CN115806625B - Chimeric antigen receptor expressed by T cell limitation and application thereof - Google Patents

Abstract

The invention relates to the field of biological medicine, in particular to a chimeric antigen receptor expressed by T cells in a limited way and application thereof. The chimeric antigen receptor includes an antigen binding domain, a linking domain, and a co-stimulatory domain; the linking domain comprises at least one of a TCR alpha, TCR beta, cd3δ, cd3ε, and a cd3γ subunit, or functional fragment thereof, that transmembrane links the antigen binding domain and the co-stimulatory domain.

Description

Chimeric antigen receptor expressed by T cell limitation and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a chimeric antigen receptor expressed by T cells in a limited way and application thereof.

Background

T cells are an important member of the vertebrate immune system and play an important role in viral infection, cancer and autoimmune processes. T cells mediate immune responses by recognizing antigenic peptides/Major Histocompatibility Complexes (MHC) through T Cell Receptors (TCRs) on their cell membranes. Most mature T cell (about 95%) TCRs consist of two heterodimeric peptide chains of α and β linked by disulfide bonds, the variable regions of tcra/β (vα and vβ) being responsible for recognition of antigen signals, tcra/β forming a TCR-CD3 receptor complex with co-receptor CD3 containing six subunits of γ/epsilon, δ/epsilon 'and ζ/ζ' for signaling, which complex determines T cell development, activation and immune response to pathogens. Eight subunits in the TCR-CD3 complex are type I transmembrane proteins, and both TCR a and TCR β comprise an extracellular domain (ECD), a membrane-proximal Connecting Peptide (CP), a transmembrane domain and a very short intracellular segment. Similarly, the subunits cd3γ, δ, and ε of each CD3 comprise an extracellular immunoglobulin domain, a short CP, a transmembrane domain, and an intracellular domain (only one ITAM), and cd3ζ has a very short extracellular domain, a transmembrane domain, and an intracellular domain (3 ITAMs).

Cell surface polyprotein complexes are synthesized in the Endoplasmic Reticulum (ER), where they are integrated and assembled. TCR-CD3 complexes are typical polyprotein complexes, whose assembly occurs on the ER membrane, then transported to the golgi apparatus for further processing modification, and finally transported to the cell membrane; wherein ER plays a very important role in the quality control of TCR-CD3 complex. Unassembled tcrα and tcrβ may reside in the Endoplasmic Reticulum (ER) and degrade in a relatively short period of time. The study found that chimeric proteins containing a TCR alpha transmembrane domain and a TCR beta transmembrane domain have a very short life cycle in ER, t _1/2 Only 10-15min; even disulfide-linked TCRab diisomers or unassembled tcra alpha, which degrade rapidly in the ER, t _1/2 And only 35-45min. Unassembled subunits are transported into the proteasome for degradation. The assembly of TCR-CD3 is mediated by ECD, CP and transmembrane regions, and strictly adheres to the following three rules: 1) TCRαβ binds first to CD3Δε, 2) then CD3γε is added to this intermediate complex, 3) finally CD3ζζ is incorporated to form the complete TCR-CD3 complex. Without cd3ζζ, the aforementioned partially assembled TCR-CD3 complex is rapidly internalized. It was found that expression of the complete TCR-CD3 complex on the cell membrane requires co-participation of all its subunits.

Complete activation of T cells requires three signals: 1) a first stimulation signal provided by peptide/MHC via TCR, 2) a second stimulation signal provided by co-stimulatory ligand via co-stimulatory receptor, 3) a third stimulation signal provided by cytokine. The binding of TCR and peptide/MHC triggers Lck to phosphorylate CD3 zeta intracellular ITAM, and ZAP-70 then binds to CD3 zeta phosphorylated tyrosine, autophosphorylating to complete activation. The activated ZAP-70 will then phosphorylate tyrosine on various adaptor molecules, such as LAT. Phosphorylated LAT activates many downstream signaling pathways involving transcription factors such as transcription factors AP-1, NF-. Kappa.B and NFAT. These downstream signaling events activate, proliferate, produce cytokines, and function as a result of T cells.

CAR-T, a chimeric antigen receptor-T cell, is an artificial engineered receptor expressed on the surface of T cell membranes, thereby conferring a certain effector function to T cells. Throughout the history of CAR-T development, the first generation of CAR-T has only the CD3 zeta chain; the second generation CAR-T has added a costimulatory domain such as CD28 or 4-1BB, such as Kymriah and Yescanta; the third generation CAR-T is added with two co-stimulatory domains such as CD28, 4-1BB and the like; the fourth generation CAR-T is added with coexpression cytokines such as IL-12 and the like on the basis of the second generation; fifth generation CAR-T is also based on the second generation, adding co-stimulatory domains that activate other signaling pathways, such as the domain of IL2-2rβ that binds STAT3/5 intracellular. The 5-generation CAR-T designs described above, while all of which are structures engineered to combine different signaling molecule domains, are limited in scope by the direct use of the original domain. The CAR-T cells in reality more or less suffer from the following problems during production and application: 1) cell depletion, 2) cytokine release syndrome, 3) short in vivo cell duration, etc. These problems are rarely found in the natural T cell immune process of the human body, and therefore the present invention has been proposed based on these observations.

Disclosure of Invention

A first object of the present invention is to provide a chimeric antigen receptor comprising an antigen binding domain, a linking domain, and a co-stimulatory domain;

the linking domain comprises at least one of a TCR alpha, TCR beta, cd3δ, cd3ε, and a cd3γ subunit, or functional fragment thereof, that transmembrane links the antigen binding domain and the co-stimulatory domain.

It is a second object of the present invention to provide an isolated nucleic acid which is expressed to give a chimeric antigen receptor as described above.

A third object of the present invention is to provide a vector containing the nucleic acid as described above.

It is a fourth object of the present invention to provide a T cell comprising a nucleic acid or vector as described above, or a chimeric antigen receptor as described above on the surface of the cell membrane.

It is a fifth object of the present invention to provide a pharmaceutical composition comprising T cells as described above.

A sixth object of the present invention is to provide the use of T cells as described above for the preparation of a medicament for the prevention and/or treatment of tumors.

The beneficial effects of the invention are as follows:

1) Because the structure needs to be assembled with other TCR-CD3 subunits to be expressed on a T cell membrane, the structure can be expressed only in T cells but not in other cells, and the characteristic of limited expression of the T cells is achieved;

2) The CAR structure can use the Signaling Domain of the TCR itself (Signaling Domain) to deliver a killing signal whose downstream Signaling pathway is more similar to that triggered by the native TCR-antigen peptide/MHC. Therefore, in theory, the structure has the potential advantages of weaker strong signals, difficult exhaustion, lower risk of CRS and safer.

3) The CAR provides a co-stimulatory domain that allows for better activation of T cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the design of various T-CARs in one embodiment of the invention; A. structural differences in the design of traditional second generation CARs and T-CARs; B.T-CAR theoretical molecular structure;

FIG. 2 shows proliferation of T-CAR group cells according to one embodiment of the present invention;

FIG. 3 is a graph showing positive rate detection of expression of T-H26B 6-gamma CAR T cells and T-H27H 4-epsilon CAR T cells CAR in one embodiment of the invention;

FIG. 4 is an illustration of the expression of T-H26B 6-. Gamma.CAR and T-H27H 4-. Epsilon.CAR in RPMI8226 and HEK293T cells according to one embodiment of the invention;

FIG. 5 is a graph showing the killing of target cells by various effector cells in one embodiment of the invention; A. killing Siha cells by cells in different target ratios; B. killing effect of cells on U937 cells by different effect target ratios;

FIG. 6 is a graph showing secretion of the cytokine IFN-gamma when various effector cells kill target cells in accordance with one embodiment of the present invention; A. secretion of killer IFN-gamma of Siha cells by effector cells; B. secretion of the killer IFN-gamma by effector cells on U937 cells.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.

The recitation of numerical ranges by endpoints of the present invention includes all numbers and fractions subsumed within that range, as well as the recited endpoint.

The present invention relates to a chimeric antigen receptor comprising an antigen binding domain, a linking domain, and a co-stimulatory domain;

the linking domain comprises at least one of a TCR alpha, TCR beta, cd3δ, cd3ε, and a cd3γ subunit, or functional fragment thereof, that transmembrane links the antigen binding domain and the co-stimulatory domain.

The present invention provides a novel CAR structure that is defined for expression in T cells. The killing function of the CAR-T cells constructed by the CAR is not weaker than that of the traditional second-generation CAR T cells, and less cytokines are secreted, so that side effects such as inflammatory factor storm and the like can be effectively avoided or reduced.

In some embodiments, the linking domain is capable of performing the function of the signaling domain of the TCR itself to transmit a killing signal.

Useful fragments are, for example, the following full length fragments or partial fragments thereof:

TRAC：UniProtKB，P01848，140aa；

//IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS//

TRBC1：UniProtKB，P01850，176aa；

//DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF//

TRBC2：UniProtKB，A0A5B9，178aa；

//DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG//

CD3δ：GenBank accession number，NP_000723，171aa；

//MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK//

CD3ε：GenBank accession number，NP_000724，207aa；

//MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI//

CD3γ：GenBank accession number，NP_000064，182aa；

//MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN//

as used herein, a "Chimeric Antigen Receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a polypeptide other than the polypeptide from which the extracellular domain was derived, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is sometimes referred to as "chimeric receptor", "T-body" or "Chimeric Immune Receptor (CIR)". An "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide that can bind to a particular antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits a signal to cause activation or inhibition of a biological process.

As used herein, a "region" or "domain" comprised in the chimeric antigen receptor refers to a region in a polypeptide that can be folded into a specific structure independently of the other regions. These "regions" or "domains" may be sequences of murine or other animal origin, preferably human origin.

As used herein, "antigen recognition domain" refers to a domain capable of specifically recognizing and binding an antigen, including but not limited to: single chain variable fragments, alpaca antibodies, ligands, etc., recognize a single or two antigen targets, respectively. "Single chain variable fragment (sc-Fv)" refers to a single chain polypeptide derived from an antibody that retains the ability to bind an antigen. Examples of sc-Fv include antibody polypeptides formed by recombinant DNA techniques and wherein the Fv regions of the immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via a spacer sequence. Various methods of preparing sc-Fv are known, including those described in: U.S. patent No. 4694778; science, volume 242, pages 423-442 (1988); nature, volume 334, page 54454 (1989); and Science, volume 242, pages 1038-1041 (1988).

In some embodiments, the chimeric antigen receptor does not contain a hinge region, such as the range region of CD8 a.

In some embodiments, the chimeric antigen receptor does not contain a CD28 transmembrane region.

In some embodiments, the co-stimulatory domain includes any one of CD28, 4-1BB, OX40, ICOS, CD27, CD40-MyD88, DAP12, DAP10, 2B4, or any combination thereof;

in some embodiments, the co-stimulatory domain is 4-1BB and the amino acid sequence is SEQ ID NO:1 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).

In some embodiments, the antigen recognition domain region specifically recognizes a tumor antigen. "tumor antigen" refers to a biological molecule that is antigenic, and its expression has been recently recognized as being associated with cellular canceration. Detection, for example, immunological detection of tumor antigens can be used to distinguish between cancerous cells and their parent cells. Tumor antigens in the present invention include tumor-specific antigens (antigens that are present only in tumor cells but not in other normal cells) and tumor-associated antigens (antigens that are also present in other organs and tissues or in heterogeneous and allogeneic normal cells, or antigens that are expressed during development and differentiation). In some embodiments, the tumor is a hematological tumor or a solid tumor, in some embodiments, the solid tumor comprises: bone, bone connection, muscle, lung, trachea, heart, spleen, artery, vein, capillary, lymph node, lymphatic vessel, lymph fluid, mouth, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gall bladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, oviduct, uterus, vagina, external pudendum, scrotum, testis, vas deferens, penis, eye, ear, nose, tongue, skin, brain stem, medulla oblongata, spinal cord, cerebrospinal fluid, tumors produced by lesions in any of nerves, thyroid, parathyroid, adrenal gland, pituitary gland, pineal gland, islet, thymus, gonad, sublingual gland. In particular, it is preferred that the contemplated tumor can be targeted, for example, cholangiocarcinoma, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-hodgkin's lymphoma, multiple myeloma, renal cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, and urinary bladder cancer.

In some embodiments, the antigen recognition domain specifically recognizes and binds to at least one of the following antigen molecules:

alpha-fetoprotein, alpha-actin-4, A3, antigen specific for the A33 antibody, ART-4, B7H3, ba 733, BAFF-R, BAGE, BCMA, brE3 antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m, CCL19, CCL21, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32B, CD33, CD37, CD38, CD40 8234, CD45, CD46, CD52, CD54, CD55, CD56, CD59, CD64, CD66a/B/c/e, CD67, CD70L, CD, CD79a, CD79B, CD80, CD83, CD95, CD123, CD126, CD 132; CD133, CD138, CD147, CD154, CD319, CD371, CDC27, CDK-4/m, CDKN2A, CLL1, CTLA4, CXCR7, CXCL12, HIF-1α, colon specific antigen p (CSAp), CEA, CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, ep-CAM, fibroblast growth factor, flt-1, flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO- β, GPRC5D, HLA-DR, HM1.24, human Chorionic Gonadotropin (HCG) and subunits thereof, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), 70-2M, HST-2, ia, IGF-1R, IFN- γ, IFN- α, IFN- β, IFN- λ, IL-4R, IL-6R, IL-13-65-R, IL-3723-R, IL-37-18, IL-12, IL-8, and the like, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor 1, igkappa, IL1RAP, lewis Y, LMP1, KC4 antigen, KS-1 antigen, KS1-4, le-Y, LDR/FUT, macrophage migration inhibitory factor, MAGE-3, MART1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MMG49, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, NKG2D, pancreatic cancer mucin, PD-L1, placental growth factor p53, PLAGL2, prostatectomy phosphatase, PSA, PRAME, PSMA, plGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, ROR1, T101, SAGE, S100, survivin, TAC, TAG-72, tenascin, TRAIL receptor, TNF-alpha, tn antigen, thomson-Fredendrin antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factor C3, C3a, C3B, C5a, C5, angiogenesis markers, bc1-2, bc1-6, kras, oncogene markers, and oncogene products.

In some embodiments, the antigen recognition domain is a single chain antibody of B7H3, preferably having an amino acid sequence of SEQ ID NO:2 (EIVLTQSPATLSLSPGERATLSCSASSSVSYMQWYQQKPGLAPRLLIYDTSKLTSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKRTVGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQGLEWIGGINPNTGGTTYNQKFNGRVTMTRDTSISTAYMELSSLRSEDTAVYYCTRPYRDDGGFHWYFDVWGQGTLVTVSS).

In some embodiments, the antigen recognition domain is a single chain antibody of CLL1, preferably having an amino acid sequence of SEQ ID NO:3 (DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSS).

In some embodiments, the CD3 epsilon amino acid sequence is SEQ ID NO:4 (DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI).

In some embodiments, the CD3 γ amino acid sequence is SEQ ID NO:5 (QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN).

In some embodiments, the antigen binding domain and the linking domain are directly linked by a linking peptide, e.g., SEQ ID NO:6 (GSTSGSGKPGSGEGSTKG).

In some embodiments, the N-terminus of the antigen binding domain is further provided with a signal peptide.

Useful signal peptides are, for example:

in addition, there are:

Human OSM:MGVLLTQRTLLSLVLALLFPSMASM

VSV-G:MKCLLYLAFLFIGVNC

Mouse Ig Kappa:METDTLLLWVLLLWVPGSTGD

Human IgG2 H:MGWSCIILFLVATATGVHS

BM40:MRAWIFFLLCLAGRALA

Human Chymotrypsinogen:MAFLWLLSCWALLGTTFG

Human trypsinogen-2:MNLLLILTFVAAAVA

Human IL-2:MYRMQLLSCIALSLALVTNS

Gaussia luc:MGVKVLFALICIAVAEA

Albumin(HSA):MKWVTFISLLFSSAYS

Influenza Haemagglutinin:MKTIIALSYIFCLVLG

Human insulin:MALWMRLLPLLALLALWGPDPAAA

Silkworm Fibroin LC:MKPIFLVLLVVTSAYA

it will be appreciated that the "region" or "domain" or "functional fragment" referred to herein, or other polypeptides (e.g. connecting peptides and signal peptides), may be selected from the exemplified sequences corresponding to each of them, e.g. SEQ ID NO: 1-5, or a sequence having at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity thereto, and retaining substantially similar sequence corresponding to the desired function.

Substantially similar sequences also retain the desired activity of the polypeptide. Substitutions generally considered to be conservative substitutions are those in aliphatic amino acids Ala, val, leu and Ile with each other, the exchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the exchange between amide residues Asn and Gln, the exchange of basic residues Lys and Arg, and the exchange between aromatic residues Phe, tyr. Further, substantially similar sequences also include modified sequences that differ little from the desired function, such as phosphorylation, glycosylation, ubiquitination, and the like.

The invention also relates to isolated nucleic acids, the expression of which results in chimeric antigen receptors as described above.

The invention also relates to vectors containing the nucleic acids as described above.

The term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). In some embodiments, the vectors of the invention comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal Ribosome Entry Sites (IRES) and other expression control elements (e.g., transcription termination signals, or polyadenylation signals, and poly U sequences, etc.).

The vector may also be a composition, e.g., different nucleic acids of different segments may be located on different vectors.

In some specific embodiments of the present disclosure, the vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a CRISPR/CAS plasmid.

The invention also relates to T cells containing a nucleic acid as described above or a vector as described above, or having a chimeric antigen receptor as described above expressed on the surface of the cell membrane.

In some embodiments, the T cell is any one of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a MAIT cell, a γδ T cell.

According to a further aspect of the invention, it also relates to a pharmaceutical composition comprising T cells as described above.

The pharmaceutical composition may also include a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" includes any material that, when combined with an active ingredient, allows the ingredient to remain biologically active and non-reactive with the subject's immune system. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, and any of various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are Phosphate Buffered Saline (PBS) or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well known conventional methods (see, e.g., remington's Pharmaceutical Sciences, 18 th edition, a. Gennaro, mack Publishing co., easton, PA,1990; and Remington, the Science and Practice of Pharmacy, 21 st edition, mack Publishing, 2005).

According to a further aspect of the invention, it also relates to the use of a T cell as described above for the preparation of a medicament for the prevention and/or treatment of tumors.

According to a further aspect of the invention, it also relates to the use of a host cell as described above for the preparation of a medicament for prophylaxis and/or therapy.

According to yet another aspect of the present invention, there is also a method of treating a tumor in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a host cell or pharmaceutical composition as described above.

A "patient" is a mammal, including but not limited to humans, monkeys, pigs and other farm animals, sports animals, pets, primates, horses, dogs, cats, rodents (including mice, rats, guinea pigs), etc.

Embodiments of the present invention will be described in detail below with reference to examples.

Example 1 design of chimeric antigen receptor for T-CAR and construction of lentiviral expression vector

This example uses single chain antibodies (scFv) as antigen binding domains against CLL1 and B7H3 antibodies, binding to various subunits of signal peptide, TCR/CD3 complex, different co-stimulatory domains, constructing chimeric antigen receptor T-CAR targeting either CLL1 or B7H3 for novel T cell-restricted expression, common co-stimulatory domains including 4-1BB, CD28, DAP10, etc. The schematic diagram is shown in fig. 1. After obtaining the amino acid sequences of the individual subunits of the TCR-CD3 complex, full gene synthesis was performed at guangzhou Ai Ji biotechnology limited, connecting ScFv and co-stimulatory domains, and constructed into lentiviral expression vectors.

The amino acid sequence of H27H4-CD3 epsilon-bb used in this example is:

//MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSSGSTSGSGKPGSGEGSTKGDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL//

the amino acid sequence of H26B6-CD3 gamma-bb is:

//MDMRVPAQLLGLLLLWLRGARCEIVLTQSPATLSLSPGERATLSCSASSSVSYMQWYQQKPGLAPRLLIYDTSKLTSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKRTVGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTEYTMHWVRQAPGQGLEWIGGINPNTGGTTYNQKFNGRVTMTRDTSISTAYMELSSLRSEDTAVYYCTRPYRDDGGFHWYFDVWGQGTLVTVSSGSTSGSGKPGSGEGSTKGQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL//

example 2: lentivirus package

The lentiviral expression vectors in the examples were individually lentivirally packaged using a four plasmid system, comprising the following steps:

(1) The four-plasmid system expresses gag/pol, rev, VSV-G required by lentiviral vector packaging and the T-CAR expression vector constructed by the invention respectively: transiently transfecting 293T cells with the four plasmids, wherein the DNA content is 2 mug/mL;

(2) Mixing the plasmid with PEI transfection reagent, adding into serum-free DMEM, mixing, standing for 15min, adding the mixture into T75 culture flask paved with 293T cells, mixing gently, and heating at 37deg.C and 5% CO ₂ Culturing the cells in a cell culture box for 6 hours;

(3) After 6 hours, the fresh medium was changed, the culture was continued, and 10mM sodium butyrate solution was added, and after 72 hours, the culture supernatant of lentivirus was collected for purification detection.

(4) The detection of viral titers was performed using a lentiviral titer ELISA detection kit (beijing bolone, BF 06203).

Example 3: lentivirus infection of T cells

Taking 10ml of whole blood from each volunteer, diluting peripheral blood with normal saline at a ratio of 1:1, adding Ficoll into a centrifuge tube, slowly adding diluted peripheral blood, centrifuging at 1500rpm for 30min, gently sucking the PBMC layer, and transferring into another centrifuge tube; PBMCs were washed with physiological saline several times, CD 3T cells were sorted using CD3 sorting beads (methone), and cultured in X-VIVO medium (300 IU/mL IL-2), and PBMCs were separated and then purified at a bead ratio=1: 1, and adding CD3-CD28 stimulating magnetic beads for stimulation (Thermo), and carrying out virus infection after 48 hours.

The infection efficiency of the slow virus on T cells is improved by utilizing the retroNectin, 30 mug of retroNectin is coated in a 6-hole plate and placed in a cell incubator at 37 ℃ for 2 hours; after blotting retroNectin and blocking the coating with Hank's solution containing 2.5% BSAIs placed in a cell incubator at 37 ℃ for 0.5h; sucking the blocking solution, washing a 6-hole plate by using Hank's solution containing 2% hepes, adding an X-VIVO culture medium, adding a proper amount of lentiviral solution, and centrifuging for 2h at 2000 g; the supernatant was discarded and 1X 10 was added ⁶ Is centrifuged at 1000g for 10min at 37℃and 5% CO ₂ And culturing in a cell culture box with a certain humidity, and repeating the process on the second day. The beads were removed on day 7. Counts were taken every two days and X-VIVO was replaced with 300IU/mL and cell concentration was maintained at 0.5X10 ⁶ -1×10 ⁶ /mL, continuous culture to day7 post virus infection. The T-CAR T cells were evaluated for expansion by counting with a Countstar IC1000 automatic cytometer.

As can be seen from fig. 2, viral infection did not negatively affect T cell proliferation, even though T-CAR group cells proliferated at a much faster rate than the control cells.

Example 4: expression of T-CARs in T cells

We prepared T-H26B 6-ycar T cells (targeting B7H 3) and T-H27H 4-epsilon CAR T cells (targeting CLL 1) and sampled for detection at day 5 and day7, respectively, after virus infection.

Cells were collected by centrifugation at 300g for 5min, washed with PBS containing 1% BSA, then incubated with cells with a solution containing 2.5ug/mL of CLL1-Fc protein (ACRO) and B7H3-Fc protein (ACRO) at room temperature for 40min, followed by washing with PBS containing 1% BSA, then incubated with a solution of the coat-pAb-to Hu IgG (PE) secondary antibody at room temperature for 40min in the absence of light, washed with PBS containing 1% BSA, then resuspended with 200uL of PBS, and finally flow-examined for T-CAR expression.

As can be seen in fig. 3, all 2T-CARs can be highly expressed on primary T cell membranes. On day 5 (day 5) after virus infection, the positive rate of T-H26B 6-. Gamma.CAR was 82.1% and that of T-H27H 4-. Epsilon.CAR was 82.7%; at Day7, the positive rate of T-H26B 6-. Gamma.CAR was 78.9%, and the positive rate of T-H27H 4-. Epsilon.CAR was 82.2%. These results demonstrate that all 2T-CARs can be stably expressed in primary T cells.

Example 5: expression of T-CARs in non-T cells

To demonstrate that T-CARs cannot be expressed in non-T cells, the present invention infects RPMI8226 cells (human multiple myeloma cells, belonging to B cells) and HEK293T cells (human embryonic kidney cells) with T-H26B 6-gamma CAR lentiviral vectors and T-H27H 4-epsilon CAR lentiviral vectors, and collects cells for flow detection on day 5 post-infection. Cells were first trypsinized, centrifuged at 300g for 5min to remove supernatant, washed with PBS containing 1% BSA for one time, then incubated with cells with 2.5ug/mL of CLL1-Fc protein (ACRO) and B7H3-Fc protein (ACRO) respectively for 40min at room temperature, then washed with PBS containing 1% BSA for one time, then incubated with a solution of the coat-pAb-to Hu IgG (PE) secondary antibody for 40min at room temperature in the absence of light, washed with PBS containing 1% BSA for one time, then resuspended with 200uL of PBS, and finally subjected to flow assay for T-CAR expression, the results of which are shown in FIG. 4.

As can be seen from the figure, neither RPMI8226 cells nor HEK293T cells express T-H26B 6-gamma CAR and T-H27H 4-epsilon CAR.

Example 6: killing function of T-CAR T cells in vitro

Target cells SiHa-luc and U937-luc stably expressing luciferase were seeded into black opaque 96-well plates, 2 ten thousand cells/well, and then at an effective target ratio=3: 1 and 10:1, and respectively inoculating effector cells into the wells, wherein the effector cells comprise Control T cells, T-H26B 6-gamma CAR T cells, T-H27H 4-epsilon CAR T cells, H26B6-CAR T cells and H27H4-CAR T cells. Incubating in an incubator for 4-6h after inoculation, adding an equal volume of luciferase substrate (Promega), incubating for 5min at room temperature in a dark place, and detecting on a multifunctional enzyme-labeled instrument. The killing rate is calculated according to the formula: % = (1-RLU _{Experimental group} /RLU _{Target Only group} )×100％。

As can be seen from fig. 5, T-CAR T does not kill target cells less than conventional second generation CAR T cells. Both T-CAR T cells and conventional CAR T cells are capable of specifically killing target cells.

Example 7: cytokine release function of T-CAR T

Target cells SiHa-luc and U937-luc stably expressing luciferase were seeded into round bottom 96-well plates, 2 ten thousand cells/well, then at 3:1 and 10:1, respectively inoculating effector cells into the wells, wherein the effector cells comprise Control T cells, T-H26B 6-gamma CAR T cells, T-H27H 4-epsilon CAR T cells, H26B6-CAR T cells and H27H4-CAR T cells. Incubating in an incubator for 24h after inoculation, centrifuging for 15min with 500g, collecting supernatant, performing ELISA to detect IFN-gamma content, processing according to the specification of IFN-gamma detection kit (4A bio), and detecting on a multifunctional enzyme-labeled instrument.

As can be seen in fig. 6, both T-CAR T cells and conventional CAR T cells can specifically secrete IFN- γ killer target cells. In addition, T-CAR T cells secrete less amount of IFN- γ relative to traditional CAR T cells.

In combination with the results of example 6, T-CAR T secreted less IFN- γ in cases where the killing effect was not weaker than traditional CAR T; this suggests that T-CAR T has lower CRS risk.

In summary, the present invention provides a novel CAR structure that is restricted to expression in T cells. The killing function of the CAR-T cells constructed by using the CAR is not weaker than that of the traditional second-generation CAR T cells, and less cytokines are secreted.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.

Sequence listing

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Claims (8)

1. A chimeric antigen receptor comprising an antigen binding domain, a linking domain, and a co-stimulatory domain;

the linking domain is CD3 epsilon, which transmembrane links the antigen binding domain and the co-stimulatory domain;

the co-stimulatory domain is 4-1BB, and the amino acid sequence is SEQ ID NO:1 is shown in the specification;

the antigen binding domain is a single-chain antibody of B7H3, and the amino acid sequence of the single-chain antibody is SEQ ID NO:2 is shown in the figure;

the CD3 epsilon amino acid sequence is SEQ ID NO: 4.

2. The chimeric antigen receptor according to claim 1, the N-terminus of the antigen binding domain further provided with a signal peptide.

3. An isolated nucleic acid which is expressed to give the chimeric antigen receptor according to any one of claims 1 to 2.

4. A vector comprising the nucleic acid of claim 3.

5. A T cell comprising the nucleic acid of claim 3 or the vector of claim 4, or having the chimeric antigen receptor of any one of claims 1 to 2 expressed on the surface of the cell membrane.

6. The T cell of claim 5, wherein the T cell is any one of a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a MAIT cell, a γδ T cell.

7. A pharmaceutical composition comprising the T cell of claim 5 or 6.

8. Use of the T cell of claim 5 or 6 in the manufacture of a medicament for the prevention and/or treatment of a tumor.