CN108070607B

CN108070607B - Chimeric antigen receptor targeting CD19-41BB-tEGFR and application thereof

Info

Publication number: CN108070607B
Application number: CN201610987904.7A
Authority: CN
Inventors: 黄飞; 金涛; 王海鹰; 何凤; 史子啸
Original assignee: Shanghai Hrain Biotechnology Co ltd
Current assignee: Shanghai Hengrun Dasheng biopharmaceutical Co.,Ltd.
Priority date: 2016-11-10
Filing date: 2016-11-10
Publication date: 2020-12-08
Anticipated expiration: 2036-11-10
Also published as: CN108070607A

Abstract

The present invention relates to chimeric antigen receptors targeting CD19 and uses thereof. In particular, the invention provides a polynucleotide sequence selected from: (1) a polynucleotide sequence comprising the coding sequence of a single chain antibody against CD19, the coding sequence of a human CD8 α hinge region, the coding sequence of a human CD8 transmembrane region, the coding sequence of a human 41BB intracellular region, the coding sequence of a human CD3 ζ intracellular region, and optionally the coding sequence of a fragment of EGFR comprising extracellular domain III and extracellular domain IV, linked in sequence; and (2) the complement of the polynucleotide sequence of (1). The invention also provides a related fusion protein, a vector containing the coding sequence, and applications of the fusion protein, the coding sequence and the vector.

Description

Chimeric antigen receptor targeting CD19-41BB-tEGFR and application thereof

Technical Field

The invention belongs to the field of chimeric antigen receptors, and particularly relates to a CD 19-targeted chimeric antigen receptor and application thereof.

Background

Chimeric Antigen Receptor-T cell (CAR-T) T cell refers to a T cell that is genetically modified to recognize a specific Antigen of interest in an MHC non-limiting manner and to continuously activate expanded T cells. The international cell therapy association (interna) in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors besides surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. CAR-T cell back-infusion therapy is the most clearly effective form of immunotherapy in current tumor therapy. A large number of studies show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and remarkably improve the survival condition of patients.

Chimeric Antigen Receptors (CARs) are a core component of CAR-T, conferring on 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 (usually the scFV fragment from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The choice of antigen of interest is a key determinant for the specificity, efficacy of the CAR and safety of the genetically engineered T cells themselves.

With the continuous development of Chimeric Antigen Receptor T cell (CAR-T) technology, CAR-T can be divided into four generations.

The first generation CAR-T cells consist of an extracellular binding domain-single chain antibody (scFV), a transmembrane domain (TM), and an intracellular signaling domain-immunoreceptor tyrosine-activated motif (ITAM), wherein the chimeric antigen receptor portions are linked as follows: scFv-TM-CD3 ζ. Although some specific cytotoxicity could be seen in the first generation CARs, it was found to be less effective when summarized in 2006 in clinical trials. The reason for this is that the first generation CAR-T cells are rapidly depleted in the patient and have poor persistence (persistence) such that CAR-T cells that have already apoptotic when they have not yet come into contact with a large number of tumour cells can elicit an anti-tumour cytotoxic effect, but they secrete less cytokines but have a shorter survival time in vivo and cannot elicit a persistent anti-tumour effect. [ Cancer Res.2007,67(22):11029-

Optimization of T cell activation signaling regions in CAR design of second generation CAR-T cells remains a hotspot of research. Complete activation of T cells relies on dual signaling and cytokine action. Wherein the first signal is a specific signal initiated by the recognition of an antigen peptide-MHC complex on the surface of an antigen presenting cell by the TCR; the second signal is a co-stimulatory signal. Second generation CARs were introduced as early as 1998 (J Immunol.1998; 161(6): 2791-7.). The 2 nd generation CAR adds a costimulatory molecule in the intracellular signal peptide region, namely the costimulatory signal is assembled into the CAR, and can better provide an activation signal for CAR-T cells, so that the CAR can simultaneously activate the costimulatory molecule and the intracellular signal after identifying tumor cells, double activation is realized, and the proliferation and secretion capacity of the T cells and the anti-tumor effect can be obviously improved. The first well-studied T cell costimulatory signal receptor was CD28, which was capable of binding to a B7 family member on the surface of target cells. Co-stimulation of CD28 promotes T cell proliferation, IL-2 synthesis and expression, and enhances T cell resistance to apoptosis. Costimulatory molecules such as CD134(OX40) and CD137(4-1BB) are subsequently presented to increase the cytotoxicity and proliferative activity of T cells, maintain T cell responses, prolong T cell survival, and the like. Such second generation CARs produced unexpected results in subsequent clinical trials, with shaking frequently triggered since 2010 based on clinical reports of second generation CARs, with complete remission rates of up to 90% and above, especially for relapsed, refractory ALL patients.

The third generation CAR signal peptide region integrates more than 2 costimulatory molecules, which can lead the T cells to continuously activate and proliferate, lead the cytokines to be continuously secreted, and lead the capability of the T cells to kill tumor cells to be more remarkable, namely, the new generation CAR can obtain stronger anti-tumor response (Mol ther.2005,12(5):933 941.). Most typically, UPen Carl June is added with a stimulating factor of CD137(4-1BB) under the action of a stimulating factor of CD 28.

The fourth generation of CAR-T cells are supplemented with cytokines or co-stimulatory ligands, for example the fourth generation CAR can produce IL-12, which can modulate the immune microenvironment-increase the activation of T cells, while activating innate immune cells to function to eliminate target antigen negative cancer cells, thus achieving a bi-directional regulatory effect. [ Expert Opin Biol ther.2015; 15(8):1145-54.].

CD19 is a glycoprotein of 95kDa on the surface of B cells, expressed from early stages of B cell development until it differentiates into plasma cells. CD19 is one of the members of the immunoglobulin (Ig) superfamily, and is one of the components of the B cell surface signal transduction complex, involved in the regulation of the signal transduction process of the B cell receptor. In a mouse model deficient in CD19, there was a marked reduction in the number of B cells in peripheral lymphoid tissues and a reduction in vaccine and mitogen responses accompanied by a reduction in serum Ig levels. It is generally accepted that expression of CD19 is restricted to B cell lines (B-cell lines) and not expressed on the surface of pluripotent hematopoietic stem cells. CD19 is also expressed on the surface of most B cell lymphomas, mantle cell lymphomas, ALLs, CLLs, hairy cell leukemias, and a fraction of acute myeloid leukemia cells. Thus, CD19 is a very valuable immunotherapeutic target in the treatment of leukemia/lymphoma. Importantly, the feature that CD19 is not expressed on the surface of most normal cells other than B cells, including pluripotent hematopoietic stem cells, allows CD19 to be a safe therapeutic target, minimizing the risk of patients developing autoimmune diseases or irreversible bone marrow toxic injuries. Currently, antibodies or scFv fragments against CD19 have been developed and demonstrated promise for their application in mouse models and human/primate animals.

In recent years, the field of CD19CAR T cells has been competitive, and several large pharmaceutical companies have also established partnerships with research institutions. Pediatric and adult relapsed or refractory acute B-cell lymphomas have a complete remission rate of approximately 90% after treatment with CD19CAR T cells expressing CD28 or 4-1 BB. Recently, CD19CAR T cell therapy has an overall remission rate of 50% -100% in diffuse large B cell lymphoma, follicular lymphoma or chronic lymphoma. CD19CAR T cells have clinical advantages in treating multiple myeloma patients, since terminally differentiated plasma cells do not express CD19, malignant B cell precursors continue to give rise to malignant plasma cells.

It is well known that for T cell activation, two important signalling pathways are required, the first being the binding of the MHC complex to the T cell receptor and the second requiring a costimulatory signal for the binding of CD80 or CD86 to CD28 on T cells. The 4-1BB signaling pathway, activated by a T cell receptor pathway, can increase the proliferation of activated T cells and the secretion of cytokines. This improvement in the integration of CD28 or 4-1BB by second generation CARs enhances the replication and survival ability of engineered T cells.

While the two second generation CARs of different costimulatory molecules are widely used clinically, there have been many studies on the functional differences between them. In vitro, CD28 or 4-1BB CARs have similar anti-tumor capabilities, but preclinical studies in vivo have shown that 4-1BB CAR engineered T cells may have greater proliferative and survival capabilities. In particular, clinical studies have shown that both second generation CAR-engineered T cells are able to continue to proliferate in vivo, although CAR-engineered T cells comprising a 4-1BB co-stimulatory molecule are able to survive longer. In clinical studies with acute lymphoma leukemia, Davila et al reported CD19-28Z CAR T cells that survived in vivo for 1 to 3 months. Similarly, CD19-CAR T cells (co-stimulatory molecule of CD 28) from NCI reported a maximum survival time of 68 days. While in another study with CAR T cells of CD19-4-1BB, CART cell survival reached 68% at 6 months, in this study, the most symptomatic patients with B-cell hypoplasia lasted two years, showing a continuous and lasting functional role for CD19-4-1 BB's CART cells.

Why CD19-4-1BB-CART has a longer survival in patients compared to CD19-CD 28-CART. An article published in Nature Medicine has been studied about this phenomenon. The studies in the article indicated that phosphorylation of the background of CD3 ζ, i.e. activation of the background (which is mainly caused by the accumulation of SCFV itself from CARs), leads to early depletion of CART cells (with the exception of the less potent CD19 CARs); whereas CARs incorporating CD28 co-stimulatory molecules exacerbate this depletion effect on T cells, CARs incorporating 4-1BB co-stimulatory molecules attenuate this effect. To better explore the molecular mechanisms by which 4-1BB signaling can reduce T cell failure, researchers also compared the difference in 4-1BB-CART cells and CD28-CART cell transcriptome, with the failure-associated indicators for 4-1BB-CART cell expression being lower than for CD28-CART cells. This study explains, on one side, why CD19-4-1BB CART cells survived more permanently than CD19-CD28 CART cells in clinical studies. The co-stimulation factor used in the patent is 4-1BB, and the pre-clinical experimental result shows that the co-stimulation factor has longer in-vivo survival time.

Studies have shown that the long-term presence of CD19CAR T cells has both the advantage of having a surveillance effect on the disease and the disadvantage of causing long-term defects in B cells. In addition to the signaling domain of the CAR itself, other factors can affect the lifespan of the CAR T, such as the cell culture system, the mode of gene transfer, the promoter of gene expression, the function and phenotype of the infused T cells, as well as the age of the patient, the type of disease affected, and the treatment regimen that has been experienced.

One advantage of CAR-T cells is that they are active drugs, and once infused, physiological mechanisms regulate T cell balance, memory formation, and antigen-driven expansion. However, this treatment is not complete and T cells can miss the target and attack other tissues or expand too much beyond what is needed for treatment. Given that CAR-T cells have been included in the standard therapeutic range, it is very useful to design patient or drug-controlled turn-on or turn-off mechanisms to regulate the presence of CAR-T cells. For technical reasons, the shutdown mechanism is more easily applied to T cells. As one of them, the iCas9 system is under clinical study. When the cell expresses the iCas9, the small molecule compound can induce the iCas9 precursor molecule to form a dimer and activate an apoptosis pathway, thereby achieving the purpose of removing the cell. Small molecule AP1903 has been used to induce iCas9 dimers and clear T cells in graft versus host disease, demonstrating the feasibility of this approach (Clin Cancer Res.2016Apr 15; 22(8): 1875-84.).

In addition, it is also possible to use clearing antibodies that have been used clinically to allow CAR-T cells to express proteins to which these antibodies are directed, such as tEGFR, and to clear the corresponding CAR-T cells by administration of antibody drugs after the therapeutic-related toxic response has developed or after the therapy has been completed (Sci Transl Med.2015; 7: 275ra 22.). Based on the consideration of safety, the car-t cells introduce a safety switch, namely tEGFR, and the constructed CD19-tEGFR can safely control the expression of the tEGFR in vivo in real time. Our patent uses the scFV heavy and light chains of CD19 as the CAR structure, and also introduces the tfegfr structure. tEGFR lacks the extracellular N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity, but retains the native amino acid sequence, is localized to the type I transmembrane cell surface, and has a spatial conformation that tightly binds to the pharmaceutical grade anti-EGFR monoclonal antibody cetuximab (BLOOD.2011Aug 4; 118(5): 1255-63.). Major functions of tfegfr: can be used as a marker on the cell surface, is also suitable for the in vivo tracking of T cells and can be detected by flow and immunohistochemistry; it can also be cleared in vivo by tuximab. The tEGFR structure introduced by the invention can be well traced in a CAR-T cell body, and more importantly, the structure can be used as a safety switch of the CAR-T cell: i.e., when it is not desired to do so, Tulcizumab can be added, safe and effective control of the effects of infused CAR-T cells directed against the CD19 target in vivo. Lays a good foundation for clinical experiments and clinical treatment.

Disclosure of Invention

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

(1) a polynucleotide sequence comprising the coding sequence of a single chain antibody against CD19, the coding sequence of a human CD8 α hinge region, the coding sequence of a human CD8 transmembrane region, the coding sequence of a human 41BB intracellular region, the coding sequence of a human CD3 ζ intracellular region, and optionally the coding sequence of a fragment of EGFR comprising extracellular domain III and extracellular domain IV, linked in sequence; and

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

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 as set forth in amino acids 1-21 of SEQ ID NO 2. In one or more embodiments, the light chain variable region of the anti-CD 19 single chain antibody has the amino acid sequence as shown in 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 as shown in amino acids 144-263 of SEQ ID NO. 2. In one or more embodiments, the amino acid sequence of the human CD8 α hinge region is depicted as amino acids 264-310 of SEQ ID NO 2. In one or more embodiments, the amino acid sequence of the transmembrane region of human CD8 is as shown in SEQ ID NO 2 at amino acids 311-332. In one or more embodiments, the amino acid sequence of the intracellular domain of human 41BB is as shown in amino acids 333-380 of SEQ ID NO 2. In one or more embodiments, the amino acid sequence of the intracellular domain of human CD3 ζ is as set forth in amino acids 381-491 of SEQ ID NO 2. In one or more embodiments, the fragment of EGFR contains or consists of the extracellular domain III, the extracellular domain IV, and the transmembrane region of EGFR. In one or more embodiments, the fragment of EGFR comprises or consists of the amino acid sequence at position 310-646 of human EGFR. In one or more embodiments, the human tEGFR amino acid sequence is as set forth in amino acids 539-873 of SEQ ID NO 2. In one or more embodiments, the polynucleotide sequence further comprises a coding sequence for a GM-CSF receptor alpha chain signal peptide disposed N-terminal to the EGFR fragment. In one or more embodiments, the amino acid sequence of the signal peptide of the α chain of the GM-CSF receptor is as shown in amino acids 517-538 of SEQ ID NO 2. In one or more embodiments, the polynucleotide sequence further comprises a coding sequence for a linker sequence linking the GM-CSF receptor alpha chain signal peptide to the intracellular domain of human CD3 ζ. In one or more embodiments, the amino acid sequence of the linker sequence is as depicted in amino acids 492 and 516 of SEQ ID NO 2.

In one or more embodiments, the coding sequence for the signal peptide preceding the coding sequence for the anti-CD 19 single chain antibody is as set forth in nucleotide sequences 1-63 of SEQ ID NO. 1. In one or more embodiments, the light chain variable region encoding sequence of the anti-CD 19 single chain antibody is as shown in SEQ ID NO. 1, nucleotide sequences 64-384. In one or more embodiments, the coding sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody is as shown in the nucleotide sequences at positions 430-789 of SEQ ID NO. 1. In one or more embodiments, the coding sequence for the human CD8 α hinge region is as shown in nucleotide sequence 790-930 of SEQ ID NO. 1. In one or more embodiments, the coding sequence for the transmembrane region of human CD8 is as shown in SEQ ID NO 1, nucleotide sequence 931 and 996. In one or more embodiments, the coding sequence of the intracellular region of human 41BB is as shown in the nucleotide sequence at position 997-1140 of SEQ ID NO. 1. In one or more embodiments, the coding sequence for the intracellular domain of human CD3 ζ is as set forth in nucleotide sequences SEQ ID No. 1, position 1144-1476. In one or more embodiments, the coding sequence of the linker sequence linking the signal peptide of the α chain of the GM-CSF receptor and the intracellular domain of human CD3 ζ is as shown in nucleotide sequence 1141-1473 of SEQ ID NO: 1. In one or more embodiments, the coding sequence of the signal peptide of the alpha chain of the GM-CSF receptor is shown as the nucleotide sequence at position 1552-1631 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the fragment of EGFR is as shown in nucleotide sequence 1632-2625 of SEQ ID NO: 1. In one or more embodiments, the polynucleotide sequence encodes an amino acid sequence as set forth in positions 22-491 of SEQ ID NO. 2, or encodes an amino acid sequence as set forth in positions 22-516 of SEQ ID NO. 2, or encodes an amino acid sequence as set forth in SEQ ID NO. 2. In one or more embodiments, the polynucleotide sequence comprises or consists of the nucleotide sequence shown in SEQ ID NO. 1, positions 1-1634 of SEQ ID NO. 1, positions 64-1476 of SEQ ID NO. 1, or positions 64-2628 of SEQ ID NO. 1, or the nucleotide sequence shown in SEQ ID NO. 1, positions 1-1476 of SEQ ID NO. 1, positions 64-1476 of SEQ ID NO. 1, or positions 64-2628 of SEQ ID NO. 1.

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

(1) a coding sequence comprising a fusion protein of an anti-CD 19 single chain antibody, a human CD8 α hinge region, a human CD8 transmembrane region, a human 41BB intracellular region, and a human CD3 ζ intracellular region, linked in sequence, and optionally, an extracellular domain III and extracellular domain IV-containing fragment of EGFR; and

(2) a fusion protein derived from (1) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (1) and retaining the activity of activated T cells;

preferably, the anti-CD 19 single chain antibody is anti-CD 19 monoclonal antibody FMC 63.

In one or more embodiments, the fusion protein further comprises a signal peptide at the N-terminus of the anti-CD 19 single chain antibody. In one or more embodiments, the amino acid sequence of the signal peptide is as set forth in amino acids 1-21 of SEQ ID NO 2. In one or more embodiments, the light chain variable region of the anti-CD 19 single chain antibody has the amino acid sequence as shown in amino acids 22-132 of SEQ ID NO. 1. In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single chain antibody can be shown as amino acids 148-264 of SEQ ID NO. 1. In one or more embodiments, the amino acid sequence of the human CD8 α hinge region is depicted as amino acids 265-311 of SEQ ID NO 1. In one or more embodiments, the amino acid sequence of the transmembrane region of human CD8 is depicted as amino acids 312-333 of SEQ ID NO 1. In one or more embodiments, the amino acid sequence of the intracellular domain of human 41BB is as shown in amino acids 334-381 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of the intracellular domain of human CD3 ζ is as set forth in amino acids 382-492 of SEQ ID NO: 1. In one or more embodiments, the fragment of EGFR contains or consists of the extracellular domain III, the extracellular domain IV, and the transmembrane region of EGFR. In one or more embodiments, the EGFR fragment comprises or consists of the amino acid sequence at position 310-646 of human EGFR. In one or more embodiments, the amino acid sequence of the EGFR fragment is as set forth in amino acids 539-873 of SEQ ID NO 1. In one or more embodiments, the fusion protein further comprises a GM-CSF receptor alpha chain signal peptide disposed N-terminal to the EGFR fragment. In one or more embodiments, the amino acid sequence of the signal peptide of the α chain of the GM-CSF receptor is as shown in amino acids 517-538 of SEQ ID NO 2. In one or more embodiments, the fusion protein further comprises a linker sequence linking the GM-CSF receptor alpha chain signal peptide to the intracellular domain of human CD3 ζ. In one or more embodiments, the amino acid sequence of the linker sequence is as depicted in amino acids 492 and 516 of SEQ ID NO 2. In one or more embodiments, the amino acid sequence of the fusion protein is as set forth in amino acids 22-491 of SEQ ID NO. 2 or as set forth in amino acids 22-516 of SEQ ID NO. 2, or as set forth in 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 initiation site, a 3 'LTR, a 5' LTR, a polynucleotide sequence described herein, and optionally a selectable marker.

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

In a fifth aspect, the invention provides a genetically modified 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, or stably expressing a fusion protein as described herein and optionally a fragment of EGFR comprising extracellular domain III, extracellular domain IV and optionally a transmembrane region.

In a sixth aspect, the invention provides a pharmaceutical composition comprising a genetically modified 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 T cell.

In an eighth aspect, the invention provides the use of a polynucleotide sequence, fusion protein, nucleic acid construct, retrovirus, or genetically modified T cell as described herein, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment of a CD 19-mediated disease.

In one or more embodiments, the CD 19-mediated disease is leukemia, lymphoma.

Drawings

FIG. 1 is a schematic representation of a CD19-CAR retroviral expression vector (CD 19-BBz). SP: a signal peptide; VL: a light chain variable region; and Lk: joint (G)₄S)₃(ii) a VH: a heavy chain variable region; h: a CD8 a hinge region; TM: the CD8 transmembrane domain.

FIG. 2 is a partial sequencing peak plot of the CD19-CAR retroviral expression vector (CD 19-BBz).

FIG. 3 is a schematic representation of the CD19-tEGFR-CAR retroviral expression vector (CD19 CAR-tEGFR). SP: a signal peptide; VL: a light chain variable region; and Lk: joint (G)₄S)₃(ii) a VH: a heavy chain variable region; h: a CD8 a hinge region; TM: the CD8 transmembrane region; 2A: P2A peptide.

FIG. 4 is a partial sequencing peak plot of the CD19-CAR retroviral expression vector (CD 19-BBz).

FIG. 5 shows the expression efficiency of CD19-BBz and CD19-BBz-tEGFR CART by a flow cytometer after retroviral infection of T cells for 72 hours.

FIG. 6 is a graph of the degranulation of CD107a by co-culturing 5-day-old CD19-BBz and CD19-BBz-tEGFR CART cells with target cells for 5 hours.

FIG. 7 shows IFN γ secretion from 5-day-old prepared CD19-BBz and CD19-BBz-tEGFR CART cells co-cultured with target cells for 5 hours.

FIG. 8 shows the killing effect on tumor cells after 5-day preparation of CD19-BBz and CD19-BBz-tEGFR CART cells co-cultured with target cells for 5 hours.

FIG. 9 is the detection of the tEGFR brake function (ADCC) in vitro by CD19-BBz-tEGFR CART cells.

Detailed Description

The present invention provides a Chimeric Antigen Receptor (CAR) that targets CD 19. The CAR comprises an anti-CD 19 single chain antibody, a human CD8 α hinge region, a human CD8 transmembrane region, a human 41BB intracellular region, a human CD3 ζ intracellular region, and optionally, an EGFR fragment comprising extracellular domain III and extracellular domain IV, connected in sequence.

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 known in the art.

Optionally, the light chain variable region and the heavy chain variable region may be linked together by a linker sequence. Such single chain antibodies that may be exemplified include, but are not limited to, FMC63, SJ25C 1. In certain embodiments, the monoclonal antibody is a monoclonal antibody having clone number FMC 63. In certain embodiments, the light chain variable region of the anti-CD 19 single chain antibody has the amino acid sequence 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 as shown in amino acid residues 144-263 of SEQ ID NO: 2.

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

The human CD8 transmembrane region suitable for use in the present invention can be the various human CD8 transmembrane region sequences commonly used in the art for CARs. In certain embodiments, the amino acid sequence of the transmembrane region of human CD8 is depicted as amino acids 311-332 of SEQ ID NO 2.

The 41BB suitable for use in the present invention can be any of the various 41 BBs known in the art for use in CARs. As an illustrative example, the present invention uses the 41BB shown in the amino acid sequence 333-380 of SEQ ID NO: 2.

The intracellular domain of human CD3 ζ suitable for use in the present invention may be various intracellular domains of human CD3 ζ conventionally used in CARs in the art. In certain embodiments, the amino acid sequence of the intracellular domain of human CD3 ζ is as set forth in amino acids 381-491 of SEQ ID NO 2.

The above-mentioned portions forming the fusion protein of the present invention, such as the light chain variable region and the heavy chain variable region of the anti-CD 19 single-chain antibody, the human CD8 α hinge region, the human CD8 transmembrane region, 41BB, and the human CD3 ζ intracellular region, may be directly linked to each other, or may be linked by a linker sequence. The linker sequence may be one known in the art to be suitable for use with antibodies, for example, a G and S containing linker sequence. Typically, the linker contains one or more motifs which repeat back and forth. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are adjacent in the linker sequence with no intervening amino acid residues between the repeats.The linker sequence may comprise 1, 2,3, 4 or 5 repeat motifs. The linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, and is usually 2 to 20, such as 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), and the like. As an example, the linker may consist of the amino acid sequence of any one of SEQ ID NO 7-18. In certain embodiments, the anti-CD 19 single chain antibody of the invention consists of (GGGGS) between the light chain variable region and the heavy chain variable region_nAnd (b) connecting, wherein n is an integer of 1-5.

In certain embodiments, the amino acid sequence of the CAR of the invention is as set forth in SEQ ID NO 2 amino acids 22-491 or in SEQ ID NO 2 amino acids 1-491. In certain embodiments, the CAR of the invention further comprises within its amino acid sequence extracellular domain III and extracellular domain IV-containing fragments of EGFR, as described below, signal peptides thereof, and linker sequences.

It will be appreciated that in gene cloning procedures it is often necessary to design appropriate cleavage sites which will introduce one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest. In order to construct a fusion protein, facilitate expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside of a host cell, or facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, for example, including, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-terminus or the 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, B, gE and Ty 1. These tags can be used to purify proteins.

The invention also encompasses a CAR as represented by the amino acid sequence at positions 22-491 of SEQ ID NO. 2, a CAR as represented by the amino acid sequence at positions 22-538 of SEQ ID NO. 2, a CAR as represented by the amino acid sequence at positions 1-491 of SEQ ID NO. 2 or a mutant of a CAR as represented by 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 (e.g., activating T cells) of the CAR. Sequence identity between two aligned sequences can be calculated using, for example, BLASTp from NCBI.

Mutants also include: an amino acid sequence as shown in positions 22-491 of SEQ ID NO 2, an amino acid sequence as shown in positions 22-538 of SEQ ID NO 2, an amino acid sequence as shown in positions 1-491 of SEQ ID NO 2 or an amino acid sequence as shown in SEQ ID NO 2 having one or several mutations (insertions, deletions or substitutions) while still retaining the biological activity of the CAR. The number of mutations usually means within 1-10, such as 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For example, conservative substitutions with amino acids of similar or similar properties are not typically used in the art to alter the function of a protein or polypeptide. "amino acids with similar or analogous properties" include, for example, families of amino acid residues with analogous 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 more sites with another amino acid residue from the same side chain species in the polypeptide of the invention will not substantially affect its activity.

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

The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding the fusion proteins described herein is as set forth in nucleotides 64 to 1473 of SEQ ID NO. 1, or as set forth in nucleotides 1 to 1473 of SEQ ID NO. 1.

In certain embodiments, the polynucleotide sequences of the invention further comprise nucleotide sequences encoding fragments of EGFR.

The EGFR suitable for use in the present invention may be an EGFR known in the art, e.g., from human. EGFR contains N-terminal extracellular domains I and II, extracellular domain III, extracellular domain IV, transmembrane, juxtamembrane domain and tyrosine kinase domain. The present invention preferably uses a truncated EGFR ("tfegfr", i.e., a fragment of EGFR as described herein), particularly a truncated EGFR that does not include its intracellular regions (membrane proximal domain and tyrosine kinase domain). In certain embodiments, EGFR that does not include an intracellular region may be further truncated to include no extracellular domains I and II. Thus, in certain embodiments, the EGFR used in the present invention contains or consists of the extracellular domain III, the extracellular domain IV and the transmembrane region of EGFR. In certain embodiments, the tEGFR comprises or consists of the amino acid sequence at positions 310 and 646 of the human EGFR, wherein the amino acid sequence at positions 310 and 480 is the extracellular domain III of the human EGFR, the amino acid sequence at positions 481 and 620 is the extracellular domain IV of the human EGFR, and the amino acid sequence at positions 621 and 646 is the transmembrane region of the human EGFR. In certain embodiments, the extracellular domains III and IV of the amino acid sequence of tEGFR have an amino acid sequence as set forth in amino acids 539-873 of SEQ ID NO 2

To promote the expression of tEGFR, a leader sequence may also be placed at its N-terminus. In certain embodiments, the invention uses a signal peptide from the α chain of the GM-CSF receptor ("GMCSFR"). In certain embodiments, the amino acid sequence of the signal peptide is as set forth in amino acids 517-538 of SEQ ID NO 2.

In addition, the signal peptide and the coding sequence for tEGFR can be linked to the coding sequence for the intracellular domain of human CD3 ζ in the CAR of the invention by the coding sequence for the P2A polypeptide. In one or more embodiments, the amino acid sequence of the P2A peptide is set forth as amino acids 492-516 of SEQ ID NO 2.

Thus, in certain embodiments, a polynucleotide sequence of the invention comprises a coding sequence for a CAR of the invention, a coding sequence for a P2A polypeptide, a coding sequence for a signal peptide from the α chain of the GM-CSF receptor, and a coding sequence for tfegfr. In certain embodiments, the polynucleotide of the invention has the sequence shown as nucleotides 64 to 2628 of SEQ ID NO. 1 or as shown in SEQ ID NO. 1.

The invention also relates to nucleic acid constructs comprising the polynucleotide sequences described herein, and one or more control sequences operably linked to these sequences. The polynucleotide sequences of the invention can be manipulated in a variety of ways to ensure expression of the fusion proteins (CAR and/or tfegfr). The nucleic acid construct may be manipulated prior to insertion into the vector, depending on the type of expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate 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 which shows 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 control 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 sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. 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 a polynucleotide sequence of the invention is typically achieved by operably linking the polynucleotide sequence to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The polynucleotide sequences of the present 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 in the form of a viral vector. Viral vector technology is well known in the art and is 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 can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

Generally, suitable vectors comprise 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; WO 01/29058; and U.S. Pat. No. 6,326,193).

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

An 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 level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the EB virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, inducible promoters are also contemplated. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter during periods of expression and turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells 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 separate 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 a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

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

Methods for introducing and expressing genes into cells are known in the art. The vector may be readily introduced into a host cell by any method known in the art, for example, mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a 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 of introducing polynucleotides into host cells 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 gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Thus, in certain embodiments, the invention also provides a retrovirus for activating T cells, the virus comprising a retroviral vector as 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 from various sources. 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 50ng/ml) of CD3 antibody prior to culturing in an appropriate amount (e.g., 30-80 IU/ml, such as 50IU/ml) of IL2 medium for use.

Thus, in certain embodiments, the invention provides a genetically modified T cell comprising a polynucleotide sequence as described herein, or comprising a retroviral vector as described herein, or infected with a retrovirus as described herein, or prepared by a method as described herein, or stably expressing a fusion protein as described herein and optionally a tfegfr.

The CAR-T cells of the invention can undergo robust in vivo T cell expansion and sustained at high levels in the blood and bone marrow for extended 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 into a central memory-like state in vivo upon encountering and subsequently depleting target cells expressing a surrogate antigen.

The invention also includes a class of cell therapies in which T cells are genetically modified to express a CAR and optionally a tfegfr as described herein, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing tumor cells of the recipient. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained 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.

Thus, the diseases that can be treated with the CARs, their coding sequences, nucleic acid constructs, expression vectors, viruses, and CAR-T cells of the invention are preferably CD 19-mediated diseases.

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 relevant cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise CAR-T cells 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 compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount 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", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Dosage of individual cells/kg body weight. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676, 1988). Optimal for a particular patientThe dosage and treatment regimen 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 composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is 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 can 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 immunosuppressive agents. For example, treatment may be combined with radiation or chemotherapeutic agents known in the art for the treatment of CD19 mediated diseases.

Herein, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, 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 and 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.

The invention adopts the gene sequence of anti-CD 19 antibody (specifically scFV derived from clone number FMC 63), searches the gene sequence information of human CD8 alpha hinge region, human CD8 transmembrane region, human 41BB intracellular region and human CD3 zeta intracellular region from NCBI GenBank database, synthesizes the gene segment of chimeric antigen receptor anti-CD 19scFv-CD8 hinge region-CD 8TM-41BB-CD3 zeta and anti-CD 19scFv-CD8 hinge region-CD 8TM-41BB-CD3 zeta-GMCSFR leader sequence-tEGFR, and inserts into the retroviral vector. The recombinant plasmid packages the virus in 293T cells, infects T cells, and causes the T cells to express the chimeric antigen receptor. The invention realizes the transformation method of the T lymphocyte modified by the chimeric antigen receptor gene based on a retrovirus transformation method. The method has the advantages of high transformation efficiency, stable expression of exogenous genes, and capability of shortening the time for in vitro culture of T lymphocytes to reach clinical level number. On the surface of the transgenic T lymphocyte, the transformed nucleic acid is expressed by transcription and translation. The CAR-T cell prepared by the invention has strong killing function on specific tumor cells, and the killing efficiency exceeds 80% under the condition that the effective target ratio is 5: 1. Furthermore, the CARs of the invention also carry a tEGFR module, the spatial conformation of which is tightly bound to the pharmaceutical grade anti-EGFR monoclonal antibody cetuximab, which can serve as a marker on the cell surface, while also being suitable for in vivo tracking of T cells (detectable by flow and immunohistochemistry); it may also be cleared in vivo by tuximab, i.e., tuximab may be added when the CAR of the invention is not desired to function, safely and effectively controlling the CAR-T cells to function in vivo. Thus, the CARs of the invention also have in vivo tracing and safety switching functions.

The present invention is described in further detail by referring 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 limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Example 1: determination of the sequence of the CD19scFv-CD8-41BB-CD3 zeta Gene

The sequence information of human CD8 alpha hinge region and transmembrane region, human 41BB intracellular region and human CD3 zeta intracellular region is searched from NCBI website database, the cloning number of the anti-CD 19 single-chain antibody is FMC63, and the sequences are subjected to codon optimization on website http:// sg.

And connecting the sequences in sequence according to the sequences of the anti-CD 19scFv, the human CD8 alpha hinge region and transmembrane region, the human 41BB intracellular region gene and the human CD3 zeta intracellular region gene by adopting overlap PCR (polymerase chain reaction), and introducing different enzyme cutting sites at the connection positions of the sequences to form complete CD19-CAR gene sequence information.

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

The recombinant plasmid is sent to Shanghai Biotechnology Limited company for sequencing, and the sequencing result is compared with the sequence of the synthesized CD19-CAR to verify whether the sequence is correct. The sequencing primer is as follows:

sense of justice AGCATCGTTCTGTGTTGTCTC

Antisense TGTTTGTCTTGTGGCAATACAC

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

Example 2: determination of the sequence of the CD19CAR-GMCSFR leader-tEGFR Gene

The sequence information of the EGFR extracellular region gene of a human is searched from an NCBI website database, and the sequence is subjected to codon optimization on a website http:// sg.idtdna.com/site, so that the EGFR extracellular region gene is more suitable for human cell expression under the condition of no change of an encoding amino acid sequence.

The sequences are connected in sequence by adopting overlapping PCR according to the CD19CAR, 2A, GMCSFR leader and tEGFR of the embodiment 1, and different enzyme cutting sites are introduced at the joints of the sequences to form complete CD19CAR-tEGFR gene sequence information.

The recombinant plasmid is sent to Shanghai Biotechnology Limited company for sequencing, and the sequencing result is compared with the sequence of the synthesized hCD19CAR-tEGFR to verify whether the sequence is correct. The sequencing primer is as follows:

sense of justice AGCATCGTTCTGTGTTGTCTC

Antisense TGTTTGTCTTGTGGCAATACAC

The plasmid map constructed in this example is shown in FIG. 3. FIG. 4 shows a partial sequencing peak plot of the retroviral expression plasmid.

Example 3: construction of viral vectors comprising the nucleic acid sequence of the CAR molecule

The nucleotide sequence of the CAR molecule prepared in example 1 was double-digested with NotI (NEB) and EcoRI (NEB), ligated with T4 ligase (NEB) and inserted into the NotI-EcoRI site of the retroviral RV vector, transformed into competent e.coli (DH5 α), and after correct sequencing, the plasmid was extracted and purified using the plasmid purification kit from Qiagen, and 293T cells were transfected with plasmid calcium phosphate for plasmid purification for retroviral packaging experiments.

Example 4: retroviral packaging

1. Day 1 293T cells should be less than 20 passages, but overgrown. Plating the cells in 0.6 x 10^6cells/ml, adding 10ml of DMEM medium into a 10cm dish, fully mixing the cells, and culturing at 37 ℃ overnight;

2. on day 2, 293T cells are transfected to a confluence of about 90% (usually, plating for about 14-18 h); plasmid complexes were prepared with amounts of each plasmid RV-CD19-BBz-tEGFR 12.5ug, Gag-pol 10ug, VSVg 6.25ug, CaCl₂ 250ul，H₂O is 1ml, and the total volume is 1.25 ml; in another tube, an equal volume of HBS to plasmid complex was added, and the plasmid complex was vortexed for 20 seconds. Adding the mixture into a 293T dish gently along the edge, culturing at 37 ℃ for 4h, removing the culture medium, washing with PBS once, and adding the preheated fresh culture medium again;

3. day 4: after transfection for 48h, the supernatant was collected, filtered through a 0.45um filter, split-charged and stored at-80 ℃, and preheated fresh DMEM medium was added continuously.

Example 5: retroviral infection of human T cells

1. Separating with Ficcol separation solution (tertiary sea of Tianjin) to obtain relatively pure CD3+ T cells, and adjusting cell density to 1 × 10 with medium containing 5% AB serum X-VIVO (LONZA)⁶and/mL. Cells were seeded at 1 ml/well into pre-human 50ng/ml CD3 antibody (North China)Jing Tongli Haiyuan) and 50ng/ml 41BB antibody (Beijing Tongli Haiyuan), then 100IU/ml interleukin 2 (Beijing double Lut) is added, and the virus infection is achieved after 48 hours of stimulated culture.

Every other day after T cell activation culture, the non-tissue treated plates were coated with 250. mu.l/well of a 24-well plate by Retronectin (Takara) diluted with PBS to a final concentration of 15. mu.g/ml. Protected from light and kept at 4 ℃ overnight for use.

And 3, after the T cells are activated and cultured for two days, taking out 2 coated 24-well plates, sucking and removing the coating solution, adding HBSS containing 2% BSA, and sealing at room temperature for 30 min. The volume of blocking solution was 500. mu.l per well, and the blocking solution was aspirated and the plate washed twice with HBSS containing 2.5% HEPES.

4. Adding the virus solution into each well, adding 2ml of virus solution into each well, centrifuging at 32 ℃ for 2000g, and centrifuging for 2 h.

5. The supernatant was discarded, and activated T cells were added to each well of a 24-well plate at 1X 10⁶The volume is 1ml, and the culture medium is T cell culture medium added with IL-2200 IU/ml. Centrifuge at 30 ℃ for 10min at 1000 g.

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

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

8. After the cells are infected, the density of the cells is observed every day, and a T cell culture solution containing IL-2100 IU/ml is supplemented at a proper time to maintain the density of the T cells at 5 x 10⁵Cells were expanded at around/ml.

Thus, CART cells infected with the retroviruses shown in example 4, respectively, were obtained, and named CD19CART cells (expressing the CD19CAR of example 1) and CD19-tEGFR CART cells (expressing the CD19CAR and teegfr of example 2), respectively.

Example 6: flow cytometry for detecting expression of CAR protein on surface of T lymphocyte after infection

CAR-T cells and NT cells (control) 72 hours post infection were collected by centrifugation, washed 1 time with PBS, supernatant discarded, added with the corresponding antibody and washed 30min in the dark with PBS, resuspended, and CAR detected by flow cytometry (anti-mouse IgG F (ab') antibody (jackson immunoresearch)).

FIG. 5 shows that the expression efficiency of CD19-tEGFR CAR + reaches 69.316% and the expression efficiency of CD19-tEGFR CAR + reaches 22.86% 72 hours after T cells are infected with the retrovirus prepared in example 3.

Example 7: detection of CD107a expression following coculture of CAR-T cells with target cells

1. Adding CART/NT cells 2 x 10 to each V-bottom 96-well plate⁵Individual and target cells (Raji)/control cells (K562)2 x 10⁵Each cell was resuspended in 200. mu.l of IL-2-free X-VIVO complete medium, BD GolgiStop (containing monesin, 1. mu.l of BD GolgiStop per 1ml of medium) was added to each well, 2. mu.l of CD107a antibody (1:50) was added to each well, incubated at 37 ℃ for 4 hours, and the cells were collected.

2. The samples were centrifuged to remove the medium, washed once with PBS, 400g, and centrifuged at 4 ℃ for 5 minutes. The supernatant was discarded, and appropriate amounts of specific surface antibodies CD3, CD4, and CD8 were added to each tube, and the volume of the suspension was 100ul, followed by incubation for 30 minutes on ice in the absence of light.

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

4. The appropriate amount of PBS was resuspended and CD107a was detected by flow cytometry.

Shown in fig. 6. Figure 6 shows that the percentage of CD107a expression in CD8 positive cells after co-culture with Raji cells was 80.8% and 74.2% for CD19CART cells and CD 19-tfegfr CART cells, respectively; the percentage of CD107a expression in CD4 positive cells after co-culture with Raji cells was 63.7% and 57.3% for CD19CART cells and CD 19-tfegfr CART cells, respectively.

Example 8: IFN gamma secretion detection after CAR-T cell co-culture with target cells

1. Taking prepared CAR-T cells, resuspending the CAR-T cells in Lonza culture medium, and adjusting the cell concentration to be 1 × 10⁶/mL。

2. The experimental group contained 2X 10 target cells (Raji) or negative control cells (K562) per well⁵2X 10 CAR-T/NT cells ⁵200. mu.l of Lonza medium without IL-2. Mix well and add to 96-well plate. BD GolgiStop (containing monesin, 1. mu.l BD GolgiStop per 1ml of medium) was added thereto, and after mixing well, the mixture was incubated at 37 ℃ for 5 hours. Cells were collected as experimental groups.

3. Cells were washed 1 time with 1mL of PBS per tube and centrifuged at 300g for 5 minutes. 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. Using 1 XBD Perm/Wash^TMbuffer washes cells 2 times, 1 mL/time.

5. Staining with intracellular factor, taking appropriate amount of IFN-gamma cytokine fluorescent antibody or negative control, and performing BD Perm/Wash^TMbuffer diluted to 50. mu.l. Resuspending the fixed and disrupted cells thoroughly with the antibody dilution, incubating at 4 ℃ in the dark for 30min, 1 XBD Perm/Wash^TMbuffer 1 mL/wash cells 2 times, then use PBS heavy suspension.

6. And (4) detecting by using a flow cytometer.

Shown in fig. 7. Figure 7 shows that the percentage of INF- γ secretion in CD8 positive cells after co-culture with Raji cells was 4.92% and 4.41% for CD19CART cells and CD 19-tfegfr CART cells, respectively; the percentage of INF- γ secretion in CD4 positive cells after co-culture with Raji cells was 8.03% and 12.8% for CD19CART cells and CD 19-tfegfr CART cells, respectively.

Example 9: detection of tumor-specific cell killing after Co-culture of CAR-T cells with target cells

K562 cells (negative control cells as target cells without CD19 target protein) were resuspended in serum-free medium (1640) adjusted to a cell concentration of 1X 10⁶Perml, the fluorescent dye BMQC (2,3,6,7-tetrahydro-9-bromomethyl-1H,5Hquinolizino (9,1-gh) coumarins) was added to a final concentration of 5. mu.M.

2. Mixing, and incubating at 37 deg.C for 30 min.

3. Centrifugation was carried out at 1500rpm for 5min at room temperature, the supernatant was discarded and the cells resuspended in cytotoxic medium (phenol red-free 1640+ 5% AB serum) and incubated for 60min at 37 ℃.

4. Fresh cytotoxic Medium cells were washed twice and resuspended in fresh cytotoxic Medium at a density of 1X 10⁶/ml。

Raji cells (containing CD19 target protein, as target cells) were suspended in PBS containing 0.1% BSA at a concentration of 1X 10⁶/ml。

6. The fluorescent dye CFSE (fluorescent dye) (CFSE) was added to a final concentration of 1. mu.M.

7. Mixing, and incubating at 37 deg.C for 10 min.

8. After the incubation was completed, FBS in an equal volume to the cell suspension was added 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 10⁶/ml。

10. Effector T cells were washed and suspended in cytotoxic medium at a concentration of 5X 10⁶/ml。

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

CD 19-BBz-tfegfr CAR-T and negative control effector T cells, according to T cell: the target cells were cultured in 5ml sterile test tubes (BD Biosciences) at a ratio of 5:1, 1: 1. In each co-culture group, the target cells were Raji cells (100,000 cells) (50. mu.l), and the negative control cells were K562 cells (100,000 cells) (50. mu.l). A panel was set up to contain only Raji target cells and K562 negative control cells.

13. The co-cultured cells were incubated at 37 ℃ for 5 h.

14. After incubation was complete, cells were washed with PBS and immediately followed by rapid addition of 7-AAD (7-aminoactomycin D) at the concentrations recommended by the instructions and incubation on ice for 30 min.

15. The Flow-type detection is directly carried out without cleaning, and the data is analyzed by Flow Jo.

16. Assay the ratio of live Raji target cells to live K562 negative control cells after co-culture of T cells and target cells was determined using 7AAD negative live cell gating.

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

percent target cell survival ═Number of viable cells of Raji/Number of viable cells in K562。

b) The% cytotoxic killer cells is 100-the% calibrated target cell survival, i.e. (ratio of Raji viable cell number when no effector cells were present-Raji viable cell number when effector cells were present)/K562 viable cell number.

The results are shown in fig. 8. Figure 8 shows that at an effective target ratio of 5:1, the killing efficiency of CD19CART cells and CD19-tEGFR CART cells against target cell Raji is 80% and 83%, respectively.

Example 10: detection of the tEGFR brake in vitro function (ADCC) of CD19-BBz-tEGFR CART cells

NK cells (Meitian whirlpool NK isolation kit) were isolated from PBMC as effector cells, co-cultured with CD19-BBz CART or CD19-BB-tEGFR CART at a ratio of 1:1, with or without Cetuximab (Cetuximab, Erbitux) at a final concentration of 10ug/mL, and after 4h, CD3 and CAR expression were flow-assayed.

Percent ADCC (1-mab group CAR positive rate/no antibody group CAR positive rate) 100%

The results are shown in fig. 9. Figure 9 shows that the ADCC effect of CD19-tEGFR CART cells on NK cells was 43%.

Sequence listing

<110> Shanghai Hengrunheng Dasheng Biotech Co., Ltd

<120> chimeric antigen receptor targeting CD19-41BB-tEGFR and use thereof

<160> 1

<170> PatentIn version 3.3

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<211> 2625

<212> DNA

<213> Artificial sequence

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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 atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaggt tcagtgtcgt gaagagaggc 1020

cggaagaagc tgctgtacat cttcaagcag cctttcatga ggcccgtgca gactacccag 1080

gaggaagatg gatgcagctg tagattccct gaagaggagg aaggaggctg tgagctgaga 1140

gtgaagttct cccgaagcgc agatgcccca gcctatcagc agggacagaa tcagctgtac 1200

aacgagctga acctgggaag acgggaggaa tacgatgtgc tggacaaaag gcggggcaga 1260

gatcctgaga tgggcggcaa accaagacgg aagaaccccc aggaaggtct gtataatgag 1320

ctgcagaaag acaagatggc tgaggcctac tcagaaatcg ggatgaaggg cgaaagaagg 1380

agaggaaaag gccacgacgg actgtaccag gggctgagta cagcaacaaa agacacctat 1440

gacgctctgc acatgcaggc tctgccacca agacgagcta aacgaggctc aggcgcgacg 1500

aactttagtt tgctgaagca agctggggat gtagaggaaa atccgggtcc catgttgctc 1560

cttgtgacga gcctcctgct ctgcgagctg ccccatccag ccttcctcct catcccgcgg 1620

aaggtgtgca atggcatagg cattggcgag tttaaagatt ctctgagcat aaatgctacg 1680

aatattaagc atttcaagaa ttgtacttct attagtggcg acctccatat tcttccggtt 1740

gccttcaggg gtgactcttt cacccacaca cctccattgg atccacaaga acttgacatc 1800

ctgaagacgg ttaaagagat tacaggcttc ctccttatcc aagcgtggcc cgagaacaga 1860

acggacttgc acgcctttga gaacctcgaa ataatacggg gtcggacgaa gcaacacggc 1920

caatttagcc ttgcggttgt tagtctgaac attacttctc tcggccttcg ctctttgaaa 1980

gaaatcagcg acggagatgt catcattagt ggaaacaaga acctgtgcta cgcgaacaca 2040

atcaactgga agaagctctt cggtacttca ggccaaaaga caaagattat tagtaacaga 2100

ggagagaata gctgtaaggc taccggacaa gtttgtcacg ccttgtgtag tccagagggt 2160

tgctggggac cggaaccaag ggattgcgtc agttgccgga acgtgagtcg cggacgcgag 2220

tgtgtggata agtgcaatct tctggaaggg gaaccgcgag agtttgtaga aaattccgaa 2280

tgtatacagt gtcatcccga gtgtcttcca caagcaatga atatcacatg tacagggagg 2340

ggtcctgata actgtatcca atgtgcacac tacatagatg gtcctcactg tgtaaagacg 2400

tgccccgccg gagtaatggg tgaaaacaac accctcgtgt ggaagtacgc cgatgccggg 2460

catgtctgtc atttgtgtca tcccaactgc acatatggct gtaccggtcc tggattggag 2520

ggctgtccaa caaacgggcc gaaaataccg agtatcgcaa caggcatggt gggagcactt 2580

ttgcttctcc tcgttgtcgc cctgggcatc ggcttgttca tgtga 2625

<160> 1

<170> PatentIn version 3.3

<210> 2

<211> 873

<212> PRT

<213> Artificial sequence

<400> 2

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 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser Val

325 330 335

Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Asn Pro Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu

515 520 525

Leu Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly

530 535 540

Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn

545 550 555 560

Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile

565 570 575

Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu

580 585 590

Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly

595 600 605

Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala

610 615 620

Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln

625 630 635 640

Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg

645 650 655

Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys

660 665 670

Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr

675 680 685

Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys

690 695 700

Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys

705 710 715 720

Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg

725 730 735

Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg

740 745 750

Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu

755 760 765

Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys

770 775 780

Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys

785 790 795 800

Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala

805 810 815

Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly

820 825 830

Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile

835 840 845

Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val

850 855 860

Val Ala Leu Gly Ile Gly Leu Phe Met

865 870

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 3

agcatcgttc tgtgttgtct c 21

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 4

tgtttgtctt gtggcaatac ac 22

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 5

agcatcgttc tgtgttgtct c 21

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 6

tgtttgtctt gtggcaatac ac 22

<210> 7

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> linker sequence

<400> 7

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 polynucleotide, the sequence of which is selected from the group consisting of:

(1) a polynucleotide sequence comprising the coding sequence of a single chain antibody against CD19, the coding sequence of a human CD8 α hinge region, the coding sequence of a human CD8 transmembrane region, the coding sequence of a human 41BB intracellular region, the coding sequence of a human CD3 ζ intracellular region, and the coding sequence of a fragment of EGFR comprising extracellular domain III and extracellular domain IV, joined in sequence; and

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

wherein, the amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is shown as the amino acids 22-128 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 the amino acid at the 144-position 263 of SEQ ID NO:2,

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

the amino acid sequence of the transmembrane region of the human CD8 is shown as the amino acid at the 311-332 position of SEQ ID NO. 2;

the amino acid sequence of the intracellular region of the human 41BB is shown as the 333-380 amino acid of SEQ ID NO. 2;

the amino acid sequence of the intracellular region of the human CD3 zeta is shown as the amino acid 381-491 of the SEQ ID NO 2;

the amino acid sequence of the EGFR fragment is shown as the amino acid No. 539-873 of SEQ ID NO: 2.

2. The polynucleotide of claim 1, wherein said polynucleotide further comprises a coding sequence for a signal peptide prior to the coding sequence for said anti-CD 19 single chain antibody.

3. The polynucleotide of claim 2, wherein the amino acid sequence of said signal peptide is as set forth in amino acids 1-21 of SEQ ID No. 2.

4. The polynucleotide of claim 1, wherein said polynucleotide further comprises a coding sequence for a GM-CSF receptor alpha chain signal peptide disposed N-terminal to said EGFR fragment.

5. The polynucleotide of claim 4, wherein the amino acid sequence of the signal peptide of the α chain of the GM-CSF receptor is as shown in amino acids 517-538 of SEQ ID NO 2.

6. The polynucleotide of claim 4, wherein said polynucleotide further comprises a coding sequence for a linker sequence linking said GM-CSF receptor alpha chain signal peptide to said intracellular domain of human CD3 ζ.

7. The polynucleotide of claim 6, wherein the amino acid sequence of said linker sequence is as set forth in amino acids 492 and 516 of SEQ ID No. 2.

8. The polynucleotide of claim 2, wherein the coding sequence for said signal peptide preceding the coding sequence for said anti-CD 19 single chain antibody is as set forth in nucleotide sequence nos. 1-63 of SEQ ID No. 1.

9. The polynucleotide of claim 1, wherein the coding sequence for the light chain variable region of said anti-CD 19 single chain antibody is as set forth in nucleotide sequences 64-384 of SEQ ID No. 1;

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

the coding sequence of the human CD8 alpha hinge region is shown as the nucleotide sequence at the 790 nd and 930 th positions of SEQ ID NO. 1;

the coding sequence of the transmembrane region of the human CD8 is shown as the nucleotide sequence of No. 931 and No. 996 of SEQ ID NO 1;

the coding sequence of the human 41BB intracellular region is shown as the nucleotide sequence at position 997-1140 of SEQ ID NO. 1;

the coding sequence of the intracellular region of human CD3 zeta is shown as the nucleotide sequence at the 1141-position 1473 of SEQ ID NO. 1;

the coding sequence of the EGFR fragment is shown as the nucleotide sequence of 1632-position 2625 of SEQ ID NO 1; or

The polynucleotide sequence codes an amino acid sequence shown as 22 th to 491 th positions of SEQ ID NO. 2, or codes an amino acid sequence shown as 22 th to 516 th positions of SEQ ID NO. 2, or codes an amino acid sequence shown as SEQ ID NO. 2; or

The polynucleotide sequence comprises a nucleotide sequence shown in SEQ ID NO. 1 and SEQ ID NO. 1 from 1 st to 1473 rd sites, a nucleotide sequence shown in SEQ ID NO. 1 from 64 th to 1473 rd sites, or a nucleotide sequence shown in SEQ ID NO. 1 from 64 th to 2625 th sites, or consists of a nucleotide sequence shown in SEQ ID NO. 1 and SEQ ID NO. 1 from 1 st to 1473 rd sites, a nucleotide sequence shown in SEQ ID NO. 1 from 64 th to 1473 rd sites, or a nucleotide sequence shown in SEQ ID NO. 1 from 64 th to 2625 th sites.

10. The polynucleotide of claim 4, wherein the coding sequence for the α -chain signal peptide of GM-CSF receptor is as shown in nucleotide sequences 1552-1631 of SEQ ID NO. 1.

11. The polynucleotide of claim 6, wherein the coding sequence for said linker sequence linking said signal peptide from the α chain of the GM-CSF receptor to the intracellular domain of human CD3 ζ is as shown in nucleotide sequence Nos. 1474 and 1551 of SEQ ID NO. 1.

12. A fusion protein comprises a coding sequence of a fragment containing an extracellular domain III and an extracellular domain IV of an anti-CD 19 single-chain antibody, a human CD8 alpha hinge region, a human CD8 transmembrane region, a human 41BB intracellular region, a human CD3 zeta intracellular region and an EGFR which are connected in sequence, wherein the amino acid sequence of a light chain variable region of the anti-CD 19 single-chain antibody is shown as amino acids 22-128 of SEQ ID NO:2, the amino acid sequence of a heavy chain variable region of the anti-CD 19 single-chain antibody is shown as amino acids 144-263 of SEQ ID NO:2, and the amino acid sequence of a hinge region of the human CD8 alpha hinge region is shown as amino acid 310 of 264-310 of SEQ ID NO: 2; the amino acid sequence of the transmembrane region of the human CD8 is shown as the amino acid at the 311-332 position of SEQ ID NO. 2; the amino acid sequence of the intracellular region of the human 41BB is shown as the 333-380 amino acid of SEQ ID NO. 2; the amino acid sequence of the intracellular region of the human CD3 zeta is shown as the amino acid 381-491 of the SEQ ID NO 2; and the amino acid sequence of the EGFR fragment is shown as the amino acid No. 539-873 of SEQ ID NO: 2.

13. The fusion protein of claim 12, further comprising a signal peptide at the N-terminus of the anti-CD 19 single chain antibody.

14. The fusion protein of claim 12, wherein the signal peptide has the amino acid sequence shown as amino acids 1-21 of SEQ ID No. 2.

15. The fusion protein of claim 12, further comprising a GM-CSF receptor alpha chain signal peptide disposed N-terminal to the EGFR fragment.

16. The fusion protein of claim 15, wherein the signal peptide of the α chain of the GM-CSF receptor has the amino acid sequence as shown in SEQ ID NO 2 at amino acids 517 and 538.

17. The fusion protein of claim 15, further comprising a linker sequence linking the GM-CSF receptor alpha chain signal peptide to the intracellular domain of human CD3 ζ.

18. The fusion protein of claim 17, wherein the amino acid sequence of the linker sequence is as shown in amino acids 492 and 516 of SEQ ID NO 2.

19. The fusion protein of claim 12, wherein the amino acid sequence of the fusion protein is as set forth in amino acids 22 to 491 of SEQ ID No. 2, or as set forth in amino acids 22 to 516 of SEQ ID No. 2, or as set forth in amino acids 1 to 516 of SEQ ID No. 2, or as set forth in SEQ ID No. 2.

20. A nucleic acid construct comprising the polynucleotide of any one of claims 1-11.

21. The nucleic acid construct of claim 20, wherein said nucleic acid construct is a vector.

22. The nucleic acid construct of claim 20, wherein the nucleic acid construct is a retroviral vector comprising a replication initiation site, a 3 'LTR, a 5' LTR, and the polynucleotide of any one of claims 1-11.

23. A retrovirus comprising the polynucleotide of any one of claims 1-11.

24. A genetically modified T-cell or a pharmaceutical composition comprising a genetically modified T-cell, wherein the cell comprises the polynucleotide of any one of claims 1-11, or comprises the nucleic acid construct of any one of claims 20-22, or is infected with the retrovirus of claim 23, or stably expresses the fusion protein of any one of claims 12-19.

25. Use of the polynucleotide of any one of claims 1-11, the fusion protein of any one of claims 12-19, the nucleic acid construct of any one of claims 20-22, or the retrovirus of claim 23 in the preparation of an agent for activating a T cell.

26. Use of the polynucleotide of any one of claims 1-11, the fusion protein of any one of claims 12-19, the nucleic acid construct of any one of claims 20-22, the retrovirus of claim 23, or the genetically modified T-cell of claim 24, or a pharmaceutical composition thereof, in the preparation of a medicament for treating a CD 19-mediated disease.

27. The use according to claim 26, wherein the CD19 mediated disease is leukemia and/or lymphoma.