CN107226866A - A kind of anti-CD24 humanized antibodies fusion protein - Google Patents
A kind of anti-CD24 humanized antibodies fusion protein Download PDFInfo
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- CN107226866A CN107226866A CN201710554362.9A CN201710554362A CN107226866A CN 107226866 A CN107226866 A CN 107226866A CN 201710554362 A CN201710554362 A CN 201710554362A CN 107226866 A CN107226866 A CN 107226866A
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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Abstract
The invention belongs to genetic engineering antibody technical field, and in particular to a kind of Preparation method and use of CD24 antibody fusion proteins.CD24 humanized antibodies hG7S and extracellular 1st 3 area of MICA molecules are passed through G by the present invention using technique for gene engineering4S flexibility peptides are connected, the stable expression hG7S MICA of mammal eukaryotic expression system;CD24 antibody fusion protein hG7S MICA in combination with the CD24 molecules and the Activating receptor NKG2D of NK cell surfaces in tumor cell surface, can remold the NK cells of NKG2D approach induction to CD24+The immunosurveillance of tumour cell, is finally reached activation body self immune system and effectively kills CD24+The purpose of tumour cell.
Description
Technical field
The invention belongs to bioengineering field, and in particular to it is a kind of it is new can simultaneously with leukocyte differentiation antigen CD24
The CD24 humanized antibody fusion proteins that (cluster of differentiation 24) and NKG2D receptor-specifics are combined
HG7-MICA, MICA is specifically showed in tumor cell surface by it using hG7 targeting, and enhancing NK cells are to people's mammary gland
The cell mediated cytotoxicity (ADCC) of the antibody dependent of cancer cell, it is to avoid due to cell surface MICA developed by molecule
Lower and the caused tumor immune escape that comes off.HG7-MICA design opens a kind of new immunotherapy of tumors strategy, is
One species specificity remolds bifunctional antibody fusion protein of the NK cells to CD24+ tumour cell immune surveillance functions.
Background technology
Current whole world Cancer Mortality and the death rate are in rising trend always.Tumor immune escape refers to tumour cell
Surface marker, which comes off, causes it to escape immunosurveillance, and this is also the bottleneck that chemicotherapy method often produces that resistance and low-security are evaluated
Place.Therefore, the key that specific molecular target and novel clinical application means are tumour personalized treatments is found.Tumour
Immunization therapy is intended to remold identification and killing ability of the body self immune system to tumour cell, utilizes " biological missile " by machine
Naturally occurring this ammunition of immunocyte is specifically raised to tumor focus position in body, the strategy have high specific with
The advantage of low side effect.
Tumor related antigen MICA
MICA (MICA, major histocompatibility complex class I chain-related gene
A it is) a kind of protein molecule of high glycosylation, belongs to non-classical HLA-I (human leucocyte antigen-I) class base
Because of family.MICA is extensive and is specifically expressed in the primary tumors cells of epithelial origin, is a kind of generally acknowledged tumour phase
Closing property antigen.NK cells or CD8+T cell is borrowed surface receptor NKG2D and part MICA/B interaction and leaned on tumour cell
Closely, combine, and then by discharging the dissolving of the induced tumor cell such as cell factor.Although confirming MICA developed by molecule in a variety of swollen
Oncocyte surface, but coming off for MICA causes tumour cell although to still fall within the MICA positives, but immunosurveillance can be avoided and led
Cause immunologic escape.Therefore a kind of new exhibition method is found to remold effects of the MICA in cancer immunosurveillance as tumour to exempt from
The key of epidemic disease therapeutic strategy, antibody is activation and a kind of conventional targeted drug for rebuilding the effect of body tumour immunity, wherein single-stranded
Antibody reaches lesions position more easily by blood-brain barrier because its molecular weight is small and is more used for such targeting and designs.Thus
It is proposed that a kind of assume:By MICA molecules and Antibody Fusion, by means of the antibodies on tumor of targets neoplastic cells surface antigen
The identification and combination of cell, MICA are anchored to tumor cell surface;Further stimulate NK thin by MICA and NKG2D combination
Born of the same parents' killing tumor cell, rebuilds the immunosurveillance by NKG2D pathway activations body itself.
NKG2D molecules are a kind of homodimer II type transmembrane proteins of c-type agglutinin receptor family.In the life of tumour
In long process, the part such as MICA, which is lowered, or proteolysis come off can suppress the cell-mediated immunoregulations of NK, and induce NKG2D's
Part can be such that tumour cell is easier by NK cell recognitions and killing in the expression of tumor cell surface.NKG2D signal pathway activateds are imitated
Answer the molecular mechanism of cell:NKG2D and its ligand binding, are aided with adaptor protein molecule DAP10 or DAP12 help, under activation
Swim signal path, final activated NK.
Tumor markers CD24 molecules
Find that leukocyte differentiation antigen CD24 (cluster of differentiation 24) is used as one through literature survey
Potential carcinogen is planted, is overexpressed in the kinds of tumors tissue such as colorectal cancer, breast cancer, liver cancer, ED-SCLC, and
Do not expressed or low expression in normal structure.The characteristics of based on CD24 molecular weight tumor cell markers, it is proposed that CD24 antibody is made
MICA molecules are connected for effective carrier, NKG2D approach is activated, the immunosurveillance of NK cells is remolded.Scientist is directed to
The type shot design antibody fusion protein, the characteristics of these tumor surface antigens all have identical with CD24:1. tumour cell
Surface height expression;2. normal tissue cell surface is not expressed or low expression level.
The fusion protein of immunocyte native ligand and antibody
There are many exploitations for being directed to antibody fusion protein at present, its design method is:1. utilize and target CD138, CEA
Single-chain antibody connection NKG2D ligand effect molecules prepare " biological missile ", effectively raise NK cells to tumor focus position, kill
Hinder tumour cell;2. using CD20 single-chain antibodies connection MICA designerantibodies fusion proteins, effectively to treat lymthoma;3. use
The Fab fragments for targetting CD20 monoclonal antibodies are connected by the mode of chemical coupling with MICA, and effectively enhancing NK cells are to CD20 positive tumors
The identification of cell and lethal effect.So far rarely has the report of NKG2D part MICA molecule related fusions.
It is connected by the CD24 humanized antibodies of independent research with MICA molecules, is built into fusion protein, can be notable
Strengthen NK cells and obtain immunosurveillance.NK cells play key effect in cancer immunosurveillance, and the activation of NK cells takes
Certainly in Activating receptors such as the NKG2D on its surface.Positive expression CD107a molecules are the labels with killing activity NK cells.
The main Activating receptor of NK cells includes NKG2D, Fc γ RIIIa and natural cytotoxic acceptor (NCRs), and immunogenic ligand is raised
And the effector function of NK cells is activated, the NK cells after activation are by discharging perforin and granzyme, while secrete cytokines
IFN-γ and TNF-α carry out mediating effect+6 cell function.Therefore, novel therapeutic means of the NK cells in specific killing tumour cell
In become a very promising weapon.
Based on above-mentioned theory basis and research practice, the present invention resists the anti-CD24 humanizations for having independent intellectual property right
Body hG7 is connected with MICA by flexible peptide, designs a kind of novel recombinant protein hG7-MICA.HG7-MICA design opens one
Plant new immunotherapy of tumors strategy.The research NK cell mediated immune surveillances that on the one hand remodeling NKG2D approach is induced, pass through activation
Body autoimmune function effectively kills tumour;On the other hand the therapeutic scheme plays targeting sexual clorminance, effectively reduces chemoradiotherapy
The toxic side effect that medicine is produced, commanding elevation will be occupied in preclinical study and clinical safety evaluation.Therefore, with autonomous
The fusion protein hG7-MICA of intellectual property exploitation, the clinical application scheme of novelty is provided for immunotherapy of tumors system.
The content of the invention
Goal of the invention
The present invention provides a kind of CD24 antibody fusion proteins hG7-MICA with antineoplastic immune curative effect.Antibody of the present invention
The feature of fusion protein is G4S flexibility peptide Linker connections CD24 humanized antibodies hG7 and MICA molecules, remains the two original
Parent activity;Mammalian cell expression system carries out protein expression, it is ensured that the bioactivity of fusion protein;Utilize hG7's
MICA is specifically showed in CD24 by targeting+Liver cancer cells Huh-7 surfaces, it is to avoid due to cell surface MICA molecule tables
Up to downward and the caused tumor immune escape that comes off;Activation NK cells simultaneously induce the cell factors such as its secretion of gamma-IFN, TNF-α,
Remold the immune surveillance function of NK cells against tumor cells;Inside and outside suppresses liver cancer cells Huh-7 growth.
Technical scheme
A kind of CD24 antibody fusion proteins, the fusion protein is with the brand-new anti-CD24 humanized antibodies of the sequence of independent research
HG7 is with based on the MICA molecules of people source, with means such as genetic recombination, being built into bifunctional antibody fusion protein hG7-MICA.
Wherein one chain is made up of humanized antibody hG7 heavy chains and MICA albumen, and its amino acid sequence table is SEQ NO.1;
Another chain is hG7 light chains, and its amino acid sequence table is SEQ NO.2.
A kind of mammalian cell CHO-s secreting, expressing methods, for secreting, expressing CD24 antibody fusion proteins hG7-MICA.
A kind of expression vector, the nucleic acid molecules containing CD24 antibody fusion protein hG7-MICA target gene.
A kind of recombinant host cell, contains above-mentioned expression vector.
CD24 antibody fusion proteins hG7-MICA application mode:HG7 humanized antibodies play targeting effect by MICA
Be showed in molecular specificity CD24 height expression tumor cell surface, by MICA half molecules optionally with NK cell surfaces
NKG2D acceptors combine, improve body microenvironment in NK cells targets identification ability and lethal effect.
Applications of the CD24 antibody fusion proteins hG7-MICA in tumor.
Invention is further illustrated:
The antibody fusion protein that CD24 is targetted in the present invention is made up of two chains, wherein a chain is by full length antibody and MICA
Albumen by flexible peptide (GGGGS) connect, another be full length antibody light chain.
It is an object of the invention to build CD24 antibody fusion proteins hG7-MICA expression vector, screen the antibody and melt
The engineering cell strain of hop protein height expression can stablize high expression and the above-mentioned CD24 antibody fusion proteins of mass purification there is provided one kind
HG7-MICA method;Another object of the present invention is that the inside and outside drug effect for setting up CD24 antibody fusion proteins hG7-MICA is commented
Valency system, assesses the feasibility of immunotherapy of tumors scheme.
The present invention is obtained the transformation of this laboratory using overlap PCR (polymerase chain reaction) technologies
The CD24 humanized antibodies obtained are aided with flexible peptide G with the extracellular 1-3 areas genes of people source MICA4S-phase is connected, and carries out clone's restructuring, structure
Antibody fusion protein hG7-MICA recombinant vectors are built, by electroporation stable transfection CHO-s cells, pressurizes and sieves through two-wheeled G418
Choosing obtains correct expression hG7-MICA stable operation cell line;Use Protein A affinity chromatography column separating purification purpose eggs
In vain, SDS-PAGE detects that purified product is that electrophoresis is pure, and Western Blot identification expression product expression and assembling are correct;SPR is real
Test the affinity of analysis antibody and antigen.
Brief description of the drawings
Fig. 1 is antigen-4 fusion protein gene recombination analysis figure, and it is 2312bp, light chain gene that recombination, which includes heavy chain gene size,
Size is about 764bp.
Fig. 2 is the structural representation of fusion protein, and the albumen is made up of two chains, wherein the CH areas of CD24 antibody and MICA
Albumen is connected by flexible peptide (GGGGS), and heavy chain is voluntarily by disulfide-bonded in expression cell with light chain and heavy chain
Fit together.
Fig. 3 is the structure qualification figure of antibody fusion protein hG7-MICA heavy chain genes.HG7 gene order has been obtained, I
Build antibody fusion protein hG7-MICA based on this.HMICA sequences are introduced at hG7 heavy chains H 3 ' ends, new weight is obtained
Chain H-MICA." H-MICA " is Overlap PCR reaction amplifications hG7-MICA heavy chain H-MICA target gene, and " H " is PCR anti-
HG7 heavy chain should be expanded, " MICA " is hMICA genetic fragments.
Embodiment
The CD24 antibody fusion proteins hG7-MICA of embodiment 1 structure
Respectively with hG7 heavy and light chain genes, MICA genes and pCA puro, pMH3 plasmid are template, design and synthesize primer
Enter performing PCR amplification.Fusion protein hG7-MICA structure is based on hG7 heavy chain genes H, then carries out over-lap PCR extension amplification
Obtain complete H-MICA genes.PCR primer detects that Ago-Gel QIAquick Gel Extraction Kit is returned with 1.0% agarose gel electrophoresis
Receive target gene.PCR expands end-product and plasmid pCApuro, pMH3 carry out double digestion with restriction enzyme, and digestion products are cut
After glue reclaim, with T4 ligases, 16 DEG C connect overnight.Bacillus coli DH 5 alpha competence is converted after connection, spread plate, next day chooses
Take monoclonal double digestion and sequencing identification.
Claims (8)
1. a kind of anti-CD24 humanized antibodies and MICA fusion proteins, with following characteristics:The antibody is with the anti-CD24 of independent research
Humanization full length antibody hG7 and MICA based on, two sections of albumen are connected using flexible peptide, CD24 antibody is built into and melts
Hop protein hG7-MICA.
2. a kind of CD24 antibody fusion proteins according to claim 1, it is characterised in that:Wherein one chain is by CD24 people source
Change heavy chain of antibody and MICA albumen composition, its amino acid sequence table is SEQ NO.1;Another chain is that CD24 humanized antibodies are light
Chain, its amino acid sequence table is SEQ NO.2.
3. a kind of nucleic acid molecules, it is characterised in that the fusion protein described in coding claim 1.
4. one group of expression vector, contains the nucleic acid described in claim 4.
5. a kind of recombinant host cell, contains the expression vector described in claim 5.
6. the application of fusion protein described in claim 1, it is characterised in that:HG7 half molecules play targeting effect by MICA half
Be showed in molecular specificity CD24 height expression tumor cell surface, by MICA half molecules optionally with NK cell surfaces
NKG2D acceptors combine, improve body microenvironment in NK cells targets identification ability and lethal effect.
7. the conjugate of fusion protein described in claim 1.
8. application of the fusion protein in tumor is prepared described in claim 1.
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN109678963A (en) * | 2018-12-18 | 2019-04-26 | 中国药科大学 | A kind of preparation and its application of the bispecific antibody for targeting CD24 and activating NK cell |
CN110964118A (en) * | 2019-11-27 | 2020-04-07 | 中国药科大学 | Bispecific fusion antibody and application thereof in tumor immunotherapy |
CN112159477A (en) * | 2020-09-16 | 2021-01-01 | 上海健康医学院 | Frizzled-7 targeted antibody fusion protein and preparation method and application thereof |
GB2596001B (en) * | 2019-02-18 | 2023-11-29 | Courier Therapeutics Inc | Bispecific fusion protein using orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP) and tumor-specific binding partner |
US11897929B2 (en) | 2016-02-05 | 2024-02-13 | Washington University | Compositions and methods for targeted cytokine delivery |
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2017
- 2017-07-05 CN CN201710554362.9A patent/CN107226866A/en active Pending
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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US11897929B2 (en) | 2016-02-05 | 2024-02-13 | Washington University | Compositions and methods for targeted cytokine delivery |
CN109678963A (en) * | 2018-12-18 | 2019-04-26 | 中国药科大学 | A kind of preparation and its application of the bispecific antibody for targeting CD24 and activating NK cell |
GB2596001B (en) * | 2019-02-18 | 2023-11-29 | Courier Therapeutics Inc | Bispecific fusion protein using orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP) and tumor-specific binding partner |
CN110964118A (en) * | 2019-11-27 | 2020-04-07 | 中国药科大学 | Bispecific fusion antibody and application thereof in tumor immunotherapy |
CN112159477A (en) * | 2020-09-16 | 2021-01-01 | 上海健康医学院 | Frizzled-7 targeted antibody fusion protein and preparation method and application thereof |
CN112159477B (en) * | 2020-09-16 | 2021-09-24 | 上海健康医学院 | Frizzled-7 targeted antibody fusion protein and preparation method and application thereof |
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