracellular N end.

The binding region of the chimeric antigen receptor and the target can be a binding region that binds to one target, or can be a bispecific antibody that binds to two targets, or can be a binding region in which two or more chimeric antigen receptors are formed across membranes and recognize different targets. For example, the immune cell expresses a chimeric antigen receptor CAR cell, whose expression of the chimeric antigen receptor can be a CAR cell that targets one target or multiple targets. In this case, the target of the chimeric antigen receptor may be one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA and EBV, and preferably the target of the chimeric antigen receptor may be CLDN 18.2.

The binding region for the chimeric antigen receptor and the target can be scFv, Fab, a combination of scFv and Fab, or the native receptor for the target; wherein, the scFv region structure can be replaced by any single-chain antibody, single-chain variable fragment (scFv) of any target point, Fab fragment or receptor of the target point, etc.; alternatively, the binding region of the chimeric antigen receptor to the target is a bispecific antibody that binds to one target or both targets.

The chimeric antigen receptor comprises a leader sequence, a binding region that recognizes a tumor-associated antigen, a hinge region and transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signal CD3 ζ. Wherein the scFv of the binding region is a scFv of an anti-idiotype antibody; the hinge region and transmembrane domain is a CD28 or CD8hinge region and transmembrane domain; the intracellular co-stimulatory domain is CD28, CD137(4-1BB), or an ICOS intracellular co-stimulatory domain. In the present invention, the above-mentioned extracellular domain of an immune cell preferably comprises an antigen binding domain targeting claudin 18.2.

The fusion protein and the chimeric antigen receptor are expressed by constructing one or more expression frames, and the expression frames are transferred into immune cells through a delivery vector, wherein the delivery mode of the vector during constructing the expression frames comprises any one of lentivirus, retrovirus, common plasmid, episome, nano delivery system, electric transduction or transposon; alternatively, the vector for transferring the gene of the immune cell into the chimeric antigen receptor may comprise: lentiviruses, retroviruses, common plasmids, episomes, nano-delivery systems, electrical transduction, transposons or other delivery systems, and the like.

Wherein, when the division between the chimeric antigen receptor and the fusion protein is in the same expression frame, the division between the chimeric antigen receptor and the fusion protein is a protein cleavage functional element; wherein, the protein cleavage functional element can be T2A, P2A, E2A, F2A or IRES; when the split between the chimeric antigen receptor and the fusion protein is in multiple expression cassettes, there is no need for split between the chimeric antigen receptor and the fusion protein and expression is performed independently or delivery is performed separately

The delivery mode of the vector when the expression frame is constructed, namely the vector for transferring the gene of the immune cell into the chimeric antigen receptor is one of lentivirus, retrovirus, common plasmid, episome, nano delivery system, electric transduction or transposon.

Immune cells of the invention include T cells, NK cells, NKT cells, macrophages, TIL cells, TCR-T cells, or other tumor killing cells.

The invention takes T cells as an example, when the chimeric antigen receptor CAR is expressed in the T cells, the chimeric antigen receptor CAR can perform antigen recognition based on antigen binding specificity or protein receptor binding; when the CAR binds to its associated antigen, the CAR-T cells will target the lysis of tumor cells, resulting in a reduction or elimination of the tumor burden in the patient.

A CAR expression vector in a T cell, which can be provided to the cell in the form of a viral vector; viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses, among others. Generally, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers; for example, retroviruses provide a convenient platform for gene delivery systems; the platform can utilize in the field of known techniques will select genes into vectors and packaging into retroviral particles, forming recombinant virus. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Wherein the CAR is an expression vector in a T cell, preferably a lentiviral vector.

In the delivery mode of the vector when constructing the expression cassette, additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence, another example is elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter. The immune fusion cell provided by the invention can be prepared into a biological preparation, and the biological preparation is a pharmaceutically acceptable carrier, diluent or excipient.

Administration of the biological agent may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The biological agent can be applied to drugs for preventing and/or treating solid tumors.

The main advantages of the immune cells of the invention include:

(1) the immune cells can express CAR (chimeric antigen receptor) and have the function of specifically recognizing tumor cells;

(2) the immune cells express fusion proteins of intracellular truncated TGF-beta II type receptors and anti-PD1, can be combined with TGF-beta II in a tumor microenvironment, reduces negative regulation and control influence of the tumor microenvironment, reduces growth inhibition and exhaustion of CAR-T cells, and enhances the tumor killing activity of the CAR-T cells;

(3) the anti-PD1 antibody part in the fusion protein can relieve the immunosuppressive effect, activate the T cell function, enhance the immune monitoring capability and killing capability of the T cell to the tumor and generate tumor immune response by combining PD-1 and blocking the combination of PD-1 with PD-L1 and PD-L2;

(4) the immune cell preparation expressing the fusion protein of the intracellular truncated TGF-beta II type receptor and the anti-PD1 is different from the combined treatment of the immune cell preparation and the fusion protein preparation, the demand for the fusion protein is low, and the fusion protein is expressed on the surface of the immune cell and only acts near the tumor due to the targeting property of the immune cell, so that the toxicity is greatly reduced;

(5) research data show that TGF-beta secretion of PD-L1 low-expression tumor tissues is very high, so that the immune cells have advantages and a wider application range compared with single PD-1 blocking treatment;

in a word, the intracellular truncated TGF-beta II type receptor and anti-PD1 fusion protein express the immune cell of the chimeric antigen receptor at the same time, and through the combination of the intracellular truncated TGF-beta II type receptor and TGF-beta II, anti-PD1 blocks the PD-1 negative regulation pathway, so as to enhance the proliferation capacity, anti-apoptosis capacity and killing capacity to tumors of the immune cell.

The following examples will representatively illustrate immune cells of the present invention in detail, taking CAR-T cells as an example.

The preparation method and function of the immune cells are verified as follows.

The immune cell culture simultaneously expresses the fusion protein of an intracellular truncated TGF-beta II type receptor and an ANTI-PD1 blocking type antibody and a chimeric antigen receptor. Constructing a membrane-expressing CAR-T cell and comprising the steps of:

s100: peripheral blood PBMC isolation and T cell culture.

Separating mononuclear cells from peripheral blood of a donor, performing density gradient centrifugation using a ficol method, and enriching T cells using a T cell sorting kit, for example, CD3 MicroBeads, human-lysoinvented or 130-097-043, and activating culture and expansion of T cells using magnetic beads coupled with anti-CD3/anti-CD 28;

for T cell culture, a TexMACS GMP Medium (Miltenyi Biotec, 170-.

S200: and (4) culturing the cell line.

Cloning a base sequence expressing CLDN18.2 into a PHBV lentiviral vector skeleton, placing the PHBV-EF 1 alpha-CLDN 18.2 under a promoter of EF1 alpha (EF-1 alpha), and transferring three plasmids, namely PHBVV-EF 1 alpha-CLDN 18.2, a lentiviral envelope Plasmid pMD2.G (Addge, Plasmid #12259) and a lentiviral packaging Plasmid psPAX2(Addge Plasmid #12260) to a lentiviral complete expression vector prepared in 293T cells by using Lipofectamine 3000; virus supernatants were collected at 48h and 72h, respectively, and ultracentrifugation concentration (Merck Millipore) was performed on the collected virus supernatants; the concentrated virus was used to infect HGC-27, resulting in a HGC-27 cell line overexpressing CLDN18.2, designated HGC-27-CLDN 18.2. The detection result of the expression of CLDN18.2 by HGC-27-CLDN18.2 cells is shown in figure 1, wherein, the detection chart corresponding to HGC-27 is a comparison chart; based on the results of detection of HGC-27-CLDN18.2 corresponding to HGC-27, as shown by the vertical graphs of the dotted line frame a part in FIG. 1 (i.e., graphs corresponding to FTTC-CLDN 18.2), the results of detection of the expression level of CLDN18.2 antigen in the FITC channel show that CLDN18.2 expression in HGC-27 is negative (the peak graph is located on the left side of the vertical line I) and CLDN18.2 expression in HGC-27-CLDN18.2 is positive (the peak graph is located on the right side of the vertical line I); as can be seen from the two vertical graphs of the dotted line box b in FIG. 1 (i.e., the graph corresponding to APC-PD-L1), the results of detecting the expression level of PD-L1 in the APC channel showed that both PD-L1 expression was positive in HGC-27 and HGC-27-CLDN18.2 (the peak graphs are located on the right side of the vertical line II).

S300: CAR structural design

The immune checkpoint antibody protein of the intracellular truncated TGF-beta type II receptor and ANTI-PD1 fusion protein and the second generation CAR were constructed in one expression cassette and split with P2A. As shown in figure 2a, the core structure of the CAR includes a secretion signal peptide sequence; CLDN18.2 targeting scFv; the CD8 transmembrane region; intracellular segment stimulation signal 4-1BB-CD3 ζ, designated P008, with the CAR-only expression cassette as control. As shown in FIG. 2b, an intracellular truncated TGF- β type II receptor and ANTI-PD1 fusion protein was added to the control CAR and designated P009. As shown in FIGS. 2a and 2b, CLDN18.2 scFv/TM/4-1BB/CD3 ζ represents a chimeric antigen receptor expressed on a cell membrane of an immune cell; and Anti-PD-1scFv & intracellular truncated TGF-beta type II receptor is expressed as receptor fusion immune checkpoint antibody protein expressed on cell membrane; the middle part of the immune checkpoint antibody protein in which the chimeric antigen receptor is fused to the intracellular truncated TGF- β type II receptor and ANTI-PD1 is cleaved by the P2A protein, and the middle parts of the ANTI-PD-1scFv & intracellular truncated TGF- β type II receptor are linked by a linker protein.

S400: immune cell preparation following CAR-T design

The constructed CAR structural plasmids P008 and P009 and a lentivirus envelope Plasmid pMD2.G (Addge, Plasmid #12259) and a lentivirus packaging Plasmid psPAX2(Addge Plasmid #12260) are respectively transformed into 293T by using Lipofectamine3000 to prepare a lentivirus complete expression vector; virus supernatants were collected at 48h and 72h, concentrated by ultracentrifugation (Merck Millipore) and titer tested, and the resulting concentrated viruses were designated Lv008 and Lv 009; the concentrated virus can be used for T cell infection, and CT008 and CT009 cells can be obtained after infection. The cell culture uses X-VIVO culture medium and adds IL-2 and human AB plasma to prepare complete culture medium to culture and expand CAR-T cells.

Second, CAR-T immune cell assay

1. CAR-T cell positive expression

And (3) sampling and detecting the cell number on 4 th, 6 th, 9 th, 11 th and 13 th days after the T cells are infected, respectively detecting the CAR positive rate of the T cells on 6 th day, and subculturing and supplementing the culture medium every 1-2 days.

The cell number detection results are shown in fig. 3a and 3b, and the proliferation rates of the three T cells (CT008, CT009, and NT, respectively) are shown in fig. 3a, which shows that the proliferation rates of the three T cells have no obvious difference and the amplification efficiency is low under the condition of no antigen stimulation; as can be seen in fig. 3 b; with antigenic stimulation, antigenic stimulation had a significant effect on the proliferation rate of CT009 cells.

As shown in fig. 4a, 4b, and 4c, the CAR positivity of the three T-cell CARs tested at day 6, in which an intracellular truncated TGF- β type II receptor and ANTI-PD1 fusion protein were added to the intracellular domain portion of the CAR structure, was as follows, with NT as a negative control, 41.95% for CT008 and 95.15% for CT 009. As shown in fig. 5a, 5b, and 5c, the PD1 showed the most significant ability to inhibit CT009, and as shown in fig. 6a, 6b, and 6c, the most significant ability to proliferate was found at CT 009. FIGS. 7a, 7b, and 7c show that smad expression in NT, CT008, and CT009 is stimulated by TGF-beta, and the results show that smad inhibitory ability in CT009 is most significant.

2. Cell killing experiment in vitro

The in vitro tumor killing function of CAR-T is verified by using HGC-27-LV035 and HGC-27 cells as positive and negative target cells respectively and adopting a flow detection method. The results of the assay are shown in FIG. 8, in contrast, CT009 of the fusion protein had the strongest killing effect on HGC27-LV035 positive target cells.

3. Cytokine release assay.

The obtained CAR-T cells and target cells (HGC27 or HGC27-LV035) were mixed at 1: mixing the mixture with the effective target ratio of 1, placing the mixture in an RPMI culture medium, co-culturing for 24h, transferring the mixture into a centrifuge tube for centrifugation, collecting supernatant after centrifugation is stopped, taking the supernatant to detect the release levels of the cytokines IFN-gamma and IL2, and detecting by using an Elisa kit (abbkine, KET6011 and KET 6014).

Results As shown in FIGS. 9a and 9b, after the CAR-T cells were co-cultured with Claudin18.2+ target cells, the CAR-T cells released large amounts of IFN-. gamma.and IL-2; indicating that CAR-T can be effectively and specifically activated by the Claudin18.2 antigen on the surface of the tumor.

4. CAR-T cell in vivo functional evaluation

24 NSG mice (weight 18-22g) with the age of 6-8 weeks are taken, after being adapted to feeding for one week, are inoculated subcutaneously with HGC27-LV035-CLDN18.2 positive tumor cell strains, each mouse is inoculated with 1 x 107 tumor cells, the animal state is closely observed, the tumor volume of the mice is measured by using a vernier caliper every three days, and when the tumor volume reaches 100mm3, the mice are randomly grouped according to the weight and the tumor size, CAR-T cells or control T cells are infused through tail veins. The details of the administration method, the administration dose and the administration route are shown in Table 1. The survival graph 10 results show that the fusion protein-expressing CAR-T cell CT009 can greatly prolong the survival of mice.

TABLE 1 animal protocol

The above examples demonstrate that: the CAR-T simultaneously expressing the fusion protein of the TGF-beta subtype (intracellular truncated TGF-beta II type receptor) receptor and the ANTI-PD1 blocking antibody has stronger proliferation capacity and in-vitro tumor killing activity on tumors compared with CAR-T not secreting other cytokines or CAR-T secreting only one cytokine.

It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.

Claims (12)

1. An immune cell expressing on the membrane of said cell a fusion protein of an intracellular truncated TGF- β type II receptor and an ANTI-PD1 blocking antibody and simultaneously expressing a chimeric antigen receptor.

2. The immune cell of claim 1, wherein the fusion protein has a structure expressed by anti-PD 1scFv, G4S x 4Linker, TGF-beta type II receptor ectodomain, TGF-beta type II receptor transmembrane domain, and TGF-beta type II receptor with 100-300 amino acids at the intracellular N-terminus in tandem.

3. The immune cell of claim 1, wherein the fusion protein and the chimeric antigen receptor are expressed by constructing one or more expression cassettes, and the expression cassettes are transferred into the immune cell by a delivery vector.

4. The immune cell of claim 3, wherein the expression cassette is constructed such that the vector is delivered in any one of a lentivirus, retrovirus, ordinary plasmid, episome, nano-delivery system, electrical transduction, or transposon.

5. The immune cell of claim 3, wherein a protein cleavage function is used between the chimeric antigen receptor and the fusion protein when the split between the chimeric antigen receptor and the fusion protein is in the same expression cassette; or the chimeric antigen receptor and the fusion protein are in multiple expression cassettes, the chimeric antigen receptor and the fusion protein do not need to be separated and are expressed independently or delivered separately.

6. The immune cell of claim 5, wherein the protein cleavage function is any one of T2A, P2A, E2A, F2A and IRES.

7. The immune cell of claim 1, wherein the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain; the extracellular domain comprises a signal peptide CD8SP and an antigen targeting sequence scFv; the transmembrane domain includes CD8 hinder and CD8 TM; the intracellular domain comprises one or more of the activation elements 4-1BB, CD28, ICOS, and CD3 ζ.

8. The immune cell of claim 1, wherein the chimeric antigen receptor is expressed as a chimeric antigen receptor that targets one or more targets; or the target of the chimeric antigen receptor comprises one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA and EBV.

9. The immune cell of claim 8, wherein the binding region of the chimeric antigen receptor to the target is scFv, Fab, or a combination of scFv and Fab; or the binding region of the chimeric antigen receptor to the target is a bispecific antibody that binds to one target or both targets.

10. The immune cell of claim 9, wherein the scFv region structure is substituted with one of any single chain antibody, single chain variable fragment, and Fab fragment of any target.

11. A biological agent comprising an immune cell according to any one of claims 1 to 10.

12. Use of the biological agent according to claim 11 for the preparation of a medicament for the prevention and/or treatment of cancer and/or tumors.

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