CAR-T cells with humanized CD19SCFV
阅读说明：本技术 具有人源化cd19 scfv的car-t细胞 (CAR-T cells with humanized CD19SCFV ) 是由 吴力军 维塔·格鲁博斯卡娅 于 2020-02-26 设计创作，主要内容包括：本发明涉及人源化CD19单链可变片段(scFv),其包括SEQ ID NO:8的氨基酸序列。本发明还涉及CD19嵌合抗原受体融合蛋白,其从N末端到C末端包括：(i)本发明的单链可变片段(scFv)、(ii)跨膜结构域、(iii)至少一个共刺激结构域,和(iv)激活结构域。本发明的人源化抗CD19scFv显示出对CD19的选择性和高亲和力结合。基于本发明的人源化scFv的CD19-CART细胞可用于治疗患有包括白血病和淋巴瘤的B细胞恶性肿瘤患者。(The present invention relates to a humanized CD19 single chain variable fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 8. The present invention also relates to a CD19 chimeric antigen receptor fusion protein comprising, from N-terminus to C-terminus: (i) a single chain variable fragment (scFv) of the invention, (ii) a transmembrane domain, (iii) at least one costimulatory domain, and (iv) an activation domain. The humanized anti-CD 19scFv of the invention showed selective and high affinity binding to CD 19. CD19-CART cells based on the humanized scFv of the present invention can be used to treat patients with B cell malignancies including leukemia and lymphoma.)
1. A humanized single chain variable fragment (scFv) against human CD19 comprising the amino acids of SEQ ID NO 8.
2. A Chimeric Antigen Receptor (CAR) comprising, from N-terminus to C-terminus:
(i) the scFv of claim 1 wherein the scFv of the human,
(ii) (ii) a transmembrane domain which is capable of,
(iii) at least one co-stimulatory domain, and
(iv) an activation domain.
3. The CAR of claim 2, wherein the co-stimulatory domain is CD 28.
4. The CAR of claim 2, wherein the activation domain is CD3 ζ.
5. The CAR of claim 5, having the amino acid sequence of SEQ ID NO 19.
6. A nucleic acid encoding the CAR of claim 2.
7. A T cell modified to express the CAR of claim 2.
8. A natural killer cell modified to express the CAR of claim 2.
Technical Field
The present invention relates to a humanized CD19ScFv (clone 6, PMC288) derived from the mouse FMC63 antibody. The invention also relates to CD19-CAR-T cells using the humanized CD19ScFv of the invention. The CD19-CAR-T cells are useful in cell therapy for treating patients with leukemia and lymphoma.
Background
Immunotherapy is becoming a very promising approach to cancer treatment. T cells or T lymphocytes are the armed force of our immune system, constantly looking for foreign antigens and differentiating abnormal cells (cancer or infected cells) from normal cells. Genetic modification of T cells using CAR (chimeric antigen receptor) constructs is the most common approach to design tumor-specific T cells. CAR-T cells targeting Tumor Associated Antigens (TAAs) can be infused into patients (called adoptive cell transfer or ACT), which represents an effective immunotherapeutic approach [1, 2 ]. The advantage of CAR-T technology over chemotherapy or antibodies is that reprogrammed T cells can proliferate and persist in the patient ("an active drug") [1, 3, 4 ].
CARs generally consist of: a monoclonal antibody-derived single chain variable fragment (scFv) at the N-terminal portion, a hinge, a transmembrane domain, and several intracellular costimulatory domains in tandem with the activating CD 3-zeta domain: (i) CD28, (ii) CD137(4-1BB), CD27, or other co-stimulatory domains. (FIG. 1) [1, 2 ]. The evolution of CARs goes from first generation (without costimulatory domains) to second generation (with one costimulatory domain) to third generation CARs (with several costimulatory domains). The generation of CARs with two co-stimulatory domains (so-called third generation CARs) leads to increased CAR-T cell killing activity and improved CAR-T cell persistence, thereby enhancing its anti-tumor activity.
FIG. 1 shows the structure of the CAR. The left panel shows the structure of the first generation CAR (no co-stimulatory domain). The middle panel shows the structure of a second generation CAR (one co-stimulatory domain CD28 or 4-BB). The right panel shows a third generation CAR (two or several co-stimulatory domains) [6 ].
Natural killer cells or NK cells are a class of cytotoxic lymphocytes that are critical to the innate immune system. NK cells exert a role similar to that of cytotoxic T cells in vertebrate adaptive immune responses. NK cells provide a rapid response against virus-infected cells, act about 3 days after infection, and respond to tumor formation.
CD19
CD19 is a B cell-type lymphocyte antigen that is expressed on ALL B cell malignancies, including Acute Lymphoblastic Leukemia (ALL), Chronic Lymphoblastic Leukemia (CLL), and non-hodgkin's lymphoma. This ubiquitous expression in leukemias and lymphomas makes this antigen an attractive target for targeting with CAR-T cells [3 ].
CD19 structure and signaling
The human CD19 protein is a 95kDa transmembrane glycoprotein consisting of 556 amino acids as shown below: 20-291-extracellular domain; 292-313-transmembrane domain; 314-556-cytoplasmic domain (extracellular domain is underlined). It belongs to the immunoglobulin superfamily of proteins and mediates B cell receptor, BCR dependent and independent signaling. CD19 binds to BCRs and other cell surface proteins to modulate intracellular signaling by binding to other kinases and binding partners. CD19 signaling is involved in Src family kinases, PI3 kinase, Abl, AKT-dependent signaling. CD19 is a biomarker for B cell mediated survival signaling and immune responses.
Drawings
FIG. 1 Structure of CAR
Figure 2 structure of humanized CD19-CAR construct. A second generation CD19-CAR was used.
Figure 3, humanized CD19-CAR-T cells killed Hela-CD19 cells. XCelligence real-time cytotoxicity assay was used to test the cytotoxicity of humanized CD19-CAR-T and mouse CD19-41BB-CD3 CAR-T cells as described [4 ]. Normalized cell indices are shown on the Y-axis and time in hours on the X-axis. Right side, top to bottom, individual target cells; then effector cells added to the target cells: t cells, mock CAR-T cells, mouse (M) CD19-CAR-T cells, humanized (H) CD19 CAR-T cells.
FIG. 4, humanized CD19-CAR-T cells secrete IFN- γ against Hela-CD19 positive cells. *: p <0.05, IFN- γ secretion by humanized CAR-T cells and mouse CD19-CAR-T cells compared to T cells, mock CAR-T cells in Hela-CD19 cells.
Figure 5, shows by imaging that humanized CD19-CAR-T cells and mouse CD19-CAR-T cells significantly reduced the growth of mouse xenografts.
Figure 6, humanized CD19-CAR-T cells significantly reduced the total flux (imaging signal) of Raji-luciferase xenograft tumors in the NSG mouse model. Humanized CAR-T cells and mouse CD19-CAR-T cells showed total flux at about baseline. P <0.0001, CD19-CAR-T cells versus mock, PBS-treated mice.
Figure 7, humanized CD19-CAR-T cells significantly prolonged survival in Raji xenograft tumor mouse model. Kaplan-Meier survival curves (Kaplan-Meier curve) show that humanized (h) CD19-CAR-T cells and mouse (m) CD19-CAR-T cells significantly prolonged the survival of mice compared to mock CAR-T cells. CAR-T cells were injected intravenously at 1X 10^7 cells/mouse. P ═ 0.0072, hCD19-41Bb-CD3 CAR-T versus mock CAR-T cells (lower panel); p-0.0294, mCD19-41BB-CD3 CAR-T versus mock-CAR-T cells (upper panel).
Figure 8, humanized CD19-CAR-T cells (clone 6 antibody) and mouse CD19-CAR-T cells (FMC63 antibody) were detected in the blood of mice after CAR-T cells were injected into the mice. Detecting mouse CD19-CAR-T cells with a rabbit anti-FMC 63 antibody; goat anti-human (Fab)2The antibody detects humanized CD19-CAR-T cells. Controls were mock CAR-T cells.
Detailed Description
Definition of
As used herein, a "Chimeric Antigen Receptor (CAR)" is a receptor protein that has been engineered to confer a new ability to target a particular protein to T cells. The receptors are chimeric in that they combine antigen binding and T cell activation functions into a single receptor. A CAR is a fusion protein that includes an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. "Chimeric Antigen Receptors (CARs)" are sometimes referred to as "chimeric receptors", "T-bodies" or "Chimeric Immunoreceptors (CIRs)". "extracellular domain capable of binding to an antigen" means any oligopeptide or polypeptide that can bind to an antigen. By "intracellular domain" is meant any oligopeptide or polypeptide known to act as a domain that transmits a signal to activate or inhibit a biological process in a cell.
As used herein, a "CDR" is a complementarity determining region of a VH or VL chain of an antibody that is critical for binding to an antigen.
As used herein, a "humanized antibody" is an antibody from a non-human species whose protein sequence has been modified to increase its similarity to naturally occurring antibody variants in humans.
As used herein, "domain" means a region in a polypeptide that folds into a particular structure independently of other regions.
As used herein, "single chain variable fragment (scFv)" means a single chain polypeptide derived from an antibody that retains antigen binding ability. Examples of scfvs include antibody polypeptides, formed by recombinant DNA techniques, and 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 engineering scfvs are known to those skilled in the art.
As used herein, "tumor antigen" means an antigenic biomolecule, the expression of which results in cancer.
The present inventors have designed humanized CD19scFv starting from the heavy and light chain variable regions of a mouse monoclonal antibody derived from hybridoma cell line FMC63 [6 ]. The inventors have prepared humanized CD19-CAR-T cells to target cancer cells that overexpress the CD19 antigen. The humanized CD19-CAR-T cells of the invention secrete high levels of IFN- γ against leukemic cancer cells. The humanized CD19-CAR-T cells of the invention killed Hela-CD19 positive target cells, but did not kill control Hela cells.
The present invention relates to humanized anti-human CD19 antibodies comprising humanized V having the amino acid sequence of SEQ ID NO 5, respectivelyLAnd humanized V having the amino acid sequence of SEQ ID NO 7H。
In one embodiment, the humanized anti-human CD19 antibody is a single chain variable fragment (scFv). The ScFv may be VH-linker-VLOr VL-linker-VH. Preferred scFv is VL-linker-VH。
The present invention also relates to a chimeric antigen receptor fusion protein comprising, from N-terminus to C-terminus: (i) a single chain variable fragment (scFv) directed against CD19 (the invention), (ii) a transmembrane domain, (iii) at least one costimulatory domain, and (iv) an activation domain.
In one embodiment, the CAR structure is as shown in figure 2.
In one embodiment, the co-stimulatory domain is selected from the group consisting of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40 and DAP 10. A preferred co-stimulatory domain is CD 28.
A preferred activation domain is CD3 ζ (CD 3Z or CD3 zeta).
The transmembrane domain may be derived from a native polypeptide, or may be artificially designed. The transmembrane domain derived from a native polypeptide may be obtained from any membrane-bound or transmembrane protein. For example, transmembrane domains of the T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR may be used. Artificially designed transmembrane domains are polypeptides that include primarily hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. Alternatively, a short oligopeptide linker or polypeptide linker (e.g., a linker of 2-10 amino acids in length) may be provided between the transmembrane domain and the intracellular domain. In one example, a linker sequence having a glycine-serine contiguous sequence may be used.
The invention provides nucleic acids encoding a CD 19-CAR. The nucleic acid encoding the CAR can be prepared from the amino acid sequence of the particular CAR by conventional methods. The base sequence encoding the amino acid sequence can be obtained from the NCBI RefSeq ID or GenBank accession number described above for the amino acid sequence of each domain, and the nucleic acid of the invention can be prepared using standard molecular biology and/or chemical procedures. For example, a nucleic acid can be synthesized based on a base sequence, and a DNA fragment obtained from a cDNA library is combined by using Polymerase Chain Reaction (PCR) to prepare the nucleic acid of the present invention.
A nucleic acid encoding a CAR of the invention can be inserted into a vector and the vector introduced into a cell. For example, viral vectors such as retroviral vectors (including tumor 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. It is preferable to use a viral vector which lacks replication ability and thus does not replicate in infected cells by itself.
For example, when a retroviral vector is used, an appropriate packaging cell can be selected based on the LTR sequence and the packaging signal sequence possessed by the vector, and retroviral particles can be produced using the packaging cell. Examples of packaging cells include PG13(ATCC CRL-10686), PA317(ATCC CRL-9078), GP + E-86 and 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 retrovirus and which can be used for packaging the packaging cell production of retroviral vectors, can be widely purchased from a number of companies.
CAR-T cells activate cells by binding the CAR to a specific antigen, transmitting a signal into the cell. Activation of the CAR-expressing cell varies depending on the kind of host cell and the intracellular domain of the CAR, and can be confirmed based on, for example, release of cytokines, improvement of cell proliferation rate, change of cell surface molecules, and the like as indicators. For example, release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells destroys target cells that express the antigen. In addition, the release of cytokines or changes in cell surface molecules stimulate other immune cells such as B cells, dendritic cells, NK cells and macrophages.
The CAR-expressing cells can be used as a therapeutic agent for a disease. The therapeutic agent includes the CAR-expressing cell as an active ingredient, and it may further include a suitable excipient.
The humanized CD19-ScFv (clone 6 antibody) of the present invention has the advantage over the mouse CD19-ScFv (FMC63) that humanized CD19scFv is less immunogenic to humans. The humanized CD19-CAR-T cells of the invention extended survival of mouse models of Raji xenograft tumors better than mouse CD19-CAR-T cells.
The humanized CD19ScFv herein can be used in immunotherapeutic applications: toxin/drug-conjugated antibodies, monoclonal therapeutic antibodies, and CAR-T cell immunotherapy.
Humanized CD19-CAR-T cells using the humanized CD19ScFv herein effectively target the CD19 antigen in CD 19-positive cancer cell lines.
Humanized CD19-CAR-T cells can be used in combination with different chemotherapies: checkpoint inhibitors, targeted therapies, small molecule inhibitors, and antibodies.
Humanized CD19-CAR-T cells can be used clinically for CD19 positive cancer cells.
Modifications to co-stimulatory domains such as CD28, 4-1BB, etc., can be used to increase the efficacy of CAR-T cells. Tag-conjugated humanized CD19scFv can be used for CAR generation.
Humanized CD19-CAR-T cells can be used with different safety switches (e.g., T-EGFR, RQR (rituximab-CD 34-rituximab), inducible caspase-9, etc.).
Third generation CAR-T or other costimulatory signaling domains can be used with the same humanized CD19-scFv to make CD 19-CAR-T.
Humanized CD19 CARs can be combined with CARs that target other tumor antigens or tumor microenvironments (e.g., VEGFR-1-3, PDL-1) to generate bispecific antibodies with CD19 and CD3 or other antigens for therapy.
Humanized CD 19-CARs can be used to make other types of cells, such as CAR-Natural Killer (NK) cells, CD 19-CAR-macrophages, and other CD19-CAR hematopoietic cells, which can target CD 19-positive cancers. The invention provides T cells, NK cells, macrophages or hematopoietic cells modified to express CD 19-CAR.
Humanized CD19-CAR-T cells can be used against cancer stem cells or tumor initiating cells that are most resistant to chemotherapy and form aggressive tumors.
The following examples further illustrate the invention. These examples are only intended to illustrate the invention and should not be construed as limiting.
Examples
The inventors have prepared a humanized CD19-ScFv-CAR construct inside a lentiviral vector, cloned into the Xba I and Eco R I sites of the lentiviral vector. The lentiviral CAR construct contained a humanized CD19ScFv (clone 6) -41BB-CD3 ζ fragment inserted between the Xba I and Eco RI cloning sites.
Lentiviruses were prepared in 293T cells and titers were determined by RT-PCR. Equal doses of lentivirus were then used for transduction of T cells.
Example 1 humanized CD 19VHAnd VLAnd scFv sequences
The inventors have humanized CD19scFv derived from the mouse CD19 FMC63 scFv clone [6], and selected clone 6 based on the efficacy of the in vivo data. The structure of the humanized CD19scFv is: VL-linker-VH.
SEQ ID NO 2 is humanized CD 19VLThe nucleotide sequence of (a). SEQ ID NO 4 is VHThe nucleotide sequence of (a). SEQ ID NO 3 is a nucleotide sequence encoding a linker.
SEQ ID NO 5 is humanized CD 19VLThe amino acid sequence of (a). SEQ ID NO 7 is humanized CD 19VHThe amino acid sequence of (a). SEQ ID NO 6 is the amino acid sequence of the linker. The CDR regions are underlined.
SEQ ID NO 8 shows the amino acid sequence of the humanized CD19scFV of clone 6 (V)L-linker-VH)
Example 2 humanized CD19-CAR sequences
The scheme for humanizing the CD19-CAR construct is shown in figure 2. Lentiviral vectors with EF1a promoter were used for cloning of the humanized scFv CAR sequence.
The following nucleotide and amino acid sequences show the CD8 leader-humanized CD19 ScFv-CD8 hinge-TM 8-41BB-CD3 ζ of the present invention. CAR structures include the human CD8 signal peptide, humanized CD19scFv (V)L-linker-VH) CD8 hinge, CD8 Transmembrane (TM), 41BB Co-stingingKinase and CD3 ζ activation domain (fig. 2).
< CD8 leader >
Nucleotide, its preparation and use
Amino acids
<Humanized CD19scFV, VL-linker-VH>
See example 1
< CD8 hinge >
Nucleotide, its preparation and use
Amino acids
< CD8 transmembrane >
Nucleotide, its preparation and use
Amino acids
< 41-BB-Co-stimulatory Domain >
Nucleotide, its preparation and use
Amino acids
<CD3ζ>
Nucleotide, its preparation and use
Amino acids:
< EcoRI restriction site >
gaattc
The amino acid sequence of the humanized CD19-4-1BB-CD3-CAR protein is shown below: the signal peptide is in italics and the linker is underlined; the CD8 hinge is bold; CD8 TM is italicized underlined; 41BB is in bold italics; CD3 is underlined and bold.
Example 3 preparation of CAR lentivirus
DNA encoding the CAR was synthesized and subcloned into a third generation lentiviral vector with EF1a promoter. All CAR lentiviral constructs were sequenced in both directions to confirm the CAR sequence and used for lentiviral production. Will be 1 × 107A single HEK293FT cell (Thermo Fisher) was seeded into a T75 flask and cultured overnight, then transfected using CalPhos transfection kit (Takara, Mountain View, Calif.) with the addition of pPACKH1 Lentivector packaging mix (System Biosciences, Palo Alto, Calif.), and 10 μ g of each lentiviral vector. The next day the medium was replaced with fresh medium and after 48 hours the lentivirus containing medium was collected. By separating at 2100gThe medium was cleared of cell debris for 30 minutes. Viral particles were collected by centrifugation at 112,000g for 100 min, suspended in AIM V medium, aliquoted and frozen at-80 ℃. The titer of the virus preparation was determined by quantitative RT-PCR according to the manufacturer's protocol and 7900HT thermal cycler (Thermo Fisher) using the Lenti-X qRT-PCR kit (Takara). The lentivirus titer is>1×108pfu/ml。
Example 4 preparation and expansion of CAR-T cells
PBMC at 1X 106Cells/ml were suspended in AIM V-Albumax medium (Thermo Fisher) containing 10% FBS and 300U/ml IL-2(Thermo Fisher), mixed with an equal number (1:1 ratio) of CD3/CD28 magnetic beads (Thermo Fisher), and cultured in untreated 24-well plates (0.5 ml/well). Lentiviruses were added to the culture at multiplicity of infection (MOI)5 at 24 and 48 hours, along with 1. mu.l of TransPlus transduction enhancer (AlStem). As T cells proliferate in the next two weeks, cells were counted every 2-3 days, and fresh medium with 300U/ml IL-2 was added to the culture to maintain cell density at 1-3X 106Individual cells/ml.
Example 5 flow cytometry
To measure CAR expression, 0.5X 106Individual cells were suspended in 100 μ l buffer (PBS containing 0.5% BSA) and incubated with 1 μ l human serum (Jackson Immunoresearch, West Grove, PA) on ice for 10 min. Mu.l Allophycocyanin (APC) -labeled anti-CD 3(eBioscience, san Diego, Calif.) and 2. mu.l Phycoerythrin (PE) -labeled goat anti-mouse or anti-human F (ab) were then added2Or isotype control antibody, and the cells were incubated on ice for 30 minutes. Cells were washed with 3ml buffer, then suspended in buffer and assayed using FACSCalibur (BD biosciences). CD3 stained cells were analyzed in comparison to f (ab)2 staining or isotype control staining.
Example 6 humanized CD19-CAR-T cells expressing CAR.
We designed humanized CD19-CAR-T cells with the humanized CD19-CAR construct shown in example 2. We used mock ScFv with an irrelevant ScFv and generated mock-CAR-T cells as negative controls.
Following transduction of humanized CD19 CAR into T cells with lentivirus, e.g. [4]]As described above, goat anti-human F (ab)2The antibody detects the humanized CD19-CAR construct by FACS analysis. About 60% of CAR-T cells Using anti-human F (ab)2The antibody was positive by FACS analysis, indicating that the CAR was expressed and detectable.
Example 7 humanized CD19-CAR-T cells killed Hela-CD19 cells but not Hela cells.
We incubated the humanized effector CD19-CAR-T cells of the invention with the target Hela-CD19 target cells and Hela (CD19 negative) control cells and performed a real-time cytotoxicity assay (RTCA) as described in [4 ].
Humanized CD19-CAR-T cells specifically killed Hela-CD19 cells because the number of target cells (cellular index) was significantly reduced by CAR-T cells, but not by T cells or by mock CAR-T cells (fig. 3).
In separate experiments, humanized CD19-CAR-T cells did not kill CD19 negative Hela cells, and there was no significant difference between T cells, mock CAR-T cells, and CAR-T cells in killing CD19 negative Hela cells. This confirms that the humanized CD19-CAR-T cells have high specificity for targeting the CD19 antigen, killing only CD19 positive cells but not CD19 negative Hela cells (data not shown).
Example 8 humanized CAR-T cells target Hela-CD19 cells to secrete IFN- γ.
We collected the supernatant after co-incubation of humanized CD19-CAR-T cells with the target Hela-CD19 and performed IFN- γ assays as described in [5 ]. Humanized CD19-CAR-T cells secreted significantly higher IFN- γ to Hela-CD19 than secreted by T cells and mock CAR-T cells (fig. 4). Humanized CD19-CAR-T cells secreted IFN- γ at levels comparable to mouse CD19-CAR-T cells, indicating their similar activity in secreting IFN- γ (figure 4).
Example 9 humanized CD19-CAR-T cells reduced Raji xenograft tumor growth in vivo.
We will read Raji-luciferase positive cellsThe cells were injected intravenously into NSG-mice, followed by injection of 1X 10 cells the next day7Humanized CD19-CAR-T cells, e.g. [4]]The method is as follows. We used the IVIS system to detect the growth of Raji xenograft tumor cells by imaging. The humanized CD19-CAR-T cells significantly reduced the growth of Raji xenografts compared to control PBS and mock-CAR-T cells (fig. 5). The reduction in Raji tumor growth was more pronounced with humanized CD19-CAR-T cells compared to mouse CD19-CAR-T cells (fig. 5).
Quantitative imaging at day 14 is shown in figure 6, which indicates that both mouse and humanized CD19-CAR-T cells significantly reduced the total flux of imaging signal (in photons/sec).
Example 10 humanized CD19-CAR-T cells prolonged Raji xenograft tumor model mouse survival time compared to mice treated with mouse CD19-CAR-T cells.
The kaplan-meier survival curve of the results of example 9 show that: mice survived significantly increased after injection of humanized CD19-CAR-T cells (clone 6) compared to control mock-treated mice. The survival of humanized CD19-CAR-T cells (p 0.0072) (figure 7, lower panel) was more significantly increased compared to mouse CD19-CAR-T cells (p 0.0294) relative to mock CAR-T cells (7, upper panel). This demonstrates the advantage of higher efficacy of humanized CD19-CAR-T cells compared to mouse CD19-CAR-T cells.
Example 11 FACS detection of CD19-CAR-T cells after in vivo injection into mice with CD19 or FAB antibodies
After 7 days of injection of humanized CD19-CAR-T cells or mouse CD19-CAR-T cells into mice with Raji xenograft tumor cells, mouse blood was collected to test for the presence of CD19-CAR-T cells. The rabbit anti-FMC 63 antibody was used to detect mouse CD19-CAR-T cells. Goat anti-human (Fab'2) antibody (Promab Biotechnologies, Inc.) was used to detect humanized CD19-CAR-T cells.
The results are shown in fig. 8. Both humanized CD19-CAR-T cells and mouse CD19-CAR-T cells were readily detected 7 days after injection into mice. The humanized CD19-CAR-T cells were superior in persistence to mouse-CAR-T cells. The persistence of CAR-T cells in vivo is consistent with a significant reduction in Raji xenograft tumor growth.
Reference to the literature
1.Maus,M.V.；Haas,A.R.；Beatty,G.L.；Albelda,S.M.；Levine,B.L.；Liu,X.； Zhao,Y.；Kalos,M.；June,C.H.T cells expressing chimeric antigen receptors can cause anaphylaxis in humans.Cancer Immunol Res 2013,1,26-31.
2.Maus,M.V.；Grupp,S.A.；Porter,D.L.；June,C.H.Antibody-modified t cells: Cars take the front seat for hematologic malignancies.Blood 2014,123,2625-2635.
3.Eshhar,Z.；Waks,T.；Gross,G.The emergence of t-bodies/car t cells. Cancer J 2014,20,123-126.
4.Berahovich,R.；Xu,S.；Zhou,H.；Harto,H.；Xu,Q.；Garcia,A.；Liu,F.； Golubovskaya,V.M.；Wu,L.Flag-tagged cd19-specific car-t cells eliminate cd19-bearing solid tumor cells in vitro and in vivo.Front Biosci(Landmark Ed) 2017,22,1644-1654.
5.Golubovskaya,V.；Berahovich,R.；Zhou,H.；Xu,S.；Harto,H.；Li,L.；Chao, C.C.；Mao,M.M.；Wu,L.Cd47-car-t cells effectively kill target cancer cells and block pancreatic tumor growth.Cancers(Basel)2017,9.
6.Nicholson,et al.,Molecular Immunology,1997,34:1157-1165.
Sequence listing
<110> Promega Biotech Co
PROMAB BIOTECHNOLOGIES, Inc.
<120> CAR-T cells with humanized CD19SCFV
<130> 119995-8023.WO01
<150> US 62/814,234
<151> 2019-03-05
<160> 19
<170> PatentIn version 3.5
<210> 1
<211> 556
<212> PRT
<213> Intelligent people
<400> 1
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Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu
405 410 415
Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu
420 425 430
Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly
435 440 445
Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu
450 455 460
Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser
465 470 475 480
Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly
485 490 495
Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln
500 505 510
Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala
515 520 525
Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp
530 535 540
Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg
545 550 555
<210> 2
<211> 321
<212> DNA
<213> Intelligent people
<400> 2
gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attacctgcc gcgcgagcca ggatattagc aaatatctga actggtatca gcagaaaccg 120
ggcaaagcgc cgaaactgct gatttatcat accagccgcc tgcatagcgg cgtgccgagc 180
cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240
gaagattttg cgacctatta ttgccagcag ggcaacaccc tgccgtatac ctttggcggc 300
ggcaccaaag tggaaattaa a 321
<210> 3
<211> 54
<212> DNA
<213> Artificial sequence
<220>
<223> composition
<400> 3
ggctccacct ctggatccgg caagcccgga tctggcgagg gatccaccaa gggc 54
<210> 4
<211> 360
<212> DNA
<213> Intelligent people
<400> 4
gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60
agctgcgcgg cgagcggcgt gagcctgccg gattatggcg tgagctgggt gcgccaggcg 120
ccgggcaaag gcctggaatg ggtgagcgtg atttggggca gcgaaaccac ctattataac 180
agcgcgctga aaagccgctt taccattagc cgcgataaca gcaaaaacac cctgtatctg 240
cagatgaaca gcctgcgcgc ggaagatacc gcggtgtatt attgcgcgaa acattattat 300
tatggcggca gctatgcgat ggattattgg ggccagggca ccctggtgac cgtgagcagc 360
<210> 5
<211> 107
<212> PRT
<213> Intelligent people
<400> 5
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105
<210> 6
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> composition
<400> 6
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 7
<211> 120
<212> PRT
<213> Intelligent people
<400> 7
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 8
<211> 245
<212> PRT
<213> Intelligent people
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln
115 120 125
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg
130 135 140
Leu Ser Cys Ala Ala Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Phe
180 185 190
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
195 200 205
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
225 230 235 240
Val Thr Val Ser Ser
245
<210> 9
<211> 63
<212> DNA
<213> Intelligent people
<400> 9
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 10
<211> 21
<212> PRT
<213> Intelligent people
<400> 10
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
20
<210> 11
<211> 135
<212> DNA
<213> Intelligent people
<400> 11
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 12
<211> 45
<212> PRT
<213> Intelligent people
<400> 12
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 13
<211> 72
<212> DNA
<213> Intelligent people
<400> 13
atctacatct gggcgcccct ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 14
<211> 24
<212> PRT
<213> Intelligent people
<400> 14
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 15
<211> 126
<212> DNA
<213> Intelligent people
<400> 15
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 16
<211> 42
<212> PRT
<213> Intelligent people
<400> 16
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 17
<211> 342
<212> DNA
<213> Intelligent people
<400> 17
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgctaat ag 342
<210> 18
<211> 112
<212> PRT
<213> Intelligent people
<400> 18
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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> 19
<211> 489
<212> PRT
<213> Intelligent people
<400> 19
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
20 25 30
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
35 40 45
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
50 55 60
Pro 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 Phe Thr Leu Thr Ile
85 90 95
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
100 105 110
Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
115 120 125
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
130 135 140
Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
145 150 155 160
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Leu Pro
165 170 175
Asp Tyr Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
180 185 190
Trp Val Ser Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala
195 200 205
Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
210 215 220
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
225 230 235 240
Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp
245 250 255
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro
260 265 270
Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
275 280 285
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
290 295 300
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
305 310 315 320
Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys
325 330 335
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
340 345 350
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
355 360 365
Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser
370 375 380
Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu
385 390 395 400
Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
405 410 415
Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln
420 425 430
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr
435 440 445
Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
450 455 460
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
465 470 475 480
Leu His Met Gln Ala Leu Pro Pro Arg
485