CN115725001A - CD 19-targeted chimeric antigen receptor, nucleic acid, recombinant vector and CAR-T cell - Google Patents

Abstract

The present application relates to a chimeric antigen receptor, nucleic acid, recombinant vector and CAR-T cell targeting CD19. The chimeric antigen receptor targeting CD19 comprises an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain and a signal transduction domain which are sequentially connected from an amino terminal to a carboxyl terminal and target CD19, wherein the costimulatory domain is 4-1BB, and the signal transduction domain is ITAM1 of CD3 zeta. The CAR-T cells containing the chimeric antigen receptor targeting CD19 have strong in vitro anti-depletion ability.

Description

CD 19-targeted chimeric antigen receptor, nucleic acid, recombinant vector and CAR-T cell

Technical Field

The invention relates to the technical field of immunotherapy, and particularly relates to a CD 19-targeted chimeric antigen receptor, a nucleic acid, a recombinant vector and a CAR-T cell.

Background

In recent years, chimeric Antigen Receptor (CAR) T cell therapy has become one of the most potential forms of tumor immunotherapy, showing surprising therapeutic effects in a variety of malignancies, especially hematologic tumors. At present, a plurality of CAR-T products are marketed in the market, and 2 CAR-T products targeting CD19 are marketed in China in 2021.

Currently marketed CAR-T targeting CD19 is composed mainly of three functional domains, an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain consists of an antigen binding domain responsible for recognizing and binding to CD19 and a Hinge region (Hinge) for ligation. The intracellular Domain is composed of a Costimulatory Domain (Costimulatory Domain) and a Signaling Domain (Signaling Domain). The costimulatory domain comprises mainly CD28 or 4-1BB, and the signaling domain is the intracellular domain of the CD3 zeta chain, containing 3 immunoreceptor tyrosine activation motifs (ITAM 1, ITAM2 and ITAM 3).

CAR-T, with CD28 as co-stimulatory molecule, started to proliferate vigorously after injection (Alexander I.salt et al 2018), but could persist in vivo for up to 3 months (Brentjens et al 2013; davila et al 2014; lee et al 2015), with shorter duration of action. Thus, although CD 19-targeted CAR-T cells have a high rate of complete remission for the treatment of B-cell lymphomas, most patients who obtain remission show disease relapse within a few years (Maude, s.l et al, 2018), with the rate of relapse of B-ALL (acute B-lymphocytic leukemia) varying from 21% to 45% and increasing with increasing follow-up time (Cheng et al, 2019). One of the partial causes of therapy failure has been reported to be due to premature CAR-T cell depletion (poorebrahim.m et al, 2020.

During clinical treatment, patients cannot be completely relieved or relapse due to the long-term exposure and the gradual loss of function of CD 19-targeted CAR-T cells taking CD28 as a co-stimulation domain, which act on inflammation and cancer cells and are easy to deplete. Therefore, how to improve the anti-exhaustion capacity of the CAR-T cells targeting CD19 is one of the problems that improvement of the therapeutic effect is urgently needed to solve. There are studies to date showing that, in the CAR-T cells using CD28+ CD3 ζ, T cell depletion is more likely to occur, and it was found that active CAR-T cells containing only ITAM-1 domain (ITAM 1 of CD28+ CD3 ζ) have stronger antitumor activity in vivo and longer persistence than active CAR-T cells containing only ITAM-1 domain (ITAM 1 of CD28+ CD3 ζ) in a mouse tumor model, although their antigen-stimulated proliferation ability and killing ability against target cells are weaker than those of CD28+ CD3 ζ (ITAM 1 to 3) in vitro.

Disclosure of Invention

In view of the above, there is a need to provide a chimeric antigen receptor capable of enhancing the anti-exhaustion ability of a conventional CD 19-targeted CAR-T cell after antigen stimulation, in response to the problem that the anti-exhaustion ability of the CAR-T cell is weak.

A chimeric antigen receptor targeting CD19, comprising an antigen binding domain targeting CD19, a hinge region, a transmembrane domain, a costimulatory domain and a signaling domain connected in sequence from amino terminus to carboxy terminus, wherein the costimulatory domain is 4-1BB and the signaling domain is ITAM1 of CD3 zeta.

The CD 19-targeting chimeric antigen receptor has 4-1BB as a co-stimulatory domain and ITAM1 of CD3 zeta as a signaling domain, thereby improving the anti-exhaustion capacity of CAR-T cells containing the CD 19-targeting chimeric antigen receptor. In addition, CAR-T cells containing the above chimeric antigen receptor targeting CD19 are more potent in vitro in killing and proliferation. One aspect of the present application provides a CD 19-targeting chimeric antigen receptor, comprising an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain of CD19, which are sequentially connected from an amino terminus to a carboxy terminus, wherein the costimulatory domain is 4-1BB and the signaling domain is ITAM1 of CD3 ζ.

In one embodiment, the hinge region is selected from the hinge region of at least one protein: CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

The transmembrane domain is selected from transmembrane regions of at least one of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2 Rbeta, IL2 Rgamma, IL7 Ralpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, ITA-1, ITGAM, CD11b, ITGAX, CD11C, ITGB1, CD29, ITGB2, ITGB7, TNFR2, ACAR 1, NKGAM 6, TAMGW 6, NKGL 2, NKGA 6, TARG, CD11 bp, NKGA 6, NKGA 1, CD11 bp, CTGA 6, CTGB 1, ITGAD, CTGL 4, and NKGL 6.

In one embodiment, the antigen binding domain targeting CD19 is a single chain antibody targeting CD19, the amino acid sequence of the heavy chain variable region of the single chain antibody targeting CD19 is shown as SEQ ID No.2, and the amino acid sequence of the light chain variable region of the single chain antibody targeting CD19 is shown as SEQ ID No. 3.

In one embodiment, the hinge region is a CD8 a hinge region having an amino acid sequence as set forth in SEQ ID No. 4.

In one embodiment, the transmembrane domain is a CD 8. Alpha. Transmembrane region having the amino acid sequence shown in SEQ ID No. 5.

In one embodiment, the amino acid sequence of 4-1BB is shown in SEQ ID NO. 6.

In one embodiment, the amino acid sequence of ITAM1 of CD3 ζ is as set forth in SEQ ID No. 7.

In one embodiment, the CD 19-targeted chimeric antigen receptor further comprises a signal peptide at the amino-terminus linked to the CD 19-targeted antigen binding domain.

In one embodiment, the amino acid sequence of the chimeric antigen receptor targeting CD19 is shown as SEQ ID No. 8.

In one embodiment, the isolated nucleic acid is used to express a CD 19-targeted chimeric antigen receptor of any one of claims 1 to 6.

In one embodiment, the isolated nucleic acid comprises a nucleic acid fragment having a nucleotide sequence set forth in SEQ ID No. 9.

Another aspect of the present application provides a recombinant vector comprising the isolated nucleic acid of claim 7 or 8.

The application also provides a CAR-T cell comprising the isolated nucleic acid of claim 7 or 8 or transformed with the recombinant vector of claim 9.

The application also provides the use of a CD 19-targeting chimeric antigen receptor of any one of claims 1 to 6, an isolated nucleic acid of claim 7 or 8, a recombinant vector of claim 9, or a CAR-T cell of claim 10 in the preparation of a medicament for the treatment of cancer.

The application also provides a pharmaceutical composition comprising an expression vector for expressing the CD 19-targeted chimeric antigen receptor of any one of claims 1 to 6 or the CAR-T cell of claim 10, and a pharmaceutically acceptable adjuvant.

Drawings

FIG. 1 is a schematic representation of the structures of 1326-CAR from example 1 (ITAM 1) and control 1502-CAR (ITAM 1+ ITAM2+ ITAM 3).

FIG. 2 is 1326-CAR-T and control 1502-CAR-T cultured to D4 days in the examples versus RS4 at different effective target ratios; 11 killing effect of cells.

Figure 3 is the Ki67 expression level on CAR-T cells of 1326-CAR-T and control 1502-CAR-T in example 1, respectively, for different time periods after one antigen stimulation in vitro.

FIG. 4 is a graph of the detection of Apo in the CAR-T cells of 1326-CAR-T and control 1502-CAR-T in example 1 at different time periods after in vitro antigen stimulation.

FIG. 5 is the expression level of Tim3 after one antigen stimulation of 1326-CAR-T and control 1502-CAR-T in example 1, respectively, in vitro.

Detailed Description

To facilitate an understanding of the present application, which is described more fully below, may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The term "and/or" includes any and all combinations of one or more of the associated listed items.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the present application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

Interpretation of terms

"T cell" refers to a T lymphocyte (T lymphocyte) which is a bone marrow-derived lymphoid stem cell, and which is differentiated and matured in the thymus, and then distributed to immune organs and tissues throughout the body through lymph and blood circulation to exert an immune function. Mature T cells distribute to thymus dependent area of peripheral immune organ through blood stream to colonize, and can be recycled through lymphatic vessel, peripheral blood and tissue fluid, etc., to exert functions of cellular immunity and immunoregulation, etc. The recycling of T cells facilitates the wide exposure to antigenic substances entering the body, enhances the immune response and maintains the immunological memory for a longer period. There are many different markers on the cell membrane of T cells, mainly surface antigens and surface receptors, which are giant protein molecules bound to the cell membrane.

The "single chain antibody" is a recombinant antibody having a minimum form (about 27 kDa) in which an antibody heavy chain variable domain (VH) and a light chain variable domain (VL) are linked by a flexible short peptide (linker) consisting of 10 to 25 amino acids. Essentially, scFv is a fusion protein and retains the specificity of the original immunoglobulin for the antigen. The small molecular size of scFv brings the advantages of strong penetrating power in tumor, rapid degradation in blood, small negative feedback in human body and the like, which lays a foundation for the clinical application of scFv.

One embodiment of the present application provides a CD 19-targeting chimeric antigen receptor, which comprises an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain and a signaling domain of CD19, which are sequentially connected from an amino terminus to a carboxy terminus, wherein the costimulatory domain is 4-1BB and the signaling domain is ITAM1 of CD3 ζ.

In some embodiments, the antigen binding domain targeted to CD19 is a single chain antibody targeted to CD19. Furthermore, the amino acid sequence of the heavy chain variable region of the single-chain antibody targeting CD19 is shown in SEQ ID No.2, and the amino acid sequence of the light chain variable region of the single-chain antibody targeting CD19 is shown in SEQ ID No. 3. In particular, the amount of the solvent to be used,

the amino acid sequence shown in SEQ ID NO.2 is as follows: EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO. 2)

The amino acid sequence shown in SEQ ID NO.3 is: DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRF SGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO. 3)

In some embodiments, the hinge region is selected from the hinge region of at least one protein: CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154. It is understood that in other embodiments, the hinge region is not limited to the above, and may be other polypeptides that can function as a hinge region.

In some embodiments, the transmembrane domain is selected from the transmembrane regions of at least one of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2 Rbeta, IL2 Rgamma, IL7 Ralpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, ITA-1, ITGAM, CD11b, ITGAX, CD11C, ITGB1, CD29, ITGB2, ITGB7, TNFR2, ACAR 1, NKGAM 6, TAMGW 6, NKGL 2, NKGA 6, TARG, CD11 bp, NKGA 6, NKGA 1, CD11 bp, CTGA 6, CTGB 1, ITGAD, CTGL 4, and NKGL 6. It will be appreciated that in other embodiments, the transmembrane domain is not limited to the above, but may be other polypeptides that may act as transmembrane domains.

In some embodiments, the hinge region of the above chimeric antigen receptor targeting CD19 is a CD8 a hinge region and the transmembrane domain is a CD8 a transmembrane domain. Furthermore, the hinge region of the chimeric antigen receptor targeting CD19 is a CD8 alpha hinge region with an amino acid sequence shown as SEQ ID No.4, and the transmembrane domain is a CD8 alpha transmembrane region with an amino acid sequence shown as SEQ ID No. 5.

Specifically, the amino acid sequence shown as SEQ ID NO.4 is: KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT RGLDFACD (SEQ ID NO. 4)

The amino acid sequence shown as SEQ ID NO.5 is: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 5)

In some embodiments, the amino acid sequence of 4-1BB as the co-stimulatory domain is shown in SEQ ID No. 6. Specifically, the amino acid sequence shown as SEQ ID NO.6 is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO. 6)

In some embodiments, the amino acid sequence of ITAM1 of CD3 ζ as the signaling domain is set forth in SEQ ID No. 7. Specifically, the amino acid sequence shown as SEQ ID NO.7 is RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ (SEQ ID NO. 7)

In some embodiments, the hinge region of the chimeric antigen receptor targeting CD19 is a CD8 α hinge region having an amino acid sequence shown in SEQ ID No.4, the transmembrane domain is a CD8 α transmembrane region having an amino acid sequence shown in SEQ ID No.5, the amino acid sequence of 4-1BB is shown in SEQ ID No.6, and the amino acid sequence of ITAM1 of CD3 ζ is shown in SEQ ID No. 7.

In some embodiments, the CD 19-targeted chimeric antigen receptor further comprises a signal peptide at the amino-terminus and linked to the CD 19-targeted antigen binding domain. Optionally, the amino acid sequence of the signal peptide is as shown in SEQ id No. 1. Specifically, the amino acid sequence shown as SEQ ID NO.1 is: MALPVTALLLPLALLLHAARP. It will be appreciated that in other embodiments, the signal peptide is not limited to the above, and may be other polypeptides that can act as a signal peptide.

In some embodiments, the amino acid sequence of the chimeric antigen receptor targeting CD19 is set forth in SEQ ID No. 8.

Wherein the amino acid sequence shown in SEQ ID NO.8 is

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKP

DGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK

LEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK

GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS

YAMDYWGQGTSVTVSSAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL

DFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE

EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ(SEQ ID NO.8)

It is understood that the various polypeptide fragments of the present application (e.g., signal peptide, single chain antibody, hinge region, transmembrane domain, co-stimulatory domain, and signal transduction domain) can be independently selected from the same or different species sources, such as murine (mouse, rat), rabbit, sheep, goat, horse, chicken, bovine, canine, and human. Further, the species source is human.

In addition, an embodiment of the present application provides an isolated nucleic acid for expressing a CD 19-targeted chimeric antigen receptor of any of the above embodiments.

In some embodiments, the isolated nucleic acid comprises a nucleic acid fragment having a nucleotide sequence set forth in SEQ ID No. 9. It is understood that nucleic acid sequences capable of expressing the same protein have various forms due to degeneracy of codons, and the above are codon-optimized nucleic acid sequences for achieving high expression of a protein of interest, but the specific sequence form is not limited thereto.

In addition, an embodiment of the present application provides a recombinant vector comprising the isolated nucleic acid of any of the above embodiments. It is understood that the nucleic acid may be DNA or RNA.

In some embodiments, the recombinant vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a CRISPR/CAS plasmid. Preferably, the recombinant vector is a lentiviral vector. The lentivirus vector can effectively integrate the exogenous gene onto a host chromosome, thereby achieving the effect of persistently expressing the target sequence. Can effectively infect various cells such as neuron cells, liver cells, cardiac muscle cells, tumor cells, endothelial cells, stem cells and the like in the aspect of infection capacity, thereby achieving good gene therapy effect. For some cells which are difficult to transfect, such as primary cells, stem cells, undifferentiated cells and the like, the lentiviral vector is used, so that the transduction efficiency of the target gene can be greatly improved, the probability of integrating the target gene into the host cell genome is greatly increased, and the long-term and stable expression of the target gene can be conveniently and quickly realized. It will be appreciated that the type of carrier is not limited thereto and may be adjusted according to specific needs. In addition, the vector may contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, etc., and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

In addition, an embodiment of the present application provides a CAR-T cell comprising a nucleic acid of any of the above embodiments or transformed with a recombinant vector of any of the above embodiments.

In some implementations, the CAR-T cell is of any one of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a MAIT cell, and a γ δ T cell. Optionally, the CAR-T cell is a CD3+ T cell, a CD3+ CD4+ T cell, or a CD3+ CD8+ T cell.

Use of a CD 19-targeting chimeric antigen receptor of any of the above embodiments, an isolated nucleic acid of any of the above embodiments, a recombinant vector of any of the above embodiments, or a CAR-T cell of any of the above embodiments in the preparation of a medicament for the treatment of cancer.

In addition, an embodiment of the present application further provides a pharmaceutical composition comprising an expression vector for expressing the CD 19-targeted chimeric antigen receptor of any of the above embodiments or the CAR-T cell of any of the above embodiments, and a pharmaceutically acceptable adjuvant.

In some embodiments, the excipients include one or more of diluents, preservatives, buffers, disintegrants, antioxidants, suspending agents, colorants, and excipients.

Optionally, the diluent is selected from one or more of polyethylene glycol, propylene glycol, vegetable oil and mineral oil. In a specific example, the preservative is selected from one or more of sorbic acid, methyl sorbate, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, benzyl paraben, sodium methyl paraben, benzoic acid, and benzyl alcohol. In a particular example, the buffering agent is selected from one or more of sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium tartrate, and sodium acetate. In a specific example, the antioxidant is selected from one or more of ethylenediaminetetraacetic acid, disodium salts of ethylenediaminetetraacetic acid, dibutylhydroxytoluene, glycine, inositol, ascorbic acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid, and sodium metabisulfite.

It is noted that a pharmaceutically acceptable excipient herein refers to a material that is compatible with the other ingredients of the pharmaceutical formulation and is suitable for contact with the tissue or organ of the recipient (e.g., human or animal). There is no or little complication of toxicity, irritation, allergic response, immunogenicity, or other problems when used.

In addition, an embodiment of the present application further provides a method for preparing the CAR-T cell, comprising the following steps: collecting peripheral blood mononuclear cells; separating T cells (e.g., CD3+ T cells) from the peripheral blood mononuclear cells; and infecting T cells with a recombinant vector (e.g., virus) containing the isolated nucleic acid of any of the above embodiments, followed by expansion culture.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

Example 1

1. Design CAR

The CD19 is taken as a target, a CAR of a scFv region (abbreviated as 'FMC 63 scFv') of a monoclonal antibody FMC63 targeting CD19 from a mouse source, a human CD8 alpha transmembrane region and a hinge region, a full length of a human CD3 zeta chain serving as a signaling region and a 4-1BB sequence serving as a costimulatory molecule is named as CD19.BBZCAR.1502 and abbreviated as '1502-CAR', and the full length of the 1502-CAR is 1502bp. On the basis of 1502-CAR, the intracellular CD3 zeta chain is modified. A first ITAM (abbreviated as ITAM1 or CD3 zeta ITAM 1) in 3 Immunoreceptor Tyrosine Activation Motifs (ITAMs) in a CD3 zeta chain is selected, the length of the first ITAM is 56 amino acid residues, a novel CAR structure is formed, the total length of the sequence is 1326bp, the novel CAR structure is named as CD19.BBZ1CAR.1326, abbreviated as 1326-CAR, and the structure is shown in figure 1.

In the 1502-CAR and 1326-CAR described above, the amino acid sequence of the CD8 α signal peptide is shown in SEQ id No. 1: MALPVTALLLPLALLLHAARP (SEQ ID NO. 1)

The amino acid sequence of the heavy chain variable region of FMC63 scFv is shown as SEQ ID NO.2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.3, the amino acid sequence of the humanized CD8 alpha hinge region is shown as SEQ ID NO.4, the amino acid sequence of the humanized CD8 alpha transmembrane region is shown as SEQ ID NO.5, the amino acid sequence of the 4-1BB costimulatory molecule is shown as SEQ ID NO.6, and the amino acid sequence of the ITAM1 is shown as SEQ ID NO. 7.

In addition, the nucleotide sequence of the 1326-CAR is shown as SEQ ID NO. 9:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCA

GATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTC

AGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATA

CATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCA

TTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCG

GAGGGGGGACTAAGTTGGAAATAACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGAT

CTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCAC

TGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGT

GGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCA

AGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTAC

TGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTC

TCCTCAGCGGCCGCAAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGT

CGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGC

TGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGG

TTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAG

TACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACT

GAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGA

GCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGTAA(SEQ ID NO.9)

1502-CAR has a nucleotide sequence shown in SEQ ID NO. 10:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCGGCCGCAAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO.10)

2. packaging of lentiviruses

After the cDNA sequences of 1326 and 1502 were synthesized, restriction endonuclease cleavage and T4 DNA ligase ligation were performedInoculating and cloning to slow virus plasmid pSin-EF _α SOX2-puro (purchased from Addgene, plasmid # 16577) replacing the original SOX2 fragment, constituting the lentiviral Plasmid pSin-EF containing the CAR sequence _α -1502-CAR-puro and pSin-EF _α -1326-CAR-puro. The constructed lentiviral plasmids, lentiviral packaging Plasmid pCMV8.91 (from Addgene) and pMD.2G Plasmid (from Addgene, plasmid # 12259) were then transfected into 293FT cells using calcium phosphate transfection reagent (Invitrogen: K278001) at a mass ratio of 3. After 36h of plasmid transfection, the lentivirus-containing culture medium supernatant was harvested and finally the collected supernatant was concentrated by centrifugation using ultracentrifugation. The titer of the concentrated lentivirus is determined by qPCR to reach 10 ⁸ TU/mL above for subsequent use.

3. Preparation of CAR-T cells

10mL of leukocyte-enriched blood collected by a hemocytometer was separated from peripheral whole blood or machine-collected blood to obtain CD3 with a purity of 99% or more using a Rosettesep Human T Cells enrichment cocktail kit from STEMCELL Techogies Co ⁺ And (4) the T cells after separation can be frozen in liquid nitrogen or directly used.

D0 day: taking a certain amount of CD3 ⁺ T cells in CAR-T Medium (GT-T551H 3+5w/v% CTS) ^TM Immune Cell SR +1w/v% non-essential amino acids +1w/v% glutamine +1

w/v% Heps +1w/v% sodium pyruvate +1w/v% streptomycin-penicillin +1w/v% GlutaMAX +46-50Unit IL-2), and magnetic beads (Dynabeads Human T-Activator CD3/CD28, invitrogen) were added at a rate of 1:1. Finally, 5% CO at 37 ℃% ₂ The culture box is used for culturing for 24 hours.

D1 day: six-well plates were coated with a fibronectin solution from TAKARA (overnight at 4 ℃). After coating, the supernatant was removed and a calculated volume of lentivirus was added to the six well plate and centrifuged at 2000g for 2h at 32 ℃. After centrifugation, the supernatant was removed and CD3 cultured to D1 was harvested ⁺ Adding T cells and magnetic beads into the six-well plate, adding Polybrene to make its final concentration 4 μ g/ml, and processing at 32 deg.C and 1200gCentrifuge under the conditions for 2h. After centrifugation, the six well plates were placed at 37 ℃ and 5% CO ₂ The cultivation was continued for 24h.

D2 days: all cells were collected and centrifuged at 350g for 5min at ambient temperature. After centrifugation, the supernatant was discarded, supplemented with a volume of fresh CAR-T medium to resuspend the cells, and seeded into T25 flasks to achieve a cell density of 0.5X 10 ⁶ And/ml. Finally placing the cells at 37 ℃,5% ₂ The cultivation was continued for 48h.

CAR-T cells cultured to D4 days were harvested.

4. Antigen stimulation experiments

To carry out the study of the killing, proliferation and anti-depletion capacity of CAR-T cells in vitro, we performed one antigen stimulation experiment on them during in vitro culture. Which comprises the following steps:

a portion of the cells was cultured for 4 days D (D0 for antigen stimulation test) at 4X 10 ⁵ (ii) CAR-T cells with RS4;11 acute leukemia cells were measured according to 1:1 proportion, and co-culturing for d14 days. Then, D0 (D4) CAR-T cells not stimulated by the antigen (as a control) and D7 and D14 CAR-T cells after stimulation by the antigen were taken to perform experiments such as killing, proliferation, exhaustion, and the like.

6. Characterization data and effect data

(1) The killing ability of the 1326-CAR-T cell on the target cell in vitro is better than that of the 1502-CAR-T cell.

Two CAR-T cells with different ratios of RS4; after 11 cells were co-cultured for 72h, RS4 in the co-culture system was examined by flow cytometry; 11 cells remained, and the results are shown in FIG. 2. In FIG. 2, the ordinate represents the killed leukemia cell line RS4 in the co-culture system; 11 percent of cells, abscissa represents 1326-CAR-T and 1502-CAR-T (effector cells) to RS4;11 (target cells) ratio of effective targets in coculture. As can be seen in FIG. 2, the effective target ratio of 1326-CAR-T to 1502-CAR-T is 2:1 to 1:2, all RS4 can be obtained; 11 cell killing. The effective target ratio is 1: both 5 and 1; 11 cells were killed in total, whereas 1502-CAR-T cells were not. Wherein, the effective target ratio is 1: at 10, the killing rate of the 1326-CAR-T cell to the target cell in vitro can reach 100%, and the killing rate of the 1502-CAR-T cell to the target cell is about 70%. Therefore, the killing capacity of the 1326-CAR-T cell on the target cell in vitro is obviously better than that of the 1502-CAR-T cell.

(2) The overall proliferation of 1326-CAR-T in vitro after antigen stimulation was better than that of 1502-CAR-T.

Clinical trials have demonstrated that CAR-T cell therapy targeting CD19 can be very effective in inducing remission in patients with CD19+ B cell lymphoid cancer. The enormous disease burden can be eliminated by injecting CAR-T cells at very small doses, thus demonstrating the importance of CAR-T cell expansion in vivo for therapeutic efficacy (Maude et al, 2014, lee DW et al 2015. We performed one antigen stimulation on 1326-CAR-T cells and 1502-CAR-T cells cultured to D4 (D0 of antigen stimulation experiment) in the same number. Detecting CAR with cell proliferation marker "Ki67" and apoptosis probe "Apo" from the same number of cells before antigen stimulation and d7 and d14 after antigen stimulation ⁺ Proliferation and apoptosis of T, the results are shown in fig. 3 and 4. In figure 3, the abscissa represents the time (days) of the cytometric assay of the two CAR-ts before and after one antigen stimulation; d0 data are CAR-T cells before receiving antigen stimulation, as controls; the ordinate represents the percentage of CAR-T cells in proliferative state to all CAR + T cells. In FIG. 4, the abscissa represents the time (days) for which two CAR-Ts were cytometrically tested before and after one antigen challenge; d0 data are CAR-T cells before receiving antigen stimulation, as controls; the ordinate represents the percentage of CAR-T cells in the apoptotic state to all CAR + T cells.

As can be seen from FIG. 3, the proportion of cells in the proliferative state in d0, 1326-CAR-T cells before antigen stimulation was higher than 1502-CAR-T, but the proportion of the 1326-CAR-T cells in the proliferative state of d7 and d14 after antigen stimulation was slightly lower or almost identical to 1502-CAR-T.

As can be seen in FIG. 4, the number of 1326-CAR-T cells in the apoptotic state at d0 before antigen stimulation and at d7 and d14 after antigen stimulation was less than 1502-CAR-T cells on the same number of days of culture.

Taken together with FIGS. 3 and 4, the 1326-CAR-T is present in a higher proportion of cells in the proliferative state than 1502-CAR-T when not stimulated by antigen in vitro. After antigen stimulation in vitro, the proportion of cells in the apoptotic state was low for 1326-CAR-T, although slightly lower or almost identical to 1502-CAR-T. Thus, the proliferation and apoptosis profile was combined, indicating that the overall proliferation profile of 1326-CAR-T cells before and after antigen stimulation was better than 1502-CAR-T.

(3) 1326-CAR-T has better anti-exhaustion ability after antigen stimulation in vitro than 1502-CAR-T.

It is reported in the literature that longer follow-up periods indicate that remission is not sustained for the majority of patients over a long period without further consolidation of treatment. There are studies that suggest that CAR-T cell persistence is important for sustained maintenance of remission, and that improving CAR-T persistence in vivo is likely to improve patient response rates and remission persistence. Therefore, we studied the persistence (depletion) of 1326-CAR-T cells and 1502-CAR-T cells, including in particular: we performed one antigen stimulation on the same number of 1326-CAR-T cells and 1502-CAR-T cells cultured to D4 (D0). The same number of cells were taken at d0 before antigen stimulation, and at d7 and d14 after antigen stimulation, respectively, and the expression level of the cell-exhaustion marker "Tim3" was examined by flow cytometry to reflect the exhaustion of 1326-CAR-T, the results are shown in fig. 5. In figure 5, the abscissa represents the time (days) of cytometric assay of the two CAR-ts before and after one antigen stimulation; d0 data are CAR-T cells before receiving antigen stimulation, as controls; the ordinate represents the percentage of CAR-T cells in the depleted state to all CAR + T cells.

As can be seen from FIG. 5, although the cell proportion in the depleted state in the d0, 1326-CAR-T cells before and after antigen stimulation was slightly higher than that of 1502-CAR-T, the CAR-T proportion in the depleted state of 1326-CAR-T was lower than that of 1502-CAR-T at the same time at d7 and d14 days after antigen stimulation in vitro.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present application, so as to understand the technical solutions of the present application in detail and in detail, but not to be construed as limiting the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present application are all within the scope of the claims appended to the present application. Therefore, the protection scope of the present patent application shall be subject to the content of the appended claims, and the description and the drawings shall be used for explaining the content of the claims.

Claims (12)

1. A CD 19-targeting chimeric antigen receptor comprising, connected in sequence from amino terminus to carboxy terminus, an antigen binding domain targeting CD19, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain, wherein the costimulatory domain is 4-1BB and the signaling domain is ITAM1 of CD3 ζ.

2. The CD19 targeted chimeric antigen receptor according to claim 1, wherein said hinge region is selected from the group consisting of a hinge region of at least one protein: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; and/or

The transmembrane domain is selected from the transmembrane regions of at least one of the following proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, ICOS, GITR, CD40, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2 Rbeta, IL2 Rgamma, IL7 Ralpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, ITA-1, ITGAM, CD11b, ITGAX, CD11C, ITGB1, CD29, ITGB2, ITGB7, TNFR2, ACAR 1, NKGAM 6, TAMGW 6, NKGL 2, NKGA 6, TARG, CD11 bp, NKGA 6, NKGA 1, CD11 bp, CTGA 6, CTGB 1, ITGAD, CTGL 4, and NKGL 6.

3. The CD 19-targeted chimeric antigen receptor according to claim 1, wherein the CD 19-targeted antigen binding domain is a CD 19-targeted single-chain antibody, the amino acid sequence of the heavy chain variable region of the CD 19-targeted single-chain antibody is shown as SEQ ID No.2, and the amino acid sequence of the light chain variable region of the CD 19-targeted single-chain antibody is shown as SEQ ID No. 3.

4. The chimeric antigen receptor targeting CD19 according to claim 3, wherein said hinge region is a CD8 a hinge region having the amino acid sequence shown in SEQ ID No. 4;

and/or the transmembrane domain is a CD8 alpha transmembrane region with the amino acid sequence shown as SEQ ID NO. 5;

and/or the amino acid sequence of the 4-1BB is shown as SEQ ID NO. 6;

and/or the amino acid sequence of ITAM1 of CD3 zeta is shown in SEQ ID NO. 7.

5. The CD19 targeted chimeric antigen receptor according to any one of claims 1 to 4, wherein said CD19 targeted chimeric antigen receptor further comprises a signal peptide at the amino terminus linked to the CD19 targeted antigen binding domain.

6. The CD19 targeted chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the CD19 targeted chimeric antigen receptor is shown as SEQ ID No. 8.

7. An isolated nucleic acid for expressing a CD 19-targeted chimeric antigen receptor of any one of claims 1 to 6.

8. The isolated nucleic acid of claim 7, wherein the isolated nucleic acid comprises a nucleic acid fragment having a nucleotide sequence set forth in SEQ ID No. 9.

9. A recombinant vector comprising the isolated nucleic acid of claim 7 or 8.

10. A CAR-T cell comprising the isolated nucleic acid of claim 7 or 8 or transformed with the recombinant vector of claim 9.

11. Use of a CD 19-targeted chimeric antigen receptor according to any one of claims 1 to 6, an isolated nucleic acid according to claim 7 or 8, a recombinant vector according to claim 9 or a CAR-T cell according to claim 10 for the preparation of a medicament for the treatment of cancer.

12. A pharmaceutical composition comprising an expression vector for expressing a CD 19-targeted chimeric antigen receptor according to any one of claims 1 to 6 or a CAR-T cell according to claim 10, and a pharmaceutically acceptable excipient.

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