WO2019128999A1 - Method for obtaining car-t cells with high positive rate - Google Patents

Abstract

The present invention relates to a method for obtaining CAR-T cells with a high positive rate, comprising the step of incubating CAR-T cells using a CD28 antibody and an antigen targeted by a chimeric antigen receptor that is expressed by the CAR-T cells. Using the method of the present invention, a moderately positive ratio of CAR-T cells can be obtained initially, wherein same helps with the proliferation thereof, and due to the memory regarding a specific antigen, the ratio of CAR-T cells in the population will be increasingly higher, such that a large number of CAR-T cells with a high positive rate are finally obtained.

Description

Method for obtaining high positive rate CAR-T cells Technical field

The present invention relates to a method of obtaining high positive rate CAR-T cells.

Background technique

Cancer has become the number one killer of human health. The fast pace of life, huge work pressure, unhealthy eating habits, and poor environment are all accomplices of cancer, making cancer's high incidence and rejuvenation trend more and more obvious. At present, the commonly used treatments are very limited, and it is still necessary to explore a more effective treatment to improve the survival rate and quality of life of cancer patients.

Immunotherapy for malignant tumors has developed rapidly in recent years and has achieved remarkable clinical results. Since 2011, Nature and the top magazine of clinical oncology JCO have published the same article "The era of tumor immunotherapy has arrived" (Nature.2011;480(7378):480; J Clin Oncol.2011;29(36) :4828), tumor immune cell therapy ushered in a new round of research boom.

As one of the important branches of tumor immunotherapy, chimeric antigen receptor T cell therapy has achieved very good curative effect in malignant hematological tumors, and the complete remission rate of relapsed and refractory B cell leukemia is over 90%. In August 2017, the US FDA approved Novartis's tissue lecleucel chimeric antigen receptor T cell (CAR-T cell) treatment for the treatment of childhood and young adult patients with acute lymphoblastic leukemia (ALL), becoming the first approved market. CAR-T drug. Immediately in October of the same year, the US FDA announced the approval of Kite Pharma's CAR-T therapy, Yescarta, for the treatment of adult patients with certain types of large B-cell lymphoma. The approval of CAR-T drugs has taken CAR-T treatment to a new level.

A chimeric antigen receptor is a synthetic receptor that typically comprises an extracellular antigen binding domain, a transmembrane hinge region, and an intracellular signal transduction region. By the identification of a single-chain fragment variable (scFv) and an intracellular signal domain of an antibody-associated antigen (TAA), an immunoreceptor tyrosine-based activation Motifs, ITAM)" genetic recombination in vitro. It is then introduced into T cells by a virus or other vector system such that the genetically engineered T cells are referred to as chimeric antigen receptor T cells (i.e., CAR-T cells). After extensive expansion in vitro, CAR-T cells are returned to the patient and can exhibit potent anticancer effects in a non-MHC-restricted mode.

However, the overall efficacy of current CAR-T cell therapy is still not satisfactory, especially for solid tumors. Among them, the transfection efficiency is low, and it is difficult to obtain a high positive rate of CAR-T cells, which is an important reason. For PBMC transfection, whether it is a viral vector or a non-viral vector such as PB transposase, the transfection efficiency is difficult to break through 50%, usually only about 20%. It is generally necessary to ensure a sufficient amount of CAR-T positive cells by preparing a large number of cells.

In order to improve the positive rate of CAR-T, researchers have gone through a lot of experiments to explore. Normally, T cells are transfected with the CAR gene by CD3/CD28 antibody coating or CD3/CD28 antibody magnetic beads to stimulate T cell activation, but this stimulation method is broad-spectrum for T cells, whether transfected or not. The CAR gene will be activated. Due to competitive growth, the proportion of CAR-T cells may be continuously diluted, and the proportion of CAR-T in the resulting T cell population is very low.

In order to solve this problem, attempts have been made to activate T cells transfected with the CAR gene by using specific antigens. The method uses the binding of specific antigens to scFv as the first signal for T cell activation and transduces the signal to CAR. The T cell intracellular co-stimulation region activates the second signal to activate T cells, so that although a highly positive proportion of CAR-T cells can be obtained, the proliferation is slow and it is difficult to obtain a large number of target cells. The reason may be due to the T cell clustering effect. When T cells proliferate vigorously, they usually aggregate into clusters. Cells secrete some cytokines to promote proliferation. Because the transfection efficiency is not high, the initial amount of CAR-T cells is insufficient. very slow.

Summary of the invention

The present invention provides a method of producing a CAR-T cell, the method comprising the step of incubating the CAR-T cell with a CD28 antibody and a chimeric antigen receptor-targeted antigen expressed by the CAR-T cell.

In one or more embodiments, the antigen is an antigen specifically bound by a single chain antibody of a CAR expressed by the CAR-T cell.

In one or more embodiments, the ratio of the antigen to the CD28 antibody is from 1:5 to 3:1, preferably from 1:2 to 2:1, more preferably 1:1.

In one or more embodiments, the CAR targets mesothelin, EGFR, Her2, CD19 or mucin.

In one or more embodiments, the antigen is mesothelin, EGFR, Her2, CD19 or mucin.

In one or more embodiments, the CAR-T cells are transfected with a non-viral vector expressing the CAR.

In one or more embodiments, the method comprises:

(1) transfecting a host T cell with a non-viral vector expressing the CAR;

(2) The T cells were incubated with the CD28 antibody and the CAR-targeted antigen 4 to 8 hours after transfection.

In one or more embodiments, the stimulatory factor IL-2 is also added upon incubation.

The present invention also provides a composition for producing CAR-T cells, which comprises a CD28 antibody and a chimeric antigen receptor-targeted antigen expressed by a CAR-T cell to be prepared.

In one or more embodiments, the antigen is an antigen specifically bound by a single chain antibody of a CAR expressed by a CAR-T cell to be prepared.

In one or more embodiments, the ratio of the antigen to CD28 antibody in the composition is from 1:5 to 3:1, preferably from 1:2 to 2:1, more preferably 1:1.

In one or more embodiments, the antigen is mesothelin, EGFR, Her2, CD19 or mucin.

In one or more embodiments, the concentration of the CD28 antibody in the composition is from 2 to 8 ug/ml and the concentration of the antigen is from 1 to 10 ug/ml.

In one or more embodiments, the composition further comprises IL-2, preferably, the IL-2 is present in the composition at a level of from 50 to 1000 U/ml.

The invention also provides a kit comprising a CD28 antibody and an antigen targeted by a chimeric antigen receptor expressed by a CAR-T cell to be prepared.

In one or more embodiments, the kit further comprises IL-2.

In one or more embodiments, the kit further comprises a vector that integrates the chimeric antigen receptor expression cassette into the host cell genome.

In one or more embodiments, the vector is a transposon vector, preferably, the transposon vector is a eukaryotic expression comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty Carrier.

In one or more embodiments, the vector is a cassette for expression of a chimeric antigen receptor between 5' LTR and 3' LTR, including a promoter sequence, a coding sequence for a chimeric antigen receptor, and a polyA tailing signal sequence.

In one or more embodiments, the kit further contains a transfection reagent and/or instrument.

The invention also provides the use of a combination of a CD28 antibody and an antigen for promoting CAR-T cell proliferation, wherein the antigen is an antigen targeted by a chimeric antigen receptor expressed by the CAR-T cell.

In one or more embodiments, the ratio of the antigen to the CD28 antibody is from 1:5 to 3:1, preferably from 1:2 to 2:1, more preferably 1:1.

In one or more embodiments, the antigen is mesothelin, EGFR, Her2, CD19 or mucin.

DRAWINGS

Figure 1: Schematic diagram of the structure of the CAR gene.

Figure 2: PBMC was transfected with meso3CAR gene by electroporation, and the positive rates of CAR-T were compared under the stimulation of antiCD3/antiCD28 and mesothelin/antiCD28, respectively.

Figure 3: CAR-T cell positive rate under specific conditions of specific antigen and CD28 antibody.

Figure 4A: RTCA detects the killing of tumor cells by meso3CAR T cells with different positive rates.

Figure 4B: Statistics on killing rates of tumor cells by meso3CAR T cells with different positive rates.

Figure 5: Flow-through detection of meso3CAR, EGFR-CAR, MUC1CAR, CD19CAR, Her2-CAR five CAR-T ratios of positive cells stimulated by antiCD3/antiCD28 and corresponding antigen/antiCD28.

Figure 6A-6B: Proportion of memory T populations in five CAR-T cells of meso3CAR, EGFR-CAR, MUC1CAR, CD19CAR, and Her2-CAR prepared by combining specific antigen with CD28 antibody. In the figure, Tem is an effector memory T cell; Tcm is a central memory T cell; Tm is a total memory T cell, that is, the sum of Tem and Tcm.

Figure 7: Proliferation of five kinds of CAR-T cells of meso3CAR, EGFR-CAR, MUC1CAR, CD19CAR, and Her2-CAR in combination with specific antigen and CD28 antibody and preparation of specific antigen alone.

Detailed ways

The inventors have established a mature preparation method for obtaining high positive rate CAR-T cells through a large amount of experiments and creative labor, and achieved more effective killing of tumor cell lines. Specifically, the present invention prepares CAR-T cells by a combination of a specific antigen and a CD28 antibody, initially obtaining a medium-positive proportion of CAR-T cells, contributing to their proliferation, and due to memory of specific antigens, the population The proportion of CAR-T cells in the process will be higher and higher, and finally a large number of high-positive CAR-T cells will be obtained.

Some of the terms related to the present invention are explained below. Other terms not explained herein have the general meaning in the art.

In the present invention, the term "expression cassette" refers to the entire element required for expression of a gene, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence.

The term "coding sequence" is defined herein as a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (eg, CAR, single chain antibody, hinge region, and transmembrane region). The boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of the 5' end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term "Fc", ie, fragment crystallizable (Fc) of an antibody, refers to a peptide located at the end of the handle of the "Y" structure of the antibody molecule, comprising the CH2 and CH3 domains of the heavy chain constant region of the antibody, and is an antibody and effect. The site of molecular or cellular interactions.

The term "costimulatory molecule" refers to a molecule that is present on the surface of an antigen presenting cell and that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal. The proliferation of lymphocytes requires not only the binding of antigens, but also the signals of costimulatory molecules. The costimulatory signal is transmitted to the T cells mainly by binding to the co-stimulatory molecule CD80 on the surface of the antigen presenting cells, and CD86 binds to the CD28 molecule on the surface of the T cell. B cells receive a costimulatory signal that can pass through a common pathogen component such as LPS, or through a complement component, or through activated antigen-specific Th cell surface CD40L.

The term "linker" or hinge is a polypeptide fragment that links between different proteins or polypeptides for the purpose of maintaining the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide. Exemplary linkers include linkers containing G and/or S, as well as, for example, Furin 2A peptide.

The term "specifically binds" refers to the reaction between an antibody or antigen-binding fragment and the antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) means that the antibody is less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, Affinity (KD) of 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or less binds to the antigen. "Specific recognition" and "targeting" have similar meanings.

The term "pharmaceutically acceptable excipient" refers to carriers and/or excipients that are compatible pharmacologically and/or physiologically to the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

The term "effective amount" refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.

The term "disease and/or condition" refers to a physical state of the subject that is associated with the disease and/or condition described herein.

The term "subject" or "patient" may refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog. , monkeys, cattle, horses, etc.

The term "chimeric antigen receptor" (CAR) is an artificially engineered receptor that anchors specific molecules (such as antibodies) that recognize tumor cell surface antigens to immune cells (such as T cells), allowing immune cells to recognize tumor antigens. Or viral antigens and cells that kill tumor cells or virus infection. CAR typically comprises, in turn, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signaling region. Generally, a polypeptide that binds to a tumor cell membrane antigen is capable of binding to a membrane antigen that is widely expressed by tumor cells with moderate affinity. The polypeptide that binds to the tumor cell membrane antigen may be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or a Fab fragment.

The term "single-chain antibody" (scFv) refers to an antibody fragment which is obtained by hinge-ligating an amino acid sequence of an antibody light chain variable region (VL region) and a heavy chain variable region (VH region), and having antigen-binding ability. In certain embodiments, the single chain antibody of interest (scFv) is from an antibody of interest. Antibodies of interest may be human antibodies, including human murine chimeric antibodies and humanized antibodies. The antibody can be secreted or membrane anchored.

"Antigen" generally refers to a substance that induces an immune response. An antigen herein refers in particular to an antigen to which a chimeric antigen receptor is targeted, particularly an antigen that specifically binds to a single chain antibody in a chimeric antigen receptor.

The present invention provides a method of producing a CAR-T cell, the method comprising the step of incubating the CAR-T cell with a CD28 antibody and a chimeric antigen receptor-targeted antigen expressed by the CAR-T cell.

The CD28 antibody is a CD28 antibody well known in the art, especially a CD28 antibody commonly used in the proliferation culture of CAR-T cells in the art.

The antigens used for incubation are different for different CAR-T cells. Specifically, the antigen should be an antigen targeted by the CAR expressed by the CAR-T cell, especially an antigen to which the single-chain antibody in the CAR can specifically bind.

Herein, the chimeric antigen receptor of interest may be directed against one or more of the following antigens: Her2, CD19, CD20, CEA, GD2 (also known as B4GALNT1, β1,4-acetyl-galactosyltransferase) 1), FR (Flavin reductase), PSMA (prostate specific membrane antigen), PMEL (premelanomeric protein), CA9 (carbonic anhydrase IX), CD171/L1-CAM, IL-13Rα2, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanoma-associated antigen E1) family protein, BAGE (B melanoma antigen family) family Protein, GAGE (growth hormone releasing factor) family protein, AFP (α-fetoprotein), MUC1 (mucin 1, cell surface related), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL- 11Rα, EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA1, ST8α-N-acetyl-ceramide α-2,8-sialic acid converting enzyme 1), PSCA (prostate stem cell antigen), FSA (also KIAA1109), PSA (also known as KLK3, kallikrein-related peptidase 3), HMGA2, fetal acetylcholine receptor, LeY (also known as FUT3), EpCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII , ERBB3, ERBB4 CA125 (also known as MUC16, mucin 16, cell surface related), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV- VCA, POA (also known as VDR, vitamin D (1,25-dihydrovitamin D3) receptor), β2-MG (β-2-microglobulin) and PROGRP (GRP gastrin releasing peptide).

In certain embodiments, the chimeric antigen receptor of interest is a chimeric antigen receptor directed against Her2, CD19, EGFR, mesothelin or mucin (Muc1). It will be understood that unless otherwise indicated, the various antigens described herein are well known in the art and the sequences are well known in the art.

Thus, an antigen suitable for use in the present invention may be a specific antibody (eg, a single chain antibody, scFv) that can be used to construct a chimeric antigen receptor to prepare a corresponding CAR-T cell antigen, including but not limited to the foregoing. Antigens include, inter alia, Her2, CD19, EGFR, mesothelin and Muc1.

Existing CAR-T cells can be incubated with the CD28 antibody and the corresponding antigen. In certain embodiments, CAR-T cells can be constructed first, and then the constructed CAR-T cells are incubated with the CD28 antibody and the corresponding antigen. Thus, in certain embodiments, the methods of the invention for preparing CAR-T cells further comprise the step of constructing CAR-T cells.

CAR-T cells can be prepared using methods routine in the art, including transfection of suitable T cells using a suitable recombinant plasmid.

The coding sequence of the chimeric antigen receptor is usually contained in the recombinant plasmid. CARs suitable for use in the present invention typically contain an optional signal peptide sequence, an amino acid sequence that specifically recognizes the antigen, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.

A signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of newly synthesized proteins to the secretory pathway, often referred to as the N-terminal amino acid sequence in the newly synthesized polypeptide chain that directs transmembrane transfer (localization) of the protein. (Sometimes not necessarily at the N-terminus), it is responsible for directing proteins into subcellular organelles with different membrane structures. The signal peptide can be a secreted signal peptide or a membrane-bound signal peptide. Suitable signal peptides include CD8 signal peptides, CD28 signal peptides or CD4 signal peptides and light chain signal peptides. The amino acid sequence of an exemplary CD8 signal peptide can be as shown in amino acid residues 1-22 of SEQ ID NO: 1, or as shown in amino acid residues 1 to 21 of SEQ ID NO: 7, the coding sequence of which is preferably as The nucleotide sequence of positions 1-66 of SEQ ID NO: 2 or the nucleotide sequence of positions 1-63 of SEQ ID NO: 8 is shown.

The amino acid sequence that specifically recognizes an antigen in a chimeric antigen receptor is typically an antibody, particularly a single chain antibody. Single chain antibodies directed against an antigen can be prepared using techniques well known in the art. In certain embodiments, a single chain antibody in a chimeric receptor antigen can be selected from a single chain antibody directed against any of the antigens described above, particularly a single strand directed against Her2, CD19, EGFR, mesothelin or Mucl antibody. In certain embodiments, the amino acid sequence of a single chain antibody directed against mesothelin is as shown in amino acid residues 23-272 of SEQ ID NO: 1; the amino acid sequence of a single chain antibody directed against EGFR is set forth in SEQ ID NO: 3 The amino acid sequence of the single-chain antibody against Her2 is shown in amino acid residues 23-264 of SEQ ID NO: 5; the amino acid sequence of the single-chain antibody against CD19 is SEQ ID NO :7 indicates the amino acid residues at positions 22-263; the amino acid sequence of the single-chain antibody against mucin is as shown in amino acid residues 23-269 of SEQ ID NO: 9. The corresponding coding sequences for these single chain antibodies can be found in the corresponding portions of SEQ ID NOs: 2, 4, 6, 8, and 10, respectively.

The hinge region refers to the region between the functional regions of the immunoglobulin heavy chain CH1 and CH2, which is rich in proline, does not form an alpha helix, is prone to stretching and is somewhat distorted, and is beneficial to the antigen binding site and antigenic epitope of the antibody. Complementary complementarity. A suitable hinge region may be selected from any one or more of the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD4. . The hinge region is preferably a hinge region that is longer than 50 amino acid residues, more preferably 80 amino acids or longer. The amino acid sequence of the exemplary IgG4 FcCH2CH3 hinge region is shown in amino acid residues 273-500 of SEQ ID NO: 1, and the coding sequence thereof is preferably as shown in SEQ ID NO: 2, bases 817-1500. The amino acid sequence of the exemplary CD8 alpha hinge region is set forth in amino acid residues 264 to 318 of SEQ ID NO: 3, and the coding sequence thereof is preferably shown in nucleotide sequence 789-954 of SEQ ID NO: 4.

The transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a CD3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region. The amino acid sequence of the exemplary CD28 transmembrane region is set forth in SEQ ID NO: 1 at 501-528, and the coding sequence thereof is preferably shown as bases 1501-1584 of SEQ ID NO: 2. The amino acid sequence of the exemplary CD8 transmembrane region is shown in amino acid residues 319 to 344 of SEQ ID NO: 3, and the coding sequence thereof is preferably shown in nucleotide sequence 955-1032 of SEQ ID NO: 4.

The intracellular co-stimulatory signal domain including the intracellular domain of the costimulatory signaling molecule may be selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), and inducible T cell costimulation. Intracellular domain of factor (ICOS) and DNAX activator protein 10 (DAP10). The amino acid sequence of the exemplary intracellular domain of CD28 is shown in amino acid residues 529-569 of SEQ ID NO: 1, and the exemplary coding sequence is shown in bases 1585-1707 of SEQ ID NO: 2. . The amino acid sequence of the intracellular domain of the exemplary CD137/4-1BB is shown in amino acid residues 333-374 of SEQ ID NO: 7, and the exemplary coding sequence is SEQ ID NO: 8 at positions 996-1122. The base sequence is shown.

The intracellular signal domain is preferably an immunoreceptor tyrosine activation motif, which may be a CD3 sputum intracellular signal domain or an FcεRI gamma intracellular signal domain. The amino acid sequence of the exemplary CD3 sputum intracellular signal domain is set forth in amino acid residues 570-681 of SEQ ID NO: 1, and exemplary coding sequences thereof are set forth in SEQ ID NO: 2, 1708-2043.

The above-mentioned various parts forming a chimeric antigen receptor, such as a signal peptide, a light chain variable region and a heavy chain variable region, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain of a single chain antibody They can be directly connected to each other or can be connected by a linker sequence. The linker sequence can be a linker sequence suitable for use in antibodies well known in the art, such as linker sequences comprising G and S. The linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, and 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The amount of glycine in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, and 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), styrene Amino acid (F), arginine (R), glutamine (Q), and the like.

The coding sequence of the chimeric antigen receptor can be obtained by PCR amplification. Specifically, primers can be designed according to the target sequence, and the relevant sequences can be amplified by using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

The coding sequences for exemplary chimeric antigen receptors are set forth in SEQ ID NOs: 2, 4, 6, 8, and 10.

After obtaining the coding sequence of the chimeric antigen receptor, it can be cloned into a suitable vector by a conventional method. Typically, the vector contains the corresponding promoter sequence and the polyA tailing signal sequence. If these sequences are not included in the vector, these sequences can be ligated to the coding sequence of the chimeric antigen receptor using methods routine in the art. A preferred vector is a non-viral vector, more preferably a vector (also referred to as an integration vector) that integrates the expression cassette of the chimeric antigen receptor into the genome of the host cell, particularly a transposon vector. In certain embodiments, the transposon vector is a eukaryotic expression vector comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty. Such transposon vectors contain the 5' inverted terminal repeat (5' LTR) of the corresponding transposon and the 3' inverted terminal repeat (3' LTR) of the corresponding transposon. The transposase can be a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system. When a transposase from a different transposition system is used, the sequences of the 5'LTR and 3'LTR in the vector are also correspondingly changed to sequences adapted to the transposition system, which can be readily determined by those skilled in the art. . Between the 5' LTR and the 3' LTR is the expression cassette for the CAR, including the corresponding promoter sequence, the coding sequence for the CAR, and the polyA tailing signal sequence.

In certain embodiments, the transposase is a transposase from a piggybac transposition system. Thus, in these embodiments, the 5' inverted terminal repeat and the 3' inverted terminal repeat of the transposon are the 5' inverted terminal repeat and the 3' inverted terminal repeat of the piggybac transposon, respectively. In certain embodiments, the transposon 5' inverted terminal repeat is SEQ ID NO: 1 as described in CN 201510638974.7, the disclosure of which is incorporated herein by reference. In certain embodiments, the transposon 3' inverted terminal repeat is as set forth in CN 201510638974.7 SEQ ID NO:4. In certain embodiments, the piggybac transposase is a transposase comprising a c-myc nuclear localization signal coding sequence. In certain embodiments, the coding sequence for the piggybac transposase is as shown in CN 201510638974.7 SEQ ID NO: 5.

The promoter of the transposase coding sequence can be a variety of promoters known in the art for controlling expression of the transposase coding sequence. In certain embodiments, the expression of the transposase coding sequence is controlled using a CMV promoter. The sequence of the CMV promoter can be as shown in CN 201510638974.7 SEQ ID NO: 6.

In certain embodiments, a recombinant plasmid containing a coding sequence for a chimeric antigen receptor is constructed using the pNB328 vector disclosed in CN 201510638974.7 as a backbone vector.

After constructing the recombinant plasmid, transfection can be carried out by conventional transfection methods in the art, including but not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation and electroporation. In certain embodiments, the vector is transfected into a cell of interest using electroporation.

The cells of interest may be various T cells well known in the art including, but not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTLs), helper T cells, suppressor/regulatory T cells, γδ T cells, and cytokines. T cells of mixed cell populations such as induced killer cells (CIK) and tumor infiltrating lymphocytes (TIL). In certain embodiments, the T cell can be derived from a PBMC of a B cell malignancy patient. In certain embodiments, the T cell is a primary cultured T cell.

After transfection for at least 4 hours, for example 4 to 8 hours or 6 to 8 hours after transfection, the resulting cells are incubated with the CD28 and antigen of the present invention while the stimulating factor IL-2 is added. The ratio of the antigen to the CD28 antibody is usually from 1:5 to 3:1, preferably from 1:2 to 2:1, more preferably 1:1 by mass. In certain embodiments, the concentration of the CD28 antibody is from 2 to 8 ug/ml, such as 5 ug/ml; the concentration of the antigen is from 1 to 10 ug/ml, such as from 2.5 to 10 ug/ml, or from 5 to 10 ug/ml. The concentration of IL-2 is usually from 50 to 1000 U/ml. Incubation conditions are conventional incubation conditions, such as a 37 ° C, 5% CO ₂ atmosphere.

When cultured for a period of time, for example, for 7 days or more, a T cell mixture having a CAR-T cell positive rate of 50% or more can be obtained. In certain embodiments, a T cell mixture having a CAR-T cell positive rate of greater than 80% can be obtained using the methods of the invention.

The present invention also provides a composition for producing CAR-T cells, which comprises a CD28 antibody and a chimeric antigen receptor-targeted antigen expressed by a CAR-T cell to be prepared. Preferably, the antigen is an antigen specifically bound by a single-chain antibody of a CAR expressed by a CAR-T cell to be prepared.

The antigen may be any of the antigens described above. In certain embodiments, the antigen is mesothelin, EGFR, Her2, CD19 or mucin.

The ratio of the antigen to the CD28 antibody in the composition is from 1:5 to 3:1, preferably from 1:2 to 2:1, more preferably 1:1. In certain embodiments, the concentration of the CD28 antibody in the composition is from 2 to 8 ug/ml and the concentration of the antigen is from 1 to 10 ug/ml.

In certain embodiments, the composition may also contain IL-2. Preferably, the IL-2 is present in the composition in an amount of from 50 to 1000 U/ml.

The composition may also contain a suitable solvent. In certain embodiments, the solvent is PBS.

Certain aspects of the invention also provide a kit comprising a CD28 antibody and an antigen as described herein. The CD28 antibody and antigen described in the kit may be packaged separately or in the form of a mixture. Accordingly, in certain embodiments, the kits of the invention comprise a composition described herein. In certain embodiments, the kit further comprises IL-2.

The kit may also contain a vector that integrates the chimeric antigen receptor expression cassette into the host cell genome. The vector may be a vector described herein, preferably the vector is a transposon vector; more preferably, the transposon vector is a eukaryotic expression comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty Carrier.

In certain embodiments, the vector is a blank vector. A blank vector can be used by a person skilled in the art to construct a recombinant vector containing the coding sequence of the chimeric antigen receptor, and then the CD28 antibody and the corresponding antigen contained in the kit are used for preparation of the corresponding CAR-T cells. The blank vector can be, for example, the pNB328 vector described herein or other vectors conventionally used in the art to construct CAR. At this time, the kit may further contain a suitable reagent for constructing the recombinant vector using the blank vector.

In certain embodiments, the vector is a vector comprising a coding sequence for a CAR. At this time, the antigen contained in the kit should be an antigen which can be specifically bound or recognized by the single-chain antibody of the CAR.

In certain embodiments, the kit may also contain transfection reagents and/or instruments.

The invention also provides the use of a combination of a CD28 antibody and an antigen described herein for promoting CAR-T cell proliferation. Preferably, in the application, the ratio of the antigen to the CD28 antibody is from 1:5 to 3:1, preferably from 1:2 to 2:1, more preferably 1:1 by mass. Preferably, the antigen is mesothelin, EGFR, Her2, CD19 or mucin.

Embodiments of the present invention will be described in detail below with reference to the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. If the reagents or instruments used are not the manufacturer, they are all conventional products that can be purchased through the market.

Example 1: Construction of recombinant plasmid

The gene sequence of meso3CAR, EGFRCAR, HER2CAR, MUC1CAR and CD19CAR was synthesized by Shanghai Jierui Biotech Co., Ltd. as shown in SEQ ID NOs: 2, 4, 6, 8 and 10, respectively. The structural pattern is shown in Figure 1, where mesoCAR The hinge region of CD19CAR is the IgG4 CH2CH3 hinge region, and the hinge regions of the other three CARs are the CD8 alpha hinge region.

The above gene sequences were separately inserted into the pNB328 vector EcoRI and SalI restriction sites, and the recombinant plasmids were named pNB328-meso3CAR, pNB328-EGFR-CAR, pNB328-HER2-CAR, pNB328-MUC1-CAR and pNB328, respectively. -CD19CAR.

The structure and sequence of the pNB328 vector are described in CN 201510638974.7, the entire disclosure of which is incorporated herein by reference.

Example 2: Comparison of the positive rate of meso3CAR T cells prepared by antiCD3/antiCD28 and mesothelin/antiCD28

1. Meso3CAR T cell construction: Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll separation. The PBMCs were cultured for 2-4 h, in which the unattached suspension cells were the initial T cells, and the suspension cells were collected into a 15 ml centrifuge tube, centrifuged at 1200 rmp for 3 min, and the supernatant was discarded. Add physiological saline, centrifuge at 1200rmp for 3min, discard the physiological saline, and repeat this step; take two 1.5ml centrifuge tubes, add 5 × 10 ⁶ cells per tube, number a, b, 1200rmp centrifugation for 3min, discard the supernatant, take electricity The kit (from Lonza), a and b tubes were added to the electrophoresis reagent in a total amount of 100 ul, and 6 ug of the recombinant plasmid pNB328-meso3CAR constructed in Example 1 was added, and the cells were resuspended and resuspended; the mixture was transferred to an electric rotor. Put in the electro-rotation instrument (from Lonza company), select the U-014 program, and carry out electric shock; use the micro-pipette in the kit to transfer the transferred cell suspension to the six-well plate with the culture solution (including 2% FBS). AIM-V medium), mixed, placed in a 37 ° C, 5% CO ₂ incubator.

2. Antibody or antigen coating method: Add CD3 and CD28 antibody or mesothelin antigen and CD28 antibody to PBS at a concentration of 5 ug/ml, mix well, and add to the six-well plate in an amount of 1 ml per well, 4 Allow to stand overnight at °C or 37 ° C for 4 hours. Discard the liquid during use.

3. Culture and amplification: After two hours of CAR-T cell electroporation, the cells of the two wells were added together, mixed and divided into two portions, one portion was placed in a six-well plate coated with antiCD3/antiCD28 antibody, and the other was Into a six-well plate coated with mesothelin antigen and CD28 antibody, 5 × 10 ⁶ to 1 × 10 ⁷ cells / well, and added the stimulation factor IL-2 (500 U / ml), 37 ° C, 5% The CO _{2 was} cultured for 4 to 5 days, and the growth of T cells was observed to obtain T cells expressing the meso3CAR gene.

4. Detection of positive rate of CAR-T cells: The positive rate of cell flow detection on the 7th and 15th day was collected. Specific methods of operation: Collect cells, 1 × 10 ⁶ per tube, add 1 ug of primary anti-meso-Biotin (with biotin label on mesothelin antigen), and incubate at 4 ° C for 30 minutes. Wash twice with saline, add 1 ul of secondary anti-streptomycin-PE, and incubate for 30 minutes at 4 °C. Wash the saline twice, and check it on the machine. The secondary antibody was added as a negative control, and the results are shown in Fig. 2.

Example 3: Comparison of CAR-T cell positive rate under different antigen and CD28 antibody ratio conditions

1 ug, 2.5 ug, 5 ug, 10 ug of mesothelin antigen and the same amount of CD28 antibody (5 ug/ml) were used for plating overnight at 4 ° C in a refrigerator. Meso3CAR T cells were constructed as described in Example 2. The electroporated cells were cultured under different ratios, and the positive rate of CAR T cells was detected on the 15th day. The detection method was as described in Example 2, and the results are shown in FIG.

Example 4: Comparison of the killing effect of meso3CAR T cells with different positive rates on tumor cell lines

The killing effect of meso3CAR T cells with different positive rates on tumor cell lines was detected by real-time label-free cell function analyzer. Specifically, ovarian cancer cell line SKOV3 with high expression of mesothelin was selected as the target cell, and different positive rates were detected by using Essen's real-time label-free cell function analyzer (RTCA) (10%, 30%, 50%, 70). % and 90%, meso3CAR T cells with different positive rates can be adjusted by in vitro killing activity of meso3CAR T cells by adding Mock T cells transfected with pNB328 empty vector in high positive rate meso3CAR T cells. The specific steps are as follows:

(1) Zero adjustment: Add 50μl DMEM culture solution to each well, put it into the instrument, select step 1, and adjust to zero;

(2) Target cell plating: ovarian cancer cell SKOV3 (purchased from the American Type Culture Collection ATCC) was placed in a plate containing the detection electrode at 10 ⁴ cells/50 μl per well, and left for a few minutes until the cells were stabilized. Into the instrument, start step 2, culture the cells;

(3) Adding effector cells: After 24 hours of target cell culture, stop step 2, add effect cells with various positive rates, 50 μl per well, set the effective target ratio to 4:1, start step 3, continue co-culture for 24 h, Observe the cell proliferation curve.

It was found that the higher the positive rate of meso3CAR T cells, the stronger the killing result, as shown in Figures 4A and 4B.

Example 5: Comparison of the positive rates of five kinds of CAR-T cells of meso3CAR, EGFR-CAR, MUC1CAR, CD19CAR and Her2CAR prepared by antiCD3/antiCD28 and specific antigen/antiCD28

Using the method described in Example 2, the plasmids used were pNB328-meso3CAR, pNB328-EGFR-CAR, pNB328-MUC1-CAR, pNB328-CD19CAR, pNB328-HER2-CAR to construct meso3CAR, EGFR-CAR, MUC1CAR, CD19CAR, respectively. , Her2CAR five kinds of CAR-T cells.

The corresponding CAR-T was cultured and amplified with antiCD3/antiCD28 and specific antigen/antiCD28, respectively, and the amplification method was the same as that described in Example 2. The specific antigens were mesothelin antigen, EGFR, mucin, CD19 and Her2, the concentration of each antigen was 5 ug/ml, and the concentration of CD28 antibody was 5 ug/ml.

Various CAR-T cells were collected on the 15th day, and the positive rate was detected by flow, and the detection method was the same as that described in Example 2. The primary antibodies used were meso-Biotin, EGFR-Biotin, MUCl-Biotin, CD19-Biotin, Her2-Biotin.

The results showed that the positive rates of various CAR-T cells were significantly increased under the preparation conditions of the combination of specific antigen and CD28 antibody, as shown in FIG. 5 .

Example 6: Comparing the ratio of memory T cells in meso3CAR, EGFR-CAR, MUC1CAR, CD19CAR, and Her2CAR five CAR-T cells prepared by antiCD3/antiCD28 and specific antigen/antiCD28

Five kinds of CAR-T cells of meso3CAR, EGFR-CAR, MUClCAR, CD19CAR, and Her2CAR were constructed as described in Example 2, and these five CAR-T cells were cultured and expanded as described in Example 5. On day 15, cells were harvested and the memory T ratio was detected by flow using CD45RO, CCR7 antibody.

As a result, as shown in Figs. 6A and 6B, CAR-T cells in the selective amplification system produced a higher proportion of memory T cells.

Example 7: Proliferation of five kinds of CAR-T cells of meso3CAR, EGFR-CAR, MUC1CAR, CD19CAR and Her2-CAR under the conditions of preparation of specific antigen combined with CD28 antibody and specific antigen alone

Five kinds of CAR-T cells, meso3CAR, EGFR-CAR, MUClCAR, CD19CAR and Her2CAR, were constructed as described in Example 4. The culture was carried out under the conditions of preparation of a specific antigen in combination with a CD28 antibody (concentration of 5 ug/ml) and a specific antigen alone (concentration: 10 ug/ml). The initial cell amount was the same, and the number of cells on the 15th day was counted and counted.

The results showed that the amplification efficiency of CAR-T cells under the conditions of preparation of specific antigen and CD28 antibody was significantly increased, as shown in FIG. 7 .

Claims (12)

1. A method of producing CAR-T cells, the method comprising the step of incubating the CAR-T cells with a CD28 antibody and a chimeric antigen receptor-targeted antigen expressed by the CAR-T cells; preferably The antigen is an antigen specifically bound by a single-chain antibody of a CAR expressed by the CAR-T cell.
2. The method according to claim 1, wherein the ratio of the antigen to the CD28 antibody is from 1:5 to 3:1, preferably from 1:2 to 2:1, more preferably 1: by mass. 1.
3. The method according to claim 1 or 2, wherein the chimeric antigen receptor targets the following antigens: Her2, CD19, CD20, CEA, GD2, FR, PSMA, PMEL, CA9, CD171/L1-CAM , IL-13Rα2, MART-1, ERBB2, NY-ESO-1, MAGE family proteins, BAGE family proteins, GAGE family proteins, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2 , IL-11Rα, EGP-2, EGP-40, FBP, GD3, PSCA, FSA, PSA, HMGA2, fetal acetylcholine receptor, LeY, EpCAM, mesothelin, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 , CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC, AFU, EBV-VCA, POA, β2-MG and PROGRP; preferably targeting mesothelin, EGFR, Her2, CD19 or Mucin.
4. The method according to any one of claims 1 to 3, wherein the CAR-T cells are transfected with a non-viral vector expressing the CAR;

   Preferably, the non-viral vector is a transposon vector, preferably a eukaryotic expression vector comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty.
5. The method of any of claims 1-4, wherein the method comprises:

   (1) transfecting a host T cell with a non-viral vector expressing the CAR;

   (2) incubated the T cells with the CD28 antibody and the CAR-targeted antigen after 4 to 8 hours of transfection;

   Preferably, the stimulating factor IL-2 is also added during the incubation.
6. A composition for the preparation of CAR-T cells, characterized in that the composition comprises a CD28 antibody and a chimeric antigen receptor-targeted antigen expressed by a CAR-T cell to be prepared; preferably, said The antigen is an antigen specifically bound by a single-chain antibody of CAR expressed by CAR-T cells to be prepared.
7. The composition according to claim 6, wherein said antigen is selected from the group consisting of: Her2, CD19, CD20, CEA, GD2, FR, PSMA, PMEL, CA9, CD171/L1-CAM, IL-13Rα2, MART- 1. ERBB2, NY-ESO-1, MAGE family proteins, BAGE family proteins, GAGE family proteins, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11Rα, EGP- 2. EGP-40, FBP, GD3, PSCA, FSA, PSA, HMGA2, fetal acetylcholine receptor, LeY, EpCAM, mesothelin, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125, CA15-3, CA19- 9. CA72-4, CA242, CA50, CYFRA21-1, SCC, AFU, EBV-VCA, POA, β2-MG and PROGRP; preferably targeting mesothelin, EGFR, Her2, CD19 or mucin.
8. The composition according to claim 6 or 7, wherein the ratio of the antigen to the CD28 antibody in the composition is from 1:5 to 3:1, preferably from 1:2 to 2 by mass. :1, more preferably 1:1

   Preferably, the concentration of the CD28 antibody in the composition is 2-8 ug/ml, and the concentration of the antigen is 1-10 ug/ml;

   Preferably, the composition further comprises IL-2, preferably, the concentration of IL-2 is from 300 to 1000 U/ml.
9. A kit comprising a CD28 antibody and an antigen targeted by a chimeric antigen receptor expressed by a CAR-T cell to be prepared; preferably, the antigen is selected from the group consisting of :Her2, CD19, CD20, CEA, GD2, FR, PSMA, PMEL, CA9, CD171/L1-CAM, IL-13Rα2, MART-1, ERBB2, NY-ESO-1, MAGE family protein, BAGE family protein, GAGE Family proteins, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11Rα, EGP-2, EGP-40, FBP, GD3, PSCA, FSA, PSA, HMGA2, Fetus Acetylcholine receptor, LeY, EpCAM, mesothelin, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC, AFU, EBV -VCA, POA, β2-MG and PROGRP; preferably targeting mesothelin, EGFR, Her2, CD19 or mucin.
10. The kit of claim 9 wherein said kit further comprises:

    IL-2;

    A vector for integrating a chimeric antigen receptor expression cassette into a host cell genome, wherein the vector is an empty vector or a vector containing the expression cassette; preferably, the vector is a transposon vector, preferably, The transposon vector is a eukaryotic expression vector comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty; and/or

    Transfection reagents and / or instruments.
11. The use of a combination of a CD28 antibody and an antigen for promoting CAR-T cell proliferation, wherein the antigen is an antigen targeted by a chimeric antigen receptor expressed by the CAR-T cell;

    Preferably, the antigen is selected from the group consisting of: Her2, CD19, CD20, CEA, GD2, FR, PSMA, PMEL, CA9, CD171/L1-CAM, IL-13Rα2, MART-1, ERBB2, NY-ESO-1, MAGE Family proteins, BAGE family proteins, GAGE family proteins, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11Rα, EGP-2, EGP-40, FBP, GD3, PSCA , FSA, PSA, HMGA2, fetal acetylcholine receptor, LeY, EpCAM, mesothelin, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21 -1, SCC, AFU, EBV-VCA, POA, β2-MG and PROGRP; preferably targeting mesothelin, EGFR, Her2, CD19 or mucin;

    Preferably, in the application, the ratio of the antigen to the CD28 antibody is from 1:5 to 3:1, preferably from 1:2 to 2:1, more preferably 1:1 by mass.
12. The CAR-T cell population prepared by the method of any one of claims 1-5.

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