WO2021155670A1 - Bispecific chimeric antigen receptor for treating hematological tumor complicated with hiv infection - Google Patents

Abstract

Disclosed are a bispecific chimeric antigen receptor for treating a HIV infection complicated with a hematological tumor, and a method for constructing a recombination gene thereof and the use thereof. The present invention relates to the fields of medicine and biology. The chimeric antigen receptor comprises an anti-HIV gp120 single-chain antibody and an anti-CD19 single-chain antibody; furthermore, the bispecific chimeric antigen receptor comprises a signal peptide, a CD8 hinge region, a CD28 transmembrane region, CD28-ICD, a 4-1BB co-stimulatory domain and a CD3ζ intracellular signaling domain.

Description

Bispecific chimeric antigen receptor for the treatment of hematological tumors complicated by HIV infection

This application requires China to be submitted on February 5, 2020, with the application number 202010081004.2, and the title of the invention "a bispecific chimeric antigen receptor, gene, construction method and application for the treatment of hematological tumors combined with HIV infection" The priority of the patent application, the entire content of which is incorporated in the present disclosure by reference.

Technical field

The present disclosure relates to the field of medical biology, in particular to a bispecific chimeric antigen receptor for the treatment of hematological tumors combined with HIV infection, its coding nucleotides, construction methods, recombinant lentiviral vectors, immune cells and their applications, and treatment of blood The method of tumor combined with HIV infection.

Background technique

After the human body is infected with HIV, CD4 ⁺ T lymphocytes are attacked by the virus, which leads to the reduction of the body's immune capacity and is prone to various viral infections or malignant tumor diseases. Lymphoma is one of them. The risk of AIDS-related lymphoma is 165 times that of the general population, and malignant lymphoma is currently one of the main causes of death in AIDS patients.

For HIV combined with malignant tumor diseases, the treatment plan is generally anti-tumor combined with anti-retroviral therapy (anti-retroviral therapy, ART). Among them, highly active anti-retroviral therapy (HAART) has been considered as a more effective treatment.

However, HAART also faces many challenges: (1) Patients must take the drug for life, which requires high economic costs; (2) Serious side effects; (3) The emergence of drug-resistant strains; (4) More importantly, , CART cannot completely remove the virus. This is mainly because the drug is only effective on the virus in replication, and is not effective on the latent virus "reservoir" (reservoir) established by HIV in the early stage of infection. Once the antiretroviral treatment is interrupted, the pre-integration virus in the virus reservoir is reactivated, and the viremia in almost all patients will rebound rapidly. The treatment of lymphoma is mainly chemotherapy, which has large side effects and is prone to recurrence, and there is no available therapy. However, HIV patients with malignant tumors are generally seriously ill, the patient's immune system suffers severe damage, the disease progresses rapidly, and the patient's mortality rate is high.

Therefore, there is an urgent need to find a treatment plan that can effectively treat HIV combined with malignant tumors while improving one or more of the above shortcomings or challenges.

Public content

In one aspect, a bispecific chimeric antigen receptor is provided, wherein the chimeric antigen receptor comprises an anti-HIV gp120 single-chain antibody and an anti-CD19 single-chain antibody.

Optionally, the bispecific chimeric antigen receptor further includes: signal peptide, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain. At least one.

Optionally, from the N-terminus to the C-terminus, the bispecific chimeric antigen receptor sequentially includes: signal peptide SP1, the anti-HIV gp120 single-chain antibody, Strep II, connecting peptide, and the anti-CD19 single-chain antibody Antibody, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain.

Optionally, the bispecific chimeric antigen receptor includes a first CAR and a second CAR,

Wherein, the first CAR includes: signal peptide SP1, anti-HIV gp120 antigen-specific single-chain antibody, Strep II, connecting peptide, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain;

Wherein, the second CAR includes: signal peptide SP2, anti-CD19 single-chain antibody, CD8 hinge region, CD8 transmembrane region, CD28-ICD, and CD3ζ intracellular signaling domain; and

Wherein, the first CAR and the second CAR are connected via a self-cleaving short peptide.

Optionally, the signal peptide is a CD8 signal peptide and a CSF2RA signal peptide; and/or

The connecting peptide is 3×G ₄ S; and/or

The self-cleaving short peptides are P2A short peptides.

In another aspect, there is provided an encoding nucleotide that encodes any of the bispecific chimeric antigen receptors in the above aspects. Preferably, the nucleotide sequence of the encoding nucleotide is shown in SEQ ID NO: 37 or SEQ ID NO: 38.

In yet another aspect, there is provided a recombinant lentiviral vector comprising any one of the coding nucleotides in the above aspects. Preferably, the carrier has a PTK881-EF1α carrier backbone.

In yet another aspect, an immune cell is provided, wherein the immune cell is transfected with any of the above-mentioned recombinant lentiviral vectors. Preferably, the immune cells are T cells. More preferably, the T cells are at least one of γδ T cells from a healthy donor and CD8+ T cells derived from cord blood.

In another aspect, there is provided a method for constructing any one of the encoding nucleotides in the above aspects, which comprises:

1) Synthesize the signal peptide-anti-HIV gp120 single-chain antibody encoding nucleotide SP1-N6, and the signal peptide-anti-CD19 single-chain antibody encoding nucleotide SP2-FMC83-28Z, and clone the synthesized encoding nucleotides into pUC57 vector;

2) Using the human cDNA library as a template, amplify CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, CD8 transmembrane region, CD3ζ intracellular signal transduction domain, Strep II, connection Peptides and P2A encoding nucleotide fragments;

3) Using Overlap PCR technology to combine the SP1-N6, Strep II, connecting peptide, SP2-FMC63-28Z, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signal The coding nucleotide fragments of the conduction domain are sequentially connected to obtain the coding gene C5-CAR of the chimeric antigen receptor; or

The SP1-N6 and Strep II, connecting peptide, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signal transduction domain encoding nucleosides were respectively combined using Overlap PCR technology. The acid fragments are sequentially connected to obtain the fragment SP1-CD3ζ; the P2A and the nucleic acids of the SP2-FMC63-28Z, CD8 hinge region, CD8 transmembrane region, CD28-ICD and CD3ζ intracellular signaling domain are sequentially connected To obtain the fragment P2A-SP2-CD3ζ; and, the fragment SP1-CD3ζ and P2A-SP2-CD3ζ were ligated to obtain the coding gene C6-CAR.

Preferably, the nucleic acid fragment of the connecting peptide is 3×G ₄ S. More preferably, the nucleotide fragment encoding the connecting peptide 3×G ₄ S comprises the nucleotide sequence shown in SEQ ID NO:23. Preferably, the single-chain antibody N6 comprises the amino acid sequence shown in SEQ ID NO:35. Preferably, the single-chain antibody FMC63-28Z comprises the amino acid sequence shown in SEQ ID NO:36. Preferably, the nucleotide fragment encoding the signal peptide SP1 comprises the nucleotide sequence shown in SEQ ID NO:7. Preferably, the nucleotide fragment encoding the signal peptide SP2 comprises the nucleotide sequence shown in SEQ ID NO:9. Preferably, the coding nucleotide fragment of Strep II comprises the nucleotide sequence shown in SEQ ID NO:11. Preferably, the nucleotide fragment encoding the CD8 hinge region in the C5-CAR comprises the nucleotide sequence shown in SEQ ID NO:13. Preferably, the nucleotide fragment encoding the CD28 transmembrane region in the C5-CAR comprises the nucleotide sequence shown in SEQ ID NO:15. Preferably, the CD28-ICD encoding nucleotide fragment in the C5-CAR comprises the nucleotide sequence shown in SEQ ID NO:17. Preferably, the coding nucleotide fragment of the 4-1BB costimulatory domain in the C5-CAR comprises the nucleotide sequence shown in SEQ ID NO:19. Preferably, the coding nucleotide fragment of the CD3ζ intracellular signaling domain in the C5-CAR comprises the nucleotide sequence shown in SEQ ID NO:21. Preferably, the P2A encoding nucleotide fragment in the C6-CAR comprises the nucleotide sequence shown in SEQ ID NO:25. Preferably, the nucleotide fragment encoding the CD8 hinge region in the C6-CAR comprises the nucleotide sequence shown in SEQ ID NO:27. Preferably, the nucleotide fragment encoding the CD8 transmembrane region in the C6-CAR comprises the nucleotide sequence shown in SEQ ID NO:29. Preferably, the CD28-ICD encoding nucleotide fragment in the C6-CAR comprises the nucleotide sequence shown in SEQ ID NO:31. Preferably, the coding nucleotide fragment of the CD3ζ intracellular signaling domain in the C6-CAR comprises the nucleotide sequence shown in SEQ ID NO:33. More preferably, the SP1-N6 comprises the nucleotide sequence shown in SEQ ID NO:1; the SP2-FMC83-28Z comprises the nucleotide sequence shown in SEQ ID NO:2.

Preferably, each of the above-mentioned nucleotide fragments consists of, or essentially consists of, each specific nucleotide sequence associated with the nucleotide fragment.

In yet another aspect, any bispecific chimeric antigen receptor, any encoding nucleotide, any recombinant lentiviral vector and/or any immune cell in the above aspects are provided for use in the preparation of Application in medicines, preparations or pharmaceutical compositions for treating hematological tumors combined with HIV infection. Preferably, the hematological tumor is a CD19-positive B lymphocyte malignant tumor. Preferably, the medicament, preparation or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In yet another aspect, any bispecific chimeric antigen receptor, any encoding nucleotide, any recombinant lentiviral vector and/or any immune cell in the above aspects are provided for the treatment of blood Application of tumor combined with HIV infection. Preferably, the hematological tumor is a CD19-positive B lymphocyte malignant tumor.

In another aspect, there is also provided a method for treating hematological tumors combined with HIV infection, which comprises: administering any one of the bispecific chimeric antigen receptors and any one of the encoding nuclei to a subject in need thereof. Glucosinolates, any recombinant lentiviral vector and/or any immune cell. Preferably, the hematological tumor is a CD19-positive B lymphocyte malignant tumor.

In one or more embodiments of the present disclosure, a broad-spectrum neutralizing antibody against HIV envelope protein and a specific antibody against CD19, a hematological tumor targeting marker, are used. They can be combined in series to form scFv, or they can be combined to generate two CAR molecules in parallel, which are further loaded on γδ T cells and CD8+ T cells. One or more embodiments of the present disclosure achieve at least one or more of the following beneficial effects: firstly, there is no need to draw blood from HIV-infected persons; secondly, CAR molecules targeting two malignant cells can be prepared only once by transduction Furthermore, the presence of high-load tumors in the body will stimulate the proliferation of CAR-T cells and accelerate the elimination of HIV-infected cells; finally, chemotherapy and HAART drugs are not required, and there are no side effects.

Description of the drawings

The following drawings form a part of this specification and are included to further illustrate certain aspects of the invention. The present invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments given herein.

Figure 1 shows a schematic diagram of the structure of C5-CAR according to some embodiments of the present disclosure;

Figure 2 shows a schematic structural diagram of C6-CAR according to some embodiments of the present disclosure;

Figure 3 shows a plasmid map of PTK881-EF1α-C5 according to some embodiments of the present disclosure;

Figure 4 shows a plasmid map of PTK881-EF1α-C6 according to some embodiments of the present disclosure;

Figure 5 shows the cell transduction efficiency test results, that is, the cell transduction efficiency test results of C5-CAR-γδT and C6-CAR-γδT prepared according to the foregoing embodiments of the present disclosure are shown sequentially from top to bottom;

Figure 6 shows the cell transduction efficiency test results, that is, the cell transduction efficiency test of C5-CAR-CD8+T and C6-CAR-CD8+T prepared according to the foregoing embodiments of the present disclosure from top to bottom. result;

Figure 7 shows the results of in vitro tumor killing efficiency of C5-CAR-γδT prepared according to the foregoing embodiments of the present disclosure;

Figure 8 shows the results of in vitro tumor killing efficiency of C5-CAR-CD8+T cells prepared according to the foregoing embodiments of the present disclosure;

Figure 9 shows the results of in vitro tumor killing efficiency of C6-CAR-γδT prepared according to the foregoing embodiments of the present disclosure;

Figure 10 shows the results of in vitro tumor killing efficiency of C6-CAR-CD8+T cells prepared according to the foregoing embodiments of the present disclosure;

Figure 11 shows the test results of in vitro tumor killing efficiency of γδT cells;

Figure 12 shows the results of in vitro tumor killing efficiency of CD8+ T cells.

Figure 13 shows the comparison of the killing efficiency of gp41-CD19CAR-γδT, C5-CAR-γδT and C6-CAR-γδT in vitro.

Figure 14 shows the comparison of the killing efficiency of gp41-CD19CAR-CD8+T, C5-CAR-CD8+T and C6-CAR-CD8+T in vitro.

Detailed ways

Before describing the present disclosure in detail, it should be understood that the present disclosure is not limited to the specific exemplified materials or process parameters, as they can of course be changed. It should also be understood that the terms used herein are only to describe some specific embodiments of the present invention, and are not intended to limit the use of alternative terms to describe the present invention.

For all purposes, all publications, patents, and patent applications (whether above or below) cited herein are hereby incorporated by reference in their entirety.

Unless the content clearly indicates otherwise, nouns without quantitative word modification used in this specification and the appended claims represent one or more types. The term "comprises/includes" or its variants as used herein should be understood to include any whole (for example, features, elements, characteristics, characteristics, methods/processing steps or limitations) or groups of wholes (for example, multiple features, Multiple elements, multiple features, multiple characteristics, multiple methods/processing steps, or multiple limitations), but do not exclude any other whole or group of wholes. Therefore, the term "comprising/comprising" as used herein is inclusive and does not exclude additional unquoted wholes or method/processing steps.

In some embodiments of any of the compositions and methods provided herein, "consisting essentially of" or "consisting of" may be used in place of "comprising/including". The phrase "essentially composed of" as used herein requires specified wholes or steps and those wholes or steps that do not significantly affect the features or functions of the claimed invention. The term "consisting of" as used herein is only used to refer to only the cited whole (such as features, elements, characteristics, characteristics, methods/processing steps or limitations) or groups of wholes (such as multiple features, multiple elements). , Multiple features, multiple characteristics, multiple methods/processing steps, or multiple limitations).

As used herein, the term "highly active anti-retroviral therapy (HAART)", also known as cocktail therapy, refers to the combined use of several (usually three or four) antiretroviral therapy Viral drugs are used to treat retroviral infections.

As used herein, the term "treatment" encompasses the treatment of a disease state in mammals, especially in humans, and includes: (a) preventing the occurrence of a disease state in a mammal, especially when such a mammal is susceptible to the disease state but When the disease state has not been diagnosed; (b) inhibiting the disease state, that is, preventing its development; and/or (c) reducing the disease state, that is, promoting the regression of the disease state.

As used herein, the term "administration" means to provide a substance to a subject in a pharmacologically usable manner.

As used herein, the term "connected" or "connected" means to chemically associate one entity (e.g., a moiety) with another entity.

When conceiving a clinical application, it is necessary to prepare the pharmaceutical composition in a form suitable for the intended administration. Generally, this will require the preparation of a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, metamorphic or other adverse reactions when administered to animals or humans. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and reagents for pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active substance of the present disclosure, its use in a therapeutic composition is contemplated. Additional or supplementary active ingredients can also be incorporated into the medicament, pharmaceutical composition or formulation of the present disclosure.

As mentioned earlier, malignant lymphoma is currently one of the main causes of death in AIDS patients. For HIV combined with malignant tumor diseases, the treatment plan is generally anti-tumor combined with antiretroviral therapy (ART) therapy. However, at present, the best plan and drugs for this comorbidity have not been found in clinical practice. The high-efficiency antiretroviral therapy (HAART), which is considered to be more effective, has many shortcomings: (1) patients must take the drug for life, which requires high economic costs; (2) severe side effects; (3) emergence of drug-resistant strains (4) More importantly, cART cannot completely remove the virus, mainly because the drug is only effective on the virus in replication, but is not effective on the latent virus "reservoir" (reservoir) established by HIV in the early stage of infection. Therefore, there is an urgent need to choose a treatment plan that can solve one or more of the above-mentioned challenges to effectively treat HIV-infected malignant lymphoma.

Unexpectedly and surprisingly, the inventors have discovered that the above-mentioned problems and limitations can be overcome by implementing one or more aspects disclosed herein. The invention is illustrated in the following examples.

In order to effectively treat patients with hematological tumors combined with HIV infection, the present disclosure provides a bispecific chimeric antigen receptor and its encoding gene for the treatment of hematological tumors combined with HIV infection. -Cell Immunotherapy (CAR-T) therapy has a very good effect of relieving, alleviating and curing the condition of patients with hematological tumors and HIV infection, and has broad clinical prospects.

In some embodiments, the present disclosure provides a bispecific chimeric antigen receptor for the treatment of hematological tumors combined with HIV infection. The bispecific chimeric antigen receptor comprises an anti-HIV gp120 single-chain antibody and an anti-CD19 single-chain antibody. Chain antibody.

The present disclosure provides an HIV gp120 and CD19 dual target modified CAR-T cell by optimizing the design of HIV gp120 and CD19-specific single-chain antibodies, which can specifically bind to the HIV gp120 antigen and CD19. In the cell killing test of HIV infection and CD19-positive B lymphocyte malignant tumors, CAR-T therapy with HIV gp120 and CD19 dual target modified CAR-T cells can achieve one CAR-T cell killing two different types Malignant cells can achieve the effect of one CAR-T in treating two diseases at the same time.

Further, the bispecific chimeric antigen receptor also includes CSF2RA signal peptide and CD8 signal peptide, CD28 transmembrane region and CD8 transmembrane region, CD28-ICD, 4-1BB costimulatory domain and CD3ζ intracellular signal transduction Guide domain.

Further, the bispecific chimeric antigen receptor is sequentially spliced from N-terminal to C-terminal with signal peptide SP1, anti-HIV gp120 single-chain antibody, Strep II, connecting peptide, anti-CD19 single-chain antibody, CD8 hinge region, CD28 Transmembrane region, CD28-ICD, 4-1BB costimulatory domain and CD3ζ intracellular signaling domain.

Further, the bispecific chimeric antigen receptor consists of the first CAR from the N-terminus to the C-terminus: signal peptide SP1, anti-HIV gp120 antigen-specific single-chain antibody, Strep II, connecting peptide and CD28 transmembrane region, CD28 -ICD, 4-1BB costimulatory domain and CD3ζ intracellular signaling domain and the second CAR: signal peptide SP2, anti-CD19 single chain antibody, CD8 transmembrane region, CD28-ICD and CD3ζ intracellular signaling domain, The first CAR and the second CAR are sequentially formed in parallel through P2A.

Furthermore, the signal peptides are preferably CSF2RA and CD8 signal peptides; the connecting peptide is 3×G ₄ S; and the self-cleaving short peptides are preferably P2A short peptides.

In some embodiments, the present disclosure also provides nucleotides encoding bispecific chimeric antigen receptors.

Further, the nucleotide sequence of the encoding nucleotide of the bispecific chimeric antigen receptor is as shown in SEQ ID NO: 37 or as shown in SEQ ID NO: 38.

In some embodiments, the present disclosure provides a recombinant lentiviral vector. The lentiviral vector uses the PTK881-EF1α vector as a backbone and contains the aforementioned bispecific chimeric antigen receptor-encoding nucleotides.

In some embodiments, the present disclosure provides an immune cell transfected with the aforementioned recombinant lentiviral vector.

In some embodiments, the present disclosure provides a method for constructing a bispecific chimeric antigen receptor encoding nucleotide.

Further, the construction method includes the following steps:

1) Gene synthesis as shown in SEQ ID NO: 1 signal peptide-anti-HIV gp120 single chain antibody encoding nucleotide SP1-N6, and as shown in SEQ ID NO: 2 signal peptide-anti-CD19 single chain antibody encoding nucleus Nucleotide SP2-FMC83-28Z, and clone the synthesized above-mentioned coding nucleotides into pUC57 vector;

2) Using the human cDNA library as a template, design primers to amplify fragments CD8 hinge region, CD28 transmembrane region, CD28 intracellular domain (ICD), 4-1BB costimulatory domain, CD8 transmembrane region, CD3ζ intracellular signal Transduction domain, Strep II, connecting peptide 3×G ₄ S, P2A are obtained by primer complementation;

3) Using Overlap PCR technology to combine SP1-N6, Strep II, connecting peptide 3×G ₄ S, SP2-FMC63-28Z with CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, CD3ζ The intracellular signal transduction domains are amplified and connected sequentially to obtain the coding gene C5-CAR of the chimeric antigen receptor. The schematic diagram of the structure is shown in Figure 1. The overlap PCR technology is used to separate SP1-N6 with Strep II and connecting peptide 3. ×G ₄ S, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain were amplified sequentially into fragments SP1-CD3ζ, P2A and SP2-FMC63-28Z , CD8 hinge region, CD8 transmembrane region, CD28-ICD, CD3ζ intracellular signal transduction domains are amplified sequentially into fragments P2A-SP2-CD3ζ, fragments SP1-CD3ζ and P2A-SP2-CD3ζ are finally connected to the coding gene C6- CAR, its structure diagram is shown in Figure 2.

Preferably, the amino acid sequence of the single-chain antibody N6 is shown in SEQ ID NO: 35, the amino acid sequence of the single-chain antibody FMC63-28Z is shown in SEQ ID NO: 36, the signal peptide SP1, signal peptide SP2, and Strep II. The nucleotide sequence of the connecting peptide 3×G ₄ S is shown in SEQ ID NO: 7, 9, 11, and 23 respectively; CD8 hinge region, CD28 transmembrane domain, CD28-ICD, 4- The nucleotide sequences of 1BB costimulatory domain and CD3ζ intracellular signaling domain are shown in SEQ ID NO: 13, 15, 17, 19, 21, respectively; the nucleotide sequence of P2A in C6-CAR is shown in SEQ ID NO : 25, the nucleotide sequences of the CD8 hinge region, CD8 transmembrane domain, CD28-ICD, and CD3ζ intracellular signaling domain connected by single-chain antibody FMC63-28Z are as shown in SEQ ID NO: 27, 29, 31, 33 shown.

In some embodiments, the present disclosure provides the use of bispecific chimeric antigen receptors, their encoding nucleotides, recombinant lentiviral vectors, and immune cells in the preparation of drugs or preparations for the treatment of hematological tumors combined with HIV infection. Further, the hematological tumor is a CD19+ positive B-lymphocyte malignant tumor.

Example

The solutions of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present disclosure, and should not be regarded as limiting the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

Example 1: Construction of PTK881-EF1α-C5 and PTK881-EF1α-C6 plasmids

1. Sequences SP1-N6 (nucleotide sequence SEQ ID NO: 1), SP2-FMC63-28Z (nucleotide sequence SEQ ID NO: 2) were gene synthesized by Suzhou Jinweizhi Biotechnology Company, and the synthesized sequence was cloned into pUC57 vector.

2. Using the human cDNA library as a template, design primers to amplify fragments CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, CD8 transmembrane region, CD3ζ intracellular signaling domain, and Strep II, connecting peptide 3×G ₄ S, P2A are obtained by primer complementation.

3. Using Overlap PCR technology to combine SP1-N6, Strep II, connecting peptide 3×G ₄ S, SP2-FMC63-28Z with CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, CD3ζ The intracellular signal transduction domains are amplified and connected sequentially to obtain the coding gene C5-CAR of the chimeric antigen receptor. The schematic diagram of the structure is shown in Figure 1, and its nucleotide sequence is SEQ ID NO: 37; the overlap PCR technology is adopted. SP1-N6 and Strep II, connecting peptide 3×G ₄ S, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain were sequentially amplified into Fragment SP1-CD3ζ, P2A and SP2-FMC63-28Z, CD8 hinge region, CD8 transmembrane region, CD28 costimulatory domain, CD3ζ intracellular signal transduction domain were sequentially amplified into fragments P2A-SP2-CD3ζ, fragment SP1- CD3ζ and P2A-SP2-CD3ζ were finally linked to form the coding gene C6-CAR. The schematic diagram of the structure is shown in Figure 2, and its nucleotide sequence is SEQ ID NO:38.

Among them, C5-CAR and C6-CAR are single-chain antibodies ScFv-N6 and ScFv-FMC63-28Z with signal peptides that can recognize the surface of hematological tumor cells infected by HIV virus. Single-chain antibody ScFv-N6: the VH amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO: 4. Single-chain antibody ScFv-FMC63-28Z: the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO: 6. The amino acid sequences of the single-chain antibodies ScFv-N6 and ScFv-FMC63-28Z are shown in SEQ ID NO: 35 and SEQ ID NO: 36.

The nucleotide sequence and amino acid sequence of signal peptide SP1 are shown in SEQ ID NO: 7, SEQ ID NO: 8, and the nucleotide sequence and amino acid sequence of signal peptide SP2 are shown in SEQ ID NO: 9, SEQ ID NO: 10. As shown, the nucleotide sequence and amino acid sequence of Strep Ⅱ are shown in SEQ ID NO: 11, SEQ ID NO: 12, and _{the nucleotide sequence and amino acid sequence of the connecting peptide 3×G 4} S are shown in SEQ ID NO: 23, SEQ ID NO: 23 and SEQ ID NO: 12. ID NO: 24 is shown. The nucleotide sequence and amino acid sequence of the CD8 hinge region in C5-CAR are shown in SEQ ID NO: 13 and SEQ ID NO: 14, and the nucleotide sequence and amino acid sequence of CD28 transmembrane domain in C5-CAR are shown in SEQ ID. NO:15, SEQ ID NO:16, the nucleotide sequence and amino acid sequence of CD28-ICD in C5-CAR are shown in SEQ ID NO:17, SEQ ID NO:18, C5-CAR 4-1BB The nucleotide sequence and amino acid sequence of the stimulation domain are as shown in SEQ ID NO: 19 and SEQ ID NO: 20, and the nucleotide sequence and amino acid sequence of CD3ζ in C5-CAR are as shown in SEQ ID NO: 21 and SEQ ID. NO: shown in 22.

The nucleotide sequence and amino acid sequence of P2A in C6-CAR are shown in SEQ ID NO: 25 and SEQ ID NO: 26. The single-chain antibody scFv-N6 in C6-CAR is linked to Strep Ⅱ and the connecting peptide 3×G ₄ S The nucleotide sequence and amino acid sequence corresponding to the CD8 hinge region, CD28 transmembrane domain, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain are the same as C5-CAR, C6-CAR The nucleotide sequence and amino acid sequence of the CD8 hinge region linked by the single-chain antibody scFv-FMC63-28Z are shown in SEQ ID NO:27 and SEQ ID NO:28. The single-chain antibody scFv-FMC63-28Z in the C6-CAR is linked The nucleotide sequence and amino acid sequence of the transmembrane region of CD8 are shown in SEQ ID NO: 29 and SEQ ID NO: 30. The nucleotide sequence of CD28-ICD linked by single-chain antibody scFv-FMC63-28Z in C6-CAR The amino acid sequence is shown in SEQ ID NO: 31 and SEQ ID NO: 32. The nucleotide sequence and amino acid sequence of CD3ζ linked to the single-chain antibody scFv-FMC63-28Z in C6-CAR are shown in SEQ ID NO: 33, SEQ ID. NO: 34 shows.

3. The plasmid PTK881-Kan was digested with EcoR I and BamH I restriction enzymes. The product was subjected to 0.8% agarose gel electrophoresis, and the gel was tapped and recovered, and placed in an Eppendorf tube, using Axygen's agarose gel The recovery kit recovers the corresponding fragments, and determines the purity and concentration of the product.

4. Add the recovered fragments of the vector with C5-CAR and C6-CAR to Eppendorf tube at a molar ratio of 1:2, Exnase II ligase (Vazyme) and homologous recombinase 5×CE II buffer, and react at 37°C for 0.5 hours; Take out 10μL of the ligation solution and add 100μL of DH5α competent cells to ice bath for 30min and heat shock at 42°C for 90s. After completion, add 500μL of soc medium and incubate at 37°C and 220rpm for 2 hours; after 2 hours, centrifuge the Eppendorf tube at 4000g for 1min to remove 400μL of excess liquid . Spread the remaining liquid on an LB plate containing kanamycin and culture for 12 hours at 37°C; pick a single colony on the plate and inoculate it into 5mL LB liquid medium at 37°C and 220rpm for 12 hours.

5. Use Axygen small extraction kit to extract plasmids to obtain plasmids PTK881-EF1α-C5-1 and PTK881-EF1α-C6-1, and send them to Shenggong Bioengineering (Shanghai) Co., Ltd. after the first-generation sequencing verification is correct, then carry out the containing The DH5α strain of plasmid PTK881-EF1α-C5-1 and PTK881-EF1α-C6-1 are preserved. The complete map of PTK881-EF1α-C5-1 is shown in Figure 3, and the complete map of PTK881-EF1α-C6-1 is shown in Figure 4.

Example 2: Preparation and sequencing of plasmids

1. Preparation of plasmid

The DH5α strains containing plasmids PTK881-EF1α-C5-1 and PTK881-EF1α-C6-1 were respectively inoculated into 250mL LB culture medium containing 100μg/mL kanamycin, and cultured overnight at 37°C and 220rpm. The culture broth was centrifuged at 6000g for 20 min at 4°C, and the supernatant was discarded.

Take out Buffers P1 in EndoFree plasma megakit (Qiagen), add 120 mL of pre-cooled Buffers P1 to the E. coli pellet obtained by centrifugation, cover the centrifuge bottle cap, vigorously shake the centrifuge bottle to completely disperse the E. coli pellet in Buffers P1.

Add 120mL Buffers P2 to the centrifuge bottle, cover the bottle and place it on the roller mixer, slowly increase the speed to 50 rpm, mix thoroughly, and leave it at room temperature for 5 minutes.

Add 120mL Buffers P3 to the centrifuge bottle, cover the bottle and place it on the roller mixer, slowly increase the speed to the maximum speed of the roller mixer 70 rpm, and mix thoroughly until a white, non-sticky, fluffy mixture is formed. Centrifuge at 9000g for 15 min at 4°C.

Pour 50mL Buffer FW into the QIAfilter Cartridge, pour the supernatant obtained from the centrifugation into the QIAfilter Cartridge, and gently stir and mix. The mixed liquid is suction filtered into the corresponding glass bottle that has been marked.

Add 20mL Buffer ER to each glass bottle, mix upside down 6 times, and incubate at -20°C for 30 min.

Put the labeled mega columns on the corresponding shelf, add 35mL Buffers QBT to each mega column to balance, and gravity will make it drain out.

Pour all the liquid in the glass bottle into the corresponding labeled mega column in batches. After the liquid in the column is exhausted, add 200 mL Buffer QC to each mega column in batches for cleaning. After the liquid in the column is exhausted, pour the waste liquid in the waste liquid collection tray into a 50 mL clean centrifuge tube.

Then add 40mL Buffer QN to each mega column, use a 50mL clean centrifuge tube to collect the effluent, invert 6 times to mix, and aliquot 20mL into another clean labeled 50mL centrifuge tube.

Add 14mL of isopropanol (normal temperature) to each 50mL centrifuge tube, and mix upside down 6 times. Centrifuge at 15000g for 50min at 4°C.

Aspirate the supernatant in the ultra-clean workbench, and rinse each tube with 3.5mL Endotoxin-free water. Do not wash away the sediment at the bottom. Centrifuge at 15000g for 30 min at 4°C. Put the BufferTE in the EndoFree plasma megakit into the oven to preheat.

The supernatant after centrifugation is sucked up in the ultra-clean workbench, and dried in the ultra-clean workbench (the residual ethanol is evaporated, the time is about 10min).

Take out BufferTE in the oven, add 1mL Buffer TE to each tube in the ultra-clean workbench, blow 10 times with a gun, and put it in a 65°C oven. During this time, tap the tube wall continuously to dissolve the precipitate completely. Centrifuge at 4000g for 1 min at 4°C to shake the liquid on the tube wall to the bottom of the tube and mix by pipetting.

In the ultra-clean workbench, transfer all the liquid to an EP tube with no endotoxin, no pyrogen, and no nuclease corresponding label. Aspirate 2μL, measure the plasmid concentration with a micro-spectrophotometer, and label it on the corresponding EP tube to obtain plasmids PTK881-EF1α-C5-1 and PTK881-EF1α-C6.

2. Sequencing of the target gene

Take 20μL (500ng) of plasmid DNA and send it for sequencing. According to the original seed sequence, check whether the target gene of the product produced by the plasmid has changed. Under a stable process, the target gene will not be changed during the fermentation, culture and amplification of the working seed. If it changes, it can be used for the next step of production and correct protein expression.

Example 3. Preparation and live drop detection of Lenti3-C5 and Lenti3-C6 lentiviral vectors

1. Preparation of lentiviral vector

^{Insert 130.0～140.0×10 6} 293T cells (Takara) into 2 multi-layer cell culture flasks (Hyperflask), and a total of 560mL DMEM complete medium (50mL fetal bovine serum, 5mL Antibiotic-Antimycotic (100×)), Incubate for 24 hours in an incubator containing 5% CO _{2 at 37°C.} 320μg plasmids (PTK881-EF1α-C5: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5:6 and PTK881-EF1α-C6: BZ1 plasmid: BZ2 plasmid: BZ3 plasmid=12:10:5 : 6) Add DMEM basal medium to two 960μg PEI tubes, vortex and shake, and equilibrate at room temperature for 10 minutes. Mix the above two tubes of 35mL PEI and plasmid mixture with 525mL DMEM complete medium, respectively, and change them into the above multi-layer cell culture flask. After placing the multi-layer cell culture flask in an _{incubator containing 5% CO 2} at 37° C. for 3 days, the cell culture supernatant was collected.

After centrifuging the supernatant at 4000 rpm (or 3000 g) for 30 minutes, cryonase enzyme (Takara) was added to the supernatant after centrifugation and placed at 4°C. After 6 hours, the lentiviral supernatant was suction filtered using a 0.22μm filter membrane, and centrifuged at 30000g for 2.5h at 4°C. Remove the supernatant, add 1mL T cell culture medium to resuspend the pellet. After resuspension, save 20μL for virus activity titer detection, and the remaining lentivirus concentrate is divided into aliquots, labeled as Lenti3-C5, Lenti3-C6, and stored at -80°C for later use.

2. Detection of activity titer of lentiviral vector

Principle: The anti-Strep tag Ⅱ antibody is labeled with fluorescein, and the anti-Strep tag Ⅱ antibody can specifically bind to the Strep tag Ⅱ in the CAR, and the fluorescent signal detected by the flow cytometer indirectly reflects the CAR in the 293T cell The expression of the situation.

^{Method: Insert 5.0×10 5} cells/well of 293T cells in a 6-well plate, add 0.1 μL, 0.5 μL, and 1 μL of lentivirus concentrate to each well, and set up a negative control. Place the culture in an incubator containing 5% CO _{2 at 37°C.} Three days later, 293T cells were collected with Versene solution (Gibco) and sent to flow cytometry to detect the proportion of CAR-positive 293T cells, and converted to obtain the activity titers of Lenti3-C5 and Lenti3-C6 lentivirus concentrates.

The current activity titer of the lentivirus concentrate is in the range of 1×10 ⁸ to 10×10 ⁸ (TU/mL). The results of detection and analysis are shown in Table 1.

Table 1 Detection and analysis results of Lenti3-C5 and Lenti3-C6 lentivirus activity titer

Sample serial number Activity titer (TU/mL) Lenti3-C5 1.8×10 ⁸ Lenti3-C6 2.1×10 ⁸

Example 4. Preparation of CAR-γδT and CAR-CD8+ T cells

1. Preparation of CAR-γδ T cell preparations:

Collect 200 mL of peripheral blood from healthy donors, and use Ficoll lymphocyte separator to separate mononuclear cells. After counting, a suitable amount TCRγδ + T Cell Isolation Kit, human ( Miltenyi Biotec) TCRγδ + T cells were sorted, and to 1.0 ~ 2.0 × 10 ⁶ cells / mL density activated broth (OpTmizer ^TM CTS ^TM T- cells in γδT Cell Expansion Basal Medium, OpTmizer ^TM CTS T-Cell Expansion Supplement (Invitrogen), 500～1000IU/mL IL-2 (Shuanglu Pharmaceutical), IL-75～20ng/mL, Zolidine phosphonic acid 5μM), Activate γδ T cells.

After 24 hours, Lenti3-C5 and Lenti3-C6 lentiviral vectors were added at MOI of 5 for transduction, mixed and incubated in a CO2 incubator, 4 hours later, supplemented with appropriate amount of γδT cell activation medium for culture.

After 24 hours of lentiviral transduction, change the transduced C5-CAR-γδT and C6-CAR-γδT cells into the γδT cell activation medium, and adjust the viable cell density to 1.0-2.0×10 ⁶ /mL, and continue the culture expansion. Add 3 days, observe and count every day, and carry out supplementation and expansion culture according to the counted number of cells, and always keep the cell culture density at 1.0-2.0×10 ⁶ /mL. Starting from day 4, supplement γδ T cell expansion medium (OpTmizer ^TM CTS ^TM T-Cell Expansion Basal Medium, OpTmizer ^TM CTS T-Cell Expansion Supplement (Invitrogen), 500～1000IU/mL IL-2 (Shuanglu Pharmaceuticals), IL-75-20ng/mL), and adjust the viable cell density to 1.0-2.0×10 ⁶ /mL, and expand the culture to 14 days.

Collect C5-CAR-γδT and C6-CAR-γδT cells according to the expected cell dosage, resuspend them in 100 mL normal saline containing 2% human albumin, transfer the cells back to the infusion bag, and heat seal to make C5-CAR -γδT, C6-CAR-γδT cell preparation finished product.

2. Preparation of CAR-CD8+ T cell preparations

Collect 60 mL of umbilical cord blood from healthy donors, and use Ficoll lymphocyte separator to separate mononuclear cells. After counting, use an appropriate amount of CD8+T Cell Isolation Kit human (Miltenyi) to sort CD8 positive cells, and in the T cell complete culture medium (OpTmizer ^TM CTS ^TM T-Cell Expansion ^{) at a density of 1.0～2.0×10 6 cells/mL} the ^{basal medium, OpTmizer TM CTS T-} cell Expansion Supplement (Invitrogen), 500 ~ 1000IU / mL of IL-2 (SL Pharmaceutical)) culture, while per 10 ⁶ cells was added 25μl Dynabeads Human T-Activator CD3 / CD28 (Invitrogen) activates T cells.

After 24 hours, Lenti3-C5 and Lenti3-C6 lentiviral vectors were added at MOI of 3 for transduction, mixed and incubated in a CO2 incubator, 4 hours later, supplemented with an appropriate amount of T cell complete medium for culture.

24 hours after lentiviral transduction, change the transduced C5-CAR-CD8+T and C6-CAR-CD8+T cells into fresh T cell complete culture medium, and adjust the viable cell density to 1.0-2.0×10 ⁶ /mL , Continue to culture and expand for 10-20 days, observe and count every day, and carry out supplemental expansion culture according to the counted number of cells, and always maintain a cell culture density of 1.0-2.0×10 ⁶ /mL.

Collect C5-CAR-CD8+T and C6-CAR-CD8+T cells according to the expected cell dosage, resuspend them in 100mL normal saline containing 2% human albumin, transfer the cells back to the infusion bag, and heat seal them to prepare Into finished products of C5-CAR-CD8+T and C6-CAR-CD8+T cell preparations.

3. Detection of transduction efficiency of CAR-γδT and CAR-CD8+T cells

Take 1.0×10 ⁶ CAR-γδT and CAR-CD8+T cells and incubate them with FITC-Strep II for 30 minutes at room temperature. After washing twice with saline, measure the FITC fluorescence signal by flow cytometer and measure the ratio of FITC positive cells. , Which reflects the ratio of CAR-positive cells in the total cells. C5-CAR-γδT, C5-CAR-CD8+T, C6-CAR-γδT, C6-CAR-CD8+T cell transduction efficiency test results are shown in Figure 5 and Figure 6, respectively. Figure 5 and Figure 6 show that C5-CAR-γδT, C5-CAR-CD8+T, C6-CAR-γδT, C6-CAR-CD8+T cells were successfully prepared.

Example 5. In vitro functional detection of CAR-γδT and CAR-CD8+ T cells

In vitro tumor killing test:

The anti-tumor function of CD8+T, γδT, CAR-γδT, CAR-CD8+T cells were tested in vitro by calcein detection method. Select to construct stable transfected cells 293T-gp160+, Raji cells as positive target cells, and 293T cells as negative target cells.

Take an appropriate amount of the above-mentioned 293T-gp160+, Raji, and 293T target cells, and ^{add calcein-acetohydroxymethyl ester (Calcein-AM) to 1×10 6} /mL cell suspension (PBS, 5% fetal bovine serum) to the end The concentration is 25μM, and incubate for 30min in an incubator. At room temperature, after washing twice, resuspend the cells to 0.5×10 ⁵ /mL. Add negative target cells and positive target cells to 96-well plates. Positive target cells are divided into three groups. Group 1: Add 5000 293T to each well -gp160+; the second group: add 5000 Raji cells per well; the third group add 2500 293T-gp160+ and 2500 Raji cells per well. Add T, γδT, C5-CAR-γδT, C5-CAR-CD8+T, C6-CAR-γδT, C6-CAR-CD8+T cells according to the effective target ratio of 25:1, 5:1, and 1:1 , Incubate at 37°C for 2 to 3 hours. After the incubation is completed, take the supernatant, measure the fluorescence intensity of calcein, and calculate the percentage of target cell lysis based on the spontaneous release control and the maximum release control. The results of percentage lysis of target cells are shown in Figure 7 to Figure 12. The results show that C5-CAR-γδT and C6-CAR-γδT cells can significantly promote the lysis of gp160 and CD19 positive target cells (293T-gp160+, Raji), which is against negative targets. Cells (293T) also have a certain killing effect; γδT cells promote the lysis of both positive and negative target cells. C5-CAR-CD8+T and C6-CAR-CD8+T cells can significantly promote the lysis of gp160 and CD19-positive target cells (293T-gp160+, Raji), but have little effect on negative target cells.

In addition, the in vitro killing efficiency of gp41-CD19CAR-γδT and C5-CAR-γδT and C6-CAR-γδT were compared when 293T-gp160+ (gp160 is the full length of the envelope protein gp120 and gp41 connected in sequence) as target cells. When 293T-gp160+ is used as the target cell, the killing efficiency of gp41-CD19CAR-CD8+T, C5-CAR-CD8+T, and C6-CAR-CD8+T in vitro is shown in Figure 13 and Figure 14 respectively. The results showed that the CAR-T killing effect of the gp120-CD19 combination was better than that of the gp41-CD19 combination.

Although the embodiments of the present disclosure have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present disclosure. Those of ordinary skill in the art can comment on the foregoing within the scope of the present disclosure. The embodiment undergoes changes, modifications, substitutions, and modifications.

Claims (12)

A bispecific chimeric antigen receptor, wherein the chimeric antigen receptor comprises an anti-HIV gp120 single-chain antibody and an anti-CD19 single-chain antibody. The bispecific chimeric antigen receptor according to claim 1, further comprising: signal peptide, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain At least one of them. The bispecific chimeric antigen receptor according to claim 1 or 2, wherein, from the N-terminus to the C-terminus, the bispecific chimeric antigen receptor sequentially includes: signal peptide SP1, the anti-HIV gp120 Single-chain antibody, Strep II, connecting peptide, the anti-CD19 single-chain antibody, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain. The bispecific chimeric antigen receptor according to claim 1 or 2, wherein the bispecific chimeric antigen receptor comprises a first CAR and a second CAR, Wherein, the first CAR includes: signal peptide SP1, anti-HIV gp120 antigen-specific single-chain antibody, Strep II, connecting peptide, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain; Wherein, the second CAR includes: signal peptide SP2, anti-CD19 single-chain antibody, CD8 hinge region, CD8 transmembrane region, CD28-ICD, and CD3ζ intracellular signaling domain; and Wherein, the first CAR and the second CAR are connected via a self-cleaving short peptide. The bispecific chimeric antigen receptor according to any one of claims 2 to 4, wherein the signal peptide is a CD8 signal peptide and a CSF2RA signal peptide; and/or The connecting peptide is 3×G ₄ S; and/or The self-cleaving short peptides are P2A short peptides. An encoding nucleotide, which encodes the bispecific chimeric antigen receptor according to any one of claims 1 to 5, wherein preferably, the nucleotide sequence of the encoding nucleotide is as SEQ ID NO: 37 or SEQ ID NO: 38. A recombinant lentiviral vector comprising the encoding nucleotide of claim 5, wherein preferably, the vector has a PTK881-EF1α vector backbone. An immune cell, wherein the immune cell is transfected with the recombinant lentiviral vector of claim 7; preferably, the immune cell is a T cell; more preferably, the T cell is a healthy donor γδ T cell And at least one of CD8+ T cells derived from cord blood. The method for constructing the encoding nucleotide of claim 5, which comprises: 1) Synthesize the signal peptide-anti-HIV gp120 single-chain antibody encoding nucleotide SP1-N6, and the signal peptide-anti-CD19 single-chain antibody encoding nucleotide SP2-FMC83-28Z, and clone the synthesized encoding nucleotides into pUC57 vector; 2) Using the human cDNA library as a template, amplify CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, CD8 transmembrane region, CD3ζ intracellular signal transduction domain, Strep II, connection Peptides and P2A encoding nucleotide fragments; 3) Using Overlap PCR technology to combine the SP1-N6, Strep II, connecting peptide, SP2-FMC63-28Z, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signal The coding nucleotide fragments of the conduction domain are sequentially connected to obtain the coding gene C5-CAR of the chimeric antigen receptor; or Using Overlap PCR technology to separate the coding nucleosides of SP1-N6 and Strep II, connecting peptide, CD8 hinge region, CD28 transmembrane region, CD28-ICD, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain The acid fragments are sequentially connected to obtain the fragment SP1-CD3ζ; the P2A and the nucleic acids of the SP2-FMC63-28Z, CD8 hinge region, CD8 transmembrane region, CD28-ICD and CD3ζ intracellular signaling domain are sequentially connected To obtain the fragment P2A-SP2-CD3ζ; and linking the fragment SP1-CD3ζ with P2A-SP2-CD3ζ to obtain the coding gene C6-CAR, Among them, preferably, The nucleic acid fragment of the connecting peptide is 3×G ₄ S, and more preferably, the _{nucleotide fragment encoding the connecting peptide 3×G 4} S comprises the nucleotide sequence shown in SEQ ID NO: 23; and/ or The single-chain antibody N6 comprises the amino acid sequence shown in SEQ ID NO: 35; and/or The single-chain antibody FMC63-28Z includes the amino acid sequence shown in SEQ ID NO: 36; and/or The coding nucleotide fragment of the signal peptide SP1 includes the nucleotide sequence shown in SEQ ID NO: 7; and/or The nucleotide fragment encoding the signal peptide SP2 includes the nucleotide sequence shown in SEQ ID NO: 9; and/or The encoding nucleotide fragment of Strep II includes the nucleotide sequence shown in SEQ ID NO: 11; and/or The nucleotide fragment encoding the CD8 hinge region in the C5-CAR includes the nucleotide sequence shown in SEQ ID NO: 13; and/or The nucleotide fragment encoding the CD28 transmembrane region in the C5-CAR includes the nucleotide sequence shown in SEQ ID NO: 15; and/or The nucleotide fragment encoding CD28-ICD in the C5-CAR includes the nucleotide sequence shown in SEQ ID NO: 17; and/or The coding nucleotide fragment of the 4-1BB costimulatory domain in the C5-CAR comprises the nucleotide sequence shown in SEQ ID NO: 19; and/or The coding nucleotide fragment of the CD3ζ intracellular signaling domain in the C5-CAR includes the nucleotide sequence shown in SEQ ID NO: 21; and/or The P2A encoding nucleotide fragment in the C6-CAR includes the nucleotide sequence shown in SEQ ID NO: 25; and/or The nucleotide fragment encoding the CD8 hinge region in the C6-CAR includes the nucleotide sequence shown in SEQ ID NO: 27; and/or The nucleotide fragment encoding the CD8 transmembrane region in the C6-CAR includes the nucleotide sequence shown in SEQ ID NO: 29; and/or The nucleotide fragment encoding CD28-ICD in the C6-CAR includes the nucleotide sequence shown in SEQ ID NO: 31; and/or The coding nucleotide fragment of the CD3ζ intracellular signaling domain in the C6-CAR includes the nucleotide sequence shown in SEQ ID NO: 33; and/or More preferably, the SP1-N6 includes the nucleotide sequence shown in SEQ ID NO: 1; and the SP2-FMC83-28Z includes the nucleotide sequence shown in SEQ ID NO: 2. The bispecific chimeric antigen receptor according to any one of claims 1 to 5, the coding nucleotide according to claim 6, the recombinant lentiviral vector according to claim 7, or according to claim 8. The application of the immune cells in the preparation of drugs or preparations for the treatment of hematological tumors combined with HIV infection, preferably, the hematological tumors are CD19-positive B lymphocyte malignancies. The bispecific chimeric antigen receptor according to any one of claims 1 to 5, the coding nucleotide according to claim 6, the recombinant lentiviral vector according to claim 7, or according to claim 8. The immune cells are used for the treatment of hematological tumors combined with HIV infection. Preferably, the hematological tumors are CD19-positive B lymphocyte malignancies. A method for the treatment of hematological tumors combined with HIV infection, which comprises: administering the bispecific chimeric antigen receptor according to any one of claims 1 to 5, and the method according to claim 6 to a subject in need thereof Encoding nucleotides, the recombinant lentiviral vector according to claim 7 and/or the immune cell according to claim 8, Among them, preferably, the hematological tumor is a CD19-positive B-lymphocyte malignant tumor.

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