WO2021073626A1

WO2021073626A1 - Chimeric antigen receptor and t cells expressing chimeric antigen receptors therein

Info

Publication number: WO2021073626A1
Application number: PCT/CN2020/121674
Authority: WO
Inventors: 芦志华; 杜宝华; 刘永峰; 王方圆; 戴卫国; 朱滨
Original assignee: 北京门罗生物科技有限公司
Priority date: 2019-10-17
Filing date: 2020-10-16
Publication date: 2021-04-22
Also published as: CN114616239A; CN114616239B

Abstract

The present invention discloses a chimeric antigen receptor and T cells expressing the chimeric antigen receptor therein, and specifically discloses a chimeric antigen receptor targeting CD3, the chimeric antigen receptor comprises a CD3 binding domain; a hinge region and a transmembrane domain; a co-stimulatory domain; and a signal transduction domain. The present invention further discloses nucleic acid molecules encoding the chimeric antigen receptor and an expression vector, host cells and a pharmaceutical composition and a kit comprising the same. The chimeric antigen receptor of the present invention can be used in immunotherapy and anti-transplant rejection reaction, and has a huge market application value.

Description

Chimeric antigen receptor and T cell expressing the chimeric antigen receptor

Technical field

The present invention relates to the field of immunotherapy, and relates to chimeric antigen receptors and T cells expressing the chimeric antigen receptors.

Background technique

Immunotherapy is becoming a very promising cancer treatment method. T cells or T lymphocytes are the armed forces of our immune system, which constantly look for foreign antigens and distinguish abnormal (cancer or infected cells) from normal cells. Genetic modification of T cells with CAR is a common method for designing tumor-specific T cells. CAR-T cells targeting tumor-associated antigens can be infused into patients (called adoptive T cell therapy), which represents an effective immunotherapy method. Compared with chemotherapy or antibodies, the advantage of CAR-T technology is that reprogrammed engineered T cells can proliferate and persist in the patient's body, acting like living drugs.

CAR-T therapy for tumor immunotherapy, CAR-T, full name Chimeric Antigen Receptor T-Cell Immunotherapy, namely chimeric antigen receptor T-cell immunotherapy; the principle is that T cells modified by chimeric antigen receptors can specifically Recognizing tumor-associated antigens enables effector T cells to have higher targeting, killing activity and durability than conventionally used immune cells, and can overcome the tumor local immunosuppressive microenvironment and break the host's immune tolerance state. Chimeric antigen receptor (CAR) is the core component of CAR-T, which gives T cells the ability to recognize tumor antigens in an HLA-independent manner. This makes CAR-modified T cells more capable of recognizing than natural T cell surface receptor TCR. Broad goals. CAR (Chimeric Antigen Receptor) is mainly composed of three functional domains, namely the extracellular domain, transmembrane domain and intracellular domain. The extracellular domain is composed of a single-chain variable fragment (scFv) of a monoclonal antibody that is responsible for recognizing and binding antigens and a hinge region (Hinge) that functions as a connection. The intracellular domain consists of a costimulatory domain (Costimulatory Domain) and a signal transduction domain (Signaling Domain). Successful use of CAR-T targeting CD19 in patients with advanced CLL and ALL (Porter et al., 2011, N Engl J Med, 365: 725-33; Kalos et al., 2011, Science Transl Med, 3:

95ra73; Grupp and Kalos, 2013, N Engl J Med, 368:1509-18) showed that these cells can eradicate huge tumor burden after a single infusion, and the relief effect has lasted up to 3 years, thus emphasizing CAR T cells The great potential of therapy.

So far, there have been no reports of CAR-T targeting CD3 and its application in the treatment of malignant tumors.

Summary of the invention

One of the objectives of the present invention is to provide the application of CD3 in preparing a chimeric antigen receptor targeting CD3.

The second objective of the present invention is to provide the application of an antibody against CD3 or an antigen-binding fragment thereof in preparing a chimeric antigen receptor targeting CD3.

The third objective of the present invention is to provide a chimeric antigen receptor targeting CD3 or a CAR-T containing the same.

The fourth object of the present invention is to provide the application of the aforementioned CD3-targeting chimeric antigen receptor or CAR-T cell in immunotherapy.

The fifth object of the present invention is to provide the application of the aforementioned CD3-targeting chimeric antigen receptor or CAR-T cell in anti-transplant rejection.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

According to the first aspect of the present invention, the present invention provides the use of CD3 in preparing a chimeric antigen receptor targeting CD3.

According to the second aspect of the present invention, the present invention provides the use of an antibody against CD3 or an antigen-binding fragment thereof in the preparation of a chimeric antigen receptor targeting CD3.

Antigen-binding fragments can be Fab fragments (Fab), F(ab') ₂ fragments, double-chain antibodies, tri-chain antibodies, quadru-chain antibodies, single-chain variable region fragments (scFv), or disulfide stabilized variable region fragments (dsFv). In a preferred embodiment, the antigen-binding fragment is a scFv. The scFv is a truncated Fab fragment that contains the V domain of the antibody heavy chain connected to the variable (V) domain of the antibody light chain via a synthetic peptide, which can be generated using conventional recombinant DNA technology.

According to the third aspect of the present invention, the present invention provides a chimeric antigen receptor targeting CD3, the chimeric antigen receptor comprising a CD3 binding domain.

Further, the chimeric antigen receptor includes from N-terminal to C-terminal: a CD3 binding domain, a hinge region, a transmembrane domain, and a signal transduction domain.

Optionally, the chimeric antigen receptor includes from N-terminus to C-terminus: a CD3 binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signal transduction domain.

In a specific embodiment of the present invention, the chimeric antigen receptor includes from N-terminus to C-terminus: CD3 binding domain, hinge region, transmembrane domain, costimulatory domain, and signal transduction domain.

The CD3 binding domain can comprise any antigen binding portion of a CD3 antibody. For example, the CD3 binding domain can be a Fab fragment (Fab), F(ab') ₂ fragment, double-chain antibody, tri-chain antibody, four-chain antibody, single-chain variable region fragment (scFv), or disulfide stabilized Variable region fragment (dsFv). In a preferred embodiment, the CD3 binding domain is scFv. The scFv is a truncated Fab fragment that contains the V domain of the antibody heavy chain connected to the variable (V) domain of the antibody light chain via a synthetic peptide, which can be generated using conventional recombinant DNA technology. However, the CD3 binding domain used in the CAR of the present invention is not limited to these exemplary types of antibody fragments.

The CD3 binding domain may comprise a light chain variable region and/or a heavy chain variable region. In an embodiment of the present invention, the heavy chain variable region includes one or more of complementarity determining region (CDR) 1, CDR2, and CDR3. In a preferred embodiment, the CD3 binding domain comprises a human heavy chain CDR1, a human heavy chain CDR2, and a human heavy chain CDR3.

The heavy chain CDR1 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 50 Amino acid sequence of

The heavy chain CDR2 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 40, SEQ ID NO: 51 Amino acid sequence of

The heavy chain CDR3 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 41, SEQ ID NO: 52 The amino acid sequence.

In an embodiment of the present invention, the light chain variable region comprises complementarity determining region (CDR) 1, CDR2, and CDR3. In a preferred embodiment, the CD3 binding domain comprises a human light chain CDR1, a human light chain CDR2, and a human light chain CDR3.

The light chain CDR1 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 42, SEQ ID NO: 53 Amino acid sequence of

The light chain CDR2 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 54 Amino acid sequence of

The light chain CDR3 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 55 The amino acid sequence.

In an embodiment of the invention, the CD3 binding domain comprises a heavy chain variable region and a light chain variable region. In a preferred embodiment, the CD3 binding domain comprises a human heavy chain variable region and a human light chain variable region. The heavy chain variable region of the CD3 binding domain has an amino acid sequence shown in any of SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 56 An amino acid sequence with at least 95% sequence identity. The light chain variable region of the CD3 binding domain has an amino acid sequence shown in any of SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID NO: 46, SEQ ID NO: 57 An amino acid sequence with at least 95% sequence identity.

In the embodiment of the present invention, the variable region of the light chain and the variable region of the heavy chain may be connected by a linker. The linker can comprise any suitable amino acid sequence. In an embodiment of the present invention, the linker may include SEQ ID NO: 9 or its homologous sequence. The homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

In an embodiment of the present invention, the CD3 binding domain includes a CD3 binding domain that has a combination with any one of SEQ ID NO: 10, SEQ ID NO: 25, SEQ ID NO: 36, SEQ ID NO: 47, SEQ ID NO: 58 The amino acid sequence shown is an scFv with an amino acid sequence of at least 95% sequence identity.

In an embodiment of the present invention, the CD3 binding domain may also include a leader sequence, or called a signal peptide sequence. The leader sequence can be located at the amino terminus of the variable region of the light chain or the variable region of the heavy chain. Preferably, the leader sequence is located at the amino terminus of the variable region of the heavy chain. The leader sequence can include any suitable leader sequence. For example, the CD3 binding domain may include a leader sequence that contains SEQ ID NO: 11 or a homologous sequence thereof. The homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more. In the embodiments of the present invention, although the leader sequence can promote the expression of the CAR on the cell surface, the presence of the leader sequence in the expressed CAR may not be necessary for the CAR to function. In an embodiment of the present invention, after the CAR is expressed on the cell surface, all or part of the leader sequence can be excised from the CAR.

In an embodiment of the present invention, the CD3 binding domain containing the signal peptide sequence or the leader sequence has the same characteristics as SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 38, SEQ ID NO: 49, SEQ ID NO : The amino acid sequence shown in any one of 60 has an amino acid sequence with at least 95% sequence identity.

The function of the hinge region is to promote the binding of the antigen receptor to the antigen; the transmembrane domain is used to immobilize the CAR. In an embodiment of the present invention, the hinge region is a human hinge region, and the transmembrane domain is a human transmembrane domain. The hinge region and the transmembrane domain may comprise the hinge region and the transmembrane domain of any one or more of the following molecules: CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154. In a specific embodiment of the present invention, the selected hinge region and transmembrane domain may comprise the amino acid sequence of SEQ ID NO: 12 or its homologous sequence. The homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

The signaling domain can also be called the T cell activation domain, which provides the first signal for T cell activation. The most commonly used signaling domain is the CD3ζ intracellular domain. In an embodiment of the present invention, the intracellular domain of CD3ζ may include the amino acid sequence of SEQ ID NO: 13 or a homologous sequence thereof. The homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

The costimulatory domain provides the second signal of T cell activation, and includes the intracellular domain of costimulatory factors, including CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, NKG2C, B7-H3 . In an embodiment of the present invention, the costimulatory domain may include an amino acid sequence containing SEQ ID NO: 14 or a homologous sequence thereof. The homology between the homologous sequence and the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4 % Or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

In a specific embodiment of the present invention, the chimeric antigen receptor of the present invention includes SEQ ID NO: 15, SEQ ID NO: 26, SEQ ID NO: 37, SEQ ID NO: 48, SEQ ID NO: 59 The amino acid sequence shown.

The CAR of the embodiments of the present invention may comprise synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanoic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetamidomethyl-cysteine, Trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine Acid, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1, 2,3,4-Tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N '-Dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-( 2-Amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

The CAR of the embodiments of the present invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, a disulfide bridge, or converted into an acid addition salt and/or optionally Ground dimerization or multimerization.

The CAR of the embodiment of the present invention can be obtained by methods known in the art. The CAR can be prepared by any suitable method for preparing polypeptides or proteins. Suitable methods for de novo synthesis of polypeptides and proteins are known in the art. In addition, you can use, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (No. 4

Version), the standard recombination method described in Cold Spring Harbor Laboratory Press (2012), using the nucleic acid recombination described herein to produce CAR. Alternatively, the CAR described herein can be synthesized commercially by a company. In this regard, the CAR of the present invention may be synthetic and/or recombinant.

According to the fourth aspect of the present invention, the present invention provides a nucleic acid molecule encoding the aforementioned chimeric antigen receptor or its component parts.

The nucleic acid molecule of the present invention may include one or more of the leader sequence, CD3 binding domain, hinge region and transmembrane domain, signal transduction domain, costimulatory domain, and chimeric antigen receptor as described herein. Nucleotide sequence.

"Nucleic acid" as used herein includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule", and generally means a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthetic or natural Obtained from a source (for example, isolated and/or purified), it may contain natural, non-natural or altered nucleotides, and it may contain natural, unnatural or altered internucleotide linkages, Such as phosphoramidate bond or phosphorothioate bond, which replaces the phosphodiester existing between the nucleotides of the unmodified oligonucleotide. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and/or substitutions. However, in some cases it may be suitable for the nucleic acid to contain one or more insertions, deletions, inversions, and/or substitutions.

The nucleic acid of the embodiment of the present invention may be a recombinant. The term "recombinant" as used herein refers to (i) molecules constructed outside living cells by linking natural or synthetic nucleic acid segments with nucleic acid molecules that can replicate in living cells, or (ii) from the above (i) ) The molecules produced by the replication of those molecules described in. For the purposes herein, replication can be in vitro replication or in vivo replication.

The nucleic acid may consist essentially of one or more designated nucleotide sequences as described herein, so that other components (such as other nucleotides) do not substantially alter the biological activity of the encoded CAR.

The recombinant nucleic acid may be a nucleic acid having a non-naturally occurring sequence or a sequence prepared by artificial combination of two originally separated segments of the sequence. This artificial combination is usually accomplished by chemical synthesis, or more usually by artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques, such as those described in Green et al. above. The nucleic acid can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green et al. above. For example, naturally-occurring nucleotides or different modified nucleotides designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed during hybridization (such as phosphorothioate derivatives and acridine) can be used. Pyridine substituted nucleotides) to chemically synthesize nucleic acids. Examples of modified nucleotides that can be used to produce nucleic acids include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine Pyrimidine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D galactosyl Q Nucleoside (beta-D-galactosylqueosine), creatinine, N 6-isopentene adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyl Adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5 -Methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine (beta-D-mannosylqueosine), 5'-methoxycarboxymethyluracil, 5-methoxyurea Pyrimidine, 2-methylthio-N 6-isopentene adenine, uracil-5-glycolic acid (v), wybutoxosine, pseudouracil, Q nucleoside (queosine), 2-mercaptocell Pyrimidine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-hydroxyacetate, 3-(3-amino-3 -N-2-carboxypropyl)uracil and 2,6-diaminopurine.

The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any CAR described herein. Alternatively, the nucleotide sequence may comprise any sequence of degenerate nucleotide sequences or a combination of degenerate sequences.

Embodiments of the present invention also provide an isolated or purified nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that is identical to any nucleic acid described herein under stringent conditions. Sequence of nucleotide sequences that hybridize to.

Nucleotide sequences that hybridize under stringent conditions can hybridize under highly stringent conditions. "Highly stringent conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. Highly stringent conditions include combining polynucleotides containing exact complementary sequences or polynucleotides containing only some scattered mismatches with random sequences that happen to have small regions (e.g. 3-10 bases) of matching nucleotide sequences. Distinguish the conditions. Such small complementary regions are easier to melt than full-length complements of 14-17 bases or more, and highly stringent hybridization makes them easier to distinguish. Relatively highly stringent conditions will include, for example, low-salt and/or high-temperature conditions, such as those provided by about 0.02-0.1M NaCl or equivalent at a temperature of about 50-70°C. Such highly stringent conditions tolerate very few (if any) mismatches between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any CAR of the invention described herein. It is generally believed that more stringent conditions can be caused by adding an increased amount of formamide.

In an embodiment of the invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding a CAR. Without being bound by a specific theory or mechanism, it is believed that the codon optimization of the nucleotide sequence increases the translation efficiency of mRNA transcripts. Codon optimization of a nucleotide sequence can involve replacing the natural codon with another codon that encodes the same amino acid, but can be translated from a tRNA that is more readily available in the cell, thereby improving translation efficiency. The optimization of the nucleotide sequence can also reduce the secondary mRNA structure that interferes with translation, thereby improving translation efficiency. In this regard, the nucleic acid encoding the CAR may comprise the codon-optimized nucleotide sequence of any one of SEQ ID NO: 16-25.

The present invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 95% or more, such as about 96%, about 97%, about 98%, or about 99% identical to the nucleotide sequence of any nucleic acid described herein Nucleotide sequence.

According to the fifth aspect of the present invention, the present invention provides a recombinant expression vector containing the aforementioned nucleic acid molecule. In an embodiment, the nucleic acid of the present invention can be incorporated into a recombinant expression vector. In this regard, embodiments of the present invention provide a recombinant expression vector comprising any nucleic acid of the present invention. For the purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct, when the construct contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and is sufficient to make When the mRNA, protein, polypeptide or peptide is expressed in the host cell when the carrier is contacted with the cell, it allows the cell to express the mRNA, protein, polypeptide or peptide. The carrier of the present invention as a whole is not naturally occurring.

However, part of the carrier may be naturally occurring. The recombinant expression vector of the present invention may contain any type of nucleotides, including but not limited to DNA and RNA, it may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and it may contain natural , Unnatural or altered nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages or both types of linkages. Preferably, non-naturally occurring or altered nucleotides or internucleotide linkages do not interfere with the transcription or replication of the vector.

In an embodiment, the recombinant expression vector of the present invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and amplification or for expression or for both, such as plasmids and viruses. The vector can be selected from: pUC series (Fermentas Life Sciences, Glen Burnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX Series (Clontech, Palo Alto, CA). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, such as a retroviral vector. In an embodiment of the present invention, the vector is a gamma retroviral vector, a lentiviral vector or a transposon.

In embodiments, standard recombinant DNA techniques such as those described in Green et al. above can be used to prepare the recombinant expression vector of the present invention. The circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus and the like.

Recombinant expression vectors can contain regulatory sequences, such as transcription and translation start and stop codons, depending on the situation and considering whether the vector is DNA-based or RNA-based, which is important for the type of host cell into which the vector is to be introduced (such as bacteria, fungi) , Plant or animal) is specific. Recombinant expression vectors may contain restriction sites to facilitate cloning.

The recombinant expression vector may contain one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include resistance to antimicrobial agents (for example, resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic hosts to provide prototrophs, and the like. Suitable marker genes for the expression vector of the present invention include, for example, neomycin/G418 resistance gene, hygromycin resistance gene, histidine resistance gene, tetracycline resistance gene, and ampicillin resistance gene.

The recombinant expression vector may comprise a natural or non-natural promoter operably linked to the following sequence: a nucleotide sequence encoding the CAR of the present invention or a nucleotide sequence that is complementary or hybridizing to the nucleotide sequence encoding the CAR of the present invention sequence. The choice of promoter, such as strong, weak, inducible, tissue-specific and development-specific, is within the abilities of those of ordinary skill in the art. Similarly, the combination of nucleotide sequence and promoter is also within the abilities of those of ordinary skill in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, or a promoter present in the long terminal repeat of murine stem cell virus.

The recombinant expression vector of the present invention can be designed for transient expression, stable expression, or both. In addition, the recombinant expression vector can be prepared for constitutive expression or inducible expression.

In addition, the recombinant expression vector can be prepared to contain a suicide gene. The term "suicide gene" as used herein refers to a gene that causes the death of cells expressing the suicide gene. A suicide gene may be a gene that imparts sensitivity to an agent (e.g., a drug) to the cell in which the gene is expressed, and causes cell death when the cell comes into contact with the agent or is exposed to the agent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

According to the sixth aspect of the present invention, the present invention provides a host cell comprising the aforementioned chimeric antigen receptor, the aforementioned nucleic acid molecule, or the aforementioned recombinant expression vector.

In an embodiment of the present invention, there is also provided a host cell comprising any of the recombinant expression vectors described herein. The term "host cell" as used herein refers to any type of cell that can contain the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacteria or protozoa. The host cell may be a cultured cell or a primary cell, that is, a cell directly isolated from an organism such as a human. The host cell may be an adherent cell or a suspension cell, that is, a cell that grows in suspension. Suitable host cells are known in the art, and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5α cell. For the purpose of producing CAR, the host cell may be a mammalian cell. The host cell may be a human cell. Although the host cell can be any type of cell, can be derived from any type of tissue, and can be at any stage of development, the host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell can be a B cell, a natural killer (NK) cell or a T cell.

For the purpose of this document, the T cell may be any T cell, such as a cultured T cell, such as a primary T cell; or a T cell derived from a cultured T cell line, such as Jurkat, SupT1, etc.; or a T cell obtained from a mammal T cells. If obtained from a mammal, T cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. T cells can be any type of T cell and can be at any stage of development, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cells) Toxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc.

T cells can be autologous or allogeneic. "Autologous" means that the cells to be used in the treatment method or use (ie, to be transduced with a nucleic acid or vector) are derived or obtained from the subject to be subjected to the treatment method. Therefore, autologous cells are obtained from the subject, transduced with nucleic acid or vector, and returned to the same subject.

"Allogeneic" means that the cells to be used in the treatment method or use (ie, to be transduced with a nucleic acid or vector) are derived or obtained from a different subject than the subject to be treated. Therefore, allogeneic cells are obtained from the first subject, transduced with the nucleic acid or vector, and administered to the second subject.

It should also be understood that the host cell of the present invention may contain more than one nucleic acid or vector. In particular, the cells of the present invention may contain 2, 3, 4, or 5 or more nucleic acids or vectors, each of which express a different chimeric antigen receptor molecule. Therefore, the cells of the present invention may contain different chimeric antigen receptor molecules that are capable of binding CD3, for example at the same or different positions of CD3. In this regard, the cell of the present invention may comprise a chimeric antigen receptor molecule comprising an scFv that binds to CD3 and a chimeric antigen receptor molecule that comprises a ligand that binds to CD3.

In addition, in addition to the expressed chimeric antigen receptor of the present invention, the cell of the present invention may contain at least one other receptor (especially exogenous) (for example, multiple receptors), which may be combined with the CAR The method is used to bind the target. In such methods, the binding of CAR and at least one other receptor to the target cell may be required to stimulate the immune response against the target cell (for example, each CAR/receptor may only provide immune cells Part of the signal of stimulation, which alone may not be sufficient for immune cell stimulation, but together allows immune cell stimulation). In the case where the cells of the present invention are T cells, both CAR binding to CD3 and at least one other receptor binding to its ligand on CD3 expressing cells may be necessary to stimulate T cells. At least one other receptor can be another CAR molecule.

Additional receptors can be used in combination with the CAR of the present invention, where the two receptors bind to different targets and induce different effects to treat diseases. Therefore, the effects of the two receptors can be completely independent of each other, but together can present an effective treatment for the disease.

According to the seventh aspect of the present invention, the present invention provides a method for preparing the aforementioned host cell, the method comprising introducing the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector into the host cell, and The host cell is cultured under conditions suitable for the cell to express the nucleic acid molecule or vector.

According to the eighth aspect of the present invention, the present invention provides a cell population, which includes the aforementioned host cell.

The cell population may be a heterogeneous population including host cells containing any of the recombinant expression vectors in addition to at least one other cell, and the other cells are, for example, host cells (such as T cells) that do not contain any recombinant expression vectors or in addition to Cells other than T cells, such as B cells, macrophages, neutrophils, red blood cells, liver cells, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the cell population may be a substantially homogeneous population, wherein the population mainly comprises host cells containing a recombinant expression vector (for example, consisting essentially of host cells containing a recombinant expression vector). The population may also be a clonal cell population, in which all cells of the population are clones of a single host cell containing a recombinant expression vector, so that all cells of the population contain the recombinant expression vector. In one embodiment of the present invention, the cell population is a clonal population comprising host cells containing the recombinant expression vector described herein.

In embodiments of the present invention, the number of cells in the population can be rapidly expanded. The expansion of the number of CAR-expressing cells can be accomplished by any of a variety of methods known in the art as described below, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent Application Publication No. 2012/0244133 ; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990).

CARs, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) are collectively referred to as "CAR materials" hereinafter.

According to the ninth aspect of the present invention, the present invention provides a pharmaceutical composition comprising the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, or the aforementioned The cell population described.

Further, the pharmaceutical composition includes a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier can be any of those conventionally used for the particular CAR material of the invention under consideration. The method of preparing the applicable composition is known or obvious to those skilled in the art, and is described in more detail in, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press (2012). Preferably, the pharmaceutically acceptable carrier is a carrier that has no harmful side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the specific CAR material of the invention and the specific method used to administer the CAR material of the invention. Therefore, there are a variety of suitable formulations of the pharmaceutical composition of the present invention. Suitable formulations may include any of those formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or intraperitoneal administration. More than one route can be used to administer the CAR material of the present invention, and in some cases, a particular route can provide a more direct and effective response than another route.

Preferably, the CAR material of the present invention is administered by injection, for example intravenously. When the CAR material of the present invention is a host cell (or a population thereof) expressing the CAR of the present invention, the pharmaceutically acceptable carrier for the injected cells may include any isotonic carrier, such as, for example, physiological saline (containing about 0.90% w /v NaCl water, containing about 300mOsm/L NaCl water, or about 9.0g NaCl per liter of water, NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), containing about 5% dextrose water or lactated Ringer's solution. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

The pharmaceutical composition of the present invention may also include other pharmaceutically active agents or drugs administered in combination with the CAR material of the present invention, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, Cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

When the CAR material of the present invention is administered with one or more additional therapeutic agents, the one or more additional therapeutic agents can be co-administered to the mammal. "Co-administration" means to administer one or more additional therapeutic agents and the CAR material of the present invention sufficiently close in time so that the CAR material of the present invention can enhance the effect of the one or more additional therapeutic agents, vice versa. In this regard, the CAR material of the present invention can be administered first, followed by one or more additional therapeutic agents, and vice versa. Alternatively, the CAR material of the present invention and one or more additional therapeutic agents can be administered simultaneously. Additional therapeutic agents that can enhance the function of CAR-expressing cells can include, for example, one or more cytokines or one or more antibodies (e.g., antibodies that inhibit PD-1 function).

It is contemplated that the CAR materials and pharmaceutical compositions of the present invention can be used in methods of treating or preventing conditions in mammals. Without being bound by a specific theory or mechanism, the CAR material of the present invention has biological activities, such as the ability to recognize CD3, so that when expressed by cells, CAR can mediate an immune response against CD3-expressing cells. In this regard, the embodiments of the present invention provide a method of treating or preventing a condition in a mammal, which comprises administering to the mammal an amount of the CAR, nucleic acid, recombinant expression of the present invention in an amount effective to treat or prevent the condition in the mammal Any of the vector, host cell, cell population and/or pharmaceutical composition. The condition can be any condition characterized by the expression or overexpression of CD3. In a preferred embodiment, the condition is cancer.

For the purpose of the method of the present invention for administering a host cell or cell population, the cell may be a cell allogeneic to a mammal or an autologous cell. In the "autologous" method of administration, cells are removed from the mammal, stored (and optionally modified), and returned to the same mammal. In the "allogeneic" method of administration, the mammal receives cells from a genetically similar but different donor. Preferably, the cell is mammalian autologous. In an embodiment of the invention, the cells administered to the mammal have undergone gene editing.

The mammal mentioned herein can be any mammal. The term "mammal" as used herein refers to any mammal, including but not limited to mammals of the Rodent order, such as mice and hamsters; and mammals of the Lagomorph order, such as rabbits. Mammals can be from the order Carnivora, including the cat family (cats) and canine family (dogs). Mammals can be from the order Artiodactyla, including Bovidae (bovine) and Suidae (pigs); or from the order Perissodactyla, including Equididae (horse). Mammals can be from the order of the Primates, Ceboids, or Simoids (monkeys), or from the order of the Apes (humans and apes). Preferably, the mammal is a human.

The terms "treatment" and "prevention" as used herein and the words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are different degrees of treatment or prevention that one of ordinary skill in the art considers to have potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide treatment or prevention of conditions in mammals at any level in any amount. In addition, the treatment or prevention provided by the method of the present invention may include the treatment or prevention of one or more conditions or symptoms of the disease (for example, cancer) being treated or prevented. In addition, for purposes herein, "prevention" can encompass delaying the onset of a disease, such as cancer or its symptoms or conditions. Alternatively or additionally, "prevention" can encompass delaying the recurrence of a disease, such as cancer or its symptoms or conditions.

According to the tenth aspect of the present invention, the present invention provides a kit comprising the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, and the aforementioned cell Group, or the aforementioned pharmaceutical composition.

Some components of the kit can be packaged in an aqueous medium or lyophilized form. The container device of the kit usually includes at least one vial, test tube, flask, bottle, syringe or other container device, in which the components can be placed, preferably appropriately divided. If there are multiple components in the kit, the kit will usually also contain a second, third or other additional container, where the additional components can be placed in the container separately. However, various combinations of components may be included in the vial. The kits of the present invention will generally also include a device for containing commercially available sealed restriction components. Such containers may include injection- or blow-molded plastic containers in which the required vials are held.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, and the sterile aqueous solution is particularly useful. In some cases, the container device itself may be a syringe, pipette, and/or other similar device from which the formulation may be applied to the infected area of the body, injected into the animal, and/or even applied to other parts of the kit Components and/or mixing with them.

However, the components of the kit can be provided as dry powder. When the reagents and/or components are provided as a dry powder, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent can also be provided in another container device. The kit may also include a second container device for containing a sterile pharmaceutically acceptable buffer and/or other diluent.

According to the eleventh aspect of the present invention, the present invention provides an immunotherapy method comprising administering the aforementioned host cell, the aforementioned cell population or the aforementioned drug to a person in need combination.

Further, the applicable diseases of the method include autoimmune diseases and cancer.

The cancer includes acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Hodgkin's lymphoma, neuroblastoma, Ewing's sarcoma, multiple myeloma, myelodysplastic syndrome, BPDCN, glioma, Or other solid tumors: including pancreatic cancer, lung cancer, colorectal cancer, breast cancer, bladder cancer.

According to the twelfth aspect of the present invention, the present invention provides a method for resisting transplant rejection, the method comprising administering the aforementioned host cell, the aforementioned cell population or the aforementioned Pharmaceutical composition.

Further, the transplant rejection reaction includes graft versus host reaction and host versus graft reaction.

According to the thirteenth aspect of the present invention, the present invention provides the use of the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector in the preparation of the aforementioned host cell or the aforementioned cell population.

According to the fourteenth aspect of the present invention, the present invention provides the use of the aforementioned nucleic acid molecule or the aforementioned recombinant expression vector in the preparation of CAR or CAR-T.

According to the fifteenth aspect of the present invention, the present invention provides the use of the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector or the aforementioned host cell in the preparation of the aforementioned pharmaceutical composition.

According to the sixteenth aspect of the present invention, the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, or the aforementioned drug combination The application of the substance in the preparation of the aforementioned kit.

According to the seventeenth aspect of the present invention, the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, and the aforementioned pharmaceutical composition The application of the aforementioned kit in the aforementioned immunotherapy.

According to the eighteenth aspect of the present invention, the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, and the aforementioned pharmaceutical composition The application of the aforementioned kit in the aforementioned anti-graft rejection.

According to the nineteenth aspect of the present invention, the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, or the aforementioned drug combination The application of drugs in immunotherapy.

According to the twentieth aspect of the present invention, the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, or the aforementioned drug combination The application of drugs in anti-graft rejection.

According to the twenty-first aspect of the present invention, the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, the aforementioned cell population, or the aforementioned Application of the pharmaceutical composition in the preparation of drugs for immunotherapy.

According to the 22nd aspect of the present invention, the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, or the aforementioned cell population, or the aforementioned The application of the pharmaceutical composition in the preparation of a medicament for resisting transplantation rejection.

According to the twenty-third aspect of the present invention, the present invention provides the aforementioned nucleic acid molecule, the aforementioned recombinant expression vector, the aforementioned host cell, or the aforementioned cell population, or the aforementioned Application of the pharmaceutical composition in the preparation of anti-cancer drugs.

The cancer of the present invention can be any cancer, including but not limited to acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Hodgkin's lymphoma, neuroblastoma, Ewing sarcoma, multiple myeloma, bone marrow Dysplasia syndrome, BPDCN, glioma, or other solid tumors: including pancreatic cancer, lung cancer, colorectal cancer, breast cancer, bladder cancer.

Preferably, the cancer is lymphoma. In a particularly preferred embodiment, the cancer is T cell lymphoma (such as, for example, anaplastic large cell lymphoma (ALCL), peripheral T cell lymphoma-non-specific (PTCL-NOS), angioimmunoblastic T cell Lymphoma (AITL) and other T-cell lymphomas). Preferably, the cancer is characterized by the expression or overexpression of CD3.

Description of the drawings

Figure 1 shows a schematic diagram of the LV-CD3CAR plasmid constructed by the present invention;

Figure 2 shows the result of detecting the transduction rate of lentivirus LV-CD3CAR-291 by flow cytometry;

Figure 3 shows the result of detecting the transduction rate of lentivirus LV-CD3CAR-cel by flow cytometry;

Figure 4 shows the result graph of detecting the transduction rate of lentivirus LV-CD3CAR-OKT31 by flow cytometry;

Figure 5 shows the result of using flow cytometry to detect the TCR knockout effect in LV-CD3CAR-291-T;

Figure 6 shows the result of using flow cytometry to detect the TCR knockout effect in LV-CD3CAR-cel-T;

Figure 7 shows the result of using flow cytometry to detect the TCR knockout effect in LV-CD3CAR-OKT31-T;

Figure 8 shows the results of using flow cytometry to detect the killing effect of LV-CD3CAR-291-T cells on Jurkat-GFP cells;

Figure 9 shows the results of using flow cytometry to detect the killing effect of LV-CD3CAR-cel-T cells on Jurkat-GFP cells;

Figure 10 shows the results of using flow cytometry to detect the killing effect of LV-CD3CAR-OKT31-T cells on Jurkat-GFP cells;

Figure 11 shows the results of using flow cytometry to detect the killing effect of LV-CD3CAR-sp34-T cells on Jurkat-GFP cells;

Figure 12 shows the result of using flow cytometry to detect the killing effect of LV-CD3CAR-UCHT1-T cells on Jurkat-GFP cells;

Figure 13 shows a result diagram of using animal models to study the effect of LV-CD3CAR-cel-T cells constructed in the present invention on tumors;

Figure 14 shows a statistical graph of fluorescence intensity in mice;

Figure 15 shows a statistical chart of the survival time of mice;

Figure 16 shows a statistical diagram of the clearance effect of LV-CD3CAR-T on CD3 positive cells.

Detailed ways

The following examples further illustrate the invention. The following examples are only to illustrate the present invention and should not be construed as restrictive.

Example 1 CAR expression targeting CD3

1. Synthesis of nucleic acid molecules targeting CD3 CAR

Synthesize a nucleic acid molecule that can express a CAR targeting CD3, with the sequence code CD3CAR-291 (SEQ ID NO: 61); CD3CAR-cel (SEQ ID NO: 62); CD3CAR-OKT31 (SEQ ID NO: 63); CD3CAR-sp34 (SEQ ID NO: 64); CD3CAR-UCHT1 (SEQ ID NO: 65);

3. Construction of LV-CD3CAR expression plasmid

Insert the CD3CAR into the expression vector pLVX-Puro according to the restriction enzyme digestion method (the linear sequence of the vector is shown in SEQ ID NO: 66) to construct the LV-CD3CAR expression plasmid. The schematic diagram of the LV-CD3CAR plasmid is shown in Figure 1 (intracellular co- The stimulation domain is 4-1BB, EGFR D III-D VI can be used as a marker for CAR expression detection and a suicide gene for CAR-T cells, increasing the safety of the product). Restriction site: XbaI, EcoRI. Transform, plate, and sequence to confirm that the plasmid is constructed successfully. Large-scale extraction of plasmids to obtain endotoxin-free expression plasmids for packaging lentivirus.

4. LV-CD3CAR lentivirus packaging

The steps of PEI transfection method (for T75 culture flask) are as follows:

(1) Day1 resuscitated 293T/17 cells to 1*T75 with a volume of 15ml;

(2) On day3, pass 293T/17 cells to 1*T225, the volume of the medium is 45ml;

(3) day5 the 293T / 17 cells were passaged into the 3 * T225, a seeding density of approximately 6 * 10 ⁷ cells / T225 flask;

(4) Virus packaging will be performed in the afternoon of day6. Observe the cell status before transfection, and proceed to transfection when the confluence is about 90%. The culture medium in the bottle was discarded, replaced with 15ml fresh DMEM medium (without antibiotics), and cultured for 30 minutes.

Solution A: Take LV-CD3CAR expression plasmid 17.7μg, helper plasmid pRSV-REV 8.8μg, helper plasmid pMDLg/pRRE 8.8μg and helper plasmid pMD2.G 4.4μg, the transfection ratio is 4:2:2:1, total The amount is 40μg, after mixing, dilute to 0.75ml with serum-free DMEM, and let stand at room temperature for 5min after mixing.

Solution B preparation: Take 630μl DMEM, and then add 120μl PEI working solution (1mg/ml, stored at 4℃), mix well, and let stand for 5min at room temperature.

Add solution B to solution A drop by drop, mix gently, and incubate at room temperature for 20 minutes. Add the mixture dropwise to the cells, mix gently, and incubate in 5% carbon dioxide for 17 hours.

(5) Discard the original medium on the morning of day 7 and add 15 ml of DMEM medium without serum and antibiotics. After culturing for 31 hours, the virus is harvested. After that, the medium is added for 24 hours and the virus is harvested again. The cell supernatant was collected and centrifuged at 2000 rpm for 5 min. Then transfer the supernatant to a high-speed centrifuge tube, balance, and then centrifuge at 30000g at 4°C for 4h, aspirate the supernatant, add 500μl of sterile PBS buffer to resuspend the virus particles, mix 200μl/little and place at -80°C Store in the refrigerator.

5. T cell separation

Obtain blood samples from healthy donors from central blood stations or hospitals. Patients who have passed the tests for the following diseases (not limited to these tests). Including: hepatitis A, hepatitis B, hepatitis C, AIDS, syphilis antibodies, tuberculosis, genetic diseases, etc. Use Miltenyi's Pan T Cell Isolation Kit human (Order no.130-096-535) to isolate T cells according to the provided protocol.

6. T cell activation

T cell complete medium preparation: OpTmizer ^TM CTS ^TM T-cell Expansion SFM + 5% CTS Immune Cell SR + 1% L-glutamine + 10ng/ml IL-7/15.

The starting cell number is 3M+Human T-Activator CD3/CD28 Dynabeads 75ul. The initial cell concentration is 1M/ml. Cultivate in a 37°C incubator. Activate for 48 hours.

7. T cell gene editing

The CRISPR/cas9 system was used to design sgRNA and knock out TCR by electrotransformation. Cas9 protein and sgRNA were purchased from ThermFisher Company.

Electric transfer system:

Power transfer conditions: 1600V, 10ms, 3pulses

Among them, the TCR sgRNA sequence is as follows:

cagggttctggatatctgt (SEQ ID NO: 67)

8. LV-CD3CAR lentiviral transduction

T cell gene editing 12 hours later, LV-CD3CAR lentivirus transduction, virus MOI: 3-20, polybrene 1.5μl (5-10μg/ml). After 6-12 hours, remove the medium containing the lentivirus and replace with fresh medium for CAR-T cell expansion.

9. CAR-T cell expansion

After replacing the fresh medium, in the continuous presence of IL-7/15, with 1M/ml as the starting cell density for cell passage, check the cell density and viability every 2 days, and supplement with fresh medium and cytokines. Keep the cell density at 1M/ml.

10. Detection of TCR knockout efficiency of CAR-T cells

After 48 hours of electroporation, flow cytometry was used to detect the TCR knockout effect. The results are shown in Figure 2-4, the TCR knockout rate reached 80-90%. LV-CD3CAR-291-T (knock out LV-CD3CAR-291 transfected T cells, CAR-T cells) cells have a small amount of CD3/αβTCR/γδ, and TCR-positive cells are less than 1%. LV-CD3CAR-cel-T (knock out T cells transfected with LV-CD3CAR-cel, CAR-T cells) cells did not have CD3/αβTCR/γδTCR positive cells. LV-CD3CAR-OKT31-T (knock out the T cells transfected with LV-CD3CAR-OKT31, CAR-T cells) cells have a small amount of CD3/αβTCR/γδ, and the TCR-positive cells are less than 1%. In the figure, PanT represents untreated T cells, PanT TCRKO represents TCR knock-out T cells, and LV-CD3CAR-291-T, LV-CD3CAR-cel-T, LV-CD3CAR-OKT31-T represent CAR-T cells.

11. LV-CD3CAR lentiviral transduction rate detection

2-7 days after lentivirus transduction, the transduction rate was measured by flow cytometry. The results are shown in Figures 5-7. After 3 days of lentiviral transduction, the CAR expression rate is not less than 50%. In the figure, PanT represents untreated T cells, PanT TCRKO represents TCR knocked out T cells, and LV-CD3CAR- 291-T, LV-CD3CAR-cel-T, LV-CD3CAR-OKT31-T represent CAR-T cells.

Example 2 In vitro killing function detection of CAR-T cells

1. The Jurkat-GFP cell line is co-cultured with the CAR-T cells prepared in Example 1, and the E/T (Jurkat-GFP: CAR-T) ratio is 8:1, 4:1, 2:1, and 1:1 respectively. , 0.5:1, 0:1.

2. The grouping is as follows:

Jurkat-GFP group: 0.5M per well, three multiple wells;

PanT TCRKO (T cell knockout TCR) group: 0.5M per well, three duplicate wells;

PanT TCRKO (T cell knockout TCR) + Jurkat-GFP group: 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1;

LV-CD3CAR-T(CAR-T)+Jurkat-GFP group: 8:1, 4:1, 2:1, 1:1, 0.5:1, 0:1;

There are three multiple holes for each ratio.

3. After 24 hours, the GFP fluorescence of Jurkat-GFP cells was detected by flow cytometry.

4. Results

The results are shown in Figure 8-12, CAR-T (LV-CD3CAR-291-T, LV-CD3CAR-cel-T, LV-CD3CAR-OKT31-T, LV-CD3CAR-sp34-T, LV-CD3CAR-UCHT1 -T)) When the cells are E:T=2:1, 90%-100% of CD3/TCR-positive Jurkat-GFP cells can be killed in one day.

Example 3 Detection of the killing function of CAR-T cells in vivo

1. Steps

1. Construction of NPG mouse tumor model by Jurkat-Fluc cell line

NPG mice aged 5-8 weeks, all female, were injected with 1×10 ⁶ Jurkat-Fluc cells through the tail vein. One week later, the biofluorescence test confirmed that the NPG mouse tumor model was successfully constructed.

2. After one week, the NPG mice were divided into tumor model group (negative control group), LV-CD3CAR-cel-T group, LV-TCRCAR-T group, a total of three groups, each with three mice.

^{3. Infusion of 1×10 7} CAR-positive cells via the tail vein of NPG mice. The observation period is 8 weeks.

4. Observe the biofluorescence intensity, weight, status, and survival time of each group of NPG mice every week.

2. Results

The results of in vivo effectiveness are shown in Figure 13. The LV-CD3CAR-T group significantly inhibited tumor growth, and the biofluorescence intensity was significantly lower than that of the tumor group. The survival time of mice in the LV-CD3CAR-T group was significantly prolonged. The mice in the experimental group were still alive during the observation period.

The results of in vivo effectiveness are shown in Figure 14: The fluorescence intensity of mice in the LV-CD3CAR-T group was significantly lower than that of the tumor model group, reflecting that the tumor burden of the LV-CD3CAR-T group was significantly lower than that of the tumor model group, proving that LV-CD3CAR -T can effectively kill tumor cells in mice.

The results of in vivo effectiveness are shown in Figure 15: the survival time of mice in the LV-CD3CAR-T group is significantly better than that of the tumor model group, which can indicate that the LV-CD3CAR-T group inhibits tumor growth, slows down the development of the disease, and can significantly prolong the growth of the disease. The life span of the mouse.

In vitro and in vivo experiments prove that the CD3-targeted CAR-T constructed in the present invention can effectively kill CD3 positive cells, and can be used to treat T cell-derived lymphocytic leukemia and T cell-derived lymphoma.

Example 4 Functional Test of CAR-T Cells to Inhibit Transplant Rejection

1. Steps

1. Obtain PBMC from allogeneic healthy blood donors, extract Pan T cells (untreated T cells), and count;

2. Cell count to determine the number of LV-CD3CAR-cel-T;

3. Flow cytometry to determine the transduction rate of LV-CD3CAR-cel-T;

4. Mix the number of LV-CD3CAR-cel-T and Pan T cells at the ratio of 0.5:1, 1:1 and 2:1 respectively;

5. Flow cytometric detection of the number of CD3 positive cells at 0h, 24h, and 48h.

2. Results

The results are shown in Figure 16, that the CAR-T cells constructed in the present invention can effectively eliminate CD3 positive cells in the allogeneic body. Therefore, the CD3CAR-T of the present invention can be used to inhibit the occurrence of transplant rejection. In Figure 16: UCAR-T stands for LV-CD3CAR-T.

All references cited herein, including publications, patent applications, and patents, are incorporated by reference to the same extent as if each reference is individually and clearly indicated to be incorporated by reference, and is shown in its entirety herein.

The terms "a/a" and "an" and "the" and similar referents are used in the context of describing the present invention (especially in the context of the following claims) The use of) is interpreted as covering both the singular and the plural, unless otherwise specified herein or the context is clearly contradictory. The terms "comprising", "having", "including" and "containing" are interpreted as open-ended terms (ie, meaning "including but not limited to") unless otherwise noted. The description of the numerical range herein is only intended as a shorthand method for separately referring to each independent numerical value falling within the range, unless otherwise specified herein, and each independent numerical value is incorporated into the specification as if it is individually described herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradictory to the context. The use of any or all of the examples or exemplary language (such as "such as") provided herein is only intended to better clarify the present invention, and does not limit the scope of the present invention unless otherwise stated. None of the language in the specification should be construed as indicating that any unclaimed element is necessary for the practice of the present invention.

The preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the present invention. After reading the foregoing description, changes to those preferred embodiments may become apparent to those of ordinary skill in the art. The inventor expects those skilled in the art to apply such changes as appropriate, and the inventor intends to practice the present invention in a manner different from that specifically described herein. Therefore, as permitted by applicable laws, the present invention includes all modifications and equivalents of the subject matter described in the claims appended hereto. In addition, the present invention encompasses any combination of all possible changes of the above-mentioned elements, unless otherwise indicated herein or otherwise clearly contradictory to the context.

Claims

Application of CD3 in preparing chimeric antigen receptors targeting CD3.
The application of an antibody against CD3 or an antigen binding fragment thereof in the preparation of a chimeric antigen receptor targeting CD3.
A chimeric antigen receptor targeting CD3, characterized in that the chimeric antigen receptor includes a CD3 binding domain.
The chimeric antigen receptor of claim 3, wherein the chimeric antigen receptor comprises from N-terminus to C-terminus:

CD3 binding domain;

Hinge region and transmembrane domain;

Costimulatory domain

Signal transduction domain.
The chimeric antigen receptor of claim 4, wherein the CD3 binding domain comprises the antigen binding portion of a CD3 antibody.
The chimeric antigen receptor according to claim 5, wherein the antigen binding portion includes a light chain variable region and/or a heavy chain variable region, and the heavy chain variable region includes CDR1, CDR2, and CDR3. The light chain variable region includes one or more of CDR1, CDR2, and CDR3.
The chimeric antigen receptor according to claim 6, wherein the variable region of the heavy chain comprises CDR1, CDR2 and CDR3; and the variable region of the light chain comprises CDR1, CDR2 and CDR3.
The chimeric antigen receptor according to claim 7, wherein the heavy chain CDR1 has the same sequence as SEQ ID NO: 1, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: The amino acid sequence shown in any one of 50 has an amino acid sequence with at least 95% sequence identity;

The heavy chain CDR2 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 40, SEQ ID NO: 51 Amino acid sequence of

The heavy chain CDR3 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 30, SEQ ID NO: 41, SEQ ID NO: 52 Amino acid sequence of

The light chain CDR1 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 42, SEQ ID NO: 53 Amino acid sequence of

The light chain CDR2 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 54 Amino acid sequence of

The light chain CDR3 has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 44, SEQ ID NO: 55 The amino acid sequence.
The chimeric antigen receptor according to claim 8, characterized in that the heavy chain variable region has the same sequence as SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID The amino acid sequence shown in any one of NO: 56 has an amino acid sequence with at least 95% sequence identity; the light chain variable region has the same sequence as SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 35, SEQ ID The amino acid sequence shown in any one of NO: 46 and SEQ ID NO: 57 has an amino acid sequence with at least 95% sequence identity.
The chimeric antigen receptor according to claim 9, wherein the CD3 binding domain has a combination with SEQ ID NO: 10, SEQ ID NO: 25, SEQ ID NO: 36, SEQ ID NO: 47, and SEQ ID NO: 47. ID NO: The amino acid sequence shown in any one of 58 has an amino acid sequence with at least 95% sequence identity.
The chimeric antigen receptor according to claim 10, wherein the CD3 binding domain further comprises a signal peptide sequence, and the signal peptide sequence is located at the amino terminus of the variable region of the heavy chain.
The chimeric antigen receptor according to claim 11, wherein the amino acid sequence of the signal peptide sequence is shown in SEQ ID NO: 11.
The chimeric antigen receptor according to claim 12, wherein the CD3 binding domain has a combination with SEQ ID NO: 16, SEQ ID NO: 27, SEQ ID NO: 38, SEQ ID NO: 49, SEQ ID NO: The amino acid sequence shown in any one of 60 has an amino acid sequence with at least 95% sequence identity.
The chimeric antigen receptor of claim 3, wherein the hinge region and transmembrane domain comprise the hinge region and transmembrane domain of any one or more of the following molecules: CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, CD154.
The chimeric antigen receptor according to claim 14, wherein the sequence of the hinge region and the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 12 or its homologous sequence; the homologous sequence is identical to the original The sequence homology is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more Above, 99.6% or above, 99.7% or above, 99.8% or above, or 99.9% or above.
The chimeric antigen receptor according to claim 15, wherein the hinge region and transmembrane domain comprise the amino acid sequence shown in SEQ ID NO: 12.
The chimeric antigen receptor of claim 3, wherein the signal transduction domain comprises an intracellular domain of CD3ζ.
The chimeric antigen receptor according to claim 17, wherein the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 13 or its homologous sequence; the homology of the homologous sequence to the original sequence Preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more Above, 99.7% or above, 99.8% or above, or 99.9% or above.
The chimeric antigen receptor according to claim 18, wherein the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO: 13.
The chimeric antigen receptor according to claim 3, wherein the costimulatory domain comprises an intracellular domain of a costimulatory factor, and the costimulatory factor includes CD27, CD28, 4-1BB, OX40, CD30, CD40 , ICOS, NKG2C, B7-H3.
The chimeric antigen receptor according to claim 20, wherein the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 14 or a homologous sequence thereof; the homology of the homologous sequence to the original sequence is preferably 95% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more , 99.7% or more, 99.8% or more, or 99.9% or more.
The chimeric antigen receptor of claim 21, wherein the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 14.
The chimeric antigen receptor according to any one of claims 3-22, wherein the chimeric antigen receptor comprises SEQ ID NO: 15, SEQ ID NO: 26, SEQ ID NO: 37, SEQ ID NO: 48, SEQ ID NO: 59 shown in the amino acid sequence.
A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 3-23.
A recombinant expression vector comprising the nucleic acid molecule of claim 24.
A host cell, characterized in that the host cell comprises the chimeric antigen receptor of any one of claims 3-23, the nucleic acid molecule of claim 24, or the recombinant expression of claim 25 Carrier.
A method for preparing the host cell according to claim 26, wherein the method comprises introducing the nucleic acid molecule according to claim 24 or the recombinant expression vector according to claim 25 into the cell, and expressing the cell in a suitable cell. The cell is cultured under the conditions of the nucleic acid molecule or vector.
A cell population, characterized in that the cell population comprises the host cell of claim 26.
A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the chimeric antigen receptor according to any one of claims 3-23, the nucleic acid molecule according to claim 24, and the recombinant protein according to claim 25 An expression vector, the host cell of claim 26, or the cell population of claim 28.
A kit, characterized in that the kit comprises the chimeric antigen receptor according to any one of claims 3-23, the nucleic acid molecule according to claim 24, and the recombinant expression vector according to claim 25 The host cell of claim 26, the cell population of claim 28, and the pharmaceutical composition of claim 29.
A method of immunotherapy, characterized in that the method comprises administering the host cell according to claim 26, the cell population according to claim 28, and the pharmaceutical composition according to claim 29 to a person in need.
The method according to claim 31, wherein the applicable diseases of the method include autoimmune diseases and cancer.
The method of claim 32, wherein the cancer comprises acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Hodgkin’s lymphoma, neuroblastoma, Ewing’s sarcoma, multiple myeloma , Myelodysplastic syndrome, BPDCN, glioma, or other solid tumors: including pancreatic cancer, lung cancer, colorectal cancer, breast cancer, bladder cancer.
A method for resisting transplant rejection, characterized in that the method comprises administering the host cell according to claim 26, the cell population according to claim 28, and the pharmaceutical composition according to claim 29 to a person in need .
The method of claim 34, wherein the transplant rejection reaction comprises a graft-versus-host reaction and a host-versus-graft reaction.
An anti-cancer gene therapy, characterized in that the method comprises administering the nucleic acid molecule according to claim 24, the recombinant expression vector according to claim 25 or the pharmaceutical composition according to claim 29 to a person in need.
The method of claim 36, wherein the cancer targeted by anti-cancer gene therapy includes acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, Hodgkin’s lymphoma, neuroblastoma, Ewing’s sarcoma, Multiple myeloma, myelodysplastic syndrome, BPDCN, glioma, or other solid tumors: including pancreatic cancer, lung cancer, colorectal cancer, breast cancer, bladder cancer.
The use of the nucleic acid molecule of claim 24 or the recombinant expression vector of claim 25 in the preparation of the host cell of claim 26 or the cell population of claim 28.
The use of the nucleic acid molecule of claim 24 or the recombinant expression vector of claim 25 in the preparation of CAR or CAR-T.
The nucleic acid molecule of claim 24, the recombinant expression vector of claim 25, the host cell of claim 26, or the cell population of claim 28 in the preparation of the pharmaceutical composition of claim 29 application.
The nucleic acid molecule of claim 24, the recombinant expression vector of claim 25, the host cell of claim 26, the cell population of claim 28, or the pharmaceutical composition of claim 29 are in preparation Application in the kit described in claim 30.
The nucleic acid molecule of claim 24, the recombinant expression vector of claim 25, the host cell of claim 26, the cell population of claim 28, or the pharmaceutical composition of claim 29 in preparation Used in immunotherapy drugs.
The nucleic acid molecule of claim 24, the recombinant expression vector of claim 25, the host cell of claim 26, the cell population of claim 28, or the pharmaceutical composition of claim 29 when preparing an anti- Application of drugs for transplant rejection.
The nucleic acid molecule of claim 24, the recombinant expression vector of claim 25, the host cell of claim 26, the cell population of claim 28, or the pharmaceutical composition of claim 29 when preparing an anti- Application of cancer drugs.
The use according to claim 44, wherein the cancer includes acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, Hodgkin’s lymphoma, neuroblastoma, Ewing’s sarcoma, multiple myeloma , Myelodysplastic syndrome, BPDCN, glioma, or other solid tumors: including pancreatic cancer, lung cancer, colorectal cancer, breast cancer, bladder cancer.