Chimeric antigen receptor and modified cells carrying the same

Abstract

The present disclosure relates to a chimeric antigen receptor (CAR), which includes an extracellular domain, which has a CD19-specific antigen binding domain to be arranged on the plasma membrane surface of a modified T cell, and the antigen binding domain includes a variable light chain region and a variable heavy chain region, and the variable light chain region and the variable heavy chain region are connected in an end-to-end manner by a joint; an intracellular domain for regulating the activation of modified T cells; a transmembrane domain, anchored on the plasma membrane of the modified T cell and between the extracellular domain and the intracellular domain. Preferably, the extracellular domain is connected to the intracellular domain by an IgG2 spacer.

Description

Technical Field

[0001] The present disclosure relates to engineered chimeric antigen receptors that are capable of interacting with cluster of differentiation 19 molecules (CD 19) expressed on the surface of B lymphocytes or cells of the B lymphocyte lineage. In addition, the present disclosure also includes genetically engineered cells, particularly immunogenic T cells, wherein these cells are endowed with the ability to express and carry chimeric antigen receptors. Background Art

[0002] B-cell acute lymphoblastic leukemia (B-cell ALL) is the most common childhood cancer and has an overall good prognosis, with up to 90% of patients surviving 5 years after diagnosis with ALL. However, for those who relapse during treatment, or who have chemotherapy-refractory disease, the outlook is poor. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) still offers the opportunity for cure, but unfortunately, relapse is common after leukemia treatment with allo-HSCT. Patients with ALL who relapse after allo-HSCT are extremely difficult to manage, and many researchers have advocated for donor lymphocyte infusions as an effective adjunct in addition to chemotherapy, although available data do not support its use in patients with overtly active disease. Therefore, new treatment strategies for relapsed / refractory B-cell ALL are critical. In this setting, CD19 is an attractive target for immunotherapy because its expression is almost ubiquitous in the blasts of B-cell ALL, but it is restricted to normal cells of the B lineage. 2 Subsequently, chimeric antigen receptor (CAR) T cell preparations were developed by modifying the T cell receptor of the CD19 antigen for the treatment of refractory B-cell ALL and advanced B-cell lymphoma. 3

[0003] The basic structure of CAR-modified T cells consists of an extracellular antigen-binding domain from a monoclonal antibody and a signaling domain from a T cell receptor to provide the ability to target specific surface antigens. Second- and third-generation CARs contain one or two co-stimulatory molecules, such as CD28, 4-1BB, or OX40. 4 Promotes in vivo expansion. 5 In addition to co-stimulatory molecules, another element of CAR, the spacer domain (CH2CH3), appears to play an important role in mediating antitumor activity. CARs with mutated CH2 regions or without CH2 regions have been reported to confer T cell persistence and more effective antitumor responses in murine models. 4 Other researchers have demonstrated the role of CAR spacer length in the binding of the receptor to its target antigen. 6.7Adoptive T cell transfer of such CAR-modified T cells has become a new frontier in cancer immunotherapy. This field has rapidly entered multiple clinical trials and has led to the US FDA approval of the first two CAR T cell preparations for the treatment of B-cell ALL and refractory lymphoma. 8 In fact, CD19 CAR T cells have shown remarkable anti-tumor effects in some trials, with up to 80% of patients achieving complete remission (CR). 5,9,10 However, emerging safety issues regarding CAR T-cell therapy should be carefully considered. Toxicity after CAR T-cell infusion includes cytokine release syndrome (CRS), neurotoxicity, and “on-target / off-target tumor” recognition, which are the most serious toxicities and may become life-threatening or even fatal adverse events. 11 Abolition of these adverse events has become another challenge to the successful implementation of CAR T cells as part of standard treatment for relapsed / refractory ALL.

[0004] Despite the stunning clinical responses and regulatory approval of both products, it is important to remember that, in addition to the potential for life-threatening adverse effects related to the formulation, there are other issues associated with large-scale commercialization of this cutting-edge therapy. First, the cost of CAR T cells is much higher than the cost of producing CAR T cells. 12 Second, the production of fresh lymphocyte preparations and final CAR T-cell products involving long-distance transportation will create difficult logistical problems. These problems will undoubtedly prompt local medical (research) centers to develop their own CAR T manufacturing facilities in order to provide such treatment options in several different regions of each country or large countries.

[0005] Further research on CARs, including international patent publication WO / 2018 / 231871, discloses engineered T cells that are configured to express CARs when endogenous expression of T cell antigens is reduced or knocked down. Mcginness et al. detailed another similar study on genetically engineered hematopoietic cells expressing one or more Krebs cycle regulatory polypeptides and, optionally, a chimeric receptor polypeptide capable of binding to a target antigen of interest in international patent publication WO2020037066. Mcginness et al. further described another genetically engineered hematopoietic cell expressing one or more lactate regulatory factors capable of binding to a target antigen of interest in international patent publication WO2020051493. Although both WO2020037066A1 and WO2020051493A1 teach the potential use of any hinge or spacer domain derived from CD28, CD16A, CD8a or immunoglobulin G (IgG) in assembling a CAR capable of binding to a specific antigen, the appropriate type of immunoglobulin G (IgG) is never identified in these disclosures.

[0006] Given the deficiencies found in previous studies conducted as described above, there is a need to explore other alternatives for therapeutic CAR systems that could be used to obtain improved performance. Summary of the invention

[0007] The present disclosure is directed to providing a CAR that is configured to bind to a specific antigen expressed on the surface of a given cell type or cell lineage, thereby enabling the subject to obtain a beneficial outcome, particularly a therapeutic outcome.

[0008] A further object of the present disclosure relates to a CAR that is specifically designed to recognize and bind to CD 19 expressed on the surface of B lymphocytes or B lineage cells, suitable for treating B cell ALL. Specifically, the disclosed CAR utilizes a modified hinge or spacer domain to more effectively kill B lymphocytes or B lineage cells to treat diseases such as B cell ALL.

[0009] Yet another object of the present disclosure is to obtain T cells capable of expressing and presenting the disclosed CARs, which binding to compatible antigens available on the cell surface via the CAR results in the initiation of one or more preferred immune responses to eradicate cancer cells associated with B cell lymphoma.

[0010] The present disclosure provides a viral vector incorporating one or more polynucleotide sequences encoding the peptides of the disclosed CAR. The disclosed vector is designed to express the encoded peptides in compatible transduced cells, which are further assembled into the disclosed CAR.

[0011] The present disclosure satisfies at least one of the aforementioned purposes in whole or in part, wherein one of the embodiments of the present disclosure is a chimeric antigen receptor (CAR), which comprises an extracellular domain, the extracellular domain having an antigen binding domain to be placed on the surface of a modified T cell plasma membrane, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connected in an end-to-end manner by a linker; an intracellular domain for regulating the activation of modified T cells; a transmembrane domain, anchored on the plasma membrane of the modified T cell and between the extracellular domain and the intracellular domain. The variable heavy chain region is connected to the intracellular domain by a spacer region, and the spacer region comprises an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID No. 1. Preferably, the spacer region is based on immunoglobulin (Ig) G2.

[0012] For more embodiments, the variable light chain region comprises an amino acid sequence having at least 90% similarity to SEQ ID No.2.

[0013] For many embodiments, the variable heavy chain region comprises an amino acid sequence having at least 90% similarity to SEQ ID No.3.

[0014] In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID No.4.

[0015] Typically, in some embodiments, the extracellular domain comprises the amino acid sequence shown in SEQ ID No.5.

[0016] Furthermore, in more embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID No.6.

[0017] Preferably, in some embodiments of the disclosed CAR, the intracellular domain comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from the CD3 zeta chain, wherein the CD28 signaling domain and the cytoplasmic signaling domain may comprise the amino acid sequences shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

[0018] In more embodiments, the disclosed CAR further comprises a leader sequence previously linked to the variable light chain region, and the leader sequence has an amino acid sequence as shown in SEQ ID No.9.

[0019] Another aspect of the present disclosure relates to a genetically engineered T cell capable of expressing a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular domain having an antigen binding domain to be placed on the surface of the modified T cell plasma membrane, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connected end-to-end by a linker; an intracellular domain for regulating the activation of the modified T cells; a transmembrane domain anchored on the plasma membrane of the modified T cells and between the extracellular domain and the intracellular domain. Preferably, the variable heavy chain region of the chimeric antigen receptor in the engineered T cell is connected to the intracellular domain by a spacer region, the spacer region comprising an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID No. 1.

[0020] According to several embodiments of the engineered T cells, the extracellular domain comprises the amino acid sequence shown in SEQ ID No.5.

[0021] According to further embodiments of the engineered T cells, the intracellular domain comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from the CD3 zeta chain.

[0022] According to multiple embodiments of the engineered T cells, the chimeric antigen receptor further comprises a leader sequence, which is pre-linked to the variable light chain region, and the leader sequence has an amino acid sequence as shown in SEQ ID No.9.

[0023] Another aspect of the present disclosure relates to a viral vector capable of promoting the expression of a peptide encoding a chimeric antigen receptor in transduced T cells. The viral vector substantially comprises the polynucleotide sequence shown in SEQ ID No. 10. Preferably, the viral vector is a lentiviral vector.

[0024] Preferably, in some embodiments of the viral vector, the encoded peptide comprises at least 90% similarity to the amino acid sequence shown in SEQ ID No.11. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagrams of several embodiments of CD19CAR, wherein each construct comprises a CD19 scFV, a predetermined range of IgG2 spacer, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3ζ cytoplasmic domain;

[0026] Figure 2 is a graph showing the number of viable T cells after transduction with CARs with short spacers, showing a logarithmic phase after day 1 of transduction (similar to untransduced cells), while CARs with full and intermediate spacers enter a logarithmic proliferation phase 2 days after transduction;

[0027] Figure 3 is a graph showing the percentage of transduction efficiency measured using QPCR, wherein the expression of CARCD28-CD3ζ domain constructs in genomic DNA was detected in CD19CAR T cells derived from different healthy donors using QPCR targeting primers specific for the CD28-CD3ζ region, wherein * and # represent statistically significant differences with P<.05 when compared to NT and CAR full spacer constructs, respectively;

[0028] Figure 4 is an agarose gel image after QPCR using specific primers targeting CD28-CD3ζ (product size is 281 bp);

[0029] Figure 5 is a graph illustrating the anti-tumor activity of CAR T cells, using a CFSE-7-AAD-based cytotoxicity assay to study the ratio of effector to target cells (E / T) associated with CD19 CAR T of different spacer lengths, wherein Raji, RS4, and Sup-B15 (CD19 expressing cells) were used as target cells, Jurkat cell line and PHA primitive cells (CD 19 non-expressing cells) were used as negative controls, and non-transduced T cells (NT) were used as controls, wherein the data represent three independent experiments performed on CAR T cells generated from five independent donors (*, #, and $ represent P < .05, respectively, compared with NT, full-length, and intermediate spacer lengths, while ** and *** represent P < .01 and .001, respectively, compared with NT);

[0030] Figure 6 is a gel image showing CD 19CAR T cells detected in peripheral blood (PB) and bone marrow (BM) samples examined on day 14 of CAR T cell infusion (D indicates day, +ve indicates transduced cells, and -ve represents non-transduced cells); and

[0031] Figure 7 shows the amino acid sequences of (a) SEQ ID No. 1, (b) SEQ ID No. 2, (c) SEQ ID No. 3, (d) SEQ ID No. 4, (e) SEQ ID No. 5, (f) SEQ ID No. 6, (g) SEQ ID No. 7, (h) SEQ ID No. 8, and (i) SEQ ID No. 9;

[0032] Figure 8 shows (a) a polynucleotide sequence represented by SEQ ID No. 10, encoding (b) an embodiment of a CAR represented by SEQ ID No. 11, which polynucleotide sequence can be incorporated into a viral vector. DETAILED DESCRIPTION

[0033] Hereinafter, the present invention will be described according to preferred embodiments and with reference to the attached description and drawings. However, it should be understood that the description with reference to the preferred embodiments and drawings of the present disclosure is only for the convenience of discussing the various disclosed embodiments, and it is conceivable that those skilled in the art can design different modifications without departing from the scope of the claims.

[0034] As used herein, in the context of component concentrations, conditions, other measurements, and the like, the term "approximately" or "about" means + / - 5% of the stated value, or + / - 4% of the stated value, or + / - 3% of the stated value, or + / - 2% of the stated value, + / - 1% of the stated value, or + / - 0.5% of the stated value, or + / - 0% of the stated value.

[0035] As used herein, the term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, cDNA or DNA. The term generally refers to oligonucleotides greater than 30 nucleotide residues in length.

[0036] The term "chimeric antigen receptor" or "CAR" used throughout the specification refers to an artificial construct or complex assembled from one or more peptides, comprising multiple domains, at least two of which are of different origins.

[0037] One aspect of the present disclosure relates to a CAR, which can be used as follows Figure 1 Various arrangements or configurations generally shown in the different embodiments shown. In particular, the disclosed CAR comprises an extracellular domain having an antigen binding domain to be placed on the surface of the modified T cell plasma membrane, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connected in an end-to-end manner by a linker; an intracellular domain for regulating the activation of modified T cells; a transmembrane domain, anchored on the plasma membrane of the modified T cell and between the extracellular domain and the intracellular domain. It is important to note that the disclosed CAR is CD 19-specific, which is ubiquitously expressed on the cell surface of B lymphocytes or B lymphocyte lineages. Therefore, engineered T cells carrying CD19-specific CARs should help identify cells of the B lymphocyte lineage through engineered T cells and their coupling. The coupling of engineered T cells and B lymphocyte lineage cells may lead to the activation of T cells and the release of corresponding immunomodulatory cytokines, ultimately killing and eradicating the identified B lymphocyte lineage cells. The above interactions between engineered T cells and B lymphocyte lineage cells enable the disclosed CAR to enable engineered T cells to function as a means of treating B cell ALL, similar diseases and / or any complications associated therewith. Although the CAR described below generally refers to a second generation CAR, some embodiments of the disclosed CAR may include a third or fourth generation CAR.

[0038] Reference again Figure 1 , the variable heavy chain region of the disclosed CAR is connected to the intracellular domain via a spacer. The spacer is generally used to provide sufficient space between the antigen binding domain and the membrane of the transduced cell so that the antigen binding domain can be folded in nature without interference from the plasma membrane or any cellular components installed on the membrane. Although it has been previously reported that the length of the spacer may play a role in determining the efficiency of T cells carrying CAR for leukemia treatment, the inventors of the present disclosure have found that the correct length of the spacer actually affects the transduction efficiency, which may be another important factor in determining the efficiency of engineered T cell therapy. Specifically, the spacer of the disclosed CAR is derived from IgG2, or more specifically, the spacer is derived from human IgG2. In some other embodiments of the disclosed CAR, the spacer may be a continuous fragment or region derived from human IgG2, with or without further modification. For more embodiments of the disclosed CAR, the spacer includes a hinge region, a CH3 region, and a CH2 region between the hinge region and the CH3 region. In addition, the inventors of the present disclosure have found that the use of a spacer region derived from an IgG2-based hinge region, a CH2 region, and a modified CH3 region of about 132 amino acids in length can enable the disclosed CAR to have better clinical results in anti-B lymphocyte lineage cancer cells. More preferably, the spacer region comprises an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID No.1.

[0039] For some embodiments, the antigen binding domain is a single chain variable fragment.

[0040] As previously described, the antigen binding domain is essentially composed of a variable light chain region and a variable heavy chain region connected in series by a linker. More specifically, the linker used in some embodiments can be a flexible GS linker, which is essentially composed of glycine (Gly) and serine (Ser) residues. More preferably, G4S or (Gly-Gly-Gly-Gly-Ser) resistant to cellular protease activity is used. nThe flexible linker fuses the variable heavy chain region and the variable light chain region together, where n can be from 1 to 10. (G4S)4 is preferably used in some embodiments of the disclosed CAR to fully separate the light chain region and the heavy chain region without causing any undesirable interactions between the two separated regions, but still retains the anti-CD-19 properties of the antigen binding domain that remains in the extracellular domain. Therefore, in many preferred embodiments, the linker comprises an amino acid sequence as shown in SEQ ID No.4. The linker used connects the amino terminus of the variable light chain region to the carboxyl terminus of the variable heavy chain region. As for the variable light chain region and the heavy chain region, these domains are generally derived from the light chain and heavy chain of the immunoglobulin of the house mouse, respectively, with or without further modification according to the embodiments of the disclosed CAR. Preferably, the variable light chain region comprises an amino acid sequence having at least 90% similarity to SEQ ID No.2. Similarly, the variable heavy chain region comprises an amino acid sequence having at least 90% similarity to SEQ ID No.3. More specifically, the extracellular domain spanning the variable light chain region, the linker, and the variable heavy chain region comprises an amino acid sequence as shown in SEQ ID No.5.

[0041] According to further embodiments, the disclosed CAR may also include a leader sequence or a leader peptide sequence that assists the expression of CAR on the membrane of engineered T cells after secretion from the Golgi complex. Preferably, the leader peptide sequence is pre-linked to the variable light chain region to guide the secreted chimeric peptide to the surface membrane for expression. The leader sequence may include an amino acid sequence, but is not limited to, an amino acid sequence as shown in SEQ ID No.9.

[0042] In addition, in more embodiments of the disclosed CAR, the intracellular domain is preferably a TCR zeta chain or structure, which is configured to promote the transfer of stimulation signals to activate T cells when the antigen binding domain reacts with CD 19 located on B lymphocyte lineage cells. More specifically, the intracellular domain comprises a human CD28 signaling domain, which is coupled to a cytoplasmic signaling domain derived from a human CD3 ζ-chain. Preferably, the CD28 signaling domain comprises the amino acid sequence shown in SEQ ID No.7, and the cytoplasmic signaling domain comprises the amino acid sequence shown in SEQ ID No.8.

[0043] According to various embodiments of the disclosed CAR, the transmembrane domain comprises an amino acid sequence as shown in SEQ ID No. 6, which is preferably derived from human CD28 specifically found on the surface of T cells.

[0044] According to another aspect of the present disclosure, genetically engineered T cells capable of expressing CAR are configured to couple with CD 19 found on the cell surface of B lymphocyte lineage cells, and CAR binds to CD 19, triggering a cascade of immunomodulatory responses that cause the binding cells of the B lymphocyte lineage to be killed or eradicated. The inventors of the present disclosure have endeavored to disclose an effective means of treating B cell-ALL by assembling a CD 19-specific chimeric antigen receptor for cells through cell therapy. As described above, in view of the application of a spacer region incorporating an IgG2-modified CH3 domain, the disclosed engineered T cells have better transduction efficiency and killing efficiency than the B lymphocyte lineage cells identified or identified. Preferably, the disclosed engineered T cells contain multiple chimeric antigen receptors, which essentially contain an extracellular domain having an antigen binding domain to be arranged on the surface of the modified T cell plasma membrane, wherein the antigen binding domain comprises a variable light chain region and a variable heavy chain region connected in an end-to-end manner by a linker; an intracellular domain for regulating the activation of the modified T cells; and a transmembrane domain, which is anchored on the plasma membrane of the modified T cells and is between the extracellular domain and the intracellular domain. In order to improve the transduction efficiency and killing efficiency of target cells of the B lymphocyte lineage, the variable heavy chain region is connected to the intracellular domain by a spacer region, and the spacer region comprises an amino acid sequence having at least 90% similarity to the sequence shown in SEQ ID No. 1.

[0045] In multiple embodiments, the disclosed engineered T cell CAR may include a leader sequence that is pre-linked to the variable light chain region. Preferably, the leader sequence has an amino acid sequence as shown in SEQ ID No.9, so that the secreted peptide or polypeptide of the CAR can be effectively expressed on the surface of the engineered T cell. It is important to note that the disclosed engineered T cells can be a tool for treating B cell leukemia patients through cell therapy, thereby resolving any complications associated with the use of standard chemotherapy drugs. Nevertheless, the disclosed engineered T cells can also be used for leukemia treatment together with predetermined chemotherapy drugs to produce the desired therapeutic results while ensuring that the side effects caused by the chemotherapy drugs are minimized.

[0046] According to several embodiments of the disclosed engineered T cells, the extracellular domain comprises the amino acid sequence shown in SEQ ID No. 5, which spans the variable light chain region, the linker and the variable heavy chain region, and comprises the amino acid sequences shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0047] As described above, the intracellular domain of the CAR expressed in the disclosed engineered T cells comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from the CD3 ζ-chain.

[0048] Another aspect of the present disclosure relates to a viral vector capable of promoting peptide expression, which corresponds to one or more embodiments of the CAR disclosed above. In particular, the disclosed viral vector encodes a chimeric antigen receptor that can be expressed in compatible transduced T cells under predetermined culture conditions. The disclosed viral vector comprises a polynucleotide sequence or base sequence as shown in SEQ ID No.10. The coding sequence of SEQ ID No.10 can be further modified to obtain a CAR with improved or altered properties for the treatment of B cell ALL, similar diseases and / or any complications associated therewith. Therefore, more embodiments of the disclosed viral vectors include a polynucleotide sequence or base sequence having at least 90% similarity as shown in SEQ ID No.10. The disclosed viral vector can be used to generate CD 19-specific chimeric antigen receptor T cells, which in turn can be used as a treatment method for B cell leukemia patients through any established leukemia cell treatment regimen. In addition, the peptide encoded by the disclosed vector may contain at least 90% similarity to the amino acid sequence shown in SEQ ID No.11. Preferably, the disclosed viral vector is a lentiviral vector.

[0049] Likewise, one aspect of the present disclosure relates to genetically engineered T cells as disclosed or described above for use as a medicament for the treatment of B-cell ALL.

[0050] The following examples are intended to further illustrate the present invention and are not meant to limit the disclosure to the specific examples described therein.

[0051] Example 1

[0052] The two CAR constructs, CAR-CD19-TM28-Z and CAR-PSCA-CD28TM-CD28-CD3ζ, were kindly provided by the Texas Cell and Gene Therapy Center, Baylor College of Medicine Cell, Texas Children's Hospital, and Houston Methodist Hospital. They were linked together to produce the second-generation anti-CD19 scFv-CD28-CD3ζ. Anti-CD 19scFv-CD28-CD3ζ was used as a template to prepare modified CAR constructs with different spacer lengths "intermediate" and "short". The intermediate and short constructs have CH2 and CH2CH3 deletions, respectively. The three CAR constructs were subcloned into a lentiviral vector backbone plasmid (pSin-EF2-Puro; Addgene, Watertown, MA, USA).

[0053] Example 2

[0054] HEK293T cells were seeded onto poly-D-lysine-coated plates (5 × 10 cells per 10-cm dish) before co-transfection with envelope (pMD2.G) and packaging (psPAX2) plasmids with the pSIN-CD19 CAR vector. 6 cells). The next day, HEK 293T cells were transfected using FuGENE HD transfection reagent (Promega, Madison, WI, USA). Supernatants were collected 48 and 72 hours after transfection and filtered through a 0.45-μm polyethersulfone filter (Merck Millipore, Burlington, MA, USA) to remove cell debris. Lenti-X concentrators (Takara, Shiga, Japan) were used to concentrate lentivirus according to the manufacturer's instructions. In brief, the supernatant was mixed with a Lenti-X concentrator at a ratio of 1:3 (Lenti-X concentrator: supernatant), and the mixture was incubated at 4°C for 30 minutes. Next, the mixture was centrifuged at 1500 x g for 45 minutes at 4°C. The pellet was suspended in Dulbecco's phosphate-buffered saline (DPBS) (GIBCO; Thermo Fisher Scientific, Inc, Waltham, MA, USA) and stored at -80°C until use.

[0055] Example 3

[0056] Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors and isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA). The experiment was approved by the Human Rights Ethics Review Committee for Research with Human Subjects, Faculty of Medicine, Ramatibodi Hospital, Mahidol University (Protocol ID 03-59-11). PBMCs were then cultured in TexMACS GMP medium (MACS; Miltenyi Biotec) containing 5% heat-inactivated human AB serum (Sigma-Aldrich Pte Ltd) and recombinant human IL2200 U (GMP grade, CellGenix; CellGenix Inc). Raji cell line (Burkitt's lymphoma) was kindly provided by Assistant Professor Dachrit Nilubol. Jurkat cell line (acute T lymphoblastic leukemia) was kindly provided by Professor Kovit Pattanapanyasat. RS4 and Sup-B15 cell lines (acute lymphoblastic leukemia) were purchased from the American Type Culture Collection (Manassas, VA, USA). Raji, RS4, and Jurkat cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (GIBCO) and 1% penicillin / streptomycin (GIBCO), while Sup-B15 was cultured in Iscove's modified Dulbecco's medium (Hyclone, GE Healthcare Life Sciences), Dulbecco's medium containing 20% ​​FBS, 1% penicillin / streptomycin, and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich).

[0057] To generate allogeneic phytohemagglutinin-stimulated (PHA) blasts, 5×10 blasts were stimulated with 5 μg / mL PHA (Invitrogen; Thermo Fisher Scientific, Inc) in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin. 5 PBMCs were added at a total of 100 U / mL IL-2 (PeproTech, Rocky Hill, NJ, USA) on day 2. PHA blasts were cultured for 7 days.

[0058] The culture medium and IL-2 were replaced every 2-3 days. All cell cultures were maintained at 37°C in a fully humidified atmosphere containing 5% CO2 in air.

[0059] Example 4

[0060] T cells were stimulated with anti-human CD3 (MACS; Miltenyi Biotec) and anti-CD28 (MACS; Miltenyi Biotec) monoclonal antibodies and then cultured in TexMACS GMP medium containing 5% heat-inactivated human AB serum and recombinant human IL2200 U (GMP grade). Transduction was performed on day 3.

[0061] Activated T cells were transduced using RetroNectin (Takara, Shiga, Japan). RetroNectin was used to coat 24-well plates at a concentration of 20 μg / mL in DPBS. The plates were incubated at room temperature for 2 hours and blocked with 2% heat-inactivated human AB serum (Sigma-Aldrich) in DPBS. The plates were then washed once with DPBS. The lentivirus was added to 500 μL of complete TexMACS GMP culture medium and added to the coated wells. The coated plates containing the virus were centrifuged at 2000 x g for 1.5 hours at 32°C. Next, 105 activated T cells in 500 μL of complete TexMACS GMP culture medium with IL-2 were added to each well at a final concentration of 100 U / mL. Complete culture medium and IL-2 were replaced every 2-3 days. Transduced T cells were cultured for 6 days for cytotoxicity assays.

[0062] Example 5

[0063] DNA was extracted from transduced CD 19CAR T cells and non-transduced T cells using GenUP gDNA kit (Biotechrabbit, Hennigsdorf, Germany). According to the manufacturer's instructions, quantitative polymerase chain reaction (QPCR) was performed using QPCR Green Master MixLRox (Biotechrabbit) to determine the copy number of the transgene, and this operation was performed using CLX96 real-time PCR detection system (Bio-Rad, Singapore). The present disclosure uses primers specific to CD28CD3ζ domains so that regardless of whether DNA is from cells transduced with full-length, medium-length or short-length interval domains, PCR will display the same product. Each reaction contains 100ng genomic DNA, and each experiment is performed in triplicate. CAR copy number quantification is based on comparison with a standard curve. The genomic DNA of non-transduced T cells is added with a plasmid containing a transgene for serial dilution to obtain a standard curve.

[0064] To investigate the effectiveness of targeted anti-CD19 CARs, all constructs in the present disclosure including scFv, transmembrane and signaling domains were identical, each encoding a CD28 transmembrane, a CD28 signaling domain and a CD3ζ-derived signaling domain ( Figure 1 ). T cell proliferation was detected after transduction. The lentiviral transduction process did not have any adverse effects on T cell growth and viability. The CAR full and middle spacer constructs entered logarithmic growth 2 days after transduction, while the CAR short spacer construct entered logarithmic proliferation within 24 hours after transduction ( Figure 2 ). CAR construct expression was measured by QPCR as an indicator of transduction efficiency. CAR copy number was calculated for all constructs before co-culture with target cells. All constructs expressed different transduction efficiencies, approximately 13%, 41%, or 21% for full-spacing, intermediate-spacing, or short-spacing, respectively ( Figure 3 ). Agarose gel electrophoresis was performed on QPCR products of genomic DNA from CAR T cells and non-transduced T cells (NT) cells. The size of each CAR product was approximately 281 bp, and as expected, NT control cells did not express the CAR construct, as shown in Figure 2. Figure 4 shown.

[0065] Example 6

[0066] Cytotoxicity assays were performed using flow cytometry-based analysis. Effector cells (T cells) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen; Thermo Fisher Science, Inc) at a concentration of 1 mM. Effector cells were co-cultured overnight with all target cells at a ratio of 1: 1, 10: 1, and 20: 1 (effect: target). Control samples with target cells only were used to assess spontaneous cell death. The next day, cells were collected and stained with 5 μL 7-AAD (7-aminoactinomycin D) (eBioscience; Thermo Fisher Scientific, Inc). Data analysis was performed by BD FACSVerse flow cytometer (BD Bioscience; San Jose, CA) and BD FACSuite software (BD Bioscience). Target cells were selected as CFSE-negative cell populations. Target cell death was determined as CFSE-negative with 7-AAD-positive cell populations. The percentage of cytotoxic activity was calculated according to the following equation:

[0067]

[0068] Example 7

[0069] The present disclosure monitors CD19 CAR T cells in peripheral blood and bone marrow samples by RT-PCR using the same primers specific for the CD28CD3ζ domain as described in Section 2.5.

[0070] Briefly, 2 million cells from bone marrow aspirate samples were incubated with premixed antibody combinations at room temperature for 15 minutes. Then, red blood cell lysis was completed by adding lysis buffer and incubating for 10 minutes. All cells were washed twice and resuspended in 0.5% paraformaldehyde for collection using BD LSRFortessa (Beckton Dickinson, San Jose, CA, USA). The minimal residual disease (MRD) panel is a 14-color panel containing 27 leukemia-related markers, including CD37, CD164, CD58, CD304, CD20, CD81, CD200, CD24, CD10, CD38, CD44, CD79b, CD49f, CD10, CD123, CD73, CD13, CD33, CD15, CD86, CD66c, NG2 (7.1), CD72, CD19, CD10, CD34, and CD45. For analysis, immature B cells were gated by using CD 19, CD79a, CD 10, CD34, CD45, FSC and SSC after double discrimination. The immunophenotypic pattern of gated immature B cells was compared with the hematogones template. The remaining mature B cells were used as an internal control. MRD was considered a positive result when two or more abnormalities were present compared with the normal template and the MRD pattern at diagnosis. The lower limit of detection was 0.01% of all nucleated cells.

[0071] To validate the increased cytotoxicity of CD 19 CAR T cells, cytotoxicity experiments were performed on three B cell AFF cell lines, Raji, RS4, and Sup-B15. Specific cytotoxicity of unsorted CD 19 CAR T cells and lymphoma cells was assessed using a flow cytometry-based assay. Effector cells were CD 19 CAR T cells of three different constructs—full-length, mid-length, and short-length, as Figure 1 As shown. NT was used as negative control T cells. All three constructs of CAR T cells showed significantly increased cytotoxicity against all B cell lines compared to NT (35-50% vs. <10%). Different target cells showed different responses to CD19 CAR T cells. Compared with NT ( Figure 5 A) All constructs expressed high levels of killing of Raji cells, while the short-spacer CAR showed RS4 ( Figure 5 B) and Sup-B15( Figure 5 C) The highest cytotoxicity level of the cell line. In contrast, NT and all CAR T cell constructs showed the highest cytotoxicity against Jurkat ( Figure 5 D) and PHA embryonic cells ( Figure 5E) There was no significant difference in the tumor killing ability, indicating that CAR T cells are CD19 specific.

[0072] In recent years, several clinical trials have shown impressive results in CD19 CAR T cell therapy for patients with B-cell malignancies. 17-20 Nevertheless, the development of CAR design is an ongoing process to optimize clinical efficacy and minimize the toxicity of this treatment in the clinic. Different co-stimulatory molecules such as CD28, 4-IBB, and OX40 have been used to test the efficacy of CAR T cells against tumor cells. Other structural units, such as the CH2CH3 spacer domain, have been reported to play a role in the persistence and survival of CAR T cells in vivo. 2 Even if different constructs differ significantly in the costimulatory domain, such changes may contribute to the observed differences in overall therapeutic efficacy in patients with various B-cell malignancies, as well as differences in the ability of CAR T constructs to elicit varying degrees of clinical adverse events.

[0073] Several researchers 7,21-25 It has been shown that the length of the spacer is a key issue for the efficiency of CAR T cells. In this study, the disclosure studies the second generation anti-CD 19 CAR T cells comprising three different lengths of spacers; full length (CH2CH3 interval), "middle" (- / CH3 interval) and "short" (- / - interval). The disclosure expresses all three different lengths of spacer CAR constructs on T cells, wherein the middle length shows the highest transduction efficiency.

[0074] All three CAR constructs had comparable levels of anti-tumor efficacy and CD 19-specific activity. We evaluated the in vitro killing activity of T cells expressing each construct against three different target cell lines with positive CD19 expression (Raji, RS4, and Sup-B15) and one CD 19-negative cell line (Jurkat) as a control. The present disclosure found that CD 19 CAR T cells with complete, intermediate, or short spacers did not have a higher percentage of specific Jurkat cell lysis compared to NT, while all three types of CD 19 CAR T cells had a higher percentage of specific lysis of Raji, RS4, and Sup-B15 cells compared to the killing effect of NT, with an E:T ratio of 10:1. At an E:T ratio of 20:1, the difference in killing effects of complete, intermediate, and short CD19 CAR T cells can be clearly observed. Therefore, modified T cells with any of the three constructs exhibit CD 19-specific activity. Our results are consistent with Similar results were found previously reported by et al. 4This suggests that CD19CAR T cells expressing CAR constructs with or without a CH2CH3 spacer have specific activity in vitro but lack anti-leukemic activity in vivo, with some of the CD19-independent toxicity being related to the IgG1-derived CH2CH3 spacer. In the absence of the CH2 domain, the CAR construct eliminated leukemia in vivo without overt toxicity.

[0075] Interestingly, although the killing effects of all three CAR constructs were comparable, short CD 19 CAR T cells showed the highest killing efficiency against all target cells expressing CD 19, despite their lower transduction efficiency than intermediate CD 19 CAR T cells. For Raji cells, both full CD 19 CAR T cells and intermediate CD 19 CAR T cells showed similar partial specific lysis as short CD 19 CAR T cells. However, their killing efficiency against RS4 and Sup-B 15 cells, which express relatively less CD19 on their cell surface, was lower than their killing efficiency against Raji cells. The difference in CAR T cell killing efficacy may be the effect of the presence of target antigens and the intensity of antigen expression. 26 However, modified T cells expressing any of the three constructs were more efficient in eradicating CD19-expressing cells compared to NT. We also observed that modified CAR T cells expressing any of the three constructs did not kill allogeneic PHA naive cells. Therefore, the disclosed CAR-expressing T cells appear to be specific for cells expressing CD19 and have minimal killing effects on CD19-negative allogeneic cells.

[0076] CRS after CAR T cell infusion is one of the most serious toxicities. 27-29 Associated with progressive immune activation. 26, 30 Ravanpay et al. have designed second-generation CAR constructs with three spacing variants: full, intermediate, and short. They report that all three spacing variants had equivalent tumor lytic activity. Short-spacing CAR T cells induced the most potent effector cytokine production—interleukin-2, interferon-γ, and tumor necrosis factor-α levels, 31 However, fully compartmentalized CAR T cells lack in vivo efficiency due to FcγR binding to mouse macrophages. 4,22 Therefore, the present disclosure selects intermediate CD19 CAR T cells to avoid CRS.

[0077] Example 9

[0078] An 11-year-old boy with B-cell ALL had a bone marrow relapse prior to participation in a compassionate clinical CAR CD19 T-cell program. He was diagnosed with B-cell ALL 8 years earlier (initial white blood cell count of 1.3x10 6 cells / μL). His cytogenetic study showed 46,XY. He was treated with the standard-risk ThaiPOG regimen. 13 Two years later, he had a bone marrow relapse and he received a relapsed ALL regimen (modified SJCRH relapsed ALL regimen). 14 One month after he completed the modified SJCRH regimen, he experienced a second bone marrow relapse and subsequently received the relapsed ALL regimen following the UK ALL regimen. 15 After he achieved CR, he received a haploidentical HSCT from his mother. One year later, he had a central nervous system (CNS) relapse with an intracranial mass. He was treated with a bortezomib-based regimen. 16 A new CR was achieved and he also received intrathecal chemotherapy and craniospinal irradiation. Six months after completing treatment for CNS involvement, he had another systemic bone marrow relapse with a 30% blast rate. Re-induction chemotherapy consisted of a combination of bortezomib, cytarabine, and etoposide. 16 Under a compassionate use investigational new drug (IND) grant, he obtained a 0.52x10 6 cells / kg body weight (0.8x10 6 The dose of 100 total T cells / kg; 65% transfection efficiency (intermediate length interval construct) was fixed with haploid CD19 CART cells. The only side effect after CAR T cell treatment was a low-grade fever, but there were no clinical symptoms of graft-versus-host disease or severe CRS, so it was decided to just observe and closely follow the patient until his low-grade fever subsided. On day 30 after CAR T cell infusion, he was recorded as having achieved an MRD-negative CR, which has lasted for more than 10 months as shown in Table 1.

[0079] Sample days %CD19 % leukemic blasts %Normal B cells Relapse (11 days) 32 27 5 0 4.1 0.18 4.5 7 0.5 0.5 0 15 0 0 0 30 0.05 0 0.05 154 1 0 1 188 3 0 3

[0080] Note: Day 0 = day of CD19CAR19 T-cell infusion

[0081] Table 1

[0082] Regarding a patient with B-cell ALL, the inventors of the present disclosure gave him a cycle of chemotherapy before CAR CD 19 T cell therapy to reduce leukemic blasts and induce an immunosuppressive state, favoring haploid CAR T cell infusion. He then received haploid CD 19 CAR T cells under a compassionate use request. The present disclosure demonstrates the induction of CR and a sustained, unmaintained remission of refractory leukemia that remained MRD negative for 10+ months. At least 2 weeks after CD19 CAR T cell infusion, genetically engineered CD19 CAR T cells were detected in peripheral blood and bone marrow samples by RT-PCR ( Figure 6 With these favorable findings, the present disclosure plans to study allogeneic CD19 CAR T cells in patients with relapsed / refractory B-cell ALL as a bridge therapy to allo-HSCT in a phase I-II setting, followed by a post-transplant CAR T cell boost as a consolidation therapy for these high-risk patients, as the present disclosure is concerned about the possible deleterious effects of pre-transplant conditioning chemotherapy on the CAR T population administered as salvage therapy prior to HSCT.

[0083] It should be understood that the present invention can be embodied in other specific forms and is not limited to the sole embodiment described above. However, modifications and equivalents of the disclosed concepts such as those readily apparent to those skilled in the art are intended to be included within the scope of the appended claims.

[0084] References

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[0115] 31. Ravanpay AC, Gust J, Johnson AJ, et al. EGFR806-CAR T cells selectively target a tumor-restricted EGFR epitope in glioblastoma. Onco-target. 2019; 10(66):7080-7095. Sequence Listing <110> Mahidol University <120> Chimeric antigen receptor and modified cells carrying the same <130> PC06222 <150> TH 2001002539 <151> 2020-05-08 <160> 11 <170> SIPOSequenceListing 1.0 <210> 1 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> Synthetic consrtuct <400> 1 Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser 1 5 10 15 Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser 20 25 30 Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu 35 40 45 Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu 65 70 75 80 Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu 85 90 95 Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Ala Pro 130 135 140 Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro 145 150 155 160 Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu 165 170 175 Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala 180 185 190 Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val 195 200 205 Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr 210 215 220 Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 245 250 <210> 2 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 2 Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser 1 5 10 15 Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser 20 25 30 Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu 35 40 45 Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu 65 70 75 80 Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu 85 90 95 Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg 100 105 110 <210> 3 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 3 Leu Gln Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 1 5 10 15 Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 20 25 30 Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 35 40 45 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 50 55 60 Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 65 70 75 80 Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 85 90 95 Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 4 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Glu Val Gln 20 <210> 5 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 5 Ser Arg Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser 1 5 10 15 Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser 20 25 30 Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu 35 40 45 Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu 65 70 75 80 Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu 85 90 95 Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Ala Pro 130 135 140 Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro 145 150 155 160 Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu 165 170 175 Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala 180 185 190 Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val 195 200 205 Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr 210 215 220 Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 245 250 <210> 6 <211> 27 <212> PRT <213> Homo sapiens <400> 6 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 7 <211> 41 <212> PRT <213> Homo sapiens <400> 7 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 8 <211> 112 <212> PRT <213> Homo sapiens <400> 8 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 9 <211> 19 <212> PRT <213> Homo sapiens <400> 9 Put Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys <210> 10 <211> 1754 <212> DNA <213> Artificial Sequence <220> <223> synthetic construct <400> 10 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgctct 60 agagacatcc agatgacaca gactacatcc tccctgtctg cctctggg agacagagtc 120 accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180 ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240 tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300 caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360 ggggggacca agctggagct gaaacgtggt ggtggtggtt ctggtggtgg tggttctggc 420 ggcggcggct ccggtggtgg tggctccgag gtgcagctgc agcagtctgg acctggcctg 480 gtggcgccct cacagagcct gtccgtcaca tgcactgtct caggggtctc attacccgac 540 tatggtgtaa gctggattcg ccagcctcca cgaaagggtc tggagtggct gggagtaata 600 tggggtagtg aaaccacata ctataattca gctctcaaat ccagactgac catcatcaag 660 gacaactcca agagccaagt tttcttaaaa atgaacagtc tgcaaactga tgacacagcc 720 attactact gtgccaaaca ttattactac ggtggtagct atgctatgga ctactggggc 780 caagggacca cggtcaccgt ctcctcgtac gtcaccgtct cttcacagga tccggagcgc 840 aaatgttgtg tcgagtgcccc accgtgccca gggcagcccc gagaaccaca ggtgtacacc 900 ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 960 ggcttctacc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacac 1020 tacaagacca cacctcccat gctggactcc gacggctcct tcttcctcta cagcaagctc 1080 accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1140 gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa aaaaaagctt 1200 aaatttggg tgctggtggt ggttggtgga gtcctggctt gctatagctt gctagtaaca 1260 gtggccttta ttattttctg ggtgaggagt aagaggagca ggctcctgca cagtgactac 1320 atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 1380 ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 1440 gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1500 tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1560 aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1620 agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1680 ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1740 cgctaagcat gcaa 1754 <210> 11 <211> 581 <212> PRT <213> Artificial Sequence <220> <223> synthetic construct <400> 11 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Ser Arg 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 Leu Lys 115 120 125 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Gly Leu 145 150 155 160 Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val 165 170 175 Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys 180 185 190 Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr 195 200 205 Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys 210 215 220 Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala 225 230 235 240 Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met 245 250 255 Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Tyr Val Thr 260 265 270 Val Ser Ser Gln Gly Ser Glu Arg Lys Cys Cys Val Glu Cys Pro Pro 275 280 285 Cys Pro Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 290 295 300 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 305 310 315 320 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 325 330 335 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 340 345 350 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 355 360 365 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 370 375 380 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Lys Leu 385 390 395 400 Lys Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 405 410 415 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 420 425 430 Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 435 440 445 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 450 455 460 Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 465 470 475 480 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 485 490 495 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 500 505 510 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 515 520 525 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 530 535 540 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 545 550 555 560 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 565 570 575 Ala Leu Pro Pro Arg 580

Claims

1. A chimeric antigen receptor (CAR), comprising: An extracellular domain having an antigen binding domain to be placed on the surface of a modified T cell plasma membrane, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connected in an end-to-end manner by a linker; an intracellular domain for regulating activation of the modified T cell, the intracellular domain comprising a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from the CD3 ζ-chain; and A transmembrane domain, anchored on the plasma membrane of the modified T cell and between the extracellular domain and the intracellular domain, wherein the transmembrane domain is connected to the extracellular domain via a spacer; in, The extracellular domain is an amino acid sequence as shown in SEQ ID No. 1, and Wherein, the spacer region is based on IgG2 and is composed of a hinge region and a CH3 region.

2. The chimeric receptor according to claim 1, characterized in that The linker is an amino acid sequence as shown in SEQ ID No.

4.

3. The chimeric receptor according to claim 1, characterized in that The transmembrane domain is an amino acid sequence as shown in SEQ ID No.

6.

4. The chimeric receptor according to claim 1, characterized in that The CD28 signaling domain is an amino acid sequence as shown in SEQ ID No.

7.

5. The chimeric receptor according to claim 1, characterized in that The cytoplasmic signal transduction domain is an amino acid sequence as shown in SEQ ID No.

8.

6. The chimeric receptor according to claim 1, characterized in that It also comprises a leader sequence, which is pre-connected to the variable light chain region, and the leader sequence is an amino acid sequence as shown in SEQ ID No.

9.

7. The chimeric receptor according to claim 1, characterized in that The antigen binding domain is a single chain variable fragment.

8. A genetically engineered T cell capable of expressing a chimeric antigen receptor, the chimeric antigen receptor comprising: An extracellular domain having an antigen binding domain to be placed on the surface of a modified T cell plasma membrane, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connected in an end-to-end manner by a linker; an intracellular domain for regulating activation of the modified T cell, the intracellular domain comprising a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from the CD3 ζ-chain; and A transmembrane domain, anchored on the plasma membrane of the modified T cell and between the extracellular domain and the intracellular domain, wherein the transmembrane domain is connected to the extracellular domain via a spacer; in, The extracellular domain is an amino acid sequence as shown in SEQ ID No. 1, and Wherein, the spacer region is based on IgG2 and is composed of a hinge region and a CH3 region.

9. The engineered T cell according to claim 8, characterized in that The chimeric antigen receptor further comprises a leader sequence, which is pre-linked to the variable light chain region, and the leader sequence is an amino acid sequence as shown in SEQ ID No. 9.

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