CN109970859A - Glypican-3 specific antibody and its specific C AR-T cell - Google Patents

Abstract

The present invention relates to Glypican-3 specific antibody and its specific C AR-T cells.In particular it relates to a kind of anti-human GPC-3 monoclonal antibody, the VL it includes the sum of the VH with amino acid sequence shown in SEQ ID NO:5 with amino acid sequence shown in SEQ ID NO:6.A kind of CAR-T cell the invention further relates to Chimeric antigen receptor fusion protein and comprising the fusion protein, it include: (i) single-chain antibody of the invention (scFv) from N-terminal to C-terminal, (ii) transmembrane domain, (iii) at least one costimulation structural domain, and (iv) activation domain.

Description

Glypican-3 specific antibody and its specific C AR-T cell

Technical field

The present invention relates to field of gene, relate more specifically to a kind of Monophosphoinositideproteoglycans proteoglycans-3 (Glypican- 3, GPC 3) specific antibody and its specific C AR-T cell, can be used for the adoptive immunity gene therapy of tumour.

Background technique

Immunization therapy is a kind of method of very promising treating cancer.T cell or T lymphocyte are that immune system has The weapon of effect, they can routinely help us to distinguish normal cell and exotic antigen or abnormal cell, as cancer or by The cell of infection.The Chimeric antigen receptor T cell (CAR-T) of gene modification is to design the common approach of tumor specific T cells. By the CAR-T cell input patient (referred to as adoptive cellular transfer or ACT) of target tumor related antigen (TAA), this represent one The effective immunotherapy method of kind.Compared with chemotherapy or antibody technique, the advantages of CAR-T technology, is that the T cell reprogrammed can Continue with proliferation and in patient's body the presence of (" drug living ").

CAR (Chimeric antigen receptor) be usually the single-chain antibody (scFv) as derived from a monoclonal antibody by hinge and Transmembrane domain is connected to the intracellular signal structural domain of variable number: the CD3- ζ structural domain of individual cells activation；And and CD3- ζ structural domain connected CD28, CD137 (4-1BB) or other costimulation domains (can also be replaced with CD27 signal domain CD28 or CD137 structural domain) (Fig. 1).The development of CAR from the first generation (not having costimulation domain) to the second generation (have a costimulation domain) to Third generation CAR (has multiple costimulation domains).CAR (the i.e. so-called third generation CAR) meeting with multiple costimulation domains generated Increased cell lysis activity, and the significant duration for improving CAR-T cell are generated, the anti-tumor activity of enhancing is shown.

However, the research of Chimeric antigen receptor at present is there are also many insufficient, ask there is also high recurrence rate, safety are low etc. Topic.Therefore, there is an urgent need in the art to develop, specificity is good, curative effect is stable, the Chimeric antigen receptor of Small side effects.

Summary of the invention

The purpose of the present invention is to provide Glypican-3 specific antibody and its specific C AR-T cells.

In the first aspect of the present invention, a kind of anti-human GPC-3 monoclonal antibody is provided, the antibody includes

(i) amino acid sequence heavy chain variable region as shown in SEQ ID NO:5；With

(ii) amino acid sequence light chain variable region as shown in SEQ ID NO:6.

In another preferred example, the preceding 55-200 amino acid region knot of the N-terminal of the antibody and people GPC-3 albumen It closes.

In another preferred example, the antibody is selected from: animal sources antibody, chimeric antibody, humanized antibody or its group It closes.

In another preferred example, the antibody is double-chain antibody or single-chain antibody.

In another preferred example, the antibody is monoclonal antibody.

In the second aspect of the present invention, a kind of single-chain antibody is provided, the single-chain antibody includes

(i) amino acid sequence heavy chain variable region as shown in SEQ ID NO:5；With

(ii) amino acid sequence light chain variable region as shown in SEQ ID NO:6.

In another preferred example, the single-chain antibody also includes the connector between VH and VL.

In another preferred example, the amino acid sequence of the connector is as shown in SEQ ID NO.:7.

In another preferred example, the amino acid sequence of the connector is sequence shown in 3-5 duplicate SEQ ID NO.:7 Column.

In another preferred example, the single-chain antibody has amino acid sequence shown in SEQ ID NO:4.

In another preferred example, the preceding 55-200 amino acid area of the N-terminal of the single-chain antibody and people GPC-3 albumen Domain combines.

In the third aspect of the present invention, a kind of Chimeric antigen receptor CAR is provided, which is characterized in that the CAR includes Single-chain antibody described in second aspect of the present invention.

In another preferred example, the CAR includes: from N-terminal to C-terminal

(i) single-chain antibody as claimed in claim 3,

(ii) transmembrane domain,

(iii) at least one costimulation structural domain, and

(iv) activation domain.

In another preferred example, the CAR has following formula I structure:

L-scFv-H-TM-C-CD3ζ (I)

In formula,

Each "-" independently is link peptide or peptide bond；

L is optional signal peptide sequence；

ScFv is single-chain antibody as claimed in claim 3；

H is optional hinge area；

TM is transmembrane domain；

C is costimulatory signal molecule；

CD3 ζ is the endochylema signal transduction sequence derived from CD3 ζ.

In another preferred example, the L be albumen selected from the group below signal peptide: CD8, GM-CSF, CD4, CD137, Or combinations thereof.

In another preferred example, the L is the signal peptide in the source CD8.

In another preferred example, the H is the hinge area of albumen selected from the group below: CD8, CD28, CD137 or its group It closes.

In another preferred example, the H is the hinge area in the source CD8.

In another preferred example, the TM be albumen selected from the group below transmembrane region: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or its group It closes.

In another preferred example, TM includes the transmembrane region in the source CD8 and/or the transmembrane region in the source CD28.

In another preferred example, the C be albumen selected from the group below costimulatory signal molecule: OX40, CD2, CD7, CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/ CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

In another preferred example, C includes the costimulatory signal molecule in the source 4-1BB and/or the costimulation letter in the source CD28 Number molecule.

In the fourth aspect of the present invention, a kind of recombinant protein is provided, the recombinant protein includes

(i) as described in the first aspect of the invention single-chain antibody described in antibody or second aspect of the present invention；And

(ii) sequence label of optional assistance expression and/or purifying.

In another preferred example, the sequence label includes 6His label.

In another preferred example, the recombinant protein (or polypeptide) includes fusion protein.

In another preferred example, the recombinant protein is monomer, dimer or polymer.

The fifth aspect of the present invention provides a kind of antibody drug conjugates, and the antibody drug conjugates contain:

(a) as described in the first aspect of the invention single-chain antibody described in antibody or second aspect of the present invention；And

(b) coupling moiety being coupled with the antibody moiety, the coupling moiety are selected from the group: detectable marker, medicine Object, toxin, cell factor, radionuclide, enzyme, or combinations thereof.

In another preferred example, the antibody moiety and the coupling moiety are carried out even by chemical bond or connector Connection.

In the sixth aspect of the present invention, a kind of nucleic acid molecules, the nucleic acid molecule encoding first aspect present invention are provided Chimeric antigen receptor (CAR) described in the single-chain antibody of the antibody, second aspect of the present invention, third aspect present invention, this Antibody drug conjugates described in recombinant protein described in invention fourth aspect or fifth aspect present invention.

In the seventh aspect of the present invention, a kind of carrier is provided, the carrier contains described in sixth aspect present invention Nucleic acid molecules.

In another preferred example, the carrier is selected from the group: DNA, RNA, plasmid, slow virus carrier, adenovirus vector, Retroviral vector, transposons, or combinations thereof.

In another preferred example, the carrier is slow virus carrier.

In the eighth aspect of the present invention, a kind of host cell is provided, the host cell contains the 7th side of the invention Carrier described in face is integrated with nucleic acid molecules described in the sixth aspect present invention of external source or the expression present invention the in chromosome CAR described in antibody described in one side, the single-chain antibody for expressing second aspect of the present invention or expression third aspect present invention.

In another preferred example, the cell is isolated cell and/or the cell is genetically engineered cell.

In another preferred example, the cell is mammalian cell.

In another preferred example, the cell is T cell.

In another preferred example, the host cell is the immunocyte of engineering.

In another preferred example, the immunocyte of the engineering includes T cell or NK cell, and preferably (i) is embedding It closes antigen receptor T cell (CAR-T cell)；Or (ii) Chimeric antigen receptor NK cell (CAR-NK cell).

In the ninth aspect of the present invention, a kind of method of preparation engineering immunocyte is provided, the engineering is exempted from Epidemic disease cell expresses CAR described in third aspect present invention, comprising the following steps: by nucleic acid molecules described in sixth aspect present invention Or carrier transduction described in seventh aspect present invention enters T cell or NK is intracellular, to obtain the engineering immunocyte.

In another preferred example, the method further includes carrying out function and validity to the engineering immunocyte of acquisition The step of detection.

In the tenth aspect of the present invention, a kind of preparation is provided, the preparation contains and resists described in first aspect present invention Body, the single-chain antibody of second aspect of the present invention, Chimeric antigen receptor, fourth aspect present invention institute described in third aspect present invention Cell described in antibody drug conjugates described in the recombinant protein or fifth aspect present invention stated or eighth aspect present invention, And pharmaceutically acceptable carrier, diluent or excipient.

In another preferred example, the preparation is pharmaceutical composition.

In another preferred example, the preparation is liquid formulation.

In another preferred example, the dosage form of the preparation is injection.

In another preferred example, the concentration of CAR-T cell described in the preparation is 1 × 10³-1×10⁸A cell/ml, Preferably 1 × 10⁴-1×10⁷A cell/ml.

In the eleventh aspect of the present invention, antibody, second party of the present invention described in a kind of first aspect present invention are provided Chimeric antigen receptor described in the single-chain antibody in face, third aspect present invention, recombinant protein described in fourth aspect present invention or The purposes of cell described in antibody drug conjugates described in fifth aspect present invention or eighth aspect present invention, is used to prepare The drug or preparation of prevention and/or treating cancer or tumour.

In another preferred example, the tumour is selected from the group: neoplastic hematologic disorder, solid tumor, or combinations thereof.

In another preferred example, the neoplastic hematologic disorder is selected from the group: acute myelocytic leukemia (AML), multiple marrow Tumor (MM), chronic lymphocytic leukemia (CLL), acute lymphatic leukaemia (ALL), diffusivity large B cell lymphoid tumor (DLBCL), or combinations thereof.

In another preferred example, the solid tumor is selected from the group: gastric cancer, gastric cancer peritoneum transfer, liver cancer, leukaemia, kidney Tumour, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical carcinoma, oophoroma, lymph cancer, nose Pharynx cancer, adrenal tumor, tumor of bladder, non-small cell lung cancer (NSCLC), glioma, carcinoma of endometrium, carcinoma of testis, knot are straight Intestinal cancer, urinary cancer, thyroid cancer, or combinations thereof.

In another preferred example, the solid tumor is selected from the group: oophoroma, celiothelioma, lung cancer, cancer of pancreas, breast cancer, liver Cancer, carcinoma of endometrium, or combinations thereof.

In the twelveth aspect of the present invention, a kind of reagent for being used to prepare cell described in eighth aspect present invention is provided Box, the kit contain container, and nucleic acid molecules described in the sixth aspect present invention in container or the present invention the Carrier described in seven aspects.

In the thirteenth aspect of the present invention, cell or the 6th side of the invention described in a kind of eighth aspect present invention are provided The purposes of preparation described in face, for prevention and/or treating cancer or tumour.

In the fourteenth aspect of the present invention, a kind of method for treating disease is provided, including is applied to object in need for the treatment of Preparation described in the cell described in suitable eighth aspect present invention or sixth aspect present invention.

In another preferred example, the disease is cancer or tumour.

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 shows the structure of CAR.The left side is first generation CAR (not having costimulation domain), and centre is second generation CAR (one Costimulation domain C D28 or 4-BB), the right is third generation CAR (two or more costimulation structural domain).

Fig. 2 shows the structure of GPC-3 albumen.HS, heparin sulfate, GPI, the glycosyl-phosphatidyl inositol of diagram albumen are repaired Decorations.

Fig. 3 shows the structure of GPC-3CAR construction.Second generation CAR is used herein as to be illustrated as representative.Pr, table Show GPC-3 antibody of the invention；Pu indicates published GPC-3 antibody.

Fig. 4 shows that the dyeing of GPC-3 antibody confirms expression of the GPC-3 in GPC-3 positive HCC cell line.First antibody is GPC-3, negative control, isotype IgG2, secondary antibody Alexa647.Fig. 4 A shows Hep-3B and Huh-7HCC (GPC-3 It is positive), SMMC7721 (GPC-3- is negative) cell line.Fig. 4 B is shown to be existed using GPC-3 antibody flow cytomery GPC-3 Expression in Hep-G2, GPC-3 positive cell.

Fig. 5 shows the amplification in vitro of GPC-3CAR-T cell.Published GPC-3 is as positive control.Promab's GPC-3CAR-T and published CAR-T cell showed efficient amplification in vitro (> 80 times) at 12 days.It include: public The GPC3-28-Z CAR-T cell opened, Promab GPC-3-28-Z CAR-T cell；Non- GPC-3-28-Z CAR-T cell；It is non- Transducing T cell.Z=CD3 ζ.

Fig. 6 shows the transduction of Fab antibody dyeing detection GPC-3CAR-T cell.3 independent experiments are shown in Fig. 6 Representativeness experiment.

Fig. 7 shows that GPC-3-CAR-T cell has high cell to GPC-3 positive cell (Hep-G2 and Huh-7 cell) Toxicity, to GPC-3 negative cells (SMMC7721 cell) no cytotoxicity.Fig. 7 A shows Hep-G2GPC-3 positive cell Experimental result, the cytotoxicity of GPC-3CAR-T cell are higher than control T cell.Fig. 7 B shows GPC-3 positive Huh-7 cell Experimental result, the cytotoxicity of GPC-3-CAR-T cell are higher than control T cell.Fig. 7 C shows that GPC-3 feminine gender SMMC7721 is thin The experimental result of born of the same parents, GPC-3 cytotoxicity are identical as control T cell.Pu-GPC-3 discloses GPC3, P3-GPC-3-Promab GPC-3 sequence.

Specific embodiment

The present inventor by depth studying extensively, be surprised to find that for the first time a kind of Glypican-3 specific antibody and Its specific C AR-T cell.In particular it relates to a kind of anti-human GPC-3 monoclonal antibody, it includes with SEQ ID VL of the sum of the VH of amino acid sequence shown in NO:5 with amino acid sequence shown in SEQ ID NO:6.Antibody of the invention is special Property targeting GPC-3 masculine liver cancer cell in GPC-3, without target normal liver tissue mouse monoclonal anti-human's antibody.This hair A kind of GPC-3-CAR-T cell of the bright cancer cell for further relating to targeting overexpression GPC-3 tumour antigen.CAR-T of the invention is thin Born of the same parents have high cell toxicity to several liver cancer cells (HCC), inactive to the liver cell of GPC-3 feminine gender.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 " 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 " 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.

As used herein, " Chimeric antigen receptor (CAR) " is a kind of fusion protein, and it includes can combine the extracellular of antigen Structural domain, with extracellular domain derived from the not transmembrane domain of homopolypeptide and at least one intracellular domain." inosculating antibody Original receptor (CAR) " is also referred to as " Chimerical receptor ", " T-body " or " chimeric immunity receptor (CIR) ".Described " can be in conjunction with anti- Former extracellular domain " is any oligopeptides or polypeptide referred in conjunction with a certain antigen." intracellular domain " refers to known work Any oligopeptides or polypeptide for transmitting signal to activate or inhibit the structural domain of intracellular biological process.

As used herein, " structural domain " refers in polypeptide the region for independently of other regions and being folded into specific structure.

As used herein, " tumour antigen " refers to that, with antigenic biomolecule, expressing leads to cancer.

GPC-3

GPC-3 is Monophosphoinositideproteoglycans proteoglycans-3 albumen, is a kind of film correlation heparan sulfate proteoglycan.GPC- 3 highly express in embryonic tissue such as developmental intestines and mesoderm tissues, express and lower in most of adult tissues, but It is overexpressed in hepatocellular carcinoma (HCC) and lung cancer.GPC-3 plays a crucial role in cell grows, is proliferated and shifts, and especially exists In hepatocellular carcinoma (HCC).The overexpression of GPC-3 shows related with hepatocellular carcinoma and patients with lung cancer prognosis mala.GPC-3 is to represent The valuable HCC molecular target of property.With shRNA or target GPC-3 CAR-T or humanization cytotoxic antibody or Vaccine carrys out silencing GPC-3, and the proliferation that will lead to HCC weakens.Recently, there is researcher in clinical test, utilize identification liver cancer The GC33 humanization GPC-3 antibody of patient's GPC-3 PROTEIN C terminal epitopes, is tested GPC-3 target spot, is applying weekly In the case where 2.5-20mg/kg, dose-limiting toxicity is not shown.Recently, researcher moves in preclinical xenogenesis It plants in mouse model, tests the immunotoxin conjugation of identification protein N terminal (HN3 antibody) and C- terminal epitopes (YP7) GPC-3 antibody.N-terminal antibody blocking WNT signal transduction, and C-terminal antibody does not have, and and pseudomonas exotoxin A (PE38) two antibody of segment composition show anti-tumor activity.

GPC-3 structure

People GPC-3 albumen is made of 580 amino acid, molecular weight 65.6kDa.GPC-3 gene is located at chromosome On Xq26.2.GPC-3 is attached to cell membrane by glycosyl-phosphatidyl inositol (GPI) anchor point.The structure of GPC-3 albumen such as Fig. 2 institute Show.

Furin cuts GPC-3 albumen between R358 and S359 amino acid, forms two subunits: the end N- of 40kDa and The C-terminal of 30kDa.Mature GPC-3 heterodimer is expressed on cell surface as glycosyl-phosphatidyl inositol (GPI).GPI anchor There are two the HS chains (Fig. 2) for being connected to C- terminal region (close to cell membrane) for point albumen tool.

GPC-3 signal

Cell surface Monophosphoinositideproteoglycans proteoglycans-3 (GPC3) passes through its heparin sulfate (HS) side chain and WNT and heparin (combining growth factor such as fibroblast growth factor (FGF) and hepatocyte growth factor (HGF)) formation compound, and The receptor-mediated signal transduction of liver cancer (HCC) cell moderate stimulation.

Antibody of the invention

The present invention relates to a kind of anti-human GPC-3 monoclonal antibodies comprising sequence VH and ammonia as shown in SEQ ID NO:5 Base acid sequence VL as shown in SEQ ID NO:6.The preceding 55-200 of anti-human GPC-3 monoclonal antibody and people GPC-3 protein N terminal A amino acid combines.

In another preferred example, anti-human GPC-3 monoclonal antibody is single-chain antibody (scFv).

As used herein, term " antibody " refers to immunoglobulin, is by two identical heavy chains and two identical light chains Four peptide chain structures being formed by connecting by interchain disulfide bond.The amino acid of immunoglobulin heavy chain constant region forms and puts in order Difference, therefore its antigenicity is also different.Accordingly, immunoglobulin can be divided into five classes, or be the not isotype of immunoglobulin, That is IgM, IgD, IgG, IgA and IgE, the heavy chain constant region corresponding to different immunoglobulin like protein be referred to as α, δ, ε, γ and μ.IgG represents most important one kind in immunoglobulin, and due to chemical structure and biological function difference, it can be divided into 4 again Subclass: IgG1, IgG2, IgG3 and IgG4.Light chain is divided into κ or λ chain by the difference of constant region.The Asia of different immunoglobulin like protein Unit structure and 3-d modelling are known to those skilled in the art.

As used herein, " single-chain antibody (scFv) " refers to the single chain polypeptide from antibody, remains in conjunction with antigen Ability.The example of scFv include by recombinant DNA technology formed antibody polypeptides, wherein heavy chain immunoglobulin (H chain) and gently The area Fv of chain (L chain) segment is connected via intervening sequence.It is well-known to those skilled in the art for preparing the various methods of scFv.

Heavy chain of antibody and light chain are very big close to the sequence variation of about 110 amino acid of N-terminal, are variable region (area V)；It is close Remaining amino acid sequence of C-terminal is relatively stable, is constant region (area C).Variable region includes 3 hypervariable regions (HVR) and 4 sequence phases To the conservative area FR (FR).The amino acid sequence of 4 FR is relatively conservative, does not participate in association reaction directly.It determines 3 hypervariable regions Determine the specificity of antibody, also known as complementarity-determining region (CDR).Every light chain variable region (LCVR) and heavy chain variable region (HCVR) At, the sequence being arranged successively from aminoterminal to c-terminus be FR1, CDR1 by 3 CDR regions and 4 FR district's groups, FR2, CDR2, FR3,CDR3,FR4.3 CDR regions of light chain, i.e. light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3；3 of heavy chain CDR region, i.e. heavy chain hypervariable region (HCDR), refer to HCDR1, HCDR2 and HCDR3.Invent the antibody or antigen-binding fragment The cdr amino acid residue in the area LCVR and HCVR meets known Kabat coding rule (LCDR1-3, HCDR2- in quantity and position 3), or meet the coding rule (HCDR1) of kabat and chothia.Four areas FR in native heavy and light chain variable region are big It is in beta sheet configuration in cause, is connected by three CDR of formation connection ring, part β-pleated sheet structure can be formed in some cases.Often CDR in chain is by the area FR firmly against the antigen-binding site for together forming antibody together and with the CDR of another chain. Be which Amino acid profile FR or CDR region domain can be determined by comparing the amino acid sequence of the antibody of same type.It is constant Area does not participate in the combination of antibody and antigen directly, but they show different effector functions, such as participate in the dependence of antibody In the cytotoxicity of antibody.

As used herein, term " antigen-binding fragment " refers to the Fab segment with antigen-binding activity, Fab ' segment, F (ab ') 2 segment or single Fv segment.Fv antibody contains antibody heavy chain variable region, light chain variable region, but does not have constant region, and have There is the minimum antibody fragment of whole antigen binding sites.In general, Fv antibody also includes that polypeptide between VH and VL structural domain connects Head, and structure needed for being capable of forming antigen binding.

As used herein, term " antigenic determinant " refers to discontinuous on antigen, by antibody of the present invention or antigen binding fragment The three-dimensional space site of section identification.

The present invention not only includes complete antibody, further includes the segment or antibody and other sequences with immunocompetent antibody Arrange the fusion protein formed.Therefore, the invention also includes the segments of the antibody, derivative and analogue.

In the present invention, antibody includes mouse prepared by the technology known to those skilled in the art, chimeric, humanization Or full people antibody.Recombinant antibodies, such as chimeric and humanization monoclonal antibody, including people's and inhuman portion Point, DNA recombinant technique well known in the art can be used.

As used herein, term " monoclonal antibody " refers to the antibody of the clones secrete derived from individual cells source.Monoclonal Antibody is high degree of specificity, for single epitope.The cell may be eukaryon, protokaryon or bacteriophage gram Grand cell strain.

As used herein, term " chimeric antibody " is the area the C gene splicing of the area the V gene by murine antibody and human antibody For mosaic gene, it is inserted into carrier, the antibody molecule of transfection host cell expression.Both the height for having remained parent murine antibody is special Property and affinity, and make Fc sections of its source of people can effective mediating biologic effector function.

As used herein, term " humanized antibody " is a kind of anti-variable region forms of modification of mouse of the present invention, has and be originated from The CDR region of (or being substantially originated from) non-human antibody (preferably mouse monoclonal antibody), and it is originated from human antibody sequence substantially The area FR and constant region；The CDR region sequence that mouse resists is grafted on different types of human germline antibody's frame sequence.Because of CDR Sequence is responsible for most antibody-antigene interaction, it is possible to it is specific natural that simulation is expressed by construction of expression vector The recombinant antibodies of existing antibody characteristic.

In the present invention, antibody can be monospecific, bispecific, tri-specific or more multiple specifics.

The preparation of antibody

Any method for being suitable for generating monoclonal antibody can be used in generating anti-GPC-3 antibody of the invention.For example, can be with Animal is immunized with connection or naturally occurring GPC-3 homodimer or its segment.Suitable methods of vaccination can be used, Including adjuvant, immunostimulant, booster immunization inoculation is repeated, one or more approach can be used.

The GPC-3 of any suitable form all can serve as immunogene (antigen), special to GPC-3 inhuman anti-for generating Body screens the biological activity of the antibody.Excitation immunogene can be overall length at acquaintance GPC-3, including natural homologous Dimer, or the peptide containing single/multiple epitope.Immunogene can be used alone, or one or more exempt from known in the art Epidemic focus reagents recombination uses.Immunogene can be purified by natural origin, or be generated in the cell of genetic modification.Coding The DNA of immunogene can be (such as cDNA) of genome or non genome on source.Suitable genetic carrier can be used The DNA of encoding immunogens is expressed, the carrier includes but is not limited to adenovirus vector, gland relevant viral vector, baculoviral load Body, material and non-virus carrier.

Full humanized antibody can be selected from any kind of immunoglobulin, including IgM, IgD, IgG, IgA and IgE.? In the present invention, antibody is IgG antibody, uses IgG4 hypotype.It is anti-by the screening of the biological characteristis described in Examples below Body is easily achieved the optimization of required constant domain sequence, to generate required biological activity.

Equally, any sort light chain can use in the Compounds and methods for of this paper.Specifically, κ, λ chain or its change Body can be used in the compound of the present invention and method.

The sequence of antibody of the present invention or the DNA molecular of its segment can use routine techniques, for example utilize PCR amplification or gene The methods of group library screening obtains.In addition, can also be fused together the coded sequence of light chain and heavy chain, single-chain antibody is formed.

Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.

In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.Then the DNA sequence dna can be introduced this In field in known various existing DNA moleculars (or such as carrier) and cell.

The invention further relates to the carriers comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence.This A little carriers can be used for converting host cell appropriate, allow it to expression protein.

Host cell is the various host cells of this field routine, as long as being able to satisfy makes above-mentioned recombinant expression carrier steadily It voluntarily replicates, and the entrained nucleic acid can be by effective expression.Specifically, host cell can be prokaryotic cell, such as Bacterial cell；Or low eukaryocyte, such as yeast cells；Or higher eucaryotic cells, such as mammalian cell.It is preferred dynamic Object cell includes (but being not limited to): CHO-S, CHO-K1, HEK-293 cell.

Preferred host cell includes E.coli TG1 or BL21 cell (expression single-chain antibody or Fab antibody), or CHO-K1 cell (expression overall length IgG antibody)

Heretofore described can be carried out the step of converting host cell with recombinant DNA with technology well known in the art.It obtains The transformant obtained can express the polypeptide of coded by said gene of the invention with conventional method culture, transformant.According to host used Cell is cultivated under suitable conditions with conventional medium.

In general, culture converts resulting host cell under conditions of being suitble to antibody expression of the present invention.Then with routine Immunoglobulin purification step, such as albumin A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange layer The routine well known to those skilled in the art such as analysis, hydrophobic chromatography, sieve chromatography or affinity chromatography isolates and purifies means and purifies To antibody of the invention.

Gained monoclonal antibody can be identified with conventional means.For example, the binding specificity of monoclonal antibody is available immune Precipitating or external combine test (such as radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) (ELISA)) to measure.

Chimeric antigen receptor (CAR)

The invention further relates to a kind of Chimeric antigen receptor fusion proteins, include: (i) anti-GPC-3 from N-terminal to C-terminal Single-chain antibody (scFv) (present invention), (ii) transmembrane domain, (iii) at least one costimulation structural domain, and (iv) activation Domain.

The present inventor is prepared for the GPC-3-ScFv- for hematologic malignancies (leukaemia, lymthoma and myeloma) CD28-CD3-CAR-T (GPC-3-CAR-T) cell shows High Fragmentation activity to the cancer cell for being overexpressed GPC-3.This hair Bright people provide prove GPC-3-CAR-T cell cultivate in vitro in the data that effectively expand.

Compared with the T cell that do not transduce, GPC-3-CAR-T cell has higher cell to the HCC cell of the GPC-3 positive Toxicity.GPC-3-CAR-T cell is to GPC-3 feminine gender HCC cell no cytotoxicity.

Compared with published GPC-3 antibody, the advantages of GPC-3 monoclonal antibody of the invention or GPC-3 single-chain antibody Be: antibody of the present invention has high degree of specificity to GPC-3 positive cancer cell, therefore cytotoxicity clinically is smaller.Therefore, GPC-3 antibody is very effective in many clinical applications as therapeutic agent.

Mouse anti human GPC-3 monoclonal antibody of the invention in conjunction with the GPC-3 in GPC-3 positive cancer cell, without with GPC-3 in GPC-3 negative cells is combined

Specifically, Chimeric antigen receptor of the invention (CAR) includes extracellular domain, transmembrane domain and intracellular Structural domain.Extracellular domain includes target-specific binding members (also referred to as antigen-binding domains).Intracellular domain includes Costimulatory signal conducting region and ζ chain part.Costimulatory signal conducting region refers to one of the intracellular domain including costimulatory molecules Part.Costimulatory molecules are cell surface molecule required for effective response of the lymphocyte to antigen, rather than antigen receptor Or their ligand.

Between the extracellular domain and transmembrane domain of CAR, or CAR cytoplasmic domain and transmembrane domain it Between, it may be incorporated into connector.As used herein, term " connector ", which is often referred to play, is connected to the extracellular of polypeptide chain for transmembrane domain Structural domain or any oligopeptides or polypeptide of cytoplasmic domain effect.Connector may include 0-300 amino acid, preferably 2 to 100 Amino acid and most preferably 3 to 50 amino acid.

In of the invention one preferable embodiment, the extracellular domain of CAR provided by the invention includes targeting The antigen-binding domains of GPC-3.CAR of the invention can be carried out when expressing in T cell based on antigen-binding specificity Antigen recognizing.When its combine its associated antigen when, influence tumour cell, cause tumour cell not grow, be prompted to death or with Other modes are affected, and the tumor load of patient is caused to reduce or eliminate.Antigen-binding domains are preferably and from costimulation The intracellular domain fusion of one or more of molecule and ζ chain.Preferably, antigen-binding domains and 4-1BB signal pass The intracellular domain fusion of transduction domain and the combination of CD3 ζ signal domain.

As used herein, " antigen-binding domains " " single chain antibody fragments " refer both to the Fab piece with antigen-binding activity Section, Fab ' segment, F (ab ')₂Segment or single Fv segment.Fv antibody contains antibody heavy chain variable region, light chain variable region, but does not have There is constant region, and there is the minimum antibody fragment of whole antigen binding sites.In general, Fv antibody also includes VH and VL structural domain Between peptide linker, and structure needed for being capable of forming antigen binding.Antigen-binding domains are usually scFv (single- chain variable fragment).Be typically of size of a complete antibody 1/6 of scFv.Single-chain antibody is preferably by one One amino acid chain sequence of nucleotide chain coding.As preferred embodiment of the invention, the scFv includes specific recognition The antibody of tumour high-expression antigen, preferably single-chain antibody.

For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include the extracellular domain for being fused to CAR Transmembrane domain.In one embodiment, using naturally with the associated transmembrane domain of one of structural domain in CAR. In some instances, transmembrane domain may be selected, or modified by amino acid replacement, to avoid by such structural domain knot It is bonded to the transmembrane domain of identical or different surface membrane protein, to minimize mutual with other members of receptor complex Effect.

Intracellular domain in CAR of the invention includes the signal transduction structural domain of 4-1BB and the signal transduction knot of CD3 ζ Structure domain.

CAR-T cell

As used herein, term " CAR-T cell of the invention ", " GPC-3CAR-T of Promab ", " CAR- of Promab T cell ", " Pr GPC-3CAR-T cell " are used interchangeably, and refer to that the CAR-T comprising GPC-3 single-chain antibody of the invention is thin Born of the same parents.

Preferably, CAR-T cell of the present invention has CD8-GPC-3ScFv MT28-CD28-CD3 δ structure, including people CD8 Signal peptide, GPC-3scFv (VH- connector 3x (G4S)-VL), CD8 hinge area, CD28 transmembrane region, activation domain CD3 ζ (Fig. 3, Pr). Specifically, the coded sequence of CD8 boot sequence is as shown in SEQ ID NO.:8.GPC-3scFv(V_HConnector-V_L)-CD28-CD3- The coded sequence of zeta is as shown in SEQ ID NO.:9, the position the 1-744 institute of the coded sequence of GPC-3scFv such as SEQ ID NO.:9 Show, and connector (coded sequence of 3x (G4S) as shown in the position 364-408 of SEQ ID NO.:9, the code sequence of CD28-CD3-zeta Column are as shown in the position 745-1452 of SEQ ID NO.:9.GPC-3-CAR (CD8-GPC-3ScFv MT28-CD28- of the invention CD3 δ) amino acid sequence as shown in SEQ ID NO.:10.

Carrier

The nucleic acid sequence of coding expectation molecule is obtained using the recombination method being known in the art, and is such as passed through Library is screened from the cell of expressing gene, by obtaining the gene from the known carrier including the gene, or passes through utilization The technology of standard is directly separated from cell and tissue comprising the gene.Optionally, interested gene can be synthesized life It produces.

Present invention provides the carriers for being wherein inserted into expression cassette of the invention.Derived from retrovirus such as slow virus Carrier is the suitable tools for realizing long-term gene transfer because they allow long-term, the stable integration of transgenosis and its in son It is proliferated in cell.It is more than the excellent of the carrier from oncogenic retrovirus such as murine leukemia virus that slow virus carrier, which has, Point, because of their transducible non-proliferative cells, such as liver cell.They also have the advantages that low immunogenicity.

Simplified summary typically operatively connects expression cassette or nucleic acid sequence of the invention to promoter, and is incorporated into Expression vector.The carrier is suitable for replicating and integrating eukaryocyte.Typical cloning vector includes that can be used for adjusting desired nucleic acid Transcription and translation terminator, initiation sequence and the promoter of sequence expression.

The gene delivery protocols of standard can also be used in expression construct of the invention, are used for nucleic acid immunization and gene therapy. The method of gene delivery is well known in the art.See such as U.S. Patent number 5,399,346,5,580,859,5,589, 466, it is incorporated to by reference of text herein.In another embodiment, the present invention provides gene therapy vectors.

The nucleic acid can be cloned into the carrier of many types.For example, the nucleic acid can be cloned into such carrier comprising but It is not limited to plasmid, phasmid, phage-derived object, animal virus and clay.Specific carrier interested includes expression vector, answers Carrier, probe generation vectors and sequencing vector processed.

Further, expression vector can be supplied to cell in the form of viral vectors.Viral vector technology is in the art It is well known and in (2001, Molecular Cloning:A the Laboratory Manual, Cold such as such as Sambrook Spring Harbor Laboratory, New York) and other virology and molecular biology manual in be described.It can Virus as carrier includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpesviral and slow virus.In general, Suitable carrier includes the replication orgin to work at least one organism, promoter sequence, convenient restriction enzyme sites With one or more selectable labels (for example, WO01/96584；WO01/29058；With U.S. Patent number 6,326,193).

Many systems based on virus are developed, for gene transfer to be entered mammalian cell.For example, reverse transcription disease Poison provides the convenient platform for gene delivery system.The gene of selection is inserted using the technology being known in the art Enter carrier and is packaged into retroviral particle.It is thin that the recombinant virus then can be separated and be transferred to internal or external object Born of the same parents.Many retroviral systems are well known in the art.In some embodiments, using adenovirus vector.It is many Adenovirus vector is well known in the art.In one embodiment, using slow virus carrier.

Additional promoter element, such as enhancer, the frequency that adjustable transcription starts.Normally, these are located at In the region 30-110bp of beginning site upstream, although having shown that many promoters recently also and including the function in initiation site downstream Element.Interval between promoter element is often flexible, to protect when element is squeezed or moves relative to another Hold promoter function.In thymidine kinase (tk) promoter, the interval between promoter element, which can be increased, separates 50bp, activity Just begin to decline.Depending on promoter, showing discrete component can cooperate or independently work, to start transcription.

One example of suitable promoter is instant early stage cytomegalovirus (CMV) promoter sequence.The promoter sequence The strong constitutive promoter sequence for being operably coupled to any polynucleotide sequence high level expression thereon can be driven by being classified as Column.Another example of suitable promoter is -1 α of the elongation growth factor (EF-1 α).It is also possible, however, to use other composing types open Promoter sequences, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary cancer viral (MMTV), people are immune scarce It falls into the long end of virus (HIV) and repeats (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Ai Baisitan- The instant early promoter of Ba Er (Epstein-Barr) virus, Rous sarcoma virus promoter and people's gene promoter, it is all Such as, but not limited to, actin promoter, Myosin promoter, ferroheme promoter and creatine kinase promoter.Further Ground, the present invention should not limited to the application of constitutive promoter.Inducible promoter is also contemplated as a part of the invention.It lures The use of conductivity type promoter provides molecular switch, can be when such expression is desired, and opening is operably connected The expression of the polynucleotide sequence of inducible promoter, or expression is closed when expression is undesirable.Inducible promoter Example includes but is not limited to metallothionein promoter, Glucocorticoid promoter, progesterone promoter and tetracycline promoter.

In order to assess the expression of CAR polypeptide or part thereof, the expression vector for being introduced into cell also may include selectable mark Any of gene or reporter or both are remembered, in order to from the cell mass for seeking to be transfected or infect by viral vectors Middle identification and selection expression cell.In other respects, selectable label can be carried on independent section of DNA and for corotation Contaminate program.The flank of selectable label and both reporters can all have adjusting sequence appropriate, so as in host It is expressed in cell.Useful selectable marker includes such as antibiotics resistance gene, such as neo etc..

Reporter is used to identify the cell of potential transfection and for evaluating the functionality for adjusting sequence.Normally, it reports Gene is following gene: it is not present in recipient organism or tissue or is expressed by recipient organism or tissue, and its Polypeptide is encoded, the expression of the polypeptide is clearly showed that by some property such as enzymatic activitys for being easy detection.It is had been incorporated into DNA After recipient cell, the expression of reporter is measured under the suitable time.Suitable reporter may include coding fluorescence Plain enzyme, beta galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or Green Fluorescent Protein gene (for example, Ui-Tei etc., 2000FEBS Letters479:79-82).Suitable expression system is well known and using known technology system It is standby or commercially obtain.In general, the construct quilt with minimum 5 flanking regions of the reporter expression of display highest level It is accredited as promoter.Such promoter region can be connected to reporter and adjust promoter-driving turn for evaluating reagent The ability of record.

Gene is introduced into cell and is well known in the art the method that gene expression enters cell.In expression vector In content, carrier can be easily introduced into host cell by any method in the art, for example, mammal, bacterium, ferment Female or insect cell.For example, expression vector can be transferred to host cell by physics, chemistry or biological means.

It include calcium phosphate precipitation by the physical method that polynucleotides introduce host cell, lipofection, particle bombardment, micro- Injection, electroporation etc..Production includes that the method for the cell of carrier and/or exogenous nucleic acid is well known in the present art.See example Such as Sambrook (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory,New York).It is calcium phosphate transfection by the preferred method that polynucleotides introduce host cell.

It include using DNA and RNA carrier by the biological method that interested polynucleotides introduce host cell.Virus carries Body, especially retroviral vector have become the most widely used side by gene insertion mammal such as people's cell Method.Other viral vectors may originate from slow virus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus etc..See example Such as U.S. Patent number 5,350,674 and 5,585,362.

It include dispersion system of colloid by the chemical means that polynucleotides introduce host cell, such as macromolecular complex is received Rice glue capsule, microballoon, pearl；With the system based on lipid, including oil in water emulsion, micella, mixed micelle and liposome.As external Exemplary colloid system with internal tool for transmitting (delivery vehicle) is liposome (for example, artificial membrane vesicle).

Using non-viral delivery system, exemplary tool for transmitting is liposome.Consider to use lipid formulations, Nucleic acid is introduced host cell (external, in vitro (ex vivo) or in vivo).On the other hand, which can be related to lipid Connection.Nucleic acid associated with lipid can be encapsulated into the aqueous interior of liposome, be dispersed in the lipid bilayer of liposome, through with Both associated connection molecule is attached to liposome for liposome and oligonucleotides, falls into liposome, with lipid bluk recombination, divides It is dispersed in the solution comprising lipid, mixes with lipid, combine with lipid, be included in lipid as suspension, be included in micella In or with micella it is compound or otherwise associated with lipid.Lipid associated with composition, lipid/DNA or lipid/ Expression vector is not limited to any specific structure in solution.For example, they may be present in bilayer structure, as micella or With " (collapsed) of collapse " structure.They can also simply be distributed in the solution, it is possible to create size or shape are not Uniform aggregation.Lipid is fatty material, can be the natural lipid occurred or synthesize.For example, lipid includes lipid droplet, Derivative such as fatty acid, alcohols, amine, amino in cytoplasm and comprising long-chain fat race hydrocarbon and they naturally occurs in it In such of alcohols and aldehydes compound.

It is preferably carried out in mode at of the invention one, the carrier is slow virus carrier.

Preparation

It is single-stranded anti-containing antibody, second aspect of the present invention described in first aspect present invention that the present invention provides a kind of Chimeric antigen receptor described in body, third aspect present invention, recombinant protein described in fourth aspect present invention or the present invention the 5th It is cell described in antibody drug conjugates described in aspect or eighth aspect present invention and pharmaceutically acceptable carrier, dilute Release agent or excipient.In one embodiment, the preparation is liquid formulation.Preferably, the preparation is injection.It is preferred that Ground, the concentration of CAR-T cell described in the preparation are 1 × 10³-1×10⁸A cell/ml, more preferably 1 × 10⁴-1×10⁷It is a Cell/ml.

In one embodiment, the preparation may include buffer such as neutral buffered saline, sulfate buffered saline Etc.；Carbohydrate such as glucose, mannose, sucrose or glucan, mannitol；Protein；Polypeptide or amino acid are such as Glycine；Antioxidant；Chelating agent such as EDTA or glutathione；Adjuvant (for example, aluminium hydroxide)；And preservative.The present invention Preparation be preferably formulated for intravenously applying.

Therapeutic application

GPC-3 antibody of the invention can be used for immunization therapy application selected from the group below: toxin/drug coupling Ab, therapeutic Monoclonal antibody, humanization GPC-3 antibody, CAR-T immunization therapy, or combinations thereof.

GPC-3-CAR-T comprising GPC-3 antibody of the present invention can the GPC-3 such as efficient targeting HCC, liver cancer, lung cancer it is positive thin GPC-3 in born of the same parents system.

GPC-3-CAR-T of the invention can be with different types of chemotherapy (immunologic test point inhibitor), targeted therapy, small The use in conjunction such as molecule inhibitor, antibody.

GPC-3 antibody of the invention can be modified by direct mutagenesis, for regulating and controlling affinity；It can also be used for source of people Change the generation with full-length human antibody.

GPC-3-CAR-T cell of the invention can be used clinically for the treatment of GPC-3 positive cell.

(epitope is located at the N-terminal area of GPC-3 segment to the -terminal amino acid of GPC-3 antibody target GPC-3 of the invention Domain: the 55-200 amino acid), since the protein fragments of its targeting are closer in tumour cell, with CAR-T and other environment C- end structure compared to more advantageous.The modification in co-activation domain: can use CD28, and 4-1BB etc. improves its effect.It can benefit CAR is generated with label conjugation GPC-3scFV.

Third generation CAR-T or other co-activation signal domains can be used for identical GPC-3-scFv in CAR.

GPC-3 with other target other tumour antigens or tumor microenvironment (VEGFR-1-3) CAR or double scFv-CAR groups Closing can be with use in conjunction, to enhance the activity that GPC-3-CAR is used alone.

The present invention include the cell (for example, T cell) transduceed with the slow virus carrier (LV) for encoding expression cassette of the present invention into Capable therapeutic application.The T cell of transduction can targets neoplastic cells marker GPC-3, synergistic activation T cell causes T cell Immune response, to significantly improve its killing-efficiency to tumour cell.

Therefore, it answers present invention provides T cell-mediation of the stimulation to the target cell of mammal group or tissue is immune The method answered comprising following steps: CAR-T cell of the invention is applied to mammal.

In one embodiment, the present invention includes a kind of cell therapy, separation patient's Autologous T cells (or heterologous confession Body), activate and carry out genetic modification generate CAR-T cell, be subsequently injected into it is same in patient body.This mode suffers from the anti-place of graft Main disease probability is extremely low, and antigen is identified in a manner of no MHC limitation by T cell.In addition, a kind of CAR-T can treat expression, this is anti- Former all cancers.Unlike antibody therapy, CAR-T cell can replicate in vivo, generate and can lead to the long-term of continued tumor control Persistence.

In one embodiment, CAR-T cell of the invention can undergo firm internal T cell to extend and sustainable prolong Long time quantum.In addition, the immune response that CAR is mediated can be a part of adoptive immunotherapy step, wherein it is thin to modify T by CAR- Born of the same parents induce the immune response to the antigen-binding domains specificity in CAR.For example, the CAR-T cell of anti-GPC-3 causes anti-table Up to the specific immune response of the cell of GPC-3.

Although data disclosed herein specifically disclose including anti-GPC-3scFv, hinge and transmembrane region and 4-1BB and The slow virus carrier of CD3 ζ signal transduction structural domain, but this invention generally should be construed as including to each in construct component part A any amount of variation.

Medicable cancer includes that not by vascularization or substantially there are no by the tumour and vascularization of vascularization Tumour.Cancer may include non-physical knurl (such as haematological tumours, such as leukaemia and lymthoma) or may include solid tumor.With this The cancer types of the CAR treatment of invention include but is not limited to that cancer, enblastoma and sarcoma and certain leukaemia or lymphoid malignant are swollen Tumor, benign and malignant tumour and malignant tumor, such as sarcoma, cancer and melanoma.Also include adult lesion/cancer disease and pediatric tumor/ Cancer.

Hematologic cancer is the cancer of blood or marrow.The example of hematology (or hematogenous) cancer includes leukaemia, packet Including acute leukemia, (such as acute lymphoblastic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and pulpefaction are thin Born of the same parents' property, promyelocyte, grain-monocyte type, monocarpotic cellularity and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukaemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymph Tumor, hodgkin's disease, non Hodgkin lymphom (painless and high-grade form), Huppert's disease, Walden Si Telun Family name's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumor is the abnormal mass of the tissue usually not comprising tumour or fluid zone.Solid tumor can be benign or malignant 's.Different types of solid tumor names (such as sarcoma, cancer and lymthoma) with the cell type for forming them.Solid tumor such as meat The example of tumor and cancer include fibrosarcoma, myxosarcoma, embryonal-cell lipoma celiothelioma, lymphoid malignancy, cancer of pancreas oophoroma,.

CAR- modification T cell of the invention also is used as the vaccine to mammal Ex vivo immunization and/or in vivo Type.Preferably, mammal is behaved.

For Ex vivo immunization, at least one of the following occurs in vitro before cell application is entered mammal: i) Amplifying cells, ii) nucleic acid that will encode CAR introduces cell and/or iii) Cell Cryopreservation.

In vitro program is well known in the present art, and is being discussed more fully below.Briefly, cell is from the food in one's mouth Separate in newborn animal (preferably people) and with the carrier for expressing CAR disclosed herein carry out gene modification (that is, ex vivo transduction or turn Dye).The cell of CAR- modification can be administered to mammalian subject, to provide treatment benefit.Mammalian subject can be The cell of people and CAR- modification can be self relative to recipient.Optionally, cell can be different base of the same race relative to recipient Cause, isogenic (syngeneic) or xenogenesis.

Other than for Ex vivo immunization using based on the vaccine of cell, present invention provides vivo immunizations to cause For the composition and method of the immune response of antigen in patient.

The present invention provides the methods for the treatment of tumour comprising is administered to a effective amount of present invention of subject for needing it CAR- modification T cell.

The T cell of CAR- modification of the invention can be administered alone or as pharmaceutical composition and diluent and/or and its His component such as IL-2, IL-17 or other cell factors or cell mass combine application.Briefly, pharmaceutical composition of the invention Object may include target cell group as described herein, with one or more pharmacy or physiologically acceptable carriers, diluent or tax Shape agent combines.Such composition may include buffer such as neutral buffered saline, sulfate buffered saline etc.；Carbon hydrate Object such as glucose, mannose, sucrose or glucan, mannitol；Protein；Polypeptide or amino acid such as glycine；It is anti-oxidant Agent；Chelating agent such as EDTA or glutathione；Adjuvant (for example, aluminium hydroxide)；And preservative.Composition of the invention is preferably matched System is for intravenously applying.

The mode that pharmaceutical composition of the invention can be suitable for the disease of (or prevention) to be treated is applied.The quantity of application It will be determined by such factor with frequency, although such as the illness of patient and the type of patient disease and severity --- it is appropriate Dosage can be determined by clinical test.

When pointing out " effective quantity in immunology ", " antitumor effective quantity ", " tumour-inhibition effective quantity " or " therapeutic dose ", The precise volume of the present composition to be administered can be determined that the age of consideration patient (object), weight, tumour are big by doctor The individual difference of small, infection or metastasis degree and illness.It can usually point out: the pharmaceutical composition including T cell described herein It can be with 10⁴To 10⁹A cell/kg weight dosage, preferably 10⁵To 10⁶A cell/kg weight dosage (including those ranges Interior all integer values) application.T cell composition can also be with these dosage multiple applications.Cell can be by using immune treatment Well known injection technique in method (see such as Rosenberg etc., NewEng.J.of Med.319:1676,1988) application.For The optimal dose and therapeutic scheme of specific patient can by monitor patient disease indication and therefore adjustment for the treatment of by medical domain Technical staff is readily determined.

The application of object composition object can carry out in any convenient manner, including pass through spray-on process, inject, swallow, is defeated Liquid, implantation or transplanting.Compositions described herein can by subcutaneous, intradermal, tumor, in knot, in spinal cord, intramuscular, pass through vein Patient is administered in interior (i.v.) injection or peritonaeum.In one embodiment, T cell composition of the invention by intradermal or Subcutaneous injection is administered to patient.In another embodiment, T cell composition of the invention is preferably applied by i.v. injection With.The composition of T cell can be injected directly into tumour, lymph node or infection position.

In some embodiments of the present invention, using method described herein or it is known in the art other by T cell The cell for extending to the method activation and extension of therapeutic level, in conjunction with any amount of related form of therapy (for example, it Before, simultaneously or after) be administered to patient, the form of therapy includes but is not limited to be treated with following reagent: the reagent Such as antiviral therapy, cidofovir and interleukin 2, cytarabine (also being known as ARA-C) or he to MS patient Pearl monoclonal antibody treats or to the method pearl monoclonal antibody in distress treatment of psoriatic or to the other treatment of PML patient.Further implementing In mode, T cell of the invention can with below in conjunction with using: chemotherapy, radiation, immunosuppressor, such as, cyclosporin, sulphur azoles Purine, methopterin, mycophenolate and FK506, antibody or other immunotherapeutic agents.In further embodiment, this hair Bright cell composition and bone-marrow transplantation utilize chemotherapeutics such as fludarabine, external beam radiation therapy (XRT), ring phosphinylidyne Amine is administered to patient in conjunction with (prior to, concurrently with, or after for example).For example, in one embodiment, object can undergo high agent Quantify the standard care treated, carries out autologous peripheral blood stemcell transplant later.In some embodiments, after the transfer, object receives The injection of the immunocyte of extension of the invention.In an additional embodiment, the cell of extension in surgery operation consent or Surgical site infections application.

The dosage for being administered to the above treatment of patient will become with the exact properties for the treatment of illness and the recipient for the treatment of Change.The practice that people's applied dose ratio can receive according to this field is implemented.In general, treatment or each course for the treatment of every time, can by 1 × 10⁶It is a to 1 × 10¹⁰A modified T cell of the present invention (e.g., CAR-T20 cell) is applied for example, by the mode of venous re-transfusion For patient.

Main advantages of the present invention include:

(a) GPC-3 in antibody target GPC-3 masculine liver cancer cell of the invention, without targeting normal liver tissue.

(b) antibody of the present invention has high degree of specificity to GPC-3 positive cancer cell, the cytotoxicity in clinical use compared with It is small.

(c) CAR-T cell of the invention has high cell toxicity to several liver cancer cells (HCC), to the liver of GPC-3 feminine gender It is cell inactivated.

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, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.

GPC-3CAR construct is cloned into Xba I and the EcoR I site of slow virus carrier by inventor, thus in slow disease CD19CAR construct is generated in poisonous carrier.The XbaI and EcoRI of pCD510-FMC63-28z slow virus CAR construct clone position It include GPC-3ScFv-CD28-CD3zeta Insert Fragment between point.

Slow virus generates in 293T cell, determines titre by RT-PCR.The slow virus of equivalent is then used for T cell Transduction, as described in method part.

The production of embodiment 1CAR slow virus

Slow virus is prepared by following steps:

1st day:

1. by 5x 10⁶Culture dish of the HEK293FT cell inoculation to 100 mm dias；

2nd day:

2. checking to ensure that the cell confluency of 70%-90%；

3. the culture dish for each 100 mm dia prepares transfection composite, process is as follows:

A. in 1.5ml pipe A: by 2.5 μ g CAR (Chimeric antigen receptor) DNA plasmids (plasmid) and 20 μ L slow virus Packaged combination (ALSTEM, cat#VP100；See Appendix B3) it is diluted to the training of 0.5ml DMEM or Opti-MEM serum-free It supports in base, is gently mixed；

B. in 1.5ml pipe B: 30 μ L Nanofect transfection reagents (ALSTEM, cat.no.NF100) are diluted to In 0.5ml DMEM or Opti-MEM serum free medium, it is gently mixed；

C. the NanoFect/DMEM in pipe B is added in DNA/DMEM solution (pipe A), vortex 5-10 seconds, by DMEM- Plasmid-NanoFect mixture is cultivated 15 minutes at room temperature；

4. the transfection composite that step 3 obtains all is dripped on cell plates, rotate back and forth so that transfection composite is uniform Ground disperses onboard；

5.37 DEG C of humidification 5%CO₂The cell is incubated overnight in incubator；

3rd day:

6. replacing the supernatant of above-mentioned transfection composite with 10mL fresh culture, and supplement 20 μ L ViralBoost (500X,ALSTEM,cat.no.VB100)；

7.37 DEG C are cultivated 24 hours；

4th day:

8. the culture solution supernatant containing slow virus is collected into, 50mL is sterile to be had in lid conical centrifuge tube, and is placed in ice On；

9. supernatant is centrifuged 15 minutes under the conditions of 4 DEG C 3000 turns, with sedimentation cell fragment；

10. utilizing the supernatant after 0.45 μm of low protein binding sterilizing filter filtering clarification；

11. measuring concentration/titre of slow virus by quantitative RT-PCR, Lenti-X qRT-PCR Titer Kit is utilized (Clontech；Catalog number 631235), the virus concentration for measuring HEK293 in supernatant (is located in advance by DNaseI Any possible remaining Plasmid DNA of reason removal)；

12. above-mentioned virus can be used for infecting, purifying, or be stored in -80 DEG C of spare, preferably aliquots as virus stock solution used Individually storage, to reduce the loss of multigelation bring virus titer.

2 slow virus packaging system of embodiment

The description of product

Name of product: SuperLenti^TM Lentivirus Packaging System

Specification:

Production for lentiviral particle, it usually needs three kinds of ingredients: 1) slow virus carrier containing target foreign gene, 2) package carrier comprising all required virus structural proteins, 3) expression vesicular stomatitis virus (VSV) glycoprotein (G).The Three generations's slow virus packaging system provides maximum biological safety, because slow virus Rev gene is as opposite other structures The separate carrier of gene provides, and further obviates the possibility in the virion that carrier is inversely recombinated to replication capacity Property.Third generation slow virus package combination only supports the Lentiviral of chimeric 5'LTR, wherein HIV promoter by CMV or RSV substitution, therefore keep it unrelated with TAT.

SuperLenti slow virus package combination is a kind of instant third generation slow virus packaging system based on HIV, Wherein element needed for plasmid expression lentivirus production allows to create the invalid slow virus of the duplication based on HIV-1, is dividing Or it is delivered in nondividing mammalian cell and expresses target foreign gene.

Catalog number: VP 100；

Specification: 200 μ L；

Transport: room temperature；

Storage and safety: the product can -20 DEG C storage-stable 6 months, avoid iterative cycles as far as possible, it should dispense At the dosage of first use.

Application method:

For the slow virus packaging of 100mm culture dish, the expression of 2.5 μ g slow virus is mixed with 20 μ l slow virus package combinations Carrier.

For the slow virus packaging of 150mm culture dish, the expression of 5 μ g slow virus is mixed with 40 μ L slow virus package combinations and is carried Body.

Quality control: the slow virus packaging of every batch of is under transfection experiment by using under human embryos kidney 293 cell The test of progress.

Only used for research.It is not used in diagnosis or treatment procedure.

The separation of peripheral blood mononuclear cells (PBMC) in 3 whole blood of embodiment

Whole blood (Stanford University Hospital Blood Center, Stamford, California) acquisition (is depended on from single individual or multiple individuals In with blood demand), it is placed in the Heparin vacutainers (Becton Dickinson company) of 10mL.

Note: should handle blood in two hours after blood sampling, to ensure cell yield maximum.Blood can be at room temperature (interior) storage was stayed overnight, for processing in second day；However, cell yield has some losses.Blood should not store at 4 DEG C In blank pipe.

In 50ml conical centrifuge tube, about 10ml anticoagulated blood is mixed with sterile phosphate buffer (PBS), total volume For 20ml, (PBS, pH7/4 are free of Ca²⁺/Mg²⁺)。

In another sterile 50mL conical centrifuge tube, absorption 15mL Ficoll-Paque PLUS (GE Healthcare, 17-1440-03).Blood/PBS of 20ml is gently laminated to the surface of Ficoll very much, and at room temperature with 400xg from Heart sample 30-40min, without braking.

The careful cellular layer comprising peripheral blood mononuclear cells (PBMC) for drawing diluting plasma and Ficoll boundary, avoids Suck Ficoll.In order to ensure completely removing Ficoll, blood platelet and plasma protein, PBMC is washed twice with PBS, total volume is 40ml, and at room temperature with 200xg centrifugation 10 minutes.Then cell is counted with hemacytometer.If the PBMC of washing is vertical Use, then with CAR-T culture medium (AIM V-AlbuMAX (BSA) (Life Technologies), containing 5%AB serum with 1.25 μ g/mL amphotericin Bs (Gemini Bioproducts, Woodland, CA), 100U/mL penicillin and 100 μ g/mL chains Mycin) it washed once.If PBMC freezed, washing cell is resuspended in transfer insulation bottle, -80 DEG C, holding 24 is small When, it is then stored in liquid nitrogen.

4 peripheral blood mononuclear cells T cell activation of embodiment

If isolated cell (is washed, no Ca with 1xPBS (pH7.4) using the PBMC of fresh separated²⁺/Mg²⁺) use (AIM V-AlbuMAX (BSA) (Life Technologies), contains 5%AB serum and 1.25 μ g/mL two to CAR-T culture medium Property mycin B (Gemini Bioproducts, Woodland, CA), 100U/mL penicillin and 100 μ g/mL streptomysins) washing one It is secondary, it does not use Human Inter Leukin-2 (huIL-2) (Invitrogen), concentration is 5 × 10⁵A cell/mL.Be free of huIL- It washed once in 2 CAR-T culture medium, finally use the (1000 × stock of huIL2 containing 30U/mL；Invitrogen CAR-T) Culture medium is resuspended floating to final concentration of 5 × 10⁵A cell/mL.

If using freezing PBMC, 9mL preheating (37 DEG C) DMEM culture medium (Life Technologies, contains 10%FBS, 100u/mL penicillin and 100 μ g/mL streptomysins) in, it thaws and cell (1 × 10 is resuspended⁷Cell/mL), concentration is 5×10⁵A cell/mL.It 300xg centrifuge cell 5 minutes, then washed once in the CAR-T culture medium without huIL-2, most It is resuspended afterwards with the CAR-T culture medium of the huIL2 containing 30U/mL floating to final concentration of 5 × 10⁵A cell/mL.

Before activation, magnetic bead (Invitrogen) the sterile 1xPBS of 1mL of anti-human CD28 and CD3 antibody will be conjugated (pH7.4) (pearl is separated from solution using Magnetic rack) washing three times, is then resuspended in (30U.mL in CAR-T culture medium HuIL-2), final concentration of 2 × 10⁷Pearl/mL.

Then the magnetic bead of 25uL is added in the PBMC of 1mL, with the magnetic cell of 1:1 than mixing PBMC and magnetic bead.

Required amount of aliquot is added in each hole of the low attachment in 12 holes or untreated cell culture plate, and 37 CO at DEG C₂In the presence of cultivate 24 hours, then carry out viral transduction.

Embodiment 5T cell transduction and amplification

After PBMC activation, by cell at 37 DEG C, 5%CO₂Lower culture 24 hours.

It thaws on ice slow virus.Every hole 1x10⁶A cell simultaneously adds 5x10⁶A slow virus and 2 μ L/mL Transplus training It supports base (Alstem, Richmond, CA) (finally diluting 1:500).It repeats to add virus after cell culture 24 hours.

Then cell cultivated 12-14 days to (total incubation time depends in the fresh culture containing 30U/ml IL-2 The final amt of required CAR-T cell).

Every 2-3 days analysis cell concentrations, culture medium diluting cells suspension was added to 1 × 10⁶Cell/mL.

Transduction verifying-the cell dyeing of embodiment 6

Cell is washed, in FACS buffer solution (phosphate buffer (PBS), in buffer plus 0.1% Sodium azide and 0.4% Bovine serum albumin(BSA)) in suspend, then cell press 1x10⁶Equal part point is stored to each test tube.

With normal goats IgG Fc (lifetechnologies) receptor blocking, it is diluted normal to add 100 μ L 1:1000 Goat IgG is incubated for 10 minutes on ice to each pipe.

1.0ml FACS buffer solution is added in every pipe, is sufficiently mixed and with 300g centrifugation 5 minutes.

2 antibody of polyclonal goat anti-mouse-F (ab) (Life Technologies) inspection of biotin labeling is added Survey CD19scFv；The normal polyclonal goat IgG antibody (Life Technologies) that biotin labeling is added is used as isotype Control.(1:200 dilution, reaction volume are 100 μ l).

Cell is incubated for 25 minutes at 4 DEG C, and washed once with FACS buffer solution.

The suspension cell and by the way that the 100 diluted normal mouses of μ l 1:1000 are added into each pipe in FACS buffer solution IgG blocks cell.It is incubated for 10 minutes in ice.Cell is washed with FACS buffer solution and is resuspended in 100 μ l FAC buffers.

The then Streptavidin (BD Pharmingen, San Diego, CA) with phycoerythrin (PE) label and other algae The CD3 (eBiocience, San Diego, CA) of azurin (APC) label dyes cell.By 1.0 μ l PE and APC points It Jia Ru not pipe 2 and 3.

Cell collection is carried out using flow cytometer BD FacsCalibur (BD Biosciences), and soft with FlowJo Part (Treestar, Inc.Amland, OR) is analyzed.

7 cell toxicity test of embodiment (real-time ACEA)

Toxicity test is carried out using ACEA instrument, the real-time ACEA test direction program according to manufacturer is operated.

The cytotoxicity of real-time detection cell, using 5:1 dilution effect cell and target cell.The results show that of the invention GPC-3-CAR-T cell has high cell toxicity effect to the HCC cell of the GPC-3 positive.GPC-3-CAR-T cell of the invention To GPC-3 feminine gender HCC cell no cytotoxicity.

The sequencing of the hybridoma variable region of 8 GPC-3, GPC-3 variable chain of embodiment and CAR construct

Mouse anti human GPC-3 monoclonal antibody is generated using hybridoma.It generates for the end GPC-3 albumen N- The hybridoma of 150 amino acid (55 to 200 amino acid).The hybridoma technology of use is standard, and is to retouch elsewhere It states.55-200 amino acid of the end antibody test extracellular domain N- is IgG2 type.We survey this antibody Sequence.The sequence of VH, VL and scFv are as described in Example 9.

The sequencing of hybridoma positive colony is carried out using the conventional method of this field.According to sequencing result, can analyze really Determine the variable region sequences of heavy chain and light chain.ScFv clone can then be synthesized.

The sequence of 9 GPC-3 VH of embodiment, VL and scFv

The sequence that sequencing obtains GPC-3 scFv is carried out by the hybridoma clone to the GPC-3 positive.The knot of GPC-3scFv Structure is VH- connector-VL.

The nucleotide sequence (SEQ ID NO:1) of runic expression VH；Nucleotide sequence (the SEQ ID of underscore expression VL NO:2)；Between sequence (italic) be encode G4S connector (GGGGS) nucleotide sequence (SEQ ID NO:3).

GATGTGCAGCTTCAGGAGTCAGGACCTGTCCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACT GTCACTGGCTACTCCATCACCAGTAATTATAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGAT GGGCTACATACACTACAGTGGTAGCACTAAGTTCAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACAT CCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAAGGGAGGG GATTTATGGTTACGACGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

GACATTGTGCTGACACAGTCTCCTGCTTCCTTACCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGC AGGGCCAGTGAAAGTGTCAGTACATCTATCTATAATTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAA ACTCCTCATCAAGTATGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACT TTTCCCTCAACATCCATCCTGTGGAGGAGGAGGATTCTGCAACATATTTCTGTCAGCACAGTTGGGAGATTCCGCTC ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG

GPC-3 scFv albumen: 238aa/26.3kD (SEQ ID NO:4)

DVQLQESGPVLVKPSQSLSLTCTVTGYSITSNYSWHWIRQFPGNKLEWMGYIHYSGSTKFNPSLKSRISITRDTSKN QFFLQLNSVTTEDTATYYCAKGGDLWLRRYFDVWGAGTTVTVSS

DIVLTQSPASLPVSLGQRATISCRASESVSTSIYNYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGS GSGTDFSLNIHPVEEEDSATYFCQHSWEIPLTFGAGTKLELKR

In protein sequence, runic indicates the amino acid sequence (SEQ ID NO:5) of VH；The amino acid of underscore expression VL Sequence (SEQ ID NO:6)；Between sequence (italic) be G4S connector (GGGGS) amino acid sequence (SEQ ID NO:7)

10 GPC-3-CAR sequence of embodiment

The schematic diagram of GPC-3-CAR construct is as shown in Figure 3.Slow virus carrier LentiCMV-MCS-EF1a-puro is used for Clone all scFv CAR sequences.

The structure of GPC-3 ScFv MT28-CD28-CD3 δ of the invention includes people's CD8 signal peptide, GPC-3scFv (VH- Connector 3x (G4S)-VL), CD8 hinge area, CD28 transmembrane region, activation domain, CD3 ζ (Fig. 3, Pr).Specifically, CD8 boot sequence Coded sequence is as shown in SEQ ID NO.:8.GPC-3scFv(V_HConnector-V_L)The coded sequence of-CD28-CD3-zeta such as SEQ Shown in ID NO.:9, the coded sequence of GPC-3 scFv is as shown in the position 1-744 of SEQ ID NO.:9, the connector (volume of 3x (G4S) Code sequence as shown in the position 364-408 of SEQ ID NO.:9, the coded sequence of CD28-CD3-zeta such as SEQ ID NO.:9's Shown in 745-1452.The amino acid sequence of GPC-3-CAR (CD8-GPC-3 ScFv MT28-CD28-CD3 δ) of the invention is such as Shown in SEQ ID NO.:10.

In an embodiment of the present invention using the GPC-3 CAR published as control, the knot of disclosed GPC-3CAR Structure includes people's CD8 signal peptide, GPC-3scFv (VL connector 3x (G4S) VH), CD8 hinge area, CD28 transmembrane region, activation domain, CD3 ζ (Fig. 3, Pu).

Embodiment 11 carries out GPC-3 antibody in GPC-3 positive cell line using flow cytometry and western blot The detection of GPC-3

Mouse anti human GPC-3 monoclonal antibody is produced using hybridoma.The end antibody combination extracellular domain N- 55-200 amino acid.The antibody is sequenced.The sequence of VH, VL and scFv are as described in Example 10.

In flow cytometry, GPC-3 high expression in GPC-3 hepatocellular carcinoma with positive (HCC), and in GPC-3 feminine gender HCC In do not express.

Specifically, using GPC-3 monoclonal antibody, SMMC-7721-GPC-3 negative cells system and Hep-3B, Hep- are carried out The dyeing of G2 and Huh-7 hepatocellular carcinoma cells system.The results show that SMMC-7721 cell line, and > 25% GPC-3 are positive thin Born of the same parents system dye-free (Fig. 4 A).Identical high GPC-3 dyeing (Fig. 4 B) is observed in HepG2 cell line.

12 GPC-3-CAR-T cell of embodiment effectively expands in culture

The GPC-3 antibody of acquisition and the sequence of GPC-3 single-chain antibody scFv are as described in Example 10.GPC-3scFv sequence (embodiment 10) is inserted into CD28 the and CD3 ζ structural domain of CAR structure, recycles slow virus that CAR transduces into T cell.GPC-3- CAR cell effectively expands (Fig. 5) in vitro.

Embodiment 13 utilizes GPC3-CAR lentivirus construct transfecting T cells, it was demonstrated that the expression on GPC-3

In order to detect transduction, CAR-T cell is dyed using anti-FAB antibody.Compared to non-transduction control cell, the staining cell Strain shows higher transduction efficiency (Fig. 6)

14 GPC-3-CAR of embodiment is higher than non-transduction T cell to the cytotoxicity of GPC-3 positive HCC cell, to GPC-3 Negative HCC cell no cytotoxicity.

Real-time cell toxicity test shows that GPC-3-CAR cell has height to GPC-3 positive Hep-G2 and Huh-7 cell Cytotoxicity (Fig. 7).It is to no cytotoxicity in GPC-3 negative cells system SMMC7721.

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 table

<110>Cao Wei

<120>Glypican-3 specific antibody and its specific C AR-T cell

<130> P2017-1504

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 363

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

gatgtgcagc ttcaggagtc aggacctgtc ctggtgaaac cttctcagtc actttcactc 60

acctgcactg tcactggcta ctccatcacc agtaattata gctggcactg gatccggcag 120

tttccaggaa acaaactgga atggatgggc tacatacact acagtggtag cactaagttc 180

aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240

ctgcagttga attctgtgac tactgaggac acagccacat attactgtgc aaagggaggg 300

gatttatggt tacgacggta cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360

tca 363

<210> 2

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

gacattgtgc tgacacagtc tcctgcttcc ttacctgtat ctctggggca gagggccacc 60

atctcatgca gggccagtga aagtgtcagt acatctatct ataattatat gcactggtac 120

caacagaaac caggacagcc acccaaactc ctcatcaagt atgcatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttttccct caacatccat 240

cctgtggagg aggaggattc tgcaacatat ttctgtcagc acagttggga gattccgctc 300

acgttcggtg ctgggaccaa gctggagctg aaacgg 336

<210> 3

<211> 15

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

ggtggcggtg gttct 15

<210> 4

<211> 238

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Asp Val Gln Leu Gln Glu Ser Gly Pro Val Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asn

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile His Tyr Ser Gly Ser Thr Lys Phe Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Lys Gly Gly Asp Leu Trp Leu Arg Arg Tyr Phe Asp Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile

115 120 125

Val Leu Thr Gln Ser Pro Ala Ser Leu Pro Val Ser Leu Gly Gln Arg

130 135 140

Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Thr Ser Ile Tyr

145 150 155 160

Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu

165 170 175

Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe

180 185 190

Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His Pro Val

195 200 205

Glu Glu Glu Asp Ser Ala Thr Tyr Phe Cys Gln His Ser Trp Glu Ile

210 215 220

Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

225 230 235

<210> 5

<211> 121

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

Asp Val Gln Leu Gln Glu Ser Gly Pro Val Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asn

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile His Tyr Ser Gly Ser Thr Lys Phe Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Lys Gly Gly Asp Leu Trp Leu Arg Arg Tyr Phe Asp Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 6

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Thr Ser

20 25 30

Ile Tyr Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ser Ala Thr Tyr Phe Cys Gln His Ser Trp

85 90 95

Glu Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105 110

<210> 7

<211> 5

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 7

Gly Gly Gly Gly Ser

1 5

<210> 8

<211> 84

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

tctagagccg ccaccatggc cttaccagtg accgccttgc tcctgccgct ggccttgctg 60

ctccacgccg ccaggccggc tagc 84

<210> 9

<211> 1452

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gatgtgcagc ttcaggagtc aggacctgtc ctggtgaaac cttctcagtc actttcactc 60

acctgcactg tcactggcta ctccatcacc agtaattata gctggcactg gatccggcag 120

tttccaggaa acaaactgga atggatgggc tacatacact acagtggtag cactaagttc 180

aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240

ctgcagttga attctgtgac tactgaggac acagccacat attactgtgc aaagggaggg 300

gatttatggt tacgacggta cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360

tcaggtggcg gtggttctgg tggcggtggt tctggtggcg gtggttctga cattgtgctg 420

acacagtctc ctgcttcctt acctgtatct ctggggcaga gggccaccat ctcatgcagg 480

gccagtgaaa gtgtcagtac atctatctat aattatatgc actggtacca acagaaacca 540

ggacagccac ccaaactcct catcaagtat gcatccaacc tagaatctgg ggtccctgcc 600

aggttcagtg gcagtgggtc tgggacagac ttttccctca acatccatcc tgtggaggag 660

gaggattctg caacatattt ctgtcagcac agttgggaga ttccgctcac gttcggtgct 720

gggaccaagc tggagctgaa acggctcgag aagcccacca cgacgccagc gccgcgacca 780

ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgagccgg 840

ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgccagtga taagcccttt 900

tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 960

tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 1020

atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 1080

gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1140

cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1200

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag 1260

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1320

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1380

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1440

taataggaat tc 1452

<210> 10

<211> 508

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

Ser Arg Ala Ala Thr Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro

1 5 10 15

Leu Ala Leu Leu Leu His Ala Ala Arg Pro Ala Ser Asp Val Gln Leu

20 25 30

Gln Glu Ser Gly Pro Val Leu Val Lys Pro Ser Gln Ser Leu Ser Leu

35 40 45

Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asn Tyr Ser Trp His

50 55 60

Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile

65 70 75 80

His Tyr Ser Gly Ser Thr Lys Phe Asn Pro Ser Leu Lys Ser Arg Ile

85 90 95

Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn

100 105 110

Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Lys Gly Gly

115 120 125

Asp Leu Trp Leu Arg Arg Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr

130 135 140

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Pro

165 170 175

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

180 185 190

Val Ser Thr Ser Ile Tyr Asn Tyr Met His Trp Tyr Gln Gln Lys Pro

195 200 205

Gly Gln Pro Pro Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser

210 215 220

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser

225 230 235 240

Leu Asn Ile His Pro Val Glu Glu Glu Asp Ser Ala Thr Tyr Phe Cys

245 250 255

Gln His Ser Trp Glu Ile Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

260 265 270

Glu Leu Lys Arg Leu Glu Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro

275 280 285

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

290 295 300

Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

305 310 315 320

Asp Phe Ala Ser Asp Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

325 330 335

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

340 345 350

Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn

355 360 365

Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr

370 375 380

Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser

385 390 395 400

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

405 410 415

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

420 425 430

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys

435 440 445

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

450 455 460

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

465 470 475 480

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

485 490 495

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505

Claims (10)

1. a kind of anti-human GPC-3 monoclonal antibody, which is characterized in that the antibody includes

(i) amino acid sequence heavy chain variable region as shown in SEQ ID NO:5；With

(ii) amino acid sequence light chain variable region as shown in SEQ ID NO:6.

2. antibody as described in claim 1, which is characterized in that the preceding 55-200 of the N-terminal of the antibody and people GPC-3 albumen A amino acid region combines.

3. a kind of single-chain antibody, which is characterized in that the single-chain antibody includes

(i) amino acid sequence heavy chain variable region as shown in SEQ ID NO:5；With

(ii) amino acid sequence light chain variable region as shown in SEQ ID NO:6.

4. single-chain antibody as claimed in claim 3, which is characterized in that the single-chain antibody also includes connecing between VH and VL Head.

5. single-chain antibody as claimed in claim 3, which is characterized in that the single-chain antibody has shown in SEQ ID NO:4 Amino acid sequence.

6. single-chain antibody as claimed in claim 3, which is characterized in that the N-terminal of the single-chain antibody and people GPC-3 albumen Preceding 55-200 amino acid region combine.

7. a kind of Chimeric antigen receptor CAR, which is characterized in that the CAR includes single-chain antibody as claimed in claim 3.

8. CAR as claimed in claim 7, which is characterized in that the CAR includes: from N-terminal to C-terminal

(i) single-chain antibody as claimed in claim 3,

(ii) transmembrane domain,

(iii) at least one costimulation structural domain, and

(iv) activation domain.

9. a kind of nucleic acid molecules, which is characterized in that nucleic acid molecule encoding antibody described in claim 1, claim 3 The Chimeric antigen receptor (CAR) of single-chain antibody, claim 7.

10. a kind of preparation, which is characterized in that the preparation contain antibody described in claim 1, claim 3 it is single-stranded anti- Body, the Chimeric antigen receptor (CAR) of claim 7 and pharmaceutically acceptable carrier, diluent or excipient.