Anti-human CD3E antibody and application thereofTechnical field
The application relates generally to genetic engineering and antibody drug field；In particular to anti-human CD3E antibody art andIts purposes.The application develops new anti-human CD3E antibody, and provides the antibody and preventing or treating the purposes in tumour.
Background technique
CD3 molecule is people's immune t-cell surface markers antigen, is made of 4 kinds of subunits, respectively CD3 ε (CD3E), CD3 δ(CD3D), CD3 γ (CD3G) and CD3 ζ (CD3Z), they are transmembrane protein.Passed through by 6 polypeptide chains that 4 kinds of subunits formCharge adsorption effect is centered around around T cell receptor (T cell receptor, TCR), forms the TCR- containing 8 polypeptide chainsCD3 compound.This compound has the function of T cell activation signal transduction and stablizes TCR structure.The cytoplasmic domain of CD3 moleculeWith referred to as immunity receptor Tyrosine Activating Motifs (immunoreceptor tyrosine-based activationMotif, ITAM) consensus, due to TCR specific recognition and combine MHC (major histo-compatibilityComplex) the Antigenic Peptide of molecule submission, so that ITAM in tyrosine protein kinase p561ck phosphorylation CD3 molecule in T cellTyrosine residue, then raise contain SH2 (Scr homology 2) structural domain tyrosine protein kinase (such as ZAP-70).ITAM phosphorylation and combination ZAP-70 are one of the important biochemical reactions of t cell activation early signal conductive process.Therefore, CD3The function for the activation signal that molecule has conduction TCR identification antigen to generate.
AntiCD3 McAb E antibody can be capable of providing t cell activation in conjunction with the CD3E subunit in the TCR receptor complex of T cell surfaceThe first signal (being integrated to TCR similar to the MHC- peptide complexes on antigen presenting cell), be conducive to the activation of T cell.AndAnd include the antibody of the bispecific for CD3E and tumor cell surface antigen (TAA), T cell may be implemented in tumour cellThe enrichment on periphery improves T cell to the killing-efficiency of tumour cell, is of great significance for oncotherapy.
Summary of the invention
In a first aspect, it includes the amino containing HCDR1, HCDR2 and HCDR3 this application provides the antibody for combining people CD3EThe heavy chain variable region of acid sequence and light chain variable region containing LCDR1, LCDR2 and LCDR3 amino acid sequence, wherein
The HCDR1 amino acid sequence is GFKFSGY, and the HCDR2 amino acid sequence is WFDGSR, the HCDR3 ammoniaBase acid sequence is QMGYWHFGL, and the LCDR1 amino acid sequence is RASQGINNSLT, and the LCDR2 amino acid sequence isGASNRET, the LCDR3 amino acid sequence are QQWLKLPPT；Or
The HCDR1 amino acid sequence is GFTFSNA, and the HCDR2 amino acid sequence is KDKSNNYA, the HCDR3Amino acid sequence is VHYGVRFFYTMDV, and the LCDR1 amino acid sequence is RPSQSLVHNNGNTYLS, the LCDR2 aminoAcid sequence is KVSNRFS, and the LCDR3 amino acid sequence is GQGTQYPFT；Or
The HCDR1 amino acid sequence is GFTFSNA, and the HCDR2 amino acid sequence is KSKSDNYA, the HCDR3Amino acid sequence is VHYGAYYGVDA, and the LCDR1 amino acid sequence is RSSQSLVHSNRNTYLN, the LCDR2 amino acidSequence is KVSNRFS, and the LCDR3 amino acid sequence is GQGTHAPYA；
Wherein HCDR and LCDR amino acid sequence is defined according to Chothia.
In some embodiments of first aspect, the amino acid sequence of the heavy chain variable region of the antibody such as SEQ IDShown in NO:19,21 or 23.
In some embodiments of first aspect, the amino acid sequence of the light chain variable region of the antibody such as SEQ IDShown in NO:20,22 or 24.
In some embodiments of first aspect, the amino acid sequence of the heavy chain variable region of the antibody such as SEQ IDShown in NO:19, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:20；Or
The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:21, the light chain variable of the antibodyThe amino acid sequence in area is as shown in SEQ ID NO:22；Or
The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:23, the light chain variable of the antibodyThe amino acid sequence in area is as shown in SEQ ID NO:24.
Second aspect, this application provides the antibody for combining people CD3E, wherein the amino of the heavy chain variable region of the antibodyAny one of acid sequence and SEQ ID NO:19,21 or 23 have at least 90% consistency, and the antibody is lightAny one of amino acid sequence and SEQ ID NO:20,22 or 24 of chain variable region have at least 90% consistency.
In some embodiments of first aspect and second aspect, the antibody is monoclonal antibody.
In some embodiments of first aspect and second aspect, the antibody is whole antibody, Fab segment, F (ab ')2Segment or Single-Chain Fv Fragment of Murine (scFv).
In some embodiments of first aspect and second aspect, the antibody is human antibody.
In some embodiments of first aspect and second aspect, the antibody also includes selected from IgG1 hypotype, IgG2The heavy chain constant region of hypotype or IgG4 hypotype.
In some embodiments of first aspect and second aspect, the antibody also includes selected from κ hypotype or λ hypotypeConstant region of light chain.
In some embodiments of first aspect and second aspect, the antibodies mediate t cell activation.
The third aspect, this application provides nucleic acid molecules, encode antibody described in first aspect or second aspect or itsAntigen-binding portion thereof.
Fourth aspect, this application provides bispecific antibodies, it includes the first arm for people CD3E and are directed to tumourSecond arm of cell surface antigen (TAA), wherein first arm for people CD3E includes:
Amino acid sequence is the HCDR1 of GFKFSGY, and amino acid sequence is the HCDR2 of WFDGSR, and amino acid sequence isThe HCDR3 of QMGYWHFGL, amino acid sequence are the LCDR1 of RASQGINNSLT, and amino acid sequence is the LCDR2 of GASNRET, ammoniaBase acid sequence is the LCDR3 of QQWLKLPPT；Or
Amino acid sequence is the HCDR1 of GFTFSNA, and amino acid sequence is the HCDR2 of KDKSNNYA, and amino acid sequence isThe HCDR3 of VHYGVRFFYTMDV, amino acid sequence are the LCDR1, amino acid sequence KVSNRFS of RPSQSLVHNNGNTYLSLCDR2, amino acid sequence be GQGTQYPFT LCDR3；Or
Amino acid sequence is the HCDR1 of GFTFSNA, and amino acid sequence is the HCDR2 of KSKSDNYA, and amino acid sequence isThe HCDR3 of VHYGAYYGVDA, amino acid sequence are the LCDR1 of RSSQSLVHSNRNTYLN, and amino acid sequence is KVSNRFS'sLCDR2, amino acid sequence are the LCDR3 of GQGTHAPYA；
Wherein HCDR and LCDR amino acid sequence is defined according to Chothia.
In some embodiments of fourth aspect, first arm for people CD3E includes amino acid sequence such as SEQHeavy chain variable region shown in ID NO:19,21 or 23.
In some embodiments of fourth aspect, first arm for people CD3E includes amino acid sequence such as SEQLight chain variable region shown in ID NO:20,22 or 24.
In some embodiments of fourth aspect, first arm for people CD3E includes:
Amino acid sequence heavy chain variable region as shown in SEQ ID NO:19, amino acid sequence is as shown in SEQ ID NO:20Light chain variable region；Or
Amino acid sequence heavy chain variable region as shown in SEQ ID NO:21, amino acid sequence is as shown in SEQ ID NO:22Light chain variable region；Or
Amino acid sequence heavy chain variable region as shown in SEQ ID NO:23, amino acid sequence is as shown in SEQ ID NO:24Light chain variable region.
5th aspect, this application provides bispecific antibodies, it includes the first arm for people CD3E and are directed to tumourSecond arm of cell surface antigen (TAA), wherein first arm for people CD3E includes: amino acid sequence and SEQ IDAny one of NO:19,21 or 23 have the heavy chain variable region of at least 90% consistency, and amino acid sequence and SEQAny one of ID NO:20,22 or 24 have the light chain variable region of at least 90% consistency.
In some embodiments of fourth aspect and the 5th aspect, first arm for people CD3E includes to be directed to peopleThe Fab segment or Single-Chain Fv Fragment of Murine (scFv) of CD3E.
In some embodiments of fourth aspect and the 5th aspect, first arm for people CD3E also includes to be selected fromThe heavy chain constant region of IgG1 hypotype, IgG2 hypotype or IgG4 hypotype.
In some embodiments of fourth aspect and the 5th aspect, first arm for people CD3E also includes to be selected fromThe constant region of light chain of κ hypotype or λ hypotype.
In some embodiments of fourth aspect and the 5th aspect, the tumor cell surface antigen (TAA) includesHER2、BCMA、CD123、GPC3、FAP、VEGFR、EGFR、PD-L1、CD19、CD125、HGFR、FGFR2、IGF-1R、SALL4Or LASEP1.In some embodiments of fourth aspect and the 5th aspect, the bispecific antibody is able to achieve T cell swollenThe enrichment on oncocyte periphery.
In some embodiments of fourth aspect and the 5th aspect, the bispecific antibody can improve T cell to swollenThe killing-efficiency of oncocyte.
6th aspect, this application provides pharmaceutical compositions, and it includes antibody described in first aspect or second aspect, orBispecific antibody and pharmaceutically acceptable excipient, diluent or carrier described in person's fourth aspect or the 5th aspect.
In some embodiments, described pharmaceutical composition is for preventing or treating tumour.
In some embodiments, the tumour is malignant tumour.
7th aspect, this application provides antibody described in first aspect or second aspect or fourth aspect or the 5Bispecific antibody described in aspect is preparing the purposes in the drug for preventing or treating tumour.
In some embodiments, the tumour is malignant tumour.
Eighth aspect, this application provides the methods prevented or treat tumour, including give first to individual in needAntibody described in aspect or second aspect perhaps fourth aspect or the 5th aspect described in bispecific antibody or the 7th sidePharmaceutical composition described in face.
In some embodiments, the tumour is malignant tumour.
Detailed description of the invention
Fig. 1 shows the binding ability of elisa assay anti-human CD3E monoclonal antibody (bacteriophage scFv) and different CD3E.
Fig. 2 shows the binding ability of elisa assay different anti-human CD3E monoclonal antibodies and CD3E recombinant protein.
Fig. 3 shows the binding ability of flow cytometry different anti-human CD3E monoclonal antibodies and different genera PBMC.
Fig. 4 shows the bioactivity testing result of different AntiCD3 McAb E monoclonal antibody activation T cells.
Amino acid sequence explanation
SEQ ID NO:1 shows the amino acid sequence of recombined human (homo sapiens) CD3E extracellular region (huCD3E-ECD)Column.
SEQ ID NO:2 shows the amino acid sequence of recombined human (homo sapiens) CD3D extracellular region (huCD3D-ECD)Column.
SEQ ID NO:3 display recombination machin (Macaca fascicularis) CD3E extracellular region (mfCD3E-ECD)Amino acid sequence.
SEQ ID NO:4 display recombination machin (Macaca fascicularis) CD3D extracellular region (mfCD3D-ECD)Amino acid sequence.
SEQ ID NO:5 shows the amino acid sequence of mouse (mus musculus) CD3E extracellular region (mCD3E-ECD).
SEQ ID NO:6 shows the amino acid sequence of mouse (mus musculus) CD3D extracellular region (mCD3D-ECD).
SEQ ID NO:7 shows the amino acid sequence of His label (His).
SEQ ID NO:8 shows the amino acid sequence of the Fc section (mFc) of mouse (mus musculus) IgG antibody 2a.
SEQ ID NO:9 shows the amino acid sequence of the Fc segment mutant (FcK) of human IgG1's antibody.
SEQ ID NO:10 shows the amino acid sequence of the Fc segment mutant (FcH) of human IgG1's antibody.
SEQ ID NO:11 shows the amino acid sequence of people (homo sapiens) IgG1 subtype heavy chain constant region.
SEQ ID NO:12 shows the amino acid sequence of people (homo sapiens) IgG2 subtype heavy chain constant region.
SEQ ID NO:13 shows the amino acid sequence of people (homo sapiens) IgG4 subtype heavy chain constant region.
SEQ ID NO:14 shows the amino acid sequence of people (homo sapiens) κ hypotype constant region of light chain.
SEQ ID NO:15 shows the amino acid sequence of people (homo sapiens) λ hypotype constant region of light chain.
SEQ ID NO:16 shows the amino acid sequence of anti-human CD3E single-chain antibody S1C3, the difference of SEQ ID NO:19 and 20Show the amino acid sequence of itself VH and VL sequence.
SEQ ID NO:17 shows the amino acid sequence of anti-human CD3E single-chain antibody S1F6, the difference of SEQ ID NO:21 and 22Show the amino acid sequence of itself VH and VL sequence.
SEQ ID NO:18 shows the amino acid sequence of anti-human CD3E single-chain antibody S4C1, the difference of SEQ ID NO:23 and 24Show the amino acid sequence of itself VH and VL sequence.
Detailed description of the Invention
Present inventor has obtained new anti-human CD3E antibody by antibody engineering technology.In multiple sides of the applicationFace provides new anti-human CD3E antibody or its antigen-binding fragment, encodes the antibody or the nucleic acid point of its antigen-binding fragmentSon, the host cell comprising the nucleic acid molecules or carrier, prepares and purifies the antibody at the carrier comprising the nucleic acid moleculesThe medicine and biological applications of method and the antibody or its antigen-binding fragment.According to the variable region of antibody provided by the present applicationAmino acid sequence, the antibody molecule of overall length can be constructed as drug for preventing or treating tumour.
Unless otherwise specified, the implementation of the application uses molecular biology, microbiology, the cell biological of this field routine, biochemistry and immunological technique.
Unless otherwise specified, the meaning that there are term use herein those skilled in the art to be generally understood.
Definition
Term " antibody " as used herein is to refer to be located at via at least one in the variable region of immunoglobulin moleculesAntigen recognition site be specifically bound to the immunoglobulin molecules of target.Target includes but is not limited to carbohydrate, morePolynucleotide, lipid, polypeptide etc.." antibody " used herein not only includes complete (i.e. overall length) antibody, but also is wrappedInclude its antigen-binding fragment (such as Fab, Fab', F (ab')2, Fv), its variant, the fusion protein comprising antibody moiety, peopleSource antibody, chimeric antibody, double antibody, linear antibodies, single-chain antibody, multi-specificity antibody (such as bispecific antibody) and appointWhat he includes that the modification of the immunoglobulin molecules of the antigen recognition site of required specificity configures, the glycosylation including antibodyVariant, the amino amino sequence variants of antibody and the antibody of covalent modification.
In general, complete or overall length antibody includes two heavy chains and two light chains.Each heavy chain contains heavy chain region of variability(VH) and the first, second and third constant region (CH1, CH2 and CH3).Each light chain contains light chain region of variability (VL) and constant region(CL).The antibody of overall length can be any kind of antibody, such as IgD, IgE, IgG, IgA or IgM (or above-mentioned subclass), butAntibody needs not belong to any specific classification.According to the antibody amino acid amino acid sequence of the constant domain of heavy chain, can will exempt fromEpidemic disease globulin is appointed as different classifications.In general, immunoglobulin is there are five types of main classification: IgA, IgD, IgE, IgG andIgM, and have in these classifications it is several can be further discriminated between into again subclass (homotype), such as IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2.Heavy-chain constant domains corresponding to different immunoglobulin class are referred to as α, δ, ε, γ and μ.NoThe sub-unit structure and three-dimensional structure of generic immunoglobulin are well known.
Term " antigen-binding fragment or antigen-binding portion thereof " as used herein refers to the complete antibody being responsible in conjunction with antigenA part of molecule or region.Antigen binding domain may include heavy chain region of variability (VH), light chain region of variability (VL) or both.Each of VH and VL usually contain three complementary determining regions CDR1, CDR2 and CDR3.
Well known to those skilled in the art, complementary determining region (CDR usually has CDR1, CDR2 and CDR3) is right in variable regionThe maximum region of affinity and specific effect of antibody.Common definition mode that there are two types of the cdr amino acid sequences of VH or VL,That is Chothia definition and kabat definition.(refering to such as Kabat, " Sequences of Proteins ofImmunological Interest ", National Institutes of Health, Bethesda, Md. (1991)；A1-Lazikani et al., J.Mol.Biol.273:927-948 (1997)；And Martin et al.,Proc.Natl.Acad.Sci.USA86:9268-9272(1989)).It, can be with for giving the variable region amino acid sequence of antibodyIt is defined according to Chothia or Kabat defines to determine the cdr amino acid sequence in VH and VL amino acid sequence.In the applicationEmbodiment in, define cdr amino acid sequence using Chothia.
For giving the variable region amino acid sequence of antibody, variable region amino acid sequence can be analyzed in several waysMiddle cdr amino acid sequence, such as can use online software Abysis and determine (http://www.abysis.org/).
The example of antigen-binding fragment includes but is not limited to: (1) Fab segment can be with VL-CL chain and VH-CH1The monovalent fragment of chain；(2)F(ab')2Segment, can be tool, there are two the bivalent fragment of Fab' segment, two Fab' segmentsIt is connected by the disulphide bridges (i.e. the dimer of Fab') of hinge area；(3) the Fv segment in the domain VL and VH of the single armed with antibody；(4)ScFv (scFv) can be the single victory peptide chain being made of the domain VH and the domain VL via victory peptide connector；And (5)(scFv)2, may include two domains VH and two domains VL connected by victory peptide connector, which is via disulphide bridgesIt is combined with two domains VH.
Term " bispecific antibody " as used herein be with the antibody for combining two kinds of different antigenic capacities, can be byTwo Fc segments and two antigen-binding portions merged respectively with it composition.
Term " specific binding " as used herein refers to the nonrandom association reaction between two molecules, such as antibody is extremelyThe combination of epitope.
Term " monoclonal antibody " as used herein refers to by the antibody of the antibody population acquisition of basic homogeneity, that is, in addition to canIt can be identical there are each antibody for other than abiogenous mutation, forming group in a small amount of individual.Dan Ke described hereinGrand antibody particularly including " chimeric " antibody, wherein a part of heavy chain and/or light chain with from specific species or belong to specificOrresponding amino acid sequence in the antibody of antibody class or subclass is identical or homologous, and the remaining part of heavy chain and/or light chain and comeDerived from another species or the orresponding amino acid sequence that belongs in the antibody of another antibody class or subclass is identical or homologous, and also wrapsThe segment for including such antibody, as long as they can show desired biological activity (U.S. Patent number 4,816,567；WithMorrison et al., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984)).
Term " tumour " as used herein refers to the neoplasm or entity lesion formed by abnormal cell growth.Tumour can be withIt is benign, premalignant or pernicious.
Term " malignant tumour " as used herein refers to or describes the physiological condition of mammal, is typically featured in thatNot modulated cell growth.Exemplary malignant tumour includes: cancer, solid tumor, melanoma sarcoma, neoplastic hematologic disorder, reproduction cellTumor and enblastoma.More specific examples of malignant tumour include: kidney, including Small Cell Lung Cancer, non-small cell lung cancer, lung glandThe lung cancer of cancer and lung squamous cancer, bladder cancer, breast cancer, cervical carcinoma, colon cancer, liver cancer (hepatic carcinoma), including stomach and intestineThe gastric cancer of cancer, prostate cancer, cancer of pancreas, peritoneal cancer, hepatocellular carcinoma, spongioblastoma, oophoroma, liver cancer (liverCancer), urinary tract cancer, hepatoma, the carcinoma of the rectum, colorectal cancer, endometrium or uterine cancer, salivary-gland carcinoma, squamous cellCancer (for example, dermoid cancer), carcinoma of vulva, thyroid cancer, cancer of anus, carcinoma of penis, melanoma, Huppert's disease and BCell lymphoma, the cancer of the brain and head and neck cancer and associated transitions stove.
Term " neoplastic hematologic disorder " as used herein refer to due to the growing multiplication of abnormal cell it is uncontrolled caused by, moreThe origin position of these abnormal cells is marrow in number situation, this is also exactly the place that blood cell generates.Exemplary bloodLiquid tumour includes each quasi-leukemia, Huppert's disease and malignant lymphoma.More specific examples of neoplastic hematologic disorder include: urgencyProperty lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelocytic leukemia (AML),Chronic granulocytic leukemia (CML), hairy cell (HCL), T cell prolymphocytic leukemia, bulky grain leachingBar chronic myeloid leukemia, juvenile myelomonocytic leukaemia, B cell prolymphocytic leukemia, Hugh Burkitt leukaemia and atHuman T-cell's property leukaemia, non-Hodgkin lymphoma, B cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphTumor, primary macroglobulinaemia (Macroglobulinemia), splenic marginal zone lymthoma, thick liquid cellTumor, extranodal marginal zone B cell lymphoma, MALT lymthoma, knot inner peripheral area B cell lymphoma (NMZL), follicular lymphoma,Lymphoma mantle cell, diffusivity large B cell lymphoid tumor, mediastinum (thymus gland) large B cell lymphoid tumor, intravascular large B cell lymphoma,Lymphoma primary effusion, Burkitt lymphoma, B cell chronic lymphocytic lymphoma, classical Hodgkin lymphoma, knotSection property lymphocytic predominance Hodgkin lymphoma, adult T cell lymphoma, knot external nose type NK/T cell lymphoma, enteropathy type TSyndrome, primary skin are thanked in cell lymphoma, liver and spleen t cell lymphoma, mother cell NK cell lymphoma, mycosis fungoides, matchSkin CD30 positive T cell lymphocytic hyperplasia disease, lymphoma primary cutaneous anaplastic large cell, lymphomatoid papulosis, blood vessel are exempted fromEpidemic disease mother cell t cell lymphoma non-refers in particular to type lymphoma peripheral T cell and primary cutaneous type.
Term " solid tumor " as used herein refers to and can be laid one's hand on by clinical examination such as x ray photograph, CT scan, B ultrasound or palpationAnd the tangible lump arrived.Clinically the solid tumor of diagnosis and treatment is divided to pernicious and two kinds benign.Malignant solid tumor includes: children Huo QijinLymthoma: lymphocytic predominance, nodular sclerosis, mixed cell type, lymphocytic depletion；Children's non-Hodgkin's lymphTumor: preceding lymphoblastic lymphoma, small non-cleaved cell lymphoma (Hugh Burkitt/non-Burkitt lymphoma), the leaching of diffusivity large B cellBar tumor, primary cutaneous type etc.；Children's tumor of kidney: the nephroblastoma (Wilms tumor), clear cell carcinoma of kidney, kidney are horizontalLine flesh sample tumor, Renal clear cell sarcoma, kidney primitive neuroectodermal tumors etc.；Primary Neuroblastoma in Children: neuroblastoma, sectionCellular neural blastoma, ganglioneuroma；Children's extracranial germ cell tumor: mature teratoma, immature teratoma, interior embryoSinus tumor (yolk sac tumor), seminoma, dysgerminoma, chorioepithelioma, embryonal carcinoma etc.；Osteosarcoma and chondrosarcoma；Children Rhabdomyosarcoma: embryo type, acinus type, pleomorphic type etc.；Children soft tissue sarcoma: fibrosarcoma, malignant fibrous histiocytoma are thinBorn of the same parents' tumor, embryonal-cell lipoma, leiomyosarcoma, angiosarcoma, lymphangioendothelial sarcoma, malignant schwannoma, alveolar soft part sarcoma, onSkin sample sarcoma, clear cell sarcoma, malignant mela noma, synovial sarcoma, rush fibroproliferative small circle cell tumor etc.；You Wenshi familyRace's sarcoma: ewing's sarcoma, primitive neuroectodermal tumors；Children's liver neoplasm: (embryo type, does not divide at fetal type hepatoblastomaChange type), hepatocellular carcinoma；Retinoblastoma；Other tumours: Posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroidCancer, thymoma, pulmonary blastoma, pancreatoblastoma, islet-cell tumour, ileocecus class cancer, celiothelioma etc..Benign solid tumor packetIt includes: lymphangioma, hemangioma, thyroglossal cyst etc..
In a first aspect, it includes the amino containing HCDR1, HCDR2 and HCDR3 this application provides the antibody for combining people CD3EThe heavy chain variable region of acid sequence and light chain variable region containing LCDR1, LCDR2 and LCDR3 amino acid sequence, wherein
The HCDR1 amino acid sequence is GFKFSGY, and the HCDR2 amino acid sequence is WFDGSR, the HCDR3 ammoniaBase acid sequence is QMGYWHFGL, and the LCDR1 amino acid sequence is RASQGINNSLT, and the LCDR2 amino acid sequence isGASNRET, the LCDR3 amino acid sequence are QQWLKLPPT；Or
The HCDR1 amino acid sequence is GFTFSNA, and the HCDR2 amino acid sequence is KDKSNNYA, the HCDR3Amino acid sequence is VHYGVRFFYTMDV, and the LCDR1 amino acid sequence is RPSQSLVHNNGNTYLS, the LCDR2 aminoAcid sequence is KVSNRFS, and the LCDR3 amino acid sequence is GQGTQYPFT；Or
The HCDR1 amino acid sequence is GFTFSNA, and the HCDR2 amino acid sequence is KSKSDNYA, the HCDR3Amino acid sequence is VHYGAYYGVDA, and the LCDR1 amino acid sequence is RSSQSLVHSNRNTYLN, the LCDR2 amino acidSequence is KVSNRFS, and the LCDR3 amino acid sequence is GQGTHAPYA；
Wherein HCDR and LCDR amino acid sequence is defined according to Chothia.
In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody such as SEQ ID NO:19,21 orShown in person 23.
In some embodiments, the amino acid sequence of the light chain variable region of the antibody such as SEQ ID NO:20,22 orShown in person 24.
In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:19,The amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:20；Or
The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:21, the light chain variable of the antibodyThe amino acid sequence in area is as shown in SEQ ID NO:22；Or
The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO:23, the light chain variable of the antibodyThe amino acid sequence in area is as shown in SEQ ID NO:24.
Second aspect, this application provides the antibody for combining people CD3E, wherein the amino of the heavy chain variable region of the antibodyAny one of acid sequence and SEQ ID NO:19,21 or 23 have at least 90% consistency, and the antibody is lightAny one of amino acid sequence and SEQ ID NO:20,22 or 24 of chain variable region have at least 90% consistency.
In some embodiments of first aspect and second aspect, the antibody is monoclonal antibody.
In some embodiments of first aspect and second aspect, the antibody is whole antibody, Fab segment, F (ab ')2Segment or Single-Chain Fv Fragment of Murine (scFv).
In some embodiments of first aspect and second aspect, the antibody is human antibody.
In some embodiments of first aspect and second aspect, the antibody also includes selected from IgG1 hypotype, IgG2The heavy chain constant region of hypotype or IgG4 hypotype.
In some specific embodiments of first aspect and second aspect, the heavy chain constant region is IgG1 hypotype.
In some embodiments of first aspect and second aspect, the antibody also includes selected from κ hypotype or λ hypotypeConstant region of light chain.
In some specific embodiments of first aspect and second aspect, the constant region of light chain is κ hypotype.
In some embodiments of first aspect and second aspect, the antibodies mediate t cell activation.
The third aspect, this application provides nucleic acid molecules, encode antibody described in first aspect or second aspect or itsAntigen-binding portion thereof.
In some embodiments, the nucleic acid molecules are operably connected to regulation amino acid sequence, regulate and control amino acidSequence can be identified with the transformed host cell of the carrier.
Fourth aspect, this application provides bispecific antibodies, it includes the first arm for people CD3E and are directed to tumourSecond arm of cell surface antigen (TAA), wherein first arm for people CD3E includes:
Amino acid sequence is the HCDR1 of GFKFSGY, and amino acid sequence is the HCDR2 of WFDGSR, and amino acid sequence isThe HCDR3 of QMGYWHFGL, amino acid sequence are the LCDR1 of RASQGINNSLT, and amino acid sequence is the LCDR2 of GASNRET, ammoniaBase acid sequence is the LCDR3 of QQWLKLPPT；Or
Amino acid sequence is the HCDR1 of GFTFSNA, and amino acid sequence is the HCDR2 of KDKSNNYA, and amino acid sequence isThe HCDR3 of VHYGVRFFYTMDV, amino acid sequence are the LCDR1, amino acid sequence KVSNRFS of RPSQSLVHNNGNTYLSLCDR2, amino acid sequence be GQGTQYPFT LCDR3；Or
Amino acid sequence is the HCDR1 of GFTFSNA, and amino acid sequence is the HCDR2 of KSKSDNYA, and amino acid sequence isThe HCDR3 of VHYGAYYGVDA, amino acid sequence are the LCDR1 of RSSQSLVHSNRNTYLN, and amino acid sequence is KVSNRFS'sLCDR2, amino acid sequence are the LCDR3 of GQGTHAPYA；
Wherein HCDR and LCDR amino acid sequence is defined according to Chothia.
In some embodiments of fourth aspect, first arm for people CD3E includes amino acid sequence such as SEQHeavy chain variable region shown in ID NO:19,21 or 23.
In some embodiments of fourth aspect, first arm for people CD3E includes amino acid sequence such as SEQLight chain variable region shown in ID NO:20,22 or 24.
In some embodiments of fourth aspect, first arm for people CD3E includes:
Amino acid sequence heavy chain variable region as shown in SEQ ID NO:19, amino acid sequence is as shown in SEQ ID NO:20Light chain variable region；Or
Amino acid sequence heavy chain variable region as shown in SEQ ID NO:21, amino acid sequence is as shown in SEQ ID NO:22Light chain variable region；Or
Amino acid sequence heavy chain variable region as shown in SEQ ID NO:23, amino acid sequence is as shown in SEQ ID NO:24Light chain variable region.
5th aspect, this application provides bispecific antibodies, it includes the first arm for people CD3E and are directed to tumourSecond arm of cell surface antigen (TAA), wherein first arm for people CD3E includes: amino acid sequence and SEQ IDAny one of NO:19,21 or 23 have the heavy chain variable region of at least 90% consistency, and amino acid sequence and SEQAny one of ID NO:20,22 or 24 have the light chain variable region of at least 90% consistency.
In some embodiments of fourth aspect and the 5th aspect, first arm for people CD3E includes to be directed to peopleThe Fab segment or Single-Chain Fv Fragment of Murine (scFv) of CD3E.
Since bispecific antibody has for two kinds of not two of synantigen different antigen-binding portions, and antigen-binding portionIt can be single-chain antibody (scfv) or two kinds of forms of Fab segment.
Herein, bispecific antibody is also described as tool there are two " arm ", it, can will be double for example, using centre as boundarySpecific antibody is divided into two arms.The arm of bispecific antibody can (Fab segment be single-stranded anti-by Fc segment and antigen-binding portionBody) composition.For the arm being made of Fc segment and Fab segment, structure is similar to common antibody, containing complete heavy chain withLight chain, therefore the structure of such arm can be expressed as Fc+Fab, can also be expressed as the heavy chain (heavy chain variable region in Fc+FabWith CH1 segment)+light chain (chain moiety in Fab).When antigen-binding portion of two arms all containing Fab pieces, thusThe structure of the bispecific antibody of formation is close to natural antibody.
In some embodiments of fourth aspect and the 5th aspect, first arm for people CD3E also includes to be selected fromThe heavy chain constant region of IgG1 hypotype, IgG2 hypotype or IgG4 hypotype.
In some specific embodiments of fourth aspect and the 5th aspect, the heavy chain constant region is IgG1 hypotype.
In some embodiments of fourth aspect and the 5th aspect, first arm for people CD3E also includes to be selected fromThe constant region of light chain of κ hypotype or λ hypotype.
In some specific embodiments of fourth aspect and the 5th aspect, the constant region of light chain is κ hypotype.
In some embodiments of fourth aspect and the 5th aspect, the tumor cell surface antigen (TAA) includesHER2、BCMA、CD123、GPC3、FAP、VEGFR、EGFR、PD-L1、CD19、CD125、HGFR、FGFR2、IGF-1R、SALL4Or LASEP1.
In some embodiments of fourth aspect and the 5th aspect, the bispecific antibody is able to achieve T cell swollenThe enrichment on oncocyte periphery.
In some embodiments of fourth aspect and the 5th aspect, the bispecific antibody can improve T cell to swollenThe killing-efficiency of oncocyte.
6th aspect, this application provides pharmaceutical compositions, and it includes antibody described in first aspect or second aspect, orBispecific antibody and pharmaceutically acceptable excipient, diluent or carrier described in person's fourth aspect or the 5th aspect.
In some embodiments, described pharmaceutical composition is for preventing or treating tumour.
In some embodiments, the tumour is malignant tumour.
In some embodiments, described pharmaceutical composition also may include one of following or a variety of: lubricant, such as slidingMountain flour, magnesium stearate and mineral oil；Wetting agent；Emulsifier；Suspending agent；Preservative, such as benzoic acid, sorbic acid and calcium propionate；IncreaseEdulcorant and/or flavoring agent etc..
In some embodiments, the pharmaceutical composition in the application can be formulated as tablet, pill, pulvis, pastille, the wine made of broomcorn milletThe forms such as agent, suspension, emulsion, solution, syrup, suppository or capsule.
In some embodiments, it can use the medicine group that any physiologically acceptable administration mode delivers the applicationClose object, these administration modes include but is not limited to: oral administration, parenteral administration, nose administration, rectally, in peritonaeum toMedicine, intravascular injection, subcutaneous administration, percutaneous dosing, inhalation etc..
It in some embodiments, can be pharmaceutically acceptable with optionally by mixing reagent with the desired purityCarrier, excipient etc., the pharmaceutical composition of therapeutical uses is formulated in the form of lyophilized preparation or aqueous solution for storing.
7th aspect, this application provides antibody described in first aspect or second aspect or fourth aspect or the 5Bispecific antibody described in aspect is preparing the purposes in the drug for preventing or treating tumour.
In some embodiments, the tumour is malignant tumour.
Eighth aspect, this application provides the methods prevented or treat tumour, including give first to individual in needAntibody described in aspect or second aspect perhaps fourth aspect or the 5th aspect described in bispecific antibody or the 7th sidePharmaceutical composition described in face.
In some embodiments, the tumour is malignant tumour.
In other respects, the application also provide encode the isolated nucleic acid molecules of antibody of the present invention or its light chain or heavy chain withAnd the carrier comprising the nucleic acid molecules, the host cell comprising the carrier and the method for generating the antibody.SomeIn embodiment, the nucleic acid molecules are operably connected to regulation amino acid sequence, and regulation amino acid sequence can be by with instituteState the transformed host cell identification of carrier.In some embodiments, generate antibody method include culture host cell withConvenient for expressing nucleic acid.In some embodiments, the method for generating antibody further includes recycling antibody from host cell culture medium.
It is deposited in addition, the antibody of specific binding people CD3E as described herein can also be used for CD3E in detection biological sample?.Detection method based on antibody is well known in the art, and including such as ELISA, immunoblotting, radio-immunityTest, immunofluorescence, immunoprecipitation and other the relevant technologies.
It should be appreciated that be discussed in detail above only for making those skilled in the art more clearly understand present context,And it is not intended to limit in any way.Those skilled in the art can carry out various changes and change to the embodimentChange.
Embodiment
Following embodiment is merely to illustrate rather than limits the purpose of the application range.
Embodiment 1: the preparation of recombinant protein
Use a variety of different recombinant proteins during preparing AntiCD3 McAb E monoclonal antibody, including recombined human CD3E extracellularArea (huCD3E-ECD, SEQ ID NO:1), people CD3D extracellular region (huCD3D-ECD, SEQ ID NO:2), machin CD3E born of the same parentsOutskirt (mfCD3E-ECD, SEQ ID NO:3), machin CD3D extracellular region (mfCD3D-ECD, SEQ ID NO:4), mouseCD3E extracellular region (mCD3E-ECD, SEQ ID NO:5), mouse CD3D extracellular region (mCD3D-ECD, SEQ ID NO:6).TheseRecombinant protein has a large amount of posttranslational modification (such as glycosylation or disulfide bond), thus utilizes mammalian cell expression systemIt would be even more beneficial to keep the structure and function of recombinant protein.In addition, purifying for convenience, the recombinant protein of non-antibody class is in C-terminalIt is added to His label (SEQ ID NO:7) or the Fc section (mFc, SEQ ID NO:8) of mouse antibodies IgG2a.In addition, thinCellular surface, CD3E can form heterodimer with CD3D or CD3G, therefore anti-in order to prepare the CD3E with native conformationOriginal, we used Fc segment mutant (FcK, the SEQ ID of human IgG1's antibody based on KIH (Knob-into-Hole) technologyNO:9 or FcH, SEQ ID NO:10), by CD3E fusion in the N-terminal for the Fc (FcK) being mutated containing Knob, CD3D fusion is existedThe N-terminal of Fc (FcH) containing Hole mutation, prepares CD3E and CD3D heterodimer.In preparation and reorganization antibody, heavy chain of antibodyConstant region can be the IgG1 hypotype (SEQ ID NO:11), IgG2 hypotype (SEQ ID NO:12) or IgG4 hypotype (SEQ of peopleID NO:13), constant region of light chain can be the κ hypotype (SEQ ID NO:14) or λ hypotype (SEQ ID NO:15) of people.
According to the amino acid sequence of the various purpose recombinant proteins of Uniprot database, design and synthesize above-mentioned various heavyThe gene (including His label, mFc Fc encoding gene) of histone.Using conventional molecular biological technology by each of synthesisKind recombinant protein gene cloning is then utilized to suitable carrier for expression of eukaryon (such as pcDNA3.1 of invitrogen company)Liposome (such as 293fectin of invitrogen company) or other transfection reagents (such as PEI) are by the recombinant protein of preparationExpression plasmid is transfected into HEK293 cell (such as HEK293F of invitrogen company), trains under serum free suspension condition of cultureIt supports 3-5 days.Then culture supernatant is harvested by modes such as centrifugations.
The recombinant protein of His tag fusion expression utilizes metal chelate affinity chromatography column (the HisTrap FF of such as GE companyDeng) a step purifying is carried out to the recombinant protein in culture supernatant.The recombinant protein and recombinant antibodies of mFc amalgamation and expression are usedProteinA/G affinity column (the Mabselect SURE of such as GE company) carries out a step purifying.Then desalting column is utilized(the Hitrap desaulting of such as GE company) by recombinant protein save buffer exchange be PBS buffer solution (pH7.0) orOther suitable buffers.When necessary, antibody samples can be filtered with degerming, then packing is stored in -20 DEG C.
Embodiment 2: Antibody library is presented using bacteriophage and screens anti-human CD3E monoclonal antibody
The recombined human CD3ECD3D heterodimer (huCD3ECD3D) prepared with embodiment 1 is sieved for antigen using solid phaseSelecting Policy Filtering that the bacteriophage of people's single-chain antibody library is presented, (experimental technique process can be found in Chinese patent application theEmbodiment 1 in No. 201510097117.0), the final human single chain variable fragments antibody S1C3 for obtaining 3 plants of specific binding people CD3E(amino acid sequence of SEQ ID NO:16, VH and VL be respectively SEQ ID NO:19 and 20), S1F6 (SEQ ID NO:17, VHAmino acid sequence with VL be respectively SEQ ID NO:21 and 22), the S4C1 (amino acid sequence of SEQ ID NO:18, VH and VLRespectively SEQ ID NO:23 and 24).Using the website NCBI online tool IGBLAST to the light of 3 source of people single-chain antibody moleculesHeavy chain variable amino acid sequence carries out homology analysis, and the VH of S1C3 comes from IGHV3-33 as the result is shown, and VL comes from IGKV1-33；The VH of S1F6 comes from IGHV3-15, and VL comes from IGKV2-29；The VH of S4C1 comes from IGHV3-15, and VL comes from IGKV2-30.
With the recombined human CD3ECD3D heterodimer (huCD3ECD3D) of preparation, machin CD3ECD3D heterodimer(mfCD3ECD3D), mouse CD3ECD3D heterodimer (mCD3ECD3D), people CD3E (huCD3E-his), people CD3D(huCD3D-his), machin CD3E (mfCD3E-his) and mouse CD3E (mCD3E-his) are coated with 96 hole elisa plates (1 respectivelyμ g/ml, 100 holes μ l/), 4 DEG C of coatings are overnight.It is closed 1 hour using confining liquid PBS-0.1%Tween20-3% milk at 37 DEG CAfterwards, the bacteriophage supernatant (bacteriophage scFv) of recombination AntiCD3 McAb E monoclonal antibody S1C3, S1F6 and S4C1,100 μ l/ are separately added intoHole, 37 DEG C combine 1 hour.Wash elisa plate with PBST buffer, be added the anti-M13 antibody of HRP (Sinobiological,11973-MM05T-H), it combines 1 hour for 37 DEG C.Elisa plate is washed with PBST buffer, OPD substrate developing solution, 5- is then addedThe H of 1M is used after ten minutes2SO4Color development stopping measures OD value in 492nm/630nm double wave strong point using microplate reader.ELISAResult (Fig. 1) display is analyzed, S1C3 identifies huCD3ECD3D heterodimer, fails to see others' CD3E subunit (huCD3E-his),Intersect with machin CD3E and mouse CD3E without kind；S1F6 identifies people CD3E subunit (huCD3E-his), with huCD3E-hisAnd huCD3ECD3D has clear binding signal, there are kinds to intersect with machin CD3E, nonrecognition mouse CD3E；S4C1 identificationPeople CD3E subunit (huCD3E-his) has clear binding signal with huCD3E-his and huCD3ECD3D, with machin CD3EThere are kind intersection, nonrecognition mouse CD3E.
Embodiment 3: the analysis of anti-human CD3E monoclonal antibody combination CD3E recombinant protein
Using conventional molecular biological method, the light chain and heavy chain of single-chain antibody S1C3, S1F6 and S4C1 will be encoded respectivelyGene cloning to carrier for expression of eukaryon, preparation and reorganization human IgG1's-κ form human antibody.
With the recombined human CD3E (huCD3E-his), people CD3D (huCD3D-his), machin CD3E (mfCD3E- of preparationHis it) is coated with respectively in 96 hole elisa plates (1 μ g/ml, 100 holes μ l/) with mouse CD3E (mCD3E-his), 4 DEG C of coatings are overnight.BenefitWith confining liquid PBS-0.1%Tween20-3% milk after 37 DEG C are closed 1 hour, it is separately added into each recombination AntiCD3 McAb E monoclonalAntibody S1C3, S1F6 and S4C1 (1.0 hole μ g/100 μ l/), 37 DEG C combine 1 hour.Elisa plate is washed with PBST buffer, is addedEnter HRP mouse anti-human igg (bioss, bsm-0297M-HRP), 37 DEG C combine 1 hour.Elisa plate is washed with PBST buffer, soOPD substrate developing solution is added afterwards, the H of 1M is used after 5-10 minutes2SO4Color development stopping, using microplate reader in 492nm/630nm double waveStrong point measures OD value.Elisa assay result (Fig. 2) display, S1C3 cannot identify that the independent subunit of CD3E, S1F6 and S4C1 are sameWhen identify people CD3E and machin CD3E, intersect with mouse CD3E without kind.
Embodiment 4: anti-human CD3E monoclonal antibody combination PBMC analysis
In 96 orifice plate of V-type respectively by anti-human CD3E monoclonal antibody S1C3, S1F6 and S4C1 (concentration is 10 μ g/ml)Mix that (cell is diluted to 10*10 with human PBMC, rhesus macaque PBMC and mouse PBMC6/ ml, by antibody and cell with each 100 μ l/The amount in hole mixes in equal volume), in 4 DEG C of incubation 60min；It is washed 2 times with PBS buffer solution；With goat anti-human IgG antibodies-FITC (1:400 dilute, 100 holes μ l/) it is incubated with 30min in 4 DEG C, Flow cytometry is carried out after washing 2 times with PBS buffer solution.As a result(Fig. 3 and table 1) shows that S1C3 can be in conjunction with human PBMC, but cannot combine the PBMC of Monkeys and mice；S1F6 and S4C1 are sameWhen identification people and rhesus macaque PBMC, but mouse PBMC cannot be combined.
The combination of table 1. AntiCD3 McAb E monoclonal antibody and people, Monkeys and mice PBMC
| MFI(FITC) | It compares (being not added with antibody) | S1C3 | S1F6 | S4C1 |
| Human PBMC | 11860 | 58155 | 18401 | 16730 |
| Rhesus macaque PBMC | 3470 | 3430 | 20431 | 18564 |
| Mouse PBMC | 3818 | 3426 | 2550 | 1454 |
Experimental result in integrated embodiment 3 and embodiment 4 it can be concluded that S1C3 to fail to see others' CD3E independentSubunit, but can normally combine the CD3ECD3D heterodimer on human T-cell surface；In embodiment 3 molecular level S1C3 notIn conjunction with machin CD3E, S1C3 is not in conjunction with rhesus macaque cell surface CD3E in embodiment 4, therefore S1C3 and machin and perseveranceKind intersection is all not present in river monkey；S1C3 and mouse CD3E is also not present kind and intersects；S1F6 and S4C1 can identify people CD3EIndependent subunit has kind to intersect with machin and rhesus macaque CD3E, nonrecognition mouse CD3E.
Embodiment 5: the affinity analysis of anti-human CD3E monoclonal antibody combination CD3E
The affinity of each AntiCD3 McAb E monoclonal antibody (S1C3, S1F6 and S4C1) is measured using Biacore X100plus.Amino coupled kit, human antibody capture the related reagents such as 10 × HBS-EP of kit, CM5 chip and pH7.4 and consumptive material is equalPurchased from GE healthcare.According to the specification in kit, using the method for amino coupled by anti-human Fc sections of antibody couplingTo CM5 chip surface, antibody protein is then diluted to suitable concentration (S1C3:1 μ g/ml, S1F6:3 μ g/ml, S4C1:1 μ g/Ml), 10 μ l/min sample introduction 30s guarantees the antibody of 100RU or so by the antibody capture of anti-human Fc.By huCD3ECD3D orMfCD3ECD3D is arranged a series of concentration gradient (100nM, 33.3nM, 11.1nM, 3.7nM and 1.23nM), 30 μ l/min intoSample 120s dissociates 600s, is regenerated with the 10mM Glycine-HCl of pH1.7 to chip surface, 25 DEG C of each monoclonals of measurementThe affinity of antibody.It assesses software 2.0.1 editions using Biacore X100 to analyze biacore data, fitting result pointNot as shown in table 2 and table 3.
The affinity constant of 2. AntiCD3 McAb E monoclonal antibody combination huCD3ECD3D of table
| Antibody type | Kon | Koff | KD |
| S1C3 | 8.143E+4 | 3.563E-3 | 4.375E-8 |
| S1F6 | 7.396E+4 | 1.503E-3 | 2.032E-8 |
| S4C1 | 1.333E+5 | 1.558E-3 | 1.169E-8 |
The affinity constant of 3. AntiCD3 McAb E monoclonal antibody combination mfCD3ECD3D of table
| Antibody type | Kon | Koff | KD |
| S1C3 | It is unbonded | - | - |
| S1F6 | 2.601E+4 | 7.353E-4 | 2.827E-8 |
| S4C1 | 2.497E+4 | 1.539E-3 | 6.163E-8 |
Embodiment 6: the analysis of anti-human CD3E mediated monoclonal antibody t cell activation
The Analysis on Biological Activity of AntiCD3 McAb E monoclonal antibody activation T cell is carried out based on Jurkat Dual cell line.It is anti-CD28 monoclonal antibody peridium concentration be 4 μ g/ml, AntiCD3 McAb E monoclonal antibody initial concentration be 30 μ g/ml, 2 times of gradient dilutions,Anti- CD28 monoclonal antibody is mixed in equal volume with the AntiCD3 McAb E monoclonal antibody of various concentration, is laid on by totally 10 concentration gradientsIn 96 orifice plates, 3 multiple holes are arranged in each concentration, and 50 holes μ l/ are placed in clean bench and air-dry overnight.It second day willCell is resuspended as 1*10 with the culture medium without screening pressure for Jurkat Dual cell count6A cell/ml, 100 holes μ l/It being added in 96 orifice plates, continues to cultivate 20h, 1500rpm is centrifuged 5min, 50 μ l supernatants is taken to be added in 96 new orifice plates, then plusEnter 50 μ l Quanti luc detection liquid, using microplate reader all-wave length readings, testing result as shown in figure 4, S1C3, S1F6 andS4C1 can activate Jurkat Dual cell.EC50Being worth (table 4) shows S1C3, S1F6 and S4C1 to Jurkat Dual cellActivation Activity without significant difference.
The EC of 4. AntiCD3 McAb E monoclonal antibody activation Jurkat Dual cell of table50Value
| S1C3 | S1F6 | S4C1 |
| EC50(M) | 3.679*10-9 | 2.724*10-9 | 3.432*10-9 |
Sequence table
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Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
260 265 270
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
275 280 285
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
305 310 315 320
Ser Leu Ser Pro Gly Lys
325
<210> 13
<211> 327
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 13
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 14
<211> 107
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 14
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 15
<211> 106
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 15
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
20 25 30
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
35 40 45
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65 70 75 80
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 16
<211> 240
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 16
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Phe Asp Gly Ser Arg Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Gly Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
130 135 140
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
145 150 155 160
Gly Ile Asn Asn Ser Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala
165 170 175
Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Glu Thr Gly Val Pro
180 185 190
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
195 200 205
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp
210 215 220
Leu Lys Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
225 230 235 240
<210> 17
<211> 251
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 17
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 Phe Thr Phe Ser Asn Ala
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Gln Ile Lys Asp Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Arg Tyr Val His Tyr Gly Val Arg Phe Phe Tyr Thr Met Asp
100 105 110
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr
130 135 140
Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile
145 150 155 160
Ser Cys Arg Pro Ser Gln Ser Leu Val His Asn Asn Gly Asn Thr Tyr
165 170 175
Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile
180 185 190
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly
195 200 205
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala
210 215 220
Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly Thr Gln Tyr Pro Phe
225 230 235 240
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
245 250
<210> 18
<211> 249
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 18
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gln Ile Lys Ser Lys Ser Asp Asn Tyr Ala Thr Tyr Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser
130 135 140
Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys
145 150 155 160
Arg Ser Ser Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu Asn
165 170 175
Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile Tyr Lys
180 185 190
Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
195 200 205
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
210 215 220
Val Gly Val Tyr Tyr Cys Gly Gln Gly Thr His Ala Pro Tyr Ala Phe
225 230 235 240
Gly Gln Gly Thr Lys Leu Glu Ile Lys
245
<210> 19
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 19
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Phe Ser Gly Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Phe Asp Gly Ser Arg Lys Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gln Met Gly Tyr Trp His Phe Gly Leu Trp Gly Arg Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 20
<211> 107
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 20
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 Gly Ile Asn Asn Ser
20 25 30
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Asn Arg Glu Thr 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 Trp Leu Lys Leu Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 21
<211> 124
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 21
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 Phe Thr Phe Ser Asn Ala
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Gln Ile Lys Asp Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Arg Tyr Val His Tyr Gly Val Arg Phe Phe Tyr Thr Met Asp
100 105 110
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 22
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 22
Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Pro Ser Gln Ser Leu Val His Asn
20 25 30
Asn Gly Asn Thr Tyr Leu Ser Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly
85 90 95
Thr Gln Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 23
<211> 122
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 23
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
20 25 30
Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gln Ile Lys Ser Lys Ser Asp Asn Tyr Ala Thr Tyr Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly Val Asp Ala Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 24
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 24
Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Arg Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Gly
85 90 95
Thr His Ala Pro Tyr Ala Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110