WO2018121474A1 - Anticorps à domaine unique pour identifier un complexe formé par une molécule hla-a2 et un polypeptide à chaîne courte rmfpnapyl - Google Patents

Anticorps à domaine unique pour identifier un complexe formé par une molécule hla-a2 et un polypeptide à chaîne courte rmfpnapyl Download PDF

Info

Publication number
WO2018121474A1
WO2018121474A1 PCT/CN2017/118283 CN2017118283W WO2018121474A1 WO 2018121474 A1 WO2018121474 A1 WO 2018121474A1 CN 2017118283 W CN2017118283 W CN 2017118283W WO 2018121474 A1 WO2018121474 A1 WO 2018121474A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
single domain
domain antibody
Prior art date
Application number
PCT/CN2017/118283
Other languages
English (en)
Chinese (zh)
Inventor
古明珠
高斌
吕丽慧
刘莹
梁猛
Original Assignee
天津天锐生物科技有限公司
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 天津天锐生物科技有限公司 filed Critical 天津天锐生物科技有限公司
Publication of WO2018121474A1 publication Critical patent/WO2018121474A1/fr

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
    • 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
    • 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
    • C07K16/2833—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 against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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®

Definitions

  • the invention belongs to the technical field of tumor immunotherapy, and particularly relates to a single domain antibody which recognizes a complex formed by an HLA-A2 molecule and a RMFPNAPYL short peptide.
  • the Wilms tumor gene (WT1) was discovered and cloned in Wilms tumors by different methods from Call, Bonetta and Gessler in 1990.
  • the gene is located on human chromosome 11p13 and is lowly expressed in normal tissues. High expression in tumors (Gessler et al. Nature. 1990; 346 (6280): 194-197; Menssen et al. Leukemia. 1995; 9(6): 1060-1067; Oji et al. Jpn J Cancer Res. 1999; 90(2): 194-204).
  • the effect of WT1 on hematopoiesis is characterized by abnormal expression or mutation in a subset of acute leukemia, which regulates its transcription by interacting with various hematopoietic regulatory factors, thereby participating in the formation of leukemia such as proliferation, differentiation and apoptosis of hematopoietic cells.
  • CAR-T has achieved good results in the treatment of CD19-positive hematoma
  • antibody-targeted CARs can only target these antigens expressed on the surface of tumor cells without exception, and many tumor-associated antigens are expressed only inside the cells.
  • TCR can recognize both extracellular antigens and intracellular antigens that are presented to the cell surface by antigen processing, TCR usually needs to isolate specific ⁇ TCR genes from T cell clones, which is difficult and reproducible.
  • transgenic TCRs have potential safety hazards associated with TCR mismatches, and the two strands of exogenous TCR may be mismatched with endogenous TCR subunits to form a new TCR.
  • This mismatched TCR creates an unknown specificity that may target normal tissues, resulting in a severe graft-versus-host response.
  • Ochi et al. integrated the siRNA that inhibits endogenous TCR into the TCR expression cassette expressing the foreign recognition WT1 antigen, used to modify T cells, and carried out adoptive treatment for leukemia, confirming that the expression of the WT1 transgene is recognized by the endogenous TCR inhibition state.
  • TCR T cells have a stronger tumor suppressor function.
  • T cell immunity and mature antibody technology can be combined to develop a novel T cell modification treatment that combines the advantages of antibodies and T cells. technology.
  • the Kurilsky laboratory of the Pasteur Institute successfully produced monoclonal antibodies in 1993 that specifically recognize T cell epitope polypeptides that bind to MHC class I molecules.
  • phage display technology was used to obtain antibodies that specifically recognize influenza virus polypeptides and inhibit infection of the corresponding viruses.
  • HLA-A2 transgenic mice used HLA-A2 transgenic mice to obtain hybridoma cells that secrete antibodies that recognize the OVA antigen-producing leader polypeptide H-2Kb complex.
  • This TCR-specific antibody is used to localize and quantify complexes of specific peptides and MHC molecules, providing a new tool for studying the mechanism of antigen processing and presentation.
  • TCR-like antibodies Such antibodies that recognize the polypeptide MHC complex, known as TCR-like antibodies, are sometimes referred to as TCR-mimic antibodies because of their T cell receptor function.
  • TCR-mimic antibodies To facilitate and modify the differentiation of T-cells from CAR-T and TCR-T, such antibodies with MHC polypeptide complex specificity were named MAR (MHC Antigen Receptor).
  • MAR MHC Antigen Receptor
  • MAR MHC Antigen Receptor
  • MAR MHC Antigen Receptor
  • MAR MHC Antigen Receptor
  • MAR as a novel molecule for recognizing MHC polypeptide complexes can recognize tumor extracellular antigens by recognizing the function of natural T cell receptors and adopting the self-heterologous recognition mechanism obtained during the evolution of life.
  • the intratumoral antigen formed by antigen processing has a wider application, especially for solid tumor antigens, and has unique advantages.
  • Modified T cells are a class of molecules used in tumor therapy.
  • MAR-NK/MAR-T not only kills HLA-A2 positive melanoma cells, but also kills HLA-A2 positive melanoma cells.
  • the growth of human melanoma in immunodeficient mice can be inhibited (zhang et al: Immunol Cell Biol 2013, 91(10): 615-24; zhang et al: Sci Rep 2014, 4: 3571). It was first confirmed that MAR modified effector cells can inhibit the growth of tumor cells in animals and lay a foundation for further clinical research to treat cancer.
  • Oka et al. used the overexpressed tumor antigen WT1 as a target molecule for MHC-like antigen-restricted CTLS action for T cell-mediated leukemia immunotherapy (Oka Y, st al: Current Cancer Drug, 2002). They selected and synthesized four non-negative peptide chains derived from WTI protein and containing HLA-A2 molecular binding anchors, Db126 peptide, WH187 peptide, Db235 peptide (CMTWNQMNL residues 235-243) and WH242 peptide (NLGATLKGV). Residus 242-250), the results demonstrate that two of the peptides Db126 and WH187 bind to HLA-A2 molecules in vitro, and induce the production of WT1 peptide-specific CTL.
  • WT1 (126) is an HLA-A2 restricted dominant epitope derived from nephroblastoma, and the following HLA-A2/WT1 (126) (RMFPNAPYL) are abbreviated as HLA-A2/RMFPNAPYL.
  • WT1 is a well-recognized tumor suppressor gene directly associated with nephroblastoma, which is overexpressed in leukemia, lymphoma, and various solid tumors. Dao et al. used a phage display scFV library to screen for scFV antibodies that specifically recognize the HLA-A2/RMFPNAPYL complex.
  • This antibody specifically binds to T2 cells loaded with RMFPNAPYL polypeptide, and also binds to HLA-A2 and WT1-positive leukemia cell line BV173 JMN.
  • Complete whole human antibodies were prepared using the information obtained from scFV.
  • the above cells can be used as target cells to induce ADCC, and the growth of these tumor cells can be inhibited (Dao et al., Sci Transl Med. 2013 Mar. 13; 5 (176): 176ra33; WO 2012/135854). Zhao et al. used phage display to obtain antibodies recognizing HLA-A2/RMFPNAPYL.
  • a first object of the invention is to provide a single domain antibody that recognizes HLA-A2/RMFPNAPYL.
  • the single domain antibody recognizing HLA-A2/RMFPNAPYL comprises a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, and the single domain antibody is any one of the following (a) to (d):
  • the complementarity determining region CDR1 of the single domain antibody is as follows (a1) or (a2) or (a3):
  • the complementarity determining region CDR2 of the single domain antibody is as follows (a4) or (a5) or (a6):
  • the complementarity determining region CDR3 of the single domain antibody is as follows (a7) or (a8) or (a9):
  • the complementarity determining region CDR1 of the single domain antibody is as follows (b1) or (b2) or (b3):
  • the complementarity determining region CDR2 of the single domain antibody is as follows (b4) or (b5) or (b6):
  • the complementarity determining region CDR3 of the single domain antibody is as follows (b7) or (b8) or (a9):
  • the complementarity determining region CDR1 of the single domain antibody is as follows (c1) or (c2) or (c3):
  • (c3) an amino acid sequence having the same function obtained by subjecting the amino acid sequence represented by SEQ ID No. 7 to substitution and/or deletion and/or addition of one or several amino acid residues;
  • the complementarity determining region CDR2 of the single domain antibody is as follows (c4) or (c5) or (c6):
  • the complementarity determining region CDR3 of the single domain antibody is as follows (c7) or (c8) or (c9):
  • the complementarity determining region CDR1 of the single domain antibody is as follows (d1) or (d2) or (d3):
  • the complementarity determining region CDR2 of the single domain antibody is as follows (d4) or (d5) or (d6):
  • the complementarity determining region CDR3 of the single domain antibody is as follows (d7) or (d8) or (d9):
  • the single domain antibody is as follows (e1) or (e2):
  • Another object of the present invention is to provide a derivative of the above single domain antibody.
  • the derivative provided by the present invention is any one of the following (f1) to (f9):
  • the fusion protein is obtained by directly fusing the single domain antibody of claim 1 or 2 with at least one polypeptide molecule having therapeutic or recognition function, or by treating or recognizing one or more peptides with a linker peptide.
  • Functional peptide molecules are obtained by ligation.
  • the polypeptide molecule having a therapeutic or recognition function is a human Fc protein or a CD3 antibody.
  • the single domain antibody is fused to a human Fc protein or a CD3 antibody, the monovalent antibody becomes a bivalent antibody, and the affinity is improved.
  • the specific preparation method of the fusion protein comprises the steps of: introducing the coding gene of the single domain antibody and the coding gene of the human Fc protein or CD3 antibody into a host cell to obtain a recombinant cell; and culturing the recombinant cell to obtain the fusion protein. .
  • the coding gene of the single domain antibody and the coding gene of the human Fc protein or CD3 antibody are introduced into a host cell by a recombinant vector;
  • the recombinant vector is obtained by inserting a gene encoding the single domain antibody and a fragment encoding the human Fc protein or CD3 antibody into a multiple cloning site of an expression vector.
  • the gene encoding the single domain antibody and the gene encoding the human Fc protein or CD3 antibody are represented by SEQ ID No. 18 or SEQ ID No. 23 or SEQ ID No. 24. DNA molecule.
  • the expression vector is a pET22b vector or a pcDNA3.1 vector.
  • the host cell is an E. coli/DE3 cell or a 293F cell.
  • the composition may be a pharmaceutical composition containing a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of the invention may be administered in combination therapy, i.e., in combination with other agents.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like which are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound ie, an antibody, immunoconjugate or bispecific molecule or multispecific molecule
  • the active compound can be coated in a material to protect the compound from acids and other natural compounds that can inactivate the compound.
  • the role of the condition ie, an antibody, immunoconjugate or bispecific molecule or multispecific molecule.
  • compositions of the invention may comprise one or more pharmaceutically acceptable salts.
  • pharmaceutically acceptable salt refers to a salt which retains the desired biological activity of the parent compound and does not cause any unwanted toxicological effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, as well as non-toxic organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids.
  • Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chlorine Salt derived from procaine, choline, diethanolamine, ethylenediamine, procaine, and the like.
  • compositions of the invention may also contain a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbic acid palmitate Ester, butylated hydroxyanisole (BHA), butylated hydroxytoluene (DHT), lecithin, propyl gallate, alpha-tocopherol, etc.; (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA) , sorbitol, tartaric acid, phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbic acid palmitate Ester, butylated hydroxyani
  • aqueous or nonaqueous vehicles examples include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, And injectable organic esters such as ethyl oleate.
  • polyols e.g., glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the application of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by a sterilization procedure or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like.
  • compositions of the invention may be administered by one or more routes of administration using one or more methods well known in the art. Those skilled in the art will appreciate that the route and/or manner of administration will vary depending on the desired result.
  • compositions of the invention have therapeutic applications in vitro and in vivo.
  • these molecules can be administered to cells cultured in vitro or ex vivo, or administered to a human subject in vivo to treat, prevent or diagnose a variety of diseases.
  • subject as used herein includes both human and non-human animals.
  • Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. These methods are particularly suitable for treating human patients with tumors.
  • the immunoconjugate may be a conjugate obtained by coupling the above single domain antibody with a therapeutic agent such as a cytotoxin, a drug (for example, an immunosuppressive agent) or a radioactive toxin.
  • a therapeutic agent such as a cytotoxin, a drug (for example, an immunosuppressive agent) or a radioactive toxin.
  • conjugates are referred to as "immunoconjugates.”
  • An immunoconjugate comprising one or more cytotoxins is referred to as an "immunotoxin.”
  • Cytotoxins or cytotoxic agents include any agent that is detrimental to the cell (eg, kills).
  • Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecaine, mitomycin, epipodophyllotoxin, epipodophyllotoxin, vincristine, vinblastine , colchicine, doxorubicin, daunorubicin, dihydroxy anthrax dione, mitoxantrone, phosfomycin, actinomycin D, l-dehydrotestosterone, glucocorticoid, proca , tetracaine, lidocaine, propranolol and puromycin and their analogs or homologs.
  • Therapeutic agents also include, for example, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine, a burning agent ( For example, nitrogen mustard, thioepa chlorambucil, phenylalanine mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, chain Oxazomycin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (eg, daunorubicin (formerly known as daunorubicin) and trichothecene , antibiotics (eg, actinomycin D, bleomycin, phosfomycin, and amphotericin (AMC)), and anti-mitotic agents (eg, vincristine and vinblastine).
  • antimetabolites e
  • antibody-conjugated therapeutic cytotoxins include doxorubicin, calicheamicin, maytansin, auristatin, and derivatives thereof. Coupling of a cytotoxin with an antibody of the invention can be utilized in the art. Joint technology.
  • the antibodies of the invention may also be conjugated to a radioisotope to produce a cytotoxic radiopharmaceutical, also known as a radioimmunoconjugate.
  • a radioisotope that can be coupled to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine 131 , indium 111 , ⁇ 90, and ⁇ 177 .
  • Methods of preparing radioactive immunoconjugates have been established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin (TM) (IDEC Pharmaceuticals) and Bexxar (TM) (Corixa Pharmaceuticals), which are capable of producing radioimmunoconjugates using similar methods using the antibodies of the invention.
  • the antibody conjugates of the invention can be used to modify a particular biological response, and the drug moiety should not be construed as being limited to classical chemotherapeutic agents.
  • the drug moiety can be a protein or polypeptide having the desired biological activity.
  • Such proteins may include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin; proteins such as tumor necrosis factor or interferon- ⁇ ; or biological response regulators such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophages Cell colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophages Cell colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • the antibody genetically engineered antibody obtained by modifying and/or modifying the above single domain antibody or antigen binding portion thereof is also within the scope of the present invention.
  • the single domain antibodies provided herein comprise CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise a specific amino acid sequence based on a single domain antibody of the invention or a conservative modification thereof, and wherein the antibody retains an antibody of the invention It has the functional characteristics of identifying and/or combining HLA-A2/RMFPNAPYL.
  • conservative sequence modification refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into the antibodies of the invention by techniques well known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitution refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain.
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include: basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoleucine) Acid, proline, phenylalanine, methionine), ⁇ -branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, styrene Amino acids of amino acids, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine,
  • one or more amino acid residues within the CDR regions of an antibody of the invention can be substituted for other amino acid residues from the same side chain family, and the above-described functions of altered antibody retention can be detected using the in vitro affinity assays described herein.
  • CDR grafting One type of variable region engineering that can be performed is CDR grafting.
  • the antibody interacts with the target antigen primarily through amino acid residues located in the complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between the individual antibodies than the sequences outside the CDRs. Since the CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of the particular antibody present can be expressed by constructing an expression vector comprising a CDR sequence from a particular antibody present, which is grafted to From the backbone sequences of different antibodies with different properties, these backbone sequences can be obtained from public DNA databases or published references.
  • variable region modification is to mutate the amino acid sequence within the CDR1, CDR2 and/or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest.
  • Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, effects on antibody binding, or other target functional properties, which can be evaluated using the assays described herein and provided in the Examples.
  • Conservative sequence modifications are preferably introduced.
  • the mutation may be an amino acid substitution, addition or deletion, but is preferably substituted. Moreover, typically no more than 5 residues are altered within the CDR regions.
  • Engineered antibodies of the invention include, for example, antibodies whose backbone residues have been modified to improve the properties of the antibody. Such backbone modifications are generally made to reduce the immunogenicity of the antibody.
  • the backbone modification involves mutating one or more residues within the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody.
  • the framework region is positions 1-26, 36-49, 56-97, 110-120 of SEQ ID No. 13, or positions 1-26 of SEQ ID No. 14. , 36-49, 56-97, 114-124, or SEQ ID No. 15 1-26, 36-49, 56-97, 110-120, or SEQ ID No. 16 positions 1-26, 36-49, 56-97, 116-126.
  • the term "homology" as used herein may describe the degree of similarity between two or more amino acid sequences, and the percentage of homology between the first amino acid sequence and the second amino acid sequence may be determined by the formula: (first amino acid The number of amino acid residues in the sequence that is identical to the amino acid sequence at the corresponding position in the second amino acid sequence) / (total number of amino acids in the first amino acid sequence) * 100%, wherein the second amino acid sequence can only be Deletions, insertions, substitutions or additions of amino acids (compared to the first amino acid) are considered to be different.
  • the percent homology can also be obtained using known computer algorithms for sequence matching such as NCBI Blast.
  • Still another object of the present invention is to provide a biological material related to the above single domain antibody or the above derivative.
  • the biomaterial provided by the present invention is any one of the following (g1)-(g4):
  • (g3) a vector comprising the nucleic acid molecule of (g1) or (g2);
  • (g4) A host cell comprising the nucleic acid molecule of (g1) or (g2) or the vector of (g3).
  • the nucleic acid molecule is any one of the following (h1) to (h3):
  • (h1) a DNA molecule represented by SEQ ID No. 19 or SEQ ID No. 20 or SEQ ID No. 21 or SEQ ID No. 22 or SEQ ID No. 18 or SEQ ID No. 23 or SEQ ID No. 24.
  • (h2) a DNA molecule having 75% or more of the identity with the (h1) defined nucleotide sequence, and encoding the above single domain antibody or fusion protein;
  • (h3) A DNA molecule which hybridizes under stringent conditions to a (h1) or (h2) defined nucleotide sequence and which encodes the above single domain antibody or fusion protein.
  • the nucleic acid molecule may be a nucleotide sequence encoding each of the complementarity determining regions or the single domain antibody or the amino acid sequence of the fusion protein, and the specific sequence of the corresponding nucleic acid molecule can be obtained at any time by the genetic code. Due to the annexation of the genetic code, the nucleic acid molecule can be varied for different application purposes.
  • nucleic acid molecules of the invention can be obtained using conventional molecular biology techniques.
  • nucleic acids encoding antibodies can be obtained from libraries.
  • the nucleic acid sequence or at least part of the sequence in the vector can be expressed by a suitable expression system to obtain a corresponding protein or polypeptide;
  • the expression system includes bacteria, yeast, filamentous fungus, lactation Animal cells, insect cells, plant cells or cell-free expression systems.
  • Still another object of the present invention is to provide a novel use of the above single domain antibody or the above derivative or the above biological material.
  • the present invention provides the use of the above single domain antibody or the above derivative or the above biological material in any of the following (i 1) to (i4):
  • the product is a drug.
  • Tumors that can be treated with the antibodies of the invention include, but are not limited to, tumors such as melanoma, breast cancer, prostate cancer, lung cancer, ovarian cancer, thyroid cancer, liver cancer, bladder cancer, or gastric cancer.
  • the T cells can be modified in vitro by using the antibody or fusion protein of the present invention to obtain T cells after arming, and the T cells are amplified after being armed, and then returned to the subject, and the armed T cells can be specific. Identify tumors for in vivo tumor immunotherapy. Modification of T cells can be achieved by conventional methods well known to those skilled in the art.
  • the HLA-A2/RMFPNAPYL of the present invention is an HLA-A2/RMFPNAPYL antigen complex, which refers to a complex of the specific polypeptide RMFPNAPYL from WT1 and the antigen molecule HLA-A2.
  • the HLA-A2/RMFPNAPYL antibody referred to in this patent is a single domain antibody.
  • a single domain antibody differs from SCFV in that it contains only one variable region of the antibody heavy chain, approximately half the size of SCFV, and is the smallest fully functional antigen-binding fragment with weak immunogenicity; easier to penetrate through the vessel wall Solid tumors are beneficial to the treatment of tumors.
  • Single domain antibodies against HLA-A2/RMFPNAPYL have not been reported.
  • the present invention is directed to the technical problems existing in the prior art, and the antibodies with higher affinity are screened from the phage single domain library by three rounds of biological panning, and the obtained antibodies are cloned into a prokaryotic/eukaryotic expression vector, and the human The Fc or CD3 antibody is fused and expressed, and the host cell is transfected to obtain a bispecific antibody.
  • the complex does not bind, and not only recognizes the synthetic HLA-A2/RMFPNAPYL complex, but also binds to the natural antigen-processed HLA-A2/RMFPNAPYL complex expressed on the surface of tumor cells, in order to further develop the biological agent for treating tumors. Laid the foundation.
  • Figure 1 shows the results of the first plate ELISA test and data analysis after three rounds of panning.
  • Figure 1A shows the first plate ELISA assay after three rounds of panning. All wells of the ELISA were coated with the antigen HLA-A2/RMFPNAPYL, and after binding to the single cloned phage of the panning sieve, the anti-phage secondary antibody labeled with HRP was added.
  • Figure 1B shows the analysis of the first plate ELISA test data after three rounds of panning. The ordinate is the light absorption value of each hole at 650 nm, and the abscissa is 96 holes.
  • Figure 2 shows the results of ELISA and data analysis of the second plate after three rounds of panning.
  • Figure 2A is a second plate ELISA assay after three rounds of panning. All wells of the ELISA were coated with the antigen HLA-A2/RMFPNAPYL.
  • Figure 2B shows the analysis of the first plate ELISA test data after three rounds of panning. The ordinate is the light absorption value of each hole at 650 nm, and the abscissa is 96 holes.
  • Figure 3 shows the results of ELISA and data analysis of different antigens by four different sequences of HLA-A2/RMFPNAPYL single domain antibody.
  • Figure 3A shows ELISA detection of different antigens by four different sequences of HLA-A2/RMFPNAPYL single domain antibodies.
  • Line 1 is M5-H1, line 2 is MA control;
  • line 3 is M4 control;
  • line 4 is M5-G3;
  • line 5 is M5-F4;
  • Figure 3B shows ELISA detection data analysis of different antigens by four different sequences of HLA-A2/RMFPNAPYL single domain antibodies.
  • the ordinate is the light absorption at 650 nm; the abscissa 1, 2, 3, 4 is the HLA-A2/ITDQVPFSV antigen, the HLA-A2/NLVPMVATV antigen, the HLA-A2/RMFPNAPYL antigen, and the HLA-A2/SLLMWITQC antigen, respectively.
  • a, b, c, d, e, f, g are M5-H1, MA control, M4 control, M5-G3, M5-F4, MB control and M5-C3, respectively.
  • Figure 4 is a reduced SDS-PAGE after purification of a single domain antibody expressed in pET22b. Marker's strips are from 14, 25, 30, 40, 50, 70, 100, 120, 160 KD. Line1 is M5-H1, line2 is M5-G3, and line3 is M5-F4.
  • Figure 5 is a schematic representation of a plasmid map of Fc expressing a single domain antibody in pET22b.
  • the single domain antibody is ligated to the Fc by linker (G4S).
  • Figure 6 is a reduced SDS-PAGE after purification of the M5-H1-Fc fusion protein in pET22b. Marker's strips are from 14, 25, 30, 40, 50, 70, 100, 120, 160 KD. Line1 is a reduced M5-H1-Fc.
  • Figure 7 is a schematic diagram of a plasmid map in which a single domain antibody is fused to express Fc in pcDNA3.1.
  • the single domain antibody is ligated to the Fc by a restriction enzyme, and the single domain antibody is preceded by a signal peptide and a kozak sequence.
  • Figure 8 is a diagram showing the reduction and non-reduction electrophoresis of SDS-PAGE after purification of M5-F4 and Fc fusion protein. Marker's strips are from 14, 25, 30, 40, 50, 70, 100, 120, 160 KD. Line1 and Line2 are the reduced M5-F4-Fc and the non-reduced M5-F4-Fc, respectively.
  • Figure 9 is a graph showing the specific detection data of three monovalent single domain antibodies for different antigens.
  • the ordinate is the light absorption at 650 nm, and the abscissa 1, 2, 3, 4 is the HLA-A2/ITDQVPFSV antigen, HLA-A2/NLVPMVATV antigen, HLA-A2/RMFPNAPYL antigen, HLA-A2/SLLMWITQC antigen.
  • the three samples were monovalent antibodies M5-H1, M5-G3, M5-F4.
  • Figure 10 is a graph showing the specificity detection and data analysis of two Fc fusion antibodies for different antigens.
  • the ordinate has a light absorption value at 650 nm, and the abscissas 1, 2, 3, and 4 are HLA-A2/ITDQVPFSV antigen, HLA-A2/NLVPMVATV antigen, HLA-A2/RMFPNAPYL antigen, and HLA-A2/SLLMWITQC antigen, respectively.
  • Two samples were M5-H1-Fc, M5-F4-Fc.
  • Figure 11 is a FACS detection of specific recognition of HLA-A2/RMFPNAPYL at the cellular level by the M5-F4-Fc fusion protein.
  • Negative control 1 negative was BV-173 cells without primary antibody plus secondary antibody goat anti-human Fc (FITC)
  • negative control 2 (BM) was BV-173 cells plus Fc fusion protein without binding to HLA-A2/RMFPNAPY BM and secondary antibody
  • the experimental group was BV-173 cells plus M5-F4-Fc fusion protein and secondary antibody.
  • Figure 12 is a graph showing the intermolecular interaction and affinity constant of the fusion protein M5-H1-Fc and HLA-A2/RMFPNAPYL complex.
  • the abscissa is time
  • the ordinate is the response value (RU) of the intermolecular interaction
  • 1 is the reference channel
  • 2 is the experimental channel.
  • Figure 13 is a graph showing the intermolecular interaction and affinity constant of the fusion protein M5-F4-Fc and HLA-A2/RMFPNAPYL complex.
  • the abscissa is time
  • the ordinate is the response value (RU) of the intermolecular interaction
  • 1 is the reference channel
  • 2 is the experimental channel.
  • Figure 14 shows the specific recognition and killing of single domain antibody fusion proteins at the cellular level.
  • the abscissa is the concentration of the antibody M5-H1-antiCD3, and the ordinate is the killing efficiency.
  • the HLA-A2/RMFPNAPYL complex in the following examples refers to a complex of the specific polypeptide RMFPNAPYL from WT1 and the antigen molecule HLA-A2, described in the literature "Kohrt HE, Muller A, Baker J, Goldstein MJ, Newell E ,Dutt S, et al.Donor immunization with WT1 peptide augments antileukemic activity after MHC-matched bone marrow transplantation.Blood.2011 Nov 10;118(19):5319–5329.[PubMed:21868578]” and “Borbulevych OY,Do P, Baker BM.
  • M13KE phage purchased from NEB#N0316S
  • AlwnI purchased from NEB
  • AfeI purchased from NEB
  • the synthetic gene fragment was also digested with AlwnI and AfeI (purchased from NEB). They are then ligated together using T4 ligase. After ligation, TG1 was transfected to obtain helper phage BM13.
  • the synthetic gene sequences are as follows: CCA GCC GGC CTT TCT GAG GGG TCG ACT ATA GAA GGA CGA GGG GCC CAC GAA GGA GGT GGG GTA CCC GGT TCC GAG GGT.
  • pUC19 (purchased from NEB) was digested with HindIII (purchased from NEB) and NdeI (purchased from NEB), and a heavy chain artificial single domain antibody sequence based on the DP47 antibody sequence was added.
  • the single domain antibody expression framework the single domain antibody is fused to the GIII protein, and the Myc and VSV-G tags are added in the middle for purification or identification, and the phage display vector pBG3 is constructed.
  • E. coli strain CJ236 (purchased from NEB) lacks functional uracil deoxyribonucleoside triphosphatase and uracil-N glycosidase to produce a uracilized single-stranded DNA template.
  • the pBG3 plasmid was transfected into CJ236 and plated on agar plate containing Carbenicillin (50 ⁇ g/ml) and chloramphenicol (15 ⁇ g/ml) and cultured overnight. A single colony screened on the plate was selected into 3 ml of 2 ⁇ TY broth medium (containing the same concentration of the above-mentioned double antibody), and cultured at 37 ° C, 250 rpm overnight.
  • the phage-containing supernatant was precipitated with 5% PEG (PEG800 and 300 mM NaCl adjusted to a concentration of 5%), then resuspended in PBS, and ssDNA was prepared using QIAprep Spin M13 kit (purchased from Qiagen).
  • the synthetic oligonucleotide strand was added to 100 ul of 50 mM Tris-HCl (Tris base was purchased from Soleil, adjusted to pH 7.5 with hydrochloric acid) to obtain a phosphorylation system containing the following components: 5 U T4 multinuclear Glycokinase, 10 mM MgCl 2 , 1 mM ATP and 5 mM DTT. After the phosphorylation system was reacted at 37 ° C for 1 hour, a phosphorylated oligonucleotide was obtained. The phosphorylated oligonucleotide strand was purified using a PCR purification kit (purchased from Tiangen).
  • phosphorylated oligonucleotide and Uracilated ssDNA were dissolved in 50 mM Tris-HCl buffer (pH of 10 mM MgCl 2 ) . In 7.5), after heating at 90 ° C for 2 minutes, the temperature was lowered to 25 ° C at a rate of 1 ° C / min to obtain an annealing-bound phosphorylated oligonucleotide and ssDNA complex.
  • dsDNA was obtained. .
  • the dsDNA was purified using Qiaquick PCR Purification Kit (purchased from Qiagen). The purified DNA was transformed into electrotransformed competent TG1.
  • a fresh single colony of Escherichia coli TG1 (purchased from Wuhan Qiling) was picked from the basic agar medium plate, inoculated into 20 ml of 2 ⁇ TY medium, and gently shaken, and cultured at 37 ° C until the OD600 was about 0.8.
  • a well-separated single phage was selected and inoculated into a 15 ml culture tube containing 2 to 3 ml of 2 x TY medium containing 25 ⁇ g/ml kanamycin. Incubate at 37 ° C, 250 rpm for 12 to 16 h. The infected supernatant was transferred to a 1.5 ml sterile microcentrifuge tube and centrifuged at 4 ° C for 2 min at maximum speed on a microfuge. The supernatant was transferred to a new tube and stored at 4 °C.
  • the prepared phage library was inoculated into 100 ml of 2 ⁇ TY medium containing 60 ⁇ g/ml ampicillin, and shake cultured at 37 ° C, 250 rpm until the OD600 was 0.8, and BM13 was added to a concentration of 2 ⁇ 107 pfu/ml. Incubate at 37 ° C, 300 rpm for 1 h, add 25 ⁇ g / ml kanamycin, continue to culture at 37 ° C for 14 ⁇ 18h. The bacterial solution was centrifuged, and the supernatant was precipitated with 5% PEG, and then resuspended in 5% MPBS for use.
  • Single colonies obtained after three rounds of panning were picked into 96-well plates.
  • the culture supernatant was prepared according to the 1.2 Chinese library proliferation method.
  • the specific steps of the ELISA are as follows: dilute the known antigen to 1 to 10 ⁇ g/ml with a coating buffer, add 0.1 ml per well, overnight at 4 ° C; wash 3 times a day; add 0.1 ml of the sample to be tested to the above In the coated reaction well, incubate at 37 ° C for 1 hour, wash; add 0.1 ml of freshly diluted enzyme-labeled secondary antibody (anti-KM13-HRP 1:5000), incubate at 37 ° C for 60 minutes, wash; last time with DDW washing. 0.1 ml of a temporarily prepared TMB substrate solution was added to each reaction well at 37 ° C for 10 to 30 minutes. The plate was read at a wavelength of 650 nm using an advanced plate reader.
  • the light absorption values of the respective holes are as shown in FIGS. 1B and 2B.
  • A ELISA were coated with anti-HLA-A2/RMFPNAPYL; the antibodies in each well were different.
  • B Data analysis.
  • the ordinate is the light absorption value of each hole at 650 nm, and the abscissa is 96 holes, wherein 1-8 is A1, B1, C1, D1, E1, F1, G1, H1, and 9-16 is A2, B2.
  • C2, D2, E2, F2, G2, H2, and so on, 89-96 are A12, B12, C12, D12, E12, F12, G12, H12.
  • Clones with A650nm above 0.8 in Figures 1 and 2 were selected for sequencing to obtain a plurality of different amino acid sequences.
  • the clone corresponding to the amino acid sequence shown in SEQ ID No. 13 was named M5-H1, and the corresponding single domain antibody was a single domain antibody M5-H1;
  • the clone corresponding to the amino acid sequence shown in SEQ ID No. 14 was named M5-G3, the corresponding single domain antibody is a single domain antibody M5-G3.
  • the clone corresponding to the amino acid sequence shown in SEQ ID No. 15 was named M5-F4, and the corresponding single domain antibody was the single domain antibody M5-F4.
  • the clone corresponding to the amino acid sequence shown in SEQ ID No. 16 was named M5-C3, and the corresponding single domain antibody was the single domain antibody M5-C3.
  • the amino acid sequence of the single domain antibody M5-H1 is shown in SEQ ID No. 13, and the coding gene sequence is shown in SEQ ID No. 19.
  • the amino acid sequence of the CDR1 of the complementarity determining region of the single domain antibody M5-H1 is as shown in SEQ ID No. 1
  • the amino acid sequence of the CDR2 of the complementarity determining region is as shown in SEQ ID No. 2
  • the amino acid sequence of the CDR3 of the complementarity determining region is SEQ. ID No.3 is shown.
  • the amino acid sequence of the single domain antibody M5-G3 is shown in SEQ ID No. 14, and the coding gene sequence is shown in SEQ ID No. 20.
  • the amino acid sequence of the CDR1 of the complementarity determining region of the single domain antibody M5-G3 is as shown in SEQ ID No. 4
  • the amino acid sequence of the CDR2 of the complementarity determining region is as shown in SEQ ID No. 5
  • the amino acid sequence of the CDR3 of the complementarity determining region is SEQ. ID No.6 is shown.
  • the amino acid sequence of the single domain antibody M5-F4 is shown in SEQ ID No. 15, and the coding gene sequence is shown in SEQ ID No. 21.
  • the amino acid sequence of the complementarity determining region CDR1 of the single domain antibody M5-F4 is as shown in SEQ ID No. 7
  • the amino acid sequence of the complementarity determining region CDR2 is as shown in SEQ ID No. 8
  • the amino acid sequence of the complementarity determining region CDR3 is SEQ. ID No.9 is shown.
  • the amino acid sequence of the single domain antibody M5-C3 is shown in SEQ ID No. 16, and the coding gene sequence is shown in SEQ ID No. 22.
  • the amino acid sequence of the CDR1 of the complementarity determining region of the single domain antibody M5-C3 is as shown in SEQ ID No. 10
  • the amino acid sequence of the CDR2 of the complementarity determining region is as shown in SEQ ID No. 11
  • the amino acid sequence of the CDR3 of the complementarity determining region is SEQ. ID No.12 is shown.
  • FIG. 3A The ELISA results of the specificity and affinity of four different single domain antibodies for different antigens are shown in Figure 3A. After TMB was added for 20 minutes, the light absorption was measured at a wavelength of 650 nm using an advanced plate reader, and the data was organized as shown in Fig. 3B. Among them, Figure 3A: ELISA plate A and B coated antigen HLA-A2/ITDQVPFSV, C, D two lines coated antigen HLA-A2/NLVPMVATV, E, F two lines coated antigen HLA-A2/RMFPNAPYL The two lines of G and H were coated with anti-HLA-A2/SLLMWITQC; the single domain antibody added to each well was added as a culture supernatant.
  • the first antibody in the eight wells of Line 1 is M5-H1
  • the line 2 is an antibody MA which cross-reacts with four antigens
  • the line 3 is an antibody of HLA-A2/NLVPMVATV, and the positive control M4
  • Line 4 is M5-G3
  • line 5 is M5-F4
  • line 6 is the negative control MB
  • line 7 is M5-C3.
  • Each antibody has two replicate wells for the affinity detection reaction of one antigen.
  • the plate was added to the TMB for 20 minutes and then read at a wavelength of 650 nm using an advanced plate reader. The data is shown in Figure 3B.
  • the ordinate is the light absorption at 650 nm
  • the graph shows that A650 is the average of two replicate wells.
  • the abscissas 1, 2, 3, and 4 are HLA-A2/ITDQVPFSV antigen, HLA-A2/NLVPMVATV antigen, HLA-A2/RMFPNAPYL antigen, and HLA-A2/SLLMWITQC antigen, respectively.
  • a, b, c, d, e, f, g are M5-H1, MA control, M4 control, M5-G3, M5-F4, MB control and M5-C3, respectively.
  • the coding genes of the four single domain antibodies were inserted between the NcoI and NotI restriction sites of the pET22b vector, respectively, to obtain recombinant vectors.
  • the constructed recombinant vector was transformed into E. coli/DE3 (Transgen Biotech, CD601-01), and the next day, the monoclonal was picked, shaken at 37 ° C, and shaken at 220 rpm until the OD600 was about 0.5. IPTG was added (working concentration was 1 mM). ), induction was induced at 18 ° C, 220 rpm for 20 h. Detection of protein expression. The ultrasonic supernatant was purified with ProteinA and ran SDS-PAG, see Figure 4.
  • Marker's strips are from 14, 25, 30, 40, 50, 70, 100, 120, 160KD.
  • Line1 is M5-H1
  • line2 is M5-G3
  • line3 is M5-F4.
  • Single domain antibodies are approximately 14 kD in size.
  • -H1-linker-Fc the structure of which is shown in FIG.
  • the recombinant vector pET22b-M5-H1-linker-Fc was transformed into E.coli/DE3, and the monoclonal antibody was picked the next day, and cultured at 37 ° C, shaking at 220 rpm until the OD600 was about 0.5, and after adding IPTG (working concentration: 1 mM), Expression was induced for 20 hours at 18 ° C, 220 rpm. After collecting the cells, they were resuspended in PBS (pH 7.4) and then sonicated. Ultrasonic crushing conditions: 600 W, ultrasound 2 seconds, interval 6 seconds, total 10 minutes, 16 ° C. After sonication, it was centrifuged at 12,000 rpm for 10 minutes at 4 °C.
  • the single domain antibody M5-H1 fusion Fc-expressed fusion protein M5-H1-Fc was purified with proteinA and ran SDS-PAGE. See Figure 6. Marker's strips are from 14, 25, 30, 40, 50, 70, 100, 120, 160 KD. Line1 is the reduced state M5-H1-Fc, approximately 43KD.
  • the recombinant vector pcDNA3.1-M5-F4-Fc has a structure as shown in FIG.
  • the recombinant vector pcDNA3.1-M5-F4-Fc was transfected into 293F cells (ThermoFisher, A14527), cultured for 4 days, centrifuged, and the supernatant was collected and purified with Protein A.
  • the fusion protein M5-F4-Fc was purified and ran SDS-PAG, see Figure 8.
  • A is a reduced electrophoresis pattern of the purified fusion protein M5-F4-Fc
  • B is a non-reduced electropherogram of the purified fusion protein M5-F4-Fc. Marker's strips are from 14, 25, 30, 40, 50, 70, 100, 120, 160 KD.
  • Line1 and Line2 are the reduced M5-F4-Fc and the non-reduced M5-F4-Fc, respectively.
  • the single domain antibody expressed on pET22b was collected, then resuspended in PBS and sonicated. Ultrasonic crushing conditions: 600 W, ultrasound 2 seconds, interval 6 seconds, total 10 minutes, 16 ° C. After ultrasonication, the cells were centrifuged at 12000 rpm for 10 minutes at 4 ° C, and the supernatant was taken for ELISA identification (Protein A-HRP as secondary antibody) for specific antigens, and the plate was read. The data analysis was shown in Fig. 9.
  • the ordinate is the light absorption value at 650 nm
  • the abscissa 1, 2, 3, 4 is the HLA-A2/ITDQVPFSV antigen, the HLA-A2/NLVPMVATV antigen, the HLA-A2/RMFPNAPYL antigen, and the HLA-A2/SLLMWITQC antigen.
  • the three samples were ultrasonic supernatants of M5-H1, M5-G3, and M5-F4, which were separately expressed in pET22b.
  • the single-domain antibody expressed in monomeric form in pET22b can specifically recognize HLA-A2/RMFPNAPYL antigen, and does not bind to other antigen complexes.
  • its ability to bind antigen in vitro is weaker than that of the antibody displayed in phage.
  • the reason may be that the antibodies are monovalent when expressed alone, resulting in a decrease in the ability to bind antigen.
  • the specific recognition and affinity identification of the fusion protein M5-H1-Fc expressed in pET22b and the fusion protein M5-F4-Fc expressed in pcDNA3.1 were analyzed, and the data analysis is shown in FIG. Among them, the ordinate has a light absorption value at 650 nm, and the abscissas 1, 2, 3, and 4 are HLA-A2/ITDQVPFSV antigen, HLA-A2/NLVPMVATV antigen, HLA-A2/RMFPNAPYL antigen, and HLA-A2/SLLMWITQC antigen, respectively. .
  • Example 4 Fusion protein M5-F4-Fc specifically recognizes naturally processed, HLA-A2/RMFPNAPYL complexes expressed on the surface of tumor cells
  • the results are shown in Figure 11.
  • the solid phase negative is BV-173 cells without primary antibody plus secondary antibody goat anti-human Fc (FITC)
  • BM is BV-173 cells plus HLA-A2/RMFPNAPY Fc fusion protein BM and secondary antibody
  • M5- F4 is BV-173 cells plus M5-F4-Fc fusion protein and secondary antibody.
  • the results showed that the fusion protein M5-F4-Fc specifically recognized HLA-A2/RMFPNAPY with high affinity.
  • the interaction and affinity constants of the antibody fusion proteins M5-H1-Fc and M5-F4-Fc of RMFPNAPYL/HLA-A2 with RMFPNAPYL/HLA-A2 were determined by plasmon resonance technique.
  • the streptavidin-coupled sensor chip (senser chip SA) was used in the experiment, and HBS+EP+ was used as the mobile phase buffer.
  • the unrelated antigen SLLMWITQC/HLA-A2 was immobilized as a reference antigen on one channel of the sensor chip, and RMFPNAPYL/HLA-A2 was immobilized on the other channel as an experimental antigen, and the reference antigen was used to detect background binding.
  • the fusion antibodies M5-F4-Fc and M5-H1-Fc were diluted, respectively, and injected at a concentration of 10 nm to 220 nm, and the samples were simultaneously flowed through the surface of the channel to which SLLMWITQC/HLA-A2 and RMFPNAPYL/HLA-A2 were immobilized.
  • the experimental data were collected and analyzed by Biacore T200 instrument.
  • the reaction kinetic curve was fitted with a 1:1 binding model, and the binding rate constant (Ka), dissociation rate constant (Kd), and affinity constant (KD) were calculated.
  • Ka binding rate constant
  • Kd dissociation rate constant
  • KD affinity constant
  • the abscissa is time
  • the ordinate is the response value (RU) of the combination of molecules
  • 1 is the reference channel
  • 2 is the experimental channel.
  • the experimental results showed that the fusion antibody M5-H1-Fc specifically binds to RMFPNAPYL/HLA-A2, and does not cross-react with the reference antigen and does not bind.
  • the binding rate constant was 2.48E+04 (1/Ms)
  • the dissociation rate constant was 5.78E-04 (1/s)
  • the affinity constant was 2.33E-08 (M)
  • the fusion antibody M5-F4-Fc specifically binds to RMFPNAPYL/HLA-A2, does not cross-react with the reference antigen, and does not bind.
  • the binding rate constant was 6.94E+04 (1/Ms)
  • the dissociation rate constant was 4.18E-04 (1/s)
  • the affinity constant was 6.03E-09 (M), as shown in FIG.
  • the in vitro killing assay was performed using the EuTDA cytotoxicity kit (perkinelmer, AD0116). The specific steps were as follows: 2.5uL of BATDA reagent was added to 1*10E6 target cell BV173, incubated at 37 ° C for 30 minutes, and then washed with cell culture medium 3-5 Next, 2*10E4 BV173 target cells (100 uL) were plated in 96-well plates. The target protein was diluted stepwise to a concentration of 50, 5, 0.5, and 0.05 ng/uL, and 100, 10, 1, 0.1, and 0.01 ng of the target protein were added to an appropriate amount of T cells for 1-2 hours at 37 ° C.
  • the cells were plated in 96-well plates plated with target cells and incubated at 37 ° C, 5% CO 2 for 2 hours.
  • Background control T cell culture medium was mixed with BV173 cell culture medium, and incubated at 37 ° C, 5% CO 2 for 2 hours; maximum release: equal amount of BV173 target cells were added to 100 uL of 1% Triton lysate, incubated at 37 ° C, 5% CO 2 2 hours; spontaneous release: An equal amount of BV173 target cells were added to an equal amount of T cell culture medium for 2 hours at 37 ° C, 5% CO 2 .
  • killing efficiency ⁇ (experimental value - spontaneous release value) / (maximum release value - spontaneous release value) ⁇ * 100%.
  • Spontaneous release efficiency ⁇ (spontaneous release value - background control value) / (maximum release value - background control value) ⁇ * 100%.
  • the bispecific antibody M5-H1-antiCD3 can mediate the killing of HLA-A2/RMFPNAPYL-positive tumor cells by T cells.
  • the present invention provides a single domain antibody that specifically recognizes the HLA-A2/RMFPNAPYL complex and its CDR1, CDR2 and CDR3 amino acid sequences for specific recognition.
  • the present invention also provides a fusion protein that specifically recognizes the HLA-A2/RMFPNAPYL complex based on such an antibody.
  • the antibody and fusion protein of the present invention can not only recognize the artificially synthesized HLA-A2/RMFPNAPYL complex, but also can be combined with the natural antigen to process the HLA-A2/RMFPNAPYL complex expressed on the surface of tumor cells, and can be further developed into a related tumor treatment. product.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un anticorps à domaine unique pour identifier un complexe HLA-A2/RMFPNAPYL et des séquences d'acides aminés CDR1, CDR2 et CDR3 de l'anticorps. L'invention concerne également une protéine de fusion basée sur l'anticorps et capable d'identifier spécifiquement le complexe HLA-A2/RMFPNAPYL. L'anticorps et la protéine de fusion non seulement identifient un complexe HLA-A2/RMFPNAPYL synthétisé artificiellement, mais se lient également au complexe HLA-A2/RMFPNAPYL traité par combinaison d'un antigène naturel et exprimé sur la surface de cellules tumorales, et peut en outre être développé dans un produit de traitement de tumeur associé.
PCT/CN2017/118283 2016-12-28 2017-12-25 Anticorps à domaine unique pour identifier un complexe formé par une molécule hla-a2 et un polypeptide à chaîne courte rmfpnapyl WO2018121474A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201611232233.XA CN107698681B (zh) 2016-12-28 2016-12-28 一种识别hla-a2/rmfpnapyl的单域抗体
CN201611232233.X 2016-12-28

Publications (1)

Publication Number Publication Date
WO2018121474A1 true WO2018121474A1 (fr) 2018-07-05

Family

ID=61168425

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/118283 WO2018121474A1 (fr) 2016-12-28 2017-12-25 Anticorps à domaine unique pour identifier un complexe formé par une molécule hla-a2 et un polypeptide à chaîne courte rmfpnapyl

Country Status (2)

Country Link
CN (1) CN107698681B (fr)
WO (1) WO2018121474A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110872346A (zh) * 2018-08-30 2020-03-10 天津天锐生物科技有限公司 一种识别hla-a2分子与fmnkfiyei短肽形成的复合物的单域抗体
CN114057880B (zh) * 2021-11-10 2022-09-09 四川大学华西医院 一种dll3单克隆抗体

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103619882A (zh) * 2011-04-01 2014-03-05 纪念斯隆-凯特琳癌症中心 对hla-a2呈递的wt1肽特异的t细胞受体样抗体
WO2015070078A1 (fr) * 2013-11-07 2015-05-14 Memorial Sloan-Kettering Cancer Center Anticorps anti-wt1/hla renforcé par fc

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103619882A (zh) * 2011-04-01 2014-03-05 纪念斯隆-凯特琳癌症中心 对hla-a2呈递的wt1肽特异的t细胞受体样抗体
WO2015070078A1 (fr) * 2013-11-07 2015-05-14 Memorial Sloan-Kettering Cancer Center Anticorps anti-wt1/hla renforcé par fc

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VEOMETT, N. ET AL.: "Therapeutic Efficacy of an Fc-Enhanced TCR-like Antibody to the Intracellular WT1 Oncoprotein", CLINICAL CANCER RESEARCH, vol. 20, no. 15, 21 May 2014 (2014-05-21), pages 4036 - 4046, XP055170338 *

Also Published As

Publication number Publication date
CN107698681A (zh) 2018-02-16
CN107698681B (zh) 2020-07-07

Similar Documents

Publication Publication Date Title
CN107686520B (zh) 抗pd-l1纳米抗体及其应用
CN109096396B (zh) 一种抗pd-l1人源化纳米抗体及其应用
CN109096395B (zh) 阻断型cd47纳米抗体及其用途
CN107216389B (zh) 抗pd-l1纳米抗体及其编码序列和用途
JP7450594B2 (ja) 治療分子
ES2445695T3 (es) Moléculas de unión con base en fibronectina mejorada y usos de las mismas
CN109963591B (zh) B7h3抗体-药物偶联物及其医药用途
TWI402078B (zh) 抗ｃｓｆ－１ｒ抗體
WO2018121473A1 (fr) Anticorps à domaine unique capable de reconnaître l'albumine sérique humaine
CN107683289A (zh) IL13RAα2结合剂和其在癌症治疗中的用途
WO2011051327A2 (fr) Petites protéines à chaîne unique de type anticorps
WO2012176765A1 (fr) Anticorps recombinant anti-p-cadhérine humaine (cdh3)
JP2019523651A (ja) 抗ｐｓｍａ抗体およびその使用
CN112225807B (zh) 抗il5纳米抗体及其应用
WO2021219127A1 (fr) Anticorps bispécifique ciblant her2 et pd-1 et son utilisation
WO2022117040A1 (fr) Anticorps anti-b7-h3 humain et utilisation associée
WO2018121474A1 (fr) Anticorps à domaine unique pour identifier un complexe formé par une molécule hla-a2 et un polypeptide à chaîne courte rmfpnapyl
WO2018121475A1 (fr) Complexe de reconnaissance d'anticorps à domaine unique formé par une molécule hla-a2 et un peptide court sllmwitqc
CN115109156A (zh) 一种靶向bcma的纳米抗体及其应用
CN111936516B (zh) 新型抗egfr抗体多肽
CN111378040B (zh) 检测多种恶性肿瘤细胞的抗体及其应用
JP2024509557A (ja) ＮＫｐ４６に対する抗体とその応用
JP2019050737A (ja) Ｃｄ４４陽性ｔｍｅｍ−１８０陽性のがん細胞由来微粒子、これを用いた抗ｔｍｅｍ−１８０抗体療法が有効ながん患者の選別方法、選別された患者に対する抗ｔｍｅｍ−１８０抗体を含む抗がん剤、および前記方法に用いるキット
CN113461825A (zh) 一种抗pd-l2纳米抗体及其应用
WO2018121476A1 (fr) Anticorps à domaine unique reconnaissant un complexe formé par une molécule hla-a2 et un peptide court nlvpmvatv

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17888264

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17888264

Country of ref document: EP

Kind code of ref document: A1