CN112759651B - T cell containing chimeric antigen receptor modification and application thereof - Google Patents

T cell containing chimeric antigen receptor modification and application thereof Download PDF

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CN112759651B
CN112759651B CN201911060442.4A CN201911060442A CN112759651B CN 112759651 B CN112759651 B CN 112759651B CN 201911060442 A CN201911060442 A CN 201911060442A CN 112759651 B CN112759651 B CN 112759651B
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gly
ser
cells
arg
hiv
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CN112759651A (en
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丁志超
张绮
史宣玲
赵学强
张林琦
林欣
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Beijing Huaxia Medical Treatment Technology Co ltd
Tsinghua University
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Beijing Huaxia Medical Treatment Technology Co ltd
Tsinghua University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention provides a chimeric antigen receptor using a 10-1074 human single-chain antibody as an extracellular antigen binding domain, a T cell expressing the chimeric antigen receptor and application thereof in preparing a medicament, wherein the medicament can specifically eliminate cell reservoirs in HIV-infected patients, realize MHC independent recognition killing on specificity of HIV latent cells, and even eradicate AIDS.

Description

T cell containing chimeric antigen receptor modification and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a chimeric antigen receptor, a T cell expressing the chimeric antigen receptor and application of the T cell in treating HIV infection or AIDS.
Background
Human Immunodeficiency Virus (HIV), an Acquired Immune Deficiency Syndrome (AIDS) virus, is an RNA virus that infects cells of the Human immune system and is the leading culprit in inducing Acquired Immune Deficiency Syndrome (AIDS) in humans.
Because HIV directly invades the immune system of the human body and destroys the cellular immunity and humoral immunity of the human body, HIV-infected patients are often accompanied by various complications, such as meningitis, tuberculosis, cancer, hepatitis, and the like. According to the united states AIDS program (unacds) statistics, about 7610 million (6520-8800 million) of people worldwide have infected the virus since its spread. In 2016, there were a total of about 3670 million (3080-4290 million) AIDS virus infected individuals worldwide, of which 1950 million acquire antiretroviral therapy.
Currently, the treatment for HIV-infected individuals is mainly a combination of antiviral drugs (cART) including HIV replication inhibitors or antibodies to HIV, such as: patent CN103429595A discloses 2,3, 4-substituted 5,6,7, 8-tetrahydro [1] benzothieno [2,3-b ] pyridine compounds and pharmaceutically acceptable salts thereof, and relates to compositions comprising such compounds and the use of such compounds as HIV replication inhibitors. Patent CN103797029A discloses broadly neutralizing antibody sequences against epitopes of human immunodeficiency virus or HIV. Patent CN106459186A discloses a broadly neutralizing monoclonal antibody sequence directed against the HIV-1V 2ENV region. Patents CN107022027A, CN107033241A disclose HIV-1 broad spectrum neutralizing antibody sequences that specifically bind to HIV-gp 140.
Through long-term practical tests, the therapy can inhibit virus replication in AIDS patients, thereby greatly slowing the progression of AIDS diseases.
However, this therapy has the following significant drawbacks:
(1) this therapy is costly and causes the presence of a larger number of HIV carriers, and is totally dependent on antiretroviral drugs and has to bear the severe side effects and economic burden that it brings;
(2) the therapy is ineffective for part of patients, and HIV in part of patients still cannot be inhibited but is amplified in a large amount;
(3) this therapy does not target or clear the latent HIV virus present in resting CD4+ central memory T cells in the patient, and these latently infected cells are very stable and increase continuously in vivo, which will lead to an immediate viral rebound after drug withdrawal.
It follows that a key problem in curing AIDS patients is how to completely eliminate the HIV virus from the patient. Studies have shown that CD4+ central memory T cells (Tcm) account for 95% of the total number of all HIV cell repositories. Finding a clear biomarker (biomarker) of the HIV cell repository can realize accurate positioning and finally clear the HIV cell repository cells.
Chimeric Antigen Receptor (CAR) modified T cell immunotherapy is one of the latest technologies in current adoptive cell therapy technologies. Chimeric Antigen Receptors (CARs) confer the ability of T cells to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a broader range of targets relative to native T cell surface receptor TCRs. In recent years, CAR-T technology has shown significant efficacy in the treatment of acute leukemias and non-hodgkin's lymphomas, and is considered to be one of the most promising approaches to tumor treatment. Therefore, genetically modified T cells are likely to be an important way to clear HIV cell depots. Also, there have been some advances in the clearance of HIV-1 infected cells, such as: the literature: by injecting the monoclonal Antibody 10-1074 into 33 subjects, the good tolerance of 10-1074 is verified, and the highest dose can be injected with 30mg/kg, wherein at least 11 subjects have good sensitivity to 10-1074 and rapidly reduce viruses, and the safety and the activity of the monoclonal Antibody 10-1074 for human use are simultaneously proved. However, none of the prior art discloses the use of a human single chain antibody (scFv) of 10-1074 as the extracellular antigen-binding domain of a CAR molecule and its application to the clearance of HIV cell depots, even the eradication of AIDS. Therefore, the research on the function of the CAR-T cells taking the human single-chain antibody (scFv) of 10-1074 as the extracellular antigen binding domain of the CAR molecule in clearing HIV cell storage and clarifying related mechanisms has important fundamental research significance and clinical application value.
Disclosure of Invention
The inventor prepares and obtains a Chimeric Antigen Receptor (CAR) taking a single-chain antibody (scFv) of 10-1074 as an extracellular antigen binding domain through a series of creative researches, the CAR can effectively activate a T cell signal to enable the Chimeric Antigen Receptor (CAR) to have a function of killing a target cell, wherein the 10-1074 has a characteristic of stronger specific binding with HIV envelope proteins in HIV antibodies of various human sources, the 10-1074 in a plurality of scFv forms with VL-VH sequences prepared from normal 10-1074 antibodies has a characteristic of stronger specific binding with HIV envelope proteins, and the prepared CAR taking the scFv of 10-1074 as the extracellular antigen binding domain can specifically recognize HIV envelope proteins or HIV latent cells. Preferably, the CAR with the scFv of 10-1074 as the extracellular antigen-binding domain is 10-1074-CD28-4-1BB-CD3 ζ -CAR. Furthermore, the 10-1074-CD28-4-1BB-CD3 zeta-CART cell prepared by the invention can be effectively and specifically recognized and activated by HIV latent cells, has killing capacity and reduces the number of HIV viruses. Further, the present invention prepares a pharmaceutical composition comprising 10-1074-CD28-4-1BB-CD3 ζ -CART for use in eliminating the cellular pool of Human Immunodeficiency Virus (HIV) in HIV infected patients, even for the purpose of treating AIDS.
Specifically, in a first aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR) comprising an extracellular antigen-binding domain which is a 10-1074 human single chain antibody.
Preferably, the human single chain antibody comprises a heavy chain variable region, a linker and a light chain variable region.
More preferably, the heavy chain variable region is a heavy chain variable region identical to SEQ ID NO: 3, has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology.
In one embodiment of the present invention, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 3, respectively.
More preferably, the light chain variable region is substantially identical to SEQ ID NO: 2, has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology.
In one embodiment of the present invention, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2, respectively.
More preferably, the linker is an amino acid sequence comprising glycine (Gly) and/or serine (Ser), and even more preferably, the sequence of the linker is as set forth in SEQ ID NO: 4-9, or a pharmaceutically acceptable salt thereof.
In one embodiment of the invention, the amino acid sequence of the linker is as set forth in SEQ ID NO: 4, respectively.
In another embodiment of the present invention, the amino acid sequence of the human single chain antibody is as shown in SEQ ID NO: 1 or SEQ ID NO: shown at 10.
Preferably, the chimeric antigen receptor further comprises a transmembrane region selected from one or a combination of two or more of a CD3 zeta polypeptide, a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide, a CD28-4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide.
More preferably, the transmembrane region is selected from any one of the following combinations:
(1) CD8 polypeptide, CD28 polypeptide, and CD3 ζ polypeptide;
(2) CD4 polypeptide, CD8 polypeptide, CD28, 4-1BB polypeptide, and CD3 zeta polypeptide;
(3) CD4 polypeptide, CD8 polypeptide, CD28, 4-1BB polypeptide, and CD3 zeta polypeptide.
In one embodiment of the present invention, the transmembrane region is a CD28 polypeptide.
In one embodiment of the present invention, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: as shown at 14.
Preferably, the chimeric antigen receptor further comprises an extracellular hinge region selected from the group consisting of an extracellular hinge region of CD8 or IgG.
In one embodiment of the invention, the extracellular hinge region is CD 8.
In one embodiment of the present invention, the amino acid sequence of the extracellular hinge region is as set forth in SEQ ID NO: 15, respectively.
Preferably, the chimeric antigen receptor further comprises a cytoplasmic domain selected from the group consisting of CD28-4-1BB-CD3 zeta, CD28-CD3 zeta, and 4-1BB-CD3 zeta.
In one embodiment of the invention, the amino acid sequence of the cytoplasmic domain is as set forth in SEQ ID NO: shown at 16.
Preferably, the chimeric antigen receptor further comprises a gene that controls cell survival or death. More preferably, the gene controlling cell survival or death is selected from the group consisting of IL12 or TK gene. Cell death can be controlled when externally dosed.
In one embodiment of the invention, the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane region, an extracellular hinge region, and a cytoplasmic region.
In one embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is identical to SEQ ID NO: 11, has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology.
In another embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: shown at 11.
In a second aspect of the invention, there is provided an isolated immunoresponsive cell comprising a chimeric antigen receptor according to the first aspect described above.
Preferably, the immunoresponsive cell expresses a gene that controls cell survival or death. More preferably, the gene controlling cell survival or death is selected from the group consisting of IL12 or TK gene. Cell death can be controlled when externally dosed.
Preferably, the immunoresponsive cell is mixed with another cell to control the survival or death of the immunoresponsive cell. The other cells are selected from cells expressing IL12 or TK gene. More preferably, the other cell is a CAR-T cell expressing IL12 or TK gene.
Preferably, the immunoresponsive cell is selected from the group consisting of a T cell, an NK cell, a CTL, a human embryonic stem cell, a lymphoid progenitor cell and/or a T cell precursor cell.
In one embodiment of the invention, the immunoresponsive cell is a T cell, i.e., a CAR-T cell.
Wherein said immunoresponsive cell is effective in specifically recognizing and activating HIV latently competent cells, in effectively reducing the HIV virus, and in treating AIDS.
Preferably, the immunoresponsive cell is 10-1074-CD28-4-1BB-CD3 zeta-CART.
In a third aspect of the invention, there is provided a nucleic acid molecule encoding a chimeric antigen receptor according to the first aspect. Preferably, the nucleic acid molecule comprises a TK or IL12 gene fragment, which can control cell survival or death, for example by external drug administration.
Preferably, the nucleic acid molecule is identical to SEQ ID NO: 12, has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology.
In one embodiment of the invention, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: shown at 12.
In a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the third aspect described above.
Preferably, the vector is a vector substantially identical to SEQ ID NO: 13, has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology.
In one embodiment of the invention, the nucleotide sequence of the vector is as set forth in SEQ ID NO: shown at 13.
In a fifth aspect of the invention, there is provided a host cell comprising a nucleic acid molecule according to the third aspect above or a vector according to the fourth aspect above.
Preferably, the host cell is a T cell.
In a sixth aspect of the invention, there is provided a cell population comprising at least 1 host cell according to the fifth aspect above.
In a seventh aspect of the present invention, there is provided an antibody or antigen-binding portion thereof that specifically binds to the chimeric antigen receptor of the first aspect described above.
In an eighth aspect of the invention, there is provided a pharmaceutical composition comprising an effective amount of the chimeric antigen receptor of the first aspect, the immunoresponsive cell of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the population of cells of the sixth aspect and/or the antibody or antigen-binding portion thereof of the seventh aspect, and a pharmaceutically acceptable carrier.
Preferably, the pharmaceutical composition is used for treating HIV infection or AIDS.
In a ninth aspect, the present invention provides the use of the chimeric antigen receptor of the first aspect described above, the immunoresponsive cell of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the cell population of the sixth aspect, the antibody or antigen-binding portion thereof of the seventh aspect, and/or the pharmaceutical composition of the eighth aspect in the manufacture of a medicament for the treatment and/or diagnosis of an HIV infection or AIDS.
In a tenth aspect of the invention, there is provided a kit for the treatment and/or diagnosis of HIV infection or AIDS, said kit comprising a chimeric antigen receptor of the first aspect described above, an immunoresponsive cell of the second aspect, a nucleic acid molecule of the third aspect, a vector of the fourth aspect, a host cell of the fifth aspect, a population of cells of the sixth aspect, an antibody or antigen-binding portion thereof of the seventh aspect, and/or a pharmaceutical composition of the eighth aspect.
In an eleventh aspect of the invention, there is provided a method of clearing HIV-infected cells or treating AIDS, said method comprising administering to a subject an effective amount of the chimeric antigen receptor of the first aspect described above, the immunoresponsive cell of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the antibody or antigen-binding portion thereof of the seventh aspect, the cell population of the sixth aspect and/or the pharmaceutical composition of the eighth aspect.
Preferably, the method may be for therapeutic or non-therapeutic purposes. Wherein the therapeutic purpose includes, but is not limited to, clearing HIV cell depots or killing HIV latent cells, increasing or prolonging the survival of HIV-infected or AIDS patients. The non-therapeutic purpose includes but is not limited to the specific recognition of HIV envelope protein, and the specific recognition and activation of immune response cells by HIV latent cells.
In a twelfth aspect of the invention, there is provided a method of detection, said method comprising contacting a sample comprising one or more cells from said host with the chimeric antigen receptor of the first aspect described above, the immunoresponsive cell of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the antibody or antigen-binding portion thereof of the seventh aspect, the population of cells of the sixth aspect, and/or the kit of the tenth aspect, to form a complex, and detecting said complex, wherein detection of said complex is indicative of the presence of an HIV virus in said host.
The detection method of the present invention may be for therapeutic purposes or for non-therapeutic purposes. Wherein the therapeutic purpose includes, but is not limited to, diagnosing AIDS. Such non-therapeutic purposes include, but are not limited to, detecting the presence of the HIV virus, or for isolating the HIV virus.
In a thirteenth aspect of the invention, there is provided a method of increasing or prolonging survival of a subject infected with HIV or AIDS, said method comprising administering to the subject an effective amount of the immunoresponsive cell of the second aspect described above, thereby increasing or prolonging survival of the subject.
In a fourteenth aspect of the present invention, there is provided a method for clearing HIV cell depots, the method comprising administering an effective amount of the immunoresponsive cells of the second aspect described above to a subject infected with HIV or AIDS, thereby clearing HIV cell depots.
In a fifteenth aspect of the present invention, there is provided a method of specifically recognizing HIV envelope protein, said method comprising administering to a subject an effective amount of the immunoresponsive cell of the second aspect as described above.
In a sixteenth aspect of the present invention, there is provided a method of specifically recognizing and activating an immunoresponsive cell by an HIV latent cell, the method comprising administering to a subject an effective amount of the immunoresponsive cell of the second aspect described above.
In a seventeenth aspect, the present invention relates to a method of killing latent HIV cells, said method comprising administering to a subject an effective amount of the immunoresponsive cells of the second aspect as described above, thereby increasing or prolonging survival of the subject.
The method described in the present invention is a cellular immunotherapy of HIV CART.
The term "pharmaceutically acceptable carrier" as used herein refers to any formulation or carrier medium capable of delivering an effective amount of an active agent of the present invention, without interfering with the biological activity of the active agent, and without toxic side effects to the host or patient.
"homology" as used herein means that in the context of using a protein sequence or a nucleotide sequence, one skilled in the art can adjust the sequence as needed to obtain a sequence having (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology.
The term "and/or" as used herein is meant to encompass a list of items in the alternative, as well as any number of combinations of items.
The term "comprising" as used herein is intended to be open ended description that includes the recited specified ingredients or steps, as well as other specified ingredients or steps, that do not materially affect the performance of the specified composition or step.
"treating" as used herein refers to slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve complete elimination of all disease-related signs, symptoms, disorders, or diseases.
"diagnosis" as used herein refers to the determination of whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or the determination of the progression or likely progression of a disease in the future, or the assessment of a patient's response to a therapy.
An "effective amount" as described herein refers to an amount or dose of a CAR, CAR-T, nucleic acid molecule, vector, host cell, immunoresponsive cell, antibody, or antigen-binding portion thereof, population of cells, pharmaceutical composition of the invention that provides the desired treatment or prevention after administration to a patient or organ in a single or multiple doses. "preventing" as described herein refers to suppressing symptoms or delaying all the behavior of a particular symptom of stress by administering a CAR, CAR-T, nucleic acid molecule, vector, host cell, immunoresponsive cell, antibody, or antigen-binding portion thereof, population of cells, pharmaceutical composition, as described herein.
The term "subject" as used herein refers to a mammal or a human, preferably a primate.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: the results of binding of the 10-1074scFv constructed in example 1 to HIV latent cells are shown; flow-through results showed that 10-1074scFv (panel B) specifically recognized ACH2, although slightly weaker than the 10-1074 antibody (panel C), compared to the control group of Mers (panel A) antibodies.
FIG. 2: example 1 is shown for a procedure for the preparation of a 10-1074scFv-CAR, wherein the 10-1074scFv-CAR is specifically a 10-1074scFv-CD8-CD8-CD28-CD3 zeta-CAR, specifically a CAR consisting of a CD8 extracellular hinge region, a CD28 transmembrane region, a CD28-4-1BB cytoplasmic region and a CD3 ξ cytoplasmic region, and a 10-1074 scFv.
FIG. 3: a graph showing the results of the ability of 10-1074-CD28-4-1BB-CD3 ζ CAR to recognize binding to HIV envelope protein; the results show that 10-1074scFv-CAR specifically recognizes binding to HIV envelope protein gp140 compared to control FMC63 scFv-CAR.
FIG. 4A: it was shown that 10-1074-CD28-4-1BB-CD3 ζ -CART (test group) cells were efficiently specifically recognized and activated by HIV latent cells and produced IL2 cytokine, wherein the control group was CD19scFv-CD28-4-1BB-CD3 ζ -CART.
FIG. 4B: it was shown that 10-1074-CD28-4-1BB-CD3 ζ -CART (test group) cells were efficiently activated by specific recognition of HIV latent cells and produced TNF α cytokines, wherein the control group was CD19scFv-CD28-4-1BB-CD3 ζ -CART.
FIG. 4C: it was shown that 10-1074-CD28-4-1BB-CD3 ζ -CART (test group) cells were efficiently specifically recognized and activated by HIV latent cells and produced INF γ cytokines, wherein the control group was CD19scFv-CD28-4-1BB-CD3 ζ -CART.
FIG. 5: the effect of 10-1074-CD28-4-1BB-CD3 zeta-CART cells on the proportion of HIV positive cells ACH2 in the whole cell population is shown, and the results show that the 10-1074-CD28-4-1BB-CD3 zeta-CART cells can kill HIV latent cells specifically and effectively.
FIG. 6: the effect of 10-1074-CD28-4-1BB-CD3 zeta-CART (test group) cells and control group cells on P24 concentration is shown, which shows that 10-1074-CD28-4-1BB-CD3 zeta-CART cells can specifically and effectively reduce HIV virus level, wherein the control group is CD19scFv-CD28-4-1BB-CD3 zeta-CART.
FIG. 7: the effect of scFv-made CART from multiple neutralizing antibodies 3BNC117, N6 and 10-1074 (test group) versus control group cells at P24 concentrations under different effect target ratios is shown, indicating that 10-1074-CD28-4-1BB-CD3 ζ -CART cells can reduce HIV virus relatively most efficiently, where the control group is CD19scFv-CD28-4-1BB-CD3 ζ -CART.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.
Example 110 construction of 1074-CD28-4-1BB-CD3 xi-CAR
A variety of 10-1074scFv were prepared and CAR based on these scFv was generated as follows:
preparation of multiple 10-1074scFv
1. Based on the nucleotide sequences of 10-1074 (SEQ ID NO: 17), antibody heavy chain (VH) and light chain (VL) sequence synthesis was performed, and Overlap PCR primers were designed based on their sequences.
2. DNA polymerase is used for PCR amplification, 10-1074VL fragment (nucleotide sequence is SEQ ID NO: 19, amino acid sequence is SEQ ID NO: 2) and VH fragment (nucleotide sequence is SEQ ID NO: 18, amino acid sequence is SEQ ID NO: 3) are amplified, after the PCR product recovery and DNA agarose gel electrophoresis are carried out, whether the size of the PCR strip is correct is verified. Wherein, the primer used for amplifying the VH segment is a primer 10-1074-VH-F sequence: cattccgctagcCAGGTGCAGCTGCAGGAGTCAGGAC (SEQ ID NO: 20); primer op-10-1074-VH-VL-R sequence: CACTTCCGCCTCCACCAGATCCTCCACCTCCGCTAGACACTGTCACTGTTGT (SEQ ID NO: 21); the primer for amplifying the VL fragment is a primer op-10-1074-VH-VL-F sequence: TGGTGGAGGCGGAAGTGGCGGAGGGGGATCTAGCTATGTGCGACCTCTGAGCGTGG (SEQ ID NO: 22); primer 10-1074-VL-R sequence: tggtcatatgCAGCACTGTCAGTCTTGTGGCTCCTCCGA (SEQ ID NO: 23).
3. Performing Overlap PCR amplification by using the 10-1074VL and VH fragments which are correctly identified as templates and an Overlap PCR primer, performing DNA agarose gel electrophoresis verification after the Overlap PCR amplification is completed, and performing gel cutting recovery on the verified correct band to obtain 10-1074 scFv; wherein the sequence of a primer 10-1074-VH-F used for amplification is as follows: cattccgctagcCAGGTGCAGCTGCAGGAGTCAGGAC (SEQ ID NO: 24); primer 10-1074-VL-R sequence: tggtcatatgCAGCACTGTCAGTCTTGTGGCTCCTCCGA (SEQ ID NO: 25). 4. Performing double enzyme digestion on the 10-1074scFv subjected to gel recovery and the carrier with the Fc fragment (stored in a laboratory), performing DNA agarose gel electrophoresis verification after enzyme digestion, and verifying a correct band for gel recovery;
5. connecting the obtained 10-1074scFv target fragment and the carrier by using T4 ligase, transforming the target fragment into a DH5 alpha strain, and plating an ampicillin plate;
6. 3 single colonies growing on the plate are selected and sent to a company for sequencing by CMV-F, and a fragment with correct sequencing is a 10-1074scFv clone (the amino acid sequence of which is SEQ ID NO: 1 or SEQ ID NO: 10);
7. the specificity and the affinity of the prepared 10-1074scFv for binding HIV latent cells are verified;
8. and (3) expressing and purifying the his-tag-carrying 10-1074scFv, the 10-1074 antibody and the Mers antibody, respectively incubating the antibodies with activated HIV latent cell ACH2, and detecting whether the antibodies specifically bind to HIV latent cell ACH2 by anti-his PE antibody. The results of the experiment (see FIG. 1) show that 10-1074scFv specifically recognized ACH2 compared to the control Mers antibody, although this recognition was slightly weaker than that of the 10-1074 antibody.
Wherein, the linker connecting the VH segment and the VL segment is as follows:
linker sequence-1 (SEQ ID NO: 4): GGGGSGGGGSGGGGS
Linker sequence-2 (SEQ ID NO: 5): GGGGSGGGGS
Linker sequence-3 (SEQ ID NO: 6): GGGGSGGGGSGGGGSGGGGS
Linker sequence-4 (SEQ ID NO: 7): GGGGSGGGGSHMESKYGPPCPPCP
Linker sequence-5 (SEQ ID NO: 8): GGGGSGGGGSGGGGSHMESKYGPPCPPCP
Linker sequence-6 (SEQ ID NO: 9): GGGGSGGGGSGGGGSGGGGSHMESKYGPPCPPCP
Through experimental verification, the sequence shown in SEQ ID NO: 4-9 are also effective, but the Linker used in this experiment is the Linker of SEQ ID NO: 4.
(II) preparation of 10-1074scFv-CAR (exemplary use of nucleotide sequence SEQ ID NO: 17 encoding 10-1074scFv of SEQ ID NO: 1)
The preparation process and the structure diagram of the plasmid are shown in FIG. 2.
1. Overlap PCR primers were designed based on the nucleotide sequences of the CD8 IgG region, CD28 transmembrane region (TM), CD28-4-1BB cytoplasmic region, and CD3 ξ cytoplasmic region.
2. And performing PCR amplification by using DNA polymerase, respectively amplifying fragments of a CD8 IgG region, a CD28 transmembrane region, a CD28-4-1BB cytoplasmic region and a CD3 xi cytoplasmic region, and then performing PCR product recovery and DNA agarose gel electrophoresis to verify whether the size of a PCR band is correct or not.
3. Using the correctly identified CD8 IgG region, CD28 transmembrane region, CD28-4-1BB cytoplasmic region and CD3 xi cytoplasmic region fragment as a template, adopting an Overlap PCR primer to perform Overlap PCR amplification, performing DNA agarose gel electrophoresis verification after the Overlap PCR amplification is completed, verifying a correct band, performing gel cutting, and recovering to obtain a CD8-CD8TM-CD28-4-1BB-CD3 xi fragment;
4. carrying out double enzyme digestion on the 10-1074scFv fragment verified in the gel recovery step (I), the CD8-CD8TM-CD28-4-1BB-CD3 xi fragment and the lentivirus vector phase-IRES-RFP or phase-IRES-GFP (stored in a laboratory, used in the embodiment) at the same time, carrying out DNA agarose gel electrophoresis verification after enzyme digestion, and verifying a correct band for gel cutting recovery;
5. connecting the obtained enzyme digestion 10-1074scFv fragment with a CD8-CD8TM-CD28-4-1BB-CD3 xi fragment and a vector by using T4 ligase, converting the fragments into a stable 3 strain, and coating the ampicillin plate;
6. 3 single colonies growing on the plate are selected and sent to a company for sequencing, a fragment with correct sequencing is 10-1074-CAR, and an expression plasmid of the fragment is named as phage-10-1074 scFv-CAR;
wherein, the first and the second end of the pipe are connected with each other,
(1) the sequence of CD8-CD28TM-CD28-4-1BB-CD3 ξ is shown below (SEQ ID NO: 26):
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(2) the amino acid sequence of CD8LS-10-1074scFv-CD28-4-1BB-CD3 ξ CAR is shown below (SEQ ID NO: 11);
MGWSCIILFLVATATGVHSASQVQLQESGPGLVKPSETLSVTCSVSGDSMNNYYWTWIRQSPGKGLEWIGYISDRESATYNPSLNSRVVISRDTSKNQLSLKLNSVTPADTAVYYCATARRGQRIYGVVSFGEFFYYYSMDVWGKGTTVTVSSGGGGSGGGGSGGGGSSYVRPLSVALGETARISCGRQALGSRAVQWYQHRPGQAPILLIYNNQDRPSGIPERFSGTPDINFGTRATLTISGVEAGDEADYYCHMWDSRSGFSWSFGGATRLTVLHMTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(3) the nucleotide sequence of CD8LS-10-1074scFv-CD28-4-1BB-CD3 ξ CAR is shown below (SEQ ID NO: 12);
atgggatggtcatgtatcatcctttttctagtagcaactgcaaccggtgtacattccgctagcCAGGTGCAGCTGCAGGAGTCAGGACCAGGACTGGTGAAGCCTAGCGAGACACTGAGCGTGACTTGCAGCGTGTCCGGCGACAGCATGAACAACTACTATTGGACTTGGATCCGGCAGAGCCCAGGCAAAGGACTCGAGTGGATCGGCTACATCAGCGACAGGGAGAGCGCCACCTACAACCCTAGCCTGAACAGCAGGGTCGTGATCAGCAGGGACACCAGCAAGAACCAGCTGAGCCTGAAGCTGAACAGCGTGACCCCAGCCGATACCGCCGTGTACTATTGCGCCACAGCCAGAAGGGGCCAGAGAATCTACGGCGTGGTGTCCTTCGGCGAGTTCTTCTACTACTACAGCATGGACGTGTGGGGCAAGGGCACAACAGTGACAGTGTCTAGCGGAGGTGGAGGATCTGGTGGAGGCGGAAGTGGCGGAGGGGGATCTAGCTATGTGCGACCTCTGAGCGTGGCTCTGGGAGAGACAGCCAGGATCTCTTGCGGCAGACAGGCTCTGGGAAGCAGAGCAGTCCAGTGGTACCAGCACAGACCAGGACAGGCCCCTATCCTGCTGATCTACAACAACCAGGACCGGCCCAGCGGAATCCCAGAGAGATTCAGCGGCACCCCAGACATCAACTTCGGCACCAGAGCCACCCTGACAATTAGCGGCGTGGAAGCCGGAGACGAGGCCGACTACTATTGCCACATGTGGGATAGCAGGAGCGGCTTCTCTTGGAGCTTCGGAGGAGCCACAAGACTGACAGTGCTGcatatgaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggggcgggcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcTAA
(4) the nucleotide sequence of the phage-10-1074scFv-CAR is shown in SEQ ID NO: shown at 13.
Example 2 preparation of CAR-T cells (exemplified by the phage-10-1074scFv-CAR as described in SEQ ID NO: 13)
Slow virus package, concentration and titer detection
1.1 reviving and adjusting the state of 293T cells, and replating the 293T cells about 24 hours before transfection, wherein the cell fusion degree is more than 7 in packaging transfection;
1.2 plasmids psPAX2, pMD2.G required for transfection of the viral packaging system and the CAR-expressing lentiviral plasmid phage-10-1074scFv-CAR, the medium was changed to serum-free medium before transfection and the following system was performed for 15cm cell culture dishes:
60 mul PEI is added into 1ml DMEM dropwise and kept stand for 5 minutes,
another tube is taken and added
phage-10-1074scFv-CAR 15μg
psPAX2 10μg
pMD2.G 5μg
Adding DMEM to the solution to make the solution 1mL,
dropwise adding the PEI mixed solution into the packaging plasmid mixed solution, and standing for 20 minutes;
1.3, uniformly dripping the mixed solution into a cell culture medium to cover the cell culture medium on the surface of the cell, and putting the cell culture medium into an incubator;
after 1.410 hours the medium was discarded in the virus console and approximately 20mL of fresh medium was added;
collecting the upper culture medium after 1.52 days, and additionally supplementing 20mL of fresh culture medium; collecting the upper layer culture medium after another 1-2 days, and mixing the culture medium supernatants collected twice;
1.6 centrifuging the culture supernatant at 3000rpm for 10 minutes; filtering with 0.45 μm filter membrane, placing 36mL of filtered culture medium in an ultracentrifuge tube at 19500rpm for 2 hours;
1.7 carefully taking out the centrifugal tube, discarding the supernatant in a virus operating table, and inversely buckling the supernatant on paper sprayed with alcohol; adding 360 μ L1640 culture medium, and blowing the bottom block containing virus clump for at least 10 times; subpackaging the virus according to a certain volume, and quickly freezing and storing in a refrigerator at-80 ℃;
(II) isolation, activation and infection of human Primary T cells
2.1 separating fresh or frozen human blood with Ficoll liquid to obtain human PBMC, culturing with normal 1640 culture medium for 1 day;
2.2 day 2 suspension of cells in the above culture broth was transferred to the easy Sep from Stem cell TM Human T Cell Isolation Kit (Catalog #17951) states that screening negative to obtain Human primary T cells;
2.3 culturing the isolated human primary T cells in Normal 1640 Medium and adding ImmunoCult from Stem cell as described TM Human CD3/CD28/CD 2T Cell Activator (cat #10970) and IL2(Pepro Tech cat # 96-200-02-50), activated for 2 days;
2.4 infecting a number of the activated human primary T cells with lentivirus at an MOI of less than 20 and adding 8. mu.g/mL polybrene (Sigma cat # H9268) and mixing; centrifuging at room temperature of 1000-; placing the mixture in an incubator for 10 hours, and centrifuging and changing the solution;
2.5 cells were expanded and infection efficiency was measured after 3-4 days, as 10-1074-scFv CAR was constructed into the phage-IRES-GFP vector and therefore the infection efficiency was measured by the RFP tag carried by the vector itself. The gene expression localization of 10-1074-scFv CAR in human primary T cells was also examined (see example 3), and CAR-enriched positive T cells were screened for subsequent experiments.
Example 3 binding validation experiment for specific recognition of HIV envelope protein and 10-1074-CD28-4-1BB-CD3 zeta-CAR
To investigate whether the HIV envelope protein specifically recognizes the binding 10-1074-CD28-4-1BB-CD3 zeta-CAR, 10-1074-CD28-4-1BB-CD3 zeta-CAR-GFP was first transfected into 293T cells, 24 hours later, incubated with his-tagged HIV envelope protein gp140 followed by anti-his PE-tagged antibody, and the binding of HIV envelope protein to CAR-GFP positive cells was visualized by flow-through.
(I) cells and reagents
1. Cells for experiments: 293T cells, purchased from ATCC and cultured on the cell laboratory platform of the university of Qinghua.
2. Experimental HIV envelope proteins: cloning HIV envelope protein (CNE 54) gene from Chinese HIV infected person to eukaryotic vector, transferring into 293f cell for culture and production, and purifying with AKTA protein purification system.
3. Experimental anti-his PE-labeled antibody (cat # 130-.
(II) Experimental method
293T cells were plated into two wells of a six-well plate one day in advance, control FMC63scFv-CD28-4-1BB-CD3 zeta-CAR-GFP and test groups 10-1074-CD28-4-1BB-CD3 zeta-CAR-GFP were transfected into the cells, 293T was digested with TEN 24 hours later, the cells in each well were divided into two equal portions, one portion was incubated with HIV envelope protein, the other portion was incubated with HBV envelope protein for half an hour, centrifuged and washed three times with PBS, all cells were again incubated with anti-his PE labeled antibody for half an hour, centrifuged and washed three times with PBS, and examined by an up-flow analyzer.
(III) results of the experiment
The ability of 10-1074-CD28-4-1BB ζ CAR to localize to the cell membrane and specifically bind to the HIV membrane protein gp140 was tested by introducing 10-1074-scFv CAR-IRES-GFP and control FMC63-scFv CAR-IRES-GFP into human primary T cells via lentiviral vectors and incubating them with his-tagged HIV envelope protein BG 505. The results show (as in fig. 3) that 10-1074-scFv CAR can specifically recognize and bind to HIV tunica vesicularis protein BG505 compared to control FMC63-scFv CAR, and that nearly one percent of HIV tunica vesicularis protein can specifically bind to 10-1074-CD28-4-1 BB-3 ζ -CAR-GFP positive cell surface relative to control FMC63scFv-CD 28-4-BB-CD 3 ζ -CAR-GFP; whereas HBV envelope protein is unable to bind 10-1074-CD28-4-1BB-CD3 zeta CAR-GFP positive cells. Thus, HIV envelope protein specifically recognizes binding to 10-1074-CD28-4-1BB-CD3 ζ -CAR, and 10-1074-CD28-4-1BB-CD3 ζ -CAR does not recognize HBV envelope protein.
Example 4 validation of HIV-latent cells and 10-1074-CD28-4-1BB-CD3 zeta-CAR specific recognition activation
To investigate whether 10-1074-CD28-4-1BB-CD3 ζ -CART cells could be efficiently activated by HIV latent cell specific recognition, T cells were first isolated from human PBMCs, control group CD19scFv-CD28-4-1BB-CD3 ζ -CAR-RFP and test group 10-1074-CD28-4-1 BB-3 ζ -CAR-RFP were transformed into T cells after activation culture, purified control group CD19scFv-CD28-4-1BB-CD3 ζ CART cells and test group 10-1074CD28-4 ζ -1BB-CD 3-CART cells were obtained by sorting; the CART cells are co-cultured with HIV negative cells A3.01 or HIV positive cells ACH2 respectively, and after 24 hours, the ratio of INF gamma and TNF alpha positive CART cells and the levels of INF gamma, TNF alpha and IL2 secreted into the culture medium are detected.
1. Cells and reagents
(1) Cells for experiments: a3.01 and ACH2 cells, purchased from NIH AIDS reagent platform and cultured on cell experiment platform of Qinghua university; PBMCs were from cooperative hospitals.
(2) Reagents for experiments: human primary T cell isolation kit (cat #17951) and activation kit (cat #10970) were purchased from Stem cell, and cytokine IL2 (cat # 96-200-02-50) was purchased from Pepro Tech; flow antibodies TNF α (cat # 17-7349), INF γ (cat # 48-7319) and ELISA kits TNF α (cat # 88-7346-88), INF γ (cat # 88-7316-88) and IL2 (cat # 88-7025-88) were purchased from eBioscience.
2. Experimental methods
Spreading human PBMC cells into a 10cm culture dish, carrying out negative screening on suspension cells in the culture solution on day 2 according to the instruction of a T cell separation kit of Stem cells to obtain human primary T cells, and adding a T cell activator and IL2 according to the instruction; infecting the activated human primary T cells by using a certain amount of packaged lentivirus, and obtaining CART cells by flow sorting; the CART cells and HIV negative cells A3.01 or HIV positive cells ACH2 are co-cultured according to the proportion of 1:2 and 1:5 respectively, and after 24 hours, the ratio of INF gamma and TNF alpha positive CART cells and the levels of INF gamma, TNF alpha and IL2 secreted into a culture medium are detected.
3. Results of the experiment
The specific recognition and activation of 10-1074-CD28-4-1BB-CD3 zeta-CART cells by HIV latent cells are shown in FIGS. 4A, 4B and 4C, and the control group CD19scFv-CD28-4-1BB-CD3 zeta-CART can be activated by HIV negative cells A3.01 and HIV positive cells ACH2 to generate cytokines such as INF gamma and TNF alpha; the test group 10-1074-CD28-4-1BB-CD3 zeta-CART can only be activated by HIV positive cells ACH2 but not by HIV negative cells A3.01 to generate cytokines such as INF gamma and TNF alpha.
Example 5, 10-1074-CD28-4-1BB-CD3 ζ -CART cell validation experiment for effective killing of HIV latent cells
To investigate whether 10-1074-CD28-4-1BB-CD3 ζ -CART cells could kill HIV latent cells efficiently, T cells were first isolated from human PBMCs, CD19scFv-CD28-4-1BB-CD3 ζ -CAR-RFP and 10-1074-CD28-4-1BB-CD3 ζ -CAR-RFP were transformed into T cells after activation culture, and purified control group CD19scFv-CD28-4-1BB-CD3 ζ -CART cells and test group 10-1074-CD28-4-1BB-CD3 ζ -CART cells were obtained by sorting; the CART cells and HIV positive cells ACH2(GFP labeled) were co-cultured, and after 24 hours, the change in the ratio of HIV positive cells ACH2 in the whole cell population was examined.
1. Cells and reagents
(1) Cells for experiments: ACH2 cells, purchased from NIH AIDS reagent platform and cultured on cell laboratory platform of Qinghua university; PBMCs were from cooperative hospitals.
(2) Reagents for experiments: human primary T cell isolation kit (cat #17951) and activation kit (cat #10970) were purchased from Stem cell, and cytokine IL2 (cat # 96-200-02-50) was purchased from Pepro Tech; flow antibody CD4 was purchased from eBioscience.
2. Experimental method
Spreading human PBMC cells into a 10cm culture dish, carrying out negative screening on suspension cells in the culture solution on day 2 according to the instruction of a T cell separation kit of Stem cells to obtain human primary T cells, and adding a T cell activator and IL2 according to the instruction; infecting the activated human primary T cells by using a certain amount of packaged lentivirus, and obtaining CART cells by flow sorting; co-culturing the CART cell and HIV positive cell ACH2 at a ratio of 2:1, and detecting the change of the ratio of the HIV positive cell ACH2 in the whole cell population after 24 hours.
3. Results of the experiment
10-1074-CD28-4-1BB-CD3 zeta-CART cells can effectively kill HIV latent cells, as shown in FIG. 5, compared with the control group CD19scFv-CD28-4-1BB-CD3 zeta-CART, the proportion of ACH2 in the HIV positive cells co-cultured by the test group 10-1074-CD28-4-1BB-CD3 zeta-CART cells is obviously reduced, and the 10-1074-CD28-4-1BB-CD3 zeta-CART cells can effectively and specifically kill HIV latent cells.
Example 6, validation experiment of effective reduction of HIV Virus by 10-1074-CD28-4-1BB-CD3 ζ -CART cells
To investigate whether 10-1074-CD28-4-1BB-CD3 ζ -CART cells could effectively reduce HIV virus, T cells were first isolated from human PBMCs, CD19scFv-CD28-4-1BB-CD3 ζ -CAR-RFP and 10-1074-CD28-4-1BB-CD3 ζ -CAR-RFP were transformed into T cells after activation culture, and purified control group CD19scFv-CD28-4-1BB-CD3 ζ -CART cells and test group 10-1074-CD28-4-1BB-CD3 ζ -CART cells were obtained by sorting; the CART cell was co-cultured with HIV-negative cell A3.01 or HIV-positive cell ACH2(A3.01 was CD 4-positive and ACH2 was CD 4-negative), respectively, and after 24 hours, the change in the amount of HIV virus (using p24 as an index) in the supernatant of the culture medium was examined.
1. Cells and reagents
(1) Cells for experiments: a3.01 and ACH2 cells, purchased from NIH AIDS reagent platform and cultured on cell experiment platform of Qinghua university; PBMCs were from cooperative hospitals.
(2) Reagents for experiments: human primary T cell isolation kit (cat #17951) and activation kit (cat #10970) were purchased from Stem cell Co., Ltd., and cytokine IL2 (cat # 96-200-02-50) was purchased from Pepro Tech Co., Ltd.; flow antibody CD4 was purchased from eBioscience.
2. Experimental methods
Spreading human PBMC cells into a 10cm culture dish, carrying out negative screening on suspension cells in the culture solution on day 2 according to the instruction of a T cell separation kit of Stem cells to obtain human primary T cells, and adding a T cell activator and IL2 according to the instruction; infecting the activated human primary T cells by using a certain amount of packaged lentivirus, and obtaining CART cells by flow sorting; the CART cell and HIV negative cell A3.01 or HIV positive cell ACH2 are co-cultured according to the ratio of 2:1 respectively, and after 24 hours, the change of HIV virus amount (taking p24 as an index) in the culture medium supernatant is detected.
3. Results of the experiment
10-1074-CD28-4-1BB-CD3 zeta-CART cells were effective in reducing HIV virus As shown in FIG. 6, the co-culture of 10-1074-CD28-4-1BB-CD3 zeta-CART in the test group significantly reduced the level of p24 in the supernatant compared to the control group in the presence of HIV positive cells ACH 2.
Example 7, comparative experiment showing that 10-1074-CD28-4-1BB-CD3 ζ -CART cells are relatively most effective in reducing HIV virus
To investigate whether HIV CAR-T cells made of multiple neutralizing antibody derived scFv could effectively reduce HIV virus, T cells were first isolated from human PBMC, CD19scFv-CD28-4-1BB-CD3 zeta-CAR-RFP and various HIV-CD28-4-1BB-CD3 zeta-CAR-RFP were transformed into T cells after activation culture, and purified control group CD19scFv-CD28-4-1BB-CD3 zeta-CART cells and test group HIV-CD28-4-1BB-CD3 zeta-CART cells were obtained by sorting; the CART cell and HIV negative cell A3.01 or HIV positive cell ACH2(A3.01 is CD4 positive, and ACH2 is CD4 negative) are co-cultured respectively at different effective target ratios, and after 24 hours, the change of HIV virus amount (p 24 is used as an index) in culture medium supernatant is detected.
1. Cells and reagents
(1) Cells for experiments: a3.01 and ACH2 cells, purchased from NIH AIDS reagent platform and cultured on cell experiment platform of Qinghua university; PBMCs were from cooperative hospitals.
(2) Reagents for experiments: human primary T cell isolation kit (cat #17951) and activation kit (cat #10970) were purchased from Stem cell, and cytokine IL2 (cat # 96-200-02-50) was purchased from Pepro Tech; flow antibody CD4 was purchased from eBioscience.
2. Experimental methods
Spreading human PBMC cells into a 10cm culture dish, carrying out negative screening on suspension cells in the culture solution on day 2 according to the instruction of a T cell separation kit of Stem cells to obtain human primary T cells, and adding a T cell activator and IL2 according to the instruction; infecting the activated human primary T cells by using a certain amount of packaged lentivirus, and obtaining CART cells by flow sorting; the CART cell and HIV negative cell A3.01 or HIV positive cell ACH2 are co-cultured according to different proportions respectively, and after 24 hours, the change of HIV virus amount (taking p24 as an index) in culture medium supernatant is detected.
3. Results of the experiment
As shown in fig. 7, HIV CAR-T cells made from scFv of 3BNC117, N6, and 10-1074 neutralizing antibodies were all effective in down-regulating p24 levels compared to the control at different effective target ratios, and this down-regulation tended to increase with increasing effective target ratios. Wherein, under the effective target ratio condition, 10-1074-CD28-4-1BB-CD3 zeta-CART cells show stronger capability of down-regulating p24 level than HIV CAR-T cells made of other scFv neutralizing antibody.
In summary, 10-1074-CD28-4-1BB-CD3 zeta-CAR can specifically recognize HIV envelope protein, and 10-1074-CD28-4-1BB-CD3 zeta-CART cells can be relatively effectively and specifically recognized by HIV latent cells to activate and kill HIV latent cells and effectively reduce virus level.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.
Sequence listing
<110> Qinghua university
Beijing Huaxia Medical Treatment Technology Co.,Ltd.
<120> T cell containing chimeric antigen receptor modification and application thereof
<130> 1
<160> 26
<170> PatentIn version 3.5
<210> 1
<211> 255
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<213> Artificial Sequence (Artificial Sequence)
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Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met Asn Asn Tyr
20 25 30
Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu Asn
50 55 60
Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser Leu
65 70 75 80
Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly Glu
100 105 110
Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr Val
115 120 125
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr
145 150 155 160
Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln
165 170 175
Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn
180 185 190
Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro
195 200 205
Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu
210 215 220
Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser
225 230 235 240
Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu
245 250 255
<210> 2
<211> 108
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr Ala Arg Ile
1 5 10 15
Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln Trp Tyr Gln
20 25 30
His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn Asn Gln Asp
35 40 45
Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro Asp Ile Asn
50 55 60
Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly Asp
65 70 75 80
Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser Gly Phe Ser
85 90 95
Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu
100 105
<210> 3
<211> 132
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met Asn Asn Tyr
20 25 30
Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu Asn
50 55 60
Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser Leu
65 70 75 80
Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly Glu
100 105 110
Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr Val
115 120 125
Thr Val Ser Ser
130
<210> 4
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
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Gly Gly Gly Ser
20
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<211> 24
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Met Glu Ser Lys Tyr
1 5 10 15
Gly Pro Pro Cys Pro Pro Cys Pro
20
<210> 8
<211> 29
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His
1 5 10 15
Met Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
20 25
<210> 9
<211> 34
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
1 5 10 15
Gly Gly Gly Ser His Met Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
20 25 30
Cys Pro
<210> 10
<211> 255
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp Ser Met Asn Asn Tyr
20 25 30
Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr Asn Pro Ser Leu Asn
50 55 60
Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys Asn Gln Leu Ser Leu
65 70 75 80
Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val Val Ser Phe Gly Glu
100 105 110
Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly Lys Gly Thr Thr Val
115 120 125
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
Gly Gly Ser Ser Tyr Val Arg Pro Leu Ser Val Ala Leu Gly Glu Thr
145 150 155 160
Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly Ser Arg Ala Val Gln
165 170 175
Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr Asn
180 185 190
Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Thr Pro
195 200 205
Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr Ile Ser Gly Val Glu
210 215 220
Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met Trp Asp Ser Arg Ser
225 230 235 240
Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg Leu Thr Val Leu
245 250 255
<210> 11
<211> 545
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Ala Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
20 25 30
Val Lys Pro Ser Glu Thr Leu Ser Val Thr Cys Ser Val Ser Gly Asp
35 40 45
Ser Met Asn Asn Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys
50 55 60
Gly Leu Glu Trp Ile Gly Tyr Ile Ser Asp Arg Glu Ser Ala Thr Tyr
65 70 75 80
Asn Pro Ser Leu Asn Ser Arg Val Val Ile Ser Arg Asp Thr Ser Lys
85 90 95
Asn Gln Leu Ser Leu Lys Leu Asn Ser Val Thr Pro Ala Asp Thr Ala
100 105 110
Val Tyr Tyr Cys Ala Thr Ala Arg Arg Gly Gln Arg Ile Tyr Gly Val
115 120 125
Val Ser Phe Gly Glu Phe Phe Tyr Tyr Tyr Ser Met Asp Val Trp Gly
130 135 140
Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Gly Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val Arg Pro Leu Ser Val
165 170 175
Ala Leu Gly Glu Thr Ala Arg Ile Ser Cys Gly Arg Gln Ala Leu Gly
180 185 190
Ser Arg Ala Val Gln Trp Tyr Gln His Arg Pro Gly Gln Ala Pro Ile
195 200 205
Leu Leu Ile Tyr Asn Asn Gln Asp Arg Pro Ser Gly Ile Pro Glu Arg
210 215 220
Phe Ser Gly Thr Pro Asp Ile Asn Phe Gly Thr Arg Ala Thr Leu Thr
225 230 235 240
Ile Ser Gly Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys His Met
245 250 255
Trp Asp Ser Arg Ser Gly Phe Ser Trp Ser Phe Gly Gly Ala Thr Arg
260 265 270
Leu Thr Val Leu His Met Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
275 280 285
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
290 295 300
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
305 310 315 320
Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
325 330 335
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
340 345 350
Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg
355 360 365
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
370 375 380
Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr
385 390 395 400
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
405 410 415
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
420 425 430
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
435 440 445
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
450 455 460
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
465 470 475 480
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
485 490 495
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
500 505 510
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
515 520 525
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
530 535 540
Arg
545
<210> 12
<211> 1638
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattccgct 60
agccaggtgc agctgcagga gtcaggacca ggactggtga agcctagcga gacactgagc 120
gtgacttgca gcgtgtccgg cgacagcatg aacaactact attggacttg gatccggcag 180
agcccaggca aaggactcga gtggatcggc tacatcagcg acagggagag cgccacctac 240
aaccctagcc tgaacagcag ggtcgtgatc agcagggaca ccagcaagaa ccagctgagc 300
ctgaagctga acagcgtgac cccagccgat accgccgtgt actattgcgc cacagccaga 360
aggggccaga gaatctacgg cgtggtgtcc ttcggcgagt tcttctacta ctacagcatg 420
gacgtgtggg gcaagggcac aacagtgaca gtgtctagcg gaggtggagg atctggtgga 480
ggcggaagtg gcggaggggg atctagctat gtgcgacctc tgagcgtggc tctgggagag 540
acagccagga tctcttgcgg cagacaggct ctgggaagca gagcagtcca gtggtaccag 600
cacagaccag gacaggcccc tatcctgctg atctacaaca accaggaccg gcccagcgga 660
atcccagaga gattcagcgg caccccagac atcaacttcg gcaccagagc caccctgaca 720
attagcggcg tggaagccgg agacgaggcc gactactatt gccacatgtg ggatagcagg 780
agcggcttct cttggagctt cggaggagcc acaagactga cagtgctgca tatgaccacg 840
acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 900
cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 960
gcctgtgatt tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta tagcttgcta 1020
gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcagggg cgggcacagt 1080
gactacatga acatgactcc ccgccgcccc gggcccaccc gcaagcatta ccagccctat 1140
gccccaccac gcgacttcgc agcctatcgc tccaaacggg gcagaaagaa actcctgtat 1200
atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1260
tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1320
gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1380
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1440
aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1500
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1560
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1620
gccctgcccc ctcgctaa 1638
<210> 13
<211> 8687
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg 2040
gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 2100
agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 2160
aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtcgt 2220
gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg 2280
ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa 2340
gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 2400
cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg 2460
ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa 2520
ttacttccac gcccctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt 2580
gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag 2640
gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc 2700
gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt 2760
ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt 2820
ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc 2880
ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg 2940
gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg 3000
gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa 3060
tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc 3120
tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac 3180
ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat 3240
gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg 3300
atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct 3360
cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgagcggc cgcggatccg 3420
ccaccatggg atggtcatgt atcatccttt ttctagtagc aactgcaacc ggtgtacatt 3480
ccgctagcca ggtgcagctg caggagtcag gaccaggact ggtgaagcct agcgagacac 3540
tgagcgtgac ttgcagcgtg tccggcgaca gcatgaacaa ctactattgg acttggatcc 3600
ggcagagccc aggcaaagga ctcgagtgga tcggctacat cagcgacagg gagagcgcca 3660
cctacaaccc tagcctgaac agcagggtcg tgatcagcag ggacaccagc aagaaccagc 3720
tgagcctgaa gctgaacagc gtgaccccag ccgataccgc cgtgtactat tgcgccacag 3780
ccagaagggg ccagagaatc tacggcgtgg tgtccttcgg cgagttcttc tactactaca 3840
gcatggacgt gtggggcaag ggcacaacag tgacagtgtc tagcggaggt ggaggatctg 3900
gtggaggcgg aagtggcgga gggggatcta gctatgtgcg acctctgagc gtggctctgg 3960
gagagacagc caggatctct tgcggcagac aggctctggg aagcagagca gtccagtggt 4020
accagcacag accaggacag gcccctatcc tgctgatcta caacaaccag gaccggccca 4080
gcggaatccc agagagattc agcggcaccc cagacatcaa cttcggcacc agagccaccc 4140
tgacaattag cggcgtggaa gccggagacg aggccgacta ctattgccac atgtgggata 4200
gcaggagcgg cttctcttgg agcttcggag gagccacaag actgacagtg ctgcatatga 4260
ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg cagcccctgt 4320
ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg agggggctgg 4380
acttcgcctg tgatttttgg gtgctggtgg tggttggtgg agtcctggct tgctatagct 4440
tgctagtaac agtggccttt attattttct gggtgaggag taagaggagc aggggcgggc 4500
acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag cattaccagc 4560
cctatgcccc accacgcgac ttcgcagcct atcgctccaa acggggcaga aagaaactcc 4620
tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag gaagatggct 4680
gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg aagttcagca 4740
ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac gagctcaatc 4800
taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac cctgagatgg 4860
ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg cagaaagata 4920
agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg ggcaaggggc 4980
acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac gcccttcaca 5040
tgcaggccct gccccctcgc taaatcgata gatcctaatc aacctctgga ttacaaaatt 5100
tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct 5160
gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg 5220
tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc 5280
gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt 5340
cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc 5400
gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg 5460
ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg 5520
cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc 5580
ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg 5640
atctcccttt gggccgcctc cccgcctgag atcctttaag accaatgact tacaaggcag 5700
ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc 5760
aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag 5820
cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt 5880
gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca 5940
gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc 6000
agtatttata acttgcaaag aaatgaatat cagagagtga gaggcccggg ttaattaagg 6060
aaagggctag atcattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt 6120
aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 6180
ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 6240
taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 6300
cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 6360
acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 6420
ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 6480
atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa 6540
gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 6600
acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 6660
atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 6720
accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 6780
ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 6840
acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 6900
gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 6960
tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 7020
ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 7080
actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 7140
taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 7200
tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 7260
gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 7320
cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 7380
gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 7440
gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 7500
tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 7560
ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 7620
cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 7680
gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 7740
gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 7800
gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 7860
cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 7920
tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 7980
cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 8040
cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 8100
ccgcctctcc ccgcgcgttg gccgattcat taatgcagca agctcatggc tgactaattt 8160
tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag 8220
gaggcttttt tggaggccta ggcttttgca aaaagctccc cgtggcacga caggtttccc 8280
gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 8340
ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 8400
caatttcaca caggaaacag ctatgacatg attacgaatt tcacaaataa agcatttttt 8460
tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 8520
aactggataa ctcaagctaa ccaaaatcat cccaaacttc ccaccccata ccctattacc 8580
actgccaatt acctgtggtt tcatttactc taaacctgtg attcctctga attattttca 8640
ttttaaagaa attgtatttg ttaaatatgt actacaaact tagtagt 8687
<210> 14
<211> 27
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 15
<211> 45
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 16
<211> 195
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu
35 40 45
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
50 55 60
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
65 70 75 80
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
85 90 95
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
100 105 110
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
115 120 125
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
130 135 140
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
145 150 155 160
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
165 170 175
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
180 185 190
Pro Pro Arg
195
<210> 17
<211> 765
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
caggtgcagc tgcaggagtc aggaccagga ctggtgaagc ctagcgagac actgagcgtg 60
acttgcagcg tgtccggcga cagcatgaac aactactatt ggacttggat ccggcagagc 120
ccaggcaaag gactcgagtg gatcggctac atcagcgaca gggagagcgc cacctacaac 180
cctagcctga acagcagggt cgtgatcagc agggacacca gcaagaacca gctgagcctg 240
aagctgaaca gcgtgacccc agccgatacc gccgtgtact attgcgccac agccagaagg 300
ggccagagaa tctacggcgt ggtgtccttc ggcgagttct tctactacta cagcatggac 360
gtgtggggca agggcacaac agtgacagtg tctagcggag gtggaggatc tggtggaggc 420
ggaagtggcg gagggggatc tagctatgtg cgacctctga gcgtggctct gggagagaca 480
gccaggatct cttgcggcag acaggctctg ggaagcagag cagtccagtg gtaccagcac 540
agaccaggac aggcccctat cctgctgatc tacaacaacc aggaccggcc cagcggaatc 600
ccagagagat tcagcggcac cccagacatc aacttcggca ccagagccac cctgacaatt 660
agcggcgtgg aagccggaga cgaggccgac tactattgcc acatgtggga tagcaggagc 720
ggcttctctt ggagcttcgg aggagccaca agactgacag tgctg 765
<210> 18
<211> 396
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
caggtgcagc tgcaggagtc aggaccagga ctggtgaagc ctagcgagac actgagcgtg 60
acttgcagcg tgtccggcga cagcatgaac aactactatt ggacttggat ccggcagagc 120
ccaggcaaag gactcgagtg gatcggctac atcagcgaca gggagagcgc cacctacaac 180
cctagcctga acagcagggt cgtgatcagc agggacacca gcaagaacca gctgagcctg 240
aagctgaaca gcgtgacccc agccgatacc gccgtgtact attgcgccac agccagaagg 300
ggccagagaa tctacggcgt ggtgtccttc ggcgagttct tctactacta cagcatggac 360
gtgtggggca agggcacaac agtgacagtg tctagc 396
<210> 19
<211> 324
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
agctatgtgc gacctctgag cgtggctctg ggagagacag ccaggatctc ttgcggcaga 60
caggctctgg gaagcagagc agtccagtgg taccagcaca gaccaggaca ggcccctatc 120
ctgctgatct acaacaacca ggaccggccc agcggaatcc cagagagatt cagcggcacc 180
ccagacatca acttcggcac cagagccacc ctgacaatta gcggcgtgga agccggagac 240
gaggccgact actattgcca catgtgggat agcaggagcg gcttctcttg gagcttcgga 300
ggagccacaa gactgacagt gctg 324
<210> 20
<211> 37
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cattccgcta gccaggtgca gctgcaggag tcaggac 37
<210> 21
<211> 52
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cacttccgcc tccaccagat cctccacctc cgctagacac tgtcactgtt gt 52
<210> 22
<211> 56
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tggtggaggc ggaagtggcg gagggggatc tagctatgtg cgacctctga gcgtgg 56
<210> 23
<211> 39
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tggtcatatg cagcactgtc agtcttgtgg ctcctccga 39
<210> 24
<211> 37
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cattccgcta gccaggtgca gctgcaggag tcaggac 37
<210> 25
<211> 39
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tggtcatatg cagcactgtc agtcttgtgg ctcctccga 39
<210> 26
<211> 267
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val
35 40 45
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
50 55 60
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly
65 70 75 80
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
85 90 95
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
100 105 110
Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
115 120 125
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
130 135 140
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
145 150 155 160
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
165 170 175
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
180 185 190
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
195 200 205
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
210 215 220
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
225 230 235 240
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
245 250 255
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
260 265

Claims (4)

1. Use of a chimeric antigen receptor, an immunoresponsive cell comprising a chimeric antigen receptor, a nucleic acid molecule encoding a chimeric antigen receptor, or a host cell or population comprising said nucleic acid molecule, in the manufacture of a medicament for treating an HIV infection or AIDS, wherein said chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane region, an extracellular hinge region, and a cytoplasmic region;
the extracellular antigen binding domain is a human single-chain antibody of 10-1074, and the amino acid sequence of the human single-chain antibody is shown in SEQ ID NO: 1 or SEQ ID NO: 10 is shown in the figure;
the transmembrane region is a CD28 transmembrane region;
the extracellular hinge region is a CD8 polypeptide;
the cytoplasmic region is CD28-4-1BB cytoplasmic region and CD3 zeta cytoplasmic region.
2. The use according to claim 1, wherein the chimeric antigen receptor has an amino acid sequence as set forth in SEQ ID NO: shown at 11.
3. The use according to claim 1, wherein said nucleic acid molecule encoding a chimeric antigen receptor is as set forth in SEQ ID NO: shown at 12.
4. The use of claim 1, wherein said population of cells comprises at least 1 host cell comprising a nucleic acid molecule encoding a chimeric antigen receptor.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104918658A (en) * 2012-10-18 2015-09-16 洛克菲勒大学 Broadly-neutralizing anti-hiv antibodies
WO2019112347A2 (en) * 2017-12-06 2019-06-13 앱클론(주) Antibody or antigen binding fragment thereof for specifically recognizing b cell malignancy, chimeric antigen receptor comprising same, and use thereof
CN110305219A (en) * 2018-03-27 2019-10-08 清华大学 One kind preparing the purposes in cell drug comprising the T cell of Chimeric antigen receptor (CAR) modification

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104918658A (en) * 2012-10-18 2015-09-16 洛克菲勒大学 Broadly-neutralizing anti-hiv antibodies
WO2019112347A2 (en) * 2017-12-06 2019-06-13 앱클론(주) Antibody or antigen binding fragment thereof for specifically recognizing b cell malignancy, chimeric antigen receptor comprising same, and use thereof
CN110305219A (en) * 2018-03-27 2019-10-08 清华大学 One kind preparing the purposes in cell drug comprising the T cell of Chimeric antigen receptor (CAR) modification

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HIV-1-Specific Chimeric Antigen Receptors Based on Broadly Neutralizing Antibodies;Ayub Ali等;《Journal of Virology》;20160525;第90卷(第15期);摘要，Fig. 1，Table 1，第7000页左栏第4段至右栏第2段 *

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