CN115160437A - Antibody or antigen binding fragment thereof and application thereof - Google Patents

Antibody or antigen binding fragment thereof and application thereof Download PDF

Info

Publication number
CN115160437A
CN115160437A CN202210832387.1A CN202210832387A CN115160437A CN 115160437 A CN115160437 A CN 115160437A CN 202210832387 A CN202210832387 A CN 202210832387A CN 115160437 A CN115160437 A CN 115160437A
Authority
CN
China
Prior art keywords
antibody
seq
amino acid
single domain
acid sequence
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.)
Pending
Application number
CN202210832387.1A
Other languages
Chinese (zh)
Inventor
王征
徐云霞
郑雷雷
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.)
Suzhou Shengji Pharmaceutical Co ltd
Original Assignee
Suzhou Shengji Pharmaceutical Co ltd
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 Suzhou Shengji Pharmaceutical Co ltd filed Critical Suzhou Shengji Pharmaceutical Co ltd
Priority to CN202210832387.1A priority Critical patent/CN115160437A/en
Publication of CN115160437A publication Critical patent/CN115160437A/en
Pending legal-status Critical Current

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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001144—Hormones, e.g. calcitonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/6847—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 hormone or a hormone-releasing or -inhibiting factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • C07K2317/56—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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—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
    • G01N2333/575—Hormones
    • G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]

Landscapes

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

Abstract

The invention discloses an antibody or an antigen binding fragment thereof and application thereof. In particular, an antibody or antigen binding fragment thereof, comprising a heavy chain variable region (VHH) comprising the following Complementarity Determining Regions (CDRs) numbered according to Kabat, or mutations thereof: CDR1 with an amino acid sequence shown as SEQ ID NO. 9; CDR2 with amino acid sequence shown in SEQ ID NO. 13; and CDR3 having an amino acid sequence shown in SEQ ID NO. 11 or 14; wherein the mutation is an insertion, deletion or substitution of 5, 4, 3, 2 or 1 amino acid on the basis of the amino acid sequences of CDR1, CDR2 and CDR3 of the VHH, respectively. The antibody prepared by the invention has high affinity binding to the antigen containing the human alpha chain shown as SEQ ID NO. 1, and has novel sequence.

Description

Antibody or antigen binding fragment thereof and application thereof
Technical Field
The invention relates to the technical field of nano antibodies, in particular to an antibody or an antigen binding fragment thereof, and a preparation method and application of the antibody or the antigen binding fragment thereof.
Background
The anterior pituitary hormones include Human Chorionic Gonadotropin (HCG), follicle Stimulating Hormone (FSH), follicle stimulating hormone CTP fusion protein (FSH-CTP), human luteinizing hormone (hLH), hormone secreted by anterior pituitary (TSH), which are all combined by alpha and beta units through ionic bonds and hydrophobic bonds, wherein the alpha subunit is a subunit shared by the anterior pituitary hormones, and the beta subunits are different and can be distinguished in immune activity.
HCG is a glycoprotein produced by the syncytiotrophoblast of placental chorion. The detection of HCG level in serum can provide clinical basis for diagnosis, differential diagnosis and prognosis judgment of HCG-related diseases such as early pregnancy, ectopic pregnancy, hydatidiform mole, incomplete abortion, spermatogonial testicular cancer and the like.
FSH is a gonadotropin released from the anterior pituitary and is important for mature follicular development in women and sperm development in men. The determination of serum follicle stimulating hormone has important significance for understanding the endocrine function of pituitary, indirectly understanding the functional states of hypothalamus and ovary, predicting ovulation time and diagnosing and treating infertility and endocrine diseases. The FSH-CTP refers to a CTP fusion protein of FSH, which acts to extend the in vivo half-life of FSH.
hLH is a gonadotropic hormone secreted by the adenohypophysis and it acts primarily on the gonads, leading to follicle maturation, and further secretion of androgens, ovulation, and formation and maintenance of the corpus luteum. Can be clinically used for identifying the cause of amenorrhea, monitoring the ovulatory period and the like. The monitoring of the ovulation period is helpful for diagnosing infertility and researching the action mechanism of contraceptive drugs.
TSH is one of the hormones secreted by the anterior pituitary, and its main function is to control and regulate the activity of the thyroid gland. The measurement of thyroid stimulating hormone in serum (plasma) is one of the important indicators for diagnosing and treating hyperthyroidism and hypothyroidism and for studying the hypothalamic-pituitary-thyroid axis. Is an indispensable tool in diagnosing thyroid hypofunction, and differentially diagnosing primary and secondary (hypothalamic or pituitary) hypothyroidism and the like. TSH can be used as index for determining therapeutic effect in treating hyperthyroidism and hypothyroidism. In addition, it can be used to observe the storage function of pituitary TSH, and further distinguish between hypothalamic and pituitary lesions. TSH testing is a preliminary screening test to ascertain thyroid function. Small changes in free thyroid concentration will result in significant adjustment of TSH concentration in the opposite direction.
The hormone content in blood is low, and a sensitive, efficient and highly stable detection antibody is needed to effectively detect or monitor the hormone content in blood. The related antibodies of the prior art have the defects of low binding activity and the like, and no antibody capable of efficiently binding a common alpha chain such as HCG, FSH, hLH, TSH, FSH-CTP and the like exists.
An antibody naturally lacking light chains, i.e., heavy chain antibody (hcAb), is present in alpaca serum. Whereas single domain heavy chain antibodies (sdabs) refer to genetically engineered antibodies consisting of only heavy chain antibody Variable regions (Variable regions), also known as VHH antibodies (Variable domains of heavywain-chain antibodies, VHH antibodies) or nanobodies (Nb) or single domain antibodies. Compared with the traditional antibody, the single domain antibody has the advantages of small molecular weight, high stability, good water solubility and the like.
Disclosure of Invention
The invention provides an antibody or an antigen binding fragment thereof, a preparation method thereof, and application thereof in detecting the heterodimeric protein or preparing a medicament, in order to overcome the defects of poor binding activity of the antibody and the heterodimeric protein comprising an alpha chain (SEQ ID NO: 1) such as FSH, HCG, TSH, hLH, FSH-CTP and the like in the prior art. The antibody has good binding activity and high sensitivity to protein molecules (such as FSH, HCG, TSH, hLH, FSH-CTP and the like) containing the sequence shown as SEQ ID NO. 1, and the method is convenient and simple to operate when detecting the protein molecules (such as FSH, HCG, TSH, hLH, FSH-CTP and the like) containing the sequence shown as SEQ ID NO. 1.
The invention provides in a first aspect an antibody comprising a heavy chain variable region (VHH), wherein the heavy chain variable region comprises the following Complementarity Determining Regions (CDRs) numbered according to Kabat, or mutations thereof: CDR1 with amino acid sequence shown in SEQ ID NO. 9; CDR2 with an amino acid sequence shown as SEQ ID NO. 13; and, CDR3 having an amino acid sequence as set forth in SEQ ID NO. 11 or 14; wherein the mutation is an insertion, deletion or substitution of 5, 4, 3, 2 or 1 amino acid on the basis of the amino acid sequences of CDR1, CDR2 and CDR3 of the VHH, respectively.
Preferably, the CDR1 mutation is an amino acid substitution of R2N/G, T3L, F4V/A, S5D, S6V/R/G/N, Y7D/H, and/or A8N/D in the amino acid sequence shown in SEQ ID NO. 9, and the amino acid sequence is preferably shown in any one of SEQ ID NO. 12, 15, 18, 21, 22, 25; the mutation of the CDR2 is an amino acid substitution with M2T/S/N, W3Q, D6G and/or S7N on the amino acid sequence shown as SEQ ID NO. 13, and the amino acid sequence is preferably shown as any one of SEQ ID NO. 10, 16, 19, 23 and 26; the mutation of CDR3 is an amino acid substitution with L4I/T or deletion, Q5D, E6G/V and/or E7Q/S/P on the amino acid sequence shown as SEQ ID NO. 14, or an amino acid substitution with G9D and/or Y14F on the amino acid sequence shown as SEQ ID NO. 11, and the amino acid sequence is preferably shown as any one of SEQ ID NO. 17, 20, 24 and 27.
In a preferred embodiment, the heavy chain variable region comprises the sequence: a CDR1 amino acid sequence shown as SEQ ID NO. 9; a CDR2 amino acid sequence shown as SEQ ID NO. 10; and, a CDR3 amino acid sequence set forth in SEQ ID NO: 11.
In a preferred embodiment, the heavy chain variable region comprises the sequence: a CDR1 amino acid sequence as set forth in SEQ ID NO. 12; a CDR2 amino acid sequence as set forth in SEQ ID NO. 13; and, a CDR3 amino acid sequence as set forth in SEQ ID NO. 14.
In a preferred embodiment, the heavy chain variable region comprises the sequence: a CDR1 amino acid sequence shown as SEQ ID NO. 15; a CDR2 amino acid sequence shown as SEQ ID NO. 16; and, a CDR3 amino acid sequence set forth in SEQ ID NO: 17.
In a preferred embodiment, the heavy chain variable region comprises the sequence: 18, as shown in SEQ ID NO; a CDR2 amino acid sequence shown as SEQ ID NO. 19; and, a CDR3 amino acid sequence as set forth in SEQ ID NO: 20.
In a preferred embodiment, the heavy chain variable region comprises the sequence: a CDR1 amino acid sequence shown as SEQ ID NO. 21; a CDR2 amino acid sequence shown as SEQ ID NO. 13; and, a CDR3 amino acid sequence as set forth in SEQ ID NO. 14.
In a preferred embodiment, the heavy chain variable region comprises the sequence: a CDR1 amino acid sequence shown as SEQ ID NO. 22; a CDR2 amino acid sequence shown as SEQ ID NO. 23; and, a CDR3 amino acid sequence set forth in SEQ ID NO: 24.
In a preferred embodiment, the heavy chain variable region comprises the sequence: a CDR1 amino acid sequence shown as SEQ ID NO. 25; a CDR2 amino acid sequence as set forth in SEQ ID NO. 26; and, the CDR3 amino acid sequence shown as SEQ ID NO. 27.
See table 1 for details.
Table 1: CDR sequences corresponding to different antibodies (according to the Kabat definition rules)
Figure BDA0003746005440000041
It is well known to those skilled in the art that CDRs of an antibody can be defined in the art by a variety of methods, such as Kabat definition rules based on sequence variability (see Kabat et al, immunological protein sequences, fifth edition, national institute of health, besiesda, maryland (1991)), and Chothia definition rules based on the position of the structural loop region (see J Mol Biol 273 927-48, 1997), among others. In the present application, the amino acid sequences of the CDRs listed above are shown according to the Kabat definition rules, but it will be understood by those skilled in the art that the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are understood to encompass complementarity determining regions as defined by any one of the CDR definition rules known to those skilled in the art, unless otherwise specified. Although the scope of the present invention is claimed to be the sequence shown based on the Kabat definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of the present invention.
The heavy chain variable region preferably further comprises a framework region (FWR) of an alpaca antibody or a human antibody.
Preferably, the antibody or antigen-binding fragment thereof is a VHH, a heavy chain antibody, a bispecific antibody or a multispecific antibody, preferably a VHH.
More preferably, the VHH comprises an amino acid sequence as set forth in any one of SEQ ID NO 2-8.
In a preferred embodiment, the VHH is an amino acid sequence as shown in any one of SEQ ID NOs 2-8.
In a preferred embodiment, the antibody or antigen-binding fragment thereof targets a protein comprising the amino acid sequence shown in SEQ ID NO. 1, preferably the protein comprising the amino acid sequence shown in SEQ ID NO. 1 is selected from one or more of FSH, HCG, TSH, FSH-CTP and hLH.
In a second aspect, the invention provides an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to the first aspect of the invention.
The preparation method of the nucleic acid is a preparation method which is conventional in the field, and preferably comprises the following steps: obtaining the nucleic acid molecule coding the antibody by a gene cloning technology, or obtaining the nucleic acid molecule coding the antibody by an artificial complete sequence synthesis method.
Those skilled in the art know that the base sequence encoding the amino acid sequence of the above antibody may be appropriately substituted with substitutions, deletions, alterations, insertions or additions to provide a polynucleotide homologue. The polynucleotide homologue of the present invention may be produced by substituting, deleting or adding one or more bases of a gene encoding the antibody sequence within a range in which the activity of the antibody is maintained.
In a third aspect, the invention provides a recombinant expression vector comprising an isolated nucleic acid as described in the second aspect of the invention.
The recombinant expression vector can be obtained by methods conventional in the art, namely: the nucleic acid molecules described herein are constructed by ligating them into various expression vectors. The expression vector is any vector conventionally used in the art so long as it can carry the aforementioned nucleic acid molecule. Preferably, the recombinant expression vector is a plasmid, cosmid, phage, or viral vector, preferably a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
In a fourth aspect, the present invention provides a transformant comprising the recombinant expression vector according to the third aspect of the present invention in a host cell.
The preparation method of the transformant may be a preparation method conventional in the art, for example: transforming the recombinant expression vector into a host cell. The host cell of the transformant is a variety of host cells which are conventional in the art, as long as the recombinant expression vector is stably self-replicating and the nucleic acid carried by the recombinant expression vector can be efficiently expressed. The recombinant expression plasmid is transformed into a host cell to obtain a recombinant expression transformant preferred in the present invention. Wherein the transformation method is a transformation method conventional in the art, preferably a chemical transformation method, a thermal shock method or an electric transformation method.
Preferably, the host cell is a yeast such as saccharomyces cerevisiae, a mold, a bacterium, or a cellular expression system.
In a fifth aspect, the invention provides a chimeric antigen receptor comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention.
In a sixth aspect, the invention provides a genetically modified cell comprising a chimeric antigen receptor according to the fifth aspect of the invention.
Preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably immune cells such as T cells, or NK cells.
In a seventh aspect, the present invention provides a method for producing an antibody or antigen-binding fragment thereof according to the first aspect, comprising the steps of: culturing the transformant according to the fourth aspect of the present invention, and obtaining the antibody or the antigen-binding fragment thereof from the culture.
In an eighth aspect, the present invention provides an antibody drug conjugate comprising an antibody moiety comprising an antibody according to the first aspect of the present invention or an antigen-binding fragment thereof, and a conjugate moiety.
Preferably, the conjugate moiety comprises a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or combination thereof, and the antibody moiety and conjugate moiety are conjugated via a chemical bond or linker.
In a ninth aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a chimeric antigen receptor according to the fifth aspect of the invention, a genetically modified cell according to the sixth aspect of the invention and/or an antibody drug conjugate according to the eighth aspect of the invention.
Preferably:
the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; and/or the pharmaceutical composition is in a liquid dosage form, a gas dosage form, a solid dosage form and a semisolid dosage form, and/or the pharmaceutical composition can be administered through oral administration, injection administration, nasal administration, transdermal administration or mucosa administration.
The tenth aspect of the present invention provides a kit comprising kit a and kit B, wherein:
the kit a comprises an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a chimeric antigen receptor according to the fifth aspect of the invention, a genetically modified cell according to the sixth aspect of the invention, an antibody drug conjugate according to the eighth aspect of the invention and/or a pharmaceutical composition according to the ninth aspect of the invention;
the kit B contains one or more selected from the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, a radiotherapeutic agent, an immunosuppressive agent, an oncolytic agent, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.
An eleventh aspect of the invention provides a kit comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention, a chimeric antigen receptor according to the fifth aspect, a genetically modified cell according to the sixth aspect, an antibody drug conjugate according to the eighth aspect and/or a pharmaceutical composition according to the ninth aspect.
Preferably, the kit further comprises (i) a means of administering the antibody or antigen-binding fragment thereof or chimeric antigen receptor or genetically modified cell or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
In a twelfth aspect, the present invention provides a use of an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, a chimeric antigen receptor according to the fifth aspect of the present invention, a genetically modified cell according to the sixth aspect of the present invention, an antibody drug conjugate according to the eighth aspect of the present invention, a pharmaceutical composition according to the ninth aspect of the present invention, a kit according to the tenth aspect of the present invention and/or a kit according to the eleventh aspect of the present invention for the manufacture of a medicament for the treatment and/or prevention of a protein-mediated disease or condition comprising an amino acid sequence as set forth in SEQ ID No. 1.
Preferably, the protein containing the amino acid sequence shown as SEQ ID NO. 1 is selected from one or more of FSH, HCG, TSH, FSH-CTP and hLH.
In a thirteenth aspect, the invention provides the use of an antibody according to the first aspect of the invention, or an antigen-binding fragment thereof, for the detection of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1.
Preferably, the protein is selected from one or more of FSH, HCG, TSH, FSH-CTP and hLH.
In a fourteenth aspect, the present invention provides a method for detecting a protein comprising an amino acid sequence as set forth in SEQ ID NO. 1, comprising the step of detecting using an antibody or an antigen-binding fragment thereof according to the first aspect of the present invention, a chimeric antigen receptor according to the fifth aspect of the present invention, a genetically modified cell according to the sixth aspect of the present invention, an antibody drug conjugate according to the eighth aspect of the present invention, a pharmaceutical composition according to the ninth aspect of the present invention, a kit of parts according to the tenth aspect of the present invention, and/or a kit according to the eleventh aspect of the present invention.
In the present application, the term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region (VH), wherein the VH unit has polyepitopic specificity; an antibody having two or more VH regions, each VH unit binding to a different target or to a different epitope of the same target; an antibody having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently, and the like.
In this application, the term "heavy chain antibody" refers to an antibody comprising only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as HCAbs.
In the present application, the term "VHH (single domain antibody)", also called "nanobody", refers to a VHH structure cloned from a heavy chain antibody, which is the smallest unit known to bind to a target antigen.
The positive progress effects of the invention are as follows:
the antibody of the invention has good binding activity and high sensitivity to protein molecules (such as FSH, HCG, TSH, hLH, FSH-CTP and the like) containing the sequence shown as SEQ ID NO. 1. The antibody is convenient and fast in method and simple in operation when detecting protein molecules such as FSH, HCG, TSH, hLH, FSH-CTP and the like containing sequences shown in SEQ ID NO. 1.
Drawings
FIG. 1 is a diagram of a cycle of amplification electrophoresis after reverse transcription of VHH extracted RNA, where M represents Marker and lanes 1-5 represent 5 groups of RNA extractions, respectively.
FIG. 2 is a diagram of two rounds of amplification electrophoresis after reverse transcription of VHH extracted RNA, wherein M represents Marker, and lanes 1-2 represent the second round of PCR amplification electrophoresis after gel cutting recovery in lanes 1 and 2, respectively, of FIG. 1.
FIG. 3 is a histogram of VHH extracted RNA library volume titer.
FIG. 4 shows the colony PCR identification of the insertion rate of the target gene in the library.
FIG. 5 shows the results for VHH expression of different binding alpha subunits, lanes 1-7 corresponding to proteins 2-8, respectively.
Detailed Description
The invention immunizes Bactrian camels with HCG and then uses the camel peripheral blood lymphocytes to establish a VHH phage library directed against HCG heavy chain antibodies. In the subsequent test, HCG and hLH are coated on an enzyme label plate respectively, immune nano antibody phage library is screened by using phage display technology, so that specific nano antibody genes aiming at HCG and hLH shared alpha chain are obtained, the genes are transferred into escherichia coli, so that a nano antibody strain capable of being efficiently expressed in the escherichia coli is established, and gene sequence identification is carried out.
Example 1: construction of immune animal and phage library
1. Immunising an animal
Taking healthy adult alpaca, mixing HCG with an adjuvant, immunizing by adopting a subcutaneous injection mode, wherein the immunization program is shown in table 2, and collecting alpaca peripheral blood for constructing a phage display library seven days after the third boosting immunization.
TABLE 2 immunization procedure
Primary immunization Boosting of immunity 1 Boosting of immunity 2 Boosting of immunity 3
Time of immunization Day 0 Day 21 Day 42 Day 63
Immunization dose (mg) 0.5 0.25 0.25 0.25
Adjuvant Freund's incomplete adjuvant Freund's incomplete adjuvant Freund's incomplete adjuvant Freund's incomplete adjuvant
Immunization regimen Under the skin Under the skin Under the skin Under the skin
2. Alpaca lymphocyte isolation:
adding 6mL of lymphocyte separation solution into a 15mL centrifuge tube, adding an isometric whole blood sample, and centrifuging at the normal temperature of 800g for 20min; carefully sucking the white blood cells suspended in the middle layer into a new centrifugal tube, adding 2 times of PBS (phosphate buffer solution) in volume, and centrifuging at normal temperature for 15min at 800 g; carefully discarding the supernatant, adding erythrocyte lysate, and lysing erythrocytes; centrifuging at normal temperature for 15min at 450g, removing supernatant and counting according to 10 7 The lymphocytes were lysed well by adding 2mL Trizol and were ready for use.
3. Total RNA extraction
Adding 1/5 volume of chloroform into the lysate, violently shaking for 20s for full emulsification, and standing on ice for 10min; centrifuging at 4 deg.C and 12000g for 10min, and transferring the supernatant to another fresh centrifuge tube; adding isopropanol with the same volume, mixing well, and standing on ice for 10min; centrifuging at 4 deg.C and 12000g for 10min, removing supernatant, adding 75% ethanol, and mixing; centrifuging at 4 deg.C and 12000g for 10min, and removing supernatant; drying at room temperature for 5min, adding appropriate amount of RNase-free water to dissolve precipitate, and storing at-80 deg.C after RNA precipitate is completely dissolved.
4. Antibody gene amplification
The nested first round PCR system is shown in Table 3 below:
TABLE 3
Figure BDA0003746005440000101
Reaction procedure: 94 ℃ for 5min; 30 cycles of 98 deg.C, 10s,50 deg.C, 15s,72 deg.C, 1 min; after the reaction is finished, gel electrophoresis is carried out, and the target fragment of about 700bp is recovered by tapping. Wherein the Alpvh-LD sequence (5 '-3') is as follows: CTTGGTGGTCCTGGCTGC (SEQ ID NO: 28); CH (CH) 2 -the R sequence (5 '-3') is as follows: GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 29). The results are shown in FIG. 1.
Nested second round PCR is shown in Table 4 below: 94 ℃ for 5min; 30 cycles of 98 ℃ and 10s,57 ℃ and 15s, and 72 ℃ and 45 s.
TABLE 4
Figure BDA0003746005440000102
Figure BDA0003746005440000111
Wherein the sequence of AlpVh-F1 (5 '-3') (SEQ ID NO: 30) is as follows:
CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC；
the sequence of AlpVHH-R1 (5 '-3') (SEQ ID NO: 31) is as follows:
CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG；
the sequence of AlpVHH-R2 (5 '-3') (SEQ ID NO: 32) is as follows:
CATGCCATGACTCGCGGCCGGCCTGGCCGTCTTGTGGTTTTGGTGTCTTGGG。
after the second reaction, gel electrophoresis (as shown in FIG. 2), tapping to recover the target fragment, and performing double enzyme digestion.
5. Library construction
5.1 cleavage of vector and target fragment
The target fragment double cleavage system (160. Mu.L system) is shown in Table 5:
TABLE 5
Figure BDA0003746005440000112
The vector double enzyme system (160. Mu.L) is shown in Table 6:
TABLE 6
Figure BDA0003746005440000113
5.2 ligation System of vector and fragment of interest is shown in Table 7:
TABLE 7
Figure BDA0003746005440000114
Figure BDA0003746005440000121
Ligation was performed overnight at 16 ℃ and 5. Mu.L (1/10 amount) of 3M CH was added 3 COONa (pH 5.2) and 125. Mu.L (2.5 times) of cold absolute ethanol, standing at-20 deg.C for 30-60min, centrifuging at 12000g to recover precipitate, washing the precipitate with 70% cold ethanol, drying at room temperature, and dissolving in 15. Mu.L of deionized water.
6. Electric conversion
Performing electric shock transformation for 10 times, adding 1mL 2YT culture medium (preheated at 37 deg.C) into electric shock cup for resuscitation immediately after electric shock, sucking out electric shock product, washing electric shock cup with 2YT culture medium to obtain total 100ml resuscitation product, resuscitating at 37 deg.C and 180rpm for 45min, and diluting 100 μ L gradient to 10% -3 And 10 -4 The number of library transformants was determined, plated on 90mm plates, centrifuged the remainder, resuspended by adding 8mL of 2YT, and plated on 8 200mm plates. The following day F mix primer library 10 on plates for determining the number of pool transformants -4 There were 132 clones with a stock size of 1.32X 10 9 (132×1000×10 4 ) (ii) a Fnew primer library 10 -4 A total of 110 clones, 1.1X 10 9 (110×1000×10 4 ) As shown in fig. 3.
7. Colony PCR verification of insertion rate
48 clones were randomly picked from the titer plate for identification, and the results indicated that the insertion rates were all 100%, as shown in FIG. 4, where the target gene band was 700bp, and the marker bands were 5000, 3000, 2000, 1500, 1000, 750, 500, 250, 100bp at one time.
Example 2: antibody screening
1. Affinity panning
1) The HCG antigen
Figure BDA0003746005440000122
Diluting with carbonate buffer solution with pH of 9.6 to final concentration of 5 μ g/mL,adding 100 mu L/hole into enzyme-labeled hole, coating 8 holes (second round screening coating 4 holes) on each target molecule, and coating overnight at 4 ℃;
2) Discard the coating solution, wash 3 times with PBS, add 300 μ L of 3% BSA-PBS blocking solution per well, block for 1h at 37 ℃;
3) Washing with PBS for 3 times, adding 100 μ L phage library, and incubating at 37 deg.C for 1h;
5) Unbound phage were aspirated, washed 6 times with PBST and 2 times with PBS;
6) Adding 100 mu L Gly-HCl eluent, incubating at 37 deg.C for 8min, and eluting specifically bound phage; transferring the eluent into a 1.5mL sterile centrifuge tube, and quickly neutralizing with 10 mu L Tris-HCl neutralization buffer solution;
7) 10 μ L of the eluate was subjected to gradient dilution, titer was measured, and the recovery rate of panning was calculated, and the remaining eluates were mixed and amplified and purified for the next round of affinity panning, and the panning conditions were changed as shown in Table 8.
TABLE 8 affinity panning conditions
Figure BDA0003746005440000131
TABLE 9 acid wash two-round screen recovery with HCG as target protein
Figure BDA0003746005440000132
Recovery = recovery/library input; enrichment = last round yield/last round yield.
2. Amplification of post-panning libraries
1) Mixing elutriation eluate with 5mL of E.coli 2738 culture (New England Biolabs) in early logarithmic growth stage, standing at 37 deg.C for 15min, and shaking and culturing at 220r/min for 45min; centrifuging at 1000g for 15min, removing supernatant, resuspending with 500. Mu.l 2 XYT and spreading on 200mm 2 XYT-GA plate;
2) Scraping with 10ml 2 XYT liquid culture medium, adding 500 μ l suspension into 50ml 2 XYT liquid culture medium, and shaking at 37 deg.C for 30min; adding M13K07 helper phage (Addgene, cat # 119819) according to the proportion of cell: phase =1, standing at 37 ℃ for 30min, and shaking and culturing at 220r/min for 30min; subpackaging the culture in a centrifuge tube, centrifuging at 25 deg.C and 5000r/min for 10min, resuspending the cell precipitate in 50mL 2 XYT-AK liquid culture medium, and shake-culturing at 30 deg.C and 230r/min overnight;
3) Centrifuging overnight culture at 4 deg.C at 10000r/min for 20min, transferring supernatant to new centrifuge tube, adding 1/5 volume of PEG-NaCl, mixing, and culturing at 4 deg.C for more than 2 hr;
4) Centrifuging at 4 deg.C at 10000r/min for 20min, removing supernatant, resuspending the precipitate in 1mL PBS, adding 1/5 volume of PEG/NaCl, mixing, and culturing at 4 deg.C for more than 1h;
5) Centrifuging at 4 deg.C and 12000r/min for 2min, removing supernatant, suspending the precipitate in 200 μ L PBS to obtain amplification product, and determining titer for next round of panning or analysis.
3. Identification and analysis of specific phage clones
3.1 identification of phagemids
1) From the second round of panning eluate titer plate, using a sterile toothpick randomly pick 96 single clones to 1mL 2 XYT-A, 37 degrees C, 220r/min shake culture 8h.
2) Taking 200. Mu.L of the culture, and carrying out cell: phase =1:20, adding M13K07 bacteriophage, standing at 37 ℃ for 15min, and performing shaking culture at 220r/min for 45min.
3) Supplemented with 2 XYT-AK of 800. Mu.L volume, and cultured overnight at 30 ℃ with vigorous shaking.
4) The next day, centrifugation was carried out at 12000rpm for 2min, and the supernatant was collected and used for monoclonal ELISA identification.
3.2 identification of Positive phage clones
1) The HCG is mixed
Figure BDA0003746005440000141
Or a hLH antigen
Figure BDA0003746005440000142
Diluting with carbonate buffer solution with pH of 9.6 to final concentration of 2 μ g/mL, adding into enzyme-labeled well at a ratio of 100 μ L/well, and packaging at 4 deg.CIs left overnight;
2) Discarding the coating solution, washing with PBST for 3 times, adding 200 μ L of 5% skimmed milk into each well, and sealing at 37 deg.C for 1h;
3) PBST washing 3 times, each hole is added with 50 μ L phage culture liquid supernatant and 50 μ L5% skimmed milk, incubated for 1h at 37 ℃;
4) PBST was washed 6 times, and horseradish peroxidase-labeled anti-M13 antibody (Abcam, cat #: ab 235228), 1h at 37 ℃;
5) PBST wash plate 6 times. Adding TMB color development solution for color development, culturing at 37 deg.C for 7min, adding stop solution at 50 μ L/well to stop reaction, and measuring OD value at 450 nm.
Finally, 7 sequences with higher affinity were obtained, including sequence Nos. 1, 13, 24, 25, 48, 62 and 70 (Table 10). The 7 positive clones were sequenced, and the amino acid sequences corresponding to the obtained sequences are specifically shown in table 10 below.
TABLE 10HCG and hLH antigen phase ELISA identification and corresponding sequences
Figure BDA0003746005440000151
Example 3: antibody expression and binding identification
1. Sequence Synthesis and expression
The resulting sequence was expressed and screened to detect its affinity for the alpha subunit-containing antigen, and the VHH sequence 1G9E [ PMID:24739391 ] was selected as a positive control. The codon optimization of Escherichia coli preference is entrusted to Nanjing King Shirui Biotechnology GmbH, N-terminal methionine and C-terminal 6 × histidine tag (VHH-sgs-HHHHHHHH) are added, polynucleotide sequences for coding SEQ ID NO 2-8 and 1G9E are synthesized and inserted into pET32a expression vector, and the recombinant plasmid for polypeptide expression is obtained after correct sequencing. The prepared recombinant plasmid was electrically transformed into E.coli BL21 Star (DE 3) and inoculated onto LB agarose plates containing 100. Mu.g/ml ampicillin. After culturing overnight at 37 ℃ until colonies grew out, individual colonies were picked up and inoculated into 3ml of LB medium containing 100. Mu.g/ml ampicillin and cultured overnight at 37 ℃ at 250 rpm. The overnight culture was inoculated into 50ml of LB medium containing 100. Mu.g/ml ampicillin, cultured at 37 ℃ until OD600 reached 0.4 to 0.6, and 0.1mM IPTG was added and the culture was continued overnight. And finally, centrifugally collecting the bacterial precipitates of the culture, carrying out ultrasonic cell breaking treatment after the bacterial precipitates are resuspended to 100g/L by PBS, and centrifugally collecting supernatant after cell breaking. The supernatant was purified by nickel column. The purified protein was pipetted into PBS (pH 7.0) using ultrafiltration centrifuge tubes. The purity of the obtained protein was evaluated by SDS-PAGE, and as shown in FIG. 5, it was found that the purity was 97% or more for each of the 8 sequences.
2. Purified protein binding Activity evaluation
The enzyme plate was washed with 2. Mu.g/ml HCG
Figure BDA0003746005440000161
Coating at 2-8 deg.c for over night; then, the plate was washed 3 times with PBST (0.05% Tween 20 in PBS, pH 7.4), and the washed plate was blocked with 300. Mu.l of a blocking solution (1% BSA in PBST) at 37 ℃ for 2 hours, followed by washing with PBST.
Sample dilution: samples were diluted to 200ng/ml starting, 2-fold for 10 spots, and loaded into 96-well plates at 100. Mu.l/well duplicate wells.
The ELISA plate was incubated at 37 ℃ for 1H with shaking, washed with PBST, 40ng/ml of Anti-6 XHis tag antibody (HRP) (Abcam, cat # AB 1187) was added to the plate, incubated at 37 ℃ for 1H with shaking, washed with PBST, added with TMB (tetramethyllbenzidine) as a chromogenic substrate for HRP, incubated for 15min, and incubated with 2N H 2 SO 4 And (6) terminating. Absorbance at 450nm was measured with a microplate reader. EC50 values were calculated using a 4 parameter equation.
TABLE 11 EC50 values measured by ELISA
SEQ ID NO: EC50(ng/ml) SEQ ID NO: EC50(ng/ml)
2 10.28 6 13.75
3 25.90 7 14.01
4 5.05 8 23.46
5 9.46 1G9E N/A*
* Note: the ELISA detection of the 1G9E sample does not form a complete 4-parameter curve, the OD value of a high-concentration point is far lower than that of other VHHs, saturation is not achieved, and no EC50 data exist.
As shown in Table 11, VHHs of SEQ ID Nos. 2-8 showed different binding activities for FSH and HCG, which were much greater than that of 1G9E.

Claims (29)

1. A single domain antibody targeting a protein comprising an amino acid sequence as set forth in SEQ ID NO:1, said single domain antibody comprising a heavy chain variable region, characterized in that said heavy chain variable region comprises the following Complementarity Determining Regions (CDRs) numbered according to Kabat:
a CDR1 amino acid sequence shown as SEQ ID NO. 25; a CDR2 amino acid sequence as set forth in SEQ ID NO. 26; and, a CDR3 amino acid sequence set forth in SEQ ID NO: 27.
2. The single domain antibody of claim 1, wherein the heavy chain variable region further comprises a framework region (FWR) of an alpaca antibody or a human antibody.
3. The single domain antibody of claim 2, wherein said single domain antibody comprises the amino acid sequence set forth in SEQ ID NO 2.
4. The single domain antibody of any one of claims 1 to 3, wherein said protein comprising the amino acid sequence shown in SEQ ID No. 1 is selected from one or more of FSH, HCG, TSH, FSH-CTP and hLH.
5. An isolated nucleic acid encoding the single domain antibody of any one of claims 1-4.
6. A recombinant expression vector comprising the isolated nucleic acid of claim 5.
7. The recombinant expression vector of claim 6, wherein the recombinant expression vector is a plasmid, cosmid, phage, or viral vector.
8. The recombinant expression vector of claim 7, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.
9. A transformant comprising the recombinant expression vector of any one of claims 6 to 8 in a host cell.
10. The transformant of claim 9, wherein the host cell is a yeast.
11. The transformant of claim 10, wherein the yeast is a saccharomyces cerevisiae, a mold, a bacterium, or a cellular expression system.
12. A chimeric antigen receptor comprising a single domain antibody of any one of claims 1-4.
13. A genetically modified cell comprising the chimeric antigen receptor of claim 12.
14. The genetically modified cell of claim 13, wherein the genetically modified cell is a eukaryotic cell.
15. The genetically modified cell of claim 13, wherein the genetically modified cell is an isolated human cell.
16. The genetically modified cell of claim 13, wherein the genetically modified cell is an immune cell.
17. The genetically modified cell of claim 16, wherein the immune cell is a T cell or an NK cell.
18. A method of producing a single domain antibody as claimed in any one of claims 1 to 4 comprising the steps of: culturing the transformant according to any one of claims 9 to 11, and obtaining the single domain antibody from the culture.
19. An antibody drug conjugate comprising an antibody moiety comprising a single domain antibody as claimed in any one of claims 1 to 4 and a conjugate moiety.
20. The antibody drug conjugate of claim 19, wherein the conjugate moiety comprises a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, and wherein the antibody moiety and the conjugate moiety are conjugated via a chemical bond or a linker.
21. A pharmaceutical composition comprising a single domain antibody according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 12, a genetically modified cell according to any one of claims 13 to 17, or an antibody drug conjugate according to claim 19 or 20.
22. The pharmaceutical composition of claim 21, further comprising a pharmaceutically acceptable carrier; alternatively, the pharmaceutical composition is in a liquid dosage form, a gaseous dosage form, a solid dosage form, and a semisolid dosage form, or the pharmaceutical composition may be administered orally, by injection, nasally, transdermally, or mucosally.
23. A kit comprising kit a and kit B, wherein:
kit a comprising a single domain antibody according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 12, a genetically modified cell according to any one of claims 13 to 17, an antibody drug conjugate according to claim 19 or 20, or a pharmaceutical composition according to claim 21 or 22;
the kit B contains one or more selected from the group consisting of a hormonal agent, a targeted small molecule agent, a proteasome inhibitor, an imaging agent, a diagnostic agent, a chemotherapeutic agent, a radiotherapeutic agent, an immunosuppressive agent, an oncolytic drug, a cytotoxic agent, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, and a vaccine.
24. A kit comprising a single domain antibody according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 12, a genetically modified cell according to any one of claims 13 to 17, an antibody drug conjugate according to claim 19 or 20, or a pharmaceutical composition according to claim 21 or 22.
25. The kit of claim 24, further comprising (i) a means of administering said single domain antibody or said chimeric antigen receptor or said genetically modified cell or said antibody drug conjugate or pharmaceutical composition; or (ii) instructions for use.
26. Use of a single domain antibody according to any one of claims 1 to 4, a chimeric antigen receptor according to claim 12, a genetically modified cell according to any one of claims 13 to 17, an antibody drug conjugate according to claim 19 or 20, a pharmaceutical composition according to claim 21 or 22, a kit of parts according to claim 23 or a kit according to claim 24 or 25 for the manufacture of a medicament for the treatment or prevention of a protein-mediated disease or condition comprising an amino acid sequence as set forth in SEQ ID No. 1.
27. Use of a single domain antibody according to any one of claims 1 to 4 for the detection of a protein comprising the amino acid sequence shown as SEQ ID No. 1.
28. The use of claim 27, wherein the protein is selected from one or more of FSH, HCG, TSH, FSH-CTP and hLH.
29. A method of detecting a protein comprising an amino acid sequence as set forth in SEQ ID No. 1, comprising the step of detecting using a single domain antibody as defined in any one of claims 1 to 4, a chimeric antigen receptor as defined in claim 12, a genetically modified cell as defined in claims 13 to 17, an antibody drug conjugate as defined in claim 19 or 20, a pharmaceutical composition as defined in claim 21 or 22, a kit of parts as defined in claim 23 or a kit of parts as defined in claim 24 or 25.
CN202210832387.1A 2021-09-07 2021-09-07 Antibody or antigen binding fragment thereof and application thereof Pending CN115160437A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210832387.1A CN115160437A (en) 2021-09-07 2021-09-07 Antibody or antigen binding fragment thereof and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111045734.8A CN113698481B (en) 2021-09-07 2021-09-07 Antibody or antigen binding fragment thereof and application thereof
CN202210832387.1A CN115160437A (en) 2021-09-07 2021-09-07 Antibody or antigen binding fragment thereof and application thereof

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN202111045734.8A Division CN113698481B (en) 2021-09-07 2021-09-07 Antibody or antigen binding fragment thereof and application thereof

Publications (1)

Publication Number Publication Date
CN115160437A true CN115160437A (en) 2022-10-11

Family

ID=78659025

Family Applications (7)

Application Number Title Priority Date Filing Date
CN202210825845.9A Pending CN116063490A (en) 2021-09-07 2021-09-07 Mutant of VHH antibody or antigen fragment thereof and application thereof
CN202210832387.1A Pending CN115160437A (en) 2021-09-07 2021-09-07 Antibody or antigen binding fragment thereof and application thereof
CN202210831856.8A Pending CN115160436A (en) 2021-09-07 2021-09-07 High-affinity single-domain antibody or antigen-binding fragment thereof and application thereof
CN202210848964.6A Pending CN116375862A (en) 2021-09-07 2021-09-07 Single-domain antibody or antigen binding fragment thereof and application thereof
CN202210817818.7A Active CN115433276B (en) 2021-09-07 2021-09-07 Mutant of VHH antibody or antigen fragment thereof and application thereof
CN202210835397.0A Pending CN116102648A (en) 2021-09-07 2021-09-07 Single-domain antibody or antigen binding fragment thereof and application thereof
CN202111045734.8A Active CN113698481B (en) 2021-09-07 2021-09-07 Antibody or antigen binding fragment thereof and application thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202210825845.9A Pending CN116063490A (en) 2021-09-07 2021-09-07 Mutant of VHH antibody or antigen fragment thereof and application thereof

Family Applications After (5)

Application Number Title Priority Date Filing Date
CN202210831856.8A Pending CN115160436A (en) 2021-09-07 2021-09-07 High-affinity single-domain antibody or antigen-binding fragment thereof and application thereof
CN202210848964.6A Pending CN116375862A (en) 2021-09-07 2021-09-07 Single-domain antibody or antigen binding fragment thereof and application thereof
CN202210817818.7A Active CN115433276B (en) 2021-09-07 2021-09-07 Mutant of VHH antibody or antigen fragment thereof and application thereof
CN202210835397.0A Pending CN116102648A (en) 2021-09-07 2021-09-07 Single-domain antibody or antigen binding fragment thereof and application thereof
CN202111045734.8A Active CN113698481B (en) 2021-09-07 2021-09-07 Antibody or antigen binding fragment thereof and application thereof

Country Status (1)

Country Link
CN (7) CN116063490A (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114316043B (en) * 2021-12-28 2022-09-20 三优生物医药(上海)有限公司 TGF beta1 antigen binding molecule and application thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2010310895A1 (en) * 2009-10-23 2012-05-03 Garvan Institute Of Medical Research Modified variable domain molecules and methods for producing and using same
WO2012016576A1 (en) * 2010-08-04 2012-02-09 Glycotope Gmbh Improved recombinant human follicle-stimulating hormone
GB201813178D0 (en) * 2018-08-13 2018-09-26 Autolus Ltd Cell
CN109081870B (en) * 2018-09-21 2021-12-03 成都阿帕克生物科技有限公司 Nano antibody of anti-human chorionic gonadotropin beta subunit, nucleic acid molecule and application

Also Published As

Publication number Publication date
CN113698481B (en) 2022-09-20
CN115433276B (en) 2024-04-02
CN116063490A (en) 2023-05-05
CN116102648A (en) 2023-05-12
CN115160436A (en) 2022-10-11
CN115433276A (en) 2022-12-06
CN113698481A (en) 2021-11-26
CN116375862A (en) 2023-07-04

Similar Documents

Publication Publication Date Title
WO2018024237A1 (en) Anti-pd-l1 nanobody and use thereof
CN112457404B (en) Anti-human EGFR nano antibody and application
WO2017198148A1 (en) Il-13 antibody and preparation method and use thereof
WO2022199590A1 (en) Nanobody targeting bcma and application thereof
CN113698481B (en) Antibody or antigen binding fragment thereof and application thereof
CN109609466B (en) Mouse aspergillus polysaccharide hybridoma cell strain, monoclonal antibody and application
CN109628410B (en) Mouse aspergillus-resistant polysaccharide hybridoma cell strain, monoclonal antibody and application
CN114106187A (en) Specific shark single-domain antibody targeting OGT (one glass solution) and preparation method and application thereof
CN113105546B (en) Anti-recombinant human basic fibroblast growth factor nano antibody and application thereof
WO2019096219A1 (en) Humanized anti-il-13 antibody and preparation method and use thereof
WO2023279803A1 (en) Protein binding molecule of rbv and use thereof
CN114057880B (en) DLL3 monoclonal antibody
WO2023137994A1 (en) Specific nanoantibody nb3.27 against colorectal cancer-associated bacteroides fragilis toxin protein activator, and application thereof
WO2020248938A1 (en) Anti-cd25 antibody and application thereof
CN114685667A (en) Mesothelin binding molecules and uses thereof
WO2024061364A9 (en) Anti-4-1bb nanobody, preparation therefor, and use thereof
CN116789813B (en) Monoclonal antibody for resisting staphylococcus aureus alpha-hemolysin and application thereof
CN117700557B (en) Antibody or antigen binding fragment specifically binding to folate receptor alpha
CN114702590B (en) anti-c-MET nanobody, encoding nucleic acid and application thereof
WO2021160153A1 (en) Human cd47-targeting single-domain antibody and use thereof
CN114249820B (en) Alpaca-derived nanobody combined with SARS-CoV-2RBD
WO2022143552A1 (en) Pd-1 binding molecule and application thereof
CN105001331B (en) VEGF (vascular endothelial growth factor) monoclonal antibody and application thereof
CN117467014A (en) Alpaca-derived Nanobodies that bind human immunoglobulin E (IgE)
TW202317631A (en) Anti-CRTAM 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