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WO2025081589A1 - Préparation de nanocorps cd4 et son utilisation - Google Patents

Préparation de nanocorps cd4 et son utilisation Download PDF

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Publication number
WO2025081589A1
WO2025081589A1 PCT/CN2023/135020 CN2023135020W WO2025081589A1 WO 2025081589 A1 WO2025081589 A1 WO 2025081589A1 CN 2023135020 W CN2023135020 W CN 2023135020W WO 2025081589 A1 WO2025081589 A1 WO 2025081589A1
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seq
cancer
cdr3
cdr1
cdr2
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范星星
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Shenzhen Fuyuan Biotechnology Co Ltd
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Shenzhen Fuyuan Biotechnology Co Ltd
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    • 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
    • C07K16/2812—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 CD4
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70514—CD4

Definitions

  • the present invention relates to the field of biomedicine, and in particular to the preparation and application of a CD4 nano antibody.
  • CD4 is a protein present on the surface of cells of the human immune system.
  • CD4 also known as T lymphocyte cofactor receptor, is a membrane molecule in the human immune system, mainly present on the surface of helper T lymphocytes.
  • MHC-II major histocompatibility complex II
  • CD4 can also help determine whether the cell's own immune response is appropriate and maintain a balanced state of immunity.
  • CD4-expressing cells such as regulatory T cells (Treg) have been shown to suppress tumor-induced immunity, thereby impairing the efficacy of various cancer therapies and CD4 T cell lymphomas, which can lead to the continuous progression of cancer. Therefore, antibodies that can effectively target and eliminate CD4-expressing cells are needed.
  • the current types of anti-CD4 antibodies are limited, and the development of new anti-CD4 nanoantibodies is crucial.
  • One of the purposes of the embodiments of the present application includes providing a nanobody targeting CD4.
  • a nanobody targeting CD4 whose VHH comprises: a CDR1 as shown in SEQ ID NO.15, a CDR2 as shown in SEQ ID NO.16, and a CDR3 as shown in SEQ ID NO.17; or, a CDR1 as shown in SEQ ID NO.18, a CDR2 as shown in SEQ ID NO.19, and a CDR3 as shown in SEQ ID NO.20; or, a CDR1 as shown in SEQ ID NO.21, a CDR2 as shown in SEQ ID NO.22, and a CDR3 as shown in SEQ ID NO.23; or, a CDR1 as shown in SEQ ID NO.
  • the species of the framework region is cattle, horses, pigs, sheep, mice, dogs, cats, rabbits, camels, alpacas, donkeys, deer, minks, chickens, ducks, geese or humans; optionally, alpacas or humans.
  • the amino acid sequence is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, or SEQ ID NO.14.
  • amino acid sequence is shown as SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48 or SEQ ID NO.49.
  • an antibody conjugate comprising: the nanobody defined in the first aspect; and a conjugate conjugated to the nanobody.
  • the conjugate comprises a small molecule drug, a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA alkylating agent, a topoisomerase inhibitors, tubulin-binding agents or other anticancer drugs such as androgen receptor inhibitors.
  • a fusion protein comprising: the nanobody defined in the first aspect; and a cytokine linked to the nanobody.
  • the fused protein comprises different cytokines, such as IL-2 and IL-15 allosteric agonists, IL-12, IL-18 and IL-36, etc.
  • an isolated nucleic acid molecule is provided, which encodes the Nanobody described in the first aspect.
  • a vector comprising the isolated nucleic acid molecule described in the fourth aspect.
  • it comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or a retrovirus.
  • it comprises a pDisplay yeast display plasmid.
  • a host cell which comprises the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect. In some embodiments of the present application, it comprises a yeast cell.
  • a method for producing a nanobody targeting CD4 comprising the following steps: culturing the cells described in the sixth aspect; and isolating the nanobody targeting CD4 from the obtained culture product.
  • a CD4 detection kit comprising the Nanobody defined in the first aspect.
  • a method for treating a CD4-related disease in a subject comprising administering to the subject a therapeutically effective amount of the Nanobody described in the first aspect, the antibody conjugate described in the second aspect, or the fusion protein described in the third aspect.
  • a method for diagnosing a CD4-related disease in a subject comprising: obtaining a sample from the subject; and contacting the sample obtained from the subject with the Nanobody described in the first aspect; determining the presence or amount of CD4 in the sample; and correlating the presence or amount of CD4 with the presence or status of the CD4-related disease in the subject.
  • the CD4-related disease includes cancer, adaptive immune disease, autoimmune disease, inflammatory disease or infectious disease.
  • the cancer includes lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, adenocarcinoma, leukemia, myeloma or lymphoma.
  • FIG1 is a diagram showing the SDS-PAGE detection results of CD4 recombinant proteins with different tags (His and Fc tags);
  • FIG2 is an electrophoresis diagram of the amplified product of the VHH fragment
  • FIG3 is the result of diversity analysis of antibody display library
  • FIG4 shows preliminary FACS test results of candidate antibodies
  • FIG5 is the SDS-PAGE test result of the candidate antibodies
  • FIG6 is the result of ELISA test of candidate antibodies
  • FIG7 is the result of FACS detection of candidate antibodies
  • FIG8 is the ADCC functional experimental results of candidate antibodies
  • Figure 9 shows the preliminary results of FACS testing of humanized antibodies
  • Figure 10 shows the results of humanized antibody ELISA gradient dilution test
  • Figures 11 and 12 are the results of FACS gradient dilution detection of humanized antibodies
  • FIG13 is the ADCC test results of candidate humanized antibodies
  • FIG. 14 shows the affinity test results of candidate humanized antibodies.
  • the technical solution of "A, and/or, B, and/or, C, and/or, D” includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").
  • plural means one or greater than or equal to two.
  • one or more means one or greater than or equal to two.
  • suitable mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.
  • first”, “second”, “third”, “fourth”, etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features.
  • first”, “second”, “third”, “fourth”, etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.
  • the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.
  • the temperature parameters in the present invention are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ⁇ 5°C, ⁇ 4°C, ⁇ 3°C, ⁇ 2°C, and ⁇ 1°C are allowed.
  • % (w/w) and wt% both represent weight percentage, % (v/v) refers to volume percentage, and % (w/v) refers to mass volume percentage.
  • affinity refers to the strength of the non-covalent interaction between an immunoglobulin molecule (ie, an antibody) or a fragment thereof and an antigen.
  • subject includes humans and non-human animals.
  • Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians and reptiles.
  • patient or “subject” are used interchangeably herein.
  • “treating” or “treatment” of a condition includes preventing or alleviating the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or eliminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.
  • vector refers to a vehicle into which a genetic element can be operably inserted to achieve expression of the genetic element, such as production of a protein, RNA or DNA encoded by the genetic element, or replication of the genetic element.
  • the vector can be used to transform, transduce or transfect a host cell so that it expresses the carried genetic element in the host cell.
  • vectors include plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages, such as lambda phages or M13 phages; and animal viruses.
  • the vector can contain a variety of elements for controlling expression, including promoter sequences, transcription start sequences, enhancer sequences, selectable elements and reporter genes.
  • the vector can contain an origin of replication.
  • the vector can also include materials that facilitate its entry into cells, including but not limited to viral particles, liposomes or protein envelopes.
  • the vector can be an expression vector or a cloning vector.
  • the present disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
  • host cell refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.
  • CD4 an acronym for the term “cluster of differentiation 4", refers to a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and encompasses all isoforms of CD4.
  • a “CD4-related” disease or condition refers to any disease or condition associated with CD4 and/or CD4-expressing cells, such as CD4+T cells, particularly Tregs.
  • a CD4-related disease or condition is, for example, an autoimmune disease, cancer, an adaptive immune disease, an inflammatory disease, or an infectious disease.
  • cancer refers to any medical condition characterized by malignant cell growth or tumor, abnormal proliferation, infiltration or metastasis, which can be benign or malignant, and includes solid tumors and non-solid cancers (e.g., hematological malignancies), such as leukemia.
  • solid tumor refers to a solid mass of neoplastic and/or malignant cells.
  • the binding specificity and affinity of an antibody are mainly determined by the CDR sequence.
  • the amino acid sequence of the non-CDR region can be easily changed to obtain a variant with similar biological activity. Therefore, the present application also includes the "functional derivative" of the neutralizing antibody.
  • “Functional derivative” refers to a variant with amino acid substitutions, and a functional derivative retains detectable binding protein activity.
  • “Functional derivatives” may include "variants” and "fragments” because they have exactly the same CDR sequence as the neutralizing antibody described in the present invention and therefore have similar biological activity.
  • the antibodies described herein may contain one or more substitutions, deletions or insertions of amino acids relative to their CDR sequences, for example, the number of amino acid insertions, substitutions or deletions does not exceed 3, preferably 1.
  • Substitutions, deletions or insertions may be introduced into nucleic acid molecules encoding the antibodies of the invention by conventional techniques such as site-directed mutagenesis or PCR-mediated mutagenesis. In some embodiments, conservative amino acid substitutions are made at one or more sites.
  • a "conservative amino acid substitution" is one in which an amino acid residue is replaced by an amino acid residue with a similar side chain. The situation.
  • Families of amino acids with similar side chains have been defined in the prior art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, gluta
  • VH variable domain
  • CDRs complementarity determining regions
  • the present application provides a nanobody targeting CD4, whose VHH includes: CDR1 as shown in SEQ ID NO.15, CDR2 as shown in SEQ ID NO.16, and CDR3 as shown in SEQ ID NO.17; or, CDR1 as shown in SEQ ID NO.18, CDR2 as shown in SEQ ID NO.19, and CDR3 as shown in SEQ ID NO.20; or, CDR1 as shown in SEQ ID NO.21, CDR2 as shown in SEQ ID NO.22, and CDR3 as shown in SEQ ID NO.23; or, CDR1 as shown in SEQ ID NO.24
  • the antibody or fragment described herein is humanized because it comprises at least one human framework region; further optionally, the sequences of the heavy chain framework region and the light chain framework region are all antibody framework region sequences derived from humans.
  • one or more (e.g., one, two, three, four, five, or six) framework regions of the antibodies produced by the hybridoma of the present invention can be replaced with one or more (e.g., one, two, three, four, five, or six) human framework regions.
  • humanized antibodies are generally less immunogenic to humans and therefore can bring therapeutic advantages in some cases.
  • Various human framework regions are known to those skilled in the art. Methods for preparing humanized antibodies are known in the art.
  • the species origin of the framework region is cattle, horses, pigs, sheep, mice, dogs, cats, rabbits, camels, alpacas, donkeys, deer, minks, chickens, ducks, geese or humans; optionally, alpacas or humans.
  • amino acid sequence is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, or SEQ ID NO.14.
  • amino acid sequence is shown as SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48 or SEQ ID NO.49.
  • the conjugate includes a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a microtubule binding agent or other anticancer drug.
  • a clearance modifier e.g., a chemotherapeutic agent
  • a toxin e.g., a radioisotope, a lanthanide
  • a luminescent label e.g., a fluorescent label
  • an enzyme-substrate label e.g., a DNA alkylating agent
  • a topoisomerase inhibitor e.g., a microtubule binding agent or other anticancer drug.
  • microtubule binding agent e.g., a microtubule binding agent or other anticancer drug.
  • anticancer drug e.g.,
  • the present application provides an isolated nucleic acid molecule encoding the nanobody described in the first aspect.
  • the present application provides a vector comprising the isolated nucleic acid molecule described in the fourth aspect.
  • it comprises a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or a retrovirus.
  • it comprises a pDisplay yeast display plasmid.
  • the present application provides a host cell, which comprises the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect. In some examples of the present application, it comprises a yeast cell.
  • the present application provides a method for producing a nanobody targeting CD4, comprising the following steps: culturing the cells described in the fifth aspect; and isolating the nanobody targeting CD4 from the obtained culture product.
  • the present application provides the use of the nanobody defined in the first aspect in the preparation of a CD4 detection kit.
  • the present application provides a CD4 detection kit, which comprises the nanobody defined in the first aspect.
  • the present application provides a method for detecting the presence or amount of CD4 in a sample, which comprises contacting the sample with the Nanobody described in the first aspect, and determining the presence or amount of CD4 in the sample.
  • the present application provides a method for diagnosing a CD4-related disease in a subject, comprising: obtaining a sample from the subject; and contacting the sample obtained from the subject with the Nanobody described in the first aspect; determining the presence or amount of CD4 in the sample; and correlating the presence or amount of CD4 with the presence or status of the CD4-related disease in the subject.
  • the CD4-related disease includes cancer, adaptive immune disease, autoimmune disease, inflammatory disease or infectious disease.
  • the cancer includes lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, adenocarcinoma, leukemia, myeloma or lymphoma.
  • the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified.
  • acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.
  • the CD4 protein extracellular segment sequence was synthesized, a His tag was added to the C-terminus, and the antigen expression vector was constructed by subcloning into a eukaryotic expression vector. After transient transfection of 293F cells, the antigen was purified by Ni-NTA column. The purified antigen was tested with a positive control antibody (ibalizumab). ELISA test was performed. In this step,
  • CD4 protein extracellular segment sequence (SEQ ID NO.50):
  • Eukaryotic expression vector pcDNA3.4.
  • recombinant CD4 proteins with different tags were constructed respectively, and the recombinant CD4 proteins were prepared by affinity chromatography and detected by SDS-PAGE.
  • the above-prepared antigen was used to immunize one alpaca, and the immunization process was performed three times subcutaneously at multiple points. The immunization process is shown below.
  • the serum from the immunized alpaca was diluted to a limiting value according to the indicated dilution gradient, and then the ELISA test was performed using a 96-well plate pre-coated with CD4-His antigen.
  • the immune serum binds to the target protein, and the OD450 value changes gradually with the serum dilution gradient, indicating that the immunization is successful.
  • 100 mL of peripheral blood was collected to establish a single domain antibody phage display library.
  • Figure 2 shows the results of agarose gel electrophoresis of PCR products.
  • the first round of PCR obtained two PCR bands of about 1000 bp and 750 bp, and the 750 bp fragment was recovered from the gel as a template for the second round of PCR.
  • the second round of PCR obtained a band of about 450 bp, which is the VHH fragment.
  • VHH fragment was digested with SfiI and subcloned into the yeast display vector pDispay.
  • the ligation product was electroporated into yeast competent cells to construct a single domain antibody yeast display library.
  • the antibody display library capacity was calculated to be 3.1 ⁇ 10 8 . From the display library, 20 monoclones were randomly selected for sequencing to analyze the diversity of the constructed display library. According to the comparison of sequencing results, the empty rate and antibody duplication rate of the display library were no higher than 5%. See Figure 3.
  • ibalizumab as the positive control antibody, preliminary FACS testing was performed on candidate antibodies B-H11, B-D07, B-B07, B-A06, B-F04, 13-H09, 13-F12, 13-H05, 13-B01, 13-D11, 13-G02, 13-C12, 13-A03, and 13-B09.
  • the candidate antibodies were diluted in a gradient manner and incubated with the cell lines expressing the target gene.
  • the binding of the candidate antibodies to the target cells was detected by FACS at different concentrations.
  • candidate antibodies 13-A03 corresponding to B-13-A03 in FIG. 8
  • 13-H05 corresponding to B-13-H05 in FIG. 8
  • B-H11, 13-C12 corresponding to B-13-C12 in FIG. 8
  • 13-H09 corresponding to B-13-H09 in FIG. 8
  • B-A06, and B-B07 were selected for ADCC functional experiments.
  • clone B-A06 was selected for humanized design; the sequence of the designed humanized candidate antibody is as follows: B-A06-HM1 (SEQ ID NO.45):
  • test results are shown in Figure 9. According to the preliminary FACS test results, all designed humanized clones were able to bind to the target antigen; affinity testing was performed after further purification of the antibodies.

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Abstract

La présente invention concerne la préparation d'un nanocorps CD4 et son utilisation. L'anticorps ciblant CD4 comporte une CDR1, une CDR2 et une CDR3 spécifiques, et présente une affinité relativement forte pour CD4.
PCT/CN2023/135020 2023-10-19 2023-11-29 Préparation de nanocorps cd4 et son utilisation Pending WO2025081589A1 (fr)

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CN202311375378.5A CN117659192A (zh) 2023-10-19 2023-10-19 Cd4纳米抗体的制备及其应用
CN202311375378.5 2023-10-19

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