CN116023500B - Chimeric antigen receptor targeting fully humanized CD70 and application thereof - Google Patents
Chimeric antigen receptor targeting fully humanized CD70 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of tumor cell immunotherapy, and particularly relates to a chimeric antigen receptor targeting fully humanized CD70 and application thereof. The chimeric antigen receptor comprises an extracellular domain, a hinge region, a transmembrane region and an intracellular signaling domain, which are sequentially connected, wherein the extracellular domain comprises an amino acid sequence shown as sequence No.1 or a functional variant thereof. The extracellular domain also includes a CD8 signal peptide comprising an amino acid sequence as shown in sequence No.2 or a functional variant thereof. The chimeric antigen receptor and the CAR-T cell thereof can effectively kill 786-O-Luc-GFP target cells, secrete effective amounts of cytokines, can effectively inhibit the growth of tumors in vivo, and have remarkable anti-tumor effect of expressing CD70 targets.
Description
Technical Field
The invention belongs to the technical field of tumor cell immunotherapy, and particularly relates to a chimeric antigen receptor targeting fully humanized CD70 and application thereof.
Background
CAR-T is known collectively as chimeric antigen receptor T cell immunotherapy, a chimeric antigen receptor (chimeric antigen receptor, CAR) is an artificial receptor that mimics TCR function, comprising, in turn, an extracellular domain comprising ScFv, a hinge region and a transmembrane region, and an intracellular signaling domain, typically a CD3 zeta chain or fcrγ, linked to one or more co-stimulatory molecules, such as 4-1bb, CD28, icos (CD 278). The ScFv contained in the CAR molecule has the characteristic of specific recognition on tumor antigens, and T cell activated cell transfer is carried out through a hinge region and a transmembrane region. Currently, clinical CAR-T treatment is mainly applied to tumor treatment of a hematological tumor system.
CD70, one of the members of the Tumor Necrosis Factor Receptor (TNFR) superfamily, is a type II transmembrane protein that is predominantly expressed in activated T cells, B cells, natural killer cells, and dendritic cells. The receptor for CD70 is CD27, and the interaction of CD70 with CD27 promotes activation, proliferation and differentiation of T cells and B cells, playing an important role in regulating the immune response. CD70 is only transiently expressed in activated lymphocytes, but it is highly expressed in various tumor tissues, and the tumor tissues which are currently confirmed to highly express CD70 include various tumor tissues such as renal cancer, hematogenous malignant tumor, thymus tumor, ovarian cancer, glioblastoma, nasopharyngeal carcinoma and the like. Making it possible to be an effective target for tumor immunotherapy.
Compared with the traditional CAR-T target indication, the CD70 has wider application, high safety and coverage of blood system tumors and solid tumors, is a potential CAR-T therapeutic target, and no related target medicine is marketed at present. Related monoclonal antibodies and ScFv are not developed at home, development of chimeric antigen receptors targeting fully humanized CD70 is very necessary, development of chimeric antigen receptors targeting fully humanized CD70 and CAR-T cell products has great clinical application value, selection of antigen gene sequences targeted by CAR-T is a critical selection, and in view of complexity of in vivo gene expression and various uncontrollable privacy, selection of a proper gene capable of being used for CAR-T is very difficult, which is a difficulty in developing CAR-T technology.
Disclosure of Invention
In view of the above, the present invention aims to provide a chimeric antigen receptor targeting fully humanized CD70, which can accurately recognize a CD70 target and effectively kill the target cell after being combined with a carrier to transfect T cells.
The chimeric antigen receptor targeting CD70 comprises an extracellular domain, a hinge region, a transmembrane region and an intracellular signaling domain, wherein the extracellular domain comprises an amino acid sequence as shown in sequence No.1 or a functional variant thereof.
In certain embodiments, the extracellular domain comprises an amino acid sequence as shown in sequence No.1 or a functional variant thereof, i.e., a CD 70-targeting ScFv, prepared by constructing a library of fully human single chain antibodies by phage display technology, enriching for phage carrying a fragment with specific affinity by affinity panning by a suitable method, and sequencing to obtain the gene sequence. To screen human antibody targeting CD70 antigen, a single chain antibody library of whole human is constructed, normal PBMC is used as raw material, total RNA is extracted for reverse transcription cDNA, antibody variable region is amplified, heavy chain variable region VH and light chain variable region VL are connected through connecting peptide (Linker), the heavy chain variable region VH and the light chain variable region VL are constructed to phagemid vector, electric transfer to display strain is carried out, antibody library is constructed, library is panned by CD70 extracellular segment antigen, screened clone is identified, and ScFv with specific binding to CD70 antigen is finally obtained. When constructing chimeric antigen receptor or other uses, PCR artificial synthesis is carried out on ScFv to obtain a large number of CD 70-targeted ScFv, namely the extracellular domain comprising the amino acid sequence shown in sequence No.1 or functional variants thereof.
Further, the extracellular domain also includes a CD8 signal peptide comprising an amino acid sequence as shown in sequence No.2 or a functional variant thereof. The CD70 antibody is localized on the cell membrane under the guidance of the CD8 signal peptide, and as known to those skilled in the art, all other signal peptides useful in the CAR or antibody field can be used in the present invention, the extracellular segment typically comprising a CD8 a or GM-CSFR a signal peptide.
Further, the hinge may be derived from: igG, CD8, CD7, CD4 and functional variants thereof; the hinge region preferably comprises one of the following optional amino acid sequences or a functional variant thereof: the obtained chimeric antigen receptor can have better cell killing effect and better cytokine secretion stimulation by the sequence No.3, the sequence No.4, the sequence No.5 and the sequence No. 6.
Further, the transmembrane region may be derived from: CD8, CD28, CD3 epsilon, CD4, CD16, CD137, CD80, and CD86; similarly, the transmembrane region preferably comprises one of the following optional amino acid sequences or a functional variant thereof: sequence No.7, sequence No.8.
Further, the intracellular signaling domain comprises a signaling region and a co-stimulatory domain, the intracellular signaling region and co-stimulatory domain may be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2 3583, CD86 and CD127; the signaling region preferably comprises the amino acid sequence shown in sequence No.9 or sequence No.34 or a functional variant thereof; the co-stimulatory domain preferably comprises one or more of the following optional amino acid sequences or one or more of its functional variants: sequence No.10, sequence No.11, sequence No.12.
In certain embodiments, the chimeric antigen receptor comprises one of the following optional combinations:
combination 1): the hinge region comprises an amino acid sequence as shown in sequence No.3 or a functional variant thereof, the transmembrane region comprises an amino acid sequence as shown in sequence No.7 or a functional variant thereof, and the intracellular signaling domain comprises an amino acid sequence as shown in sequence No.34 or a functional variant thereof and a sequence amino acid sequence of sequence No.10 or a functional variant thereof;
combination 2): the hinge region comprises an amino acid sequence as shown in sequence No.4 or a functional variant thereof, the transmembrane region comprises an amino acid sequence as shown in sequence No.8 or a functional variant thereof, and the intracellular signaling domain comprises an amino acid sequence as shown in sequence No.34 or a functional variant thereof and the amino acid sequence as shown in sequence No.11 or a functional variant thereof;
combination 3): the hinge region comprises an amino acid sequence as shown in sequence No.6 or a functional variant thereof, the transmembrane region comprises an amino acid sequence as shown in sequence No.8 or a functional variant thereof, and the intracellular signaling domain comprises an amino acid sequence as shown in sequence No.9 or a functional variant thereof and an amino acid sequence as shown in sequence No.12 or a functional variant thereof;
combination 4): the hinge region comprises an amino acid sequence as set forth in sequence No.5 or a functional variant thereof, the transmembrane region comprises an amino acid sequence as set forth in sequence No.8 or a functional variant thereof, and the intracellular signaling domain comprises an amino acid sequence as set forth in sequence No.9 or a functional variant thereof and an amino acid sequence as set forth in sequence No.12 or a functional variant thereof;
combination 5): the hinge region comprises an amino acid sequence as shown in sequence No.6 or a functional variant thereof, the transmembrane region comprises an amino acid sequence as shown in sequence No.7 or a functional variant thereof, and the intracellular signaling domain comprises an amino acid sequence as shown in sequence No.34 or a functional variant thereof and an amino acid sequence as shown in sequence No.10 or a functional variant thereof.
In certain embodiments, the chimeric antigen receptor comprises one or more components of a natural killer cell receptor (NKR), thereby forming a NKR-CAR. The NKR component may be a transmembrane domain, hinge domain or cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR DL5B, KIR DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; natural Cytotoxic Receptors (NCR), e.g., NKp30, NKp44, NKp46; a family of Signaling Lymphocyte Activation Molecules (SLAM) for immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD2F-10; fc receptors (FcR), e.g., CD16, and CD64; and Ly49 receptors, e.g., ly49A, LY C. The NKR-CAR molecule may interact with an adapter molecule or an intracellular co-stimulatory domain (e.g., DAP 12).
The invention also aims to provide a nucleic acid sequence for encoding the chimeric antigen receptor, and the nucleic acid sequence is used as a target gene and is combined with a carrier to transfect T cells to obtain CAR-T cells.
The nucleic acid sequence encoding combination 1) in the chimeric antigen receptor comprises a sequence as shown in sequence No. 13; or nucleic acid sequence b encoding combination 2) in the chimeric antigen receptor comprises a sequence as shown in sequence No. 14; or the nucleic acid sequence c encoding combination 3) in the chimeric antigen receptor comprises a sequence as shown in sequence No. 15; or the nucleic acid sequence d encoding combination 4) in the chimeric antigen receptor comprises a sequence as shown in sequence No. 16; or the nucleic acid sequence e encoding said combination 5) comprises a sequence as shown in sequence No. 36.
Further, the chimeric antigen receptor also includes a promoter, the nucleic acid sequence of which is shown in sequence No.18 or sequence No. 19.
In certain embodiments, the 5' end of the nucleic acid sequence a is linked to a promoter, the nucleic acid sequence of which is shown in sequence No.18 or sequence No. 19; or the 5' end of the nucleic acid sequence b is connected with a promoter, and the nucleic acid sequence of the promoter is shown in sequence No. 19; or the 5' end of the nucleic acid sequence c is connected with a promoter, and the nucleic acid sequence of the promoter is shown as sequence No.18 or sequence No. 19; or the 5' end of the nucleic acid sequence d is connected with a promoter, and the nucleic acid sequence of the promoter is shown as sequence No.18 or sequence No. 19; or the 5' -end of the nucleic acid sequence e is connected with a promoter, and the nucleic acid sequence of the promoter is shown in sequence No. 19.
In certain embodiments, the 5' end of the nucleic acid sequence a is linked to the ligation signal peptide sequence a; or the 5' end of the nucleic acid sequence b is connected with a connecting signal peptide sequence b; or the 5' end of the nucleic acid sequence c is connected with a connecting signal peptide sequence c; or the 5' end of the nucleic acid sequence d is connected with a connecting signal peptide sequence d; or the 5' end of the nucleic acid sequence e is connected with a connecting signal peptide sequence e; the nucleic acid sequences of the signal peptide sequence a, the signal peptide sequence b, the signal peptide sequence c, the signal peptide sequence d and the signal peptide sequence e are shown in sequence No. 17. The 5' end of the signal peptide sequence a is connected with a promoter, and the nucleic acid sequence of the promoter is shown as sequence No.18 or sequence No. 19; or the 5' -end of the signal peptide sequence b is connected with a promoter, and the nucleic acid sequence of the promoter is shown in sequence No. 19; or the 5' end of the signal peptide sequence c is connected with a promoter, and the nucleic acid sequence of the promoter is shown as sequence No.18 or sequence No. 19; or the 5' end of the signal peptide sequence d is connected with a promoter, and the nucleic acid sequence of the promoter is shown as sequence No.18 or sequence No. 19; or the 5' -end of the signal peptide sequence e is connected with a promoter, and the nucleic acid sequence of the promoter is shown in sequence No. 19.
Further, to exert the tumor-specific killing ability of the CAR in a tumor environment with a high background, a CD70 interfering RNA sequence may be added to the structure of the CAR in the present invention. Specifically, the nucleic acid sequence (with or without a promoter) encoding the chimeric antigen receptor further includes a CD70 interfering RNA, the CD70 interfering RNA sequence being as shown in sequence No. 20.
In certain embodiments, to overcome the tumor microenvironment in a solid tumor, a hypoxia promoter may be added to the structure of the CAR of the invention.
In certain embodiments, the 5' end of the nucleic acid sequence d or the signal peptide sequence d is linked to a promoter, the nucleic acid sequence of which is shown in sequence No.18 or sequence No. 19; the 3' end of the nucleic acid sequence d is connected with a CD70 interference RNA sequence, and the CD70 interference RNA sequence is shown in sequence No. 20.
The invention also aims at providing an expression vector comprising the nucleic acid sequence of the chimeric antigen receptor.
Further, the expression vector is selected from any one of a lentiviral expression vector, a retrovirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector and a plasmid.
In certain embodiments, the lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine Infectious Anemia Virus (EIAV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).
In certain embodiments, the vector comprises a left (5 ') retroviral LTR, a Psi (ψ) packaging signal, a central polypurine tract/DNA FLAP (cPPT/FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR encompassed by the invention, and a right (3') retroviral LTR.
In certain embodiments, the CAR comprises a hepatitis b virus posttranscriptional regulatory element (HPRE) or a Woodchuck Posttranscriptional Regulatory Element (WPRE) and an optimized woodchuck posttranscriptional regulatory element (oPRE).
In certain embodiments, the nucleic acid sequence encoding the foregoing chimeric antigen receptor comprises an optimized Kozark sequence.
In certain embodiments, the gene expression vector may comprise a secreted anti-PD-1 ScFv; in certain embodiments, the gene expression vector comprises a PD-1 conjugated transduction peptide (e.g., a PD-1-CD28-CD137-CD3 signal structure); in certain embodiments, the gene expression vector may comprise multiple CAR combinations, such as 2CAR combinations targeting different antigens or different recognition sites of the same antigen.
The present invention also aims at providing an engineered cell transduced with the nucleic acid sequence of any one of the chimeric antigen receptors or the expression vector of any one of the foregoing; preferably, the cell is a T cell, a T cell precursor or an NK cell.
The present invention aims to provide a cell preparation comprising an engineered cell according to any one of the preceding.
Further, the cell preparation also includes other agents that enhance the activity of CAR expression.
In certain embodiments, the active agent is an immunosuppressant such as cyclosporine (cycloporin), azathioprine (azathioprine), methotrexate (methotrexa), mycophenolic acid ester (mycophenolic acid) and FK506, antibodies or other immune scavengers (immunodepleting agents) such as CAMPATH, anti-CD 3 antibodies or other antibody therapies, cyclophosphamide (cytoxan), fludarabine (fludarabine), cyclosporine (cycloporin), FK506, rapamycin (rapamycin), mycophenolic acid (mycophenolic acid), steroids (steroids), FR901228, cytokines and radiation.
In certain embodiments, the agent that enhances the activity of the CAR-expressing cell can be an agent that blocks an inhibitory molecule. Inhibitory molecules such as PD1 may, in some embodiments, reduce the ability of CAR-expressing cells to mount an immune effector response. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD 276), B7-H4 (VTCN 1), HVEM (TNFRSF 14 or CD 270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFR beta) and TGFR beta. The extracellular domain of the inhibitory molecule may be fused to a transmembrane domain and an intracellular signaling domain, such as a PD1 CAR.
Further, a variety of additional therapeutic agents may be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include P D-1 inhibitors such as nivolumab pembrolizumab (nivolumab) pembrolizumab (pembrolizumab), ab Fu Shan antibody (pimelizumab) and avilamab (Atezolizumab).
In particular, the method comprises the steps of, additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to, ibrutinib (ibretinib) ofatuzumab (ofa tuximab) rituximab (rituximab) trastuzumab (trastuzumab) t ra s tuz uma bem ta ns i ne imatinib (imatinib) cetuximab (c e t u x i m a b) panitumumab (pa n i t u m u m a b) cetuximab (catheter) thiostuzumab (ibutnimab), tibetab (ibrituximab), oxfamoxamide, toximab (tositumomab), rituximab (brentuximab), alemtuzumab (alemtuzumab), getuzumab (gemtinib), erlotinib (erlotinib), gefitinib (gefitinib), adatinib (vanneatinib), altinib (panitumumab), alfuzotinib (panitude), alfuzotinib (panitumumab), valatinib (valatinib), panitumumab (valatinib), panitutinib (valatinib), panitumumab (valatinib), panitutinib (panitumumab), panitutinib) and (valatinib) panitumumab (valatinib), sorafenib (sorafenib), sunitinib (supininib), tivozanib, toceranib, vandetanib, emtrictinib (entrectinib), cabozantinib (cabozantinib), imatinib (imatinib), dasatinib (dasatinib), nilotinib (nilotinib), panatinib (ponatinib), radatinib (radoditinib), bosutinib (bosutinib), letatinib (lesatinib), ruxotinib (ruxolitinib), pacritinib, cobitinib (cobitinib), semitinib (seletinib), trametinib (tramtinib), bimetinib, aletinib), ceritinib (ceritinib), crizotinib (crizotinib), apride (abatinib), interleukin.
Further, the cell preparation may also be used in combination with some therapeutic means, which may be surgery, chemotherapy, radiation.
The invention aims at further providing a pharmaceutical composition, which comprises the nucleic acid sequence of the chimeric antigen receptor or the expression vector or the engineered cell.
The invention aims to provide the application of the chimeric antigen receptor or the nucleic acid sequence of the chimeric antigen receptor or the expression vector of the chimeric antigen receptor or the engineered cell of the chimeric antigen receptor or the cell preparation of the engineered cell of the chimeric antigen receptor or the pharmaceutical composition of the chimeric antigen receptor in preparation of antitumor drugs.
Specifically, the antitumor drugs include, but are not limited to, an anti-renal cancer drug, an anti-hematological malignancy drug, an anti-thymus tumor drug, an anti-ovarian cancer drug, an anti-glioblastoma drug, and an anti-nasopharyngeal cancer drug.
In the present invention, the term "functional variant" is generally meant to include an amino acid sequence that has substantially the same function as it (e.g., may possess the properties of the chimeric antigen receptor) and has at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity thereto. In certain embodiments, the variant of the amino acid sequence has substantially the same function as it.
The invention has the beneficial effects that
The chimeric antigen receptor and the CAR-T cell provided by the invention can effectively kill 786-O-Luc-GFP target cells and can secrete effective amounts of cytokines.
The chimeric antigen receptor and the CAR-T cell provided by the invention can effectively inhibit the growth of in-vivo tumors, and have remarkable anti-tumor effect on CD 70-expressing targets.
Drawings
Figure 1 shows the in vitro cell killing results of different ScFv CAR-T formulations.
FIG. 2 shows the results of in vitro IFN-gamma cytokine secretion from different ScFv CAR-T formulations.
FIG. 3 shows the results of in vitro IL-2 cytokine secretion by different ScFv CAR-T formulations.
FIG. 4 shows the change curves of in vivo imaging fluorescence values of different ScFv CAR-T formulations.
Figure 5 is a tumor volume growth curve for different ScFv CAR-T formulations.
FIG. 6 is a photograph of an in vivo image of different ScFv CAR-T formulations.
FIG. 7 shows the results of in vitro positive rate assays for different CAR-T formulations containing CD70 (1) ScFv.
FIG. 8 shows the in vitro cell killing results of different CAR-T formulations containing CD70 (1) ScFv.
FIG. 9 shows the in vitro factor secretion results for different CAR-T formulations containing CD70 (1) ScFv.
Figure 10 is a graph showing the results of in vivo CAR-positive rate detection of different CAR-T formulations containing CD70 (1) ScFv.
FIG. 11 shows the change in fluorescence values of in vivo imaging of different CAR-T formulations containing CD70 (1) ScFv.
FIG. 12 is a graph showing the in vivo tumor volume growth curves of different CAR-T formulations containing CD70 (1) ScFv.
FIG. 13 is a photograph of an in vivo image of different CAR-T formulations containing CD70 (1) ScFv.
FIG. 14 shows the results of in vitro positive rate detection of CAR-8 and CAR-9.
Figure 15 is an in vitro cell killing result of CAR-8 and CAR-9.
Figure 16 is the in vitro factor secretion results for CAR-8 and CAR-9.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
In the embodiment of the invention, the preparation method of the humanized CD70 comprises the following steps:
(1) Construction of fully human Single chain antibody library
And (3) performing PBMC separation on the Ficoll separating liquid, and slowly adding the Ficoll separating liquid into the collected normal human blood, so that a clear separation interface between the Ficoll separating liquid and the normal human blood is maintained. The 50mL centrifuge tube containing the blood and the separation solution was centrifuged at about 15℃for 20min. After centrifugation, the whole liquid level is divided into four layers, wherein the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, the middle layer is Ficoll liquid, and the junction of the upper layer and the middle layer is provided with a white cloud layer narrow band mainly comprising PBMC, namely a PBMC cell layer. The PBMCs were aspirated with a new sterile pasteur pipette to obtain isolated PBMCs.
Total RNA was extracted by conventional methods and reverse transcribed into cDNA. Based on the similarity of heavy chain and light chain germline gene sequences, degenerate primers are designed at both ends of the variable region (Li Xiaolin, construction and preliminary screening of a high-capacity non-immune humanized Fab phage antibody library, university of Chinese medical science, innova 6 month in 2007), heavy chain variable region gene fragments and light chain variable region gene fragments of antibodies are obtained by PCR, scFv nucleic acid fragments are obtained by amplification by a conventional overlap PCR method (PCR method refers to "molecular cloning Experimental guidelines (Molecular Cloning: A Laboratory Manual)" (third edition), U.S. Joe Sambrook, david russell. Science publishers), and ScFv nucleic acid fragments are connected with a phagemid vector pComb3xss, and the products are transformed into TGI strains by an electroconverter to obtain a fully human single chain antibody library.
(2) Phage display fully human single chain antibody library preparation
Resuscitates the library broth in fresh LB liquid medium, adds VSCM13 helper phage to the complex of infection with VCSM 13:bacteria=50:1, mixes well, and continues culture in shaker. The culture was centrifuged to discard the supernatant, and the pellet was resuspended in ampicillin-kanamycin-resistant SOB medium overnight. The bacterial solution was centrifuged at 8000rpm at 4℃for 20min, the supernatant was collected and added to a 1/5 volume of 20%PEG 8000 2.5mmol/L NaCl solution, incubated on ice for 1 hour, centrifuged at 12000rpm at 4℃for 30min, and the precipitated phage was resuspended in PBS and filtered with a 0.22 μm filter.
(3) CD70 antigen panning
Incubating CD70 protein with Fc label with proteoG magnetic beads to prepare CD 70-proteoG coupled magnetic beads, pumping the coupled magnetic beads into the prepared fully human single chain antibody library phage panning, and performing 3-4 rounds of co-incubation, washing and eluting panning process to obtain the specific monoclonal antibody against antigen.
(4) Screening of Positive clones
After the panning is finished, the monoclonal bacterial plaques finally taken out of the warehouse are picked for ELISA detection and screening, the detection result is shown in figure 1, phage clones which are combined with the CD70 antigen are obtained, the scFv targeting the CD70 is obtained through panning, and the specific binding capacity of the human CD70 antigen is provided. The amino acid Sequence of the ScFv targeting CD70 is shown as Sequence No.1, a plurality of pieces are selected, and only the piece can successfully construct CAR
In the embodiment of the invention, the antigen panning is specifically implemented as follows:
(1) Closing: dissolving 5% skimmed milk powder with PBS, filtering to obtain sealing solution, respectively suspending phage and CD70-protein G coupled magnetic beads with appropriate amount of sealing solution, and tumbling and mixing;
(2) Co-incubation: placing CD70-protein G coupled magnetic beads on a magnetic rack, discarding supernatant, and re-suspending the magnetic beads with phage, and performing rolling co-incubation;
(3) Cleaning: placing the magnetic bead phage mixed solution in a magnetic frame, removing the supernatant, adding a cleaning solution to clean the magnetic beads, and determining the cleaning times according to different numbers of panning rounds;
(4) Eluting: placing the cleaned magnetic beads on a magnetic frame, drying the supernatant, adding Gly-HCl 4, uniformly mixing, incubating at room temperature for 7min, adding about Tris-HCl to adjust the pH to be close to neutral, finally placing the mixture on the magnetic frame, and transferring the phage supernatant to a new 1.5mL EP tube to complete one round of panning;
(5) Enrichment: inoculating phage into TGI bacterial liquid for infection, standing, centrifuging, re-suspending and precipitating, coating on a 2YTAG plate, and culturing upside down overnight;
(6) Washing the plate: the overnight cultured plates were washed from the medium to remove plaque and used as seed stock for the next round of library packaging.
In the embodiment of the invention, the specific implementation process of ELISA detection is as follows:
(1) Coating: diluting CD70 antigen with carbonate coating buffer to 1 μg/mL, adding 100 μl/well to 96-well plate, capping at 4 ℃ overnight;
(2) Obtaining phage samples: centrifuging the phage monoclonal recombinant bacteria liquid cultured overnight for 10min, and taking the supernatant as a detection sample;
(3) Closing: washing antigen coated plate on a plate washer with PBS for 3 times, adding skimmed milk powder sealing liquid into each hole, and sealing at 37deg.C;
(4) Antibody incubation: adding a phage monoclonal recombinant bacterial liquid to be detected into each hole after the plate washing machine washes the plate for 3 times, and incubating at 37 ℃;
(5) Adding a secondary antibody: plate washing machine washes the plate 3 times, add 1 per hole: 5000Anti-M13-HRP secondary antibody 100. Mu.L, 37℃incubation;
(6) Color development: washing the plate for 6 times by a plate washing machine, adding a color development liquid into each hole, and standing at room temperature in a dark place for 25min for color development;
(7) And (3) terminating: h is added into each hole 2 SO 4 Terminating the reaction;
(8) And (3) detection: placing the detection plate in an enzyme labeling instrument to detect OD 450 The absorbance was 2.5 times higher than that of the negative control, and the phage clone was positive.
In the examples of the present invention, the sequences of the structural elements of the vectors are shown in Table 1 below; 8H/7H/8hdc/G4H/G4HH2H3mt are different hinge structures; 8TM/28TM is a different transmembrane structure; 28z/BBz/28z (YFM) are different intracellular signal structures.
In the embodiment of the invention, CD70 (1)/CD 70 (2)/CD 70 (3)/CD 70 (4) are ScFvs of different targeting CD70, and the different ScFvs are CD70 ScFv, CD70 ScFv2, CD70 ScFv3 and CD70 ScFv4.
TABLE 1 sequence of vector structural elements
Example 1 plasmid construction
The ScFv sequence of the CD70 antibody is obtained through PCR amplification, then the sequence is connected to lentiviral vectors containing different promoters, different hinges, different transmembrane and different costimulatory signals in a restriction endoenzyme digestion mode, namely, the CAR T plasmid vector targeting CD70 with different structures is obtained, and after sequencing comparison verification, the structure is constructed, and the specific abbreviation and structure correspond to Table 2.
TABLE 2CAR Structure and abbreviation
Since activated T cells transiently express CD70, in order to verify the effect of T cell endogenous CD70 expression on CAR-T function, and also to prevent such CAR from binding to CD70 on body's own immune cells, and to kill body's own immune cells, CAR-T sequences comprising CD70 interfering RNAs are also designed in the examples of the present invention, as in table 2, CAR-17 and CAR-19 differ by CAR-19 plus a CD70 interfering RNA sequence. The embodiment of the invention is an experiment carried out under the condition of low background, the in-vivo and in-vitro results of the CAR-17 and the CAR-19 are not greatly different, and can be widely guessed, and if the background is higher, the effect of the CAR containing the CD70 interference RNA sequence is obviously better than that of the CAR without the CD70 interference RNA sequence. In addition to the design of the assay, in certain embodiments, a vector carrying CD 70-interfering RNA may be co-transfected with a CAR vector to achieve the objective of interfering with endogenous CD 70; in some embodiments, gene editing may also be used such as: the zinc finger technology, TELN, cas9, cas12 and other gene editing modes lead to the reduction or disruption of endogenous CD70 expression so as to inactivate, such as knocking out endogenous CD70 expression; the techniques of patent CN111826352a and patent CN113088495A may also be used in certain embodiments to cause reduced or disrupted endogenous CD70 expression and thus inactivation.
EXAMPLE 2 lentiviral preparation
The embodiment of the packaging lentivirus adopts a calcium phosphate method, which is specifically as follows: 293T cells were cultured in DMEM medium containing 10% FBS (w/v) to a preferable state, packaging plasmid (RRE: REV: 2G) and expression plasmid were added in a 1.5 centrifuge tube at a certain ratio, caCl2 and 2 XHBS were added, and after mixing, they were allowed to stand at room temperature, and then added to the treated 293T cell culture medium, and after 3-5 hours, they were again changed to 10mL of DMEM medium containing 10% FBS, and after 48 hours or 72 hours, the cell supernatant was collected and virus was purified. 15 virus particles in example 1 were obtained.
Example 3CAR-T cell preparation
Lymphocyte separation by using a gradient centrifugation method; after centrifugation, the second white lymphocyte layer was washed with physiological saline, and cultured in RPMI 1640 complete medium containing 10% fbs to obtain human PBMC cells. After the obtained PBMC cells are activated by anti-CD 3 and CD28 monoclonal antibodies for 24 hours, the activated PBMC cells are infected according to a certain multiplicity of infection (MOI), the positive rate of the CAR-T is detected on the 8 th day of virus infection, the detection method is flow detection, and the antibodies are as follows: protein-L-PE, protein-L can recognize antibody light chain, light chain of ScFv sequence of CAR antigen recognition region can be recognized by Protein-L, so that the positive rate of CAR and the expression intensity of CAR can be detected by utilizing Protein-L.
Example 4 evaluation of in vitro and in vivo pharmacodynamics of different humanized ScFv
Example 3 was used to prepare CAR-T cell formulations comprising CAR-2, CAR-3, CAR-4, CAR-5, with 4 kinds of CAR-T differing only in ScFv, the remainder being identical, verifying the feasibility of different ScFv to prepare CAR-T. CAR positive rate was measured by Protein-L and the specific results are shown in Table 3.
TABLE 3 Positive Rate of different CAR-T cell formulations
Control T is used as a Control group, a CAR-2 group, a CAR-3 group and a CAR-4 group are arranged in an experimental group, 786-O-Luc-GFP (CD 70 positive) and A549-Luc-GFP (CD 70 negative) are used as target cells, and in vitro effectiveness is verified through in vitro killing and in vitro factor secretion. The results show that the CAR-2, the CAR-3 and the CAR-4 have certain killing and factor secretion in vitro compared with the control group, but have poor effect, the CAR-5 group has higher killing and factor secretion, and detailed results are shown in figures 1, 2 and 3.
NOG mice (female, 6 weeks old) were selected at 5×10 6 Cells/dose subcutaneous injection (i.v.) 786-O-Luc-GFP Cells were modeled as tumor Cells in vivo, 42d after tumor3×10 6 The CAR-T Cells/dose-only tail intravenous injection (i.v.) was given to the different groups (Saline, control, CAR-2, CAR-3, CAR-4, CAR-5) and the results showed that the CAR-5 group showed the best antitumor effect in vivo, and the specific results are shown in fig. 4, 5 and 6; only CD70 (1) ScFv constructed CAR-T has excellent in-vivo and in-vitro curative effects.
Example 5 evaluation of in vitro efficacy of different CAR-T formulations containing CD70 (1) ScFv
To further determine the anti-tumor effect of different CAR-T formulations of CD70 (1) ScFv, reference example 3 produced different CAR-T formulations CAR-5, CAR-11, CAR-12, CAR-13, CAR-14, CAR-16 containing CD70 (1) ScFv, CAR positive rates were detected by Protein-L, see in particular fig. 7. The CAR-T preparation was tested in vitro by killing and factor secretion in vitro using Control T as a Control group, 786-O-Luc-GFP (CD 70 positive), a549-Luc-GFP (CD 70 negative) as target cells. The results show that the experimental groups have obvious killing and factor secretion detailed results compared with the control groups, and the detailed results are shown in fig. 8 and 9.
Example 6 evaluation of in vivo and in vitro efficacy of different CAR-T formulations containing CD70 (1) ScFv
To further evaluate the in vivo anti-tumor effect of different CAR-T formulations of CD70 (1) ScFv, reference example 3 prepared different CAR-T formulations CAR-5, CAR-12, CAR-13, CAR-14, CAR-16, CAR-17, CAR-19 containing CD70 (1) ScFv, CAR positive rates were detected by Protein-L, see in particular fig. 10. NOG mice (female, 6 weeks old) were selected at 5×10 6 Cells/dose subcutaneous injection (i.v.) 786-O-Luc-GFP Cells were modeled as tumor Cells in vivo at 1.5X10 d post-tumor 6 CAR-T Cells/dose-only tail intravenous injection (i.v.) different groups of CAR-T cell formulations were administered and tumor growth was observed in vivo by in vivo imaging and tumor measurement to evaluate the in vivo anti-tumor effects of CD70 (1) ScFv different CAR-T formulations. The results show that the antitumor effect in each group is obvious, and the detailed results are shown in fig. 11, 12 and 13.
Example 7
Then, whether other CAR structures are effective or not is verified again, and only in-vitro efficacy evaluation is verified, and the results are shown in figures 14-16, and the verification of CAR-8 and CAR-9 is also feasible, so that SCFV in the patent can be applicable to all CAR structures.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Sequence listing
<110> Chongqing precision biotechnology Co., ltd., chongqing precision biotechnology industry research institute Co., ltd
<120> chimeric antigen receptor targeting fully humanized CD70 and application thereof
<130> 2021-10-12
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 244
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 1
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 Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Phe Ala Ala Ser Ser Leu Gln 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 Ser Tyr Ser Thr Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Asp Ile Lys Arg Gly Ser Thr Ser
100 105 110
Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val
115 120 125
Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala Ser Val
130 135 140
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met
145 150 155 160
Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp Met Gly Val
165 170 175
Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe Lys Gly
180 185 190
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu
195 200 205
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
210 215 220
Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
225 230 235 240
Thr Val Ser Ser
<210> 2
<211> 21
<212> PRT
<213> Synthesis (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
20
<210> 3
<211> 45
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 3
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> 4
<211> 47
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 4
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
1 5 10 15
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
20 25 30
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 5
<211> 36
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 5
Ala Pro Pro Arg Ala Ser Ala Leu Pro Ala Pro Pro Thr Gly Ser Ala
1 5 10 15
Leu Pro Asp Pro Gln Thr Ala Ser Ala Leu Pro Asp Pro Pro Ala Ala
20 25 30
Ser Ala Leu Pro
35
<210> 6
<211> 228
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 6
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr
65 70 75 80
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
100 105 110
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
130 135 140
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
145 150 155 160
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
180 185 190
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220
Ser Leu Gly Lys
225
<210> 7
<211> 24
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 7
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> 8
<211> 27
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 8
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 9
<211> 112
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 9
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Ser 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> 10
<211> 42
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 10
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> 11
<211> 41
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 11
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Phe Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 12
<211> 41
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 12
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 13
<211> 1401
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 13
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta ccccctggac gttcggccaa 300
gggaccaaac tggatatcaa acgtggcagc acaagcggaa gcggcaaacc aggaagcgga 360
gaaggaagca ccaagggaga ggtgcagctg gtgcagtccg gaccagaggt gaagaagcca 420
ggagccagcg tgaaggtgtc ctgtaaggcc tctggctaca ccttcacaga ttactatatg 480
aactgggtgc ggcagatgca cggcaaggga ctggagtgga tgggcgtgat caacccatac 540
aatggcggca ccgattataa tcagaagttt aagggcagag tgaccatcac agccgacaag 600
tccacctcta cagcctacat ggagctgagc tccctgagga gcgaggacac agccgtgtac 660
tattgtgccc gctccgtgta cgactatccc tttgattatt ggggccaggg caccctggtc 720
acagtctcct caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 780
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 840
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 900
ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 960
ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1020
tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1080
aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1140
ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1200
gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1260
aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1320
cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1380
atgcaggccc tgccccctcg c 1401
<210> 14
<211> 1413
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 14
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta ccccctggac gttcggccaa 300
gggaccaaac tggatatcaa acgtggcagc acaagcggaa gcggcaaacc aggaagcgga 360
gaaggaagca ccaagggaga ggtgcagctg gtgcagtccg gaccagaggt gaagaagcca 420
ggagccagcg tgaaggtgtc ctgtaaggcc tctggctaca ccttcacaga ttactatatg 480
aactgggtgc ggcagatgca cggcaaggga ctggagtgga tgggcgtgat caacccatac 540
aatggcggca ccgattataa tcagaagttt aagggcagag tgaccatcac agccgacaag 600
tccacctcta cagcctacat ggagctgagc tccctgagga gcgaggacac agccgtgtac 660
tattgtgccc gctccgtgta cgactatccc tttgattatt ggggccaggg caccctggtc 720
acagtctcct caaagcccac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 780
atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 840
gtgcacacga gggggctgga cttcgcctgc gacttctggg tgctggtcgt ggtgggtggc 900
gtgctggcct gctacagcct gctggtgaca gtggccttca tcatcttttg ggtgaggagc 960
aagcggagca gactgctgca cagcgactat atgtttatga ctccccgccg ccccgggccc 1020
acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1080
gtgaagttca gcaggagcgc agacgccccc gcgtacaagc agggccagaa ccagctctat 1140
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1200
gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1260
ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1320
aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1380
gacgcccttc acatgcaggc cctgccccct cgc 1413
<210> 15
<211> 1956
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 15
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta ccccctggac gttcggccaa 300
gggaccaaac tggatatcaa acgtggcagc acaagcggaa gcggcaaacc aggaagcgga 360
gaaggaagca ccaagggaga ggtgcagctg gtgcagtccg gaccagaggt gaagaagcca 420
ggagccagcg tgaaggtgtc ctgtaaggcc tctggctaca ccttcacaga ttactatatg 480
aactgggtgc ggcagatgca cggcaaggga ctggagtgga tgggcgtgat caacccatac 540
aatggcggca ccgattataa tcagaagttt aagggcagag tgaccatcac agccgacaag 600
tccacctcta cagcctacat ggagctgagc tccctgagga gcgaggacac agccgtgtac 660
tattgtgccc gctccgtgta cgactatccc tttgattatt ggggccaggg caccctggtc 720
acagtctcct cagagtccaa gtacggacca ccttgcccac catgtccagc acctccagtg 780
gcaggaccaa gcgtgttcct gtttccacct aagcctaagg acaccctgat gatctctcgg 840
accccagagg tgacatgcgt ggtggtggac gtgagccagg aggatccaga ggtgcagttc 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga caaagcccag ggaggagcag 960
tttcagagca cctaccgcgt ggtgtccgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt ataagtgcaa ggtgtccaat aagggcctgc ctagctccat cgagaagacc 1080
atctctaagg caaagggaca gcccagggag cctcaggtgt acacactgcc acccagccag 1140
gaggagatga ccaagaacca ggtgtccctg acatgtctgg tgaagggctt ctatccctcc 1200
gacatcgccg tggagtggga gtctaatggc cagcctgaga acaattacaa gaccacacct 1260
ccagtgctgg actccgatgg ctctttcttt ctgtattctc ggctgaccgt ggataagagc 1320
agatggcagg agggcaacgt gttttcttgt agcgtgatgc acgaggccct gcacaatcac 1380
tacacacaga agtccctgtc tctgagcctg ggcaagttct gggtgctggt cgtggtgggt 1440
ggcgtgctgg cctgctacag cctgctggtg acagtggcct tcatcatctt ttgggtgagg 1500
agcaagcgga gcagaggcgg ccacagcgac tacatgaaca tgaccccccg gaggcctggc 1560
cccacccgga agcactacca gccctacgcc cctcccaggg acttcgccgc ctaccggagc 1620
cgggtgaagt tcagccggag cgccgacgcc cctgcctacc agcagggcca gagccagctg 1680
tacaacgagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 1740
cgggaccctg agatgggcgg caagccccgg agaaagaacc ctcaggaggg cctgtataac 1800
gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 1860
cggaggggca agggccacga cggcctgtac cagggcctga gcaccgccac caaggatacc 1920
tacgacgccc tgcacatgca ggccctgccc cctcgc 1956
<210> 16
<211> 1380
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 16
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta ccccctggac gttcggccaa 300
gggaccaaac tggatatcaa acgtggcagc acaagcggaa gcggcaaacc aggaagcgga 360
gaaggaagca ccaagggaga ggtgcagctg gtgcagtccg gaccagaggt gaagaagcca 420
ggagccagcg tgaaggtgtc ctgtaaggcc tctggctaca ccttcacaga ttactatatg 480
aactgggtgc ggcagatgca cggcaaggga ctggagtgga tgggcgtgat caacccatac 540
aatggcggca ccgattataa tcagaagttt aagggcagag tgaccatcac agccgacaag 600
tccacctcta cagcctacat ggagctgagc tccctgagga gcgaggacac agccgtgtac 660
tattgtgccc gctccgtgta cgactatccc tttgattatt ggggccaggg caccctggtc 720
acagtctcct cagcaccacc tcgggccagc gccctgcctg caccacccac cggctccgcc 780
ctgccagacc ctcagacagc atctgccctg ccagatcctc cagcagcaag cgccctgccc 840
ttctgggtgc tggtcgtggt gggtggcgtg ctggcctgct acagcctgct ggtgacagtg 900
gccttcatca tcttttgggt gaggagcaag cggagcagag gcggccacag cgactacatg 960
aacatgaccc cccggaggcc tggccccacc cggaagcact accagcccta cgcccctccc 1020
agggacttcg ccgcctaccg gagccgggtg aagttcagcc ggagcgccga cgcccctgcc 1080
taccagcagg gccagagcca gctgtacaac gagctgaacc tgggccggag ggaggagtac 1140
gacgtgctgg acaagcggag aggccgggac cctgagatgg gcggcaagcc ccggagaaag 1200
aaccctcagg agggcctgta taacgaactg cagaaagaca agatggccga ggcctacagc 1260
gagatcggca tgaagggcga gcggcggagg ggcaagggcc acgacggcct gtaccagggc 1320
ctgagcaccg ccaccaagga tacctacgac gccctgcaca tgcaggccct gccccctcgc 1380
<210> 17
<211> 63
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 17
atggctctgc cagtgacagc tctgctgctg cctctggctc tgctgctgca cgcagctaga 60
ccc 63
<210> 18
<211> 545
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 18
ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg 60
agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa 120
actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt 180
atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac 240
agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300
gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg 360
cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc 420
cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac 480
tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc 540
ctact 545
<210> 19
<211> 545
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 19
ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg 60
agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa 120
actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt 180
atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac 240
agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 300
gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg 360
cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc 420
cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac 480
tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc 540
ctact 545
<210> 20
<211> 286
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 20
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgcgtctcag cttccaccaa tcaagagttg gtggaagctg agacgc 286
<210> 21
<211> 45
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 21
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
1 5 10 15
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Arg Pro Ala Ala
20 25 30
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Asp
35 40 45
<210> 22
<211> 732
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 22
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta ccccctggac gttcggccaa 300
gggaccaaac tggatatcaa acgtggcagc acaagcggaa gcggcaaacc aggaagcgga 360
gaaggaagca ccaagggaga ggtgcagctg gtgcagtccg gaccagaggt gaagaagcca 420
ggagccagcg tgaaggtgtc ctgtaaggcc tctggctaca ccttcacaga ttactatatg 480
aactgggtgc ggcagatgca cggcaaggga ctggagtgga tgggcgtgat caacccatac 540
aatggcggca ccgattataa tcagaagttt aagggcagag tgaccatcac agccgacaag 600
tccacctcta cagcctacat ggagctgagc tccctgagga gcgaggacac agccgtgtac 660
tattgtgccc gctccgtgta cgactatccc tttgattatt ggggccaggg caccctggtc 720
acagtctcct ca 732
<210> 23
<211> 135
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 23
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 24
<211> 141
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 24
aagcccacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 60
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 120
gggctggact tcgcctgcga c 141
<210> 25
<211> 108
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 25
gcaccacctc gggccagcgc cctgcctgca ccacccaccg gctccgccct gccagaccct 60
cagacagcat ctgccctgcc agatcctcca gcagcaagcg ccctgccc 108
<210> 26
<211> 135
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 26
aagccaacca caaccccagc accaaggcca cctacacctg caccaaccat cgcaagccag 60
ccactgtccc tgaggccaga ggcaagacct gcagcaggag gcgccgtgca cacacggggc 120
ctggacttcg ccgat 135
<210> 27
<211> 684
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 27
gagtccaagt acggaccacc ttgcccacca tgtccagcac ctccagtggc aggaccaagc 60
gtgttcctgt ttccacctaa gcctaaggac accctgatga tctctcggac cccagaggtg 120
acatgcgtgg tggtggacgt gagccaggag gatccagagg tgcagttcaa ctggtacgtg 180
gatggcgtgg aggtgcacaa tgccaagaca aagcccaggg aggagcagtt tcagagcacc 240
taccgcgtgg tgtccgtgct gacagtgctg caccaggact ggctgaacgg caaggagtat 300
aagtgcaagg tgtccaataa gggcctgcct agctccatcg agaagaccat ctctaaggca 360
aagggacagc ccagggagcc tcaggtgtac acactgccac ccagccagga ggagatgacc 420
aagaaccagg tgtccctgac atgtctggtg aagggcttct atccctccga catcgccgtg 480
gagtgggagt ctaatggcca gcctgagaac aattacaaga ccacacctcc agtgctggac 540
tccgatggct ctttctttct gtattctcgg ctgaccgtgg ataagagcag atggcaggag 600
ggcaacgtgt tttcttgtag cgtgatgcac gaggccctgc acaatcacta cacacagaag 660
tccctgtctc tgagcctggg caag 684
<210> 28
<211> 72
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 28
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 29
<211> 81
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 29
ttctgggtgc tggtcgtggt gggtggcgtg ctggcctgct acagcctgct ggtgacagtg 60
gccttcatca tcttttgggt g 81
<210> 30
<211> 123
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 30
aggagcaagc ggagcagagg cggccacagc gactacatga acatgacccc ccggaggcct 60
ggccccaccc ggaagcacta ccagccctac gcccctccca gggacttcgc cgcctaccgg 120
agc 123
<210> 31
<211> 336
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 31
cgggtgaagt tcagccggag cgccgacgcc cctgcctacc agcagggcca gagccagctg 60
tacaacgagc tgaacctggg ccggagggag gagtacgacg tgctggacaa gcggagaggc 120
cgggaccctg agatgggcgg caagccccgg agaaagaacc ctcaggaggg cctgtataac 180
gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240
cggaggggca agggccacga cggcctgtac cagggcctga gcaccgccac caaggatacc 300
tacgacgccc tgcacatgca ggccctgccc cctcgc 336
<210> 32
<211> 126
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 32
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 33
<211> 123
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 33
aggagcaagc ggagcagact gctgcacagc gactatatgt ttatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 34
<211> 112
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 34
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> 35
<211> 336
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 35
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 cctcgc 336
<210> 36
<211> 1950
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 36
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240
gaagattttg caacttacta ctgtcaacag agttacagta ccccctggac gttcggccaa 300
gggaccaaac tggatatcaa acgtggcagc acaagcggaa gcggcaaacc aggaagcgga 360
gaaggaagca ccaagggaga ggtgcagctg gtgcagtccg gaccagaggt gaagaagcca 420
ggagccagcg tgaaggtgtc ctgtaaggcc tctggctaca ccttcacaga ttactatatg 480
aactgggtgc ggcagatgca cggcaaggga ctggagtgga tgggcgtgat caacccatac 540
aatggcggca ccgattataa tcagaagttt aagggcagag tgaccatcac agccgacaag 600
tccacctcta cagcctacat ggagctgagc tccctgagga gcgaggacac agccgtgtac 660
tattgtgccc gctccgtgta cgactatccc tttgattatt ggggccaggg caccctggtc 720
acagtctcct cagagtccaa gtacggacca ccttgcccac catgtccagc acctccagtg 780
gcaggaccaa gcgtgttcct gtttccacct aagcctaagg acaccctgat gatctctcgg 840
accccagagg tgacatgcgt ggtggtggac gtgagccagg aggatccaga ggtgcagttc 900
aactggtacg tggatggcgt ggaggtgcac aatgccaaga caaagcccag ggaggagcag 960
tttcagagca cctaccgcgt ggtgtccgtg ctgacagtgc tgcaccagga ctggctgaac 1020
ggcaaggagt ataagtgcaa ggtgtccaat aagggcctgc ctagctccat cgagaagacc 1080
atctctaagg caaagggaca gcccagggag cctcaggtgt acacactgcc acccagccag 1140
gaggagatga ccaagaacca ggtgtccctg acatgtctgg tgaagggctt ctatccctcc 1200
gacatcgccg tggagtggga gtctaatggc cagcctgaga acaattacaa gaccacacct 1260
ccagtgctgg actccgatgg ctctttcttt ctgtattctc ggctgaccgt ggataagagc 1320
agatggcagg agggcaacgt gttttcttgt agcgtgatgc acgaggccct gcacaatcac 1380
tacacacaga agtccctgtc tctgagcctg ggcaagatct acatctgggc gcccttggcc 1440
gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1500
aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1560
gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1620
aagttcagca ggagcgcaga cgcccccgcg tacaagcagg gccagaacca gctctataac 1680
gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1740
cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1800
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1860
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1920
gcccttcaca tgcaggccct gccccctcgc 1950
<210> 37
<211> 244
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 37
Glu Thr Thr Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Gly Ser Thr Ser
100 105 110
Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val
115 120 125
Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala Ser Val
130 135 140
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met
145 150 155 160
Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp Met Gly Val
165 170 175
Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe Lys Gly
180 185 190
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu
195 200 205
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
210 215 220
Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
225 230 235 240
Thr Val Ser Ser
<210> 38
<211> 244
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 38
Ala Ile Arg 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 Ser Ile Ser Ser Ser
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Val Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln 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 Ser Tyr Asn Thr Pro Arg
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg Gly Ser Thr Ser
100 105 110
Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val
115 120 125
Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala Ser Val
130 135 140
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Met
145 150 155 160
Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp Met Gly Val
165 170 175
Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe Lys Gly
180 185 190
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu
195 200 205
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
210 215 220
Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
225 230 235 240
Thr Val Ser Ser
<210> 39
<211> 249
<212> PRT
<213> Synthesis (Artificial Sequence)
<400> 39
Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Pro Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Ser Ser
20 25 30
Ala Asp Asp Leu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln
85 90 95
Tyr Tyr Gly Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105 110
Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
115 120 125
Thr Lys Gly Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Lys Lys
130 135 140
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
145 150 155 160
Thr Asp Tyr Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu
165 170 175
Glu Trp Met Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn
180 185 190
Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser
195 200 205
Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
210 215 220
Tyr Tyr Cys Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly
225 230 235 240
Gln Gly Thr Leu Val Thr Val Ser Ser
245
<210> 40
<211> 732
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 40
gaaacgacac tcacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180
aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240
gaagattttg cagtttatta ctgtcagcag tataataact ggcccctcac tttcggcgga 300
gggaccaagg tggagatcaa acgtggcagc acaagcggaa gcggcaaacc aggaagcgga 360
gaaggaagca ccaagggaga ggtgcagctg gtacagtccg gaccagaggt gaagaagcca 420
ggagccagcg tgaaggtgtc ctgtaaggcc tctggctaca ccttcacaga ttactatatg 480
aactgggtgc ggcagatgca cggcaaggga ctggagtgga tgggcgtgat caacccatac 540
aatggcggca ccgattataa tcagaagttt aagggcagag tgaccatcac agccgacaag 600
tccacctcta cagcctacat ggagctgagc tccctgagga gcgaggacac agccgtgtac 660
tattgtgccc gctccgtgta cgactatccc tttgattatt ggggccaggg aaccctggtc 720
acagtctcct ca 732
<210> 41
<211> 732
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 41
gccatccgga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc agctctttaa attggtatca gcagaaacca 120
gggaaagccc ctaagcttgt gatctatgct gcgtccagtt tacaaagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttacta ctgtcaacag agttacaata ccccgagaac tttcggccct 300
gggaccaagg tggagatcaa acgtggcagc acaagcggaa gcggcaaacc aggaagcgga 360
gaaggaagca ccaagggaga ggttcagctg gtacagtccg gaccagaggt gaagaagcca 420
ggagccagcg tgaaggtgtc ctgtaaggcc tctggctaca ccttcacaga ttactatatg 480
aactgggtgc ggcagatgca cggcaaggga ctggagtgga tgggcgtgat caacccatac 540
aatggcggca ccgattataa tcagaagttt aagggcagag tgaccatcac agccgacaag 600
tccacctcta cagcctacat ggagctgagc tccctgagga gcgaggacac agccgtgtac 660
tattgtgccc gctccgtgta cgactatccc tttgattatt ggggccaggg caccctggtc 720
acagtctcct ca 732
<210> 42
<211> 747
<212> DNA
<213> Synthesis (Artificial Sequence)
<400> 42
gacatccaga tgacccagtc tccagactcc ctgcctgtgt ctctgggcga gagggccacc 60
atcaattgca agtccagcca gagtcttcta tccagcgccg acgatttgaa ctacttagct 120
tggtaccagc agaaaccagg gcagcctcct aagctgctca tttactgggc atctacccgg 180
caatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240
atcaacagcc tgcaggctga agatgtggca gtttatttct gtcagcaata ttatggtact 300
cccaccttcg gccaagggac acgactggag attaaacgtg gcagcacaag cggaagcggc 360
aaaccaggaa gcggagaagg aagcaccaag ggacaggtac agctgcagca gtccggacca 420
gaggtgaaga agccaggagc cagcgtgaag gtgtcctgta aggcctctgg ctacaccttc 480
acagattact atatgaactg ggtgcggcag atgcacggca agggactgga gtggatgggc 540
gtgatcaacc catacaatgg cggcaccgat tataatcaga agtttaaggg cagagtgacc 600
atcacagccg acaagtccac ctctacagcc tacatggagc tgagctccct gaggagcgag 660
gacacagccg tgtactattg tgcccgctcc gtgtacgact atccctttga ttattggggc 720
cagggaaccc tggtcaccgt ctcctca 747
Claims (16)
1. The chimeric antigen receptor targeting the fully humanized CD70 is characterized by comprising an extracellular domain, a hinge region, a transmembrane region and an intracellular signal domain, wherein the amino acid sequence of the extracellular domain is shown as SEQ ID NO. 1.
2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the hinge region is shown in SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6.
3. The chimeric antigen receptor according to claim 2, wherein: the amino acid sequence of the transmembrane region is shown as SEQ ID NO.7 or SEQ ID NO.8.
4. A chimeric antigen receptor according to claim 3, wherein the intracellular signaling domain comprises a signaling region and a costimulatory domain, the amino acid sequence of the signaling region being shown in SEQ ID No.9 or SEQ ID No. 34; the amino acid sequence of the costimulatory domain is shown as SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12.
5. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor comprises one of the following combinations:
combination 1): the amino acid sequence of the hinge region is shown as SEQ ID NO.3, the amino acid sequence of the transmembrane region is shown as SEQ ID NO.7, and the intracellular signal domain comprises the amino acid sequence shown as SEQ ID NO.34 and the amino acid sequence shown as SEQ ID NO. 10;
combination 2): the amino acid sequence of the hinge region is shown as SEQ ID NO.4, the amino acid sequence of the transmembrane region is shown as SEQ ID NO.8, and the intracellular signal domain comprises the amino acid sequence shown as SEQ ID NO.34 and the amino acid sequence shown as SEQ ID NO. 11;
combination 3): the amino acid sequence of the hinge region is shown as SEQ ID NO.6, the amino acid sequence of the transmembrane region is shown as SEQ ID NO.8, and the intracellular signal domain comprises the amino acid sequence shown as SEQ ID NO.9 and the amino acid sequence shown as SEQ ID NO. 12;
combination 4): the amino acid sequence of the hinge region is shown as SEQ ID NO.5, the amino acid sequence of the transmembrane region is shown as SEQ ID NO.8, and the intracellular signal domain comprises the amino acid sequence shown as SEQ ID NO.9 and the amino acid sequence shown as SEQ ID NO. 12;
combination 5): the amino acid sequence of the hinge region is shown as SEQ ID NO.6, SEQ ID NO.5 or SEQ ID NO.21, the amino acid sequence of the transmembrane region is shown as SEQ ID NO.7, and the intracellular signal domain comprises the amino acid sequence shown as SEQ ID NO.34 and the amino acid sequence shown as SEQ ID NO. 10.
6. A nucleic acid molecule encoding the chimeric antigen receptor according to claim 5, wherein the nucleic acid molecule encoding said combination 1) comprises the sequence shown in SEQ ID No. 13; or the nucleic acid molecule encoding said combination 2) comprises the sequence as shown in SEQ ID NO. 14; or the nucleic acid molecule encoding said combination 3) comprises a sequence as shown in SEQ ID NO. 15; or the nucleic acid molecule encoding said combination 4) comprises the sequence as shown in SEQ ID NO. 16; or the nucleic acid molecule encoding said combination 5) comprises the sequence as shown in SEQ ID NO. 36.
7. The nucleic acid molecule of claim 6, further comprising a promoter having a nucleic acid sequence set forth in SEQ ID No.18 or SEQ ID No. 19.
8. The chimeric antigen receptor according to any one of claims 1-5, further comprising a CD70 interfering RNA, wherein the sequence of the CD70 interfering RNA is shown in SEQ ID No. 20.
9. An expression vector comprising the nucleic acid molecule of any one of claims 6-7.
10. The expression vector of claim 9, wherein the expression vector is selected from any one of a lentiviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, and a plasmid.
11. An engineered cell transduced with the nucleic acid molecule of any one of claims 6-7 or the expression vector of any one of claims 9-10.
12. The engineered cell of claim 11, wherein the cell is a T cell, a T cell precursor, or an NK cell.
13. A cell preparation comprising the engineered cell of any one of claims 11-12.
14. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1-5 or the nucleic acid molecule of any one of claims 6-7 or the expression vector of any one of claims 9-10 or the engineered cell of any one of claims 11-12.
15. Use of a chimeric antigen receptor according to any one of claims 1 to 5 or a nucleic acid molecule according to any one of claims 6 to 7 or an expression vector according to any one of claims 9 to 10 or an engineered cell according to any one of claims 11 to 12 or a pharmaceutical composition according to claim 14 for the preparation of a medicament against a tumor highly expressing a CD70 molecule.
16. Use of the chimeric antigen receptor of any one of claims 1-5 or the nucleic acid molecule of any one of claims 6-7 or the expression vector of any one of claims 9-10 or the engineered cell of any one of claims 11-12 or the pharmaceutical composition of claim 14 for the preparation of an anti-renal cancer drug, an anti-hematological malignancy drug, an anti-thymic tumour drug, an anti-ovarian cancer drug, an anti-glioblastoma drug, an anti-nasopharyngeal cancer drug.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111239065.8A CN116023500B (en) | 2021-10-25 | 2021-10-25 | Chimeric antigen receptor targeting fully humanized CD70 and application thereof |
| PCT/CN2022/137448 WO2023072307A1 (en) | 2021-10-25 | 2022-12-08 | Antigen binding fragment targeting cd70, single-chain antibody and chimeric antigen receptor, and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN104619351A (en) * | 2012-06-19 | 2015-05-13 | Ambrx公司 | Anti-CD70 antibody drug conjugates |
| CN109021106A (en) * | 2018-08-30 | 2018-12-18 | 浙江蓝盾药业有限公司 | A kind of humanization CD70 antibody LD70 and the preparation method and application thereof |
| CN110592023A (en) * | 2019-09-11 | 2019-12-20 | 浙江蓝盾药业有限公司 | Anti CD70 CAR-T cell and preparation method and application thereof |
| CN113493524A (en) * | 2020-04-03 | 2021-10-12 | 重庆精准生物技术有限公司 | Bispecific chimeric antigen receptor and application thereof |
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| EP4008728A1 (en) * | 2017-02-14 | 2022-06-08 | Kite Pharma, Inc. | Cd70 binding molecules and methods of use thereof |
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| CN104619351A (en) * | 2012-06-19 | 2015-05-13 | Ambrx公司 | Anti-CD70 antibody drug conjugates |
| CN109021106A (en) * | 2018-08-30 | 2018-12-18 | 浙江蓝盾药业有限公司 | A kind of humanization CD70 antibody LD70 and the preparation method and application thereof |
| CN110592023A (en) * | 2019-09-11 | 2019-12-20 | 浙江蓝盾药业有限公司 | Anti CD70 CAR-T cell and preparation method and application thereof |
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