CN108864286B

CN108864286B - Chimeric antigen receptor targeting CD19, method for combined expression of anti-PD 1 antibody variable region and application thereof

Info

Publication number: CN108864286B
Application number: CN201710341236.5A
Authority: CN
Inventors: 黄飞; 金涛; 王海鹰; 何凤; 史子啸
Original assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Current assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Priority date: 2017-05-16
Filing date: 2017-05-16
Publication date: 2023-08-29
Anticipated expiration: 2037-05-16
Also published as: CN108864286A

Abstract

The invention discloses a chimeric antigen receptor CD19 scFV-CD8hinge-CD28TM-CD28-CD3 zeta combined aPD1 scFV CAR-T cell and application thereof. Specifically, the present invention provides a polynucleotide sequence selected from the group consisting of: (1) Comprises a coding sequence of an anti-CD 19 single-chain antibody, a coding sequence of a human CD8 alpha hinge region, a coding sequence of a human CD28 transmembrane region, a coding sequence of a human CD28 intracellular region, a coding sequence of a human CD3 zeta intracellular region, a P2A polypeptide coding sequence, a coding sequence of a human IL2 signal peptide, and a coding sequence of an anti-human PD1 monoclonal antibody which are connected in sequence; and (2) the complement of the polynucleotide sequence of (1). The invention also provides related fusion proteins, vectors containing the coding sequences, and uses of the fusion proteins, the coding sequences and the vectors. The CD19-28z-aPD1 CAR-T cells prepared by the invention have strong killing function on specific tumor cells. The CD19-28z-aPD1 CAR-T cell prepared by the invention has the function of secreting PD1 antibodies, and can seal the PD1-PDL1 locus, so that the CAR-T cell is prevented from inhibiting the PD 1.

Description

Chimeric antigen receptor targeting CD19, method for combined expression of anti-PD 1 antibody variable region and application thereof

Technical Field

The invention belongs to the field of chimeric antigen receptors, and particularly relates to a target CD19CAR combined expression anti-PD 1 antibody variable region cell and application thereof.

Background

Chimeric antigen receptor (Chimeric Antigen Receptor-T cell, CAR-T) T cells refer to T cells that, after genetic modification, recognize a specific antigen of interest in an MHC non-limiting manner and continue to activate expansion. The annual meeting of the international cell therapy association in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors outside surgery, radiotherapy and chemotherapy, and is becoming an essential means for future tumor treatment. CAR-T cell feedback therapy is the most clearly effective form of immunotherapy in current tumor therapy. A large number of researches show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and obviously improve the survival condition of patients.

Chimeric Antigen Receptors (CARs) are the core component of CAR-T, conferring to T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. The basic design of a CAR includes a Tumor Associated Antigen (TAA) binding region (typically derived from the scFV segment of a monoclonal antibody antigen binding region), an extracellular hinge region, a transmembrane region and an intracellular signaling region. The selection of the antigen of interest is a critical determinant of the specificity, effectiveness, and safety of the genetically engineered T cells themselves of the CAR (science.1986.233 (4770): p.1318-21.).

CD19 is a 95kDa glycoprotein on the surface of B cells, which is expressed from the early stages of B cell development until it differentiates into plasma cells. CD19 is one of the members of the immunoglobulin (Ig) superfamily, which is one of the constituent elements of the B cell surface signaling complex, involved in regulating the signaling process of B cell receptors. In a mouse model of CD19 deficiency, a significant decrease in the number of B cells in peripheral lymphoid tissue occurs, as well as a decrease in the response to vaccine and mitogen, accompanied by a decrease in serum Ig levels. It is generally believed that CD19 is expressed only on B cell lines (B-cell lines) and not on the surface of pluripotent hematopoietic stem cells. CD19 is also expressed on the surface of most B-cell lymphomas, mantle cell lymphomas, all, CLLs, hairy cell leukemia, and a portion of acute myelogenous leukemia cells. Thus, CD19 is a very valuable immunotherapeutic target in the treatment of leukemia/lymphoma. Importantly, CD19 is not expressed on the surface of most normal cells other than B cells, including pluripotent hematopoietic stem cells, a feature that allows CD19 to serve as a safe therapeutic target that minimizes the risk of autoimmune disease or irreversible bone marrow toxicity damage in patients. Currently, antibodies or scFv fragments against CD19 have been developed and demonstrated in a mouse model and in human/primate animals for their application.

PD1 (programmed desath 1) was originally obtained in apoptotic T-cell hybridomas and was designated as the programmed death 1 receptor due to its association with apoptosis. PD1 receptors are expressed on the surface of T cells and on the surface of primary B cells and play a role in the differentiation and apoptosis of these cells. PD1 has two ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), respectively, belonging to the B7 family of proteins (blood.2009.114 (8): p.1537-44.). PD-L1 protein is widely expressed in association with antigen presenting cells, activated T, B cells, macrophages, placental trophoblasts, myocardial endothelium and thymic cortical epithelial cells. PD-L1 interacts with the receptor PD1 on T cells and plays an important role in the negative regulation of immune responses. Normally, when the body encounters a foreign pathogen or antigen invasion, antigen presenting cells capture the antigen, process the antigen to become an epitope that can be recognized by T cells, bind to MHC molecules and present a binding domain to the outside of the cell for T cell recognition. T cells bind to MHC molecules of antigen presenting cells via TCR, and additionally co-stimulatory signal CD28 receptors bind to B7-1 (CD 80) or B7-2 (CD 86) on the surface of the naive T cells, which receive the forward regulatory signals, which activate as effector T cells, initiating an immune response. When there is continuous antigen stimulation, to avoid excessive response, the surface of activated T cells express PD1, and combine with PD-L1 on the surface of antigen presenting cells to transmit negative regulation signals to T cells, and the proliferation of T cells is reduced or apoptosis. The research shows that the expression of PD-L1 protein can be detected in many human tumor tissues, the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells, and the combination of the expressed PD-L1 and the PD1 on the surface of the T cells can inhibit the anti-tumor activity of the T cells, so that the tumor cells can evade the monitoring and the elimination of the immune system of the organism, and the tumor generation and the tumor growth are facilitated.

The PD1/PD-L1 pathway inhibitor can block the combination of PD1 and PD-L1, block negative regulation signals, and enable T cells to restore activity, so that immune response is enhanced. The PD1/PDL1 pathway inhibitor mainly comprises anti-PD1 or anti-PD-L1 monoclonal antibody. In 7 months 2014, the Opdivo of schnobiletin was first approved in japan for the treatment of advanced melanoma as a PD1 inhibitor that was first approved for marketing worldwide. This is the first time that PD1 inhibitors show survival efficacy in phase III clinical trials with 73% to 42% survival, 40% to 14% response, and reduced adverse effects compared to the chemotherapeutic dacarbazine. Whereas the us merck Keytruda (pembrolizumab), 2014, 9 successfully entered the us market with a non-routine 1000 patient-enrolled large phase I clinical trial with the first PD-1 inhibitor identity, was approved for the treatment of advanced melanoma patients who were unable to undergo surgical excision or had developed metastases and were unresponsive to other drugs (N Engl J med.2013 Jul 11;369 (2): 134-44.).

Although the compound combination has wide prospect, the current anti-tumor drug treatment window is generally narrow, the effect of the combined drug is still difficult to predict, and the exertion of the PD1 effect is severely restricted. The rapid development of CAR-T cells provides a new opportunity for this. The CAR-T cells have strong targeting property and high specificity, can be rapidly proliferated in large quantity after being stimulated by tumor antigens, and can be limited by inhibitory signals, so that the anti-tumor capability of the CAR-T cells is greatly reduced. If the cell surface inhibitory receptor PD1 can be blocked, the CAR-T cells can be free from constraint, and the tumor killing effect can be fully exerted. Based on this, strategies that combine the use of CAR-T cells and blocking PD1/PD-L1 signals have received rapid attention from researchers (Oncominium.2014Dec 21;3 (11): e 970027.).

A series of studies was conducted by the professor Edmund Moon at the university of pennsylvania and the team he brought on against this joint application strategy. When the TCR-T cells taking NY-ESO-1 as an antigen target point kill tumor cells, the anti-PD 1 antibody is added, so that the phenomenon of T cell hypofunction can be obviously improved; accordingly, TCR-T cells have limited tumor clearance in a mouse subcutaneous tumor engrafting model, and complete tumor elimination is achieved after concurrent PD1 antibody treatment (Clin Cancer Res.2016.22 (2): p.436-47.). Meanwhile, the team designs a new generation of CAR-T cells by utilizing a genetic engineering technology, namely, a transgenic receptor PD1CD28 is inserted into the CAR-T cells through a viral vector, the structure consists of an extracellular segment of PD1 and a transmembrane segment and an intracellular segment of a co-stimulatory molecule CD28, and after the PD1 is combined with a ligand PD-L1 on the surface of tumor cells, a PD1/PD-L1 inhibition signal is converted into an activation signal, so that the power is increased for the functions of the CAR-T cells. The effect is also successfully verified in preclinical animal model research, and compared with the T cells without the PD1CD28 inserted into the CAR-T cells loaded with the PD1CD28, the CAR-T cells can generate stronger immune response to a mouse tumor model, and the survival rate of the mouse is increased.

The research initially shows the feasibility of the combined application strategy of CAR-T cells and blocking PD1/PD-L1 signals in tumor treatment, and on the basis, the best combination between the CAR-T cells and the PD1/PD-L1 signals is realized by fully utilizing the mature genetic engineering technology developed nowadays. The strategy of combining the CD19CAR with the PD1 single-chain antibody, which is designed at present, enables CAR-T cells and blocking PD1 signals to be well applied, well improves tumor immunosuppression microenvironment, and lays a foundation for future clinical experiments.

Disclosure of Invention

In a first aspect the invention provides a polynucleotide sequence selected from the group consisting of:

(1) Leader peptide coding sequence comprising a CD8 antigen, an anti-CD 19 single chain antibody coding sequence, a human CD8 alpha hinge region coding sequence, a human CD28 transmembrane region coding sequence, a human CD28 intracellular region coding sequence, a human CD3 zeta intracellular region coding sequence, and P2A polypeptide coding sequence, a human IL2 signal peptide coding sequence, coding sequences for heavy and light chain variable regions of an anti-human PD1 monoclonal antibody, and

(2) The complement of the polynucleotide sequence of (1).

In one or more embodiments, the polynucleotide sequence further comprises a coding sequence for a signal peptide prior to the coding sequence for the anti-CD 19 single chain antibody. In one or more embodiments, the amino acid sequence of the signal peptide is shown as amino acids 1-21 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-CD 19 single chain antibody is shown as amino acids 22-128 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single chain antibody is shown as amino acids 144-263 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human CD 8. Alpha. Hinge region is shown as amino acids 264-310 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human CD28 transmembrane region is shown as amino acids 311-337 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human CD28 intracellular region is shown as amino acids 338-378 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human CD3 zeta intracellular region is shown as amino acids 379-489 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of human P2A is shown as amino acids 490-515 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human IL2 signal peptide is shown as amino acids 516-535 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-PD 1 single-chain antibody is as shown in amino acids 536-648 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-PD 1 single-chain antibody is as shown in amino acids 664-770 of SEQ ID NO. 2.

In one or more embodiments, the coding sequence of the signal peptide preceding the coding sequence of the anti-CD 19 single chain antibody is shown as nucleotide sequences 1-63 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the light chain variable region of the anti-CD 19 single chain antibody is shown as nucleotide sequences 64-384 of SEQ ID NO. 1. In one or more embodiments, the heavy chain variable region of the anti-CD 19 single chain antibody has a coding sequence as set forth in nucleotide sequences 430-789 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the human CD 8. Alpha. Hinge region is shown in nucleotide sequences 790-930 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the human CD28 transmembrane region is shown as nucleotide sequence No. 931-1010 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the human CD28 intracellular region is shown as nucleotide sequences 1011-1134 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the human CD3 zeta intracellular region is as shown in nucleotide sequences 1135-1467 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of human P2A is as shown in nucleotide sequences 1368-1545 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the human IL-2 signal peptide is as shown in nucleotide sequences 1546-1605 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the heavy chain variable region of the anti-PD 1 single-chain antibody is as shown in nucleotide sequences 1606-1944 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the light chain variable region of the anti-PD 1 single-chain antibody is as shown in nucleotide sequences 1990-2310 of SEQ ID NO. 1.

In a second aspect the invention provides a fusion protein selected from the group consisting of:

(1) Fusion proteins comprising a leader peptide of a CD8 antigen, an anti-CD 19 single chain antibody, a human CD8 a hinge region, a human CD28 transmembrane region, a human CD28 intracellular region and a human cd3ζ intracellular region, and P2A polypeptide, a human IL2 signal peptide, heavy and light chain variable regions of an anti-human PD1 monoclonal antibody, which are sequentially linked; and

(2) A fusion protein derived from (1) by substitution, deletion or addition of one or more amino acids in the amino acid sequence defined in (1) and retaining the activity of activating CAR-T cells;

preferably, the anti-CD 19 single chain antibody is anti-CD 19 monoclonal antibody FMC63.

In one or more embodiments, the amino acid sequence of the signal peptide is shown as amino acids 1-21 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-CD 19 single chain antibody is shown as amino acids 22-128 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single chain antibody is shown as amino acids 144-263 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human CD 8. Alpha. Hinge region is shown as amino acids 264-310 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human CD28 transmembrane region is shown as amino acids 311-337 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human CD28 intracellular region is shown as amino acids 338-378 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human CD3 zeta intracellular region is shown as amino acids 379-489 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of human P2A is shown as amino acids 490-515 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human IL2 signal peptide is shown as amino acids 516-535 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-PD 1 single-chain antibody is as shown in amino acids 536-648 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-PD 1 single-chain antibody is as shown in amino acids 664-770 of SEQ ID NO. 2.

In a third aspect, the invention provides a nucleic acid construct comprising a polynucleotide sequence as described herein.

In one or more embodiments, the nucleic acid construct is a vector. In one or more embodiments, the nucleic acid construct is a retroviral vector comprising a replication origin site, a 3'LTR, a 5' LTR, the polynucleotide sequences described herein, and optionally a selectable marker.

In a fourth aspect the invention provides a retrovirus comprising a nucleic acid construct as described herein, preferably comprising the vector, more preferably comprising the retroviral vector.

In a fifth aspect, the invention provides a genetically modified CAR-T cell comprising a polynucleotide sequence as described herein, or comprising a nucleic acid construct as described herein, or infected with a retrovirus as described herein.

In a sixth aspect, the invention provides a pharmaceutical composition comprising a genetically modified CAR-T cell as described herein.

In a seventh aspect, the invention provides the use of a polynucleotide sequence, fusion protein, nucleic acid construct or retrovirus as described herein in the preparation of an activated CAR-T cell.

An eighth aspect of the invention is a method of genetically modified CAR-T cell activation and culture and uses thereof

In a ninth aspect, the invention provides the use of a polynucleotide sequence, fusion protein, nucleic acid construct, retrovirus, or genetically modified CAR-T cell described herein, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment of a CD19 mediated disease.

In one or more embodiments, the CD19 mediated disease is leukemia, lymphoma.

Drawings

FIG. 1 is a schematic representation of RV-CD19-28z-aPD1-CAR retroviral expression vector (CD 19-28z-aPD 1). SP: a signal peptide; VL: a light chain variable region; lk: joint (G) ₄ S) ₃ The method comprises the steps of carrying out a first treatment on the surface of the VH: a heavy chain variable region; h: a CD8 a hinge region; TM: CD8 transmembrane region.

FIG. 2 is a peak plot of partial sequencing results for RV-CD19-28z-aPD1-CAR retroviral expression vector (CD 19-28z-aPD 1).

FIG. 3 shows the expression efficiency of CD19-28z-tEGFR and CD19-28z-aPD1 CAR-T for 72 hours in retroviral infected T cells by flow cytometry.

FIG. 4 is a CD107a degranulation assay for 5 days of preparation of CD19-28z-tEGFR and CD19-28z-aPD1 CAR-T cells co-cultured with target cells for 4 hours.

FIG. 5 is a secretion assay for 4 hours IFNγ prepared 5 days of CD19-28z-tEGFR and CD19-28z-aPD1 CAR-T cells co-cultured with target cells.

FIG. 6 is a graph showing the detection of tumor cell killing after 16 hours of co-culture of 5-day-prepared CD19-28z-tEGFR and CD19-28z-aPD1 CAR-T cells with target cells.

FIG. 7 is a graph showing detection of PD1 expression on the surface of CD19-28z-tEGFR and CD19-28z-aPD1 CAR-T cells after 24 hours of co-culture with target cells after 5 days of preparation.

Detailed Description

The present invention provides a CD 19-targeting Chimeric Antigen Receptor (CAR) that combines expression of the variable region of an anti-PD 1 antibody. The CAR contains a leader peptide of a CD8 antigen, an anti-CD 19 single chain antibody, a human CD8 a hinge region, a human CD28 transmembrane region, a human CD28 intracellular region and a human CD3 zeta intracellular region, and fusion proteins of a P2A polypeptide, a human IL2 signal peptide, and an anti-human PD1 single chain antibody variable region, which are sequentially connected.

The anti-CD 19 single chain antibodies suitable for use in the present invention may be derived from a variety of anti-CD 19 monoclonal antibodies well known in the art.

Optionally, the light chain variable region and the heavy chain variable region may be linked together by a linker sequence. Exemplary such single chain antibodies include, but are not limited to, FMC63, SJ25C1. In certain embodiments, the monoclonal antibody is a monoclonal antibody with clone number FMC 63. In certain embodiments, the amino acid sequence of the light chain variable region of the anti-CD 19 single chain antibody is shown as amino acid residues 22-128 of SEQ ID NO. 2. In other embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single chain antibody is shown as amino acid residues 144-263 of SEQ ID NO. 2.

The amino acid sequence of the human CD8 alpha hinge region suitable for the present invention can be shown as amino acids 264-310 of SEQ ID NO. 2.

The human CD28 transmembrane region suitable for use in the present invention may be a variety of human CD28 transmembrane region sequences commonly used in the art for CARs. In certain embodiments, the amino acid sequence of the transmembrane region of human CD28 is shown as amino acids 311-337 of SEQ ID NO. 2.

CD28 suitable for use in the present invention can be any of a variety of CD28 known in the art for use in CARs. As an illustrative example, the present invention uses CD28 shown in the amino acid sequences 338-378 of SEQ ID NO. 2.

The human cd3ζ intracellular region suitable for use in the present invention may be various human cd3ζ intracellular regions conventionally used in the art for CARs. In certain embodiments, the amino acid sequence of the human CD3 zeta intracellular region is shown as amino acids 379-489 of SEQ ID NO. 2.

The amino acid sequence of the human P2A suitable for the invention can be shown as 490-515 th amino acid of SEQ ID NO. 2.

The amino acid sequence of the human IL-2 signal peptide suitable for the invention can be shown as 516 th to 535 th amino acids of SEQ ID NO. 2.

The amino acid sequence of the heavy chain variable region of the human anti-PD 1 single-chain antibody suitable for the invention can be shown as 536-648 amino acids of SEQ ID NO. 2.

The amino acid sequence of the light chain variable region of the human anti-PD 1 single-chain antibody suitable for the invention can be shown as amino acids 664-770 of SEQ ID NO. 2.

The above-described portions forming the fusion protein of the present invention, i.e., the leader peptide of the CD8 antigen, the anti-CD 19 single chain antibody, the human CD 8. Alpha. Hinge region, the human CD28 transmembrane region, the human CD28 intracellular region, the human CD3 zeta intracellular region, the human P2A polypeptide, the human IL2 signal peptide, the anti-human PD1 single chain antibody may be directly linked to each other or may be linked via a linker sequence. The linker sequences may be linker sequences suitable for antibodies as known in the art, such as G and S containing linker sequences. Typically, a linker contains one or more motifs that repeat back and forth. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are contiguous in the linker sequence with no amino acid residues inserted between the repeats. The linker sequence may comprise 1, 2, 3, 4 or 5 repeat motif compositions. The length of the linker may be 3 to 25 amino acid residues, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a glycine linker sequence. The number of glycine in the linker sequence is not particularly limited, and is usually 2 to 20, for example 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc. By way of example, the linker may consist of the following amino acid sequence: g (SGGGG) ₂ SGGGLGSTEF(SEQ ID NO:7)、RSTSGLGGGS(GGGGS) ₂ G(SEQ ID NO:8)、QLTSGLGGGS(GGGGS) ₂ G (SEQ ID NO: 9), GGGS (SEQ ID NO: 10), GGGGS (SEQ ID NO: 11), SSSSG (SEQ ID NO: 12), GSGSGSA (SEQ ID NO: 13), GGSGG (SEQ ID NO: 14), GGGGSGGGGSGGGGS (SEQ ID NO: 15), SSSSGSSSSGSSSSG (SEQ ID NO: 16), GSGSAGSGSAGSGSA (SEQ ID NO: 17), GGSGGGGSGGGGSGG (SEQ ID NO: 18), and the like.

In certain embodiments, the anti-CD 19 and PD1 single-chain antibodies of the invention have a heavy chain variable region-to-light chain variable region-to-heavy chain variable region (GGGS) _n And (3) a connection, wherein n is an integer of 1 to 5.

It will be appreciated that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed amino acid sequence, without affecting the activity of the sequence of interest. To construct fusion proteins, facilitate expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside of the host cell, or facilitate purification of recombinant proteins, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, for example, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-or carboxy-terminus of the fusion protein of the invention (i.e., the CAR) may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, poly-His, poly-Arg, strep-TagII, AU1, EE, T7,4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins.

The invention also includes mutants of CARs formed by sequential tandem of SEQ ID NO. 1 and SEQ ID NO. 2. These mutants include: an amino acid sequence that has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the CAR and retains the biological activity of the CAR (e.g., activates T cells). Sequence identity between two aligned sequences can be calculated using BLASTp, e.g., NCBI.

Mutants also included: an amino acid sequence having one or several mutations (insertions, deletions or substitutions) in the sequences shown in SEQ ID NO. 1 and 2, while still retaining the biological activity of the CAR. The number of mutations is generally within 1 to 10, for example 1 to 8, 1 to 5 or 1 to 3. The substitution is preferably a conservative substitution. For example, conservative substitutions with amino acids that are similar or analogous in nature typically do not alter the function of the protein or polypeptide. "similar or analogous amino acids" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, substitution of one or several sites with another amino acid residue from the same side chain class in a polypeptide of the invention will not substantially affect its activity.

The invention includes polynucleotide sequences encoding the fusion proteins of the invention. The polynucleotide sequences of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The invention also includes degenerate variants of the polynucleotide sequence encoding a fusion protein, i.e., nucleotide sequences that encode the same amino acid sequence but differ in nucleotide sequence.

The polynucleotide sequences described herein can generally be obtained using PCR amplification methods. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding the fusion proteins described herein is set forth in SEQ ID NO. 1.

The invention also relates to nucleic acid constructs comprising the coding sequences for the fusion proteins described herein, and one or more regulatory sequences operably linked to these sequences. The coding sequence of the fusion protein of the invention can be manipulated in a number of ways to ensure expression of the protein. The nucleic acid construct may be manipulated according to the expression vector or requirements prior to insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The regulatory sequence may be a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequences may also be suitable leader sequences, untranslated regions of mRNA that are important for host cell translation. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

In certain embodiments, the nucleic acid construct is a vector. Expression of the polynucleotide sequence encoding the CAR is typically achieved by operably linking the polynucleotide sequence encoding the CAR to a promoter, and incorporating the construct into an expression vector. The vector may be suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

Polynucleotide sequences encoding the CARs of the invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phagemids, phage derivatives, animal viruses and cosmids. Further, the vector is an expression vector. The expression vector may be provided to the cell as a viral vector. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

For example, in certain embodiments, the invention uses a retroviral vector comprising a replication origin site, a 3'LTR, a 5' LTR, polynucleotide sequences as described herein, and optionally a selectable marker.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the epstein barr virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter. Further, the use of inducible promoters is also contemplated. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when expressed for a period of time and switching off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cell from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequences. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at the appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or commercially available.

Methods for introducing genes into cells and expressing genes into cells are known in the art. The vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Chemical means for introducing the polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly retroviral vectors, which have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene may be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

Thus, in certain embodiments, the invention also provides a retrovirus for activating a T cell, the virus comprising a retroviral vector described herein and corresponding packaging genes, such as gag, pol and vsvg.

T cells suitable for use in the present invention may be of various types of T cells of various origins. For example, T cells may be derived from PBMCs of B cell malignancy patients.

In certain embodiments, after T cells are obtained, activation may be stimulated with an appropriate amount (e.g., 30-80 ng/ml, such as 50 ng/ml) of CD3 antibody, and then cultured in an IL2 medium containing an appropriate amount (e.g., 30-80 IU/ml, such as 50 IU/ml) for use.

The CAR-T cells of the invention can undergo robust in vivo T cell expansion and last at high levels in blood and bone marrow for prolonged amounts of time and form specific memory T cells. Without wishing to be bound by any particular theory, the CAR-T cells of the invention can differentiate in vivo into a central memory-like state upon encountering and subsequently eliminating target cells expressing the surrogate antigen.

The invention also includes a class of cell therapies in which T cells are genetically modified to express a CAR as described herein, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing the recipient's tumor cells. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to persistent tumor control.

The anti-tumor immune response elicited by the CAR-T cells can be an active or passive immune response. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-T cells induce an immune response specific for the antigen binding portion in the CAR.

The cancer that can be treated can be a non-solid tumor, such as a hematological tumor, e.g., leukemia and lymphoma. In particular, diseases treatable with the CAR, coding sequences thereof, nucleic acid constructs, expression vectors, viruses and CAR-T cells of the invention are preferably CD19 mediated diseases, in particular CD19 mediated hematological tumors.

In particular, herein, "CD19 mediated diseases" include, but are not limited to, leukemias and lymphomas, such as B-cell lymphomas, mantle cell lymphomas, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, hairy cell leukemia, and acute myelogenous leukemia.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as the relevant cytokine or cell population. Briefly, the pharmaceutical compositions of the invention may comprise a CAR-T cell as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative.

The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease.

When referring to an "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising T cells described herein may be administered at 10 ⁴ To 10 ⁹ A dose of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Dosage of individual cells/kg body weight. T cell compositions may alsoMultiple administrations at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by intravenous injection. The composition of T cells may be injected directly into the tumor, lymph node or site of infection.

In some embodiments of the invention, the CAR-T cells of the invention or compositions thereof can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressants. For example, treatment may be performed in combination with various radiotherapeutic agents, including: cyclosporine, azathioprine, methotrexate, mycophenolate, FK506, fludarabine, rapamycin, mycophenolic acid, and the like. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablation therapy with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH.

Herein, "anti-tumor effect" refers to a biological effect that can be represented by a decrease in tumor volume, a decrease in tumor cell number, a decrease in metastasis number, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

"patient," "subject," "individual," and the like are used interchangeably herein to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

Examples

The present invention is described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to include any and all variations that become apparent from the teachings provided herein.

Example 1: determination of the Gene sequence of CD19scFv-CD 8. Alpha. -CD28-CD3 zeta-P2A-aPD 1scFV

The NCBI website database searches the gene sequence information of the light chain and heavy chain variable region of the anti-CD 19 antibody, the human CD8 alpha hinge region, the human CD28 transmembrane region and intracellular region, the human CD3 zeta intracellular region and the heavy chain and light chain variable region of the anti-human PD1 antibody, and the sequences are subjected to codon optimization on the website http:// sg.idtdna.com/codonOpt, so that the coding amino acid sequence is ensured to be more suitable for human cell expression under the condition of unchanged coding amino acid sequence.

The sequences are connected by adopting overlapped PCR according to the sequences of anti-CD 19scFv, human CD8 alpha hinge region gene, human CD28 transmembrane region gene, human CD28 intracellular region gene, human CD3 zeta intracellular region gene, heavy chain and light chain variable region gene of anti-PD 1 antibody, and different enzyme cutting sites are introduced at the connection part of each sequence to form a complete CD19-CD28-aPD1 CAR gene sequence.

The nucleotide sequence of the CAR molecule was digested with NotI (NEB) and EcoRI (NEB), ligated by T4 ligase (NEB) and inserted into the NotI-EcoRI site of retroviral vector MSCV (Addgene), and transformed into competent E.coli (DH 5. Alpha.).

The recombinant plasmid was sent to Shanghai Biotechnology Co., ltd for sequencing, and the sequencing result was aligned with the proposed mCD19-CAR sequence to verify whether the sequence was correct. The sequencing primer is as follows:

sense sequence AGCATCGTTCTGTGTTGTCTC

Antisense sequence TGTTTGTCTTGTGGCAATACAC

After sequencing correctly, plasmids were extracted and purified using Qiagen's plasmid purification kit, and 293T cells were transfected with the plasmid purified by the plasmid calcium phosphate method for retrovirus packaging experiments.

The plasmid map constructed in this example is shown in FIG. 1. FIG. 2 shows a peak plot of partial sequencing results of the retroviral expression plasmid.

Example 2: retroviral packaging

1. 293T cells should be less than 20 passages on day 1, but not overgrown. 10ml of DMEM medium was added to a 10cm dish of 0.6X10cells/ml plate, and the cells were thoroughly mixed and cultured overnight at 37 ℃.

2. Day 2, transfection was performed with 293T cell fusion reaching about 90% (usually about 14-18h plating); plasmid complexes were prepared with the amounts of each plasmid being 12.5ug retrobackup, 10ug gag-pol, 6.25ug VSVg, caCl ₂ 250ul，H ₂ O is 1ml and the total volume is 1.25ml; in the other tube, HBS was added in an equal volume to the plasmid complex, and vortexed for 20s while adding the plasmid complex. The mixture was gently added to 293T dishes along the sides, incubated at 37℃for 4h, medium removed, washed once with PBS, and pre-warmed fresh medium was added again.

3. Day 4: the supernatant was collected 48h after transfection and filtered with a 0.45um filter and stored in aliquots at-80℃with continued addition of pre-warmed fresh DMEM medium.

Example 3: retrovirus infects human T cells

1. Separating with Ficcol separating solution (Tianjin, cys.) to obtain purer CD3+ T cells, and adjusting cell density to 1×10 with 5% AB serum (GEMINI) X-VIVO (LONZA) medium ⁶ /mL. Cells were inoculated at 1 ml/well into cells pre-incubated with anti-human 50ng/ml CD3 antibody (Beijing co-dried sea) and 50ng/ml CD28 antibody (Beijing co-dried sea), followed by the addition of 100IU/ml interleukin 2 (Beijing double Lu), and after 48 hours of stimulation.

Every other day after T cell activation culture, PBS was diluted to a final concentration of 15. Mu.g/ml in Retronectin (Takara) -coated Non-ti tissue treated plates, 250. Mu.l per well of 24 well plates. Light was protected from light and kept at 4℃overnight for further use.

After two days of T cell activation culture, 2 pieces of the coated 24-well plate were removed, the coating solution was removed by suction, and HBSS containing 2% BSA was added and blocked at room temperature for 30min. The blocking solution was pipetted into a volume of 500 μl per well and the plates were washed twice with HBSS containing 2.5% hepes.

4. The virus solution was added to the wells, 2ml of virus solution was added to each well, and the wells were centrifuged at 32℃and 2000g for 2 hours.

5. The supernatant was discarded and activated T cells 1X 10 were added to each well of the 24-well plate ⁶ The volume of the culture medium is 1ml, and IL-2 200IU/ml is added to the T cell culture medium. Centrifuge at 30℃for 10min at 1000 g.

6. After centrifugation, the plates were placed in a 5% CO2 incubator at 37 ℃.

7. 24h after infection, the cell suspension was aspirated, at 1200rpm,4℃and centrifuged for 7min.

8. After cell infection, observing the density of cells every day, and timely supplementing T cell culture solution containing IL-2 100IU/ml to maintain the density of T cells at 5×10 ⁵ About/ml, and the cells are expanded.

Thus, CAR-T cells each infected with the retrovirus shown in example 3 were obtained, and designated as CD 19-28-apc 1 CAR-T cells (expressing CD 19-28-apc 1 CAR of example 1), respectively.

Example 4: flow cytometry detection of expression of T lymphocyte surface CAR proteins after infection

CAR-T cells and NT cells 72 hours after infection (control) were collected separately by centrifugation, the supernatant was washed 1 time in PBS, the corresponding antibody was added to protect from light for 30min, washed in PBS, resuspended, and finally CAR (anti-mouse IgG F (ab') antibody (Jackson Immunoresearch)) was detected by flow cytometry.

The results of this example are shown in FIG. 3, in which the expression efficiency of CD19-28-aPD1 CAR+ is 42.278% and the expression efficiency of CD 19-28-tEGFRCAR+ is 46.69% after T cells are infected with the retrovirus prepared in example 3 for 72 hours. Both sets of CAR-T cells have similar expression efficiencies.

Example 5: detection of CD107a degranulation after co-culture of CAR-T cells and target cells

1. Taking a V-bottom 96-well plate, adding 2 x 10 of CAR-T/NT cells to each well ⁵ Individual and target cells (Raji or NALM 6)/control cells (K562) 2 x 10 ⁵ Complete culture of IL-2-free X-VIVO resuspended at 100ulThe cells were collected by adding BD Golgi stop (1. Mu.l BD Golgi stop per 1ml medium) to each well, adding 2ul CD107a antibody (bioleged) (1:50), and incubating at 37℃for 4 hours.

2. The samples were centrifuged to remove the medium, the cells were washed once with PBS and centrifuged at 400g for 5 min at 4 ℃. The supernatant was discarded, and an appropriate amount of specific surface antibodies CD3 (bioleged), CD4 (bioleged), CD8 (bioleged) was added to each tube, and the volume was resuspended at 100ul and incubated on ice for 30 minutes in the absence of light.

3. Cells were washed 1 time with 3mL of PBS per tube and centrifuged at 400g for 5 minutes. The supernatant was carefully aspirated.

4. The appropriate amount of PBS was resuspended and the flow cytometer detected CD3, CD4, CD8, CD107a.

The results of this example are shown in fig. 4. The percentage of CD107a degranulation in CD8 positive cells after co-culture with Raji cells was 40.1% and 55.5% for CD19-28 z-acd 1 CAR-T cells and CD19-28 z-tggfr CAR-T cells, respectively; the percentage of CD107a degranulation in CD8 positive cells after co-culture of CD19-28 z-atd 1 CAR-T cells and CD19-28 z-tggfr CAR-T cells with NALM6 cells was 30.4% and 60.3%, respectively; the percentage of CD107a degranulation in CD4 positive cells after co-culture with Raji cells was 40.9% and 56.9% for CD19-28 z-acd 1 CAR-T cells and CD19-28 z-tggfr CAR-T cells, respectively; the percentage of CD107a degranulation in CD4 positive cells after co-culture of CD19-28 z-atd 1 CAR-T cells and CD19-28 z-tggfr CAR-T cells with NALM6 cells was 38.5% and 45.5%, respectively.

Example 6: IFNgamma secretion assay after CAR-T cell co-culture with target cells

1. Taking prepared CAR-T cells, re-suspending in Lonza culture medium, and adjusting cell concentration to 1×10 ⁶ /mL。

2. Each well of the experimental group contained 2X 10 target cells (Raji or NALM 6) or negative control cells (K562) ⁵ CAR-T/NT cells 2X 10 ⁵ 100. Mu.l of Lonza medium without IL-2. After thoroughly mixing, the mixture was added to a 96-well plate. BD Golgi stop (containing monesin, 1. Mu.l BD Golgi stop per 1ml medium) was added, and after thoroughly mixing, incubated at 37℃for 4 hours. Cells were collected as an experimental group.

3. Cells were washed 1 time with 1mL of PBS per tube and centrifuged at 300g for 5 min. The supernatant was carefully aspirated or decanted.

After washing the cells with PBS, 250. Mu.l/EP tube Fixation/Permeabilization solution was added and incubated at 4℃for 20 minutes to fix the cells and rupture the membranes. With 1 XBD Perm/Wash ^TM buffer washed cells 2 times, 1 mL/time.

5. Dyeing with intracellular factor, collecting appropriate amount of IFN-gamma cytokine fluorescent antibody or negative control, and using BD Perm/Wash ^TM buffer was diluted to 50. Mu.l. The cells with fixed rupture membranes are fully resuspended by the antibody diluent, incubated for 30min at 4 ℃ in the absence of light, 1 XBD Perm/Wash ^TM buffer 1 mL/cell wash 2 times, then re-suspend with PBS.

6. And (5) detecting by a flow cytometer.

The results of this example are shown in FIG. 5, where the percentage of IFNγ expression in CD8 positive cells after co-culture with Raji cells is 28.2% and 57.4% for CD19-28z-aPD1 CAR-T cells and CD19-28z-tEGFR CAR-T cells, respectively; the percentage of ifnγ expression in CD8 positive cells after co-culture of CD19-28 z-acd 1 CAR-T cells and CD19-28 z-tggfr CAR-T cells with NALM6 cells was 29.4% and 58.9%, respectively; the percentage of ifnγ expression in CD19-28 z-acd 1 CAR-T cells and CD19-28 z-tgfcr CAR-T cells in CD4 positive cells after co-culture with Raji cells was 29.7% and 44.1%, respectively; the percentage of ifnγ expression in CD4 positive cells after co-culture of CD19-28 z-acd 1 CAR-T cells and CD19-28 z-tggfr CAR-T cells with NALM6 cells was 24.8% and 38.9%, respectively.

Example 7: detection of tumor-specific cell killing after co-culture of CAR-T cells and target cells

K562 cells (negative control cells without CD19 target protein, target cells) were resuspended in serum-free medium (1640) to a cell concentration of 1X 10 ⁶ Per ml, the fluorochrome BMQC (2, 3,6, 7-tetrahydroo-9-bromoxyyl-1H, 5Hquinolizino (9, 1-gh) was added to a final concentration of 5. Mu.M.

2. Mixing well and incubating at 37 ℃ for 30min.

3. Centrifugation at 1500rpm for 5min at room temperature, removal of supernatant and resuspension of cells in cytotoxic medium (phenol red 1640+5% AB serum free), incubation at 37℃for 60min.

4. Fresh cytotoxic medium was washed twice and resuspended in fresh cytotoxic medium at a density of 1X 10 ⁶ /ml。

Raji or NALM6 cells (containing CD19 target protein, target cells) were suspended in PBS containing 0.1% BSA to a concentration of 1X 10 ⁶ /ml。

6. Fluorescent dye CFSE (carboxyfluoresceindiacetatesuccinimidyl ester) was added to a final concentration of 1 μm.

7. Mixing well and incubating at 37 ℃ for 10min.

8. After the incubation was completed, FBS was added in an equal volume to the cell suspension, and incubated at room temperature for 2min to terminate the labeling reaction.

9. Cells were washed and resuspended in fresh cytotoxic medium at a density of 1X 10 ⁶ /ml。

10. Effector T cells were washed and suspended in cytotoxic medium at a concentration of 5X 10 ⁶ /ml。

11. In all experiments, cytotoxicity of effector T cells (CAR-T cells) infected with CD19-28z-aPD1 CAR was compared to cytotoxicity of uninfected negative control effector T cells (NT cells), and these effector T cells were from the same patient.

Cd19-28 z-acd 1 CAR-T and negative control effector T cells, according to T cells: target cells = 5:1,1:1 ratio, cultured in 5ml sterile assay tubes (BD Biosciences). In each co-cultured group, the target cells were 100,000 (50. Mu.l) Raji cells, and the negative control cells were 100,000K 562 cells (50. Mu.l). A set of cells containing only Raji or NALM6 target cells and K562 negative control cells was also set.

13. The co-cultured cells were incubated at 37℃for 16h.

14. After the incubation was completed, the cells were washed with PBS, and immediately 7-AAD (7-aminoactinomycin D) was added rapidly at the concentrations recommended in the instructions and incubated on ice for 30min.

15. The Flow machine test was directly performed without washing, and the data was analyzed with Flow Jo.

16. Analysis the proportion of viable Raji or NALM6 target cells and viable K562 negative control cells after co-culture of T cells and target cells was determined using 7AAD negative viable cell gating.

a) For each group of co-cultured T cells and target cells,

Target cell survival% =Raji or NALM6 viable cell count/K562 viable cell count。

b) Cytotoxic killer cell% = 100-calibrated target cell survival, i.e., (Raji or NALM6 viable cell count at no effector cells-Raji or NALM6 viable cell count at effector cells)/K562 viable cell count).

The results of this example are shown in fig. 6. Figure 6 shows that the killing efficiency of CD19-28z-aPD1CAR-T cells and CD19-28 z-tggfr CAR-T cells against target cells NALM6 was 96% and 95%, respectively, at an effective target ratio of 5:1.

Example 8: detection of surface PD1 expression after co-culture of CAR-T cells and target cells

1. Taking a V-bottom 96-well plate, adding 2 x 10 of CAR-T/NT cells to each well ⁵ The cells were collected by incubating with target cells (Raji or NALM 6) at 37℃for 24 hours, and the group without target cells was set as a negative control, and resuspended to 100ul of X-VIVO complete medium containing IL-2.

2. The samples were centrifuged to remove the medium, the cells were washed once with PBS and centrifuged at 400g for 5 min at 4 ℃. The supernatant was discarded, and an appropriate amount of specific surface antibodies CD3, CAR, PD1 (Biolegend) was added to each tube, and the volume was resuspended at 100ul and incubated for 30 minutes on ice in the absence of light.

4. The appropriate amount of PBS was resuspended, and the flow cytometer detected CD3, CAR, PD1, and analyzed the expression of PD1 in the CD3+CAR+ cell population.

The results of this example are shown in FIG. 7, where CD19-28z-aPD1CAR-T surface PD1 expression was lower than that of CD19-28z-tEGFR CAR-T after incubation with target cells (Raji or NALM 6).

Sequence listing

<110> Shanghai Hengrun biological technology Co., ltd

<120> CD 19-targeting chimeric antigen receptor, method for the combined expression of variable regions of anti-PD 1 antibodies, and uses thereof

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 2310

<212> DNA

<213> artificial sequence

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atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgacattc agatgactca gaccacaagc agcctcagtg cgagcctggg ggacagggtg 120

actatcagct gccgggccag ccaggacatt tccaagtacc tgaattggta ccagcagaag 180

cccgatggta ctgtgaaact cctgatatat catacttcta ggctccattc cggggttcca 240

agccgattca gtggctccgg ttccggtaca gattattccc tgaccattag caacttggaa 300

caggaggaca ttgcaacgta tttctgtcag caaggcaaca cattgcccta cacattcggg 360

ggcgggacta aactcgaaat aactggcggc gggggttctg gtggcggcgg cagcggcggt 420

ggaggatcag aagtgaagct gcaggaaagt ggccccgggc tggtagcccc aagtcagtcc 480

ctgagtgtaa cctgtacagt gagtggagtg tctcttcctg actacggggt aagttggatt 540

cggcaacctc cacgcaaggg cctggagtgg ctcggcgtga tttggggatc tgagacaact 600

tactacaatt ccgccctgaa gagcaggctg accatcatta aggacaatag caagtcacag 660

gtgtttctga agatgaactc actgcagacc gacgacaccg ccatctatta ctgcgccaaa 720

cattattatt atggcgggag ttatgctatg gactactggg gccagggcac tagcgtcacc 780

gtcagcagta ctacaactcc agcacccaga ccccctacac ctgctccaac tatcgcaagt 840

cagcccctgt cactgcgccc tgaagcctgt cgccctgctg ccgggggagc tgtgcatact 900

cggggactgg actttgcctg tgatatctac ttctgggtgc tggtcgtggt cggaggggtg 960

ctggcctgtt atagcctgct ggtgactgtc gccttcatta tcttctgggt gcggagcaag 1020

aggtctcgcg gtgggcattc cgactacatg aacatgaccc ctagaaggcc tggcccaacc 1080

agaaagcact accagccata cgcccctccc agagatttcg ccgcttatcg aagcgtgaag 1140

ttctcccgaa gcgcagatgc cccagcctat cagcagggac agaatcagct gtacaacgag 1200

ctgaacctgg gaagacggga ggaatacgat gtgctggaca aaaggcgggg cagagatcct 1260

gagatgggcg gcaaaccaag acggaagaac ccccaggaag gtctgtataa tgagctgcag 1320

aaagacaaga tggctgaggc ctactcagaa atcgggatga agggcgaaag aaggagagga 1380

aaaggccacg acggactgta ccaggggctg agtacagcaa caaaagacac ctatgacgct 1440

ctgcacatgc aggctctgcc accaagaaga gctaagcgcg gctcaggcgc gaccaacttt 1500

tctctgctta agcaggcagg cgacgtggaa gagaaccccg ggcccatgta cagaatgcag 1560

ctgttgtctt gtattgccct ttctctcgcc ctcgtaacaa attcacaagt ccaattggtg 1620

gagtctggcg gtggggtagt tcagcccggc cgatccctgc gcctggactg caaagcttct 1680

ggaattacgt tctcaaactc cggaatgcac tgggtgcggc aagcacctgg gaaagggctg 1740

gagtgggttg cggtgatttg gtacgatggc tctaagaggt actacgcaga cagcgttaaa 1800

ggcagattta ctatatcccg agataactct aaaaatacgc tcttcctcca aatgaatagc 1860

ctgagggcag aagacacagc cgtttactat tgtgctacca atgatgatta ctggggacag 1920

ggcaccctgg ttaccgtaag ttccggcggt ggtggaagtg gaggaggggg atccggaggc 1980

gggggttctg agatcgtcct gacccagtct ccagccactc tctccctgtc tccaggcgag 2040

cgcgctacac tgagttgtag agcttcccag tccgtgagca gctatctggc ctggtatcag 2100

cagaagcctg ggcaggctcc acggttgctg atttatgacg cctccaaccg cgcgactggg 2160

ataccagcta ggttttccgg atcaggcagc ggcactgatt ttacactgac catctcatct 2220

ctcgagccgg aagatttcgc cgtttactat tgtcaacaga gttcaaactg gccacggaca 2280

ttcggtcagg ggaccaaggt tgaaattaag 2310

<160> 1

<170> PatentIn version 3.3

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<213> artificial sequence

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

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

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Phe Trp Val Leu Val Val Val Gly Gly Val

305 310 315 320

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

325 330 335

Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met

340 345 350

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

355 360 365

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Phe Ser Arg Ser

370 375 380

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

385 390 395 400

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

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

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

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

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

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg Arg Ala Lys Arg Gly Ser Gly

485 490 495

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

500 505 510

Pro Gly Pro Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser

515 520 525

Leu Ala Leu Val Thr Asn Ser Gln Val Gln Leu Val Glu Ser Gly Gly

530 535 540

Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser

545 550 555 560

Gly Ile Thr Phe Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro

565 570 575

Gly Lys Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys

580 585 590

Arg Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

595 600 605

Asn Ser Lys Asn Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu

610 615 620

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

625 630 635 640

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

645 650 655

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Ala

660 665 670

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

675 680 685

Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly

690 695 700

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

705 710 715 720

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

725 730 735

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

740 745 750

Gln Ser Ser Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu

755 760 765

Ile Lys

770

<210> 3

<211> 21

<212> DNA

<213> artificial sequence

<223> primer

<400> 3

agcatcgttc tgtgttgtct c 21

<210> 4

<211> 22

<212> DNA

<213> artificial sequence

<223> primer

<400> 4

tgtttgtctt gtggcaatac ac 22

<210> 7

<211> 21

<212> PRT

<213> artificial sequence

<223> linker sequence

<400> 5

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Leu

1 5 10 15

Gly Ser Thr Glu Phe

20

Claims

1. A fusion protein comprising a leader peptide of a CD8 antigen, an anti-CD 19 single chain antibody, a human CD8 alpha hinge region, a human CD28 transmembrane region, a human CD28 intracellular region and a human CD3 zeta intracellular region, and P2A polypeptide, a human IL2 signal peptide, and heavy and light chain variable regions of an anti-human PD1 monoclonal antibody, which are sequentially linked,

the amino acid sequence of the leader peptide of the CD8 antigen is shown as amino acids 1-21 of SEQ ID NO. 2;

the amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown as 22 th-128 th amino acid of SEQ ID NO. 2;

the amino acid sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody is shown as 144 th-263 th amino acid of SEQ ID NO. 2;

the amino acid sequence of the human CD8 alpha hinge region is shown as 264 th to 310 th amino acids of SEQ ID NO. 2;

The amino acid sequence of the human CD28 transmembrane region is shown as 311-337 amino acids of SEQ ID NO. 2;

the amino acid sequence of the human CD28 intracellular region is shown as 338 th to 378 th amino acids of SEQ ID NO. 2;

the amino acid sequence of the human CD3 zeta intracellular area is shown as 379-489 amino acids of SEQ ID NO. 2;

the amino acid sequence of the P2A is shown as 490-515 th amino acid of SEQ ID NO. 2;

the amino acid sequence of the human IL2 signal peptide is shown as 516 th to 535 th amino acids of SEQ ID NO. 2;

the amino acid sequence of the heavy chain variable region of the anti-human PD1 single-chain antibody is shown as 536 th to 648 th amino acids of SEQ ID NO. 2;

the amino acid sequence of the light chain variable region of the anti-human PD1 single-chain antibody is shown as 664-770 th amino acid of SEQ ID NO. 2.

2. The fusion protein of claim 1, wherein the anti-CD 19 single chain antibody is anti-CD 19 monoclonal antibody FMC63.

3. A polynucleotide, wherein the sequence of the polynucleotide is selected from the group consisting of:

(1) Leader peptide coding sequence of CD8 antigen, anti-CD 19 single chain antibody coding sequence, human CD8 alpha hinge region coding sequence, human CD28 transmembrane region coding sequence, human CD28 intracellular region coding sequence, human CD3 zeta intracellular region coding sequence, P2A polypeptide coding sequence, human IL2 signal peptide coding sequence, coding sequence and coding sequence of heavy chain and light chain variable region of anti-human PD1 monoclonal antibody connected in sequence

(2) (1) the complement of the polynucleotide sequence;

4. The polynucleotide according to claim 3, wherein,

the coding sequence of the signal peptide in front of the coding sequence of the anti-CD 19 single-chain antibody is shown as the 1 st-63 st nucleotide sequence of SEQ ID NO. 1; and/or

The coding sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown as the 64 th-384 th nucleotide sequence of SEQ ID NO. 1; and/or

The coding sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody is shown as the 430 th-789 th nucleotide sequence of SEQ ID NO. 1; and/or

The coding sequence of the human CD8 alpha hinge region is shown as the nucleotide sequence of 790 to 930 of SEQ ID NO. 1; and/or

The coding sequence of the human CD28 transmembrane region is shown as the 931 st to 1011 st nucleotide sequence of SEQ ID NO. 1; and/or

The coding sequence of the human CD28 intracellular region is shown as the 1012 th to 1134 th nucleotide sequence of SEQ ID NO. 1; and/or

The coding sequence of the human CD3 zeta intracellular area is shown as 1135-1467 nucleotide sequence of SEQ ID NO. 1; and/or

The coding sequence of the P2A is shown as a 1368 th-1545 th nucleotide sequence of SEQ ID NO. 1; and/or

The coding sequence of the human IL2 signal peptide is shown as the 1546 th-1605 th nucleotide sequence of SEQ ID NO. 1; and/or

The coding sequence of the heavy chain variable region of the anti-PD 1 single-chain antibody is shown as the nucleotide sequence of 1606-1944 of SEQ ID NO. 1; and/or

The coding sequence of the light chain variable region of the anti-PD 1 single-chain antibody is shown as the nucleotide sequence of 1990-2310 of SEQ ID NO. 1.

5. A nucleic acid construct comprising the polynucleotide of claim 3 or 4.

6. The nucleic acid construct of claim 5, wherein the nucleic acid construct is a vector.

7. The nucleic acid construct of claim 6, wherein the nucleic acid construct is a retroviral vector comprising a replication origin site, a 3'ltr, a 5' ltr.

8. A retrovirus containing the nucleic acid construct of any one of claims 5-7.

9. A method of ex vivo activating T cells comprising the step of infecting said T cells with the retrovirus of claim 8.

10. A genetically modified T cell or a pharmaceutical composition comprising the genetically modified T cell, wherein the cell comprises the polynucleotide of claim 3 or 4, or comprises the nucleic acid construct of any one of claims 5-7, or is infected with the retrovirus of claim 8, or is produced by the method of claim 9.

11. A method of culturing the T cells obtained according to claim 10 using a CAR-T cell culture medium containing IL-2 cytokines.

12. The method of claim 11, wherein the cells are cultured for 3-5 days.

13. The method of claim 12, wherein activating T cells is adding an anti-CD 3 antibody to the culture medium, and optionally adding exogenous IL-2 to the cell culture.

14. Use of the fusion protein of any one of claims 1-2, the polynucleotide of claim 3 or 4, the nucleic acid construct of any one of claims 5-7, or the retrovirus of claim 8 in the preparation of activated T cells.

15. Use of the fusion protein of any one of claims 1-2, the polynucleotide of claim 3 or 4, the nucleic acid construct of any one of claims 5-7, the retrovirus of claim 8, or the genetically modified T cell of claim 10 in the manufacture of a medicament for the treatment of a CD19 mediated disease, which CD19 mediated disease is leukemia or lymphoma.

16. The use according to claim 15, wherein the CD19 mediated disease is selected from the group consisting of B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and acute myelogenous leukemia.