CN114478778B - anti-Tim-3 nano antibody and application thereof - Google Patents

anti-Tim-3 nano antibody and application thereof Download PDF

Info

Publication number
CN114478778B
CN114478778B CN202210337559.8A CN202210337559A CN114478778B CN 114478778 B CN114478778 B CN 114478778B CN 202210337559 A CN202210337559 A CN 202210337559A CN 114478778 B CN114478778 B CN 114478778B
Authority
CN
China
Prior art keywords
nanobody
cancer
tim
cell
vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210337559.8A
Other languages
Chinese (zh)
Other versions
CN114478778A (en
Inventor
韩根成
李葛
周洁
莫荣亮
李宇翔
王智鼎
高振芳
郝莹
陈金婕
侯春梅
冯健男
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Academy of Military Medical Sciences AMMS of PLA
Original Assignee
Academy of Military Medical Sciences AMMS of PLA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Academy of Military Medical Sciences AMMS of PLA filed Critical Academy of Military Medical Sciences AMMS of PLA
Priority to CN202210337559.8A priority Critical patent/CN114478778B/en
Publication of CN114478778A publication Critical patent/CN114478778A/en
Application granted granted Critical
Publication of CN114478778B publication Critical patent/CN114478778B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Virology (AREA)
  • Hospice & Palliative Care (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses an anti-Tim-3 nano antibody and application thereof, wherein the nano antibody has a sequence shown in SEQ ID NO: 1-3, CDR1, CDR2, CDR 3. The invention also provides a nucleic acid molecule for encoding the monoclonal antibody, an expression vector containing the nucleic acid molecule and a host cell. The nano antibody has high binding activity and neutralizing activity.

Description

anti-Tim-3 nano antibody and application thereof
Technical Field
The invention belongs to the fields of cellular immunology and genetic engineering, and relates to an anti-Tim-3 nano antibody and an application thereof.
Background
Tim-3 belongs to the immunoglobulin superfamily, is a type I transmembrane glycoprotein, and is composed of 1 membrane distal variable immunoglobulin region (IgV) and 1 membrane proximal mucin region, the former being the main structure of Tim-3 exerting inhibitory function. It was originally found on the surface of helper T cells (Thl), and was later found on CD4+ T cells, CD8+ T cells, cytotoxic lymphocytes (CTL), regulatory T cells (Treg), Tumor Infiltrating Lymphocytes (TIL), monocytes/macrophages, natural killer cells (NK) and Dendritic Cells (DC).
Tim-3 has been shown to be associated with a variety of diseases, and it was originally discovered that Tim-3 is associated with the suppression of T cell function in a variety of autoimmune diseases (autoimmune encephalomyelitis, type I diabetes and multiple sclerosis) (Kiyasu J, Miyoshi H, Hirata A, et al. Expression of programmed cell depletion potential 1 is associated with a porous with porous in substrates with a differential large B-ce111 photoma [ J ] Blood 2015, 126: 2193 and 2201.). Subsequent researches successively find that Tim-3 is up-regulated in various tumors such as gastric cancer, liver cancer, lung cancer, nasopharyngeal cancer, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, breast cancer, cervical cancer, bladder cancer and the like, and is related to poor prognosis. In China, Caoyang and the like (Caoyang, lymphoma vascular endothelial cells abnormally express Tim-3: a research study on a novel tumor immune evasion mechanism [ D ] Huazhong university of science and technology, 2009.) firstly find that the high-expression Tim-3 protein of the vascular endothelial cells of the lymphoma possibly participates in the formation and immune escape of new blood vessels of the lymphoma by in vitro and in vivo experiments, and finally promote the growth and dissemination of the lymphoma. Later on, Zhangli et al (Zhangli. PD-1 and TIM-3 expression in diffuse large B-cell lymphoma and their effect on cell proliferation and apoptosis [ D ]. Shandong university, 2016) confirmed that the expression level of TIM-3 decreased with the increase of chemotherapy treatment course, and correlated with the efficiency of chemotherapy, is an index of chemotherapy sensitivity of patients with DLBCL, and thus, becomes a factor affecting prognosis. Recent studies have shown that Tim-3 antibodies are also capable of exerting an antiviral Encephalitis effect (Li G, Tang LL, Hou CM, et al. Peripheral Injection of Tim-3 Antibody antibodies VSV Encephalitis by Enhancing MHC-I Presentation [ J ]. Front Immunol,2021, 12: 667478).
Nanobodies (Nbs) are recombinant antibody fragments of about 15 kDa found in camelids, alpaca, etc., the variable regions of heavy chain antibodies in camelids, also known as single domain antibodies (VHH), consisting of only two H chains, lacking L chains. Nbs have good biological and physicochemical properties, small volume, simple structure, easy expression, heat resistance and strong specificity. With the continuous and deep exploration of the nanometer antibody, the nanometer antibody is widely applied to various fields such as biotechnology, safety detection, clinical treatment and the like. The research on the anti-Tim-3 antibody has important significance for the detection and treatment of diseases.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide a nano antibody which has higher affinity activity and binding activity, can effectively detect Tim-3 and prevent and treat Tim-3 related diseases.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first aspect of the invention provides a nano antibody against Tim-3, wherein the amino acid sequences of CDR1, CDR2, CDR3 of the heavy chain variable region of the nano antibody are respectively as shown in SEQ ID NO: 1. 2 and 3.
Further, the heavy chain variable region further comprises a framework region FR, wherein FR1, FR2, FR3 and FR4 sequences of the framework region are set forth in SEQ ID NO: 4. 5, 6 and 7.
Further, the amino acid sequence of the heavy chain variable region of the nanobody has a sequence identical to that of SEQ ID NO: 8, preferably 95% sequence identity.
Further, the nanobody comprises all or part of an antibody heavy chain constant region.
In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a nanobody according to the first aspect of the present invention. The nucleic acid molecules of the invention can be synthesized, for example, by standard chemical synthesis methods and/or recombinant methods, or semi-synthetically produced, for example, by combined chemical synthesis and recombinant methods. Ligation of the coding sequence to transcriptional regulatory elements and/or to other amino acid coding sequences can be performed using established methods, such as restriction digest, ligation, and molecular cloning.
In a third aspect, the present invention provides a vector comprising a nucleic acid molecule according to the second aspect of the invention.
In a fourth aspect, the invention provides a host cell comprising a nucleic acid molecule according to the second aspect of the invention, or a vector according to the third aspect of the invention.
A fifth aspect of the invention provides a drug conjugate comprising:
the nanobody of the first aspect of the present invention, and
a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.
A sixth aspect of the present invention provides a composition comprising:
the nanobody of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, the host cell of the fourth aspect of the invention or the drug conjugate of the fifth aspect of the invention.
Further, the composition also comprises a pharmaceutically acceptable carrier.
The seventh aspect of the present invention provides any one of the following applications:
1) the use of a nanobody according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a host cell according to the fourth aspect of the present invention, or a drug conjugate according to the fifth aspect of the present invention for the preparation of a product for the detection of Tim-3;
2) use of a nanobody according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a host cell according to the fourth aspect of the present invention or a drug conjugate according to the sixth aspect of the present invention for the preparation of a product for the diagnosis of a Tim-3 related disease;
3) The use of the nanobody of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the vector of the third aspect of the present invention, the host cell of the fourth aspect of the present invention, the drug conjugate of the sixth aspect of the present invention, or the composition of the seventh aspect of the present invention in the preparation of a medicament for treating or preventing a Tim-3 related disease.
The Tim-3 related diseases include: bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer (kidney cancer), head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphomas, leukemias, myelomas, sarcomas, and virus-related cancers, as well as encephalitis.
An eighth aspect of the present invention provides a method of producing an anti-Tim-3 nanobody, the method comprising the steps of:
culturing the host cell of the fourth aspect of the invention under conditions suitable for the production of nanobodies, thereby obtaining a culture comprising the anti-Tim-3 nanobodies; and
isolating or recovering the anti-Tim-3 nanobody from the culture.
In a ninth aspect, the present invention provides a method for producing a recombinant cell expressing the nanobody of the first aspect of the present invention, the method comprising the steps of:
(1) introducing a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention into a competent host cell in vitro or ex vivo,
(2) recombinant host cells obtained by in vitro or ex vivo culture, and
(3) optionally selecting cells expressing and/or secreting said nanobody.
The invention has the advantages and beneficial effects that:
the invention provides a nano antibody for resisting Tim-3, which has the advantages of small volume, simple structure, easy expression, heat resistance, strong specificity, higher binding activity and neutralizing activity.
Drawings
FIG. 1 is an SDS-PAGE pattern of anti-Tim-3 nanobodies;
FIG. 2 is a graph of the binding energy of an ELISA to detect Tim-3 nm antibodies.
Detailed Description
The invention successfully obtains a group of nano antibodies through extensive and intensive research and a large amount of screening. Specifically, the invention utilizes Tim-3 antigen protein to immunize alpaca to obtain a high-quality immune nano antibody gene library. Then, the antigen molecules are coupled on an enzyme label plate, and the immune nano antibody gene library (heavy chain antibody phage display gene library) is screened by the antigen in the form by utilizing a phage display technology, so that the Tim-3 specific nano antibody gene is obtained. Experimental results show that the nano antibody obtained by the invention can be effectively combined with antigen. The present invention has been completed based on this finding.
In the present invention, the term "framework region", "framework" or "FR" refers to an amino acid sequence inserted between CDRs.
In the present invention, the terms "nanobody", "VHH antibody fragment" and "single domain antibody" are used indiscriminately and denote the variable domain of a single heavy chain of those types of antibodies found in camelids, which naturally lacks a light chain. In the absence of a light chain, nanobodies each have three CDRs, denoted as CDR1, CDR2, and CDR 3. It is the smallest antigen-binding fragment with full function. Nanobodies (VHHs) consisting of only one heavy chain variable region are typically constructed by obtaining an antibody naturally lacking the light and heavy chain constant region 1 (CH 1) and then cloning the variable region of the antibody heavy chain. The nano antibody/single domain antibody (Nanobody) is used as a novel small molecule antibody fragment and is obtained by cloning heavy chain variable regions (VHH) of camel natural heavy chain antibodies. The monoclonal antibody has excellent biological characteristics, has the molecular weight of 12-15kDa, is one tenth of that of a complete antibody, and has good tissue penetrability, high specificity and good water solubility. The nanobody according to the invention may in particular be a camel, dromedary, llama or alpaca nanobody.
In a particular embodiment of the invention, the nanobody according to the invention is an alpaca nanobody.
The term "affinity" refers to the binding capacity between a macromolecule and the antigen to which it binds, in particular between a nanobody and the antigen to which it binds, for example between the nanobody of the invention and the pathological form of Tim-3 protein as defined above.
The affinity of the nanobodies of the invention may be measured in vitro by several methods, including surface plasmon resonance or, for example, by ELISA assays, as described in the examples.
In the present invention, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the antibodies or fragments thereof of the present invention. The invention also comprises a cell surface marker or antigen combined with the nano antibody or the fragment thereof.
In the present invention, the terms "heavy chain variable region" and "VH" are used interchangeably.
In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR 3.
As is well known to those skilled in the art, the combination of CDR1, CDR2, and CDR3 is sufficient to define an antigen binding site. It is also known to those skilled in the art that two nanobodies recognizing the same antigen compete for binding to the antigen.
The invention provides a nano antibody for resisting Tim-3, which is characterized in that amino acid sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the nano antibody are respectively shown in SEQ ID NO: 1. 2 and 3.
The invention also provides a framework region of the nanobody, and the amino acid sequences of FR1, FR2, FR3 and FR4 of the framework region are respectively shown as SEQ ID NO: 4. 5, 6 and 7.
In a specific embodiment, the invention relates to a nanobody comprising or consisting of: the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 series of one of the nanobodies identified by the present inventors as defined above.
In an alternative embodiment, the nanobody according to the invention is thus a nanobody comprising or consisting of an amino acid sequence selected from the group consisting of: amino acid sequence SEQ ID NO: 8, two or three CDRs, or a conservative substitution of one or two amino acids in the CDRs. The function-conservative variants as defined above may also be found in the amino acid sequence SEQ ID NO: 8, which is not a CDR, e.g. a "framework" region, in particular a "framework" region as defined above.
In a specific embodiment of the invention, the nanobody of the invention is a nanobody comprising the amino acid sequence of SEQ ID NO: 8 of the nano antibody.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region.
In the context of the present invention, the expression "function-conservative variant" refers to a variant in which a given amino acid in a nanobody according to the present invention is replaced without impairing the overall conformation and function of the nanobody, including the replacement of one amino acid with another having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, presence of aromatic groups, etc.). Amino acids with similar properties are well known to those skilled in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchanged. Similarly, isoleucine (a hydrophobic amino acid) may be replaced with leucine, methionine, or valine. This change has little or no effect on the apparent molecular weight or isoelectric point of the nanobody. Natural amino acids may be replaced by unnatural amino acids, e.g., amino acids in the D configuration, or β or γ amino acids.
The skilled person is aware of the amino acid sequences of nanobodies of interest, which are capable of producing nanobodies according to the present invention, in particular the nanobodies as defined above, by conventional polypeptide production techniques. For example, they can be synthesized using well-known solid phase synthesis methods; preferably, a commercially available peptide synthesizer (e.g., manufactured by Applied Biosystems, Foster City, California) is used and the manufacturer's instructions are followed.
Alternatively, nanobodies according to the present invention may be synthesized by recombinant DNA techniques well known to those skilled in the art (Maniatis et al, (1982) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, NY, 51-54 and 412-430). For example, they may be obtained as DNA expression products after incorporating the DNA sequence encoding the polypeptide of interest into an expression vector, and introducing these vectors into suitable prokaryotic or eukaryotic hosts which will express the polypeptide of interest, from which they may then be isolated using techniques well known to those skilled in the art. As is well known to those skilled in the art, when a protein is synthesized by recombinant DNA techniques, its synthesis is typically linked to a tag that facilitates its purification. These tags are well known to those skilled in the art and include, for example, hexahistidine (6 His), glutathione S-transferase (GST), myc tag, or influenza Hemagglutinin (HA).
As a preferred embodiment, the nanobody of the present invention comprises a myc and/or a hexahistidine tag. The subject of the invention therefore also consists of a nanobody comprising or consisting of the amino acid sequence: the amino acid sequence is selected from the group consisting of the sequences SEQ ID NO: 8 which further comprises a myc and/or a six histidine residue tag, more preferably a myc and six histidine residue tag, at its C-terminus or N-terminus, preferably at its C-terminus. As is well known to those skilled in the art, when a protein is linked to a tag that facilitates its purification, such protein comprises a sequence between the native sequence and the tag that allows for enzymatic cleavage between the protein and the tag. Therefore, nanobodies comprising or consisting of enzymatically cleaved sequence amino acid sequences are also part of the present invention.
In another aspect, the invention provides nucleic acid molecules encoding the nanobodies of the invention.
Typically, the nucleic acid or nucleic acid molecule is a DNA or RNA molecule, which may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.
The terms "vector," "cloning vector," and "expression vector" refer to a vector into which a DNA or RNA sequence can be introduced into a host cell by transforming the host and facilitating expression (e.g., transcription and translation) of the introduced sequence.
Accordingly, the invention provides in one aspect a vector comprising a nucleic acid of the invention.
Non-limiting examples of plasmid vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027 pCAG Kosak-Cherry (L45 a) vector system, pREP Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edgesystems), pTriEx-hygro (Novagen) and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris (Pichia pastoris) include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus (Xenopus) embryos, zebrafish embryos, and a wide variety of mammalian and avian cells is the multipurpose expression vector pCS2 +.
Generally, a vector may contain one or more origins of replication (ori) and genetic systems for cloning or expression, one or more markers for selection in a host (e.g., antibiotic resistance), and one or more expression cassettes. In addition, the coding sequences contained in the vector may be linked to transcriptional regulatory elements and/or to other amino acid coding sequences using established methods. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences that ensure initiation of transcription, an Internal Ribosome Entry Site (IRES), and optionally regulatory elements that ensure termination of transcription and stabilization of the transcript. Non-limiting examples of such regulatory elements which ensure transcription initiation include promoters, translation initiation codons, enhancers, insulators, and/or regulatory elements which ensure transcription termination, which are included downstream of the nucleic acid molecule of the invention. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals, or signal sequences depending on the expression system used, which are capable of directing the expressed protein to a cellular compartment or culture medium. The vector may also contain additional expressible polynucleotides encoding one or more chaperones to facilitate proper protein folding.
Other examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses can be produced by techniques well known to those skilled in the art, for example, by transfection of packaging cells or by transient transfection of helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv + cells, 293 cells, and the like.
Thus, in a further aspect of the invention there is provided a host cell transfected, transduced or transformed with a nucleic acid and/or vector according to the invention.
The term "transformation" refers to the introduction of a "foreign" (i.e., extrinsic or extracellular) gene or DNA or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a substance of interest, typically a protein encoded by the gene or introduced sequence. Host cells that receive and express the introduced DNA or RNA have been "transformed".
The nucleic acid molecules and/or vectors of the invention can be designed to be introduced into cells by, for example, chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-dextran, nuclear transfection, non-chemical methods (electroporation, sonoporation, light transfection, gene electrotransfer, fluid delivery, or transformation that occurs naturally when cells are contacted with the nucleic acid molecules of the invention), particle-based methods (gene gun, magnetic transfection, transfections by puncture), phage-based vector methods, and viral methods.
The nucleic acids according to the invention can be used to produce nanobodies of the invention in a suitable expression system. The term "expression system" refers to a host cell and a vector which are compatible under appropriate conditions, e.g., for expressing a protein encoded by foreign DNA carried by the vector and introduced into the host cell.
Conventional expression systems include E.coli host cells and plasmid vectors, insect host cells and baculovirus vectors, as well as mammalian host cells and vectors thereof. Other examples of host cells include prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include, but are not limited to, E.coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells, and HEK293T cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, epithelial cells, neural cells, adipocytes, etc.).
In the present invention, any suitable host cell/vector system may be used for the expression of the DNA sequence encoding the antibody molecule of the present invention. Bacterial (e.g., E.coli) and other microbial systems may be used, or eukaryotic (e.g., mammalian) host cell expression systems may also be used. Such cells include, but are not limited to, mammalian cells, plant cells, insect cells, fungal cells, or cells of bacterial origin. As the mammalian cell, one selected from the group consisting of, but not limited to, CHO cell, F2N cell, CSO cell, BHK cell, Bowes melanoma cell, HeLa cell, 911 cell, AT1080 cell, a549 cell, HEK293 cell, and HEK293T cell can be preferably used as the host cell. Any cell known to those skilled in the art to be useful as a mammalian host cell may be used in the art.
The antibodies and fragments disclosed herein are expressed at good levels from host cells. Thus, the properties of the antibody and/or binding fragment are suitable for expression on a commercial scale.
The invention also relates to a method for producing a recombinant host cell expressing a nanobody according to the invention, comprising the steps of:
(1) Introducing a nucleic acid as described above into a competent host cell in vitro or ex vivo,
(2) recombinant host cells obtained by in vitro or ex vivo culture, and
(3) optionally selecting cells expressing and/or secreting said nanobody. Such recombinant host cells may be used to produce nanobodies according to the present invention.
Nanobodies of the present invention may be produced by any technique known to those skilled in the art, such as, for example, any chemical, biological, genetic or enzymatic technique, alone or in combination.
In particular, the present invention also relates to a method for preparing the nanobody of the present invention, which comprises the steps of:
(1) culturing the transduced or transfected or transformed cells according to the invention under conditions suitable to allow expression of said nanobodies, and
(2) recovering the expressed nanobody.
Nanobodies of the present invention may be suitably separated from the culture medium by conventional immunoglobulin purification methods, such as, for example, protein a-sepharose, hydroxylapatite chromatography, gel electrophoresis, affinity dialysis or chromatography.
The invention also relates to nanobodies conjugated to detectable labels.
The term "nanobody bound to a detectable label" means herein that the detectable label is bound directly or indirectly to the nanobody, e.g. by a cleavable or non-cleavable linker peptide, or incorporated into the nanobody. The detectable label may be bound to the nanobody, inter alia, by substitution (e.g., by substituting H with I at the tyrosine residue level), by complexing, or by chelation.
The term "detectable label" means herein a compound that produces a detectable signal. When it is attached to the tracer, it can monitor what the tracer becomes in the body. The detectable label can be an MRI contrast agent, a scintigraphic contrast agent, an X-rayLine imaging contrast agents, ultrasound contrast agents, optical imaging contrast agents. Examples of detectable labels include radioactive elements, fluorophores such as fluorescein, Alexa, or cyanine; chemiluminescent compounds, such as luminol; bioluminescent compounds, such as luciferase or alkaline phosphatase; and contrast agents such as nanoparticles or gadolinium. The selection of a suitable detectable label depends on the detection system used and is within the ability of the person skilled in the art. For example, when the detection system is an MRI, the detectable label is preferably an iron oxide nanoparticle or gadolinium; when the detection system is fluorescence imaging, the detectable label is preferably fluorescein, Alexa or cyanine; when the detection system is chemiluminescent imaging, the detectable label is preferably luminol; when the detection system is bioluminescent imaging, the detectable label is preferably luciferase or alkaline phosphatase; when the detection system is nuclear imaging, the detectable label is preferably a radioactive element, such as gallium (g) for PET imaging 68Ga), or technetium 99m (for SPECT imaging99mTc）。
In some embodiments, the detectable label is a radioactive element. More particularly, embodiments of the radioactive element for nuclear imaging techniques include technetium 99m (technetium)99mTc), iodine 123 (123I) Iodine 125 (1)125I) Fluorine 18 (f)18F) Gallium 68 (c)68Ga) and any other radioactive element that may be used on humans. Thus, preferably, the radioactive element is selected from99mTc、123I、125I、18F and68ga. Most preferably, the radioactive element is99mTc or68Ga, more preferably99mTc。
The anti-Tim-3 nanobodies described herein can also be conjugated to therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nucleophilicity inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In ADCs, the antibody and therapeutic agent are preferably conjugated via a cleavable linker (e.g., a peptidyl, disulfide, or hydrazone linker).
The invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, fragments of the nanobodies of the invention are also encompassed by the present invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
Nanobodies according to the present invention may be administered to a patient using any method of administration known to those skilled in the art. In particular, the nanobody may be administered, e.g., orally, by inhalation, or parenterally (in particular by intravenous injection). When the parenteral route is chosen, the nanobodies may be in the form of injectable solutions and suspensions, packaged in vials or bottles. The parenteral administration form is generally obtained by mixing the nanobody according to the present invention with buffers, stabilizers, preservatives, solubilizers, isotonizing agents and suspending agents. These mixtures can be sterilized and packaged in the form of intravenous injections, according to known techniques. The person skilled in the art can use, for example, phosphate-based buffers as buffers. Examples of suspending agents include methylcellulose, acacia gum and sodium carboxymethylcellulose. Examples of the stabilizer include sodium sulfite and sodium metabisulfite, and examples of the preservative include sodium p-hydroxybenzoate, sorbic acid, cresol and chlorocresol.
The amount of nanobody naturally administered depends on the route of administration, the height and/or weight of the patient, and the detection technique used.
The term "patient" means a human exhibiting symptoms associated with a Tim-3 related disease. Depending on the Tim-3 related disease, the disease includes solid or hematological malignancies, inflammatory diseases, and the like.
Non-limiting examples of cancers for treatment include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer (e.g., Renal Cell Carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer (or carcinoma), gastric cancer, germ cell tumor, pediatric sarcoma, sinus natural killer, melanoma (e.g., metastatic malignant melanoma, e.g., cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, cancer of the colon, and cervical cancer (or carcinoma), ovarian cancer of the head and neck, or head, or neck, or germ cell, tumor, pediatric sarcoma, natural killer, sinus natural killer, melanoma (e.g., metastatic malignant melanoma, e.g., malignant melanoma, skin or intraocular malignant melanoma), bone cancer, skin cancer, cancer of the anal region, cancer of the skin, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, cancer of the renal pelvis, neoplasms of the Central Nervous System (CNS), primary CNS lymphomas, tumor angiogenesis, spinal tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), virus-related cancers (such as Human Papillomavirus (HPV) related tumors) and cancers derived from two major blood cell lineages (i.e., myeloid cell lines (which produce granulospheres, erythrocytes, platelets, macrophages and mast cells) or lymphoid cell lines (which produce B, and B, and B, B, T, NK and plasma cells)), such as ALL types of leukemia, lymphomas and myelomas, such as acute, chronic, lymphocytic and/or myelogenous leukemia, such as acute leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL) and Chronic Myelogenous Leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [ M3V ]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [ M4E ]), monocytic leukemia (M5), erythroleukemia (M6), megakaryocytic leukemia (M7), solitary myelosarcoma, and chloroma; lymphomas, such as Hodgkin's Lymphoma (HL), non-hodgkin's lymphoma (NHL), B-cell hematologic malignancies (e.g., B-cell lymphoma), T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1 +) large cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, genuine histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, burkitt's lymphoma, follicular lymphoma, Diffuse Histiocytic Lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) and Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, Chronic Lymphocytic Leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratocarcinoma, central and peripheral nerve tumors, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma, hematopoietic tumors of lymphoid lineage, such as T cell and B cell tumors, including (but not limited to) T cell disorders such as T prolymphocytic leukemia (T-PLL), including small cell and brain cell types; large granular lymphocytic leukemia (LGL), preferably of the T cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/postthymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of said cancers. The methods described herein may also be used to treat metastatic cancer, unresectable cancer, refractory cancer, and/or recurrent cancer.
Diagnostic use
The invention relates to the use of said nanobodies in diagnostic or prognostic methods.
The term "diagnostic method" or "diagnosing" as used herein means a method by which it is possible to determine whether an individual has a pathological condition.
The term "prognostic method" or "prognosis" means herein a method that allows determining whether an individual is at risk of developing a pathological condition.
Preferably, the nanobody as defined above is used for the diagnosis or prediction of a Tim-3 related disease.
Therapeutic uses
The invention also relates to the application of the nano antibody in preparing medicines, in particular to medicines for treating Tim-3 related diseases. A method of treatment comprising administering to a patient in need thereof a therapeutically effective amount of a nanobody as defined above is also part of the present invention.
The term "treatment" of a Tim-3 related disease is intended to mean "therapeutic treatment" (or curative treatment) of a Tim-3 related disease, which includes slowing or inhibiting the progression of the Tim-3 related disease. It is also intended to mean "prophylactic treatment" of Tim-3 related diseases.
The term "preventing" is intended to mean preventing or delaying the onset or reduction in the intensity of a clinical or biochemical manifestation associated with a Tim-3 related disease.
The person skilled in the art knows by his general knowledge how to determine the clinical or biochemical manifestations associated with a given Tim-3 related disease and which are capable of improving (treating) or preventing, delaying or reducing the intensity (preventing). In the case of Tim-3 related diseases, the biological parameter of interest may be the presence and location of a pathological form of Tim-3.
The invention more particularly relates to nanobodies as defined above for the treatment and/or prevention of Tim-3 related diseases, preferably for the treatment and/or prevention of bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer (kidney cancer), head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumors, lymphomas, leukemias, myelomas, sarcomas, and virus-related cancers, as well as encephalitis.
The invention also relates to the use of nanobodies for the preparation of a medicament intended for the treatment of a Tim-3 related disease and/or the prevention of a Tim-3 related disease in a patient likely to develop a Tim-3 related disease.
The invention also relates to a method of treating a Tim-3 related disease and/or preventing a Tim-3 related disease in a patient in need thereof comprising administering to the patient in need thereof a therapeutically effective amount of a nanobody as defined above.
The nanobody according to the invention may be administered, for example, orally, by inhalation, parenterally (in particular by intravenous injection) in a suitable form. When a parenteral route is contemplated, the nanobody may be in the form of an injectable solute and suspension packaged in a vial or bottle. Generally, the form of parenteral administration is obtained by mixing the nanobody with buffers, stabilizers, preservatives, solubilizers, isotonic agents and suspending agents. These mixtures are subsequently sterilized and then packaged in the form of intravenous injections, according to known techniques. By means of a buffer, the person skilled in the art can use buffers based on organophosphates. Examples of suspending agents include methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, gum arabic, and sodium carboxymethyl cellulose. Furthermore, the stabilizers used according to the invention are sodium sulfite and sodium metabisulfite, while sodium p-hydroxybenzoate, sorbic acid, cresol and chlorocresol may be mentioned as preservatives.
The invention also relates to a pharmaceutical composition comprising the nanobody in combination with a pharmaceutically acceptable carrier.
The terms "pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce side effects, allergic reactions, or otherwise untoward reactions when administered to a mammal, particularly a human.
In the context of the present invention, the expression "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, antibacterial or antifungal agent, isotonic or absorption delaying agent, and the like. The use of such media and agents for pharmaceutically active substances is well known to those skilled in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated. Additional active ingredients may also be incorporated into the composition.
The invention also relates to the application of the nano antibody in-vitro detection of Tim-3 in a sample.
The term "sample" means herein a portion of a larger element. Preferably, the sample is a substance of biological origin. It comprises the cellular and/or other molecular entities to be characterized and/or identified, e.g., according to physical, biochemical, chemical and/or physiological characteristics, e.g., the phrase "disease sample" or variants thereof refers to any sample obtained from a subject of interest that is expected or known to comprise the cellular and/or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid (follicullar fluid), semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture fluid (tissue culture medium), tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.
The present invention will be described in further detail with reference to the drawings and examples.
The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
Example 1 construction of phage library
1. Immunization of alpaca
10 mL of blood was drawn before immunization as a negative serum control. Mixing the Tim-3 antigen and CFA in equal volume, and performing subcutaneous injection for the first immunization; after three weeks, the antigen and IFA are mixed with equal volume and then injected subcutaneously for the second immunization; next, mixing the antigen and IFA with equal volume uniformly every two weeks, and performing subcutaneous injection for the third and fourth immunization; peripheral blood was collected one week after each immunization.
2. Peripheral blood lymphocyte isolation
50mL of peripheral blood after three Tim-3 immunizations of alpaca were collected and PBMC were isolated according to the lymphocyte isolate protocol.
RNA extraction
Total RNA of isolated PBMCs was extracted with RNAiso Plus reagent.
4. Gel electrophoresis for detecting RNA purity
5. Reverse transcription
Using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, cat # 6210A), reverse transcription was performed to co-transcribe 5. mu.g of RNA, the detailed steps of which are described in the specification.
6. Concentration search of first round cDNA template of nested PCR
The cDNA stock solution obtained by the reverse transcription was diluted 5 times and amplified, and 0.5. mu.L, 1. mu.L, 2.5. mu.L, and 5. mu.L of the amplified product were added to the diluted product, respectively, and subjected to electrophoresis.
7. Nested first round PCR
Constructing a PCR reaction system for PCR amplification, and carrying out gel electrophoresis on PCR amplification products.
8. Nested PCR second round
And performing second round PCR amplification on the rubber tapping recovery product, and performing agarose gel electrophoresis analysis on the amplification product.
9. Ligation of the vector to the fragment of interest
Constructing a phage library by using a pComb3XSS Vector, carrying out single enzyme digestion on the pComb3XSS phagemid Vector through SfiI, carrying out overnight enzyme digestion at 50 ℃, then recovering a target fragment, wherein the connection molar ratio is Vector: VHH =1: 3. The pComb3XSS vector is provided with His tag and HA tag for convenient purification and detection. The carrier information is as follows:
Tag：6×His; HA
Promotor: LacZ
Resistance: Amp+
Vector backbone：pComb3H
Backbone size w/o insert (bp) 3300
OmpA：5＇-AAG ACA GCT ATC GCG ATT GCA G-3＇
g-back：5＇-GCC CCC TTATTA GCG TTT GCC ATC-3＇
10. electric conversion
Converting with motor, performing 10 times, adding 1mL 2YT culture medium (preheated at 37 deg.C) into the cuvette for resuscitation immediately after electric shock, sucking out shock product, washing the cuvette with 2YT culture medium, resuscitating at 37 deg.C and 180rpm for 45min, and diluting to 10-3And 10-4The number of library transformants was determined, plated on 90mm plates, centrifuged the rest, resuspended by adding 8mL of 2YT, plated on 8 200mm plates, and the number of library transformants was determined the next day.
11. Colony PCR validation insertion rate and library diversity analysis
Clones were randomly picked from the titer plates for characterization of the number of transformants in the library, and the primer pComb3XSS-F was sequenced.
12. Phage library packaging
1) The bacterial library was inoculated into 2X 300mL of 2YT + A + G (ampicillin Amp: 100. mu.g/mL, Glu:1%) medium until the initial OD600=0.1-0.2, cultured at 37 ℃ and 230rpm until OD600=0.8 or more;
2) adding a helper phage M13KO7 according to the OD600 value;
3) adding M13KO7, mixing, and standing at 37 deg.C for 30 min;
4) slowly shaking at 37 deg.C and 180rpm for 30 min;
5) centrifuging at 5000rpm for 10min, discarding supernatant, resuspending with equal volume of 2YT + A + K (Amp: 100. mu.g/ml, kanamycin Kan: 50. mu.g/ml) medium, standing at 30 deg.C overnight at 220 rpm;
6) the overnight culture was centrifuged at 10000rpm for 20min at 4 ℃ and the supernatant was collected and the precipitate was discarded. Changing the centrifugal cylinder, centrifuging at 4 deg.C and 10000rpm for 20min, and collecting supernatant;
7) adding 1/5 the volume of the supernatant into PEG8000/NaCl, mixing, and precipitating in ice bath for more than 2 hours;
8) centrifuging at 4 deg.C and 10000rpm for 20min, discarding the supernatant, and removing the supernatant by air separation. 1mL of 1 XPBS is suspended for precipitation, and 1/5 volume of PEG8000/NaCl is added for secondary precipitation for 1 h;
9) centrifuging at 12000rpm for 10min at 4 deg.C, discarding the supernatant, and removing the supernatant by air separation. Adding 1 XPBS to resuspend the precipitate according to the amount of the precipitate;
10) adding 100% glycerol to a final concentration of 50%, mixing, and packaging into 1.5mL EP tube, and storing at-80 deg.C;
11) 10 μ L of library phage were diluted with a 2YT gradient from 10-8And 10-9mu.L of the mixture was added to 90. mu.L of TG1, and the mixture was gently mixed. Standing at 37 ℃ for 15min, respectively coating Amp resistant plates, and culturing overnight;
12) the next day, clones were tested and phage library titers calculated.
EXAMPLE 2 screening of monoclonal antibodies
1. Affinity panning
1) Diluting the target molecule Tim-3 antigen with a carbonate buffer solution with the PH value of 9.6 to the final concentration of 5 mu g/mL, adding the diluted target molecule Tim-3 antigen into enzyme-labeled holes according to 100 mu L/hole, coating 8 holes for each target molecule (2 holes for second round screening coating), and coating overnight at 4 ℃;
2) discarding the coating solution, washing with PBS for 3 times, adding 300 mu L of 3% OVA-PBS blocking solution into each hole, and blocking for 1h at 37 ℃;
3) washing with PBS for 3 times, adding 100 muL phage library, and incubating for 1h at 37 ℃;
4) unbound phage were aspirated, washed 6 times with PBST and 2 times with PBS;
5) adding 100 mu L Gly-HCl eluent, incubating for 8min at 37 ℃, and eluting specifically combined phage;
6) transferring the eluent into a 1.5mL sterile centrifuge tube, and quickly neutralizing with 10 μ L Tris-HCl neutralization buffer;
7) and taking 10 mu L for gradient dilution, determining the titer, calculating the elutriation recovery rate, mixing the rest eluates, and then amplifying and purifying for the next round of affinity elutriation.
2. Construction of post-elutriation libraries
1) Elutriating the eluate to early stage of logarithmic growthE.coli2738 mixing 20mL of culture, standing at 37 deg.C for 30min, adding 1mL of 20% glucose, shaking at 220 r/min for 30min, and culturing at cell: phase = 1: 20 ratio of M13K07 phage and 4. mu.l Amp+Standing at 37 deg.C for 30min, supplementing 20ml 2YT liquid culture medium, and shake culturing at 220 r/min for 30 min;
2) the culture is subpackaged in a centrifuge tube, the temperature is 4 ℃, 5000 r/min and 10 min, the cell sediment is resuspended in 50 mL of 2 XYT-AK liquid culture medium, and the shaking culture is carried out overnight at the temperature of 30 ℃ and 250 r/min;
3) centrifuging the overnight culture at 4 deg.C and 10000 r/min for 20 min, transferring the supernatant to a new centrifuge tube, adding 1/5 volume of PEG-NaCl, mixing, and standing at 4 deg.C for more than 2 hr;
4) removing supernatant at 4 ℃, 10000 r/min, 20 min, suspending the precipitate in 1mL PBS, adding 1/5 volume of PEG/NaCl, mixing uniformly, and placing at 4 ℃ for more than 1 h;
5) and (4) removing the supernatant at 12000 r/min for 2 min, suspending the precipitate in 200 mu L PBS to obtain an amplification product, and determining the titer for the next round of panning or analysis.
3. Identification and analysis of specific phage clones
3.1 rescue of phagemids
1) Randomly selecting 288 clones from a plate with the titer of elutriation eluate from the first round of titer plate by using a sterilized toothpick, inoculating the clones into 1mL of 2 XYT-A, and carrying out shaking culture at 37 ℃ at 230 r/min for 8 h;
2) taking 200 muL of the culture, and performing cell: phase = 1: adding M13K07 bacteriophage at the ratio of 20, standing at 37 deg.C for 15 min, and performing shake culture at 220 r/min for 45 min;
3) supplementing 2 XYT-AK with the volume of 800 muL, and carrying out vigorous shaking culture at 30 ℃ for overnight;
4) the next day, centrifugation was carried out at 12000 rpm for 2min, and the supernatant was collected and used for monoclonal ELISA identification.
3.2 identification of Positive phage clones
1) Diluting a target molecule Tim-3 antigen with a carbonate buffer solution with a PH value of 9.6 to a final concentration of 2 mu g/mL, adding the diluted target molecule Tim-3 antigen into an enzyme-labeled hole according to 100 mu L/hole, and coating overnight at 4 ℃;
2) discarding the coating solution, washing for 3 times by PBST, adding 5% skimmed milk of 300 mu L into each hole, and sealing for 1 h at 37 ℃;
3) PBST washing 3 times, each hole adding 35 u L phage culture liquid supernatant and 65 u L5% skim milk, 37 degrees C, incubation for 1 h;
4) PBST is washed for 6 times, horseradish peroxidase-labeled anti-M13 antibody (diluted by PBS according to the ratio of 1: 10000) is added, 100 mu L/hole is treated for 1 h at 37 ℃;
5) PBST wash plate 6 times. Adding TMB color developing solution for color development, 100 μ L/well, 37 deg.C, 7min, adding stop solution to stop reaction, 50 μ L/well, and measuring optical density at 450 nm.
3.3 sequence analysis of Positive phage clones
The positive clones were sent to Tokyngxi Biotechnology Limited for sequencing.
4. Results
The nano antibody with higher antigen affinity activity is screened out through the steps, and the sequence of the nano antibody is obtained through sequence comparison, as shown in table 1.
TABLE 1 sequences of Nanobodies
Figure 252636DEST_PATH_IMAGE001
Example 3 functional study of Nanobodies
1. Expression and purification of Nanobodies
1) Chemically synthesizing the screened sequence, and cloning the sequence into a eukaryotic expression vector;
2) amplifying and extracting the plasmid;
3) transiently transfecting mammalian cells with a plasmid encoding the antibody;
4) collecting the supernatant, and purifying by affinity chromatography to obtain recombinant protein.
2. Identification of anti-human Tim-3 antibody by Coomassie brilliant blue
1) Sample preparation:
get 10
Figure 373039DEST_PATH_IMAGE002
g anti-Tim-3Ab, adding 5 Xloading buffer solution containing beta-mercaptoethanol, and boiling for 10 min. Carrying out SDS-PAGE electrophoresis on all the samples;
2) electrophoresis:
the samples were subjected to 15% SDS-PAGE gel electrophoresis (constant pressure: 60V,1.5 h);
3) coomassie brilliant blue staining
After electrophoresis is finished, putting the gel into Coomassie brilliant blue dye solution for overnight, putting the gel into destaining solution for destaining after the overnight, and scanning and storing the gel after complete destaining;
4) As a result, the
The results are shown in fig. 1, and the result of the Mas brilliant blue shows that the target position is provided with an anti-Tim-3 nano antibody strip with better purity.
3. ELISA (enzyme-Linked immunosorbent assay) for detecting combination of Tim-3 nano antibody and Tim-3 antigen
1) Diluting the Tim-3-Fc antigen to 2 mu g/ml by using carbonate coating solution with the pH value of 9.6, coating the solution on an enzyme-linked plate, and standing overnight at 4 ℃;
2) washing the plate: a full-automatic enzyme labeling plate washing machine and PBST washing liquor are used, the volume of each hole is 300 mu 1, and the plate is washed for 3 times;
3) and (3) sealing: blocking with 10% fetal calf serum and PBS blocking solution, adding 200 μ 1/well into enzyme-linked plate, and placing in wet box at 37 deg.C for 30 min;
4) washing the plate: the method is the same as the step 2);
5) sample adding: diluting the Tim-3 nano antibody and the control antibody from 1 mu g/ml to 0.0016 mu g/ml, adding 100 mu 1/well into an enzyme-linked plate, adding two wells in each case, and placing the enzyme-linked plate into a wet box at 37 ℃ for 1 h;
6) washing the plate: the method is the same as the step 2);
7) incubation of secondary antibody: 1: diluting anti-his-HRP secondary antibody at 2000, adding 100 mu 1/hole into an enzyme linked plate, and placing the enzyme linked plate into a wet box at 37 ℃ for 30 min;
8) washing the plate: the method is the same as the step 2);
9) color development: adding TMB, adding 100 mu 1/hole into the enzyme-linked plate, standing at room temperature, and observing whether the color in the plate turns blue;
10) and (4) terminating: 100 ul/well 1M H 2SO4Stopping, and detecting an OD450 value by using an enzyme label instrument;
11) as a result, the
As a result, as shown in FIG. 2, the anti-Tim-3 nanobody (1-3-1) can specifically bind to the Tim-3 fusion protein and exhibit concentration dependency.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that it would be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit of the invention, and these modifications and variations also fall within the scope of the claims of the present invention.
Sequence listing
<110> military medical research institute of military science institute of the people's liberation army of China
<120> anti-Tim-3 nano antibody and application thereof
<141> 2022-03-30
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gly Phe Arg Phe Asn Asp Tyr Ile
1 5
<210> 2
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ile Ser Gly Ser Gly Gly Thr Thr
1 5
<210> 3
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gly Ala Val Gly Gly Arg Trp Cys Ser Arg Glu Met Thr Tyr
1 5 10
<210> 4
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Thr Leu Arg Leu Ser Cys Thr Ala Ser
20 25
<210> 5
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser
1 5 10 15
Cys
<210> 6
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Thr Tyr Ala Asp Ser Val Lys Gly Arg Ala Thr Val Ser Arg Asp Asn
1 5 10 15
Ala Ala Asn Thr Val Tyr Leu Gln Met Phe Asn Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 7
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Trp Gly Lys Gly Thr Leu Val Asn Val Ser Ser
1 5 10
<210> 8
<211> 121
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Arg Phe Asn Asp Tyr
20 25 30
Ile Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Gly Ser Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Ala Thr Val Ser Arg Asp Asn Ala Ala Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Phe Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Gly Ala Val Gly Gly Arg Trp Cys Ser Arg Glu Met Thr Tyr Trp Gly
100 105 110
Lys Gly Thr Leu Val Asn Val Ser Ser
115 120

Claims (10)

1. A nanobody against Tim-3, wherein the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the nanobody are shown in SEQ ID NO: 1. 2 and 3.
2. The nanobody of claim 1, wherein the amino acid sequences of FR1, FR2, FR3 and FR4 of the framework region of the heavy chain variable region of the nanobody are respectively as shown in SEQ ID NO: 4. 5, 6 and 7.
3. The nanobody according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region of the nanobody is as shown in SEQ ID NO: shown in fig. 8.
4. The nanobody of claim 1 or 2, wherein the nanobody comprises all or part of an antibody heavy chain constant region.
5. A nucleic acid molecule comprising a nucleotide sequence encoding the nanobody of any one of claims 1 to 4.
6. A vector comprising the nucleic acid molecule of claim 5.
7. A host cell comprising the nucleic acid molecule of claim 5, or comprising the vector of claim 6.
8. A drug conjugate, comprising:
the nanobody of any one of claims 1 to 4, and
a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.
9. A composition comprising the nanobody of any one of claims 1 to 4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of claim 7 or the drug conjugate of claim 8.
10. Any one of the following applications:
1) use of a nanobody according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, a vector according to claim 6, a host cell according to claim 7, a drug conjugate according to claim 8 for the preparation of a product for the detection of Tim-3;
2) use of the nanobody of any one of claims 1 to 4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of claim 7, the drug conjugate of claim 8 for the preparation of a product for the diagnosis of a Tim-3 related disease.
CN202210337559.8A 2022-04-01 2022-04-01 anti-Tim-3 nano antibody and application thereof Active CN114478778B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210337559.8A CN114478778B (en) 2022-04-01 2022-04-01 anti-Tim-3 nano antibody and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210337559.8A CN114478778B (en) 2022-04-01 2022-04-01 anti-Tim-3 nano antibody and application thereof

Publications (2)

Publication Number Publication Date
CN114478778A CN114478778A (en) 2022-05-13
CN114478778B true CN114478778B (en) 2022-06-28

Family

ID=81488944

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210337559.8A Active CN114478778B (en) 2022-04-01 2022-04-01 anti-Tim-3 nano antibody and application thereof

Country Status (1)

Country Link
CN (1) CN114478778B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114632156B (en) * 2022-05-17 2022-08-16 中国人民解放军军事科学院军事医学研究院 Use of Tim-3 for the prevention, treatment or alleviation of pain
CN117264054B (en) * 2023-11-21 2024-02-06 中国人民解放军军事科学院军事医学研究院 IL-6 targeting nanobodies, compositions, methods and uses

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103936853B (en) * 2014-01-26 2016-08-17 中国人民解放军军事医学科学院基础医学研究所 A kind of detection TIM-3 test kit and using method thereof
CN109476751B (en) * 2016-05-27 2024-04-19 艾吉纳斯公司 Anti-TIM-3 antibodies and methods of use thereof
CN110144011B (en) * 2018-02-14 2020-06-23 上海洛启生物医药技术有限公司 Single domain antibodies against T lymphocyte immunoglobulin mucin3
KR102572804B1 (en) * 2020-02-25 2023-08-31 국립암센터 Method of providing information for diagnosing or predicting prognosis cancer measuring the TIM-3 expression level in CD11b+ cell
CN112062850B (en) * 2020-09-21 2021-04-20 中赛干细胞基因工程有限公司 TIM3 antibodies and their use in treating cancer

Also Published As

Publication number Publication date
CN114478778A (en) 2022-05-13

Similar Documents

Publication Publication Date Title
CN110144009B (en) CD47 single domain antibodies and uses thereof
CN107686520B (en) anti-PD-L1 nano antibody and application thereof
CN109096395B (en) Blocking type CD47 nano antibody and application thereof
CN114478778B (en) anti-Tim-3 nano antibody and application thereof
US11912770B2 (en) Blocking type PD-L1 single-domain camel antibody and application thereof
CN107216389B (en) anti-PD-L1 nano antibody and coding sequence and application thereof
US20220259306A1 (en) Nkg2a antibody, preparation method therefor and application thereof
JP7266117B2 (en) Antibodies or Chimeric Antigen Receptors Targeting Claudin 18.2
CN111349161B (en) Monoclonal antibody of anti-CD 19 antibody and application thereof
JP2020195398A (en) ANTIBODY AGAINST EGFRvIII (EPIDERMAL GROWTH FACTOR RECEPTOR VARIANT III) AND USE THEREOF
CN112724255A (en) Small molecule antibodies targeting carcinoembryonic antigens
CN111378040B (en) Antibody for detecting multiple malignant tumor cells and application thereof
CN114685664B (en) Single-domain antibody for resisting human B lymphocyte surface antigen CD20 and application thereof
CN107987168B (en) Single-chain double-specific antibody for resisting VEGF and EGFR and application thereof
CN113880948B (en) anti-CA 724 antibody or antigen-binding fragment thereof, and preparation method and application thereof
CN111234019B (en) anti-CTLA-4 nano antibody, pharmaceutical composition and application thereof
CN117242092A (en) VEGFA binding molecules
CN111153997B (en) anti-CTLA-4 nano antibody and application thereof in tumor treatment
CN111378039B (en) Antibody for treating malignant tumor and application thereof
CN115850511A (en) Multi-target fusion protein for resisting tumor invasion and metastasis as well as preparation method and application thereof
CN113637074A (en) NKp46 antibody, and preparation method and application thereof
CN117700557B (en) Antibody or antigen binding fragment specifically binding to folate receptor alpha
CN111153998B (en) anti-CTLA-4 nano antibody and application thereof
CN116003582B (en) Antibody for detecting coronavirus and application thereof
WO2022151958A1 (en) Human cd276-targeting monoclonal antibody and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant