CN107226866A - A kind of anti-CD24 humanized antibodies fusion protein - Google Patents

A kind of anti-CD24 humanized antibodies fusion protein Download PDF

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Publication number
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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mica
antibody
fusion protein
cell
molecules
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王旻
张娟
孙福谋
杜晓典
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China Pharmaceutical University
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China Pharmaceutical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
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  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)

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

A kind of anti-CD24 humanized antibodies fusion protein
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.
CN201710554362.9A 2017-07-05 2017-07-05 A kind of anti-CD24 humanized antibodies fusion protein Pending CN107226866A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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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