CN110305219B

CN110305219B - Use of T cell comprising Chimeric Antigen Receptor (CAR) modification for preparing cell medicament

Info

Publication number: CN110305219B
Application number: CN201810257952.XA
Authority: CN
Inventors: 赵学强; 张林琦; 林欣; 丁志超
Original assignee: Beijing Huaxia Medical Treatment Technology Co ltd; Tsinghua University
Current assignee: Beijing Huaxia Medical Treatment Technology Co ltd; Tsinghua University
Priority date: 2018-03-27
Filing date: 2018-03-27
Publication date: 2021-05-04
Anticipated expiration: 2038-03-27
Also published as: CN110305219A

Abstract

The invention discloses a T cell containing a Chimeric Antigen Receptor (CAR) modification, a preparation or a medicament prepared from the T cell, and application of the T cell in preparing a cell medicament, wherein the preparation or the medicament is used for treating and eliminating a cell reservoir of Human Immunodeficiency Virus (HIV) in an HIV-infected patient. The invention further discloses a cell immunotherapy adopting gp120 antibody CART, in particular to 3BNC117 scFv-CART, so as to realize specific MHC independent recognition and killing of HIV latent cells, thereby achieving the purpose of eradicating AIDS.

Description

Use of T cell comprising Chimeric Antigen Receptor (CAR) modification for preparing cell medicament

Technical Field

The invention relates to the field of biomedicine, in particular to application of a T cell containing a Chimeric Antigen Receptor (CAR) modification in preparation of a cell drug.

Background

Human Immunodeficiency Virus (HIV), an Acquired Immune Deficiency Syndrome (AIDS) virus, is a Lentivirus (Lentivirus) 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 patients is mainly a highly active antiretroviral therapy (HAART) consisting of a combination of antiviral drugs (cART). 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 results in the presence of a larger number of HIV carriers who are totally dependent on antiretroviral drugs and have to bear the severe side effects and economic burden they bring;

(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 HIV cell repositories. Finding a well-defined biomarker (biomarker) for HIV cell depots allows accurate localization and eventually clearance of HIV cell depots.

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. The research on the function of the CAR-T cells in clearing HIV cell repositories and the elucidation of related mechanisms has important fundamental research significance and clinical application value.

Disclosure of Invention

The inventor finds that the CAR with the scFv-41BBCD3 zeta structure can effectively activate T cell signals to kill target cells through a series of previous researches; the 3BNC117 has stronger characteristic of being specifically combined with HIV envelope protein in various human-derived HIV antibodies, the 3BNC117 prepared by the normal 3BNC117 antibody in various scFv forms with VL-VH sequence has stronger characteristic of being specifically combined with HIV envelope protein, and the prepared 3BNC117-41BBCD3 zeta CAR can specifically recognize HIV envelope protein or HIV latent cells; further, the inventor finds that the prepared 3BNC117-41BBCD3 zeta-CART cells can be effectively and specifically recognized and activated by HIV latent cells and have killing capacity. This suggests that 3BNC117-41BBCD3 ζ -CART cell drugs can be used to clear the cellular stores of Human Immunodeficiency Virus (HIV) in HIV-infected patients for the purpose of treating AIDS. Thus, the invention provides the use of 3BNC117-41BBCD3 zeta-CART in the preparation of a preparation or a medicament. According to an embodiment of the invention, the medicament is for the purpose of eliminating the cellular reservoir of Human Immunodeficiency Virus (HIV) in HIV-infected patients for the purpose of treating AIDS.

The use of 3BNC117-41BBCD3 ζ -CART in the preparation of a medicament according to the above embodiment of the invention may also have the following additional technical features:

according to an embodiment of the invention, the 3BNC117-41BBCD3 ζ CAR can specifically recognize HIV envelope protein;

according to the embodiment of the invention, the 3BNC117-41BBCD3 zeta-CART cells can be effectively and specifically recognized and activated by HIV latent cells;

according to the embodiment of the invention, the 3BNC117-41BBCD3 zeta-CART cells can kill HIV latent cells specifically and effectively.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

In particular, in a first aspect, the present invention relates to a Chimeric Antigen Receptor (CAR) comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain, characterized in that said extracellular antigen-binding domain is a human single chain antibody (scFv) of 3BNC 117.

Preferably, the Chimeric Antigen Receptor (CAR) further comprises an extracellular hinge region.

Specifically, the sequence of the human single-chain antibody (scFv) of the 3BNC117 is shown as SEQ ID NO. 1 or SEQ ID NO. 10.

In a second aspect, the present invention relates to a Chimeric Antigen Receptor (CAR) according to the first aspect, wherein the human single chain antibody (scFv) comprises a heavy chain variable region, a Linker (Linker) and a light chain variable region, wherein the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology with the amino acid sequence of SEQ ID NO. 3, and the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology with the amino acid sequence of SEQ ID NO. 2.

In a third aspect, the invention relates to a Chimeric Antigen Receptor (CAR) according to the second aspect, characterized in that the sequence of the Linker (Linker) is an amino acid sequence comprising glycine (G) and serine (Ser).

A fourth aspect of the invention relates to a Chimeric Antigen Receptor (CAR) according to the second or third aspect, characterized in that the Linker (Linker) has the sequence shown in any one of SEQ ID NOS: 4-7.

In a fifth aspect the invention relates to a Chimeric Antigen Receptor (CAR) according to any of the first to third aspects, wherein the transmembrane domain comprises one or more combinations of a CD3 ζ polypeptide, a CD4 polypeptide, a CD8 polypeptide, a CD28 polypeptide, a 4-11B polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CRLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide.

In a sixth aspect, the present invention relates to a Chimeric Antigen Receptor (CAR) according to the fifth aspect, wherein said transmembrane domain comprises the group consisting of:

(1) CD8a polypeptide, CD28 polypeptide, and CD3 ζ polypeptide;

(2) CD3 ζ polypeptide, CD4 polypeptide, CD8 polypeptide, CD28 polypeptide, and 41BB polypeptide, and CD3 ζ polypeptide;

(3) CD3 ζ polypeptide, CD4 polypeptide, CD8 polypeptide, CD28 polypeptide, and 41BB polypeptide, and CD3 ζ polypeptide.

Preferably, the sequence of the transmembrane domain is shown as SEQ ID NO. 9.

In a seventh aspect, the invention relates to a Chimeric Antigen Receptor (CAR) according to any of the preceding claims, wherein said extracellular hinge region is a CD8 or IgG extracellular hinge region.

An eighth aspect of the invention relates to a Chimeric Antigen Receptor (CAR) according to any of the preceding claims, wherein said intracellular domain is 41BBCD3 ζ or CD28CD3 ζ.

Preferably, the encoding nucleotide sequence of the Chimeric Antigen Receptor (CAR) is shown as SEQ ID NO. 12, or the amino acid sequence thereof is shown as SEQ ID NO. 13.

In a ninth aspect, the present invention relates to an isolated immunoresponsive cell comprising the Chimeric Antigen Receptor (CAR) of any one of the first to eight aspects.

In a tenth aspect, the present invention relates to the immunoresponsive cell according to the ninth aspect, wherein 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 a T cell precursor cell.

An eleventh aspect of the invention relates to an isolated nucleic acid molecule encoding the Chimeric Antigen Receptor (CAR) of any one of the first to eight aspects.

In a twelfth aspect, the present invention relates to the nucleic acid molecule according to the eleventh aspect, wherein the sequence of the nucleic acid molecule is as follows:

(1) is a nucleotide sequence comprising amino acids shown as SEQ ID NO 1, SEQ ID NO 10 or SEQ ID NO 11;

(2) is a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology with the nucleotide sequence of SEQ ID NO. 12;

or (3) is a nucleotide sequence encoding at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% homology with the amino acid sequence of SEQ ID NO. 13.

In a thirteenth aspect, the present invention relates to a vector comprising the isolated nucleic acid molecule of any one of the eleventh or twelfth aspects.

Preferably, the sequence of the vector is shown as SEQ ID NO. 14.

In a fourteenth aspect, the present invention relates to a host cell expressing the isolated nucleic acid molecule of any one of the eleventh or twelfth aspects or comprising the vector of the thirteenth aspect.

In a fifteenth aspect, the present invention relates to the host cell of the fourteenth aspect, wherein the host cell is a T cell.

In a sixteenth aspect, the present invention relates to a pharmaceutical composition comprising an effective amount of the immunoresponsive cell of any one of the ninth to tenth aspects.

A seventeenth aspect of the present invention relates to the pharmaceutical composition according to the sixteenth aspect, wherein the pharmaceutical composition is for use in the treatment of HIV infection or AIDS.

An eighteenth aspect of the invention relates to the use of a Chimeric Antigen Receptor (CAR) according to any one of the first to eight aspects, an immunoresponsive cell according to any one of the ninth to tenth aspects, a nucleic acid molecule according to the eleventh to twelfth aspects, a vector according to the thirteenth aspect, a host cell according to any one of the fourteenth to fifteenth aspects and/or a pharmaceutical composition according to any one of the sixteenth to seventeenth aspects in the manufacture of a medicament for the treatment of an HIV infection or AIDS.

A nineteenth aspect of the invention relates to a kit for treating HIV infection or AIDS, comprising the Chimeric Antigen Receptor (CAR) of any one of the first to eight aspects, the immunoresponsive cell of any one of the ninth to tenth aspects, the nucleic acid molecule of the eleventh to twelfth aspects, the vector of the thirteenth aspect, the host cell of any one of the fourteenth to fifteenth aspects, and/or the pharmaceutical composition of any one of the sixteenth to seventeenth aspects.

In a twentieth aspect, the present invention relates to a method for increasing or prolonging the survival of a patient infected with HIV or AIDS, which comprises administering to the subject an effective amount of the immunoresponsive cells of the ninth to tenth aspects, thereby increasing or prolonging the survival of the subject.

In a twenty-first aspect of the present invention, there is provided a method for clearing HIV cell depots, characterized by administering an effective amount of the immunoresponsive cells described in the ninth to tenth aspects to a subject infected with HIV or AIDS, thereby clearing the HIV cell depots.

In a twenty-second aspect, the present invention relates to a method for specifically recognizing HIV envelope protein, characterized in that an effective amount of the immunoresponsive cell according to the ninth to tenth aspects is administered to a subject.

In a twenty-third aspect of the present invention, there is provided a method for specifically recognizing and activating an immunoresponsive cell by an HIV latent cell, characterized by administering an effective amount of the immunoresponsive cell according to the ninth to tenth aspects to a subject.

In a twenty-fourth aspect, the present invention relates to a method of killing HIV latent cells, characterized in that an effective amount of the immunoresponsive cells of the ninth to tenth aspects is administered to a subject, thereby increasing or prolonging the survival of the subject.

In a twenty-fifth aspect the invention relates to a population of cells comprising a host cell according to any one of the preceding claims.

A twenty-sixth aspect of the invention relates to an antibody, or antigen-binding portion thereof, that specifically binds to the aforementioned CAR. And to pharmaceutical compositions of the aforementioned cell populations, antibodies or antigen-binding portions thereof.

A twenty-seventh aspect of the present invention relates to a kit for the treatment of HIV infection or AIDS, characterized in that it comprises a Chimeric Antigen Receptor (CAR) of any of the preceding, an immunoresponsive cell, a nucleic acid molecule, a vector, a host cell, a population of cells, an antibody or antigen-binding portion thereof, and/or a pharmaceutical composition of any of the preceding.

In a twenty-eighth aspect, the present invention is directed to a method of detecting the presence of an HIV virus in a host, the method comprising: (a) contacting a sample comprising one or more cells from the host with a Chimeric Antigen Receptor (CAR) of any one of the preceding, an immunoresponsive cell of any one of the preceding, a nucleic acid molecule of any one of the preceding, a vector of any one of the preceding, a host cell of any one of the preceding, a population of cells of any one of the preceding, an antibody or antigen-binding portion thereof of any one of the preceding, and/or a kit of any one of the preceding, thereby forming a complex, and (b) detecting the complex, wherein detection of the complex is indicative of the presence of an HIV virus in the host.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows graphs showing the results of binding of 3BNC117scFv to HIV latent cells constructed according to an embodiment of the present invention; the results show that 3BNC117scFv specifically recognizes ACH2 compared to the control Mers antibody, although this recognition ability is slightly weaker than the 3BNC117 antibody.

FIG. 2 shows a flow and schematic diagram for the preparation of 3BNC117scFv-CAR according to an embodiment of the invention; the control to which the present invention relates is the use of the same structure of FMC63scFv-CAR against CD 19.

FIG. 3 shows a graph of the results of the ability of 3BNC117-41BBCD3 ζ CAR to recognize binding to HIV envelope proteins according to embodiments of the present invention; the results show that 3BNC117scFv-CAR specifically recognizes binding to HIV capsular protein BG505 compared to control FMC63 scFv-CAR.

FIG. 4 shows a graph of the results of lentiviral vector mediated infection of human primary T cells with 3BNC117scFv-CAR according to an embodiment of the invention; the results show that 3BNC117scFv-CAR infects human primary T cells with an efficiency of about 60%.

FIG. 5 is a graph showing the results that 3BNC117-41BBCD3 ζ -CART cells can be effectively activated by specific recognition of HIV latent cells according to an embodiment of the present invention;

FIG. 6 is a graph showing the results that 3BNC117-41BBCD3 ζ -CART cells can kill HIV latent cells specifically and effectively according to an embodiment of the present invention.

Detailed Description

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.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the assays, screens, and treatments of the present invention can be made and used, and it is to be understood that these examples are intended to be illustrative only and are not to be construed as limiting the invention.

Example 1, 3 construction of BNC117-41BBCD3 ξ -CAR

Various 3BNC117 scfvs were prepared and CARs based on these scfvs were generated as follows:

(one) preparation of multiple 3BNC117scFv

1. Based on the nucleotide sequence of 3BNC117, antibody heavy chain (VH) and light chain (VL) sequence synthesis was performed, and Overlap PCR primers were designed based on their sequences.

2. And performing PCR amplification by using DNA polymerase, amplifying the VL fragment and the VH fragment of the 3BNC117, and performing PCR product recovery and DNA agarose gel electrophoresis to verify whether the size of the PCR band is correct or not.

3. Performing Overlap PCR amplification by using the 3BNC117VL and the VH fragment 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 a verified correct band to obtain 3BNC117 scFv;

4. carrying out double enzyme digestion on the 3BNC117scFv subjected to gel recovery and the carrier with the Fc fragment (stored in a laboratory), carrying out DNA agarose gel electrophoresis verification after enzyme digestion, and verifying a correct band for gel recovery;

5. connecting the obtained 3BNC117scFv target fragment subjected to enzyme digestion with a vector by using T4 ligase, converting the obtained product into a DH5 alpha strain, and coating a plate on an ampicillin plate;

6. selecting 3 single colonies growing on the plate, sending to a company for sequencing by using CMV-F, wherein a fragment with correct sequencing is 3BNC117scFv clone;

7. verifying the specificity and affinity of the prepared 3BNC117scFv for binding HIV latent cells;

8. 3BNC117scFv, 3BNC117 antibody and Mers antibody with his tag are expressed and purified, and are respectively incubated with activated HIV latent cell ACH2, and whether the antibodies specifically bind with HIV latent cell ACH2 is detected by anti-his PE antibody. The results of the experiment (see figure 1) show that 3BNC117scFv specifically recognizes ACH2 compared to the control Mers antibody, although this recognition ability is slightly weaker than that of the 3BNC117 antibody.

Wherein the 3BNC117scFv sequence comprises:

(1)3BNC117scFv-1(SEQ ID NO：1)

DIQMTQSPSSLSASVGDTVTITCQANGYLNWYQQRRGKAPKLLIYDGSKLERGVPS RFSGRRWGQEYNLTINNLQPEDIATYFCQVYEFVVPGTRLDLKRTVAAPGGGGSGGGGSGGGGSQVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVGWINPK TGQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDV WGSGTQVTVSSASTKGP

wherein,

VL sequence (SEQ ID NO: 2)

DIQMTQSPSSLSASVGDTVTITCQANGYLNWYQQRRGKAPKLLIYDGSKLERGVPS RFSGRRWGQEYNLTINNLQPEDIATYFCQVYEFVVPGTRLDLKRTVAAP

VH sequence (SEQ ID NO: 3)

QVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVGWINPKT GQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDVW GSGTQVTVSSASTKGP

Linker sequence-1 (SEQ ID NO: 4): GGGGSGGGGSGGGGS

The following Linker sequences were also effective as verified by experiments:

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

(2)3BNC117scFv-2(SEQ ID NO：10)

QVQLLQSGAAVTKPGASVRVSCEASGYNIRDYFIHWWRQAPGQGLQWVGWINPKT GQPNNPRQFQGRVSLTRHASWDFDTFSFYMDLKALRSDDTAVYFCARQRSDYWDFDVW GSGTQVTVSSASTKGPGGGGSGGGGSGGG GSDIQMTQSPSSLSASVGDTVTITCQANGYL NWYQQRRGKAPKLLIYDGSKLERGVPSRFSGRRWGQEYNLTINNLQPEDIATYFCQVYEF VVPGTRLDLKRTVAAP

Wherein, the VL sequence (SEQ ID NO: 2), the VH sequence (SEQ ID NO: 3) and the Linker sequence-1 (SEQ ID NO: 4).

The following Linker sequences were also effective as verified by experiments:

linker sequence-2 (SEQ ID NO: 5), Linker sequence-3 (SEQ ID NO: 6), Linker sequence-4 (SEQ ID NO: 7), Linker sequence-5 (SEQ ID NO: 8), Linker sequence-6 (SEQ ID NO: 9).

(II) preparation of 3BNC117scFv-CAR (exemplary use of 3BNC117scFv of SEQ ID NO: 1), the preparation process and the plasmid structure diagram are shown in FIG. 2.

1. Overlap PCR primers were designed based on the nucleotide sequence of the CD8IgG region, CD8 transmembrane region, 41BB intracellular region, and CD3 ξ intracellular region.

2. And performing PCR amplification by using DNA polymerase, respectively amplifying CD8IgG region, CD8 transmembrane region, 41BB intracellular region and CD3 xi intracellular region fragments, and 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 CD8IgG region, CD8 transmembrane region, 41BB intracellular region and CD3 xi intracellular 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-41BB-CD3 xi fragment;

4. carrying out double enzyme digestion on the CD8-CD8TM-41BB-CD3 xi fragment and the lentivirus vector phase-IRES-RFP or phase-IRES-GFP (stored in a laboratory) which are subjected to gel recovery, carrying out DNA agarose gel electrophoresis verification after enzyme digestion, and verifying a correct band for gel recovery;

5. connecting the obtained enzyme-digested fragment CD8-CD8TM-41BB-CD3 xi with a vector by using T4 ligase, converting the enzyme-digested fragment into a stable 3 strain, and coating the strain on an ampicillin plate;

6. selecting 3 single colonies growing on the plate, sending to a company for sequencing, wherein a fragment with correct sequencing is 3BNC117-CAR, and an expression plasmid of the fragment is named as phase-3 BNC117 scFv-CAR;

wherein,

(1) the sequence of CD8-CD8TM-41BB-CD3 ξ is as follows (SEQ ID NO: 11):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

(2) the nucleotide sequence of the 3BNC117scFv-41BBCD3 ξ CAR is shown below (SEQ ID NO: 12);

(3) the amino acid sequence of 3BNC117scFv-41BBCD3 ξ CAR is shown below (SEQ ID NO: 13);

(4) the nucleotide sequence of range-3 BNC117scFv-CAR is shown below (SEQ ID NO: 14).

Example 2 preparation of CAR-T cells (exemplified using the phage-3BNC117scFv-CAR as described in SEQ ID NO: 14)

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 and the CAR-expressing lentiviral plasmid phage-3BNC117scFv-CAR required for transfection of the viral packaging system, 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,

adding another tube

phage-3BNC117scFv-CAR 15μg

psPAX2 10μg

pMD2.G 5μg

DMEM was added to the solution to a volume of 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 layer 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 mul 1640 culture medium, and blowing and beating the bottom block containing the virus clumps 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^TMHuman 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^TMHuman CD3/CD28/CD2T Cell Activator (cat #10970) and IL2(Pepro Tech cat # 96-200-02-50) for 2 days;

2.4 infecting a number of the activated human primary T cells with a lentivirus amount having 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 expanding cells, detecting infection efficiency after 3-4 days, constructing 3BNC117-scFv CAR into a phase-IRES-RFP vector, packaging the vector into virus to infect human primary T cells according to experimental operation steps, and measuring the infection efficiency through an RFP label carried by the vector. The results show that 3BNC117-scFv CAR infects human primary T cells with an efficiency of about 60% (as shown in figure 4) and gene expression localization (see example 3), and CAR-enriched positive T cells were screened for subsequent experiments.

Example 3 verification of specific recognition binding of HIV envelope protein to 3BNC117-41BBCD3 ζ -CAR

In this experiment, whether the HIV envelope protein specifically recognizes and binds to 3BNC117-41BBCD3 zeta-CAR was studied, 3BNC117-41BB CD3 zeta-CAR-GFP was transfected into 293T cells, and after 24 hours, the cells were incubated with his-tagged HIV envelope protein gp120 and anti-his PE-labeled antibody in sequence, and the binding of HIV envelope protein to CAR-GFP positive cells was observed by flow-cytometry.

(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 CD19scFv-41BB CD3 ζ -CAR-GFP and experimental group 3BNC117-41BBCD3 ζ -CAR-GFP were transfected into the cells, 293T was digested with TEN 24 hours later, the cells in each well were divided equally into two 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 incubated with anti-his PE-labeled antibody for half an hour again, centrifuged and washed three times with PBS, and detected on an up-flow analyzer.

(III) results of the experiment

3BNC117-scFv CAR-IRES-GFP and control FMC63-scFv CAR-IRES-GFP were introduced into human primary T cells via lentiviral vectors and incubated with his-tagged HIV envelope protein BG505, and the ability of 3BNC117-41BB zeta CAR to localize to the cell membrane and specifically bind to HIV membrane protein gp160 was examined. The results show (as in figure 3) that 3BNC117-scFv CAR specifically recognizes binding to HIV tunica vesiculosa protein BG505 compared to control FMC63-scFv CAR, and that nearly one percent of HIV tunica vesiculosa protein can specifically bind to 3BNC117-41BBCD3 ζ -CAR-GFP positive cell surface relative to control CD19scFv-41BBCD3 ζ -CAR-GFP; whereas HBV envelope protein could not bind to 3BNC117-41BBCD3 ζ CAR-GFP positive cells. Thus, HIV envelope protein specifically recognizes binding to 3BNC117-41BBCD3 ζ -CAR, and 3BNC117-41BBCD3 ζ -CAR does not recognize HBV envelope protein.

Example 4 verification of specific recognition activation of HIV latent cells and 3BNC117-41BBCD3 ζ -CAR

In this experiment, to investigate whether 3BNC117-41BBCD3 ζ -CART cells could be effectively specifically recognized and activated by HIV latent cells, T cells were first isolated from human PBMC, control group CD19scFv-41BBCD3 ζ -CAR-RFP and experimental group 3BNC117-41BBCD3 ζ -CAR-RFP were transformed into T cells after activation culture, and purified control group CD19scFv-41BBCD3 ζ CART cells and experimental group BNC117-41BBCD3 ζ -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 are co-cultured with HIV negative cells A3.01 or HIV positive cells ACH2 according to the ratio of 1:2 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

3BNC117-41BBCD3 zeta-CART cells can be effectively and specifically recognized and activated by HIV latent cells, as shown in FIG. 5, a control group CD19scFv-41BBCD3 zeta-CART can not be activated by HIV negative cells A3.01 and HIV positive cells ACH2 to generate cytokines such as INF gamma and TNF alpha; in contrast, in the experimental group 3, BNC117-41BBCD3 ζ -CART can only be activated by HIV positive cells ACH2 but not by HIV negative cells A3.01 to produce cytokines such as INF γ and TNF α.

Example 5, 3 validation experiment of effective killing of HIV latent cells by BNC117-41BBCD3 ζ -CART cells

In this experiment, to investigate whether 3BNC117-41BB ζ -CART cells could effectively kill HIV latent cells, T cells were first isolated from human PBMC, CD19scFv-41BBCD3 ζ -CAR-RFP and BNC117-41BBCD3 ζ -CAR-RFP were transformed into T cells after activation culture, and purified control group CD19scFv-41BBCD3 ζ -CART cells and experimental group 3BNC117-41BBCD3 ζ -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 respectively cultured together, and after 24 hours, the change of the proportion of the HIV negative cell A3.01 or HIV positive cell ACH2 in the whole cell population is detected.

1. Cells and reagents

(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 the ratio of 2:1 respectively, and after 24 hours, the change of the ratio of the HIV negative cell A3.01 or HIV positive cell ACH2 in the whole cell population is detected.

3. Results of the experiment

3BNC117-41BBCD3 zeta-CART cells can effectively kill HIV latent cells, as shown in FIG. 6, the ratio of HIV negative cells A3.01 co-cultured with experimental group 3BNC117-41BBCD3 zeta-CART has no obvious change compared with the control group CD19scFv-41BBCD3 zeta-CART, but the ratio of HIV positive cells ACH2 co-cultured with experimental group 3BNC117-41BBCD3 zeta-CART has obvious reduction, which shows that 3BNC117-41BBCD3 zeta-CART cells can effectively kill HIV latent cells specifically.

It should be noted that the embodiments of the present application exemplarily employ the sequences shown in SEQ ID NOs: 1, and the invention also uses the 3BNC117scFv-1 shown in SEQ ID NO: the experiment was carried out using 3BNC117scFv-2 shown in FIG. 10, and the experiment effects of 3BNC117-41BBCD3 zeta-CART prepared by using 3BNC117scFv-2 were similar to those of 3BNC117-41BBCD3 zeta-CART prepared by using 3BNC117scFv-2, and thus the description thereof is omitted.

In conclusion, 3BNC117-41BBCD3 zeta-CAR can specifically recognize HIV envelope protein, and 3BNC117-41BBCD3 zeta-CART cells can effectively and specifically recognize and activate HIV latent cells and kill the HIV latent cells.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Sequence listing

<110> Qinghua university, Beijing Huaxia Qing medical treatment science and technology Co., Ltd

<120> use of T cell comprising Chimeric Antigen Receptor (CAR) modification for the preparation of a cellular medicament

<130> 1

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 249

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Thr Val Thr Ile Thr Cys Gln Ala Asn Gly Tyr Leu Asn Trp Tyr

20 25 30

Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Gly Ser

35 40 45

Lys Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly Arg Arg Trp Gly

50 55 60

Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu Gln Pro Glu Asp Ile Ala

65 70 75 80

Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val Val Pro Gly Thr Arg Leu

85 90 95

Asp Leu Lys Arg Thr Val Ala Ala Pro Gly Gly Gly Gly Ser Gly Gly

100 105 110

Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Leu Gln Ser Gly

115 120 125

Ala Ala Val Thr Lys Pro Gly Ala Ser Val Arg Val Ser Cys Glu Ala

130 135 140

Ser Gly Tyr Asn Ile Arg Asp Tyr Phe Ile His Trp Trp Arg Gln Ala

145 150 155 160

Pro Gly Gln Gly Leu Gln Trp Val Gly Trp Ile Asn Pro Lys Thr Gly

165 170 175

Gln Pro Asn Asn Pro Arg Gln Phe Gln Gly Arg Val Ser Leu Thr Arg

180 185 190

His Ala Ser Trp Asp Phe Asp Thr Phe Ser Phe Tyr Met Asp Leu Lys

195 200 205

Ala Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg Gln Arg

210 215 220

Ser Asp Tyr Trp Asp Phe Asp Val Trp Gly Ser Gly Thr Gln Val Thr

225 230 235 240

Val Ser Ser Ala Ser Thr Lys Gly Pro

245

<210> 2

<211> 105

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Thr Val Thr Ile Thr Cys Gln Ala Asn Gly Tyr Leu Asn Trp Tyr

20 25 30

Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Gly Ser

35 40 45

Lys Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly Arg Arg Trp Gly

50 55 60

Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu Gln Pro Glu Asp Ile Ala

65 70 75 80

Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val Val Pro Gly Thr Arg Leu

85 90 95

Asp Leu Lys Arg Thr Val Ala Ala Pro

100 105

<210> 3

<211> 129

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gln Val Gln Leu Leu Gln Ser Gly Ala Ala Val Thr Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Val Ser Cys Glu Ala Ser Gly Tyr Asn Ile Arg Asp Tyr

20 25 30

Phe Ile His Trp Trp Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Val

35 40 45

Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro Asn Asn Pro Arg Gln Phe

50 55 60

Gln Gly Arg Val Ser Leu Thr Arg His Ala Ser Trp Asp Phe Asp Thr

65 70 75 80

Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu Arg Ser Asp Asp Thr Ala

85 90 95

Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp Tyr Trp Asp Phe Asp Val

100 105 110

Trp Gly Ser Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro

<210> 4

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 5

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 6

<211> 20

<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

1 5 10 15

Gly Gly Gly Ser

20

<210> 7

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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)

<400> 8

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)

<400> 9

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> 249

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gln Val Gln Leu Leu Gln Ser Gly Ala Ala Val Thr Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Val Ser Cys Glu Ala Ser Gly Tyr Asn Ile Arg Asp Tyr

20 25 30

Phe Ile His Trp Trp Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Val

35 40 45

Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro Asn Asn Pro Arg Gln Phe

50 55 60

Gln Gly Arg Val Ser Leu Thr Arg His Ala Ser Trp Asp Phe Asp Thr

65 70 75 80

Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu Arg Ser Asp Asp Thr Ala

85 90 95

Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp Tyr Trp Asp Phe Asp Val

100 105 110

Trp Gly Ser Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

145 150 155 160

Asp Thr Val Thr Ile Thr Cys Gln Ala Asn Gly Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Gly Ser

180 185 190

Lys Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly Arg Arg Trp Gly

195 200 205

Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu Gln Pro Glu Asp Ile Ala

210 215 220

Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val Val Pro Gly Thr Arg Leu

225 230 235 240

Asp Leu Lys Arg Thr Val Ala Ala Pro

245

<210> 11

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

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 Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

65 70 75 80

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

85 90 95

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

100 105 110

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

115 120 125

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

130 135 140

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

145 150 155 160

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

165 170 175

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

180 185 190

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

195 200 205

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

210 215 220

<210> 12

<211> 1479

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattccgct 60

agcgatattc agatgaccca gtctccctct tctctgtctg catccgtggg cgacactgtg 120

accattacct gccaggccaa cggctatctc aactggtatc agcagcgcag agggaaagct 180

cccaaactcc tgatctacga tggttccaag ctggaacgcg gcgtcccaag ccgctttagc 240

ggcagaagat ggggacagga gtataacctg acaattaaca acctccagcc cgaagacatc 300

gccacctact tctgtcaagt ttatgagttc gtcgttccag gaacaaggct cgacctgaaa 360

cgcaccgtcg ctgctcctgg tggcggtggc agcggcggtg gtggttccgg aggcggcggt 420

tctcaggtgc aactcctcca gtccggcgcc gctgtgacta aaccaggagc cagcgtcagg 480

gtctcttgcg aagcctccgg ttataatatt cgcgactact ttatacactg gtggaggcaa 540

gctccaggcc agggcctgca atgggtcgga tggatcaacc caaagaccgg acaacccaac 600

aacccaagac aatttcaggg tagggtctcc ctgaccagac atgcttcctg ggacttcgat 660

acattctcat tctacatgga tctgaaagcc ctgcgcagcg atgacaccgc cgtgtacttc 720

tgtgcccgcc agcggtcaga ctactgggat ttcgatgtgt ggggttctgg cacccaggtg 780

accgttagct ctgcatcaac caaaggacca catatgacca cgacgccagc gccgcgacca 840

ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 900

ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatctacatc 960

tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat caccaaacgg 1020

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1080

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1140

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1200

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1260

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1320

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1380

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1440

tacgacgccc ttcacatgca ggccctgccc cctcgctaa 1479

<210> 13

<211> 492

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

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 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu

20 25 30

Ser Ala Ser Val Gly Asp Thr Val Thr Ile Thr Cys Gln Ala Asn Gly

35 40 45

Tyr Leu Asn Trp Tyr Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu Leu

50 55 60

Ile Tyr Asp Gly Ser Lys Leu Glu Arg Gly Val Pro Ser Arg Phe Ser

65 70 75 80

Gly Arg Arg Trp Gly Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu Gln

85 90 95

Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val Val

100 105 110

Pro Gly Thr Arg Leu Asp Leu Lys Arg Thr Val Ala Ala Pro Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

130 135 140

Leu Leu Gln Ser Gly Ala Ala Val Thr Lys Pro Gly Ala Ser Val Arg

145 150 155 160

Val Ser Cys Glu Ala Ser Gly Tyr Asn Ile Arg Asp Tyr Phe Ile His

165 170 175

Trp Trp Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Val Gly Trp Ile

180 185 190

Asn Pro Lys Thr Gly Gln Pro Asn Asn Pro Arg Gln Phe Gln Gly Arg

195 200 205

Val Ser Leu Thr Arg His Ala Ser Trp Asp Phe Asp Thr Phe Ser Phe

210 215 220

Tyr Met Asp Leu Lys Ala Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe

225 230 235 240

Cys Ala Arg Gln Arg Ser Asp Tyr Trp Asp Phe Asp Val Trp Gly Ser

245 250 255

Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro His Met

260 265 270

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

275 280 285

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

290 295 300

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

305 310 315 320

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

325 330 335

Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 14

<211> 9771

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

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 cgtgaagcgg ccgcgccacc 3420

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattccgct 3480

agcgatattc agatgaccca gtctccctct tctctgtctg catccgtggg cgacactgtg 3540

accattacct gccaggccaa cggctatctc aactggtatc agcagcgcag agggaaagct 3600

cccaaactcc tgatctacga tggttccaag ctggaacgcg gcgtcccaag ccgctttagc 3660

ggcagaagat ggggacagga gtataacctg acaattaaca acctccagcc cgaagacatc 3720

gccacctact tctgtcaagt ttatgagttc gtcgttccag gaacaaggct cgacctgaaa 3780

cgcaccgtcg ctgctcctgg tggcggtggc agcggcggtg gtggttccgg aggcggcggt 3840

tctcaggtgc aactcctcca gtccggcgcc gctgtgacta aaccaggagc cagcgtcagg 3900

gtctcttgcg aagcctccgg ttataatatt cgcgactact ttatacactg gtggaggcaa 3960

gctccaggcc agggcctgca atgggtcgga tggatcaacc caaagaccgg acaacccaac 4020

aacccaagac aatttcaggg tagggtctcc ctgaccagac atgcttcctg ggacttcgat 4080

acattctcat tctacatgga tctgaaagcc ctgcgcagcg atgacaccgc cgtgtacttc 4140

tgtgcccgcc agcggtcaga ctactgggat ttcgatgtgt ggggttctgg cacccaggtg 4200

accgttagct ctgcatcaac caaaggacca catatgacca cgacgccagc gccgcgacca 4260

ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 4320

ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatctacatc 4380

tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat caccaaacgg 4440

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 4500

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 4560

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 4620

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 4680

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 4740

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 4800

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 4860

tacgacgccc ttcacatgca ggccctgccc cctcgctaac ccccccccct aacgttactg 4920

gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat 4980

tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc 5040

ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag 5100

cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc 5160

ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac 5220

ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca 5280

aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt 5340

gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa 5400

aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgataat 5460

accatgacca tggcctcctc cgaggacgtc atcaaggagt tcatgcgctt caaggtgcgc 5520

atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 5580

tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc 5640

tgggacatcc tgtcccctca gttccagtac ggctccaagg cctacgtgaa gcaccccgcc 5700

gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 5760

aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag 5820

ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag 5880

aagaagacca tgggctggga ggcctccacc gagcggatgt accccgagga cggcgccctg 5940

aagggcgaga tcaagatgag gctgaagctg aaggacggcg gccactacga cgccgaggtc 6000

aagaccacct acatggccaa gaagcccgtg cagctgcccg gcgcctacaa gaccgacatc 6060

aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgcgcc 6120

gagggccgcc actccaccgg cgcctaaatc gatagatcct aatcaacctc tggattacaa 6180

aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 6240

cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 6300

cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 6360

tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 6420

ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 6480

cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 6540

ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat 6600

tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 6660

ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 6720

tcggatctcc ctttgggccg cctccccgcc tgagatcctt taagaccaat gacttacaag 6780

gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac 6840

tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc 6900

tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 6960

ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 7020

ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt 7080

attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggcc cgggttaatt 7140

aaggaaaggg ctagatcatt cttgaagacg aaagggcctc gtgatacgcc tatttttata 7200

ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 7260

gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 7320

acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 7380

tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 7440

agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 7500

cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 7560

aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg 7620

gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 7680

agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 7740

aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 7800

gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 7860

ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 7920

aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 7980

aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 8040

tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 8100

agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 8160

ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 8220

ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 8280

ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 8340

acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 8400

agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 8460

ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 8520

cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa 8580

gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 8640

cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 8700

gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 8760

caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 8820

aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 8880

tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 8940

gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 9000

ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 9060

atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 9120

cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 9180

caaaccgcct ctccccgcgc gttggccgat tcattaatgc agcaagctca tggctgacta 9240

atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag 9300

tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tccccgtggc acgacaggtt 9360

tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta 9420

ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg 9480

ataacaattt cacacaggaa acagctatga catgattacg aatttcacaa ataaagcatt 9540

tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 9600

gatcaactgg ataactcaag ctaaccaaaa tcatcccaaa cttcccaccc cataccctat 9660

taccactgcc aattacctgt ggtttcattt actctaaacc tgtgattcct ctgaattatt 9720

ttcattttaa agaaattgta tttgttaaat atgtactaca aacttagtag t 9771

Claims

1. The use of a chimeric antigen receptor, an immunoresponsive cell comprising said chimeric antigen receptor, in the preparation of a medicament for treating HIV infection, wherein said chimeric antigen receptor specifically binds to the HIV membrane protein gp160, and said immunoresponsive cell is effectively recognized and activated by HIV latent cells specifically and effectively killing HIV latent cells specifically;

the chimeric antigen receptor comprises an extracellular antigen binding domain, an extracellular hinge region, a transmembrane domain and an intracellular domain, wherein the extracellular antigen binding domain is a human single-chain antibody of 3BNC117, the extracellular hinge region is a CD8IgG region, the transmembrane domain is a CD8 transmembrane region, and the intracellular domain is 41BB CD3 ξ;

the human single-chain antibody comprises a heavy chain variable region, a joint and a light chain variable region, wherein the heavy chain variable region is shown as SEQ ID NO. 3, and the light chain variable region is shown as SEQ ID NO. 2.

2. The use of claim 1, wherein the linker is an amino acid sequence comprising glycine and serine.

3. The use of claim 2, wherein the linker has the sequence set forth in SEQ ID NO:4 to 6.

4. Use according to claim 1, wherein said immunoresponsive cells are selected from the group consisting of T cells, NK cells, CTLs, human embryonic stem cells, lymphoid progenitor cells and/or T cell precursor cells.

5. Use according to claim 1, characterized in that the nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor is as follows:

(1) comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 11;

(2) a nucleotide sequence shown as SEQ ID NO. 12;

or, (3) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 13.