Application of CD28 CAR in substance for simultaneously killing plasma cell tumor and T cell tumor

Abstract

The invention belongs to the technical field of immunology and biological medicine, and relates to application of CD28 CAR in substances for simultaneously killing plasma cell tumors and T cell tumors. The invention relates to application of CD28 CAR in preparation of substances for killing plasma cell tumors and T cell tumors, in particular to application in preparation or screening of CAR-T products with high safety, and the amino acid sequence of the CD28 CAR protein is shown as SEQ ID NO: 1. The CD28 CAR-T cell can safely and efficiently kill plasma cell tumors and T cell tumors at the same time, shows good amplification capacity, and does not generate a cell suicide phenomenon. According to the CAR-T cell immunotherapy, the problem of sib suicide is expected to be reduced, the killing efficiency of CAR-T cells is improved, the release of inflammatory factors is reduced, synchronous removal of various tumors is realized, key barriers of the CAR-T therapy in tumor immunotherapy are expected to be overcome, and a new hope of'healing 'is provided for patients with hematologic tumors and even solid tumors.

Description

Technical Field

[0001] The present invention belongs to the field of immunology and biomedicine technology, and relates to the application of anti-CD28 CAR in substances that simultaneously kill plasma cell tumors and T cell tumors. Background Art

[0002] Multiple myeloma (MM), the most common type of plasma cell neoplasm, is a hematologic malignancy characterized by the presence of abnormal clonal plasma cells in the bone marrow. MM often leads to end-organ damage, including anemia, renal impairment, lytic bone lesions, and hypercalcemia. Treatment options include traditional chemotherapy, glucocorticoids, and one or more additional agents, such as proteasome inhibitors (bortezomib, carfilzomib, ixazomib), immunomodulators (lenalidomide, thalidomide, pomalidomide), or monoclonal antibodies (such as daratumumab, isatuximab, and elotuzumab). Although prognosis has improved significantly in the era of targeted myeloma and immunomodulatory agents, patients often face a significantly shortened survival period due to the development of drug resistance and continued disease progression. Therefore, MM remains largely incurable and ultimately leads to relapse.

[0003] Acute T-cell lymphoblastic leukemia (T-ALL) is a subtype of T-cell neoplasm. It is a hematologic malignancy caused by the malignant proliferation of immature T cells in the bone marrow and / or lymphoid tissues, characterized by high immunophenotypic and molecular diversity. Among newly diagnosed ALL cases, T-ALL accounts for 15% in children and 25% in adults. T-ALL patients typically present with elevated peripheral blood white blood cell counts, accompanied by neutropenia, anemia, and thrombocytopenia. Furthermore, mediastinal thymic masses and central nervous system (CNS) infiltration are common in T-ALL patients, and these risk factors hinder long-term remission in T-ALL. Currently, first-line treatment utilizes multi-drug combination chemotherapy regimens and intrathecal chemotherapy, and some patients also receive cranial radiotherapy. Although traditional treatments have achieved certain results, T-ALL is still considered an aggressive subtype due to its unique clinical and biological characteristics. The prognosis of adult T-ALL patients and relapsed pediatric T-ALL patients remains poor, with event-free survival and overall survival rates of relapsed diseases less than 25%. Therefore, there is an urgent need to develop new treatments.

[0004] Patients with relapsed or refractory hematologic malignancies have limited treatment options and a poor prognosis, with a five-year overall survival rate of less than 20%. Chimeric antigen receptor T cell (CAR-T) therapy represents a revolutionary breakthrough in the treatment of hematologic malignancies, but it also faces numerous challenges. BCMA-targeted CAR-T therapy can target and kill malignant plasma cells in MM patients, thereby achieving a therapeutic effect. Similar to other CAR-T products, MM patients can eventually develop drug resistance after BCMA CAR-T therapy due to mutation or loss of tumor antigens and a suppressive immune environment.

[0005] As a specific target on the surface of T cells, CD7 CAR-T cells hold promise as a new treatment option for patients with relapsed / refractory T-ALL. However, because T cells and tumor cells share the same CD7 target, CD7 CAR-T cells can engage in fratricidal activity. Furthermore, while killing tumor cells, CD7 CAR-T cells can also kill healthy T cells, leading to treatment-related T cell immunodeficiency and an increased risk of infection. These factors, coupled with the challenges of separating malignant and normal T cells during cell product manufacturing, further complicate the development of effective targeted immune cell therapies for patients with relapsed / refractory T-ALL. While CAR-T technology has steadily advanced, and clinical results demonstrate its significant advantages in treating hematologic malignancies, these therapies present both risks and opportunities. CAR-T cell therapy also presents potential toxicity and drug resistance. Therefore, accelerating the search for new tumor targets and developing new technologies is crucial to overcome current bottlenecks. Summary of the Invention

[0006] Aiming at the potential toxicity and drug resistance problems in traditional CAR-T cell therapy, the present invention proposes the use of a novel anti-CD28 CAR in substances that kill plasma cell tumors and T cell tumors.

[0007] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions: The use of anti-CD28 CAR protein in the preparation of substances for killing plasma cell tumors and T cell tumors, especially in the preparation or screening of highly safe CAR-T products, the amino acid sequence of the anti-CD28 CAR protein is shown in SEQ ID NO: 1.

[0008] The present invention proposes that the substance for killing plasma cell tumors and T cell tumors is anti-CD28 CAR-T cells.

[0009] The present invention proposes that the plasma cell tumor cell line is a human multiple myeloma cell, and the T cell tumor cell line is a human acute T lymphocytic leukemia cell.

[0010] The present invention further proposes a method for preparing anti-CD28 CAR-T cells, which are obtained by expressing the chimeric antigen receptor CD28 on T cells, and the steps are: (1) activating and culturing the T cells; and (2) infecting the activated and cultured T cells with a polynucleotide encoding the anti-CD28 CAR through a lentivirus.

[0011] Preferably, the T cells in step (1) include natural cells separated from peripheral blood, cells cultured or passaged from natural cells, and genetically engineered T cells.

[0012] The genetically engineered T cells include, but are not limited to, cells into which genes encoding functional components other than the chimeric antigen receptor component encoding genes are introduced.

[0013] The chimeric antigen receptor targets an antigen on the target cell, which is an antigen specifically expressed by tumor cells; or an antigen highly expressed by tumor cells and lowly expressed or not expressed by normal cells.

[0014] Preferably, the antigen is CD28.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are: Experimental results from this study demonstrate that anti-CD28 CAR-T cells can safely and efficiently kill plasma cell tumors and T cell tumors, exhibiting excellent expansion capacity. No cell suicide occurred during the CAR-T production process, and the cells did not kill T cells in healthy peripheral blood mononuclear cells in vivo. This approach is expected to provide new solutions in the following areas: reducing the suicide problem, improving the killing efficiency of CAR-T cells, reducing the release of inflammatory factors, and achieving clearance of various hematologic malignancies. This breakthrough is expected to overcome a key barrier to CAR-T therapy in tumor immunotherapy, offering new hope for a cure for patients with hematologic malignancies and even solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Figure 3 shows the expansion of anti-CD28 CAR-T cells, anti-CD7 CAR-T cells, and untransduced T cells.

[0017] Figure 2 Figure 2 shows the in vitro cytotoxicity of anti-CD28 CAR-T cells and untransduced T cells against plasma cell tumor cells and T cell tumor cells. T cells were co-incubated with tumor cells at effector-target ratios (E:T) of 1:1, 1:2, 1:4, 1:8, and 1:16 for 24 hours, and the cytotoxicity was measured. Panel A used RPMI 8226 cells, and Panel B used MOLT4 cells.

[0018] Figure 3Figure 1 shows the in vivo cytotoxicity of anti-CD28 CAR-T cells against plasma cell tumor cells and T cell tumor cells. Panel A compares the effects of anti-CD28 CAR-T cells, anti-CD7 CAR-T cells, and untransduced T cells (negative control) on the survival of mice bearing RPMI 8226 NTG tumors. Panel B compares the effects of anti-CD28 CAR-T cells, anti-BCMA CAR-T cells, and untransduced T cells on the survival of mice bearing MOLT4 NTG tumors.

[0019] Figure 4 To test the in vivo safety of anti-CD28 CAR-T cells, Panel A shows the injection time points of human GFP+ T cells, untransduced T cells (negative control), anti-CD28 CAR-T cells, and anti-CD7 CAR-T cells. Panel B shows the number of human GFP+ T cells in peripheral blood on day 8 in the untransduced T cell group, anti-CD28 CAR-T group, and anti-CD7 CAR-T group. Panel C shows the level of human interferon-γ in peripheral blood on day 8 in the untransduced T cell group, anti-CD28 CAR-T group, and anti-CD7 CAR-T group. DETAILED DESCRIPTION

[0020] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] The CAR involved in the present invention refers to a recombinant polypeptide construct comprising the following domains: an extracellular domain (also referred to as an antigen binding domain) responsible for specific recognition and binding to a target antigen, a transmembrane domain, and a cytoplasmic signaling domain (also referred to as an intracellular signaling region), wherein the intracellular signaling region comprises a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule. For example, the stimulatory molecule may include (but is not limited to) a ζ chain associated with an immune cell (such as a T cell) receptor complex, and the co-stimulatory molecule may include (but is not limited to) 4-1BB (CD137) or CD28. The CAR may comprise one or more (including two) antigen binding domains.

[0023] As used herein, "specific recognition" means that the extracellular domain of the present invention binds exclusively or primarily to the target antigen and does not or substantially does not cross-react with other non-target polypeptides. The degree of specificity can be assessed using conventional immunological techniques in the art, including but not limited to immunoblotting, immunoaffinity chromatography, and flow cytometry. In the present invention, flow cytometry is preferably used to verify specific recognition. Specific criteria can be determined by those skilled in the art based on their expertise and actual application requirements.

[0024] T cells are core effector cells of adaptive immunity, mediating targeted killing through specific antigen recognition. Their primary immune activities include: 1) direct perforin-granzyme killing of target cells by cytotoxic T lymphocytes (CTLs); 2) activation of immune cells such as macrophages through the secretion of effector factors such as IFN-γ and TNF-α; and 3) induction of target cell apoptosis through death receptors such as Fas / FasL. Furthermore, memory T cells can establish long-term immune surveillance, while regulatory T cells (Tregs) maintain immune balance and prevent excessive responses. This precise recognition and killing mechanism makes them ideal vectors for tumor immunotherapy, such as CAR-T.

[0025] Conventional nucleic acid transduction methods in the art, including non-viral and viral transduction methods, can be used to establish the CAR-T cells. Non-viral transduction methods include electroporation and transposon methods. Viral transduction methods include lentivirus, adeno-associated virus, and retrovirus.

[0026] A variety of vectors for viral packaging can be used in the present invention, including, for example, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc., as well as viral vectors formed by further modification based on these viral vectors. Various types of vectors are available, as long as active CARs and immune effector cells modified therewith can be ultimately obtained.

[0027] In a specific embodiment of the present invention, CAR-T cells targeting tumor cells expressing the CD28 antigen were prepared. The extracellular binding region of the CAR was a scFv that binds to the CD28 antigen, including the CD28 costimulatory factor and the CD3ζ activation domain. This demonstrated excellent targeted killing effects.

[0028] Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial sources. The percentages of substances not otherwise specified in the following examples are by mass.

[0029] Cells: Plasma cell tumors refer to the human multiple myeloma cell line: RPMI 8226 (Cat. No. 1101HUM-PUMC000083) purchased from the National Laboratory Cell Resource Sharing Platform (Beijing); T cell tumors refer to the human acute T-lymphoblastic leukemia cell line: MOLT4 (Cat. No. CRL-1582) purchased from ATCC (American Type Culture Collection). Cells were cultured in RPMI-1640 medium (gbico) supplemented with 15% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, and 2 mM L-glutamine. HEK293T cells (National Laboratory Cell Resource Sharing Platform (Beijing), Cat. No. CBP60439) were cultured in DMEM high-glucose medium (gbico) supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, and 2 mM L-glutamine.

[0030] Reagents and Materials: Human Interferon-γ ELISA Kit, including: 1× Coating Buffer A, 1× Detection Diluent A, Wash Buffer, Antibody Human Interferon-γ Solution, Avidin-HRP Solution, TMB Substrate Solution D, Stop Solution (Biolegend, Cat. No. 430104); GFP-PE Flow Cytometry Antibody (Biolegend, Cat. No. 338003); Cell Counting Kit (MCE, Cat. No. HY-K0301); Human T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130-096-535); CFDA, SE Cell Proliferation and Tracing Assay Kit (Solarbio, Cat. No. S1076); Human CD28-PE Flow Cytometry Antibody (Biolegend, Cat. No. 983404); Human CD7-APC Flow Cytometry Antibody (Biolegend, Cat. No. 982702); Human CD269-PE Flow Cytometry Antibody (BCMA) Antibody (Biolegend, Catalog No. 357504); CD3 / CD28 T cell activation magnetic beads (ThermoFisher SCIENTIFIC, Catalog No. 11141D); apoptosis detection kit (Annexin V + PI + 4× binding buffer, Beyotime, Catalog No. C1062S); red blood cell lysis buffer (Solebio, Catalog No. R1010); 1× PBS (Solebio, Catalog No. P1020); pLTE plasmid vector (Addgene, Catalog No. 67044); CD28 CAR plasmid (pLTE-CD28 CAR, constructed by GeneGene); CD7 CAR plasmid (pLTE-CD28CAR, constructed by GeneGene); BCMA CAR plasmid (pLTE-CD28 CAR, constructed by GeneGene); psPAX2 (Addgene, Catalog No. 12260); pMD2.G (Addgene, Catalog No. 12259); IL-2 (R&D SYSTEMS, BT-002-AFL); RPMI-1640 medium (gibco, product number C11875500BT); DMEM medium (gibco, product number C11995500BT); Trypan blue staining solution (0.4%) (Solybol, product number C0040).

[0031] Experimental animals: 6-week-old female NTG immunodeficient mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd.

[0032] Data Analysis: All data were obtained from three independent replicates. Results are presented as mean ± standard error (SEM). Differences between groups were analyzed using unpaired two-tailed Student's t-test and two-way ANOVA. A P value < 0.05 was considered statistically significant. Example 1

[0033] Expansion of anti-CD28 CAR-T cells, anti-CD7 CAR-T cells, and untransduced T cells.

[0034] The expansion and suicide phenomena of anti-CD28 CAR-T cells, anti-CD7 CAR-T cells, and untransduced T cells were observed on culture days 0, 7, and 12.

[0035] The specific steps are as follows: 1. Construction of anti-CD28 CAR-T and anti-CD7 CAR-T cells Lentiviral vector construction, production, and viral infection.

[0036] The anti-CD28 CAR or anti-CD7 CAR sequence was cloned into the pLTE vector to construct a recombinant expression vector. HEK293T cells were co-transfected with 18 μg of the anti-CD28 CAR or anti-CD7 CAR plasmid, 12 μg of psPAX2, and 8 μg of the pMD2.G packaging plasmid into 18 μg of the anti-CD28 CAR or anti-CD7 CAR plasmid. The cells were cultured in DMEM medium. The medium was changed 24 hours after transfection, and the viral supernatant was collected 48 hours later and concentrated by ultracentrifugation (30,000 g for 1.5 hours; centrifuge speed 9:00, speed 3:00).

[0037] T cell isolation: Primary T cells were isolated from peripheral blood using a human T cell isolation kit and the cell density was adjusted to 1×107 / mL. T cell activation: Human T-Expander Dynabeads were added at a 1:1 ratio to T cell culture medium (X-VIVO 15 basal medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, 40 IU / mL human IL-2, 10 ng / mL human IL-15, and 10 ng / mL human IL-7) and cultured for 3 days. Viral transduction: On day 3 of activation, lentiviral transduction was performed at a multiplicity of infection (MOI) of 10. Cells were incubated with the virus (centrifuged at 1000 × g for 1 hour at 32°C). Twelve hours after transduction, the supernatant was replaced with culture medium supplemented with 300 IU / mL IL-2. Post-treatment: Remove the CD3 / CD28 beads on day 5. Change the culture medium and supplement with IL-2 every two days to maintain a cell density of 2 × 106 cells / mL. Infection efficiency typically reaches 30%. Applications: In vitro experiments or mouse transfusion should be performed on days 10-11 after activation. Expression of CD28 CARs and CD7 CARs: Analyzed using a BD LSRFortessa flow cytometer (BD Biosciences).

[0038] 2. Viable cell counting Prepare a single cell suspension by adding 0.4% trypan blue solution to the cell suspension to make the final concentration of trypan blue 0.04%. Stain for three minutes and count the living cells using a cell counting instrument.

[0039] The results showed that both anti-CD28 CAR-T cells and non-transduced T cells showed good expansion capacity; anti-CD7 CAR-T cells could not be expanded in large quantities (such as Figure 1 shown). Example 2

[0040] The killing effect of anti-CD28 CAR-T cells on plasma cell tumor cells and T cell tumor cells in vitro.

[0041] Anti-CD28 CAR-T cells or untransduced T cells were co-incubated with luciferase-expressing RPMI 8226 or MOLT4 cells at an E:T ratio of 1:1, 1:2, 1:4, 1:8, and 1:16. After 24 hours of incubation, the luciferase intensity was used to analyze the ratio of RPMI 8226 or MOLT4 cells and to detect the ability of anti-CD28 CAR-T cells to kill tumor cells.

[0042] The specific steps are as follows: Luciferase intensity analysis of CD28 CAR-T cell tumor killing effect Anti-CD28 CAR-T cells or untransduced T cells (negative control) were co-incubated with luciferase-expressing RPMI 8226 or MOLT4 cells at an E:T ratio of 1:1, 1:2, 1:4, 1:8, and 1:16. After 24 hours, the luciferase intensity was used to analyze the ratio of RPMI 8226 or MOLT4 cells to evaluate the tumor killing ability of anti-CD28 CAR-T cells.

[0043] The results showed that compared with the non-transduced T cell group, anti-CD28 CAR-T can effectively kill RPMI 8226 cells (such as Figure 2 A) and MOLT4 cells (as Figure 2 B). Example 3

[0044] In vivo killing effect of anti-CD28 CAR-T cells on plasma cell tumor cells and T cell tumor cells.

[0045] A plasma cell tumor model and a T cell tumor model were established in NTG immunodeficient mice, respectively. Different CAR-T cells were injected through the tail vein, and the differences in mouse survival were compared.

[0046] The specific steps are as follows: 1. Effect of anti-CD28 CAR-T on the survival of mice bearing plasma cell tumors On day 0, 2×10*6 RPMI 8226 cells were injected into the tail vein of NTG mice. On day 12, 3×10*5 anti-CD28 CAR-T or anti-BCMA CAR-T or untransduced T cells (negative control) were injected through the tail vein, and the survival of the mice was observed.

[0047] 2. Effect of anti-CD28 CAR-T on the survival of T cell tumor mice On day 0, 2×10*6 MOLT4 cells were injected into NTG mice through the tail vein. On day 5, 2×10*6 anti-CD28 CAR-T or anti-CD7 CAR-T or untransduced T cells (negative control) were injected through the tail vein, and the survival of the mice was observed.

[0048] The results showed that anti-CD28 CAR-T can significantly prolong the survival of mice with plasma cell tumors compared with anti-BCMA CAR-T (e.g. Figure 3 Anti-CD28 CAR-T can significantly prolong the survival of T cell tumor mice compared with anti-CD7 CAR-T (as shown in Figure 3 B). Example 4

[0049] In vivo safety of anti-CD28 CAR-T cells.

[0050] On day 0, 10*7 human GFP+ T cells were injected into the tail vein of NTG mice. On day 3, 2×10*6 anti-CD28 CAR-T or anti-CD7 CAR-T or untransduced T cells (negative control) were injected into the tail vein. On day 8, the number of human GFP+ T cells and the level of human γ interferon were detected in the peripheral blood (e.g. Figure 4 (as shown in A).

[0051] The specific steps are as follows: 1. Construction of anti-CD28 CAR-T cells and anti-CD7 CAR-T cells The specific steps are the same as those in Example 1.

[0052] 2. Construction of GFP+T cells The GFP sequence was cloned into the pLTE vector to construct a recombinant expression plasmid. 18 μg of GFP vector was co-transfected with 12 μg of psPAX2 and 8 μg of the pMD2.G packaging plasmid into HEK293T cells. Fresh medium was replaced 24 hours after transfection, and viral supernatant was collected 48 hours after transfection and concentrated by ultracentrifugation (30,000 g for 1.5 hours; centrifuge speed 9 for 1 minute and speed 3 for 2 minutes).

[0053] T cell isolation: Primary T cells were isolated from peripheral blood using a human T cell isolation kit and the cell density was adjusted to 1×107 / mL. T cell activation: Human T-Expander Dynabeads were added at a 1:1 ratio to T cell culture medium (X-VIVO 15 basal medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, 40 IU / mL human IL-2, 10 ng / mL human IL-15, and 10 ng / mL human IL-7) and cultured for 3 days. Viral transduction: On day 3 of activation, lentiviral transduction was performed at a multiplicity of infection (MOI) of 10. Cells were incubated with the virus (centrifuged at 1000 × g for 1 hour at 32°C). Twelve hours after transduction, the supernatant was replaced with culture medium supplemented with 300 IU / mL IL-2. Subsequent Processing: Remove the CD3 / CD28 beads on day 5. Change the culture medium and supplement with IL-2 every two days to maintain a cell density of 2×106 / mL. Transduction efficiency typically reaches 90%. Experimental Application: Mice were transfused 10-11 days after activation. GFP expression was analyzed using a FACS Aria II flow cytometer (BD Bioscience).

[0054] 3. Detect the number of GFP+T cells in peripheral blood using a fluorescence counter Collect 100-200 μL of blood from the mouse tail vein into an anticoagulant tube, and remove 10 μL of peripheral blood into an anticoagulant tube for subsequent detection; add 2-3 volumes of red blood cell lysis buffer, lyse on ice for 5 minutes, centrifuge (300×g, 5 min, 4°C), discard the supernatant, and wash 1-2 times with PBS; resuspend the cells in 200-300 μL of flow cytometry buffer, filter through a 35 μm filter to remove aggregates, and use a fluorescence counter to detect the number of GFP+ T cells.

[0055] 4. Detection of human interferon-γ concentration in peripheral blood by enzyme-linked immunosorbent assay A. The day before the ELISA assay, dilute the capture antibody in 1× Coating Buffer A. Add 100 μL of this capture antibody solution to all wells of the 96-well plate provided in the kit. Seal the plate and incubate overnight (16-18 hours) at 2°C to 8°C.

[0056] B. Equilibrate all reagents to room temperature (RT) before use. All standards and samples must be tested in duplicate (triplicate). A standard curve must be generated for each experiment.

[0057] C. Add at least 300 μL of wash buffer to each well and repeat the wash process four times. After each wash, invert the plate onto absorbent paper and pat dry. Repeat this procedure for all subsequent washes.

[0058] D. To block nonspecific binding and reduce background interference, add 200 μL of 1× Detection Diluent A to each well.

[0059] E. After sealing the plate, incubate on a plate shaker at room temperature for 1 hour (500 rpm, 0.3 cm circumference). All subsequent shaking and incubation steps should be performed under these conditions.

[0060] F. While the plate is sealed, prepare sample dilutions and standards in advance.

[0061] G. Wash the plate 4 times with wash buffer.

[0062] H. Add 100 μL / well of standard or sample to the appropriate wells. If dilution is required, dilute the sample with 1× Assay Diluent A before adding it to the plate.

[0063] I. Seal the ELISA plate and incubate at room temperature with shaking for 2 hours.

[0064] J. Wash the plate 4 times with wash buffer.

[0065] K. Add 100 μL of diluted detection antibody human interferon-γ solution to each well, seal the ELISA plate, and incubate at room temperature with shaking for 1 hour.

[0066] L. Wash the plate 4 times with wash buffer.

[0067] M. Add 100 μL of diluted Avidin-HRP solution to each well, seal the plate, and incubate at room temperature with shaking for 30 minutes.

[0068] N. Wash the plate five times with wash buffer. During the final wash, soak each well in wash buffer for 30 seconds to 1 minute to reduce background signal.

[0069] O. Add 100 μL of TMB substrate solution D to each well and incubate in the dark for 15 minutes (this can be adjusted based on experimental conditions). Positive wells should appear blue. The plate does not need to be sealed at this step.

[0070] P. Add 100 μL of stop solution to each well to terminate the reaction. The color of the positive wells should change from blue to yellow.

[0071] Q. Read the absorbance at 450 nm within 15 minutes. If the microplate reader can detect at 570 nm, correct the data by subtracting the absorbance at 570 nm from the absorbance at 450 nm.

[0072] The results showed that the number of human GFP+ T cells in peripheral blood was similar in the non-transduced T cell group and the anti-CD28 CAR-T group, and was higher than that in the anti-CD7 CAR-T group (e.g. Figure 4 As shown in B), the levels of human interferon-γ induced by the first two groups were lower than that of the anti-CD7 CAR-T group (as shown in Figure 4 C).

[0073] The above description is merely a preferred embodiment of the present invention and does not constitute any other form of limitation to the present invention. Any person skilled in the art may utilize the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes for application in other fields. However, any simple modification, equivalent change, and modification of the above embodiments made in accordance with the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Use of an anti-CD28 CAR protein in the preparation or screening of substances that kill plasma cell tumors and T cell tumors, wherein the amino acid sequence of the anti-CD28 CAR protein is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that The substance that kills plasma cell tumors and T cell tumors is anti-CD28 CAR-T cells.

3. The use according to claim 1 or 2, characterized in that: The plasma cell tumor cell line is a human multiple myeloma cell, and the T cell tumor cell line is a human acute T lymphocytic leukemia cell.

4. The application according to claim 2, characterized in that: The anti-CD28 CAR-T cells are obtained by expressing the chimeric antigen receptor CD28 on T cells, and the steps are: (1) activating and culturing the T cells; and (2) infecting the activated and cultured T cells with a polynucleotide encoding the anti-CD28 CAR through a lentivirus.

5. The application according to claim 4, characterized in that: The T cells in step (1) include natural cells separated from peripheral blood, cells cultured or passaged from natural cells, and genetically engineered T cells.

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