CN110577604A - Chimeric antigen receptor T cell carrying GITR costimulatory signal targeting EGFR - Google Patents

Chimeric antigen receptor T cell carrying GITR costimulatory signal targeting EGFR Download PDF

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CN110577604A
CN110577604A CN201810582487.7A CN201810582487A CN110577604A CN 110577604 A CN110577604 A CN 110577604A CN 201810582487 A CN201810582487 A CN 201810582487A CN 110577604 A CN110577604 A CN 110577604A
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曹卫
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Lixi Biotechnology Shanghai Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The present invention relates to a chimeric antigen receptor fusion protein comprising, from N-terminus to C-terminus: (i) single chain variable fragment (scFv) comprising VH and VL, wherein the scFv has low affinity for human Epidermal Growth Factor Receptor (EGFR), dissociation constant (K)D) (ii) a transmembrane domain, (iii) a GITR costimulatory domain, and (iv) an activation domain, > 50 nm.

Description

Chimeric antigen receptor T cell carrying GITR costimulatory signal targeting EGFR
Technical Field
The invention relates to the technical field of tumor treatment, in particular to chimeric antigen receptor T cells carrying GITR co-stimulatory signals to target EGFR, which can effectively attack tumor cells over-expressing EGFR but not EGFR negative tumor cells.
Background
Immunotherapy is an emerging and very promising approach to the treatment of cancer. T cells or T lymphocytes are effective weapons of the immune system, they are able to continuously search for foreign antigens and from normal cells or abnormal cells, such as cancer or infected cells. Genetically modified chimeric antigen receptor T cells (CAR-T) are a common approach to designing tumor-specific T cells. Delivery of CAR-T cells targeted to Tumor Associated Antigens (TAAs) into patients (known as adoptive cell transfer or ACT) represents an effective immunotherapeutic approach. An advantage of CAR-T technology over chemotherapy or antibody technology is that reprogrammed T cells can proliferate and persist in a patient ("live drug").
CARs (chimeric antigen receptors) are typically single chain variable fragments (scFv) derived from one monoclonal antibody linked by hinges and transmembrane domains to a variable number of intracellular signaling domains: (i) a single cell activated CD 3-zeta domain; (ii) CD28, CD137(4-1BB) or other co-stimulatory domains (CD 27 signaling domain can also be used in place of CD28 or CD137 domain) linked to the CD 3-zeta domain. The development of CARs ranges from first generation (without co-stimulatory domains) to second generation (with one co-stimulatory domain) to third generation CARs (with multiple co-stimulatory domains). The resulting CAR with multiple co-stimulatory domains (so-called third generation CAR) will produce increased cytolytic activity and significantly improve the persistence of CAR-T cells, exhibiting enhanced anti-tumor activity.
However, the current studies on chimeric antigen receptors have many defects, and have the problems of high recurrence rate, low safety and the like. Therefore, the development of chimeric antigen receptors with good specificity, stable therapeutic effect and less side effects is urgently needed in the field.
Disclosure of Invention
The invention aims to provide a chimeric antigen receptor T cell carrying a GITR co-stimulatory signal targeting EGFR.
in a first aspect of the invention, there is provided a chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus:
(i) Comprising VHAnd VLThe single chain variable fragment (scFv) of (1), wherein the scFv has low affinity for human Epidermal Growth Factor Receptor (EGFR), dissociation constant (K)D)＞50nM,
(ii) (ii) a transmembrane domain which is capable of,
(iii) A GITR co-stimulatory domain, and
(iv) An activation domain.
In another preferred embodiment, the scFv is derived from the C10 antibody and has the amino acid sequence of SEQ ID NO: 6, or at least 90% sequence identity thereto.
in another preferred embodiment, the scFv is derived from the P3-5 antibody and has at least 90% sequence identity thereto.
In another preferred embodiment, the activation domain is CD3 ζ.
In another preferred embodiment, the CAR has the structure of formula I:
L-scFv-H-TM-C-CD3ζ (I)
In the formula (I), the compound is shown in the specification,
each "-" is independently a linker peptide or a peptide bond;
L is an optional signal peptide sequence;
scFv is a peptide with low affinity for human Epidermal Growth Factor Receptor (EGFR), dissociation constant (K)D) Single chain antibodies > 50 nM;
H is an optional hinge region;
TM is a transmembrane domain;
C is a GITR costimulatory domain;
CD3 ζ is the cytoplasmic signaling sequence derived from CD3 ζ.
In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD8, GM-CSF, CD4, CD137, or a combination thereof.
In another preferred embodiment, said H is a hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, or a combination thereof.
in another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.
In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor fusion protein is shown in SEQ ID No. 2.
In a second aspect of the invention, there is provided a nucleic acid molecule encoding a fusion protein according to the first aspect of the invention.
In another preferred embodiment, the nucleic acid sequence is as set forth in SEQ ID NO: 1, or at least 90% identity thereof in each fragment.
in another aspect of the invention, there is provided a nucleic acid sequence of EGFR ScFv-CD8 hinge-CD 28 transmembrane domain-GITR domain-CD 3 ζ, the nucleic acid sequence being as set forth in SEQ ID NO: 1, or at least 90% identity thereof in each fragment.
In another aspect of the invention there is provided a protein encoded by a nucleic acid sequence according to the second aspect of the invention.
In a third aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.
In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.
In another preferred embodiment, the vector is a lentiviral vector.
In another preferred embodiment, the nucleic acid molecule is located in the Xba I and EcoR I lentiviral sites of a lentiviral vector.
In a fourth aspect of the invention, there is provided a host cell comprising a vector or chromosome of the third aspect of the invention into which has been integrated an exogenous nucleic acid molecule of the second aspect of the invention or which expresses a fusion protein of the first aspect of the invention.
In another preferred embodiment, the cell is an isolated cell, and/or the cell is a genetically engineered cell.
In another preferred embodiment, the cell is a mammalian cell.
In another preferred embodiment, the cell is a T cell.
in a fifth aspect of the invention, there is provided a method of preparing an engineered immune cell expressing a fusion protein according to the first aspect of the invention, comprising the steps of: transferring the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention into a T cell or NK cell, thereby obtaining the engineered immune cell.
In another preferred embodiment, the method further comprises the step of performing functional and effective detection on the obtained engineered immune cells.
In a sixth aspect of the invention, there is provided a formulation comprising a fusion protein according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a cell according to the fourth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.
In another preferred embodiment, the formulation is a liquid formulation.
In another preferred embodiment, the formulation is in the form of an injection.
in another preferred embodiment, the CAR-T cells are present in the formulation at a concentration of 1X103-1×108Individual cells/ml, preferably 1X104-1×107Individual cells/ml.
In a seventh aspect of the present invention, there is provided a fusion protein according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, or a cell according to the fourth aspect of the present invention for use in the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumor.
In another preferred embodiment, the tumor is an EGFR-positive tumor, preferably an EGFR-high expressing tumor.
In another preferred embodiment, the tumor is selected from the group consisting of: a hematologic tumor, a solid tumor, or a combination thereof.
In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute Myeloid Leukemia (AML), Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.
In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or a combination thereof.
In another preferred embodiment, the tumor is selected from the group consisting of: breast cancer, ovarian cancer, pancreatic cancer, brain cancer, glioblastoma, or a combination thereof.
In an eighth aspect of the invention, there is provided a kit for preparing a cell according to the fourth aspect of the invention, the kit comprising a container, and a nucleic acid molecule according to the second aspect of the invention, or a vector according to the third aspect of the invention, in the container.
In a ninth aspect of the invention there is provided a cell according to the fourth aspect of the invention, or a formulation according to the sixth aspect of the invention, for use in the prevention and/or treatment of cancer or a tumour.
In a tenth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a cell according to the fourth aspect of the invention, or a formulation according to the sixth aspect of the invention.
In another preferred embodiment, the disease is cancer or a tumor.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the structure of the CAR. Where the left is a first generation CAR (without co-stimulatory domains), the middle is a second generation CAR (one co-stimulatory domain CD28 or 4-BB), and the right is a third generation CAR (two or more co-stimulatory domains).
Fig. 2 shows the structure of EGFR and EGFRvIII proteins. Wherein EGFR comprises extracellular, transmembrane and intracellular domains. The tyrosine phosphorylation sites are marked in the tyrosine kinase and the carboxy-terminal domain (Y845-Y1175). EGFRvIII is formed by deletion of amino acids 5-273 and introduction of glycine at the junction of amino acids 5 and 274.
Figure 3 shows the structure of the EGFR CAR construct. Using the second generation CAR-T construct, the co-stimulatory domain was CD28,4-1BB or GITR.
FIG. 4 shows the in vitro expansion of EGFR-GITR-CD3zeta CAR-T cells.
Figure 5 shows FAB staining demonstrating expression of EGFR scFv in transduced T cells. Fab staining with anti-human Fab antibody was higher in EGFR-CAR-T cells than in non-transduced T cells.
FIG. 6 shows that EGFR-GITR-CD3zeta CAR-T cells are highly cytotoxic to EGFR positive cells and non-toxic to EGFR negative cancer cells. FIGS. 6A-6D show cytotoxicity of EGFR-CAR with different co-activation domains in different cancer cell lines with high expression of EGFR. FIG. 6E shows that no cytotoxicity of EGFR-CAR was observed in EGFR-1 negative MCF-7 cells. Wherein, E: t represents the ratio of effector to target cells.
Detailed Description
The present inventors have extensively and intensively studied and, for the first time, have unexpectedly found a chimeric antigen receptor fusion protein comprising, from N-terminus to C-terminus: (i) single chain variable fragment (scFv) comprising VH and VL, wherein the scFv has low affinity for human Epidermal Growth Factor Receptor (EGFR), dissociation constant (K)D) (ii) a transmembrane domain, (iii) a GITR costimulatory domain, and (iv) an activation domain, > 50 nm.
term(s) for
In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
the term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal cord or other parenteral routes of administration, e.g., by injection or infusion.
The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.
As used herein, a "Chimeric Antigen Receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric Antigen Receptors (CARs)" are also referred to as "chimeric receptors", "T-bodies" or "Chimeric Immunoreceptors (CIRs)". The term "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding an antigen. "intracellular domain" refers to any oligopeptide or polypeptide known to be a domain that transmits signals to activate or inhibit biological processes in a cell.
As used herein, "domain" or "domain" refers to a region of a polypeptide that is independent of other regions and folds into a specific structure.
as used herein, "single chain variable fragment (scFv)" refers to a single chain polypeptide derived from an antibody that retains the ability to bind antigen. Examples of scfvs include antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via a spacer sequence. Various methods for making scFv are well known to those skilled in the art.
As used herein, "tumor antigen" refers to an antigenic biomolecule, the expression of which results in cancer.
EGFR
EGFR is an epidermal growth factor receptor and is overexpressed in many types of cancers. EGFR is one of the four receptor tyrosine kinases of the Erb family, which includes ErbB2/HER-2, ErbB3/HER3 and ErbB4/HER 4. EGFR plays an important role in proliferation, growth regulation, angiogenesis, survival and metastasis.
The structure of EGFR is as follows: including the extracellular domain, the transmembrane domain, and the intracellular domain containing the tyrosine kinase domain and the carboxy-terminal tail (as shown in fig. 2). Many types of cancers, such as glioblastoma and others, have deletions of amino acids 5-273, resulting in the expression of EGFRvIII types. EGFR wild type and EGFRvIII type, both of which are critical for tumor survival signaling.
EGFR is involved in the regulation of MAP kinase, PJ3K, AKT, STAT signaling pathways. Recently, researchers have developed a variety of tyrosine kinase inhibitors, and the field of immunotherapy approaches has also developed 10 approaches to targeting EGFR signaling. CAR-T cells are useful in clinical trials against hematologic cancer targets.
recently, CAR-T cells are used for solid tumors against mesothelin, EGFR, Her-2 or other targets. Affinity-modulated Her-2 and EGFR show high specificity for cancer cells compared to normal cells, providing greater safety for CAR-T therapy.
The present invention provides CAR-T cells that target the EGFR tumor antigen, EGFR being highly overexpressed in many types of cancer, such as breast, pancreatic and glioblastoma. The present inventors made use of (i) an EGFR antibody that specifically recognizes EGFR-positive cancer cells with low affinity to make scFv, and (ii) GITR as a co-activation domain, to generate EGFR-GITR-CAR-T cells. The EGFR-GITR-CD3-CAR-T cells of the present invention have high cytotoxicity against a variety of EGFR-highly expressed cancer cell lines and are non-toxic in EGFR-negative cells.
GITR
GITR (glucocorticoid-induced TNFR family-related gene, C0357) domain belongs to the TNFR superfamily (TNFRSF) and is a costimulatory domain that provides T cell activation. The inventors have found the following advantages of CAR-T therapy using GITR. The GITR-GITRL interaction may be effective in mediating an anti-tumor immune response by promoting the expansion and activation of effector T cell populations and by suppressing T reg (T regulatory) cells, which may suppress immune activity. The dual function of GITR to activate effector cells and inhibit Treg cell suppression makes it effective for CAR-T immunotherapy.
Chimeric antigen receptors
The present invention relates to a chimeric antigen receptor fusion protein comprising, from N-terminus to C-terminus: (i) single chain variable fragment (scFv) comprising VH and VL, wherein the scFv has low affinity for human Epidermal Growth Factor Receptor (EGFR), dissociation constant (K)D) (ii) a transmembrane domain, (iii) a GITR costimulatory domain, and (iv) an activation domain, > 50 nm.
The scFv useful in the present invention have low affinity for human EFGR, i.e., its dissociation constant (K)D)>50nM, or>80nM, or>100nM, or>150nM, preferably ≧ 200nM or 250 nM. In one embodiment, the scFv is derived from C10(Liu, X.; Jiang, S.; Fang, C.; Yang, S.; Olalere, D.; Pelignot, E.C.; Cogdill, A.P.; Li, N.; RAMONs, M.; Granda, B., el. Affinity-tuned erb 2 or an egfrric anti-inflammatory receptor t cells expressed in a secreted thermal index estimated from cells) or P3-5 (Zhuou, Y.; Drummond, D.C.; Zou, H.; Hayes, M.E. E.; G.P., Kicoding. D.C.; M.P., B.371. expressing P.; Marq. expressing cells J.371. injection J.2007, n. expression of molecular expression). And has at least 90% homology to C10 or P3-5. Preferably, the scFv is at least 92%, 9S%, 98% or 99% homologous to C10 or P3-5. The CAR-T cells of the invention comprise scfvs with low affinity for EGFR, which exhibit potent anti-tumor efficacy similar to high affinity antibody cells, but do not act on normal cells expressing physiological EGFR levels, thus increasing the therapeutic index.
The invention discloses a construction method of an EGFR-GITR-CD3zeta lentivirus vector and an EGFR-GITR-CAR-T cell; the EGFR-GITR-CAR-T cells can kill EGFR-positive cancer cells but not EGFR-negative cancer cells.
The present invention utilizes low affinity EGFR antibodies and GITR to make EFGR-GITR CAR-T cell constructs. The EGFR scFv (e.g., from low affinity antibodies C10 or P3-5) were cloned into the Xba I and EcoR I sites of the lentiviral vectors.
The CAR construct of the invention (figure 2) contains the CD8 signal peptide, EGFR scFv: VH (heavy chain variable region) -linker 3x (GGGGS, SEQ ID No.:10) -VL (light chain variable region), CD8 hinge region, CD28 transmembrane domain, GITR and CD3zeta activation domain from low affinity EGFR antibodies.
The present inventors prepared EGFR-ScFv-GITR-CD3 ζ -CAR-T cells against EGFR-positive cancer cell lines, such as breast, ovarian, pancreatic, brain, and the like. The present inventors provide data demonstrating that cultured EGFR-GITR CAR-T cells can be efficiently expanded.
EGFR-GITR-CD3zeta CAR-T can target EGFR-positive and EGFR-vIII-positive cancer cells.
EGFR-GITR-CD3zeta CAR-T can be used in combination with different chemotherapeutics (checkpoint inhibitors), targeted therapies, small molecule inhibitors and antibodies.
EGFR-GITR-CD3zeta CAR-T cells can be produced for clinical use. The CARs of the invention have advantages over EGFR-CARs with other costimulatory domains.
The Tag-conjugated EGFR scFv can be used for CAR production.
For the same EGFR-scFv within a CAR, a third generation CAR-T or other co-activation signaling domain can be used.
combinations of EGFR-GITR-CAR-T with CAR-T or bis-scFv CAR targeting other tumor antigens or the tumor microenvironment (VEGFR-I-3) can be used to enhance the activity of EGFR-GITR-CAR monotherapy.
Nucleic acid molecules
The invention provides a nucleic acid encoding the CAR described above. Nucleic acids encoding a CAR can be readily prepared by conventional methods, using the amino acid sequence of a particular CAR. The nucleotide sequence encoding the amino acid sequence can be obtained using the NCBI RefSeq ID or GenBenk accession number of the amino acid sequence of each of the aforementioned domains. The nucleic acids of the invention can be prepared using standard molecular biology and/or chemistry methods. For example, a nucleic acid can be synthesized based on a base sequence, and the nucleic acid of the present invention can be prepared by fusing DNA fragments obtained from a cDNA library using Polymerase Chain Reaction (PCR).
The nucleic acid encoding the CAR of the invention can be inserted into a vector, and the vector can be introduced into a cell. For example, viral vectors such as retroviral vectors (including oncogenic retroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian viral vectors, vaccinia viral vectors or sendai viral vectors, epstein-barr virus (EBV) vectors, and HSV vectors can be used. As the viral vector, it is preferable to use a viral vector lacking the replication ability so as not to self-replicate in the infected cell. As the viral vector, it is preferable to use a viral vector lacking the replication ability so as not to self-replicate in the infected cell.
for example, when a retroviral vector is used, the method of the present invention may be carried out by selecting an appropriate packaging cell based on the LTR sequence and packaging signal sequence possessed by the vector, and preparing a retroviral particle using the packaging cell. Examples of packaging cells include PG13(ATCC CRL-10686), PA317(ATCC CRL-9078), GP + E-86, GP + envAm-12 and Psi-Crip. Also 293 cells or 293T cells with high transfection efficiency can be used for the preparation of retroviral particles. A variety of retroviral vectors based on the production of retroviruses and packaging cells are widely available from a number of companies.
The cells of the invention bind to the specific antigen via the CAR, thereby transmitting a signal into the cell, which in turn activates the cell. Activation of the CAR-expressing cell may vary depending on the kind of host cell and the intracellular domain of the CAR, and may be based on, for example, release of cytokines, increase in cell proliferation rate, change in cell surface molecules, and the like as indicators. For example, cytotoxic factors (tumor necrosis factor, lymphotoxin, etc.) are released from activated cells, thereby destroying target cells that express the antigen. In addition, cytokine release or changes in cell surface molecules can also activate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages. The CAR-expressing cells can be used as a therapeutic for a disease. The therapeutic agent comprises CAR-expressing cells as an active ingredient, and may also comprise a suitable excipient. Examples of the excipient include the above-mentioned pharmaceutically acceptable excipients (excipients for a composition comprising the nucleic acid of the present invention as an active ingredient), various cell culture media, and isotonic sodium chloride.
Carrier
Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.
The present invention also provides a vector into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.
In brief summary, an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is 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 expression constructs of the invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.
The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
Further, 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).
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.
Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp apart, and activity begins to decline. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to initiate transcription.
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 Epstein-Barr (Epstein-Barr) 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, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. 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 when such expression is desired, or turning off expression when expression is not desired. 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. Typically, the reporter gene is the following: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. 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, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.
Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell by any method known in the art, e.g., 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. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring harbor Laboratory, New York). A preferred method for introducing the polynucleotide into a host cell is calcium phosphate transfection.
Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, 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. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.
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. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).
In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use to introduce nucleic acids into host cells (ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained as a suspension in the lipid, contained in or complexed with a micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in bilayer structures, either as micelles or with a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
In a preferred embodiment of the invention, the vector is a lentiviral vector.
Preparation
The invention provides a composition comprising a CAR-T cell of the first aspect of the invention, and a pharmaceutically acceptable carrierA carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X103-1×108Individual cells/ml, more preferably 1X104-1×107individual cells/ml.
In one embodiment, the formulation 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 formulations of the present invention are preferably formulated for intravenous administration.
Therapeutic applications
The invention includes therapeutic applications of cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding expression cassettes of the invention. The transduced T cells can target the marker EGFR of the tumor cells, and synergistically activate the T cells to cause T cell immune response, so that the killing efficiency of the T cells on the tumor cells is obviously improved.
Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering to the mammal the CAR-T cells of the invention.
In one embodiment, the invention includes a class of cell therapy in which autologous T cells (or allogeneic donors) from a patient are isolated, activated, genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by T cells in an MHC-unrestricted manner. Furthermore, one CAR-T can treat all cancers expressing this antigen. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.
In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, anti-EGFR CAR-T cells elicit a specific immune response against EGFR-expressing cells.
Although the data disclosed herein specifically disclose lentiviral vectors comprising an anti-EGFR scFv, hinge and transmembrane regions, and 4-1BB and CD3zeta signaling domains, the invention should be construed to include any number of variations on each of the construct components.
treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.
Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.
preferred tumor types are EGFR-positive tumors, preferably EGFR-high expressing tumors, such as breast cancer, ovarian cancer, pancreatic cancer, brain cancer, glioblastoma, or combinations thereof.
The CAR-modified T cells of the invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.
For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) expanding the cell, ii) introducing a nucleic acid encoding the CAR into the cell, and/or iii) cryopreserving the cell.
Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic (syngeneic), or xenogeneic with respect to the recipient.
In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.
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 IL-2, IL-17 or other cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise a target cell population 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 compositions of the present invention are preferably formulated for intravenous administration.
The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.
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 106Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. 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 dosages and treatment regimens for a particular patient can be readily determined by those 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 (i.v.) 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 i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.
in certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or efavirenz therapy for psoriasis patients or other therapy for PML patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery.
The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X10 may be administered per treatment or per course of treatment61 to 1010A subject modified T cell (e.g., CAR-T20 cell) is administered to a patient, for example, by intravenous infusion.
The main advantages of the invention include:
(a) the CAR-T cells are prepared by using the EGFR antibody with low affinity, and only have high cytotoxicity on EGFR positive cancer cells, and have no cytotoxicity on EGFR negative cancer cells and normal cells which slightly express the EGFR;
(b) According to the CAR disclosed by the invention, GITR is used as a co-stimulation domain, and the EFGR-GITR CAR-T cell is prepared, so that the cytotoxicity of EGFR positive cancer cells is improved.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
In general, the inventors cloned the EGFR CAR construct into the Xba I and EcoR I sites of a lentiviral vector, thereby generating the EGFR CAR construct within the lentiviral vector. The pCD510-FMC63-28z lentiviral CAR construct contained the CDS signal peptide-EGFRscFv-CD 8 hinge region-CD 28 transmembrane domain-GITR domain-CD 3zeta between XbaI and EcoRI cloning sites.
Lentivirus was produced using 293T cells and titers were established by RT-PCR. Subsequently, T cells were transduced with the same dose of lentivirus as described in the examples. The invention uses CAR lentiviruses to transduce T cells, and also utilizes an untransduced T cell control to detect toxicity of CAR-T cells to EGFR-positive cell lines.
Example 1 production of CAR lentivirus
Lentiviruses were prepared by the following steps:
Day 1:
1. Will be 5X 106HEK293FT cells were seeded into 100mm diameter petri dishes;
Day 2:
2. Check to ensure 70% -90% cell fusion;
3. The transfection complexes were prepared for each 100mm diameter dish as follows:
a. In 1.5ml tube a: mu.g CAR (chimeric antigen receptor) DNA plasmid (plasmid) and 20. mu.L lentivirus packaging combination (ALSTEM, catalog # VP 100; see Appendix B3) were diluted into 0.5ml DMEM or Opti-MEM serum free medium and mixed gently;
b. In 1.5ml tube B: 30 μ L of Nanofect transfection reagent (ALSTEM, Cat. NF100) was diluted into 0.5ml DMEM or Opti-MEM serum-free medium and mixed gently;
c. Adding the NanoFect/DMEM in tube B to the DNA/DMEM solution (tube A), vortexing for 5-10 seconds, and incubating the DMEM-plasmid-NanoFect mixture at room temperature for 15 minutes;
4. Dropwise adding all the transfection complexes obtained in the step 3 onto the cell plate, and rotating the plate back and forth to uniformly disperse the transfection complexes on the plate;
humidification of 5% CO at 5.37 deg.C2Culturing the cells overnight in an incubator;
Day 3:
6. the supernatant of the above transfection complex was replaced with 10mL of fresh medium and supplemented with 20 μ L of ViralBoost (500X, alsem, catalog No. VB 100);
incubation at 7.37 ℃ for 24 hours;
Day 4:
8. collecting the culture solution supernatant containing the lentivirus into a 50mL sterile conical centrifuge tube with a cover, and placing the centrifuge tube on ice;
9. The supernatant was centrifuged at 4 ℃ for 15 minutes at 3000 rpm to pellet cell debris;
10. Filtering the clarified supernatant using a 0.45 μm low protein conjugate sterile filter;
11. The lentivirus concentration/titer was determined by quantitative RT-PCR, and the HEK293 virus concentration in the supernatant was determined using the Lenti-X qRT-PCR titration kit (Clontech; Cat. No. 631235) (removal of any possible residual plasmid DNA by DNaseI pretreatment);
12. the virus can be used for infection, purification, or be stored as a virus stock solution at-80 ℃ for later use, preferably stored separately in small portions to reduce the loss of virus titer caused by repeated freeze thawing.
example 2 Lentiviral packaging System
Description of the products
The product name is as follows: SuperLentiTM Lentivirus Packaging System
The specification is as follows:
For the production of lentiviral particles, three components are typically required: 1) a lentiviral vector containing a foreign gene of interest, 2) a packaging vector containing all necessary viral structural proteins, 3) an envelope vector expressing Vesicular Stomatitis Virus (VSV) glycoprotein (G). The third generation lentiviral packaging system provides the greatest biological safety, since the lentiviral Rev gene is provided as a separate vector from the other structural genes, further eliminating the possibility of reverse recombination of the vector into replication competent viral particles. The third generation lentiviral packaging mixture supports only a lentiviral expression vector for the chimeric 5' LTR, where the HIV promoter is replaced by CMV or RSV, thus making it independent of TAT.
The SuperLenti lentivirus packaging cocktail is an HIV-based, ready-to-use third generation lentivirus packaging system in which plasmids express elements required for lentivirus production, which allows the creation of HIV-1 based replication-null lentiviruses, delivering and expressing targeted foreign genes in dividing or non-dividing mammalian cells.
Catalog number for product: VP 100;
Specification: 200 mu L;
And (3) transportation: room temperature;
Storage and security: the product can be stored at-20 deg.C for 6 months. Should be dispensed in single use doses to avoid repeated freezing and thawing as much as possible. The small portions are stored in a refrigerator at-20 ℃ before use.
The using method comprises the following steps:
For 100mm dish lentiviral packaging, 2.5. mu.g of lentiviral expression vector was mixed with 20. mu.l of lentiviral packaging mixture.
For lentiviral packaging in 150mm dishes, 5. mu.g of lentiviral expression vector was mixed with 40. mu.L of lentiviral packaging mixture.
Quality control: each batch of lentivirus packaging mixture was tested by using human embryonic kidney 293 cells under transfection experimental conditions.
It is only used for research. Not used in diagnostic or therapeutic procedures.
Example 3 isolation of Peripheral Blood Mononuclear Cells (PBMC) from Whole blood
Whole blood (stanford university hospital blood center, stanford, ca) was collected from a single individual or multiple individuals (depending on the amount of blood needed) and placed in 10mL of hepain vacutainers (Becton Dickinson).
note: blood should be processed two hours after blood collection to ensure maximum cell production. Blood can be stored overnight at room temperature (indoors) for the next day of treatment; however, there is some loss in cell yield. Blood should not be stored in an empty tube at 4 ℃. Approximately 10ml of anticoagulated whole blood was mixed with sterile Phosphate Buffered Saline (PBS) in a 50ml conical centrifuge tube in a total volume of 20ml (PBS, pH7/4, Ca free2+/Mg2+). In a separate, sterile 50mL conical centrifuge tube, the pipette is moved into 15mL Ficoll-Paque PLUS (GE Healthcare, 17-1440-03). Very gently laminate 20ml of blood/PBS to the surface of the Ficoll and centrifuge the sample at 400Xg for 30-40min at room temperature without braking. The cell layer containing Peripheral Blood Mononuclear Cells (PBMCs) at the cut-off between diluted plasma and Ficoll was carefully aspirated to avoid inhalation of Ficoll. To ensure complete removal of Ficoll, platelet and plasma proteins, PBMCs were washed twice with a total volume of 40ml of PBS and centrifuged at 200xg for 10 min at room temperature. Cells were then counted using a hemocytometer. If the washed PBMCs are used immediately, they are washed once with CAR-T medium (AIM V-Albumax (BSA) (Life technologies) containing 5% AB serum and 1.25. mu.g/mL amphotericin B (Gemini Bioproducts, woodland, CA), 100U/mL penicillin and 100. mu.g/mL streptomycin). If the PBMC are to be frozen, the washed cells are resuspended in transfer insulated vials, kept at-80 ℃ for 24 hours, and then stored in liquid nitrogen.
Example 4 peripheral blood monocyte T cell activation
If freshly isolated PBMC are used, the isolated cells are washed (with 1xPBS (pH7.4) and Ca-free2+/Mg2+) Using CAR-T medium (AIM V-Albumax (BSA) (Life technologies, containing)5% AB serum and 1.25. mu.g/mL amphotericin B (Gemini Bioproducts, Wudland, CA), 100U/mL penicillin and 100. mu.g/mL streptomycin) were washed once, without human interleukin-2 (huIL-2) (Invitrogen) at a concentration of 5X 105Individual cells/mL. Washed once in CAR-T medium without huIL-2 and finally resuspended to a final concentration of 5X 10 with CAR-T medium with 300U/mL huIL2(1000 × stock; Invitrogen)5Individual cells/mL.
If frozen PBMC are used, they are cultured in 9mL of pre-warmed (37 ℃) DMEM medium (Life technologies) in the presence of 10% FBS, 100u/mL penicillin and 100. mu.g/mL streptomycin at 5X 105concentration of individual cells/mL, thawing and resuspending cells (1X 10)7cells/mL). Cells were centrifuged at 300Xg for 5 minutes, then washed once with CAR-T medium without huIL-2, and finally resuspended with 30U/mL huIL 2-containing CAR-T medium to a final concentration of 5X 105Individual cells/mL.
Prior to activation, the anti-human CD28 and CD3 antibody-conjugated magnetic beads (Invitrogen) were washed three times with 1mL sterile 1xPBS (pH7.4) (beads were isolated from solution using a magnetic rack), and then resuspended in CAR-T medium (300U.mLhuIL-2) to a final concentration of 2X 107beads/mL.
Then 25uL of magnetic beads were added to 1mL of PBMC, and PBMC and magnetic beads were mixed at a magnetic bead-to-cell ratio of 1: 1.
The desired number of aliquots were added to each well of a 12-well low-attachment or untreated cell culture plate and CO was added at 37 deg.C2Cultured in the presence for 24 hours, and then subjected to virus transduction.
Example 5T cell transduction and expansion
After PBMC activation, cells were incubated at 37 ℃ with 5% CO2and culturing for 24 hours.
The lentiviruses were thawed on ice. 1x10 per well6Adding 5X 10 cells6lentivirus and 2. mu.L/mL Transplus medium (Alstem, Richmond, Calif.) (final dilution 1: 500). The cells were cultured for an additional 24 hours before repeated addition of virus.
The cells were then cultured in fresh medium for 12-14 days in the continuous presence of 300U/ml IL-2 (total culture time depends on the final number of CAR-T cells required).
The cell concentration was analyzed every 2-3 days by adding medium to dilute the cell suspension to 1X106cells/mL.
Example 6 transduction validation-cell staining
Cells were washed and suspended in FACS buffer (phosphate buffered saline (PBS), buffer plus 0.1% sodium azide and 0.4% bovine serum albumin). The cells were then aliquoted into 1X106Small portions of (a).
Fc receptor was blocked with normal goat IgG (Life technologies), 100. mu.l of normal goat IgG at 1:1000 dilution was added to each tube, and incubated on ice for 10 min.
Add 1.0ml FACS buffer to each tube and spin pellet 300g for 5 min.
Adding biotin-labeled polyclonal goat anti-mouse F (ab)2 antibody (Life technologies) to detect CD24 scFv; biotin-labeled normal polyclonal goat IgG antibody (Life technologies) was added as isotype control. (1:200 dilution, reaction volume 100. mu.l).
Cells were incubated at 4 ℃ for 25 minutes and washed once with FACS buffer.
Cells were suspended in FACS buffer and blocked by adding 100 μ l of normal mouse IgG at 1:1000 dilution to each tube. Incubate in ice for 10 min. Cells were washed with FACS buffer and resuspended in 100 μ l FAC buffer.
cells were then stained with Phycoerythrin (PE) -labeled streptavidin (BD Pharmingen, San Diego, CA) and Allophycocyanin (APC) -labeled CD3(eBiocience, San Diego, CA). Add 1.0. mu.l PE and APC to tubes 2 and 3, respectively.
Cell collection was performed using a flow cytometer BD FacsCalibur (BD Biosciences) and analyzed with FlowJo software (Treestar, inc.
Example 7 cytotoxicity assay (real-time ACEA)
Cytotoxicity assays were performed using the ACEA instrument according to the manufacturer's protocol described below.
Real-time cell assay (RTCA) protocol
Day 1:
A. Preparation of (adherent) target cells:
1. Old medium was aspirated from the flask.
2. Add 5-10mL of conventional media (serum free) to the flask and rinse off the serum residue. Then, the medium was aspirated.
3. 0.5mL to 1mL of 0.05% trypsin-EDTA was added to the flask to detach the cells.
4. 4.5-9.5 mL of medium (FBS-containing) was added to the flask, and the medium was pipetted up and down to the bottom of the flask to disperse the cells into a single cell solution.
5. The suspended cell suspension was transferred to a 15mL tube.
6. 10 μ L of the cell suspension was removed and counted with a hemocytometer to determine the cell concentration.
7. The cell suspension was centrifuged at 1000rpm (or 300Xg) for 5 minutes at 25 ℃.
8. After centrifugation, the supernatant was aspirated, and the cell pellet was resuspended in FBS-containing medium until the concentration reached 1X105Individual cells/mL.
B. Preparing an RTCA plate:
1. Only 50uL of medium (the same as used for the cell suspension) was added to the wells to be tested.
2. the E-plate (ACEA bioscience, Inc, product number: JL-10-156010-1A) was returned to the bench.
3. Starting RTCA 2.0 software
a. Inputting layout information:
i. At a minimum, all wells are selected and then "apply" is clicked. This activates the aperture.
b. Inputting a plan:
i. Step 1-background measurement. And not changed.
Step 2 ═ monitoring of cells
Step 3 ═ short term cytotoxicity
Step 4 long term cytotoxicity (click "auto" box)
**The number of cycles per step and the interval between each cycle may be altered**
4. Click "step 1" and then start the naming experiment and measure the background of the medium.
After "step 1" was completed, the E-plate was removed from the table.
6. Add 100 μ L of cell suspension of part a to the wells (without removing the previous medium, eventually totaling 150uL in each well).
7. The cells were allowed to stand in a fume hood for 5 minutes.
8. the E-plate was returned to the bench.
9. Click on "step 2" and then resume recording as started.
Day 2-treatment:
A. Preparation of CAR-T effector cells:
1. All suspended CAR-T cells were removed from the 6-well plate and mixed well.
2. 10 μ L of the cell suspension was taken and the cell concentration was determined by a hemocytometer.
3. The cell suspension was centrifuged at 1000rpm (or 300Xg) for 5 minutes at 25 ℃.
4. The supernatant was aspirated and the cell pellet was resuspended in 2mL of cytotoxic buffer (RPMI 1640 phenol-free (Invitrogen) + 1% P/S (Invitrogen) + 5% human AB serum (Gemini Bioproducts; 100-318).
5. and (5) repeating the step (3).
6. The supernatant was aspirated and the cell pellet was resuspended in cytotoxic medium (phenol red-free RPMI1640(Invitrogen) plus 5% AB serum (Gemini Bioproducts; 100-318)) to obtain 1X106Final concentration of cells/mL.
B. Preparing an RTCA plate:
After part a is complete, proceed to step 3.
2. the E-plate was removed from the table.
3. The supernatant was carefully aspirated from each well.
4. to each well 100 μ L of cytotoxic medium was added.
5. And (5) repeating the step (3).
6. To each well 50 μ L of cytotoxic medium was added.
7. 100 μ l from part ACAR-T cell suspension (1X 10)5Individual cells) were dispensed into the desired wells of each design.
8. The E-plate is placed back on the table.
9. Click on "step 3" and then resume recording as started.
Example 8 sequence of EGFR-GITR-CD3 ζ CAR
The CAR structures of examples 8-11 are as follows: human CD8 signal peptide, human EGFR scFv (V) derived from a low affinity C10EGFR antibodyH-Linker-3x(GGGGS，SEQ ID NO.:10)-VL) A CD8 hinge region, a CD28 transmembrane region, a co-activation domain (GITR in examples 8 and 9, CD28 in example 10, or 4-1BB in example 11), a CD3 zeta-activation domain (FIG. 3). Dissociation constant K of C10EGFR antibody for A431 cellsDWas 265 nM.
The sequence of the lentiviral vector with CAR construct inserted between EcoR1 and XhoI sites is shown below. scFv was flanked by NheI and XhoI sites to allow possible recloning into other constructs.
Nucleotide sequence, SEQ ID NO: 1.
SEQ ID NO: 2 is a polypeptide corresponding to SEQ ID NO: 1 (translation sequence of SEQ ID NO: 1, EGFR-GITR-CD3zeta CAR)
The CAR construct construction scheme is shown below, showing SEQ ID NO: 1, or a sequence of each subdomain of seq id no.
1-78 of SEQ ID NO. 1 is the < huCD8 signal peptide >
1 < EGFR scFV > at positions 85-831, wherein the EGFR scFV is derived from C10, and the amino acid sequence is as follows (SEQ ID NO: 3):
SEQ ID NO. 1 < CD8> at position 838-984
SEQ ID NO. 1 < CD28TM > at position 985-1065
1 at position 1066-1239 < GITR >
SEQ ID NO. 1 < CD3zeta > at position 1240-1584
example 9 sequence of EGFR-CD28-CD3 ζ -CAR
the construct includes a human CD8 signal peptide, a human EGFR scFv (V) derived from P3-5H-Linker-3x(GGGGS，SEQ ID NO.:10)-VL) CD8 hinge region, CD28 transmembrane region, CD28 costimulatory domain, CD3zeta activating domain (fig. 3). K of P3-5 on A431 cellsDWas 88 nM.
The sequence of this construct was similar to that described in example 8, except that the human EGFR scFv was derived from P3-5.
The VH of the EGFR scFv derived from P3-5 is shown below (SEQ ID NO.:4), with the underlined amino acids being different from C10.
html was reverse translated to a sequence of 375 bases (SEQ ID No.:5) using www.bioinformatics.org/sms2/rev _ trans.
The VL of the EGFR scFv derived from P3-5 is shown below (SEQ ID NO.:6), with the underlined amino acids being different from C10.
The amino acid sequence was reverse translated into a sequence of 324 bases (SEQ ID No.:7) with the most likely codon.
Example 10 sequence of EGFR-CD28-CD3zeta-CAR
The construct includes human CD8 signal peptide, human EGFR scFv (V)H-Linker-3x(GGGGS，SEQ ID NO.:10)-VL) CD8 hinge region, CD28 transmembrane region, CD28 costimulatory domain, CD3zeta activating domain (fig. 3).
The sequence of this construct was similar to that described in example 8, except that the co-stimulatory domain was CD 28.
< CD28 coactivation domain sequence > as shown below (SEQ ID No.: 8):
Example 11 EGFR-4-1BB-CD3zeta CAR
The construct includes human CD8 signal peptide, human EGFR scFv (V)H-Linker-3x(GGGGS，SEQ ID NO.:10)-VL) A CD8 hinge region, a CD28 transmembrane region, a 4-1BB costimulatory domain, a CD3zeta activating domain (FIG. 3).
The sequence of this construct was similar to that described in example 8, except that the costimulatory domain was 4-1 BB.
<4-1BB co-stimulatory domain sequence > as shown below (SEQ ID No.: 9):
Example 12 EGFR-GITR-CD3zeta CAR-T cells demonstrate efficient expansion in culture
EGFR-GITR-CD3 ζ CAR cells were efficiently expanded in vitro (fig. 4). Within 20 days of culture, EGFR-CAR-T cells expanded more than 60-fold. After 16 days of culture, the growth of EGFR-4-1BB-CD3z-CAR-T cells was reduced. CD19-CD28-CD3zeta CAR-T, non-CD 19-CAR-T cells and untransduced T cells were also efficiently expanded in vitro.
example 13 transduction of T cells with EGFR-CAR Lentiviral constructs demonstrates expression of EGFR scFv
To detect transduction and expression of human scFv derived from C10EGFR antibody, CAR-T cells were stained with anti-human FAB antibody. Staining showed efficient transduction of lentiviral CAR. Expression of EGFR scFv was 22-32% higher than untransduced control cells (9.3%). The results are shown in FIG. 5.
example 14 EGFR-GITR-CD3zeta CAR is more cytotoxic to EGFR positive cancer cells and not cytotoxic to EGFR negative cancer cells
Real-time cytotoxicity assays showed that EGFR-GITR-CD3zeta-CAR cells were highly cytotoxic to EGFR-positive cancer cells (fig. 6). The cytotoxicity of EGFR-CAR is cancer-type dependent. For example, EGFR-GITR-CD3z-CAR-T cells had higher cytotoxicity than EGFR-CD28-CD3zeta-CAR-T cells in U87 glioblastoma cells (fig. 6A). The activity of EGFR-GITR-CD3zeta CAR-T is dose dependent; the activity ratio is from 10: 1 to 20: 1.
In SKOV-3 ovarian cancer cells, the activity of EGFR-GITR-CD3zeta CAR-T and EGFR-CD28-CD3zeta CAR-T cells were the same, but higher than that of EGFR-4-1BB-CD3zeta and mock control CAR-T cells (FIG. 6B). In SKOV-3 ovarian cancer cells, CAR-T cells containing the GITR and CD28 co-stimulatory domains are more cytotoxic than CAR-T cells containing 4-1 BB. In another ovarian cancer cell line A1847, the activity of EGFR-GITR-CD3zeta was better than that of EGFR-CD28-CD3zeta, at an E: T (effector: target cell ratio) of 30:1 (FIG. 6C).
In pancreatic cancer cells SXPC3, the cytotoxicity of EGFR-CD28-CD3 ζ was higher than that of EGFR-GITR-CD3 ζ and EGFR-41BB-CD3 ζ (FIG. 6D). There was no cytotoxicity in EGFR negative cells, MCF-7 cells (FIG. 6E).
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
sequence listing
<110> Zhang Xi Biotechnology (Shanghai) Co., Ltd
<120> chimeric antigen receptor T cells targeting EGFR carrying GITR costimulatory signals
<130> P2017-2043
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 1590
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tctagagccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60
ctccacgccg ccaggccggc tagcgaagtg cagctggtgc agagcggcgc ggaagtgaaa 120
aaaccgggca gcagcgtgaa agtgagctgc aaagcgagcg gcggcacctt tagcagctat 180
gcgattagct gggtgcgcca ggcgccgggc cagggcctgg aatggatggg cggcattatt 240
ccgatttttg gcaccgcgaa ctatgcgcag aaatttcagg gccgcgtgac cattaccgcg 300
gatgaaagca ccagcaccgc gtatatggaa ctgagcagcc tgcgcagcga agataccgcg 360
gtgtattatt gcgcgcgcga agaaggcccg tattgcagca gcaccagctg ctatggcgcg 420
tttgatattt ggggccaggg caccctggtg accgtgagca gcggtggcgg tggttctggt 480
ggcggtggtt ctggtggcgg tggttctcag agcgtgctga cccaggatcc ggcggtgagc 540
gtggcgctgg gccagaccgt gaaaattacc tgccagggcg atagcctgcg cagctatttt 600
gcgagctggt atcagcagaa accgggccag gcgccgaccc tggtgatgta tgcgcgcaac 660
gatcgcccgg cgggcgtgcc ggatcgcttt agcggcagca aaagcggcac cagcgcgagc 720
ctggcgatta gcggcctgca gagcgaagat gaagcggatt attattgcgc ggcgtgggat 780
gatagcctga acggctatct gtttggcgcg ggcaccaaac tgaccgtgct gctcgagaag 840
cccaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 900
ctgtccctgc gcccagaggc gagccggcca gcggcggggg gcgcagtgca cacgaggggg 960
ctggacttcg ccagtgataa gcccttttgg gtgctggtgg tggttggtgg agtcctggct 1020
tgctatagct tgctagtaac agtggccttt attattttct gggtgcagct tggactgcac 1080
atctggcagc tgaggagtca gtgcatgtgg ccccgagaga cccagctgct gctggaggtg 1140
ccgccgtcga ccgaagacgc cagaagctgc cagttccccg aggaagagcg gggcgagcga 1200
tcggcagagg agaaggggcg gctgggagac ctgtgggtga gagtgaagtt cagcaggagc 1260
gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1320
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1380
aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1440
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1500
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1560
caggccctgc cccctcgcta ataggaattc 1590
<210> 2
<211> 521
<212> PRT
<213> Artificial Sequence (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 Ala Ser Glu Val Gln Leu Val Gln Ser Gly Ala
20 25 30
Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser
35 40 45
Gly Gly Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro
50 55 60
Gly Gln Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr
65 70 75 80
Ala Asn Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp
85 90 95
Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Glu Gly Pro Tyr Cys Ser
115 120 125
Ser Thr Ser Cys Tyr Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu
130 135 140
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
145 150 155 160
Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Asp Pro Ala Val Ser Val
165 170 175
Ala Leu Gly Gln Thr Val Lys Ile Thr Cys Gln Gly Asp Ser Leu Arg
180 185 190
Ser Tyr Phe Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Thr
195 200 205
Leu Val Met Tyr Ala Arg Asn Asp Arg Pro Ala Gly Val Pro Asp Arg
210 215 220
Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly
225 230 235 240
Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp
245 250 255
Ser Leu Asn Gly Tyr Leu Phe Gly Ala Gly Thr Lys Leu Thr Val Leu
260 265 270
Leu Glu Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
275 280 285
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg
290 295 300
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser
305 310 315 320
Asp Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
325 330 335
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Leu
340 345 350
Gly Leu His Ile Trp Gln Leu Arg Ser Gln Cys Met Trp Pro Arg Glu
355 360 365
Thr Gln Leu Leu Leu Glu Val Pro Pro Ser Thr Glu Asp Ala Arg Ser
370 375 380
Cys Gln Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu Glu Lys
385 390 395 400
Gly Arg Leu Gly Asp Leu Trp Val Arg Val Lys Phe Ser Arg Ser Ala
405 410 415
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
420 425 430
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
435 440 445
Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln
450 455 460
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
465 470 475 480
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
485 490 495
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
500 505 510
Leu His Met Gln Ala Leu Pro Pro Arg
515 520
<210> 3
<211> 249
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Glu Gly Pro Tyr Cys Ser Ser Thr Ser Cys Tyr Gly Ala
100 105 110
Phe Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val
130 135 140
Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr Val Lys
145 150 155 160
Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Phe Ala Ser Trp Tyr
165 170 175
Gln Gln Lys Pro Gly Gln Ala Pro Thr Leu Val Met Tyr Ala Arg Asn
180 185 190
Asp Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly
195 200 205
Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala
210 215 220
Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly Tyr Leu Phe
225 230 235 240
Gly Ala Gly Thr Lys Leu Thr Val Leu
245
<210> 4
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala
20 25 30
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val Gly
35 40 45
Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln
50 55 60
Gly Arg Val Lys Ile Thr Ala Asp Glu Ser Ala Ser Thr Ala Tyr Met
65 70 75 80
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Glu Gly Pro Tyr Cys Ser Ser Thr Ser Cys Tyr Gly Ala Phe
100 105 110
Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 5
<211> 375
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaagtgcagc tggtgcagag cggcgcggaa gtgaaaaaac cgggcagcag cgtgaaaagc 60
tgcaaagcga gcggcggcac ctttagcagc tatgcgatta gctgggtgcg ccaggcgccg 120
ggccagggcc tggaatgggt gggcggcatt attccgattt ttggcaccgc gaactatgcg 180
cagaaatttc agggccgcgt gaaaattacc gcggatgaaa gcgcgagcac cgcgtatatg 240
gaactgagca gcctgcgcag cgaagatacc gcggtgtatt attgcgcgcg cgaagaaggc 300
ccgtattgca gcagcaccag ctgctatggc gcgtttgata tttggggcca gggcaccctg 360
gtgaccgtga gcagc 375
<210> 6
<211> 108
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gln Ser Val Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Lys Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Leu Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Thr Leu Val Thr Tyr
35 40 45
Ala Arg Asn Asp Arg Pro Ala Gly Val Pro Asp Arg Phe Ser Gly Ser
50 55 60
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln Ser Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu Asn Gly
85 90 95
Tyr Leu Phe Gly Ala Gly Thr Lys Leu Thr Val Leu
100 105
<210> 7
<211> 324
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cagagcgtgc tgacccagga tccggcggtg agcgtggcgc tgggccagac cgtgaaaatt 60
acctgccagg gcgatagcct gcgcagctat ctggcgagct ggtatcagca gaaaccgggc 120
caggcgccga ccctggtgac ctatgcgcgc aacgatcgcc cggcgggcgt gccggatcgc 180
tttagcggca gcaaaagcgg caccagcgcg agcctggcga ttagcggcct gcagagcgaa 240
gatgaagcgg attattattg cgcggcgtgg gatgatagcc tgaacggcta tctgtttggc 300
gcgggcacca aactgaccgt gctg 324
<210> 8
<211> 123
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 9
<211> 126
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 10
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gly Gly Gly Gly Ser
1 5

Claims (10)

1.A chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus:
(i) Comprising VHand VLthe single chain variable fragment (scFv) of (1), wherein the scFv has low affinity for human Epidermal Growth Factor Receptor (EGFR), dissociation constant (K)D)＞50nM,
(ii) (ii) a transmembrane domain which is capable of,
(iii) A GITR co-stimulatory domain, and
(iv) An activation domain.
2. The fusion protein of claim 1, wherein the scFv is derived from C10 antibody and has the amino acid sequence of SEQ ID NO: 6, or at least 90% sequence identity thereto.
3. The fusion protein of claim 1, wherein the scFv is derived from the P3-5 antibody and has at least 90% sequence identity thereto.
4. the fusion protein of claim 1, wherein the activation domain is CD3 ζ.
5. A nucleic acid molecule encoding the fusion protein of claim 1.
6. An EGFR ScFv-CD8 hinge-CD 28 transmembrane domain-GITR domain-CD 3 ζ nucleic acid sequence set forth in SEQ ID NO: 1, or at least 90% identity thereof in each fragment.
7. A protein encoded by the nucleic acid sequence of claim 6.
8. A vector comprising the nucleic acid molecule of claim 5 or 6.
9. A host cell comprising the vector or chromosome of claim 8 having integrated therein the exogenous nucleic acid molecule of claim 5 or 6 or expressing the CAR of claim 1.
10. A formulation comprising the chimeric antigen receptor of claim 1, or the cell of claim 9, and a pharmaceutically acceptable carrier, diluent, or excipient.
CN201810582487.7A 2018-06-07 2018-06-07 Chimeric antigen receptor T cell carrying GITR costimulatory signal targeting EGFR Pending CN110577604A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101355966A (en) * 2005-06-15 2009-01-28 加州大学评议会 Bispecific single chain Fv antibody molecules and methods of use thereof
WO2015142675A2 (en) * 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor
CN105142677A (en) * 2013-02-15 2015-12-09 加利福尼亚大学董事会 Chimeric antigen receptor and methods of use thereof
WO2017176525A1 (en) * 2016-04-04 2017-10-12 Promab Biotechnologies, Inc. Car having replicated binding motifs in a co-stimulatory domain
WO2018045034A1 (en) * 2016-08-30 2018-03-08 Promab Biotechnologies, Inc. Chimeric antigen receptors having gitr intracellular domain as co-stimulatory domain

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101355966A (en) * 2005-06-15 2009-01-28 加州大学评议会 Bispecific single chain Fv antibody molecules and methods of use thereof
CN105142677A (en) * 2013-02-15 2015-12-09 加利福尼亚大学董事会 Chimeric antigen receptor and methods of use thereof
WO2015142675A2 (en) * 2014-03-15 2015-09-24 Novartis Ag Treatment of cancer using chimeric antigen receptor
WO2017176525A1 (en) * 2016-04-04 2017-10-12 Promab Biotechnologies, Inc. Car having replicated binding motifs in a co-stimulatory domain
WO2018045034A1 (en) * 2016-08-30 2018-03-08 Promab Biotechnologies, Inc. Chimeric antigen receptors having gitr intracellular domain as co-stimulatory domain

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