CN112898412A

CN112898412A - High-stability Fab-like antibody and preparation method and application thereof

Info

Publication number: CN112898412A
Application number: CN202010562267.5A
Authority: CN
Inventors: 应天雷; 王春雨; 吴艳玲
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2019-12-03
Filing date: 2020-06-18
Publication date: 2021-06-04

Abstract

The invention belongs to the technical field of biology, and relates to a Fab-like antibody fragment or antigen binding fragment, a separated VH-CH3 polypeptide, a VL-CH3 polypeptide, a method for constructing the Fab-like antibody fragment or antigen binding fragment, the VH-CH3 polypeptide and the VL-CH3 polypeptide, required nucleic acid, plasmid and host cell, a corresponding medicinal composition and application thereof. The invention uses IgG1-Fab structure as template to transform through antibody engineering means, and carries out homologous replacement of antibody constant region (CL, CH1 are replaced by CH3 respectively), heavy chain variable region is connected with antibody constant region CH3 by linker, light chain variable region is connected with antibody constant region CH3 by linker, and high stability Fab antibody fragment is obtained; and the antibody with the new structure can be effectively expressed in prokaryotic cells and can be effectively expressed in eukaryotic cells and yeast cells. The high-stability Fab-like antibody fragment prepared by the invention has improved stability and antigen binding capacity, higher stability and better drug property, and has the advantages of treating and diagnosing diseases.

Description

High-stability Fab-like antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a Fab-like antibody fragment or antigen binding fragment, a separated VH-CH3 polypeptide and a VL-CH3 polypeptide, a method for constructing the Fab-like antibody fragment or antigen binding fragment, the VH-CH3 polypeptide and the VL-CH3 polypeptide, required nucleic acid, plasmid and host cell, a corresponding medicinal composition and application thereof.

Background

The prior art discloses that the monoclonal antibody medicament has the characteristics of strong targeting property, obvious curative effect, low toxic and side effects and the like, and shows excellent curative effect and wide application prospect in the treatment of various diseases. Compared with cancer treatment and immune disease treatment, monoclonal antibody medicines are relatively slow to develop and apply in the field of treatment of other diseases. The molecular weight of the full-length monoclonal antibody IgG is too large (about 150kD), so that the full-length monoclonal antibody IgG has significant defects in some clinical applications, such as poor tissue permeability caused by the excessive molecular weight and influence on the treatment effect; secondly, some steric target points cannot be reached; more importantly, the monoclonal antibody drug with high molecular weight has high production cost and high price, which seriously hinders the clinical application of the monoclonal antibody drug in the disease treatment field such as antiviral treatment and the like and becomes the bottleneck in the field. Researchers in the industry have long searched for the use of "antibody fragments" (antibody fragments) with smaller molecular weight and capable of being expressed in prokaryotic cells instead of monoclonal antibodies as a new generation of antibody drugs with low production cost and strong tissue penetration ability. Therefore, research and development of small molecule antibody fragment drugs with small molecular weight and high specificity, which can be efficiently expressed in a prokaryotic system, have become the focus of researchers.

Based on the current state of the art, the inventors of the present application intend to provide a high-stability Fab-like antibody, a preparation method and applications thereof, and particularly, to a Fab-like antibody fragment or antigen binding fragment, an isolated VH-CH3 polypeptide, a VL-CH3 polypeptide, and a preparation method thereof.

Disclosure of Invention

The invention aims to solve the problems in the prior art based on the current situation of the prior art, and prepares a novel Fab-like antibody fragment or antigen-binding fragment through computer simulation and genetic engineering modification, in particular to the Fab-like antibody fragment or antigen-binding fragment, separated VH-CH3 polypeptide, VL-CH3 polypeptide and a preparation method thereof. The Fab-like antibody fragment or the antigen binding fragment has higher stability and better antigen binding capacity, and can realize the co-expression of a prokaryotic system.

The present invention provides a Fab-like antibody fragment or antigen binding fragment, comprising:

1) heavy chain variable region, linker and antibody constant region CH3 fragments, and

2) light chain variable region, linker and antibody constant region CH3 fragments.

Preferably, the heavy chain variable region in the Fab-like antibody fragment or antigen binding fragment starts with amino acid QVQL/QMQL/EVQL/QIQL/QVTL/TVQL/EVHL/EMQL/EVEL/EDQL/QLQL/EAQL and ends with amino acid TVSS.

Preferably, the Fab-like antibody fragment or antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence that is at least 95% homologous to SEQ ID NO. 5 or SEQ ID NO. 7.

Preferably, the light chain variable region in the Fab-like antibody fragment or antigen binding fragment starts with amino acid DIQMT/AIQMT/AIRMT/DIQLT/AIQLT/vivmt/NIQMT/DIVMT/EIVMT/EIVLT/ETTLT/DVVMT/DIVLT and ends with amino acid VEIK.

Preferably, the variable region of the light chain in the Fab-like antibody fragment or antigen-binding fragment comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO 6 or SEQ ID NO 8.

Preferably, the linker in the Fab-like antibody fragment or antigen-binding fragment comprises an amino acid sequence that is at least 80% homologous to SEQ ID NO. 3 or SEQ ID NO. 4.

Preferably, the linker in the Fab-like antibody fragment or antigen binding fragment, the linker linking the heavy chain variable region and the antibody constant region CH3 fragment comprises an amino acid sequence having at least 80% homology to SEQ ID No. 4.

Preferably, the linker in the Fab-like antibody fragment or antigen binding fragment, the linker linking the light chain variable region and the antibody constant region CH3 fragment comprises an amino acid sequence having at least 80% homology to SEQ ID No. 3.

Preferably, the antibody constant region CH3 fragment of the Fab-like antibody fragment or antigen binding fragment comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO. 1 or SEQ ID NO. 2.

Preferably, the amino acid sequence of the antibody constant region CH3 fragment of the Fab-like antibody fragment or antigen binding fragment comprises:

1, wherein X1 is amino acid S or T, X2 is amino acid L or A, X3 is amino acid V or Y, or,

2, wherein X1 is amino acid W or T.

Preferably, the Fab-like antibody fragment or antigen binding fragment comprises an antibody constant region CH3 fragment, the antibody constant region CH3 fragment linked to the heavy chain variable region by a linker, comprising an amino acid sequence that is at least 95% homologous to SEQ ID No. 1.

Preferably, the antibody constant region CH3 fragment in the Fab-like antibody fragment or antigen binding fragment, the antibody constant region CH3 fragment linked to the heavy chain variable region by a linker, comprises an amino acid sequence having at least 95% homology to SEQ ID No. 1, wherein X1 is amino acid S or T, X2 is amino acid L or a, and X3 is amino acid V or Y.

Preferably, the Fab-like antibody fragment or antigen binding fragment comprises an antibody constant region CH3 fragment, the antibody constant region CH3 fragment linked to the light chain variable region by a linker, comprising an amino acid sequence that is at least 95% homologous to SEQ ID No. 2.

Preferably, the Fab-like antibody fragment or antigen binding fragment comprises an antibody constant region CH3 fragment, the antibody constant region CH3 fragment linked to the light chain variable region by a linker, comprising an amino acid sequence at least 95% homologous to SEQ ID No. 2, wherein X1 is amino acid W or T.

Preferably, the Fab-like antibody fragment or antigen binding fragment comprises:

1) a heavy chain variable region starting with amino acids QVQL/QMQL/EVQL/QIQL/QVTL/TVQL/EVHL/EMQL/EVEL/EDQL/QLQL/EAQL, ending with amino acid TVSS, a linker comprising an amino acid sequence having at least 80% homology to SEQ ID NO. 4 and an antibody constant region CH3 fragment comprising an amino acid sequence having at least 95% homology to SEQ ID NO. 1, and

2) with amino acids

DIQMT/AIQMT/AIRMT/DIQLT/AIQLT/VIWMT/NIQMT/DIVMT/EIVMT/EIVLT/ETTLT/DVVMT/DIVLT, a light chain variable region beginning with amino acid VEIK, a linker comprising an amino acid sequence at least 80% homologous to SEQ ID NO. 3 and an antibody constant region CH3 fragment comprising an amino acid sequence at least 95% homologous to SEQ ID NO. 2.

1) beginning with amino acids QVQL/QMQL/EVQL/QIQL/QVTL/TVQL/EVHL/EMQL/EVEL/EDQL/QLQL/EAQL, the heavy chain variable region ending with amino acid TVSS, a linker comprising an amino acid sequence having at least 80% homology to SEQ ID NO. 4 and an antibody constant region CH3 fragment comprising an amino acid sequence having at least 95% homology to SEQ ID NO. 1, wherein X1 in SEQ ID NO. 1 is amino acid S or T, X2 is amino acid L or A, X3 is amino acid V or Y, and

2) with amino acids

DIQMT/AIQMT/AIRMT/DIQLT/AIQLT/VIWMT/NIQMT/DIVMT/EIVMT/EIVLT/ETTLT/DVVMT/DIVLT starting from the light chain variable region ending with amino acid VEIK, a linker comprising an amino acid sequence having at least 80% homology to SEQ ID NO. 3 and an antibody constant region CH3 fragment comprising an amino acid sequence having at least 95% homology to SEQ ID NO. 2, wherein X1 in SEQ ID NO. 2 is amino acid W or T.

1) a heavy chain variable region comprising an amino acid sequence having at least 95% homology to SEQ ID NO 5 or SEQ ID NO 7, a linker comprising an amino acid sequence having at least 80% homology to SEQ ID NO 4 and an antibody constant region CH3 fragment comprising an amino acid sequence having at least 95% homology to SEQ ID NO 1, and

2) a light chain variable region comprising an amino acid sequence having at least 95% homology to SEQ ID NO 6 or SEQ ID NO 8, a linker comprising an amino acid sequence having at least 80% homology to SEQ ID NO 3 and an antibody constant region CH3 fragment comprising an amino acid sequence having at least 95% homology to SEQ ID NO 2.

1) a heavy chain variable region comprising an amino acid sequence having at least 95% homology to SEQ ID NO 5 or SEQ ID NO 7, a linker comprising an amino acid sequence having at least 80% homology to SEQ ID NO 4 and an antibody constant region CH3 fragment comprising an amino acid sequence having at least 95% homology to SEQ ID NO 1, wherein X1 in SEQ ID NO 1 is amino acid S or T, X2 is amino acid L or A, X3 is amino acid V or Y, and

2) a light chain variable region comprising an amino acid sequence having at least 95% homology to SEQ ID NO 6 or SEQ ID NO 8, a linker comprising an amino acid sequence having at least 80% homology to SEQ ID NO 3 and an antibody constant region CH3 fragment comprising an amino acid sequence having at least 95% homology to SEQ ID NO 2, wherein X1 in SEQ ID NO 2 is amino acid W or T.

The invention provides an isolated VH-CH3 polypeptide, wherein the isolated VH-CH3 polypeptide comprises a heavy chain variable region, a linker, and an antibody constant region CH3 fragment.

Preferably, the heavy chain variable region in the VH-CH3 polypeptide is an amino acid

QVQL/QMQL/EVQL/QIQL/QVTL/TVQL/EVHL/EMQL/EVEL/EDQL/QLQL/EAQL starts and ends with the amino acid TVSS.

Preferably, the heavy chain variable region in the VH-CH3 polypeptide comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO 5 or SEQ ID NO 7.

Preferably, the linker in the VH-CH3 polypeptide comprises an amino acid sequence that is at least 80% homologous to SEQ ID NO. 4.

Preferably, the antibody constant region CH3 fragment of the VH-CH3 polypeptide comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO. 1.

Preferably, the antibody constant region CH3 fragment of the VH-CH3 polypeptide comprises an amino acid sequence at least 95% homologous to SEQ ID NO 1, wherein X1 is amino acid S or T, X2 is amino acid L or A, and X3 is amino acid V or Y.

The invention provides an isolated VL-CH3 polypeptide, characterized in that the isolated VL-CH3 polypeptide comprises a light chain variable region, a linker and an antibody constant region CH3 fragment.

Preferably, the light chain variable region in the VL-CH3 polypeptide is an amino acid

DIQMT/AIQMT/AIRMT/DIQLT/AIQLT/VIWMT/NIQMT/DIVMT/EIVMT/EIVLT/ETTLT/DVVMT/DIVLT start and end with the amino acid VEIK.

Preferably, the variable region of the light chain in the VL-CH3 polypeptide comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO 6 or SEQ ID NO 8.

Preferably, the linker in the VL-CH3 polypeptide comprises an amino acid sequence that is at least 80% homologous to SEQ ID NO. 3.

Preferably, the antibody constant region CH3 fragment of the VL-CH3 polypeptide comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO. 2.

Preferably, the antibody constant region CH3 fragment of the VL-CH3 polypeptide comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO. 2, wherein X1 is amino acid W or T.

The present invention provides isolated nucleic acids encoding the Fab-like antibody fragments or antigen-binding fragments, VH-CH3 polypeptides, VL-CH3 polypeptides. The invention provides plasmids comprising the nucleic acids, optionally operably linked to regulatory sequences. The invention provides host cells comprising such vectors, as well as methods for producing and optionally recovering such Fab antibody fragments or antigen-binding fragments, VH-CH3 polypeptides, VL-CH3 polypeptides. The host cell of the invention may be any prokaryotic or eukaryotic cell, including but not limited to bacterial cells (e.g., E.coli), insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells (e.g., CHO or BHK cell lines). Other suitable host cells are known to those skilled in the art. Preferably, the host cell is an Escherichia coli cell, which has a short culture period and low production cost.

The Fab-like antibody fragment or antigen binding fragment, VH-CH3 polypeptide or VL-CH3 polypeptide provided by the invention can be specifically combined with the antigen against which the modified Fab aims, and the antigen refers to all substances which can induce the body to generate immune response. Such antigens include, but are not limited to, small molecule compounds. Such antigens include, but are not limited to, the proteins listed below, subunits, domains, motifs and epitopes belonging to the following proteins: CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32, CD33(p67 protein), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3, IL-1R, IL-2R, IL-4, IL-5, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, alpha-interferon, beta-interferon, gamma-interferon; TNF- α, TNF β 2, TNFac, TNF α β, TNF-RI, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEGI, OX40L, TRAIL receptor-1, A1 adenosine receptor, lymphotoxin β receptor, TACI, BAFF-R, EPO; LFA-3, ICAM-1, ICAM-3, EpCAM, β 1-integrin, β 2-integrin, α 4/β 7-integrin, α 2-integrin, α 3-integrin, α 4-integrin, α 5-integrin, α 6-integrin, α V-integrin, α V β 3-integrin, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, anti-idiotypic antibody (anti-id), E-selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin (VNR integrin), TGF- β 1, TGF- β 2, eotaxin 1, BLyS (B-lymphocyte stimulator), complement C5, IgE, factor VII, CD64, CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3(ErbB-3), Her4(ErbB-4), tissue factor, VEGF, VEGFR, endothelin receptor, VLA-4, hapten NP-or NIP-capping protein, T cell receptor alpha/beta, E-selectin, digoxin, placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase, transferrin receptor, carcinoembryonic antigen (CEA), CEACAM5, HMFGM, mucin MUC1, MUC18, heparanase (heparanase) I, human cardiac myosin, tumor-associated glycoprotein-72 (TAG-72), tumor-associated antigen CA, Prostate Specific Membrane Antigen (PSMA), high molecular weight melanoma-associated antigen (HMW-MAA), cancer-associated antigen, gcoprotein IIb/IIIa (GPIIb/IIIa), tumor-associated antigen expressing Lewis Y-associated carbohydrate (tumor-associated antigen expressed Lewis Y-related carbohydrate), Human Cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HIV gp140, HCMV, respiratory syncytial virus RSVF, RSVF Fgp, VNR integrin, IL-8, cytokeratin tumor-associated antigen, Hep B gp120, CMV, gpIbIIIa, HIV IIIB gp120V3 loop, Respiratory Syncytial Virus (RSV) Fgp, Herpes Simplex Virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and Clostridium capsulatum (Clostridium perfringens) toxin, Programmed death protein (Programmed death-1, PD-1).

It will be understood by those skilled in the art that the above list of target antigens refers not only to specific proteins and biomolecules, but also to the biochemical pathway or pathways in which they are involved. For example, when reference is made to CTLA-4 as the target antigen, it is meant that the ligand and receptor constitute the T cell costimulatory pathway, including CTLA-4, B7-1, B7-2, CD28, and any other undiscovered ligand or receptor is also the target object. Thus, a target antigen as used herein refers not only to a particular biomolecule, but also to a group of proteins that interact with the target antigen and a member of the biochemical pathway to which the target antigen belongs. It will be further understood by those skilled in the art that any of the above-described target antigens, ligands or receptors linked thereto, or other members of their respective biochemical pathways can be operably linked to the Fc monomer polypeptides of the invention so as to produce Fc fusions. Thus, for example, an EGFR-targeting Fc fusion can be constructed by operably linking an Fc monomer polypeptide to EGF, TGF- α, or any other ligand that may or may not be found to bind EGFR. Thus, the Fc variants of the invention can be operably linked to EGFR so as to produce Fc fusions that bind EGF, TGF α, or any other ligand that may or may not have been found to bind EGFR. Thus, virtually any polypeptide, whether a ligand, receptor, or some other protein or protein domain, including but not limited to the target antigens described above and the proteins that make up their respective biochemical pathways, can be operably linked to the Fc monomer polypeptides of the invention in order to develop Fc fusions.

More preferably, the Fab-like antibody fragment or antigen-binding fragment, VH-CH3 polypeptide or VL-CH3 polypeptide provided by the present invention is capable of specifically binding to an antigen against which the corresponding engineered Fab is directed, said antigen source including but not limited to cancer, infectious diseases (such as viral, bacterial, fungal, parasitic infections, etc.), autoimmune diseases, inflammatory disorders.

The antigen may be from a virus. The virus of the invention, for example a virus from one of the following families: retroviridae (e.g., Human Immunodeficiency Virus (HIV), human T-cell leukemia virus (HTLV)); picornaviridae (e.g., poliovirus, hepatitis a virus, hepatitis c virus, enterovirus, human coxsackievirus, rhinovirus, echovirus, foot and mouth disease virus); cadoviridae (e.g., strains of virus that cause gastroenteritis); togaviridae (e.g., equine encephalitis virus, rubella virus); flaviviridae (e.g., dengue virus, yellow fever virus, west nile virus, st louis encephalitis virus, japanese encephalitis virus, and other encephalitis viruses); coronaviridae (e.g., coronavirus, Severe Acute Respiratory Syndrome (SARS) virus); rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, Respiratory Syncytial Virus (RSV)); orthomyxoviridae (e.g., influenza virus); bunyaviridae (e.g., hantavirus, Sin Nombre virus, rift valley fever virus, bunya virus, phleboviruses, and Nairo virus); arenaviridae (e.g., hemorrhagic fever virus, Machupo virus, Junin virus); reoviridae (e.g., reoviruses, orbiviurses, and rotaviruses); binuclear glyconucleoviridae; hepadnaviridae (e.g., hepatitis b virus); parvoviridae (e.g., parvoviruses); papovaviridae (e.g., papilloma virus, polyoma virus, BK virus); adenoviridae (e.g., most adenoviruses, such as adeno-associated viruses); herpesviridae (e.g., herpes simplex virus (HSV-1 and HSV-2), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella Zoster Virus (VZV), and other herpes viruses, including HSV-6); poxviridae (e.g., variola virus, vaccinia virus, poxvirus); and iridoviridae (e.g., african swine fever virus); virinae (e.g., ebola virus, marburg virus); the caliciviridae family (e.g., norwalk virus) and unclassified viruses (e.g., the causative agent of spongiform encephalopathy, the causative agent of hepatitis delta (believed to be a defective satellite of hepatitis b virus), and astrovirus).

The antigen may be from a bacterium. The bacteria of the invention are, for example, helicobacter pylori, Borelia burgdorferi, Legionella pneumophila, mycobacteria (e.g., M.tuboculosis, M.avium, M.intracellularie, M.kansai, M.gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, neisseria meningitidis, listeria monocytogenes, streptococcus pyogenes (group a streptococcus), streptococcus agalactiae (group B streptococcus), streptococcus (the group phaeophora fuliginosus), streptococcus faecalis, streptococcus bovis, streptococcus (anaerobe), streptococcus pneumoniae, pathogenic campylobacter, enterococcus, haemophilus influenzae, bacillus anthracis, corynebacterium diphtheriae, corynebacterium, classical swine fever virus, clostridium perfringens, clostridium tetani, enterobacter aerogenes, klebsiella pneumoniae, pasteurella multocida, bacteroides, clostridium, streptomyces maltophilia, treponema pallidum, leptospira, or actinomycetes (Actinomyces israelli).

The antigen may be from a fungus. Fungi according to the invention are, for example, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis or Candida albicans.

The antigen may be from a parasite. The parasite according to the invention is for example Plasmodium falciparum (Plasmodium falciparum) or Toxoplasma gondii (Toxoplasma gondii).

The antigen may be a cancer antigen, such as a solid tumor or blood-borne cancer antigen. The solid tumor of the invention is a sarcoma or carcinoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, or another sarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, wilms' tumor, cervical cancer, testicular tumor, bladder cancer, or central nervous system tumor (e.g., glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma or retinoblastoma). The blood-borne cancers of the present invention are leukemias, such as acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytes, myelomonocytic cells, monocytes and erythroleukemia); chronic leukemias (e.g., chronic myelogenous (granulocytic) leukemia, chronic granulocytic leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, or myelodysplasia. Tumor antigens are well known in the art and include, for example, carcinoembryonic antigen (CEA), β -human chorionic gonadotropin (β -HCG), alpha-fetoprotein (AFP), lectin-reactive AFP, (AFP-L3), thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase (hTERT), RU1, RU2(AS), intestinal carboxyesterase, mut hsp70-2, M-CSF, prostaglandinase, Prostate Specific Antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prothelin, PSMA, Her2/neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), melanoma associated antigen (MAGE), ELF2M, neutrophil elastase, ephrinB2, and CD 22. It may also be any cancer-associated protein, such as IGF-I, IGF-II, IGR-IR or mesothelin.

The invention provides a medicinal composition, which is prepared by mixing a nucleic acid molecule, a plasmid and a physiologically or pharmaceutically acceptable carrier, excipient or stabilizer of a Fab-like antibody fragment or antigen binding fragment, a VH-CH3 polypeptide or VL-CH3 polypeptide or Fab-like antibody fragment or antigen binding fragment, a VH-CH3 polypeptide or VL-CH3 polypeptide, wherein the composition comprises but is not limited to a freeze-drying dosage form, an aqueous solution dosage form, a liposome or a capsule dosage form and the like. The concentration of the nucleic acid molecules, plasmids of the Fab-like antibody fragment or antigen-binding fragment, VH-CH3 polypeptide or VL-CH3 polypeptide or Fab-like antibody fragment or antigen-binding fragment, VH-CH3 polypeptide or VL-CH3 polypeptide of the invention can vary from about 0.1% to 100% by weight.

The invention provides a detection kit, which contains the antibody, the polypeptide, the nucleic acid molecule or the plasmid. The detection kit is used for detecting pathogens and tumor cells, wherein the pathogens comprise viruses, bacteria, fungi, parasite infection and the like, and the tumor cells comprise various benign tumor cells, malignant tumor cells (namely cancer cells), solid tumor cells and blood-borne cancer cells.

The invention provides a method for constructing a Fab-like antibody fragment or an antigen binding fragment, which is characterized by comprising the following steps:

(a) introducing mutations into one or more β chains of the antibody constant region CH3 domain of a Fab antibody fragment or antigen binding fragment of any one of claims 1 to 18; or

(b) Replacing a portion of the CH3 domain with a Complementarity Determining Region (CDR) or functional fragment that retains a specific binding antigen from a heterologous immunoglobulin variable region; or

(c) Both of which are present;

fab-like antibody fragments or antigen-binding fragments were constructed using the methods described above.

The invention provides a Fab-like antibody fragment or antigen-binding fragment constructed according to the method.

Drawings

FIG. 1: and (3) engineering and designing a novel high-stability Fab-like antibody fragment.

FIG. 2: a novel high-stability Fab-like antibody fragment modification and a preparation method thereof.

FIG. 3: and (3) detecting the stability of the novel high-stability Fab-like antibody fragment.

FIG. 4: the binding capacity of the novel high-stability Fab-like antibody fragment with anti-endothelin (mesothelin) and vascular endothelial growth factor (VEGF165) is detected by using a biofilm layer optical interference technique (BLI).

Detailed Description

In order that the invention may be more fully understood, some definitions are set forth below. The above definitions are intended to include grammatical equivalents.

As used herein, "antibody fragment or antigen-binding fragment" means a portion of an antibody, such as F (ab ') 2, F (ab)2, Fab', Fab, Fv, scFv (single chain Fv), single domain antibody (DAB or VHH), etc., including the above-referenced half molecule of IgG4 (van der Neut Kolfsschoten et al, Science 2007; 317 (9/14 th): 1554-1557). Regardless of structure, the antibody fragment used binds to the same antigen that is recognized by the intact antibody. The term "antibody fragment" also includes synthetic or genetically engineered proteins that function as antibodies by binding to a particular antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv" fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which the light and heavy variable regions are joined by a peptide linker ("scFv proteins"). Fragments can be constructed in different ways to produce multivalent and/or multispecific binding forms.

As used herein, "heavy chain variable region" means a region of an immunoglobulin heavy chain that varies greatly near the N-terminal amino acid sequence. The heavy chain variable region referred to herein is preferably an amino acid

As used herein, "light chain variable region" means a region of an immunoglobulin light chain that varies greatly near the N-terminal amino acid sequence. The light chain variable region referred to herein is preferably an amino acid

"linker" as used herein means a peptide that links two or more protein fragments. The linker referred to herein is preferably RQPREPQ or AQPREPQ.

As used herein, "CH 3 fragment or domain" means a polypeptide derived from an immunoglobulin CH3 fragment or domain, CH3 fragment or domain molecule comprising at least one CDR or functional fragment thereof; or may further comprise additional amino acid sequences, such as a complete hypervariable loop; may have at least a portion of one or more beta strands replaced by a CDR or functional fragment thereof; or the CH3 fragment or domain comprises one or more mutations in a loop region of the molecule. The "loop region" of the CH3 fragment or domain refers to the portion of the protein located between the β chain regions. The CH3 fragment or domain is small in molecular size, typically less than 15 kD. Its size may vary depending on the length of the CDR/hypervariable amino acid sequence inserted into the loop region, or the number of CDRs inserted and whether another molecule (e.g. an effector molecule or a tag) binds to the CH3 domain. The CH3 fragment or domain molecule may be glycosylated or unglycosylated. For example, a recombinant CH3 fragment or domain can be expressed in a suitable mammalian cell to allow glycosylation of the molecule.

"amino acid" as used herein means one of the 20 naturally occurring amino acids or any non-natural analog, which may be located at a specifically defined position. By "protein" herein is meant at least two covalently linked amino acids, which include proteins, polypeptides, oligopeptides and peptides. Proteins can be composed of naturally occurring amino acids and peptide bonds, or can be composed of synthetic peptidomimetic structures, i.e., "analogs". Thus "amino acid" or "peptide residue" as used herein means naturally occurring and synthetic amino acids. For example, for the purposes of the present invention, homophenylalanine, citrulline and norleucine are considered amino acids for the purposes of the present invention. "amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain may be in the (R) or (S) configuration. In preferred embodiments, the amino acids are present in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substitutions may be used, for example to prevent or delay in vivo degradation.

"polypeptide" as used herein means a polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acid is an α -amino acid, an L-optical isomer or a D-optical isomer may be used. The term "polypeptide" or "protein" as used herein is intended to encompass any amino acid sequence and includes modified sequences, such as glycoproteins. The term "polypeptide" is specifically intended to encompass naturally occurring proteins, as well as recombinantly or synthetically produced proteins.

By "antibody" herein is meant a protein consisting of one or more polypeptides encoded by substantially all or part of a recognized immunoglobulin gene. The recognized immunoglobulin genes, for example in humans, include kappa (. kappa.), lambda (. lamda.), and heavy chain loci, which contain a myriad of variable region genes, as well as constant region genes mu (. mu.), delta (. delta.), gamma (. gamma.), epsilon., alpha (. alpha.), which encode IgM, IgD, IgG, IgE, and IgA isotypes, respectively. Antibodies herein are meant to include full length antibodies and antibody fragments, as well as natural antibodies from any organism, engineered antibodies, or recombinantly produced antibodies for testing, therapeutic purposes, or other purposes as further specified below. The term "antibody" includes antibody fragments, as known in the art, such as Fab, Fab ', F (ab') 2, Fv, scFv or other subsequences for antigen binding of an antibody, or antibody fragments produced by modification of whole antibodies or those antibodies synthesized de novo using recombinant DNA techniques. The term "antibody" includes monoclonal as well as polyclonal antibodies. The antibody may be an antagonist, agonist, neutralizing antibody, or inhibitory antibody, or stimulatory antibody. The antibodies of the invention may be non-human, chimeric, humanized or fully human antibodies.

Specifically included within the definition of "antibody" are non-glycosylated antibodies. Preferably, as used herein, is an "aglycosylated antibody", meaning an antibody lacking a sugar attachment at position 297 of the Fc region, wherein the numbering is according to the EU system of Kabat. The non-glycosylated antibody may be a deglycosylated antibody, which is an antibody from which the Fc sugars have been removed, e.g. chemically or enzymatically. Alternatively, the aglycosylated antibody may be an aglycosylated or unglycosylated antibody, which is an antibody that does not express Fc sugars, for example by mutating one or more residues encoding the glycosylation pattern or by expressing in an organism such as a bacterium that does not attach sugars to proteins.

As used herein, "IgG" means a polypeptide belonging to the class of antibodies, which is substantially encoded by the putative immunoglobulin gamma gene. In humans, this class comprises IgG1, IgG2, IgG3 and IgG 4. In mice, this class comprises IgG1, IgG2a, IgG2b, IgG 3. By "immunoglobulin (Ig)" herein is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have many structural types including, but not limited to, full length antibodies, antibody fragments, and single immunoglobulin domains. The known Ig domains VH, C γ i, C γ 2, C γ 3, VL and CL in antibody IgG class.

As used herein, "antigen" means a compound, composition or substance that can stimulate antibody production or a T cell response in an animal, including compositions injected or absorbed into the animal, which may be proteins, carbohydrates, lipids, or other pathogens.

As used herein, "nucleic acid" means a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) via phosphodiester linkages. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs such as, but not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2' -O-methyl ribonucleotide, Peptide Nucleic Acid (PNA), and the like. For example, these polynucleotides can be synthesized using an automated DNA synthesizer. The term "oligonucleotide" generally refers to short polynucleotides, typically no more than about 50 nucleotides. It will be understood that when the nucleotide sequence is represented by a DNA sequence (i.e.A, T, G, C), this also includes RNA sequences in which "U" replaces "T" (i.e.A, U, G, C).

Conventional notation is used herein to describe nucleotide sequences: the left-hand end 5' end of the single-stranded nucleotide sequence; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5' -direction. The direction of addition of 5 'to 3' nucleotides to a nascent RNA transcript is referred to as the direction of transcription. A DNA strand having the same sequence as mRNA is called an encoding strand.

"homology" as used herein means similarity between nucleotide or amino acid sequences, otherwise referred to as sequence identity. Sequence identity is usually measured in terms of percent identity (or similarity or homology); the higher the percentage, the more similar the two sequences. Homologues or variants will have a relatively high degree of sequence identity when aligned using standard methods. Methods of sequence alignment for comparison are well known in the art. Various procedures and alignment algorithms are described in: smith and Waterman, Adv appl.math, 2:482,1981; needlema and Wunsch, J.mol.biol.48:443,1970; pearson and Lipman, proc.natl.acad.sci.u.s.a.85:2444,1988; higgins and Sharp, Gene 73: 237-; higgins and Sharp, cabaos 5: 151-153, 1989; corpet et al, Nucleic Acids Research 16: 10881-10890, 1988; and Altschul et al, Nature genet, 1994, 6: 119-129.

NCBI basic local alignment search tools (BLAST TM) (Altschul et al, J.mol.biol., 215: 403-.

"plasmid" as used herein means a plasmid artificially constructed for adaptation to laboratory procedures on the basis of a native plasmid. The nucleic acid molecule can be introduced into a host cell, thereby producing a transformed host cell. The vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication, and may also include one or more selectable marker genes and other genetic elements known in the art.

As used herein, "host cell," also referred to as recipient cell, refers to a host cell that receives a foreign gene during transformation and transduction (infection).

As used herein, "pharmaceutically acceptable carrier" means a conventional pharmaceutically acceptable carrier. Remington's Pharmaceutical Sciences, ewmar, Mack Publishing co., Easton, Pa., 15 th edition (1975), describe compositions and formulations suitable for drug delivery of one or more therapeutic compounds or molecules (e.g., one or more antibodies), as well as additional agents.

By "effective prophylactic or therapeutic dose" herein is meant an amount of a particular agent sufficient to achieve a desired effect in a subject being treated with the agent. The precise dosage will depend on the purpose of the treatment and can be determined by one skilled in the art using well known techniques. The dosage range may be 0.01-100mg/kg body weight or more, for example 0.1, 1, 10 or 50mg/kg body weight, preferably 1-10 mg/kg. As is well known in the art, adjustments may be necessary for antibody or Fc fusion degradation, systemic or local delivery, and rates of neoprotease synthesis, as well as age, body weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition, and can be determined by those skilled in the art through routine experimentation. Such agents include Fab-like antibody fragments or antigen-binding fragments, VH-CH3 polypeptides, or VL-CH3 polypeptides described herein. Ideally, a therapeutically effective amount of the antibody is an amount sufficient to prevent, treat or ameliorate an infection or disease, such as caused by HIV infection in a subject, without causing significant cytotoxic effects in the subject. A therapeutically effective amount of an agent for preventing, ameliorating and/or treating a subject will depend upon the subject being treated, the type and severity of the affliction, and the mode of administration of the therapeutic composition.

In this contextThe "beta chain" as used herein means a segment of a polypeptide chain, typically 3 to 10 amino acids long, with the backbone in an almost fully extended conformation. Typically, the beta strands are single contiguous stretches of amino acids in this extended conformation and involve hydrogen bonding with the backbone of at least one other strand so that they form a beta sheet. In β sheets, most β strands are arranged adjacent to other strands and form extensive hydrogen bonding networks with their neighbors, where the N-H group in one strand backbone forms a hydrogen bond with the C ═ O group of the adjacent strand backbone. In a fully extended beta-strand, consecutive side chains point straight up, then straight down, then straight up, etc. Adjacent beta strands in the beta sheet are aligned such that their C^αThe atoms are adjacent and their side chains point in the same direction. The "wrinkled" appearance of the beta strands is from C^αTetrahedral chemical bonding of atoms; for example, if the side chain is directed directly upwards, then this is coupled with C^vMust be slightly downward because of its bond angle of about 109.5. Pleating results in C^α _iAnd C^α _i+2Is about a distance of

Rather than the two fully extended trans peptide virtual bonds as would be expected

The "lateral" distance between adjacent C.alpha.atoms in the hydrogen bond beta chain is approximately

As used herein, "Complementarity Determining Region (CDR)" means a region within the variable region of an antibody in which amino acid residues are more likely to vary in composition and arrangement, and is also known as the hypervariable region. Among the L and H chain V regions are three hypervariable regions (HVRs) which are sterically complementary to antigenic determinants. Each antigen receptor contains six hypervariable loops: h1, H2, H3, L1, L2 and L3. For example, the H1 loop comprises the CDR1 of the heavy chain and the L3 loop comprises the CDR3 of the light chain. The CH2 and CH3 domain molecules may include grafted amino acids from antibody variable domains. The grafted amino acids comprise at least a portion of a CDR. The grafted amino acids may also include additional amino acid sequences, such as complete hypervariable loops. As used herein, a "functional fragment" of a CDR is at least a portion of a CDR that retains the ability to bind a particular antigen.

By "isolated" herein is meant that a biological component (e.g., a nucleic acid molecule or protein) has been substantially isolated or purified from other biological components in which the component naturally occurs (e.g., other biological components of a cell), such as other chromosomal and extra-chromosomal DNA, RNA, and proteins, including other antibodies. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. An isolated antibody is an antibody that is substantially separated or purified from other proteins or biological components, thereby maintaining its antigenic specificity. The term also includes nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins or fragments thereof.

"encoding" as used herein means the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, as a template for the synthesis of other polymers and macromolecules in biological processes having defined nucleotide sequences, or defined amino acid sequences and biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA produced by the gene produces the protein in a cell or other biological system. The coding strand (which has the same nucleotide sequence as the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (which serves as a transcription template, gene or cDNA) may be referred to as encoding protein. Or other products of the gene or cDNA. Unless otherwise indicated, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. It is also understood that all base sizes or amino acid sizes, and all molecular weights or molecular weight values given for a nucleic acid or polypeptide are approximate and provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprising" means "including". All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Detailed Description

Standard recombinant DNA techniques and Molecular cloning techniques used in the examples are well known in the art (published by Ausubel, F.M et al, Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience), and materials and methods suitable for the growth of microorganisms are well known in the art. The major chemical and biological reagents were purchased from KAPA Biosystems, New England Biolabs, TransGen Biotech, Thermo Fisher Scientific, OMEGA bio-tek, etc.

The present invention will be described in detail with reference to specific examples.

Example 1 engineered design of high stability Fab-like antibody fragments

The related literature indicates that the CH3 structure of the antibody has more biological action sites (the related literature PMID: 29181008; PMID: 24765095; PMID: 22518843; PMID: 23867459). Through computer Molecular simulation (PyMOL Molecular Graphics System and PDBePISA), the antibody fragment CH3 on IgG has better homology with CH1 and CL, and the Root Mean Square Deviation (RMSD) is only

Meanwhile, the invention also compares the amino terminal amino acid sequences of CH3, CH1 and CL through computer homology simulation, selects RQPREPQ (arginine, glutamine, proline, arginine, glutamic acid, prolyl) according to the side chain trend of amino acidAmino acid and glutamine) and aqprep pq (alanine, glutamine, proline, arginine, glutamic acid, proline and glutamine) link the Fab-like antibody fragment light chain variable region and heavy chain variable region, respectively, to the antibody constant region CH3 domain (shown in figure 1).

Example 2 high stability Fab-like antibody fragment engineering and preparation method

The IgG-Fab amino acid sequence is reformed, CH3 is used to replace CH1 and CL, VL-CH3 and VH-CH3 sequences are designed and synthesized, and the linker is the amino acid sequence of natural antibody (for example, IgG1-CH3 uses 341-347 amino acid QPREPQ to replace IgG1-CH1 114-120 amino acid STKGPS and IgG1-CL 109-114 TVAAPS). Wherein, the CH3 fragment is subjected to specific site mutation by a Knobs-into-Holes technology (see a document PMID: 25427258; PMID:23831709), so that the expressed protein forms a concave-convex chimeric complementary structure on a spatial conformation, random recombination pairing is reduced, the expression yield is improved, and the amino acid sequence of the mutated CH3 fragment is shown as a sequence SEQ ID NO 1 (wherein X1 is amino acid S or T, X2 is amino acid L or A, and X3 is amino acid V or Y) and a sequence SEQ ID NO 2 (wherein X1 is amino acid W or T).

The sequences of The expression Fab-like antibody fragments VL-CH3 and VH-CH3 were constructed into plasmid pComb3x (vector as given by Dr. Dennis Burton, The Scripps Research Institute), and then two recombinant plasmids were transformed into E.coli HB2151, respectively, and after culturing and inducing (see literature PMID: 28966056; PMID:23831709), The induced bacterial solution was cultured according to The following protocol 5: 5 or 4: mixing the above 6 ratios, performing lysis, purifying to obtain high purity protein (according to the procedure of nickel column purification instruction, using Ni-NTA resin (Qiagen, Valencia, CA)), and purifying by two steps

(iba-lifesciences), protein purity by SDS-PAGE, and protein concentration by spectrophotometry (Nano Vue, GE Healthcare).

The main processes of Fab-like antibody fragment modification and preparation method, and the purified target protein are shown in FIG. 2.

Example 3 stability testing of high stability Fab-like antibody fragments

Both the antibody Fab and the novel high stability Fab-like antibody fragment were diluted to a concentration of 0.25mg/ml and the stability of the protein was measured at 216nm using the apparatus Jasco J-815 Spropolarmer (Jasco International) and the protein aggregation was measured using Zetasizer Nano ZS ZEN3600(Malvern Instruments Limited, Westborough, MA, USA). Cd (circular dichroism) was used to assess the thermostability of Fab-like antibody fragments, and the aggregation properties of Fab-like antibody fragments were assessed by dls (dynamic light scattering). And standing the protein at 4 deg.C and 37 deg.C for 1, 3, and 7 days, and detecting the stability of the protein by SDS-PAGE and ELISA (the specific detection method of protein stability and aggregation characteristics is described in literature PMID 29181008; PMID 24765095; PMID 22518843; PMID 23867459; PMID 26963639); as shown in FIG. 3, the high stability Fab-like antibody fragment prepared exhibits high stability and good aggregation.

Example 4 binding Capacity of highly stable Fab-like antibody fragments to the antigens endothelin (mesothelin) and vascular endothelial growth factor (VEGF165)

The binding capacity of antibody Fab and novel high-stability Fab-like antibody fragments to the antigens endothelin (mesothelin) and vascular endothelial growth factor (VEGF165) was examined using biofilm layer optical interference technique (BLI) (see literature PMID: 29181008; PMID:28966056) and amino-coupled probe (ARG2) (purchased from Octet-RED (pall fortebio)).

The modification and preparation method of the Fab-like antibody fragment modified based on the antigen endothelin (mesothelin), namely m912-Fab and the Fab-like antibody fragment modified based on the vascular endothelial growth factor (VEGF165), namely Ranibizumab-Fab is shown in example 2, wherein the amino acid sequence m912-FabCH3-VH of the heavy chain variable region of the Fab-like antibody fragment modified based on the antigen endothelin (mesothelin), namely m912-Fab is shown in SEQ ID NO:5, and the amino acid sequence m912-FabCH3-VL of the light chain variable region is shown in SEQ ID NO: 6; the amino acid sequence of the heavy chain variable region of the Fab-like antibody fragment modified based on vascular endothelial growth factor (VEGF165), namely Ranibizumab-Fab, is shown as SEQ ID NO:7 in Ranibizumab-FabCH3-VH, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO:8 in Ranibizumab-FabCH 3-VL.

In the detection process, protein is diluted according to a 2-fold ratio, and the binding and dissociation are detected under the condition of pH 7.4 to obtain an affinity constant; the results in fig. 4 show that the high stability Fab-like antibody fragments prepared have an improved ability to bind to the corresponding antigen compared to the antibody Fab.

Example 5 expression of different sequences of high stability Fab-like antibody fragments and stability testing

Selecting the heavy chain variable region in a Fab antibody fragment or antigen binding fragment as the amino acid

QVQL/QMQL/EVQL/QIQL/QVTL/TVQL/EVHL/EMQL/EVEL/EDQL/QLQL/EAQL, starting with amino acid TVSS; light chain variable region with amino acids

DIQMT/AIQMT/AIRMT/DIQLT/AIQLT/VIWMT/NIQMT/DIVMT/EIVMT/EIVLT/ETTLT/DVVMT/DIVLT starting with the amino acid VEIK ending; the linker is the amino acid sequence of natural antibody (e.g. using amino acid QPREPQ at 341-347 position on IgG1-CH3 to replace STKGPS at 114-120 position on IgG1-CH1 and TVAAPS at 109-114 position on IgG 1-CL), using CH3 to replace CH1 and CL, and performing specific site mutation on CH3 fragment by Knobs-into-Holes technique (see literature PMID: 25427258; PMID:23831709) to construct vector. Expression and purification are carried out according to the above instructions, and finally the novel Fab-like antibody fragment is obtained. We used cd (circular dichroism) to assess the thermostability of Fab-like antibody fragments and dls (dynamic light scattering) to assess the aggregation properties of Fab-like antibody fragments. And standing the protein at 4 ℃ and 37 ℃ for 1, 3 and 7 days respectively, and detecting the stability of the protein by SDS-PAGE and ELISA; the results show that most of Fab antibody fragments have better stability and even better stability than the Fab antibody fragments before modification.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered as the protection scope of the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

1 (amino acid sequence of IgG1-CH3 mutants)

VYTLPPSRDELTKNQVSLX1CX2VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLX3SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

2 (amino acid sequence of IgG1-CH3 mutants)

VYTLPPSRDELTKNQVSLX1CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

3 (linker amino acid sequence between antigen binding fragment and IgG1-CH 3)

RQPREPQ

4 (linker amino acid sequence between antigen-binding fragment and IgG1-CH 3)

AQPREPQ

5 (amino acid sequence m912-FabCH3-VH based on the heavy chain variable region of the m912-Fab engineered Fab-like antibody fragment)

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGKNGAFDIWGQGTMVTVSS

6 (amino acid sequence m912-FabCH3-VL based on the variable region of the light chain of the engineered Fab-like antibody fragment of m 912-Fab)

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK

7 (amino acid sequence of heavy chain variable region of Fab-like antibody fragment based on Ranibizumab-Fab modification Ranibizumab-FabCH3-VH)

EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS

SEQ ID NO:8 (amino acid sequence of light chain variable region of Fab-like antibody fragment based on Ranibizumab-Fab modification Ranibizumab-FabCH3-VL)

DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK。

Sequence listing

<110> university of Compound Dan

<120> high-stability Fab-like antibody and preparation method and application thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 100

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of IgG1-CH3 mutants

<220>

<222> (19)..(19)

<223> The 'Xaa' at location 19 stands for Ser or Thr.

<220>

<222> (21)..(21)

<223> The 'Xaa' at location 21 stands for Leu or Ala.

<220>

<222> (60)..(60)

<223> The 'Xaa' at location 60 stands for Val or Tyr.

<400> 1

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

1 5 10 15

Ser Leu Xaa Cys Xaa Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

20 25 30

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

35 40 45

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Xaa Ser Lys Leu Thr

50 55 60

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

65 70 75 80

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

85 90 95

Ser Pro Gly Lys

100

<210> 2

<211> 100

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of IgG1-CH3 mutants

<220>

<221> MUTAGEN

<222> (19)..(19)

<223> The 'Xaa' at location 19 stands for Trp or Thr.

<400> 2

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

1 5 10 15

Ser Leu Xaa Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

20 25 30

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

35 40 45

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

50 55 60

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

65 70 75 80

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

85 90 95

Ser Pro Gly Lys

100

<210> 3

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker amino acid sequence between antigen binding fragment and IgG1-CH3

<400> 3

Arg Gln Pro Arg Glu Pro Gln

1 5

<210> 4

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> linker amino acid sequence between antigen binding fragment and IgG1-CH3

<400> 4

Ala Gln Pro Arg Glu Pro Gln

1 5

<210> 5

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence m912-FabCH3-VH of heavy chain variable region of Fab-like antibody fragment engineered based on m912-Fab

<400> 5

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 6

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence m912-FabCH3-VL of light chain variable region of Fab-like antibody fragment engineered based on m912-Fab

<400> 6

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 7

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Ranibizumab-FabCH3-VH amino acid sequence of heavy chain variable region of Fab-like antibody fragment modified based on Ranibizumab-Fab

<400> 7

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Ranibizumab-FabCH3-VL based on amino acid sequence of light chain variable region of Ranibizumab-Fab modified Fab-like antibody fragment

<400> 8

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

Claims

1. A Fab-like antibody fragment or antigen-binding fragment, characterized in that the Fab-like antibody fragment or antigen-binding fragment comprises:

2. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the heavy chain variable region in the Fab-like antibody fragment or antigen-binding fragment begins with amino acid QVQL/QMQL/EVQL/QIQL/QVTL/TVQL/EVHL/EMQL/EVEL/EDQL/QLQL/EAQL and ends with amino acid TVSS.

3. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the heavy chain variable region of the Fab-like antibody fragment or antigen-binding fragment comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO 5 or SEQ ID NO 7. .

4. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the light chain variable region in the Fab-like antibody fragment or antigen-binding fragment begins with amino acid DIQMT/AIQMT/AIRMT/DIQLT/AIQLT/vivmt/NIQMT/dimmt/EIVMT/EIVLT/ett/DVVMT/DIVLT and ends with amino acid VEIK.

5. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the light chain variable region in the Fab-like antibody fragment or antigen-binding fragment comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO 6 or SEQ ID NO 8.

6. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the linker in the Fab-like antibody fragment or antigen-binding fragment comprises an amino acid sequence that is at least 80% homologous to SEQ ID NO 3 or SEQ ID NO 4.

7. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the linker connecting the heavy chain variable region and the antibody constant region CH3 fragment in the Fab-like antibody fragment or antigen-binding fragment comprises an amino acid sequence having at least 80% homology to SEQ ID NO. 4.

8. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the linker connecting the variable region of the light chain and the fragment of antibody constant region CH3 comprises an amino acid sequence having at least 80% homology to SEQ ID NO. 3.

9. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the antibody constant region CH3 fragment of the Fab-like antibody fragment or antigen-binding fragment comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO. 1 or SEQ ID NO. 2.

10. The Fab-like antibody fragment or antigen binding fragment of claim 1, wherein the amino acid sequence of the antibody constant region CH3 fragment of the Fab-like antibody fragment or antigen binding fragment comprises: 1, wherein X1 is amino acid S or T, X2 is amino acid L or A, and X3 is amino acid V or Y, or, 2, wherein X1 is amino acid W or T.

11. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the antibody constant region CH3 fragment of the Fab-like antibody fragment or antigen-binding fragment, the antibody constant region CH3 fragment linked to the heavy chain variable region by a linker, comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO. 1.

12. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the Fab-like antibody fragment or antigen-binding fragment comprises an antibody constant region CH3 fragment, wherein the antibody constant region CH3 fragment linked to the heavy chain variable region by a linker comprises an amino acid sequence at least 95% homologous to SEQ ID NO:1, wherein X1 is amino acid S or T, X2 is amino acid L or A, and X3 is amino acid V or Y.

13. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the Fab-like antibody fragment or antigen-binding fragment comprises an antibody constant region CH3 fragment, wherein the antibody constant region CH3 fragment linked to the light chain variable region by a linker comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO. 2.

14. The Fab-like antibody fragment or antigen-binding fragment of claim 1, wherein the Fab-like antibody fragment or antigen-binding fragment comprises an antibody constant region CH3 fragment, wherein the antibody constant region CH3 fragment linked to the light chain variable region by a linker comprises an amino acid sequence that is at least 95% homologous to SEQ ID NO. 2, wherein X1 is amino acid W or T.

15. The Fab-like antibody fragment or antigen binding fragment of claim 1, characterized in that the Fab-like antibody fragment or antigen binding fragment comprises:

2) a light chain variable region beginning with amino acids DIQMT/AIQMT/AIRMT/DIQLT/AIQLT/vivmt/NIQMT/DIVMT/EIVMT/EIVLT/ETTLT/DVVMT/DIVLT, ending with amino acid VEIK, a linker comprising an amino acid sequence at least 80% homologous to SEQ ID No. 3 and an antibody constant region CH3 fragment comprising an amino acid sequence at least 95% homologous to SEQ ID No. 2.

16. The Fab-like antibody fragment or antigen binding fragment of claim 1, characterized in that the Fab-like antibody fragment or antigen binding fragment comprises:

2) a light chain variable region beginning with amino acids DIQMT/AIQMT/AIRMT/DIQLT/AIQLT/vivmt/NIQMT/DIVMT/EIVMT/EIVLT/ETTLT/DVVMT/DIVLT, ending with amino acid VEIK, a linker comprising an amino acid sequence having at least 80% homology to SEQ ID No. 3 and an antibody constant region CH3 fragment comprising an amino acid sequence having at least 95% homology to SEQ ID No. 2, wherein X1 in SEQ ID No. 2 is amino acid W or T.

17. The Fab-like antibody fragment or antigen binding fragment of claim 1, characterized in that the Fab-like antibody fragment or antigen binding fragment comprises:

18. The Fab-like antibody fragment or antigen binding fragment of claim 1, characterized in that the Fab-like antibody fragment or antigen binding fragment comprises:

19. An isolated VH-CH3 polypeptide, characterized in that the isolated VH-CH3 polypeptide comprises a heavy chain variable region, a linker, and an antibody constant region CH3 fragment.

20. The VH-CH3 polypeptide of claim 19, wherein the heavy chain variable region in the VH-CH3 polypeptide begins with amino acid QVQL/QMQL/EVQL/QIQL/QVTL/TVQL/EVHL/EMQL/EVEL/EDQL/QLQL/EAQL and ends with amino acid TVSS.

21. The VH-CH3 polypeptide of claim 19, wherein the heavy chain variable region in the VH-CH3 polypeptide comprises an amino acid sequence at least 95% homologous to SEQ ID No. 5 or SEQ ID No. 7.

22. The VH-CH3 polypeptide of claim 19, wherein the linker in the VH-CH3 polypeptide comprises an amino acid sequence at least 80% homologous to SEQ ID No. 4.

23. The VH-CH3 polypeptide of claim 19, characterized in that the antibody constant region CH3 fragment in the VH-CH3 polypeptide comprises an amino acid sequence having at least 95% homology with SEQ ID No. 1.

24. The VH-CH3 polypeptide of claim 19, characterized in that the antibody constant region CH3 fragment in the VH-CH3 polypeptide comprises an amino acid sequence at least 95% homologous to SEQ ID No. 1, wherein X1 is amino acid S or T, X2 is amino acid L or a, and X3 is amino acid V or Y.

25. An isolated VL-CH3 polypeptide, characterized in that the isolated VL-CH3 polypeptide comprises a light chain variable region, a linker, and an antibody constant region CH3 fragment.

26. The VL-CH3 polypeptide of claim 25, wherein the light chain variable region in the VL-CH3 polypeptide begins with amino acids DIQMT/AIQMT/AIRMT/DIQLT/AIQLT/vivmt/NIQMT/DIVMT/EIVMT/EIVLT/ettt/DVVMT/DIVLT and ends with amino acid VEIK.

27. The VL-CH3 polypeptide of claim 25, wherein the light chain variable region in the VL-CH3 polypeptide comprises an amino acid sequence having at least 95% homology with SEQ ID No. 6 or SEQ ID No. 8.

28. The VL-CH3 polypeptide of claim 25, wherein said linker in VL-CH3 polypeptide comprises an amino acid sequence having at least 80% homology to SEQ ID No. 3.

29. The VL-CH3 polypeptide of claim 25, wherein the antibody constant region CH3 fragment of the VL-CH3 polypeptide comprises an amino acid sequence that is at least 95% homologous to SEQ ID No. 2.

30. The VL-CH3 polypeptide of claim 25, wherein the antibody constant region CH3 fragment of the VL-CH3 polypeptide comprises an amino acid sequence having at least 95% homology with SEQ ID No. 2, wherein X1 is amino acid W or T.

31. A nucleic acid molecule encoding a Fab-like antibody fragment or antigen-binding fragment as claimed in claims 1 to 18, a VH-CH3 polypeptide as claimed in claims 19 to 24, and/or a VL-CH3 polypeptide as claimed in claims 25 to 30.

32. A plasmid comprising the nucleic acid molecule of claim 31.

33. A host cell comprising the plasmid of claim 19.

34. A pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a Fab-like antibody fragment or antigen-binding fragment of any one of claims 1-18, a VH-CH3 polypeptide of any one of claims 19-24, or a VL-CH3 polypeptide of any one of claims 25-30, and a pharmaceutically acceptable carrier.

35. A pharmaceutical composition comprising a prophylactically or therapeutically effective amount of the nucleic acid molecule of claim 31, or the plasmid of claim 32, and a pharmaceutically acceptable carrier.

36. A test kit comprising a Fab-like antibody fragment or antigen-binding fragment of any one of claims 1 to 18, a VH-CH3 polypeptide of any one of claims 19 to 24, a VL-CH3 polypeptide of any one of claims 25 to 30, a nucleic acid molecule of claim 31, or a plasmid of claim 32.

37. The test kit according to claim 36, characterized in that it is used for the detection of pathogens, tumor cells.

38. A method of constructing a Fab-like antibody fragment or antigen binding fragment comprising:

(c) Both of which are present;

39. A Fab-like antibody fragment or antigen binding fragment constructed according to the method of claim 38.