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CN109385400A - Co-express the immune effector cell of the Chimeric antigen receptor modification of PD-L1 blocking agent - Google Patents

Co-express the immune effector cell of the Chimeric antigen receptor modification of PD-L1 blocking agent Download PDF

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CN109385400A
CN109385400A CN201710676368.3A CN201710676368A CN109385400A CN 109385400 A CN109385400 A CN 109385400A CN 201710676368 A CN201710676368 A CN 201710676368A CN 109385400 A CN109385400 A CN 109385400A
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李宗海
潘泽雁
狄升蒙
王华茂
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Clegg Medical Co ltd
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Keji Biomedical (shanghai) Co Ltd
Shanghai Cancer Institute
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Abstract

There are the Chimeric antigen receptor of targeting GPC3 and the immune effector cell of PD-L1 blocking agent the present invention relates to expression.The present invention also provides the pharmaceutical composition comprising the immune effector cell and utilize the immune effector cell or medicine composite for curing tumour, the especially method of GPC3 positive tumor.Immune effector cell of the invention is not only effective to solid tumor cell in vitro, also has excellent fragmentation effect to solid tumor cell in vivo.

Description

Co-express the immune effector cell of the Chimeric antigen receptor modification of PD-L1 blocking agent
Technical field
The invention belongs to immunotherapy fields.More particularly it relates to co-express the chimeric antigen of PD-L1 blocking agent The immune effector cell of receptor modification.
Background technique
In recent years, t lymphocyte receptor (T Cell is depended on according to identification specificity of the CTL to target cell Receptor, TCR) discovery, by for tumor cell associated antigen the scFv of antibody and the CD3 ζ of t lymphocyte receptor or The intracellular signals such as Fc ε RI γ activation motif is fused into Chimeric antigen receptor (Chimeric antigen receptor, CAR), and By it by the modes such as such as slow-virus infection gene modification on T lymphocyte surface.This CAR T lymphocyte can be with main Histocompatibility complex (Major Histocompatibility Complex, MHC) non-limiting way is selectively by T Lymphocyte is directed to tumour cell and specifically kills tumour.
Chimeric antigen receptor includes extracellular combined area, transmembrane region and intracellular signal area.Usual extracellular region includes that can identify The scFv of tumor associated antigen, transmembrane region use CD8, the equimolecular transmembrane region of CD28, and intracellular signal area uses immunity receptor junket Propylhomoserin activation motifs (ITAM) CD3 ζ or Fc ε RI γ and costimulatory signal molecule CD28, CD27, CD137, CD134's etc. is intracellular Signaling zone.
However, due to organism, the especially complexity of the microenvironment of solid tumor shows the time of excellent results in vitro Drug is selected, can not often show corresponding effect in vivo.In other words, the in vitro results of drug candidate can not rationally indicate body Interior effect.In addition, the same antibody also has different-effect to the tumour of the different parts with identical target spot.For example, bent more Pearl monoclonal antibody be applied to HER2 positive breast cancer when have preferable therapeutic effect, but be applied to HER2 positive gastric carcinoma when, do not have but It is effective that (Fu Qiang and goes to hold monoclonal antibody Clinical advances, drug assessment, 2012,9 (27): 8- gastric cancer HER2 signal path 12)。
Although immune effector cell has tempting prospect, the curative effect in solid tumor in immunotherapy of tumors Still not significant, survival rate of the immune effector cell in tumor tissues be not poor, active high.
Therefore, this field is there is still a need for conducting further research, to further increase immune effector cell for swollen The curative effect of tumor immunization therapy especially develops the immune effector cell effective to solid tumor.
Summary of the invention
The purpose of the present invention is to provide a kind of immune effector cells that the curative effect for immunotherapy of tumors improves, especially It is the immune effector cell for having effective lethal effect to solid tumor.
In a first aspect, the present invention provides a kind of immune effector cell for targeting GPC3, the immune effector cell expression There are the Chimeric antigen receptor and PD-L1 blocking agent of targeting GPC3.
In a preferred embodiment, the tumour of the immune effector cell targeting positive expression GPC3, including but it is unlimited In liver cancer, gastric cancer, lung cancer, cancer of the esophagus, head and neck cancer, bladder cancer, oophoroma, cervical carcinoma, kidney, cancer of pancreas, cervical carcinoma, fatty meat Tumor, melanoma, adrenal, neurinoma, malignant fibrous histiocytoma, cancer of the esophagus；Preferably, the tumour is liver Cancer, gastric cancer, lung cancer, cancer of the esophagus.
In a preferred embodiment, the immune effector cell is that preferably do not having in the presence of no cell factor In the presence of IL2, the immune effector cell of excellent amplification in vitro performance is shown.
In a particular embodiment, the PD-L1 blocking agent is natural PD-1, or can be prominent in conjunction with PD-L1 Become PD-1, or natural with PD-L1 ining conjunction with or the segment of PD-1 or the antibody of anti-PD-L1 can be mutated.
In a preferred embodiment, the PD-1 is soluble PD-1 (sPD-1).
In a preferred embodiment, the antibody of the anti-PD-L1 is monoclonal antibody or polyclonal antibody.
In a particular embodiment, the PD-L1 blocking agent are as follows:
Natural PD-1, or can be with the mutation PD-1 in conjunction with PD-L1, or it being capable of natural in conjunction with PD-L1 or mutation PD- 1 segment；
Natural PD-1, or can be with the mutation PD-1 in conjunction with PD-L1, or it being capable of natural in conjunction with PD-L1 or mutation PD- The fusogenic peptide that the segment composition of the Fc section of 1 segment and the Fc section of antibody or antibody is formed；Or
Natural PD-1, or can be with the mutation PD-1 in conjunction with PD-L1, or it being capable of natural in conjunction with PD-L1 or mutation PD- 1 segment merges the fusogenic peptide to be formed with the mutant of the segment of the Fc section of antibody or the Fc section of antibody.
In a particular embodiment, the PD-L1 blocking agent is natural PD-1, or can be with the mutation in conjunction with PD-L1 PD-1, or it is capable of the segment of natural in conjunction with PD-L1 or mutation PD-1.
In a particular embodiment, the antibody is IgG1, IgG2, IgG3 or IgG4, preferably IgG4.
In a particular embodiment, the segment of the Fc section of the antibody is CH3 structural domain.
In a particular embodiment, the PD-L1 blocking agent is natural PD-1, or can be prominent in conjunction with PD-L1 Become PD-1, or natural with PD-L1 ining conjunction with or the segment of PD-1 and the fusogenic peptide of hIgG4e1-Fc can be mutated；Preferably, institute The natural PD-1 stated, or or natural in conjunction with PD-L1 or mutation PD-1 can be capable of with the mutation PD-1 in conjunction with PD-L1 It is connected between segment and the hIgG4e1-Fc via linker；It is highly preferred that the coded sequence of the linker such as SEQ Shown in ID NO:3.
In a particular embodiment, the PD-L1 blocking agent is natural PD-1, or can be prominent in conjunction with PD-L1 Become PD-1, or be capable of the structural domain formation of the CH3 of the segment and hIgG4e1-Fc of natural with PD-L1 ining conjunction with or mutation PD-1 Fusogenic peptide；Preferably, the natural PD-1, or can be with the mutation PD-1 in conjunction with PD-L1, or it can be in conjunction with PD-L1 It is connected between natural or mutation PD-1 segment and the structural domain of the CH3 via linker；It is highly preferred that described The coded sequence of liner is as shown in SEQ ID NO:3.
In a particular embodiment, the natural PD-1, or can be with the mutation PD-1 in conjunction with PD-L1, or it can Natural or mutation PD-1 segment in conjunction with PD-L1 includes PD-1 born of the same parents nucleotide sequence coded shown in SEQ ID NO:2 Outskirt.
In a particular embodiment, the natural PD-1, or can be with the mutation PD-1 in conjunction with PD-L1, or it can The coding nucleotide sequence of natural or mutation PD-1 segment in conjunction with PD-L1 includes encoding PD- shown in SEQ ID NO:1 The nucleotide sequence of PD-1 extracellular region is encoded shown in the nucleotide sequence and SEQ ID NO:2 of 1 signal peptide.
In a particular embodiment, the coding nucleotide sequence of the hIgG4e1-Fc is as shown in SEQ ID NO:5.
In a particular embodiment, the coding nucleotide sequence such as SEQ of the CH3 structural domain of the hIgG4e1-Fc Shown in ID NO:4.
In a particular embodiment, the Chimeric antigen receptor includes: to target the extracellular antigen binding domain of GPC3, across Film area and intracellular signal area.
In a particular embodiment, the extracellular antigen binding domain of the described targeting GPC3 have SEQ ID NO:15,16, LCDR1, LCDR2, LCDR3 shown in HCDR1, HCDR2, HCDR3 and SEQ ID NO:18,19,20 shown in 17.
In a particular embodiment, the extracellular antigen binding domain of the targeting GPC3 is contained shown in SEQ ID NO:21 Heavy chain variable region and SEQ ID NO:22 shown in light chain variable region.
In a particular embodiment, the extracellular antigen binding domain of the described targeting GPC3 have SEQ ID NO:14 or ScFv sequence shown in SEQ ID NO:27.
In a particular embodiment, the intracellular signal area includes: CD3 ζ, Fc ε RI γ, CD27, CD28,4-1BB, The intracellular signal region sequence or Myd88 of CD134, CD40, or combinations thereof.
In a particular embodiment, the transmembrane region includes: CD8 transmembrane region or CD28 transmembrane region.
In a particular embodiment, the Chimeric antigen receptor have SEQ ID NO:23,24,25,26,28,29, Amino acid sequence shown in 30 or 31.
In a particular embodiment, the immune effector cell includes: T lymphocyte, NK cell or NKT cell, Treg cell.
In second aspect, the present invention provides a kind of nucleic acid constructs, and the nucleic acid constructs includes being linked in sequence: PD-L1 blocking agent coded sequence, targets the coded sequence of the extracellular antigen binding domain of GPC3 in Chimeric antigen receptor, transmembrane region and Intracellular signal area coded sequence.
In a particular embodiment, the PD-L1 blocking agent coded sequence and extracellular antigen binding domain coded sequence Between with connect peptide-coding sequence connection；And/or
It further include that ribosomes is jumped between the PD-L1 blocking agent coded sequence and extracellular antigen binding domain coded sequence Jump sequence.
In the third aspect, the present invention provides a kind of expression vector, and it includes nucleic acid described in coding second aspect of the present invention Construction.
In fourth aspect, the present invention provides a kind of virus, and the virus includes expression described in third aspect present invention Carrier.
At the 5th aspect, the present invention provides nucleic acid constructs described in second aspect of the present invention or includes the nucleic acid construct The expression vector of object or the purposes of virus are used to prepare the immune effector cell of the targeting GPC3 of target tumor.
At the 6th aspect, the present invention provides the use of the immune effector cell of targeting GPC3 described in first aspect present invention On the way, it is used to prepare the pharmaceutical composition for inhibiting tumour.
In a preferred embodiment, the tumour is the tumour of positive expression GPC3, including but not limited to liver cancer, stomach Cancer, lung cancer, cancer of the esophagus, head and neck cancer, bladder cancer, oophoroma, cervical carcinoma, kidney, cancer of pancreas, cervical carcinoma, embryonal-cell lipoma, melanin Tumor, adrenal, neurinoma, malignant fibrous histiocytoma, cancer of the esophagus；Preferably, the tumour is liver cancer, gastric cancer, lung Cancer, cancer of the esophagus.
At the 7th aspect, the present invention provides a kind of for inhibiting the pharmaceutical composition of tumour comprising first party of the present invention The immune effector cell of the modification of Chimerical receptor described in face.
In a preferred embodiment, the tumour is the tumour of positive expression GPC3, including but not limited to liver cancer, stomach Cancer, lung cancer, cancer of the esophagus, head and neck cancer, bladder cancer, oophoroma, cervical carcinoma, kidney, cancer of pancreas, cervical carcinoma, embryonal-cell lipoma, melanin Tumor, adrenal, neurinoma, malignant fibrous histiocytoma, cancer of the esophagus；Preferably, the tumour is liver cancer, gastric cancer, lung Cancer, cancer of the esophagus.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 is the streaming figure of the positive rate detection of CAT-T cell；
Fig. 2A shows the amount of the sPD1 secreted in GPC3-28Z-sPD1 culture supernatant, and Fig. 2 B shows Western Blot detects the expression of results of sPD1 in supernatant；
Fig. 3 shows be incubated for altogether with target cell after GPC3-28Z-sPD1 and GPC3-28Z PD-1, TIM-3, Lag-3 Expression；
PD-L1 is raised after Fig. 4 A shows expression (blue) and IFN-γ stimulation of the PD-L1 in liver cancer cell lines Situation (yellow)；Fig. 4 B shows that the expression of GPC3 and PD-L1 in liver cancer cell lines is reaffirmed in Western Blot detection；
Fig. 5 A and Fig. 5 B show that GPC3-28Z-sPD1 is compared with GPC3-28Z after target cell is incubated for altogether, p-AKT with Bcl-xL has more obvious up-regulation, illustrates that the CAR-T for carrying soluble PD-1 enhances antitumor action；
Fig. 6 A-6F shows the external lethal effect to different tumor cell lines of GPC3-28Z-sPD1 and GPC3-28Z；
Fig. 7 shows the cytokine release of GPC3-28Z-sPD1 and GPC3-28Z；
Fig. 8 show GPC3-28Z-sPD1 and GPC3-28Z to SK-HEP-1, SK-HEP-1-GPC3 subcutaneous transplantation tumor Antineoplaston result；
Fig. 9 shows GPC3-28Z, GPC3-28Z-sPD1 to the antineoplaston result of Huh7 subcutaneous transplantation tumor；
Figure 10 shows the internal survival condition and cell factor IFN-γ secretion feelings of GPC3-28Z-sPD1 and GPC3-28Z Condition；
Figure 11 A and 11B show the ImmunohistochemistryResults Results of SK-HEP-1-GPC3 transplantable tumor pathological section；Figure 11 C and 11D Show the ImmunohistochemistryResults Results of huh7 transplantable tumor pathological section；
Figure 12 shows the amplification in vitro situation of different CAR-T cells.
Specific embodiment
Inventor after extensive and in-depth study, it was unexpectedly found that expression have targeting GPC3 Chimeric antigen receptor It is not only effective to solid tumor cell in vitro with the immune effector cell of PD-L1 blocking agent, also have to solid tumor cell in vivo Standby superior fragmentation effect, to improve survival and function of the immune effector cell in tumour.It completes on this basis The present invention.
Term
In order to which the disclosure can be more easily to understand, certain terms are defined first.As used in this application, unless originally Text is otherwise expressly specified, and otherwise each of following term should have meaning given below.It is elaborated in entire application Other definition.
Term " giving " refers to will using any one of various methods well known by persons skilled in the art and delivery system Product physics of the invention introduces subject, including intravenous, intramuscular, subcutaneously, in peritonaeum, spinal cord or other parenteral administrations way Diameter, such as by injecting or being transfused.
Term " antibody " (Ab) should include but is not limited to immunoglobulin, molecule of the antigen binding and include by two sulphur At least two weight (H) chains and two light (L) chains of key interconnection or its antigen-binding portion thereof.Every H chain includes heavy chain variable region (being abbreviated as VH herein) and heavy chain constant region.Heavy chain constant region includes three constant domains
CH1, CH2 and CH3.Every light chain includes light chain variable region (being abbreviated as VL herein) and constant region of light chain.Light chain is permanent Determining area includes a constant domain CL.The area VH and VL can be further subdivided into the hypervariable region of referred to as complementary determining region (CDR), It is scattered with the more conservative region for being known as framework region (FR).Each VH and VL includes three CDR and four FR, from amino terminal It is arranged in the following order to carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.The variable region of heavy chain and light chain Contain the binding structural domain with antigen interactions.
Immunoglobulin can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG and IgM.IgG subclass is also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3 and IgG4. " isotype " refers to by the Ab class or subclass (such as IgM or IgG1) of weight chain constant area gene coding.Term " antibody " includes example Such as, naturally occurring and non-naturally occurring Ab；Monoclonal and polyclonal Ab；Chimeric and humanization Ab；People or inhuman Ab；Quan He At Ab；With single-stranded Ab.Inhuman Ab can reduce its immunogenicity in people by recombination method humanization.Not clear In the case where explanation, and unless otherwise indicated by context, otherwise term " antibody " further includes any of above immunoglobulin Antigen-binding fragment or antigen-binding portion thereof, and including unit price and bivalent fragment or part and single-stranded Ab.
Terms used herein " PD-1 " include people PD-1 (hPD-1), variant (hPD-1 of mutation), the isotype of hPD-1 And species homologue, and there is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, the 99% phase same sex with hPD-1 Analog.Complete hPD-1 sequence can be found at GenBank accession number U64863.
" Programmed death ligand-1 (PD-L1) " is that (another kind is one of two kinds of cell surface glycoprotein ligands of PD-1 PD-L2), T cell activation and cytokine secretion are lowered when combining PD-1.The term as used herein " PD-L1 " includes people Variant, isotype and the species homologue of PD-L1 (hPD-L1), hPD-L1, and there is at least one common table with hPD-L1 The analog of position.Complete hPD-L1 sequence can be found at GenBank accession number Q9NZQ7.
Term " about " can refer to the acceptable error model of the particular value or composition that determine in those of ordinary skill in the art Value or composition in enclosing, will depend partially on how measuring or measured value or composition.
Term " the Fc section of antibody " can come from natural or wild type, be also possible to natural or wild type the area Fc The mutant of section." the Fc section of antibody " includes comprising the antibody constant region in addition to the first constant region immunoglobulin domains Polypeptide.Thus, Fc refer to IgA, IgD and IgG most latter two constant region immunoglobulin domains and IgE and IgM it is last The flexible hinge of three constant region immunoglobulin domains and these structural domain N-terminals.It may include J for IgA and IgM, Fc Chain.
" CH3 " as described herein or " CH3 structural domain " can come from natural or wild type, be also possible to natural or wild The mutant of the Fc section of raw type, the preferably mutant of the CH3 of the IgG4 of the CH3 or people of the IgG4 of people, such as hIgG4e1.
Term " mutation " or " variant " or " mutant " they include at least one amino acid modification due to comparing parent, without It is same as the antibody sequence of parent antibody sequence, the sequence and parent antibody sequence of " variant " or " mutant " have at least about 80%, preferably at least about 90%, more preferably at least about 95%, more preferably at least about 97%, more preferably at least about 98%, most preferably At least about 99% amino acid sequence identity.
GPC3 and GPC3 positive tumor
" GPC3 " or " glypican-3 " used herein are a member of Glypican family, are being regulated and controled Cell growth and differentiation aspect play an important role.The generation of GPC3 unconventionality expression and kinds of tumors, development relationship are close, such as in liver Cancer, lung cancer, breast cancer, oophoroma, kidney, thyroid cancer, gastric cancer, colorectal cancer, etc. in unconventionality expression is presented.
In the present invention, the tumour of immune effector cell targeting GPC3 positive expression.In a particular embodiment, described Tumour includes but is not limited to liver cancer, gastric cancer, lung cancer, cancer of the esophagus, head and neck cancer, bladder cancer, oophoroma, cervical carcinoma, kidney, pancreas Cancer, cervical carcinoma, embryonal-cell lipoma, melanoma, adrenal, neurinoma, malignant fibrous histiocytoma, cancer of the esophagus.Ability Field technique personnel know that some tumour cells, such as liver cancer cells are insensitive to many drugs, therefore, even if in vitro effectively Drug, effect sometimes in vivo is also bad, even without effect.Therefore, in a preferred embodiment, described herein GPC3 positive expression tumour or GPC positive tumor be liver cancer, gastric cancer, lung cancer, cancer of the esophagus.
Term " immune effector cell (or abbreviation Chimeric antigen receptor immune effector cell) of Chimeric antigen receptor modification " It is it is known in the art that it is the immune effect using genetic modification technological expression antigen (such as tumour antigen) specific chimeric receptor Cell is answered, it can targeting performance lethal effect.The immune effector cell is adjusted for example including T cell, NK cell, NKT cell Section property T cell (Regulatory cell, abbreviation Treg)." immunological effect of Chimeric antigen receptor modification is thin for conventional preparation The method of born of the same parents " is known to the skilled in the art, including it is allowed to express costimulation cellular elements intracellular domain intracellular, such as CD28, CD137, CD27, CD3 ζ (preferably CD3 ζ intracellular domain), CD8, CD19, CD134, CD20, one in Fc ε RI γ Kind is a variety of.Through them in conjunction with respective ligand, the second signal of immune effector cell is activated, enhances the proliferation of immunocyte The secreting function of ability and cell factor extends the time-to-live of immunocyte.
In a particular embodiment, the structure of Chimeric antigen receptor of the invention is successively (preferably from N-terminal to C-terminal) It include: Programmed death ligand-1 blocking agent, extracellular antigen binding domain, transmembrane region and intracellular signal area.
Described Programmed death ligand-1 (PD-L1) blocking agent can be and a variety of can lower, block, blocking, inhibiting The substance of PD-L1, as long as it can be prevented or the competitive PD-1 phase interaction interfered PD-L1 and be expressed in immune effector cell With.For example, PD-L1 blocking agent of the invention is natural PD-1, or can be with the mutation PD-1 in conjunction with PD-L1, or it can be with PD- Natural or mutation PD-1 segment or anti-PD-L1 the antibody that L1 is combined.In a particular embodiment, described procedural - 1 blocking agent of death ligand includes but is not limited to: sPD-1；The fusogenic peptide of the CH3 structural domain of sPD-1 and hIgG4e1-Fc；sPD-1 With the fusogenic peptide of hIgG4e1-Fc；Or the anti-PD-L1 antibody of specificity.
As preferred embodiment of the invention, the extracellular antigen binding domain includes the antigen of specific recognition tumour high-expression Antibody, preferably single-chain antibody.It is highly preferred that the extracellular antigen binding domain of the Chimeric antigen receptor passes through CD8 hinge Area is connected with the transmembrane region of CD8 or CD28, immediately intracellular signal area after transmembrane region.
The transmembrane region of Chimeric antigen receptor can be selected from the transmembrane region of the albumen such as CD8 or CD28.People's CD8 albumen is different dimerization Volume morphing is made of two chains of α β or γ δ.In one embodiment of the invention, transmembrane region is selected from CD8 α or CD28 Transmembrane region.In addition, CD8 α hinge area (hinge) is a flexible region, therefore, CD8 or CD28 and transmembrane region add hinge Area be used to connect the extracellular antigen binding domain of Chimeric antigen receptor CAR and intracellular signal area.
Intracellular signal area can be selected from CD3 ζ, Fc ε RI γ, CD28, CD137 (4-1BB), the intracellular signal of CD134 albumen Area, and combinations thereof.CD3 molecule is made of five subunits, and wherein 3 are contained in CD3 ζ subunit (also known as CD3zeta, abbreviation Z) ITAM motif, the motif are signal transduction areas important in TCR-CD3 complex.CD3 δ Z is truncated without ITAM motif CD3 ζ sequence, in the practice of the present invention generally as the building of negative control.Fc ε RI γ is mainly distributed on mast cell and thermophilic Alkaline surfaces of granulocytes contains an ITAM motif, similar with CD3 ζ in structure, distribution and function.Further, as described above, CD28, CD137, CD134 are costimulatory signal molecules, in the costimulation generated with its intracellular signal section after respective ligand binding Effect causes the continuous proliferation of immune effector cell (mainly T lymphocyte), and can be improved immune effector cell secretion IL- The level of the cell factors such as 2 and IFN-γ, while improving CAR immune effector cell time to live in vivo and antitumor effect Fruit.
Chimeric antigen receptor polypeptide of the invention can be selected from sequential connection as follows:
Extracellular antigen binding domain-CD8 transmembrane region -4-1BB-CD3 ζ,
Extracellular antigen binding domain-CD28a-CD28b-CD3 ζ,
Extracellular antigen binding domain-CD28a-CD28b-4-1BB-CD3 ζ,
And combinations thereof, wherein CD28a represents the transmembrane region of CD28 molecule, CD28b generation in related Chimeric antigen receptor albumen The intracellular signal area of table CD28 molecule.
The present invention also includes the nucleic acid for encoding the Chimeric antigen receptor.The invention further relates to the variations of above-mentioned polynucleotides Body, coding have the polypeptide of identical amino acid sequence or the segment of polypeptide, analogs and derivatives with the present invention.
The present invention also provides the carriers of the nucleic acid comprising above-mentioned Chimeric antigen receptor.The invention also includes include above-mentioned load The virus of body.Virus of the invention includes the infectious virus of tool after packaging, also includes comprising being packaged as with appeal Viral institute's essential component virus to be packaged.It is known in the art other to can be used for foreign gene being transduceed into immune effect The viral and its corresponding plasmid vector of cell is answered to can also be used for the present invention.
The present invention also provides the immune effector cells of chimeric antigen modification, by the chimeric antigen for having coding described of transduceing The nucleic acid of receptor is had above-mentioned comprising the recombinant plasmid containing the nucleic acid, or the virus comprising the plasmid by transduction.Ability The nucleic acid transduction method of domain routine may be used to the present invention including non-viral and viral transduction method.Based on non-viral Transduction method includes electroporation and transposons method.The Nucleofector nuclear transfection instrument of recent Amaxa company research and development can be straight It connects and foreign gene is imported into the high efficiency transduction that nucleus obtains target gene.In addition, being based on sleeping beauty transposon stand (Sleeping Beauty system) or the more common electroporation of transduction efficiency of the Transposon Systems such as PiggyBac transposons improve a lot, will [Davies JK., et has been reported in nucleofector transfection instrument and the system combined application of sleeping beauty transposon stand al.Combining CD19redirection and alloanergization to generate tumor-specific Human T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res, 2010,70 (10): OF1-10.], this method not only transduction efficiency with higher but also can be realized the site-directed integration of target gene. In one embodiment of the invention, the transduction method for realizing the immune effector cell of Chimeric antigen receptor gene modification is base In virus such as retrovirus or the transduction method of slow virus.This method has transduction efficiency high, and foreign gene can stablize table It reaches, and the advantages that in vitro culture immune effector cell reaches the time of clinical number of stages can be shortened.In the immune effect of the transgenosis Cell surface is answered, the nucleic acid of transduction is by transcription, accurate translation on its surface.Pass through the tumour cell to a variety of different cultures Cell in vitro poison is carried out it is demonstrated experimentally that the immune effector cell that chimeric antigen of the invention is modified has the tumour of high degree of specificity Cell killing efficacy (also known as cytotoxicity), and can effectively survive in tumor tissues.Therefore encoding chimera of the invention is anti- The nucleic acid of original receptor, the plasmid comprising the nucleic acid, virus and transduction comprising the plasmid have above-mentioned nucleic acid, turn of plasmid or virus Genetic immunization effector cell can be efficiently used for the immunization therapy of tumour.
The immune effector cell of chimeric antigen modification of the present invention can also carry the coding of the cell factor of external source Sequence；The cell factor includes but is not limited to: IL-12, IL-15 or IL-21 etc..These cell factors have immunological regulation Or antitumor activity, can enhancement effect T cell and activation NK cell function, or directly play antitumor action.Therefore, It preferably plays a role it will be understood by those skilled in the art that the utilization of these cell factors facilitates the immunocyte.
The immune effector cell of chimeric antigen modification of the present invention can also express other than above-mentioned Chimerical receptor Another Chimerical receptor, this receptor do not contain CD3 ζ, but the intracellular signal of the intracellular signal structural domain containing CD28, CD137 Structural domain or combination of the two.
The immune effector cell of chimeric antigen modification of the present invention can also express chemokine receptors；Described becomes Changing factor acceptor includes but is not limited to CCR2.It will be understood by those skilled in the art that the CCR2 chemokine receptors can be with So that intracorporal CCR2 competitive binding therewith, for blocking the transfer of tumour to be advantageous.
The immune effector cell of chimeric antigen modification of the present invention can also express the siRNA that can reduce PD-1 expression Or block the albumen of PD-L1.It will be understood by those skilled in the art that competitive blocking PD-L1 and its receptor PD-1's is mutual Effect, is conducive to restore antitumor t cell responses, to inhibit tumour growth.
The immune effector cell of chimeric antigen modification of the present invention can also express safety switch；Preferably, described Safety switch include: iCaspase-9, truncated EGFR or RQR8.
The immune effector cell of Chimeric antigen receptor modification of the invention can be applied to preparation pharmaceutical composition or diagnosis Reagent.The composition also may include pharmaceutically acceptable carrier in addition to including a effective amount of immunocyte.Term " pharmaceutically acceptable " refers to that it is unfavorable that they will not be generated when biomolecule ontology and composition suitably give animal or people , allergy or other adverse reactions.
The specific example that can be used as pharmaceutically acceptable carrier or some substances of its component is carbohydrate, such as lactose, Portugal Grape sugar and sucrose；Starch, such as cornstarch and potato starch；Cellulose and its derivates, as sodium carboxymethylcellulose, ethyl are fine Dimension element and methylcellulose；Tragacanth powder；Malt；Gelatin；Talcum；Solid lubricant, such as stearic acid and magnesium stearate；Sulphur Sour calcium；Vegetable oil, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cupu oil；Polyalcohol, such as propylene glycol, sweet Oil, D-sorbite, mannitol and polyethylene glycol；Alginic acid；Emulsifier, such asWetting agent, such as NaLS； Colorant；Flavoring agent；Tablet agent, stabilizer；Antioxidant；Preservative；Apirogen water；Isotonic salting liquid and phosphate buffer Deng.
Various dosage forms can be made in composition of the invention as needed, and can be by doctor according to patient category, age, weight Substantially the factors such as disease condition, administration mode determine that the dosage beneficial to patient is administered.Administration mode can be using note It penetrates or other therapeutic modalities.
Advantages of the present invention:
1. the immune effector cell that Chimeric antigen receptor of the invention is modified can effectively increase the immune effector cell Survival and function in tumour；
2. the immune effector cell that Chimeric antigen receptor of the invention is modified is not only effective to solid tumor cell in vitro, with The immune effector cell for not co-expressing the Chimeric antigen receptor modification of PD-L1 blocking agent is compared, and is had in vivo to solid tumor cell Standby superior fragmentation effect and amplification in vitro performance.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or According to the normal condition proposed by manufacturer.
The recombined lentivirus vector PRRLSIN- of embodiment 1. solubility sPD1-CH3 combination chimeric antigen (GPC3) receptor The building of cPPT.EF-1 α-sPD1-CH3-F2A-9F2-CAR
1. designing source of people sPD1, sPD1-CH3 and sPD1-Fc sequence
1) source of people sPD1 sequence design
PD-1DNA sequence reference pubmed Nucleotide database, PD-1 reference numeral NM_005018.2；PD-1's Signal peptide and extracellular fragment refer to Uniprot database.
PD-1 signal peptide sequence:
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGG(SEQ ID NO:1)。
PD-1 extracellular fragment sequence:
CCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGA AGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCC CCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTC ACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTG TGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGG CAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTG(SEQ ID NO: 2)
2) source of people sPD1-CH3 sequence design
SPD1 by the CH3 structural domain in linker and hIgG4e1-Fc, linker sequence be TATGGT (SEQ ID NO: 3), CH3 domain sequence refers to pFUSE-hIgG4e1-Fc1v01 plasmid.
The DNA sequence dna of CH3 structural domain:
CCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGT TCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGGCCTCCCGTCC(SEQ ID NO:4)
3) source of people sPD1-Fc sequence design
For sPD1 by linker and hIgG4e1-Fc structural domain, linker sequence is TATGGT.HIgG4e1-Fc structural domain Sequence reference pFUSE-hIgG4e1-Fc1v01 plasmid.
The DNA sequence dna of hIgG4e1-Fc structural domain:
CCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGT TCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGC CCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATC GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC CTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO:5)
The signal peptide sequence of PD-1, PD-1 extracellular fragment sequence, the sequence of CH3 are carried out fully synthetic and cloned into carrier T, is obtained To plasmid T-sPD1-Fc.
2. constructing GPC3-28Z-sPD1
1) amplification of sPD1-CH3 segment
Using T-sPD1-Fc plasmid as template, with upstream primer 5 '- Acgcgtcctagcgctaccggtcgccaccatgcagatcccacaggcgccc-3 ' (SEQ ID NO:6), downstream primer 5 '- Ctctcggggctgcccaccatacaccagggtttggaactggc-3 ' (SEQ ID NO:7) PCR amplification obtains sPD1 sequence； With upstream primer 5 '-tatggtgggcagccccgagagccacag-3 ' (SEQ ID NO:8), downstream primer Aaaattcaaagtctgtttcactttacccggagacagggag-3 ' (SEQ ID NO:9) amplification obtains sPD1-CH3 segment.
2) acquisition of the segment with F2A and CD8 signal peptide
Using the packaging plasmid pMDLg RRE (Addgene) of coding Protein G ag/Pol, the packaging plasmid of Rev albumen is encoded PRSV-REV (Addgene) encodes the envelope plasmid pCMV-VSV-G (Addgene) of vesicular stomatitis virus-G protein and based on empty carrier The recombinant expression carrier of the coding target gene CAR of pRRLSIN-cPPT.PGK-GFP.WPRE (Addgene).
Firstly, the technical approach cloned by common molecular, carries out empty carrier pRRLSIN-cPPT.PGK-GFP.WPRE Transformation substitutes the promoter of original vector with the promoter of -1 α of elongation factor (elongation factor-1 α, abbreviation EF-1 α), And MluI restriction enzyme site is increased between promoter and CD8 alpha signal peptide.Specifically, using ClaI/SalI (NEB) double digestion Carrier pWPT-EGFP (Addgene), recycles the DNA fragmentation of 1.1Kb, is connected to T4DNA ligase through the bis- enzymes of ClaI/SalI The carrier pRRLSIN-cPPT.PGK-GFP.WPRE cut, and convert in host strain TOP10, picked clones are reflected by bacterium colony PCR Determine positive colony and by sequencing confirmation, acquisition recombinant plasmid pRRLSIN-cPPT.EF-1 α-EGFP.WPRE.
Using upstream primer 5 '-gcaggggaaagaatagtagaca-3 ' (SEQ ID NO:32) and downstream primer 5 '- CGGCCTGGCGGCGTGGAG-3 ' (SEQ ID NO:33), using pRRLSIN-cPPT.EF-1 α-EGFP.WPRE as template, PCR The EF-1 α promoter (SEQ ID NO:3) (including Mlu I restriction enzyme site) containing CD8 alpha signal area is expanded, segment 1 is named as.
Using the plasmid of heavy chain variable region (SEQ ID NO:34) segment comprising AB1 as template, using upstream primer 5 '- Ctccacgccgccaggccggaggtgcagctggtgcag-3 ' (SEQ ID NO:36) and downstream primer 5 '- GCGGTGTCCTCGCTCCGCAGGCTGCTCAGCTCCATGTAGGCGGTG-3 ' (SEQ ID NO:37) amplifies weight chain variable Area's segment；Using the plasmid of light chain variable region (SEQ ID NO:35) segment as template, using upstream primer 5 '- GCGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGTTCTACAGCTAC-3 ' (SEQ ID NO:38) and downstream primer 5 '-CGGCGCTGGCGTCGTGGTACGTTTGATCTCCAGCTTGGTG-3 ' (SEQ ID NO:39) amplify light chain variable region Segment.Above-mentioned heavy chain and light chain variable region primer by bridging PCR, further amplify containing with upstream CD8 alpha signal peptide and under The scFv segment for swimming the AB1 of hinge area repetitive sequence, is named as segment 2.
Using upstream primer 5 '-accacgacgccagcgccg-3 ' (SEQ ID NO:40) and downstream primer 5 '- Aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3 ' (SEQ ID NO:41), respectively with pWPT- EGFP-F2A-GPC3-28Z is template (with specific reference to Chinese patent CN104140974A), and amplification obtains the piece of Hinge-28Z Section, is named as segment 3.
The segment 1, segment 2 and segment 3 of equimolar about 50ng are subjected to splicing PCR respectively, splice condition are as follows: initial denaturation: 94 DEG C, 4min；Denaturation: 94 DEG C, 40s；Annealing: 60 DEG C, 40s；Extend: 68 DEG C, 140s, carrying out 5 circulations, then overall elongation 68 DEG C, 10min, supplement archaeal dna polymerase and upstream primer 5 '-gcaggggaaagaatagtagaca-3 ' (SEQ ID NO:32) and Downstream primer 5 '-aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3 ' (SEQ ID NO:41), passes through PCR amplification 25 circulations, amplification condition is initial denaturation: 94 DEG C, 4min；Denaturation: 94 DEG C, 40s；Annealing: 60 DEG C, 40s；Extend: 68 DEG C, 140s, 68 DEG C of overall elongation, 10min.Amplification obtains AB1-28Z.
Using MluI and SalI digestion carrier pRRLSIN-cPPT.EF-1 α-EGFP.WPRE and AB1-28Z.Connected by T4 It connects enzyme to be attached, converts TOP10, choose clone and carry out PCR identification positive bacteria, send to Invitrogen and carry out sequencing confirmation sequence Correctly, plasmid pRRL-EF-1 α-AB1-28Z is obtained.
With PRRLSIN-cPPT.EF-1 α-AB1-28Z (pRRL-EF-1 α-AB1-28Z) for template, upstream primer 5 '- Caaaggcctcccgtccgtgaaacagactttgaattttgac-3 ' (SEQ ID NO:10), downstream primer 5 '- Tgcgggaagtgtacgtccgggacgggggagcgatccagctgttagt-3 ' (SEQ ID NO:11), amplification obtain CD8 α Hinge-CD28TM-AB1-28Z segment.
3) preparation of sPD1-CH3- α GPC3-28z
The segment PCR of sPD1-CH3 segment, CD8 α hinge-CD28TM-AB1-28Z are spliced.Splicing condition are as follows: pre- to become Property: 94 DEG C, 4min；Denaturation: 94 DEG C, 40s；Annealing: 60 DEG C, 40s；Extend: 68 DEG C, 150s, carrying out 5 circulations, then always prolong 68 DEG C, 10min are stretched, archaeal dna polymerase and upstream primer 5 '-are supplemented Acgcgtcctagcgctaccggtcgccaccatgcagatcccacaggcgccc-3 ' (SEQ ID NO:6) and downstream primer 5 '-tgcgggaagtgtacgtccgggacgggggagcgatccagctgttagt-3 ' (SEQ ID NO:9) carry out PCR amplification, Amplification condition are as follows: initial denaturation: 94 DEG C, 4min；Denaturation: 94 DEG C, 20s；Annealing: 58 DEG C, 30s；Extend: 68 DEG C, 1000bp/min (kod-plus)；35 circulations are carried out, then 68 DEG C of overall elongation, 10min.Obtain sPD-1-CH3-GPC3-28z, purpose band Theoretical size 2373bp, PCR amplification band meet target fragment size by agarose gel electrophoresis confirmation.
5 ' the ends of above-mentioned building segment sPD-1-CH3- α GPC3-28z have MluI restriction enzyme site, and 3 ' ends have SalI enzyme Enzyme site.By MluI and SalI double digestion, it is connected into the PRRLSIN-cPPT.EF-1 α carrier of same double digestion, is expressed The plasmid of the Chimeric antigen receptor of the targeting GPC3 of sPD-1-CH3 albumen.
The preparation of 2. slow virus of embodiment and the expression of T lymphocyte Chimeric antigen receptor
1. slow virus is packed
(1) 293T cell inoculation: according to 6 × 106Density inoculated and cultured to the 6th~10 generation 293T cell in 10cm train It supports in ware, 37 DEG C, 5%CO2Overnight incubation prepares for transfecting, and culture medium is containing 10% fetal calf serum (Gibco) without antibiosis Plain DMEM, next day prepare to transfect in Cell abundance about 70%-80%.
(2) it transfects:
To Pignus pignoris grain: the targeting GPC3's of plasmid pRRL-EF-1 α-AB1-28Z (plasmid 1) or expression sPD-1-CH3 albumen The plasmid (plasmid 2) of Chimeric antigen receptor
It is transfected using PEI, using four plasmid slow virus packaging systems, prepares PEI/DNA mixture A liquid and prepare: point Plasmid 1 or plasmid 2 (to Pignus pignoris grain) 5.4 μ g, pMDLg-pRRE 6.2 μ g, pRSV-Rev 6.2 μ g, pCMV-VSV-G are not taken 2.4μg.Plasmid is dissolved in the serum-free DMEM culture solution of 800 μ L, mixing (or 1000rpm is vortexed 5 seconds) is given.B liquid is matched System: 60 μ g PEI (1 μ g/ μ L) being dissolved in the plasma-free DMEM medium of 800 μ L and mix (or 1000rpm is vortexed 5 seconds), It is incubated at room temperature 5min.The formation of complex to be transfected: will A liquid be added B liquid in be gently mixed (i.e. plasmid dilution be added PEI it is dilute Release in liquid, cannot sequentially overturn), PEI and DNA mass ratio are about 3:1.Vortex mixed immediately is given after addition or is mixed gently, room Temperature is lower to be incubated for 20min.Then transfecting complexes 1.6ml is added dropwise in the 10cm culture dish of the culture medium of DMEM containing 11ml.4-5h After hour, liquid is changed to the 293T cell of transfection with the DMEM of 2%FBS training base.
(3) collection virus and concentration: the 293T cell after transfection is placed in 37 DEG C of incubators and is incubated for 72 hours, can be small 48 When collect viral supernatants it is primary, in 4 DEG C save, then plus 10mL2%FBS DMEM culture medium is collected when 72 hours for the second time, The viral supernatants collected with 48 hours are concentrated together.Pre-prepared PEG8000NacL solution is added in the ratio of 1:4 In viral supernatants, it is placed in room temperature, per half an hour, which turns upside down, mixes primary, 2-3 times total, 4 DEG C of precipitates overnights.Next day gives 4000rpm, 60min, 4 DEG C of refrigerated centrifuge centrifugations, it is (dense that viral pellet uses the AIM-V complete medium containing 2% human serum to be resuspended About 70-100 times of demagnification number), -80 DEG C of refrigerators freeze spare after packing.
(4) slow virus titrates: limiting dilution assay infection 293T raji cell assay Raji is packaged with the slow virus titre the of recombinant vector One day, with 1 × 105/ mL is inoculated with 293T cell in 96 well culture plates, 100 holes μ L/, in 37 DEG C, 5%CO2Culture, culture solution be containing The DMEM of 10% fetal calf serum of 6 μ g/mL polybrene, in 37 DEG C, 5%CO2It is incubated for 30min.Add the virus in 10 holes μ L/ former Liquid or the viral concentration liquid in 1 hole μ L/, 5 times of dilutions, 4 gradients, two multiple holes, 37 DEG C, 5%CO2Culture, is packaged with The slow virus of CAR-GCP3 and the slow virus for being packaged with sPD-1-CH3 and CAR.
The preparation of the T lymphocyte of the 2.CAR positive
(1) activation of T lymphocyte: counting after peripheral blood mononuclear cells (PBMC) washing, and adjustment concentration is 1 × 106/ ML, is added the magnetic bead of coating α CD3/ α CD28 antibody according to 1:2 ratio and the recombinant human il-2 for adding concentration 500U/mL stimulates training Support 48h.
(2) virus infection T cell: the polybrene that working concentration is 5 μ g/mL is added before infection to improve infection The T cell activated, weight after 1800g, 32 DEG C of centrifugation 30min is resuspended using the viral concentration liquid of MOI ≈ 5-10 infection in efficiency It is outstanding, in 5%CO2Incubator in 37 DEG C culture.
(3) the metainfective cell of amplification cultivation every other day uses 5 × 105The density of/mL is passed on, while in lymph The recombinant human il-2 of final concentration 300U/mL is added in cell culture fluid.Obtain only express CAR T cell (GPC3-28z) and Co-express the T cell (GPC3-28Z-sPD1) of sPD-1-CH3 and CAR.
3. expression of the flow cytometer detection CAR in T cell
The expression of T lymphocyte Chimeric antigen receptor: the T lymphocyte of slow-virus infection is being cultivated the 4-5 days, and collection 5 × 105T cell in EP manage, washed 2 times with the streaming cleaning solution (1%NCS adds PBS to prepare) of prior ice bath, 1:50 dilution proportion The Fab antibody of Biotin label is added, is incubated for 45min on ice, is vortexed washing three times, PE label is added in 1:200 dilution proportion Two antiantibody of chain and sistomycocin is incubated for 45min on ice, and the washing that is vortexed goes to streaming dedicated pipe, upper machine testing afterwards three times.As a result such as Shown in Fig. 1.Carry the CAR-T positive expression rate with higher same as common CAR-T of sPD-1-CH3.
Supernatant after collecting slow-virus infection T cell detects sPD1-CH3 in infection T cell supernatant by Elisa As a result as shown in Figure 2 A expression shows that the secretory volume of the PD-1 of GPC3-28Z-sPD1 is greater than 1000pg/ml, illustrates sPD1-CH3 It can preferably be secreted into extracellular.
It is thin in infection T to detect sPD1-CH3 by Western blot for cell and supernatant after collecting slow-virus infection T cell Expression in born of the same parents' cell and supernatant, it is immune heavy that the sPD-1-CH3 secreted into cells and supernatant is carried out by proterinA/G It forms sediment, as a result as shown in 2B, sPD1-CH3 can detected in T cell supernatant and cell after infection, illustrate sPD1- CH3 can preferably be secreted into extracellular.
3. aging marker of embodiment is in CAR-T cell membrane surface expression
Select the SK-HEP-1-GPC3 of the GPC3 positive as target cell, by target cell respectively in vitro culture the 11st day It co-expresses the CAR-T cell (GPC3-28Z-sPD1) of sPD1 and does not express the CAR-T cell (GPC3-28Z) of sPD1 by 1:1 ratio Example is incubated for altogether, and giving within every 24 hours target cell stimulates 1 time, totally 72 hours, collects 5 × 105T cell in EP manage, with prior ice bath Streaming cleaning solution (1%NCS adds PBS to prepare) wash 2 times, 1:50 dilution proportion is separately added into Anti-human CD279 (PD- 1)(eBioscience PerCP-eFluor○R CLONE:eBioJ105)；Anti-human CD366(TIM-3) (eBioscience APC CLONE:F38-2E2)；Anti-human CD223(Lag-3)(eBioscience eFluor○R 450 CLONE:3DS223H) straight labeling antibody, it is incubated for 45min on ice, is vortexed after washing three times, the exhaustion mark of flow cytometer detection T cell Will object PD-1, TIM-3, Lag-3, as a result as shown in figure 3, T cell is after target antigen stimulates, GPC3-28Z-sPD1 and GPC3- 28Z is compared, and the PD-1 expression of film surface is lower, is illustrated after receiving target antigen stimulation jointly, GPC3-28Z-sPD1 has anti- The only effect of T cell failure.
Expression of the embodiment 4.PD-L1 in hepatocellular carcinoma cell line
Good following HCC (Hepatocellular Carcinoma) cell of the growth conditions of culture: SK-HEP-1, SK-HEP-1-GPC3, Huh7, Hep3B, PLC/PRF/5 and HepG2, convergence degree 80-90%, wherein SK-HEP-1 GPC3 Feminine gender, remaining cell are GPC3 positive.With pancreatin digestive juice digestion process cell and cell is dispelled in 2mL EP pipe, 400g centrifugation 5min.Streaming cleaning solution washs and is resuspended cell, and 2~5 × 105/ pipe, is in charge of.
The expression of flow cytometer detection PD-L1, antibody are Anti-Human CD274 (B7-H1) PE, 5ul (0.5ug)/sample, ice 45min is bathed, washing three times, removes the straight labeling antibody of PE, carries out flow cytometry.Another group of tumour cell give IFN-γ by 10ng/ml concentration and tumour cell are incubated for stimulation 24 hours altogether, and detection PD-L1 raises situation.As a result as shown in Figure 4 A, explanation SK-HEP-1, SK-HEP-1-GPC3, Huh7, Hep3B, PLC/PRF/5 and HepG2 have the expression of PD-L1, through micro The PD-L1 of each cell line has apparent up-regulation after IFN-γ (10ng/mL) stimulation.
In order to further verify each liver cancer cell lines PD-L1 expression, using Western Blot detection PD-L1 table It reaches, as a result as shown in Figure 4 B, shows that other cells of SK-HEP-1 and the GPC3 positive of GPC3 feminine gender show the PD-L1 positive.
The tumor immune escape of embodiment 5.GPC3-28Z-sPD1 is fought
Since the expression of the phosphorylation level and Bcl-xL albumen of AKT is the important indicator of tumor immune escape, lead to The expression for crossing the AKT (p-AKT) and Bcl-xL albumen of the phosphorylation of detection T cell is swollen come the confrontation for verifying GPC3-28Z-sPD1 The case where tumor is escaped.
By GPC3-28Z, GPC3-28Z-sPD1-T cell 1 × 106As effector cell, according to 1:1 ratio respectively with Target cell Huh7, SK-HEP-1-GPC3 are incubated for 24 hours altogether, collect T lymphocyte, are then cracked T lymphocyte and are collected egg White, Western Blot detects expression of AKT, p-AKT and Bcl-xL albumen in each group after BCA method protein quantification.
Expression of results is as shown in Figure 5 A and 5B, compared with GPC3-28Z, in two different target cells, and GPC3-28Z- SPD1 has the expression of higher levels of p-AKT and Bcl-xL, illustrates that GPC3-28Z-sPD1 has stronger anti-tumor activity.
The test of 6. in vitro toxicity of embodiment
It is carried out using 96 non-radioactive cell toxicity detection kit (Promega company) of CytoTox.Specific method ginseng According to 96 non-radioactive cell toxicity detection kit specification of CytoTox.
Effector cell: it is thin that it is inoculated with Untransduced T cell, GPC3-28Z T respectively by effect target ratio 3:1,1:1 or 1:3 Born of the same parents, GPC3-28Z-sPD1T cell are in 96 orifice plates.
Target cell: it is inoculated with 50 μ L 2 × 10 respectively5SK-HEP-1, SK-HEP-1-GPC3, Huh-7, PLC/PRF/ of/mL 5, Hep3B, HepG2 are to corresponding 96 orifice plate.
Every group is respectively provided with 5 multiple holes.Culture plate is placed in cell incubator and is incubated for 18h.
Wherein each experimental group and each control group are provided that experimental group: the different Chimeric antigen receptors of each target cell+expression T lymphocyte；Control group 1: target cell maximum discharges LDH；Control group 2: the spontaneous release LDH of target cell；Control group 3: effect is thin The spontaneous release LDH of born of the same parents.Calculation formula are as follows: % cytotoxicity=[(spontaneous group of spontaneous group-target cell of experimental group-effector cell)/ (target cell maximum-target cell is spontaneous)] * 100.Experimental result is as shown in Figure 6.
The cytokines measurement of 7. cell of embodiment release
By 5 × 104CAR-T cell inoculation to 24 orifice plates, while being inoculated with 5 × 104Target cell, final volume 400ul, Do not add external source IL-2, put incubator culture and collect supernatant afterwards for 24 hours, using ELISA method detection IL-2, IFN-γ, IL-4 and The concentration of TNF-α.
Experimental result is as shown in fig. 7, there is cytokine secretion to illustrate that T cell is activated and plays lethal effect.
The antineoplaston of 8. subcutaneous transplantation tumor of embodiment is tested
1.SK-HEP-1, SK-HEP-1-GPC3 subcutaneous transplantation knurl model
1) experimental group: NOD-SCID mouse 20/24,6-8 week old is randomly divided into 3 groups, and every group 5, respectively Untransduced, GPC3-28Z control group and GPC3-28Z-sPD1 treatment group.
2) inoculation of subcutaneous transplantation tumor: trypsin digestion, which is collected, is in logarithmic growth phase and the good SK- of growth conditions HEP-1 cell, SK-HEP-1-GPC3 cell, after brine 1 time, adjustment cell density is 1 × 107/ mL, with note Emitter injects 200 μ L cell suspensions, i.e. every mouse inoculation 2 × 10 at the right side of mice subcutaneous abdomen position NOD-SCID6It is swollen Oncocyte, inoculation are denoted as the 0th day day.
3) CAR-T cell is fed back: when tumor size is about 150mm3When, i.e., the 14th day intraperitoneal injection ring phosphorus after inoculated tumour Amide (200mg/kg) is the 15th day tail vein injection 8 × 10 after inoculated tumour after injection 24 hours6/ CAR-T cell (sun F-duction rate 70-90%) or not transduced T cell control.Experimental results are shown in figure 8.
2.Huh7 subcutaneous transplantation knurl model
Referring to aforesaid operations, Huh7 subcutaneous transplantation knurl model is prepared, when tumor size is about 150mm3When, i.e. inoculated tumour 13rd day intraperitoneal injection of cyclophosphamide (200mg/kg) afterwards is the 14th day tail vein injection 7 after inoculated tumour after injection 24 hours ×106The T cell (positive transduction rate 70-90%) of/gene modification or not transduced T cell control, as a result such as Fig. 9 institute Show.
It is said according to Fig. 8 and Fig. 9 both without therapeutic effect in the negative tumor model SK-HEP-1 of GPC3 expression GPC3-28Z-sPD1 does not have non-specific killing under the premise of the bright feminine gender for target antigen；In the positive SK-HEP- of GPC3 expression In 1-GPC3 model, the two all has stronger anti-tumor activity in early days, but over time, GPC3-28Z-sPD1 With stronger tumor killing effect.
The quantity and serum I FN- γ of 9. periphery blood T cell of embodiment secrete situation
Mouse orbit takes blood that BDTruCount test tube is added, and then takes 20 μ L CD4-FITC/CD8-PE/CD3-PerCP anti- BDTruCount test tube is added in body；It draws the anticoagulation that 50 μ L and heparin mix well and CD4-FITC/CD8-PE/CD3- is added It in PerCP antibody, and mixes, is incubated at room temperature 30min；450 1 × erythrocyte cracked liquids of μ L (BD) are added, mix well, room temperature It is incubated for 15min.
Flow cytometer detection CD4+FITC and CD8+PE positive cell calculates the two positive cell number, calculation formula: cell number/μ Microballoon number/microballoon number/50 μ L of the cell number of L=FITC or the PE positive × total.
Detect the secretion of IFN-γ in serum, cell in vitro factors check of the detection method with embodiment 7.Testing result is such as Shown in Figure 10, the survival volume of periphery blood T cell is the key that can CAR-T play permanent tumor-inhibiting action, the secretion table of IFN-γ Bright T cell is sufficiently activated and plays antitumor action.
10. tumor tissues immunohistochemical staining of embodiment
The tumor cases slice for two kinds of animal models that Example 8 constructs, by the pathological section of paraffin embedding, is placed in 65 1~2h of piece is baked in DEG C baking oven, and after conventional dewaxing, aquation, the operation of aquation are carried out to sample are as follows: xylene soak 3 times, every time 10min (according to the sequence of dimethylbenzene I, II, III)；Soaked in absolute ethyl alcohol 2 times, each 5min；90% ethyl alcohol impregnates 5min； 80% ethyl alcohol impregnates 5min；70% ethyl alcohol impregnates 5min；Distilled water immersion 5min.
Slice is placed in PBS on shaking table after the completion of aquation to clean 3 times, each 5min uses 3%H2O2(being prepared with methanol) room Temperature is incubated for 10min, and with PBS buffer solution, (145rpm/min) is washed 3 times on decolorization swinging table, washs 2min every time.
Antigen hot repair is multiple: it will fill after citrate buffer solution boils and histotomy be put into citrate buffer solution, 92 DEG C ~98 DEG C of heating 10min, the cooling 1h of room temperature.With PBS buffer solution, (145rpm/min) is washed 3 times on decolorization swinging table, is washed every time Wash 5min；It is closed using 1%BSA, is incubated at room temperature 30min.
Corresponding GPC-3 antibody, PD-L1 antibody, GranB antibody, Ki67 antibody is added (with 1% in the slice closed BSA confining liquid 1:200 dilution) or blank control reagent, 4 DEG C of overnight incubations；With 0.5%PBST buffer on decolorization swinging table (145rpm/min) is washed 1 time, 5min；Then with PBS buffer solution, (145rpm/min) washs 2 times on decolorization swinging table, every time Wash 5min.
By the slice washed be added secondary antibody (Dako REALTMEnVisionTMDetection System, Peroxidase/DAB+, rabbit/mouse), 37 DEG C of incubation 1h.With 0.5%PBST buffer on decolorization swinging table (145rpm/ Min it) washs 2 times, each 5min；Then with PBS buffer solution, (145rpm/min) washs 1 time on decolorization swinging table, 5min.DAB (Dako REALTMEnVisionTMDetection System, Peroxidase/DAB+, 1:50 dilution) colour developing.
Haematoxylin is redyed to nuclei dyeing to peony, and the histotomy after redying is placed in 1% acidic alcohol differentiation liquid Differentiation 3~5 seconds；It is dehydrated after tap water flushing 20min transparent；90% ethyl alcohol impregnates 1min；100% ethyl alcohol I impregnates 1min； 100% ethyl alcohol II impregnates 1min；Xylene soak 3min；Neutral gum mounting is carried out in draught cupboard, is air-dried.
As a result as shown in figure 11, tumor biopsy CD3, Ki67, GramB expression of two kinds of liver cancer models: CD3 expression increases Illustrate that T cell viable count is more (GPC3-28Z-sPD1 treatment group CD3 expression is more) more；Ki67 expression, which is reduced, illustrates tumour cell It is inhibited by growth (GPC3-28Z-sPD1 treatment group Ki67 expression is minimum)；GramB secretion increase illustrate T cell activation after point More GramB are secreted, illustrate to kill stronger (expression of GPC3-28Z-sPD1 treatment group becomes apparent from).
The detection of embodiment 11.CAR-T cell proliferation in vitro situation
In order to verify expression PD1 after CAR-T cell target antigen stimulate post activation amplification situation difference, carried out amplification examination It tests.
By Untransduced T, tri- groups of T cells of GPC3-28Z, GPC3-28Z-sPD1 activation amplification the 4th day when and spoke It is incubated for altogether according to rear (50Gy) target cell SK-HEP-1-GPC3, additional IL2 stimulation is not added, give a target antigen stimulation weekly And continue surrounding, as a result as shown in figure 12, the amplification in vitro situation of GPC3-28Z-sPD1 is better than GPC3-28Z.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
SEQUENCE LISTING
<110>Ke Ji biological medicine (Shanghai) Co., Ltd.
Shanghai Inst. of Tumor
<120>immune effector cell of the Chimeric antigen receptor modification of PD-L1 blocking agent is co-expressed
<130> P2017-1557
<160> 41
<170> PatentIn version 3.5
<210> 1
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223>PD-1 signal coding sequence
<400> 1
atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60
<210> 2
<211> 450
<212> DNA
<213> Artificial Sequence
<220>
<223>PD-1 extracellular fragment coded sequence
<400> 2
ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 60
ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 120
gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 180
gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 240
cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 300
tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 360
gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 420
aggccagccg gccagttcca aaccctggtg 450
<210> 3
<211> 6
<212> DNA
<213> Artificial Sequence
<220>
<223>linker coded sequence
<400> 3
tatggt 6
<210> 4
<211> 321
<212> DNA
<213> Artificial Sequence
<220>
<223>DNA sequence dna of CH3 structural domain
<400> 4
cccccatgcc caccatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 60
cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 120
gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 180
gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 240
agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 300
tccaacaaag gcctcccgtc c 321
<210> 5
<211> 672
<212> DNA
<213> Artificial Sequence
<220>
<223>DNA sequence dna of hIgG4e1-Fc structural domain
<400> 5
cccccatgcc caccatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 60
cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 120
gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 180
gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 240
agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 300
tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 360
cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 420
agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 480
aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 540
ttcttcctct acagcaggct aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 600
tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 660
tctccgggta aa 672
<210> 6
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 6
acgcgtccta gcgctaccgg tcgccaccat gcagatccca caggcgccc 49
<210> 7
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 7
ctctcggggc tgcccaccat acaccagggt ttggaactgg c 41
<210> 8
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 8
tatggtgggc agccccgaga gccacag 27
<210> 9
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 9
aaaattcaaa gtctgtttca ctttacccgg agacagggag 40
<210> 10
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 10
caaaggcctc ccgtccgtga aacagacttt gaattttgac 40
<210> 11
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 11
tgcgggaagt gtacgtccgg gacgggggag cgatccagct gttagt 46
<210> 12
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 12
aagcttacgc gtcctagcgc taccggtcgc caccatgcag atcccacagg cgccctggcc 60
<210> 13
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 13
atgcaggccc tgccccctcg ctaggtcgac aatcaacctc tggattaca 49
<210> 14
<211> 243
<212> PRT
<213> Artificial Sequence
<220>
<223>AB1 scFv(9F2) amino acid sequence
<400> 14
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg
<210> 15
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223>area HCDR1 of AB1
<400> 15
Asp Tyr Glu Met His
1 5
<210> 16
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223>area HCDR2 of AB1
<400> 16
Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe Lys
1 5 10 15
Gly
<210> 17
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223>area HCDR3 of AB1
<400> 17
Phe Tyr Ser Tyr Ala Tyr
1 5
<210> 18
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223>area LCDR1 of AB1
<400> 18
Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Gln
1 5 10 15
<210> 19
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223>area LCDR2 of AB1
<400> 19
Lys Val Ser Asn Arg Phe Ser
1 5
<210> 20
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223>area LCDR3 of AB1
<400> 20
Ser Gln Ser Ile Tyr Val Pro Tyr Thr
1 5
<210> 21
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223>area VH of AB1
<400> 21
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
115
<210> 22
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<223>area VL of AB1
<400> 22
Asp Ile Val Met Thr Gln Thr 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 Gly Asn Thr Tyr Leu Gln 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 Ser Gln Ser
85 90 95
Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg
<210> 23
<211> 356
<212> PRT
<213> Artificial Sequence
<220>
<223> AB1-CD3Z
<400> 23
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
245 250 255
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
260 265 270
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
275 280 285
Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
290 295 300
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
305 310 315 320
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
325 330 335
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
340 345 350
Leu Pro Pro Arg
355
<210> 24
<211> 469
<212> PRT
<213> Artificial Sequence
<220>
<223> AB1-28z
<400> 24
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
290 295 300
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
305 310 315 320
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
325 330 335
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
340 345 350
Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
355 360 365
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
370 375 380
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
385 390 395 400
Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
405 410 415
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
420 425 430
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
435 440 445
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
450 455 460
Ala Leu Pro Pro Arg
465
<210> 25
<211> 466
<212> PRT
<213> Artificial Sequence
<220>
<223> AB1-BBZ
<400> 25
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
290 295 300
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
305 310 315 320
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
325 330 335
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
340 345 350
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
355 360 365
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
370 375 380
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
385 390 395 400
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
405 410 415
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
420 425 430
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
435 440 445
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
450 455 460
Pro Arg
465
<210> 26
<211> 511
<212> PRT
<213> Artificial Sequence
<220>
<223> AB1-28BBZ
<400> 26
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
290 295 300
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
305 310 315 320
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
325 330 335
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
340 345 350
Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
355 360 365
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
370 375 380
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
385 390 395 400
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
405 410 415
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
420 425 430
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln
435 440 445
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
450 455 460
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
465 470 475 480
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
485 490 495
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505 510
<210> 27
<211> 243
<212> PRT
<213> Artificial Sequence
<220>
<223>scFv of AB2(GC33) amino acid sequence
<400> 27
Asp Val 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 Ala Asn Thr Tyr Leu His 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 Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser
<210> 28
<211> 469
<212> PRT
<213> Artificial Sequence
<220>
<223> AB2-28Z
<400> 28
Asp Val 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 Ala Asn Thr Tyr Leu His 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 Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
290 295 300
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
305 310 315 320
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
325 330 335
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
340 345 350
Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
355 360 365
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
370 375 380
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
385 390 395 400
Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
405 410 415
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
420 425 430
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
435 440 445
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
450 455 460
Ala Leu Pro Pro Arg
465
<210> 29
<211> 511
<212> PRT
<213> Artificial Sequence
<220>
<223> AB2-28BBZ
<400> 29
Asp Val 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 Ala Asn Thr Tyr Leu His 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 Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
290 295 300
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
305 310 315 320
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
325 330 335
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
340 345 350
Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
355 360 365
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
370 375 380
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
385 390 395 400
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
405 410 415
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
420 425 430
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln
435 440 445
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
450 455 460
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
465 470 475 480
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
485 490 495
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505 510
<210> 30
<211> 356
<212> PRT
<213> Artificial Sequence
<220>
<223> AB1-CD3Z
<400> 30
Asp Val 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 Ala Asn Thr Tyr Leu His 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 Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
245 250 255
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
260 265 270
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
275 280 285
Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
290 295 300
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
305 310 315 320
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
325 330 335
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
340 345 350
Leu Pro Pro Arg
355
<210> 31
<211> 466
<212> PRT
<213> Artificial Sequence
<220>
<223> AB1-BBZ
<400> 31
Asp Val 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 Ala Asn Thr Tyr Leu His 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 Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
290 295 300
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
305 310 315 320
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
325 330 335
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
340 345 350
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
355 360 365
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
370 375 380
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
385 390 395 400
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
405 410 415
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
420 425 430
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
435 440 445
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
450 455 460
Pro Arg
465
<210> 32
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 32
gcaggggaaa gaatagtaga ca 22
<210> 33
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 33
cggcctggcg gcgtggag 18
<210> 34
<211> 441
<212> DNA
<213> Artificial Sequence
<220>
<223>the VH region nucleotide sequence of AB1
<400> 34
ggatcgatat ctgcggccta tctagccacc atgcgggtgc tgatcctgct gtggctgttt 60
accgccttcc ccggcttcct gagcgaggtg cagctggtgc agagcggcgc cgaggtgaag 120
aagcccggcg ccagcgtgaa ggtgagctgc aaggccagcg gctacacctt cagcgactac 180
gagatgcact gggtgcggca ggcccccggc cagggcctgg agtggatggg cgccatccac 240
cccggcagcg gcgacaccgc ctacaaccag cggttcaagg gccgggtgac catcaccgcc 300
gacaagagca ccagcaccgc ctacatggag ctgagcagcc tgcggagcga ggacaccgcc 360
gtgtactact gcgcccggtt ctacagctac gcctactggg gccagggcac cctggtgacc 420
gtgagcgccg ctagcaccaa a 441
<210> 35
<211> 429
<212> DNA
<213> Artificial Sequence
<220>
<223>the VL region nucleotide sequence of AB1
<400> 35
ggatcgatat ccaccatgga catgatggtg ctggcccagt tcctggcctt cctgctgctg 60
tggttcccag gcgctagatg cgacatcgtg atgacccaga cccccctgag cctgcccgtg 120
acccccggcg agcccgccag catcagctgc cggagcagcc agagcctggt gcacagcaac 180
ggcaacacct acctgcagtg gtacctgcag aagcccggcc agagccccca gctgctgatc 240
tacaaggtga gcaaccggtt cagcggcgtg cccgaccggt tcagcggcag cggcagcggc 300
accgacttca ccctgaagat cagccgggtg gaggccgagg acgtgggcgt gtactactgc 360
agccagagca tctacgtgcc ctacaccttc ggccagggca ccaagctgga gatcaaacgt 420
acggtggct 429
<210> 36
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 36
ctccacgccg ccaggccgga ggtgcagctg gtgcag 36
<210> 37
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 37
gcggtgtcct cgctccgcag gctgctcagc tccatgtagg cggtg 45
<210> 38
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 38
gcggagcgag gacaccgccg tgtactactg cgcccggttc tacagctac 49
<210> 39
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 39
cggcgctggc gtcgtggtac gtttgatctc cagcttggtg 40
<210> 40
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 40
accacgacgc cagcgccg 18
<210> 41
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 41
aatccagagg ttgattgtcg acctagcgag ggggcagggc ctgc 44

Claims (27)

1. a kind of immune effector cell for targeting GPC3, which is characterized in that the immune effector cell expression has targeting GPC3's Chimeric antigen receptor and PD-L1 blocking agent.
2. the immune effector cell of targeting GPC3 as described in claim 1, which is characterized in that the PD-L1 blocking agent is Natural PD-1, or can with the mutation PD-1 in conjunction with PD-L1, or be capable of natural in conjunction with PD-L1 or mutation PD-1 segment, Or the antibody of anti-PD-L1.
3. the immune effector cell of targeting GPC3 as claimed in claim 2, which is characterized in that the PD-L1 blocking agent are as follows:
Natural PD-1, or or natural in conjunction with PD-L1 or mutation PD-1 can be capable of with the mutation PD-1 in conjunction with PD-L1 Segment；
Natural PD-1, or or natural in conjunction with PD-L1 or mutation PD-1 can be capable of with the mutation PD-1 in conjunction with PD-L1 The fusogenic peptide that the segment composition of the Fc section of the Fc section or antibody of segment and antibody is formed；Or
Natural PD-1, or or natural in conjunction with PD-L1 or mutation PD-1 can be capable of with the mutation PD-1 in conjunction with PD-L1 Segment merges the fusogenic peptide to be formed with the mutant of the segment of the Fc section of antibody or the Fc section of antibody.
4. the immune effector cell of targeting GPC3 as claimed in claim 3, which is characterized in that the PD-L1 blocking agent is day Right PD-1, or or the segment of natural in conjunction with PD-L1 or mutation PD-1 can be capable of with the mutation PD-1 in conjunction with PD-L1.
5. as claimed in claim 3 targeting GPC3 immune effector cell, which is characterized in that the antibody be IgG1, IgG2, IgG3 or IgG4, preferably IgG4.
6. such as the immune effector cell of targeting GPC3 as claimed in claim 3 to 5, which is characterized in that the Fc of the antibody The segment of section is CH3 structural domain.
7. the immune effector cell of targeting GPC3 as claimed in claim 3, which is characterized in that the PD-L1 blocking agent is Natural PD-1, or or the segment of natural in conjunction with PD-L1 or mutation PD-1 can be capable of with the mutation PD-1 in conjunction with PD-L1 With the fusogenic peptide of hIgG4e1-Fc；Preferably, the natural PD-1, or can be with the mutation PD-1 in conjunction with PD-L1, or it can It is connected between natural or mutation PD-1 segment and the hIgG4e1-Fc in conjunction with PD-L1 via linker；More preferably Ground, the coded sequence of the linker is as shown in SEQ ID NO:3.
8. the immune effector cell of targeting GPC3 as claimed in claim 3, which is characterized in that the PD-L1 blocking agent is Natural PD-1, or or the segment of natural in conjunction with PD-L1 or mutation PD-1 can be capable of with the mutation PD-1 in conjunction with PD-L1 The fusogenic peptide formed with the structural domain of the CH3 of hIgG4e1-Fc；Preferably, the natural PD-1, or can be in conjunction with PD-L1 Mutation PD-1, or be capable of between the segment of natural in conjunction with PD-L1 or mutation PD-1 and the structural domain of the CH3 via Linker connection；It is highly preferred that the coded sequence of the liner is as shown in SEQ ID NO:3.
9. the immune effector cell of the targeting GPC3 as described in claim 2-8 is any, which is characterized in that the natural PD- 1, or can be with the mutation PD-1 in conjunction with PD-L1, or capableing of the segment of natural in conjunction with PD-L1 or mutation PD-1 includes SEQ Nucleotide sequence coded PD-1 extracellular region shown in ID NO:2.
10. the immune effector cell of the targeting GPC3 as described in claim 2-8 is any, which is characterized in that the natural PD- 1, or or the coding core of the segment of natural in conjunction with PD-L1 or mutation PD-1 can be capable of with the mutation PD-1 in conjunction with PD-L1 Nucleotide sequence includes that volume shown in the nucleotide sequence and SEQ ID NO:2 of PD-1 signal peptide is encoded shown in SEQ ID NO:1 The nucleotide sequence of code PD-1 extracellular region.
11. the immune effector cell of targeting GPC3 as claimed in claim 7, which is characterized in that the hIgG4e1-Fc's Coding nucleotide sequence is as shown in SEQ ID NO:5.
12. the immune effector cell of targeting GPC3 as claimed in claim 8, which is characterized in that the hIgG4e1-Fc's The coding nucleotide sequence of CH3 structural domain is as shown in SEQ ID NO:4.
13. the immune effector cell of targeting GPC3 as described in claim 1, which is characterized in that the Chimeric antigen receptor It include: the extracellular antigen binding domain for targeting GPC3, transmembrane region and intracellular signal area.
14. the immune effector cell of targeting GPC3 as claimed in claim 13, which is characterized in that the born of the same parents of the targeting GPC3 Exoantigen combined area have SEQ ID NO:15,16,17 shown in HCDR1, HCDR2, HCDR3 and SEQ ID NO:18, 19, LCDR1, LCDR2, LCDR3 shown in 20.
15. the immune effector cell of targeting GPC3 as claimed in claim 14, which is characterized in that the born of the same parents of the targeting GPC3 Contain light chain variable region shown in heavy chain variable region shown in SEQ ID NO:21 and SEQ ID NO:22 in exoantigen combined area.
16. the immune effector cell of targeting GPC3 as claimed in claim 13, which is characterized in that the born of the same parents of the targeting GPC3 Exoantigen combined area has scFv sequence shown in SEQ ID NO:14 or SEQ ID NO:27.
17. the immune effector cell of targeting GPC3 as claimed in claim 13, which is characterized in that the intracellular signal area packet It includes: the intracellular signal region sequence or Myd88 of CD3 ζ, Fc ε RI γ, CD27, CD28,4-1BB, CD134, CD40, or combinations thereof.
18. the immune effector cell of targeting GPC3 as claimed in claim 13, which is characterized in that the transmembrane region includes: CD8 transmembrane region or CD28 transmembrane region.
19. the immune effector cell of -18 any targeting GPC3 according to claim 1, which is characterized in that the inosculating antibody Original receptor has amino acid sequence shown in SEQ ID NO:23,24,25,26,28,29,30 or 31.
20. the immune effector cell of targeting GPC3 as described in claim 1, which is characterized in that the immune effector cell It include: T lymphocyte, NK cell or NKT cell, Treg cell.
21. a kind of nucleic acid constructs, the nucleic acid constructs includes being linked in sequence: PD-L1 blocking agent coded sequence is fitted into The coded sequence of the extracellular antigen binding domain of GPC3, transmembrane region and intracellular signal area coded sequence are targeted in antigen receptor.
22. nucleic acid constructs as claimed in claim 21, which is characterized in that the PD-L1 blocking agent coded sequence and born of the same parents To connect peptide-coding sequence connection between the coded sequence of exoantigen combined area；And/or
It further include ribosomal skip sequence between the PD-L1 blocking agent coded sequence and extracellular antigen binding domain coded sequence Column.
23. a kind of expression vector, it includes nucleic acid constructs described in coding claim 21 or 22.
24. a kind of virus, which is characterized in that the virus includes expression vector described in claim 23.
25. the purposes of nucleic acid constructs described in claim 21 or the expression vector comprising the nucleic acid constructs or virus is used In the immune effector cell for the targeting GPC3 for preparing target tumor.
26. the purposes of the immune effector cell of any targeting GPC3 of claim 1-20, is used to prepare and inhibits tumour Pharmaceutical composition.
27. a kind of for inhibiting the pharmaceutical composition of tumour, characterized in that it comprises: described in claim 1-20 is any The immune effector cell of Chimerical receptor modification.
CN201710676368.3A 2017-08-09 2017-08-09 Co-express the immune effector cell of the Chimeric antigen receptor modification of PD-L1 blocking agent Pending CN109385400A (en)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109748975A (en) * 2019-03-05 2019-05-14 南京市第一医院 A kind of bispecific chimeric antigen receptor and its application
CN110724697A (en) * 2018-07-16 2020-01-24 上海恒润达生生物科技有限公司 Chimeric antigen receptor method targeting GPC 3and CD19 double targets and application
WO2021013274A3 (en) * 2019-07-22 2021-03-11 北京助天科技发展有限公司 Chimeric antigen receptor and application thereof
WO2021170100A1 (en) * 2020-02-27 2021-09-02 Nanjing Legend Biotech Co., Ltd. Antibodies and chimeric antigen receptors targeting glypican-3 (gpc3) and methods of use thereof
CN113913385A (en) * 2021-09-01 2022-01-11 苏州璞惠卓越生物科技有限公司 Immune cell modified by inhibitory protein blocking type chimeric antigen receptor and application thereof
CN113999319A (en) * 2021-11-02 2022-02-01 深圳先进技术研究院 GPC3-targeting chimeric antigen receptor expressing PD-L1 single-chain antibody, T cell, preparation method and application
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WO2021013274A3 (en) * 2019-07-22 2021-03-11 北京助天科技发展有限公司 Chimeric antigen receptor and application thereof
WO2021170100A1 (en) * 2020-02-27 2021-09-02 Nanjing Legend Biotech Co., Ltd. Antibodies and chimeric antigen receptors targeting glypican-3 (gpc3) and methods of use thereof
WO2022028623A1 (en) 2020-08-07 2022-02-10 佧珐药业有限公司 Engineered cells and method for engineering cells
CN114316045A (en) * 2020-09-29 2022-04-12 锋宏生物医药科技(昆山)有限公司 Anti-PD-L1 antibody and its use
CN114316045B (en) * 2020-09-29 2024-07-12 英诺欧奇生物医药(苏州)有限公司 Anti-PD-L1 antibodies and uses thereof
CN115141806A (en) * 2021-03-31 2022-10-04 深圳宾德生物技术有限公司 Chimeric antigen receptor T cell targeting Her2 and expressing PD-L1 antibody, and preparation method and application thereof
WO2022214089A1 (en) 2021-04-08 2022-10-13 克莱格医学有限公司 Cellular immunotherapy use
WO2023274303A1 (en) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 Chimeric polypeptide for regulating cell physiological activity
CN113913385A (en) * 2021-09-01 2022-01-11 苏州璞惠卓越生物科技有限公司 Immune cell modified by inhibitory protein blocking type chimeric antigen receptor and application thereof
CN113999319A (en) * 2021-11-02 2022-02-01 深圳先进技术研究院 GPC3-targeting chimeric antigen receptor expressing PD-L1 single-chain antibody, T cell, preparation method and application

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