WO2022152185A1 - 靶向cd5的全人源抗体 - Google Patents

靶向cd5的全人源抗体 Download PDF

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WO2022152185A1
WO2022152185A1 PCT/CN2022/071674 CN2022071674W WO2022152185A1 WO 2022152185 A1 WO2022152185 A1 WO 2022152185A1 CN 2022071674 W CN2022071674 W CN 2022071674W WO 2022152185 A1 WO2022152185 A1 WO 2022152185A1
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antibody
antigen
seq
binding
amino acid
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PCT/CN2022/071674
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French (fr)
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谭涛超
魏巧娥
贾向印
谈佳玥
谢萌
戴振宇
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南京驯鹿医疗技术有限公司
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Priority to CN202280009712.2A priority Critical patent/CN116829600A/zh
Priority to EP22739063.0A priority patent/EP4279508A1/en
Priority to US18/261,059 priority patent/US20240092925A1/en
Publication of WO2022152185A1 publication Critical patent/WO2022152185A1/zh

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    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
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    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/62—DNA sequences coding for fusion proteins
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/13—Antibody-based
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/21—Transmembrane domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
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    • C07K2317/00—Immunoglobulins specific features
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    • 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
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    • 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
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    • 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®
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07—ORGANIC CHEMISTRY
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a fully human antibody or an antigen-binding fragment thereof that specifically binds to human CD5 antigen protein and the native CD5 antigen on the cell membrane surface.
  • CAR chimeric antigen receptor
  • CD5 is a type I transmembrane glycosylated protein that plays an important role in the negative regulation of T-cell receptor signaling and promotes the survival of normal and malignant lymphocytes.
  • CD5 is not expressed on the surface of hematopoietic stem cells, but is highly expressed on malignant T cells.
  • CD5 is one of the characteristic surface markers of malignant T-cell tumors, and 80% of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas express CD5.
  • T-ALL T-cell acute lymphoblastic leukemia
  • CD5 is also expressed on some malignant B-cell tumors.
  • CD5 monoclonal antibody has shown moderate therapeutic effect in patients with cutaneous T-cell lymphoma (CTCL) or chronic lymphocytic leukemia (CLL).
  • CTCL cutaneous T-cell lymphoma
  • CLL chronic lymphocytic leukemia
  • an antibody or antigen-binding fragment thereof targeting CD5 wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2, and HCDR3 selected from one of the following combinations:
  • amino acid sequence of HCDR1 is GFTFSHSA (SEQ ID NO: 1);
  • amino acid sequence of HCDR2 is IYARGGYT (SEQ ID NO: 2);
  • HCDR3 The amino acid sequence of HCDR3 is ARGYHLEYMVSQDV (SEQ ID NO: 3);
  • amino acid sequence of HCDR2 is ISSSGSTI (SEQ ID NO: 5);
  • HCDR3 The amino acid sequence of HCDR3 is ARVAQREGDV (SEQ ID NO: 6);
  • HCDR2 The amino acid sequence of HCDR2 is ISAYNGDT (SEQ ID NO: 8);
  • amino acid sequence of HCDR3 is ARYESMSGQDI (SEQ ID NO: 9);
  • HCDR1 The amino acid sequence of HCDR1 is GYSFSNHW (SEQ ID NO: 10);
  • amino acid sequence of HCDR2 is VYPGDSDT (SEQ ID NO: 11);
  • HCDR3 The amino acid sequence of HCDR3 is ARGGTIDGDYGGRQDF (SEQ ID NO: 12); or
  • the antibody includes a variant of the combination of CDR sequences in any one of (1)-(4), wherein the variant has at least 90% of the CDR sequences in any one of (1)-(49). Sequence identity, or a total of at least 1 and no more than 10, or no more than 5, 4, 3, 2, or 1 amino acid change in the CDR sequence.
  • amino acid sequence of the heavy chain variable region is selected from any of the following:
  • amino acid sequence of the heavy chain variable region and/or light chain variable region is selected from any of the following:
  • the antibody is a fully human antibody.
  • the antibody is a single domain antibody.
  • the antibody has a KD value for binding to the CD5 antigen, as determined by biofilm interferometry, below 10-7 M, preferably below 10-8 M.
  • the present application also provides fusion proteins comprising one or two antigen-binding functional moieties, wherein each of the antigen-binding functional moieties comprises the above-mentioned antibody or antigen-binding fragment thereof.
  • the fusion protein further includes an Fc fragment.
  • the two antigen-binding functional moieties each bind the same or different antigenic epitopes.
  • the fusion protein comprises a first antigen-binding functional portion and a second antigen-binding functional portion linked in tandem; wherein the first antigen-binding functional portion comprises a first heavy chain variable region (HCVR), wherein The first heavy chain variable region includes HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8 and HCDR3 shown in SEQ ID NO: 9; wherein the second antigen-binding functional part includes the first Double chain variable region (HCVR), the second heavy chain variable region includes HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3.
  • HCVR first heavy chain variable region
  • the first heavy chain variable region includes HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8 and HCDR3 shown in SEQ ID NO: 9
  • the second antigen-binding functional part includes the first Double chain variable region (HCVR)
  • the second heavy chain variable region includes
  • the first antigen-binding functional moiety is N-terminal to the second antigen-binding functional moiety.
  • the fusion protein comprises the heavy chain variable region sequence set forth in SEQ ID NO: 19 and the heavy chain variable region sequence set forth in SEQ ID NO: 17 linked in tandem.
  • the heavy chain variable region sequence set forth in SEQ ID NO:19 is N-terminal to the heavy chain variable region sequence set forth in SEQ ID NO:17.
  • the antigen-binding functional moiety is directly linked through a linker molecule; preferably, the linker molecule comprises the amino acid sequence shown in SEQ ID NO:21.
  • the fusion protein has an EC50 value of 1-5 nM for binding between the fusion protein and CD5 positive cells as determined by flow cytometry.
  • the present application also includes isolated nucleic acid molecules encoding the above-described antibodies or antigen-binding fragments or fusion proteins thereof.
  • the nucleic acid molecule comprises the nucleotide sequence shown in any one of SEQ ID NOs: 13-16.
  • the present application also includes an expression vector comprising the nucleic acid molecule described herein.
  • the vectors are plasmid, retroviral and lentiviral vectors.
  • the present application also includes a host cell comprising the expression vector described in the present application.
  • the present application also includes a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment or fusion protein thereof described in the present application, and a pharmaceutically acceptable carrier or diluent.
  • the application also includes a method of treating a disease or disorder by administering to a patient in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof, fusion protein, or host cell described herein, or A pharmaceutical composition to eliminate, inhibit or reduce CD5 activity, thereby preventing, alleviating, ameliorating or inhibiting a disease or disorder.
  • the present application also includes the use of the above-mentioned antibodies or antigen-binding fragments thereof, fusion proteins or the above-mentioned host cells in the preparation of a medicament for eliminating, inhibiting or reducing CD5 activity, thereby preventing, alleviating, improving or inhibiting a disease or condition.
  • the present application also includes the use of the above-described antibodies or antigen-binding fragments thereof, fusion proteins, or the above-described host cells as medicaments or in therapy, eg, to eliminate, inhibit or reduce CD5 activity, thereby preventing, alleviating, ameliorating or inhibiting a disease or disorder .
  • the disease or disorder is selected from: cancer or autoimmune disease.
  • the cancer is selected from: a malignant T cell tumor or a malignant B cell tumor T cell malignancy.
  • the malignant T-cell tumor is selected from T-cell acute lymphoblastic leukemia (T-ALL), T-cell lymphoma (eg, peripheral T-cell lymphoma or cutaneous T-cell lymphoma (CTCL)), wherein Said malignant B cell tumor is selected from chronic lymphocytic leukemia (B-CLL) or mantle cell lymphoma (B-MCL).
  • T-ALL T-cell acute lymphoblastic leukemia
  • T-cell lymphoma eg, peripheral T-cell lymphoma or cutaneous T-cell lymphoma (CTCL)
  • Said malignant B cell tumor is selected from chronic lymphocytic leukemia (B-CLL) or mantle cell lymphoma (B-MCL).
  • the present application also includes a kit for detecting CD5 protein in a sample.
  • the kit includes the antibody or antigen-binding fragment or fusion protein thereof described herein.
  • the detection can be in vitro or in vivo.
  • the present application also includes antibodies or fragments that compete for the same epitope with the antibodies or antigen-binding fragments thereof described herein.
  • the application also includes a multispecific antibody molecule, which at least includes a first functional part and a second functional part, wherein the first functional part includes the antibody or its antigen-binding fragment described in the application; the second functional part has a different binding specificity than said first functional moiety.
  • the second functional moiety has binding specificity for immune cells.
  • the second functional moiety has binding specificity for T cells.
  • the second functional moiety has binding specificity for CD7.
  • the application also includes immunoconjugates comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6 linked to a therapeutic agent.
  • the therapeutic agent is a drug.
  • the therapeutic agent is a cytotoxin.
  • the therapeutic agent is a radioisotope.
  • the present application also includes the use of the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned fusion protein, the above-mentioned multispecific antibody molecule or the above-mentioned immunoconjugate in the preparation of a medicament for eliminating, inhibiting or reducing CD5 activity.
  • the medicament is for preventing, alleviating, ameliorating or inhibiting cancer or autoimmune disease.
  • the cancer is selected from: a malignant T cell tumor or a malignant B cell tumor.
  • the present application also includes the use of the above-mentioned antibodies or antigen-binding fragments thereof, the above-mentioned fusion proteins, the above-mentioned multispecific antibody molecules or the above-mentioned immunoconjugates for the preparation of vaccines, preferably antibody vaccines, more preferably Anti-idiotypic antibody vaccine.
  • the present application also includes a vaccine preparation comprising the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned fusion protein, the above-mentioned multispecific antibody molecule or the above-mentioned immunoconjugate.
  • Figure 1 shows the general flow of the present invention for screening specific antibodies targeting CD5 from a phage antibody library.
  • Figure 2 shows the results of an enzyme-linked immunosorbent assay (ELISA) of some of the panned phage monoclones with target and control antigens.
  • ELISA enzyme-linked immunosorbent assay
  • Figure 3 shows the results of flow cytometric analysis of the binding of some phage monoclones to Raji and Jurkat cells.
  • Figures 4A-F show the results of flow cytometry analysis (peak graphs and MFI values) of the binding of screened phage monoclones #1-64 to various different CD5 positive and negative cell lines.
  • Negative Control is a negative control phage antibody clone.
  • Figures 5A-F show the results of ELISA analysis of screened phage monoclones #1-64 with CD5 antigen proteins and non-related antigens from various companies.
  • Negative control is a negative control phage antibody clone
  • anti-M13 phage mouse Ab/anti-mouse HRP Ab is a negative antibody control with only primary antibody and secondary antibody added
  • anti-mouse HRP Ab is a negative antibody with only secondary antibody added Control
  • Mouse anti-human CD5 Ab/anti-mouse HRP Ab is the positive antibody control for the target antigen (CD5-Fc-Bio)
  • anti-human IgG-HRP Ab/anti-his-HRP Ab is the positive antibody control for detecting the antigen label .
  • the bar graphs corresponding to each test antibody and the control group from left to right indicate in order from left to right the correlation with the proteins Kactus-CD5-Fc-Bio, SB-CD5-his-Bio, Acro-CD5-his, Test results for Kactus-BAFFR-his-Bio, Kactus-CD19-FC-Bio, SA.
  • Figure 6 shows the results of a binding study of the RD125 61-42-rFc single domain antibody rabbit Fc fusion protein to CD5 positive cells.
  • Antibody refers to an immunoglobulin secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.).
  • the foreign substance is correspondingly called an antigen.
  • the basic structure of a classical antibody molecule is a 4-mer consisting of 2 identical heavy chains and 2 identical light chains. According to the conservative differences in amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxy terminus. The variable regions of one heavy and one light chain interact to form the antigen binding site (Fv).
  • variable region the composition and arrangement of amino acid residues in certain regions are more variable than other regions (framework regions, FR) in the variable region, called hypervariable regions (HVR), which are actually antibodies. key site for antigen binding. Since these hypervariable regions are complementary to antigenic determinants, they are also called complementarity-determining regions (CDRs). Both heavy and light chains have three complementarity determining regions, designated HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, respectively.
  • CDRs complementarity-determining regions
  • Single chain antibody Single chain fragment variable, scFv
  • scFv single chain fragment variable
  • Single-domain antibody refers to an antibody composed only of amino acids in the variable region of a heavy chain antibody, and its molecular weight is only 12-15kDa, but it has similar or higher specificity and affinity to traditional antibodies.
  • single-domain antibodies have attracted much attention due to their stable physicochemical properties, high affinity, easy recombinant expression and preparation, and easy combination with other target or epitope antibodies.
  • a "murine antibody” is an antibody produced by a mouse against a specific antigen, usually referring to an antibody produced by mouse B lymphocytes. In most cases, the murine antibody is a monoclonal antibody produced by hybridoma cells.
  • the fully human antibody of the present application is obtained by screening a human-derived phage antibody library, which reduces the immunogenicity compared with the mouse-derived antibody, and is more beneficial to the therapeutic use of the human body.
  • antibody or antigen-binding fragment thereof in this application generally refers to any form of antigen-binding molecule that can bind to a target antigen.
  • the antigen-binding molecule can be a protein or polypeptide, including, for example, antibodies and antigen-binding fragments thereof, single Chain scFv antibodies, single domain antibodies, various fusions and conjugates based on scFv constructions such as scFv-Fc antibodies, immunoconjugates, antibody drug conjugates (ADC), multi/bispecific antibodies, chimeric antigen receptor (CAR).
  • CD5 is a type I transmembrane glycosylated protein that plays an important role in the negative regulation of T-cell receptor signaling and promotes the survival of normal and malignant lymphocytes.
  • CD5 is one of the characteristic surface markers of malignant T-cell tumors, and 80% of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas express CD5. This forms the basis for the clinical treatment of relevant tumors with antibodies targeting CD5.
  • T-ALL T-cell acute lymphoblastic leukemia
  • peripheral T-cell lymphomas express CD5. This forms the basis for the clinical treatment of relevant tumors with antibodies targeting CD5.
  • sequence identity when referring to amino acid or nucleotide sequences refers to the identity between two amino acid or nucleotide sequences (eg, a query sequence and a reference sequence) The amount of degree, usually expressed as a percentage. Typically, prior to calculating the percent identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be identical or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent at that position or mismatch.
  • the number of matched positions is divided by the total number of positions in the alignment window to obtain sequence identity.
  • the number of gaps and/or the gap length are also taken into account.
  • the published alignment software BLAST available at ncbi.nlm.nih.gov
  • BLAST can be employed to obtain optimal sequence alignments and calculate two amino acids or nucleotides by using default settings Sequence identity between sequences.
  • "at least 90% sequence identity" as referred to herein includes, but is not limited to: at least 95%, at least 98%, at least 99%, or even 100% sequence identity.
  • a small number of amino acids can be replaced, deleted, added and verified or screened for the binding ability or biological activity of the resulting product with the corresponding antigen CD5, thereby obtaining the Corresponding variants of the provided antibodies targeting CD5 are also intended to be included within the scope of the present invention.
  • a fully human antibody or antigen-binding fragment thereof of the present application may have at least 1 and no more than 10, or no more than 5, 4, 3, 2, or 1 amino acid changes in the full-length or CDR sequence.
  • an antibody light chain library (such as a human phage light chain library) can be screened by using CD5 as an antigen, so as to obtain the same
  • the heavy chain variable region matches and maintains the CD5 binding capacity of the light chain variable region.
  • Anti-CD5 antibody molecules obtainable in this manner are also included within the scope of the present invention.
  • the antigen-binding molecules of the present application may further comprise post-translational modifications.
  • post-translational protein modifications include: phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitination, biotinylation, or addition of polypeptide side chains or hydrophobic groups group.
  • modified soluble polypeptides may contain non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates.
  • a preferred form of glycosylation is the sialylation modification, which binds one or more sialic acid groups to the polypeptide. The sialic acid group improves the solubility and serum half-life of the protein, while also reducing the possible immunoheritability of the protein. See Raju et al. Biochemistry. 2001 31;40(30):8868-76.
  • pharmaceutically acceptable carrier is used to refer to substances such as solid or liquid diluents, fillers, antioxidants, stabilizers, etc., which are safe for administration, and which are suitable for humans and/or animals Administration without undue adverse side effects, while being suitable for maintaining the viability of the drug or active agent located therein.
  • a “therapeutically effective amount” refers to an amount of an active compound sufficient to elicit the biological or medical response desired by a clinician in a subject.
  • the “therapeutically effective amount” of the antibody of the present application can be determined by those skilled in the art according to the administration route, the subject's body weight, age, disease condition and other factors. For example, a typical daily dose may range from 0.01 mg to 100 mg of active ingredient per kg of body weight.
  • Modes of administration of the antibodies of the present application include, but are not limited to, injection, eg, by intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal, and the like.
  • epitope refers to a portion of a molecule that is bound by an antigen binding protein (eg, an antibody). Epitopes may comprise non-adjacent portions of the molecule (eg, in a polypeptide that are not adjacent in the main sequence of the polypeptide, but are sufficiently close to each other in the trivalent and tetravalent structure of the polypeptide to be bound by the antigen binding protein amino acid residues).
  • an antigen binding protein eg, an antibody
  • Fusion protein refers to a protein molecule composed of at least two different peptide segments that is artificially produced (eg, by genetic engineering techniques). These peptides do not exist in nature, or do not exist in the same protein molecule.
  • Examples of common fusion proteins that include antibody fragments include antibody-cytokine fusion proteins, antibody-cytotoxin fusion proteins (also known as immunotoxins), enzyme-labeled antibodies for immunodetection, chimeric antigen receptors (CARs), etc.
  • a fusion protein can include at least two single domain antibodies provided herein that can bind the same or different epitopes.
  • the KD value can be used to measure the binding affinity between an antibody and its antigen.
  • the KD value is the equilibrium dissociation constant between an antibody and its antigen, ie the ratio of k off /k on . Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody.
  • EC 50 concentration for 50% of maximal effect refers to the concentration that causes 50% of the maximal effect.
  • concentration of antibody molecules when half of the maximum detection signal such as fluorescence intensity
  • kits provided by the present application comprises one or more containers containing a large number of gene constructs encoding the polypeptides of the present application, and pharmaceutically acceptable excipients.
  • the kit may also contain instructions for use.
  • the kit may also have a notice in the form prescribed by the governmental agency regulating the manufacture, use, or sale of the drug or biological product that it has been licensed by the agency for the manufacture, use, or sale of the drug for human use.
  • the present invention uses fully human phage for antibody screening, and directly obtains fully human monoclonal antibodies. Compared with traditional hybridoma technology, the difficult step of humanization of murine antibodies is omitted, and fully human antibodies have lower immunogenicity than humanized murine antibodies.
  • Antibodies, ADC, etc.), cell therapy drugs (including CAR-T, CAR-NK, etc.), detection reagents and other applications have better potential.
  • the present invention uses the method of antigen protein panning, which can efficiently enrich the antibody that binds to recombinant CD5 and the natural structure CD5 on the cell membrane, greatly reduces the difficulty of later antibody screening, and improves the efficiency.
  • Example 1 Enrichment of specific antibody clones targeting the CD5 protein from a phage antibody library by affinity panning
  • the phage antibody library we constructed includes natural library, semi-synthetic library and single domain library.
  • Semi-synthetic phage antibody library used together with the natural library, to solve the problem that the natural library may lack CD5 high-affinity antibody clones.
  • the single-domain phage antibody library is an antibody library composed only of amino acids in the variable region of heavy chain antibodies, and its molecular weight is only 12-15kDa, but it has similar or higher specificity and affinity to traditional antibodies.
  • single-domain antibodies have attracted much attention due to their stable physicochemical properties, high affinity, easy recombinant expression and preparation, and easy combination with other target or epitope antibodies.
  • steps 1) to 6 usually 3 rounds of panning are required, until a significant increase in the recovery rate of phage (number of eluted phage/number of input phage) is observed.
  • the enriched phage pool can be used for subsequent monoclonal selection and ELISA/FACS screening.
  • Fully human phage antibody library including natural library, semi-synthetic library and single domain library;
  • Example 2 Screening from enriched phage pools using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) specific clone
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry
  • the phage pool enriched by the affinity panning step contains phage antibodies of various properties: specific clones, non-specific clones, and negative clones.
  • specific clones we need to isolate monoclones from them, package them into monoclonal phages, and perform primary screening on a large number of monoclonal clones by enzyme-linked immunoassay (ELISA) and flow cytometry (FACS), and select from them for simultaneous specific binding Monoclonal CD5 protein and CD5 positive cell line Jurkat.
  • ELISA enzyme-linked immunoassay
  • FACS flow cytometry
  • the combination of Streptavidin and Biotin makes the biotinylated target protein (CD5-Fc-Bio) closer to the native antigen conformation in the reaction solution. Clones that bound only CD5-Fc-Bio but not the control antigen CD19-Fc-Bio were identified as specific clones.
  • the primary FACS screening was performed using the CD5-positive cell line Jurkat and the CD5-negative cell line Raji, and those that only bound to Jurkat cells but not Raji cells were identified as specific clones. Through ELISA and FACS two primary screenings, we can obtain candidate antibodies that can not only bind to the recombinantly expressed CD5 protein, but also recognize the native CD5 molecule on the cell surface for subsequent further screening.
  • step 7 Add 100 ⁇ L of the cultured phage supernatant in step 1) to the wells coated with the target antigen, and bind at room temperature for 2 hours;
  • mice anti M13 primary antibody diluted 1:2000, 100 ⁇ L/well, and incubate at room temperature for 45min;
  • mice anti M13 primary antibody diluted 1:2000, 100 ⁇ L/well, after pipetting and mixing, incubate at room temperature for 45min;
  • the monoclonal phages were randomly selected from the enriched phage antibody pool and packaged into phages.
  • the binding of monoclonal phages to CD5-Fc-Bio protein and the control protein CD19-Fc-Bio was detected by phage ELISA, and CD5-specific phage antibody clones were found. .
  • ELISA results of some clones are shown in Figure 2. It can be seen from the figure that clones H1, H2, H3, H4, H5, H6 and H7 bind strongly to the target antigen CD5 (CD5-Fc-Bio), but do not bind to the control antigen CD19-Fc-Bio, with good specificity .
  • Negative phage control is a negative control phage antibody clone, which does not bind to target antigen and control antigen.
  • Anti-M13 phage mouse Ab/anti-mouse HRP Ab is a negative antibody control with only primary antibody and secondary antibody added
  • anti-mouse HRP Ab is a negative antibody control with only secondary antibody added, which does not bind to the target antigen and control antigen.
  • Mouse anti human CD5 Ab/anti-mouse HRP Ab is a positive antibody control for the target antigen (CD5-Fc-Bio), Binds to the target antigen and does not bind to the control antigen.
  • the results of FACS preliminary screening of antibody clones corresponding to ELISA are shown in FIG. 3 .
  • the H3, H4 and H7 clones bind to Jurkat, but not to Raji cells, and are specific clones; other clones are negative clones (the two cells do not bind).
  • Antibodies used for therapy must have very good target specificity and only bind to the target antigen without binding to any unrelated antigen; on the other hand, the amino acid sequence of the same antigen on different cell lines will vary (Isoforms or mutants) or binding ligands are different, and it is also necessary to investigate whether our antibodies can bind to cells positive for various target proteins.
  • CCRF-CEM cell line CD5 positive cell line
  • NALM6 cell line CD5 negative cell line
  • the rest of the reagents are the same as the FACS primary screening.
  • Antibodies used in therapy must have very good target specificity.
  • clone binds to two CD5-positive cell lines, Jurkat and CCRF-CEM, with strong or weak median fluorescence intensity (MFI), but does not bind to two CD5-negative cell lines, Raji and NALM6, with low MFI and good specificity; Clones #3, #13, #18, #23, #24, #28, #29, #40, #41, #50, #57 and #63 bind weakly or not to the positive cell line Jurkat, and bind to the positive cell line Jurkat.
  • MFI median fluorescence intensity
  • the positive cell line CCRF-CEM does not bind, so it is a negative clone; clones 52 and 53 only bind to the positive cell line Jurkat, but not CCRF-CEM, indicating that they cannot recognize the CD5 antigens of different conformations or isomers expressed by different cell lines. Does not meet the experimental needs; clone 56 can bind to both positive cell lines, but has a weaker binding to the negative cell line Raji, indicating that its binding may be non-specific binding, which does not meet the experimental needs.
  • the antibody used for treatment must have very good target specificity, and only bind to the target antigen without binding to any unrelated antigen; on the other hand, the amino acid sequence of the same antigen produced by different companies will be different (isomers or mutants), it is also necessary to investigate whether our antibodies can bind to various target proteins.
  • ELISA enzyme-linked immunoassay
  • the rest of the reagents are the same as the ELISA primary screening.
  • Antibodies used in therapy must have very good target specificity.
  • ELISA enzyme-linked immunosorbent assay
  • Negative control is a negative control phage antibody clone, which does not bind to the target antigen and control antigen, and anti-M13 phage mouse Ab/anti-mouse HRP Ab is only the first antibody and the second antibody are added.
  • the negative antibody control of anti-mouse HRP Ab is the negative antibody control with only the second antibody added, they do not bind to the target antigen and the control antigen
  • Mouse anti human CD5 Ab/anti-mouse HRP Ab is the target antigen (CD5-Fc -Bio) positive antibody control that binds to the target antigen and does not bind to the control antigen.
  • Anti-human IgG-HRP Ab/anti-his-HRP Ab is a positive antibody control for the detection of antigen tags. It binds to antigens containing Fc tags or his tags, indicating that the coated antigens have been bound to the ELISA plate.
  • Clones #1-64 bind to all three CD5 antigens, but none of the three unrelated antigens, indicating that they can bind to CD5 antigens from different companies with good specificity.
  • #42, #60, #61 and #62 were cloned as single domain antibodies, and their CDR sequences were as follows:
  • the affinity between CD5 sdAbs and antigens may have an important impact on the killing effect and survival time of CAR-T in patients.
  • ForteBio's Octet molecular interaction technology used by the Octet system is a label-free technology that provides high-throughput biomolecular interaction information in real time.
  • the instrument emits white light to the sensor surface and collects the reflected light.
  • the reflected spectrum of different frequencies is affected by the thickness of the optical film layer of the biosensor. Some frequencies of reflected light form constructive interference (blue), while others are destructive interference (red). These interferences are detected by the spectrometer and form an interference spectrum, which is displayed as the phase-shift intensity (nm) of the interference spectrum.
  • the spectrometer will detect the displacement of the interference spectrum in real time, and this displacement directly reflects the thickness of the biofilm on the sensor surface, from which the interaction of biomolecules can be obtained.
  • High-quality data for the determination of biomolecular interaction kinetic parameters (Kon, Kdis and KD) provide important information for the R&D process.
  • Anti-CD5 IgG (constructed by fusion of the VHH sequence of CD5 with human IgG4 Fc) was diluted to 20 ⁇ g/mL with loading buffer (1 ⁇ PBS, pH 7.4, 0.01% BSA and 0.02% Tween 20), and added in a biological The sensor was loaded at about 0.8 nM.
  • Binding kinetics of CD5 antigen (Acro, CD5-H52H5) were monitored at various antigen concentrations (100 to 1.563 nM) after a 60 s equilibration phase. 160s of association and 300s of dissociation were performed at each concentration, respectively.
  • Binding constants were analyzed by using a 1:1 binding site model (Biacore X-100 evaluation software).
  • Affinity refers to the strength with which a single molecule binds to its ligand, and is usually measured and reported by the equilibrium dissociation constant (KD), which can be used to assess and rank the strength of the interaction between two molecules.
  • KD equilibrium dissociation constant
  • #61, #61-42 (#61 and #42 connected by linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 21)
  • #62 and #60 can all bind to CD5 antigen
  • the affinity of #61-42 is slightly higher than that of #42, #61, #62 and #60.
  • CD5 tandem single-domain antibody rabbit FC fusion protein 61-42rFc (constructed by fusing the above #61-42 with rFc) has high affinity with 4 strains of CD5 positive cells, Kd are: 2.99 ⁇ 0.35 nM (CCRF-CEM-Luc); 4.02 ⁇ 0.92 nM (SUP-T1-Luc); 0.64 ⁇ 0.07 nM (JVM-2-Luc-CD5); 1.14 ⁇ 0.16 nM (MEC-1-CD5-Luc).
  • Kd are: 2.99 ⁇ 0.35 nM (CCRF-CEM-Luc); 4.02 ⁇ 0.92 nM (SUP-T1-Luc); 0.64 ⁇ 0.07 nM (JVM-2-Luc-CD5); 1.14 ⁇ 0.16 nM (MEC-1-CD5-Luc).
  • the CD5 tandem single domain antibody rabbit Fc fusion protein 61-42-rFc has stable, good and specific binding ability between the four tested CD5 positive cells, and the EC50 values are all 1-5nM.

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Abstract

提供了靶向CD5的全人源抗体或其抗原结合片段，其以高亲和力特异性结合CD5，且与异源抗体相比，其具有更低的免疫原性，在抗体药物、细胞治疗药物检测试剂等开发上有很好的应用潜力。

Description

靶向CD5的全人源抗体
相关申请的交叉援引
本申请要求于2021年1月12日提交至中国专利局、申请号为202110032708.5的中国专利申请的优先权，在此通过引用将其全文并入本文。
技术领域
本发明涉及与人CD5抗原蛋白和细胞膜表面天然状态的CD5抗原特异性结合的全人源抗体或其抗原结合片段。
背景技术
目前T细胞恶性肿瘤患者的临床治愈仍然很差，现在还没有比化疗更好的治疗策略，但是化疗对复发难治的患者没有获益，而且有严重的毒副作用。因此，开发新颖有效的靶向治疗策略势在必行。近年来，嵌合抗原受体(CAR)修饰的免疫细胞和抗体药物在B细胞恶性肿瘤的治疗中表现出了杰出的疗效。以此类推，开发CAR修饰的免疫细胞或者抗体药物可能有助于T细胞恶性肿瘤的治疗。
CD5是I型跨膜糖基化蛋白，在T细胞受体信号传导的负调控中起着重要作用，并促进正常和恶性淋巴细胞的存活。CD5在造血干细胞的表面不表达，但是在恶性T细胞上是高表达的。CD5是恶性T细胞肿瘤的特征性表面标志物之一，80％的T细胞急性淋巴细胞白血病(T-ALL)和外周T细胞淋巴瘤都表达CD5。另外，CD5也在一些恶性B细胞肿瘤上表达。使用CD5单抗的临床实验中，CD5单抗在患有皮肤T细胞淋巴瘤(CTCL)或慢性淋巴细胞白血病(CLL)的患者中已经显示出中等的治疗效果。
因此，开发能够对CD5表达的细胞发挥临床有效的细胞毒性、细胞抑制或免疫抑制效应，且对CD5不表达的细胞无不良影响的全人源抗体，对研发CD5表达相关的免疫治疗产品，有非常重要的意义。
发明内容
在一方面，本文提供了靶向CD5的抗体或其抗原结合片段，其中所述抗体的重链可变区包括HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3选自如下组合之一：
(1)HCDR1的氨基酸序列为GFTFSHSA(SEQ ID NO：1)；
HCDR2的氨基酸序列为IYARGGYT(SEQ ID NO：2)；
HCDR3的氨基酸序列为ARGYHLEYMVSQDV(SEQ ID NO：3)；
(2)HCDR1的氨基酸序列为GFTFSSYE(SEQ ID NO：4)；
HCDR2的氨基酸序列为ISSSGSTI(SEQ ID NO：5)；
HCDR3的氨基酸序列为ARVAQREGDV(SEQ ID NO：6)；
(3)HCDR1的氨基酸序列为GGTFSNYA(SEQ ID NO：7)；
HCDR2的氨基酸序列为ISAYNGDT(SEQ ID NO：8)；
HCDR3的氨基酸序列为ARYESMSGQDI(SEQ ID NO：9)；
(4)HCDR1的氨基酸序列为GYSFSNHW(SEQ ID NO：10)；
HCDR2的氨基酸序列为VYPGDSDT(SEQ ID NO：11)；
HCDR3的氨基酸序列为ARGGTIDGDYGGRQDF(SEQ ID NO：12)；或
所述抗体包括(1)-(4)任一项中的CDR序列组合的变体，其中所述变体(1)-(49)任一项中的CDR序列相比，具有至少90％的序列一致性，或在CDR序列上共包含至少1个且不超过10，或不超过5、4、3、2或1个氨基酸改变。
在一些实施方案中，所述重链可变区的氨基酸序列选自如下任一项：
(1)SEQ ID NO：17所示序列或与其有至少90％序列一致性的重链可变区序列；
(2)SEQ ID NO：18所示序列或与其有至少90％序列一致性的重链可变区序列；
(3)SEQ ID NO：19所示序列或与其有至少90％序列一致性的重链可变区序列；
(4)SEQ ID NO：20所示序列或与其有至少90％序列一致性的重链可变区序列。
在一些实施方案中，所述重链可变区和/或轻链可变区的氨基酸序列选自如下任一项：
(1)SEQ ID NO：17所示的重链可变区序列；
(2)SEQ ID NO：18所示的重链可变区序列；
(3)SEQ ID NO：19所示的重链可变区序列；
(4)SEQ ID NO：20所示的重链可变区序列。
在一些实施方案中，所述抗体为全人源抗体。
在一些实施方案中，所述抗体为单域抗体。
在一些实施方案中，通过生物膜干涉技术测定的所述抗体对CD5抗原的结合KD值低于10- 7M，优选低于10 -8M。
另一方面，本申请还提供了融合蛋白，其包括一个或两个抗原结合功能部分，其中每个所述抗原结合功能部分包括上述抗体或其抗原结合片段。
在一些实施方案中，所述融合蛋白还包括Fc片段。
在一些实施方案中，所述两个抗原结合功能部分分别结合相同或不同的抗原表位。
在一些实施方案中，所述融合蛋白包括串联连接的第一抗原结合功能部分和第二抗原结合功能部分；其中所述第一抗原结合功能部分包括第一重链可变区(HCVR)，所述第一重链可变区包括SEQ ID NO：7所示的HCDR1、SEQ ID NO：8所示的HCDR2和SEQ ID NO：9所示的HCDR3；其中所述第二抗原结合功能部分包括第二重链可变区(HCVR)，所述第二重链可变区包括SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2和SEQ ID NO：3所示的HCDR3。
在一些实施方案中，所述第一抗原结合功能部分位于第二抗原结合功能部分的N端。
在一些实施方案中，所述融合蛋白包括串联连接的SEQ ID NO：19所示的重链可变区序列和SEQ ID NO：17所示的重链可变区序列。
在一些实施方案中，SEQ ID NO：19所示的重链可变区序列位于SEQ ID NO：17所示的重链可变区序列的N端。
在一些实施方案中，所述抗原结合功能部分直接通过接头分子连接；优选地，所述接头分子包括SEQ ID NO：21所示的氨基酸序列。
在一些实施方案中，所述融合蛋白通过流式细胞术测定的所述融合蛋白与CD5阳性细胞之间结合的EC 50值在1-5nM。
另一方面，本申请还包括分离的核酸分子，其编码上述抗体或其抗原结合片段或融合蛋白。
在一些实施方案中，所述的核酸分子，其包括SEQ ID NO:13-16任一项所示的核苷酸序列。
另一方面，本申请还包括一种表达载体，其包含本申请所述核酸分子。在一些实施方案中，所述载体为质粒、逆转录病毒和慢病毒载体。
另一方面，本申请还包括一种宿主细胞，其包括本申请所述的表达载体。
另一方面，本申请还包括一种药物组合物，其包括本申请所述的抗体或其抗原结合片段或融合蛋白，以及药学上可接受的载体或稀释剂。
另一方面，本申请还包括一种治疗疾病或病症的方法，所述方法通过向有需要的患者施用治疗有效量的本申请所述抗体或其抗原结合片段，融合蛋白，或宿主细胞，或药物组合物来消除、抑制或降低CD5活性，从而预防、减轻、改善或抑制疾病或病症。
本申请还包括上述抗体或其抗原结合片段、融合蛋白或上述宿主细胞在制备用于消除、抑制或降低CD5活性，从而预防、减轻、改善或抑制疾病或病症的药物中的用途。
本申请还包括将上述抗体或其抗原结合片段、融合蛋白或上述宿主细胞用作药物或用于治疗中，例如用于消除、抑制或降低CD5活性，从而预防、减轻、改善或抑制疾病或病症。
在一些实施方案中，所述疾病或病症选自：癌症或自身免疫疾病。在一些实施方案中，所述癌症选自：恶性T细胞肿瘤或恶性B细胞肿瘤T细胞恶性肿瘤。
在一些实施方案中，所述恶性T细胞肿瘤选自T细胞急性淋巴细胞白血病(T-ALL)、T细胞淋巴瘤(例如：外周T细胞淋巴瘤或皮肤T细胞淋巴瘤(CTCL))，所述恶性B细胞肿瘤选自慢性淋巴细胞白血病(B-CLL)或套细胞淋巴瘤(B-MCL)。
另一方面，本申请还包括一种试剂盒，用于检测样品中CD5蛋白。所述试剂盒包括本申请所述的抗体或其抗原结合片段或融合蛋白。所述检测可以是体外的或体内的。
另一方面，本申请还包括与本申请所述的抗体或其抗原结合片段竞争相同表位的抗体或片段。
另一方面，本申请还包括多特异性抗体分子，其至少包括第一功能部分和第二功能部分，其中第一功能部分包括本申请所述的抗体或其抗原结合片段；第二功能部分具有与所述第一功能部分不同的结合特异性。
在一些实施方案中，所述第二功能部分对免疫细胞具有结合特异性。
在一些实施方案中，所述第二功能部分对T细胞具有结合特异性。
在一些实施方案中，所述第二功能部分对CD7具有结合特异性。
另一方面，本申请还包括免疫偶联物，其包括与治疗剂连接的权利要求1-6中任一项所述的抗体或其抗原结合片段。
在一些实施方案中，所述治疗剂是药物。
在一些实施方案中，所述治疗剂是细胞毒素。
在一些实施方案中，治疗剂是放射性同位素。
另一方面，本申请还包括上述抗体或其抗原结合片段、上述融合蛋白、上述多特异性抗体分子或上述免疫偶联物在制备用于消除、抑制或降低CD5活性的药物中的用途。
在一些实施方案中，所述药物用于预防、减轻、改善或抑制癌症或自身免疫疾病。
在一些实施方案中，所述癌症选自：恶性T细胞肿瘤或恶性B细胞肿瘤。
另一方面，本申请还包括上述抗体或其抗原结合片段、上述融合蛋白、上述多特异性抗体分子或上述免疫偶联物的用途，用于制备疫苗，较佳地为抗体疫苗，更佳地为抗独特型抗体疫苗。
另一方面，本申请还包括一种疫苗制品，其包括上述抗体或其抗原结合片段、上述融合蛋白、上述多特异性抗体分子或上述免疫偶联物。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
图1显示了本发明从噬菌体抗体库筛选靶向CD5的特异抗体的大体流程。
图2显示了所淘选的部分噬菌体单克隆与靶抗原和对照抗原的酶联免疫吸附测定(ELISA)结果。
图3显示了部分噬菌体单克隆与Raji和Jurkat细胞结合的流式细胞分析结果。
图4 A-F显示了所筛选的噬菌体单克隆#1～64与多种不同的CD5阳性和阴性细胞系的结合的流式细胞分析结果(峰形图和MFI值)。Negative Control为阴性对照噬菌体抗体克隆。
图5A-F显示了所筛选的噬菌体单克隆#1～64与多种不同公司的CD5抗原蛋白和非相关抗原的酶联免疫吸附测定分析结果。Negative control为阴性对照噬菌体抗体克隆，anti-M13 phage mouse Ab/anti-mouse HRP Ab为只加第一抗体和第二抗体的阴性抗体对照，anti-mouse HRP Ab为只加第二抗体的阴性抗体对照，Mouse anti human CD5 Ab/anti-mouse HRP Ab为靶抗原(CD5-Fc-Bio)的阳性抗体对照，anti-human IgG-HRP Ab/anti-his-HRP Ab为检测抗原标签的阳性抗体对照。其中，图5A-F中，每个测试抗体和对照组对应的柱形图从左至右依次表示与蛋白Kactus-CD5-Fc-Bio、SB-CD5-his-Bio、Acro-CD5-his、Kactus-BAFFR-his-Bio、Kactus-CD19-FC-Bio、SA的测试结果。
图6显示了RD125 61-42-rFc单域抗体兔Fc融合蛋白与CD5阳性细胞的结合研究结果。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
“抗体”指由浆细胞(效应B细胞)分泌、被机体免疫系统用来中和外来物质(多肽、病毒、细菌等)的免疫球蛋白。该外来物质相应地称作抗原。经典抗体分子的基本结构是由2个相同重链和2个相同轻链组成的4聚体。根据氨基酸序列的保守性差异，将重链和轻链分为位于氨基端的可变区(V)和位于羧基端的恒定区(C)。一条重链和一条轻链的可变区相互作用形成了抗原结合部位(Fv)。在可变区中，某些区域氨基酸残基的组成和排列次序比可变区内的其它区域(骨架区，FR)更易变化，称为高变区(HVR)，高变区实际上是抗体与抗原结合的关键部位。由于这些高变区序列与抗原决定簇互补，故又称为互补决定区(complementarity-determining region,CDR)。重链和轻链均具有三个互补决定区，分别称为HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3。
“单链抗体”(single chain fragment variable,scFv)，是由抗体重链可变区和轻链可变区通过短肽连接成一条肽链而构成。通过正确折叠，来自重链和轻链的可变区通过非共价键相互作用形成Fv段，因而scFv能较好地保留其对抗原的亲和活性。
“单域抗体”是指只由重链抗体的可变区氨基酸组成的抗体，其分子量仅有12-15kDa，但却具备与传统抗体相似或更高的特异性和亲和力。此外，单域抗体理化性质稳定、亲和力高、易于重组表达制备、易于与其它靶点或表位抗体组合等特点使其备受关注。
“鼠源抗体”是由鼠类针对特异抗原产生的抗体，通常指小鼠B淋巴细胞产生的抗体。在大多情况下，该鼠源抗体为杂交瘤细胞产生的单克隆抗体。本申请的全人源抗体是从人源噬菌体抗体库筛选获得，其相对于鼠源抗体降低了免疫原性，更利于人体的治疗用途。
本申请所述的“抗体或其抗原结合片段”通常是指能够与靶抗原结合的任何形式的抗原结合分子，例如该抗原结合分子可为蛋白质或多肽，包括例如抗体及其抗原结合片段、单链scFv抗体、单域抗体、基于scFv构建的各种融合物和缀合物，例如scFv-Fc抗体、免疫缀合物、抗体药物偶联物(ADC)、多/双特异性抗体、嵌合抗原受体(CAR)。
CD5是I型跨膜糖基化蛋白，在T细胞受体信号传导的负调控中起着重要作用，并促进正常和恶性淋巴细胞的存活。CD5是恶性T细胞肿瘤的特征性表面标志物之一，80％的T细胞急性淋巴细胞白血病(T-ALL)和外周T细胞淋巴瘤都表达CD5。这构成了靶向CD5的抗体在临床上治疗相关肿瘤的基础。
提及氨基酸或核苷酸序列时，术语“序列一致性(Sequence identity)”(也称为“序列同一性”)指两氨基酸或核苷酸序列(例如查询序列和参照序列)之间一致性程度的 量，一般以百分比表示。通常，在计算两氨基酸或核苷酸序列之间的一致性百分比之前，先进行序列比对(alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置，两序列中的氨基酸残基或碱基相同，则认为两序列在该位置一致或匹配；两序列中的氨基酸残基或碱基不同，则认为在该位置不一致或错配。在一些算法中，用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中，还将缺口数量和/或缺口长度考虑在内。出于本发明的目的，可以采用公开的比对软件BLAST(可在网页ncbi.nlm.nih.gov找到)，通过使用缺省设置来获得最佳序列比对并计算出两氨基酸或核苷酸序列之间的序列一致性。在一些实施方案中，本申请所述“至少90％序列一致性”包括但不限于：至少95％、至少98％、至少99％或甚至100％序列一致性的情形。
本领域技术人员可以理解的是，在本文提供的具体序列基础上，可以通过对少数氨基酸进行替换、删除、添加并验证或筛选所得产物与相应抗原CD5的结合能力或生物学活性，从而获得本文提供的靶向CD5抗体的相应变体，这些变体也应包括在本发明的范围内。例如，本申请的全人源抗体或其抗原结合片段在全长或CDR序列上，可以有至少1个且不超过10，或不超过5、4、3、2或1个氨基酸的改变。
本领域技术人员还可以理解的是，在本文提供的具体重链可变区序列基础上，可以通过以CD5作为抗原筛选抗体轻链库(如人源噬菌体轻链库)，从而获得与所述重链可变区匹配并维持CD5结合能力的轻链可变区。以此方式可获得的抗CD5抗体分子也包括在本发明的范围内。
在一些实施方案中，本申请的抗原结合分子可以进一步包含转译后修饰。转译后蛋白修饰的示例包括：磷酸化、乙酰化、甲基化、ADP-核糖化、泛素化、糖基化、羰基化、类泛素化、生物素化或加入多肽侧链或疏水基团。因此，经修饰的可溶性多肽可以包含非氨基酸成分，例如类脂、多聚糖或单糖、以及磷酸盐。糖基化的一种优选形式是唾液酸化修饰，将一个或多个唾液酸基团与多肽结合。唾液酸基团改善蛋白质的溶解性和血清半衰期，同时也降低蛋白质可能的免疫遗传性。参见Raju et al.Biochemistry.2001 31；40(30):8868-76。
提及药物组合物，所使用的“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。
“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。本申请抗体的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg活性成分。本申请的抗体的施用方式包括但不限于注射，例如通过静脉内、肌内、动脉内、皮下、腹膜内等。
“表位”是指受抗原结合蛋白(例如，抗体)约束的分子的部分。表位可以包含该分子的非相邻的部分(例如，多肽中，在该多肽的主要序列上不相邻、但在该多肽的三价和四价结构中彼此足够靠近以被抗原结合蛋白约束的氨基酸残基)。
“融合蛋白”指人为生成(例如通过基因工程技术)的由至少两个不同肽段构成的蛋白分子。这些肽段在自然界中不存在，或者不存在于同一个蛋白分子中。常见的包括抗体片段的融合蛋白的实例包括抗体-细胞因子融合蛋白、抗体-细胞毒素融合蛋白(也称为免疫毒素)、用于免疫检测的酶标抗体、嵌合抗原受体(CAR)等。在一个具体实例中，融合蛋白可包括至少两个本文提供的单域抗体，这两个单域抗体可结合相同或不同的抗原表位。
KD值可用于衡量抗体与其抗原之间的结合亲和力。KD值是抗体与其抗原之间的平衡解离常数，即k off/k on的比值。因此KD值越低(浓度越低)，抗体的亲和力越高。
EC 50(concentration for 50％of maximal effect)指引起50％最大效应的浓度。在流式细胞术中用于表示抗体分子与对应抗原或表达抗原的细胞的结合能力时，可指产生最大检测信号(如荧光强度)一半时的抗体分子浓度。EC 50值越低，则与抗原或表达该抗原的细胞的结合亲和力越大。
本申请提供的一种试剂盒，包含一个或多个容器，装有大量编码本申请多肽的基因构建体，及药学上可接受的赋形剂。该试剂盒也可以包含使用说明。试剂盒上还可以有管理生产、使用或销售药品或生物制品的政府机构所规定形式的通知，该通知表明已获人用药的生产、使用或销售机构许可。
研究概述：
本发明使用全人源噬菌体进行抗体筛选，直接获得全人源的单克隆抗体。与传统杂交瘤技术相比，省却了困难的鼠源抗体人源化步骤，而且全人源抗体比人源化的鼠源抗体具有更低的免疫原性，在抗体药物(包括单抗，双抗，ADC等)、细胞治疗药物(包括CAR-T，CAR-NK等)、检测试剂等应用上有更好的潜力。
本发明使用了抗原蛋白淘选的方法，能高效地富集的同时结合重组CD5和细胞膜上天然结构CD5的抗体，大大降低了后期抗体筛选的难度，提高了效率。
我们应用大容量噬菌体抗体库筛选全人源的CD5特异抗体，并通过ELISA和FACS实验来评估这些抗体在phage水平的特异性。最终，我们获得了若干特异性良好的全人源抗体克隆。
我们使用不同的抗体库，经过重组CD5蛋白淘选，总共挑选了184个单克隆进行酶联免疫吸附测定(ELISA)和流式细胞术(FACS)检测初筛，其中93个克隆特异结合CD5-Fc-Bio蛋白和CD5表达阳性细胞Jurkat，而不结合对照蛋白CD19-Fc-Bio和CD5表达阴性细胞Raji。测序后得到了64种不同的单克隆序列。随后，我们将这64种抗 体与多种CD5阳性(Jurkat，CCRF-CEM)和阴性细胞系(Raji，NALM6)进行流式细胞分析(FACS)鉴定，与不同公司的CD5蛋白(Kactus-CD5-Fc-Bio,Acro-CD5-his,SB-CD5-his-Bio)，非相关蛋白(Kactus-BAFFR-his-Bio，Kactus-CD19-FC-Bio，SA)进行酶联免疫吸附(ELISA)鉴定，其中49个克隆在多个细胞系和多种蛋白抗原上都表现出良好的结合力和特异性。这些克隆的获得为后续开发全人源的CD5CAR-T产品或者抗体药奠定了基础。总体项目流程如图1所示。
以下结合具体实施例详细说明本发明。
实施例1.通过亲和淘选从噬菌体抗体库富集靶向CD5蛋白的特异抗体克隆
采用合适的负淘选和正淘选策略从噬菌体抗体库中富集我们所需的特异性抗体克隆。
噬菌体抗体库的构建
我们构建的噬菌体抗体库包括天然库、半合成库和单域库。半合成噬菌体抗体库，与天然库一起使用，解决天然库可能缺乏CD5高亲和力抗体克隆的问题。单域噬菌体抗体库是只由重链抗体的可变区氨基酸组成的抗体库，其分子量仅有12-15kDa，但却具备与传统抗体相似或更高的特异性和亲和力。此外，单域抗体理化性质稳定、亲和力高、易于重组表达制备、易于与其它靶点或表位抗体组合等特点使其备受关注。
CD5蛋白淘选
以CD5-Fc-Bio作为正淘选蛋白，以CD19-Fc-Bio作为负淘选蛋白进行多轮淘选，获得富集目的抗体克隆的噬菌体池(pool)。实验步骤简述如下：
1)将SA磁珠先用封闭液封闭2h，然后再将靶抗原(CD5-Fc-Bio)与封闭好的SA磁珠结合；
2)加入噬菌体文库(含5x10 12个噬菌体颗粒)和和对照抗原一起孵育，以便扣除非特异结合Fc标签的噬菌体抗体克隆；
3)孵育后将上清转移到结合了靶抗原的SA磁珠中，继续孵育，使噬菌体和靶抗原结合；
4)用洗涤液洗涤磁珠，将未结合的噬菌体洗去；
5)用洗脱液将阳性噬菌体从靶抗原上洗脱下来，加入中和液中和；
6)以洗脱后的噬菌体重新感染宿主菌XL1-blue，扩增回收的噬菌体。留少量样品梯度稀释，感染宿主菌，涂Amp抗性平板，计算回收噬菌体数量；
7)重复步骤1)至6)，通常需要进行3轮淘选，直到观察到噬菌体的回收率(洗脱噬菌体数/投入噬菌体数)有明显上升。
富集好的噬菌体池可用于进行随后的单克隆挑选以及ELISA/FACS筛选。
主要材料和试剂：
全人源噬菌体抗体库，包含天然库，半合成库和单域库；
辅助噬菌体KO7，Thermo/Invitrogen,18311019；
Recombinant biotinylated Human CD5-Fc Protein,Kactus,CD5-HM401；
Recombinant biotinylated Human CD19 Protein-Fc,Kactus,CD2-HE121
BeaverBeads TM Streptavidin,海狸生物,22307-10；
High binding ELSIA plate,Costar，#3590
封闭液：PBS+3％BSA
漂洗液：PBS+0.1％Tween20
洗脱液：0.2M Glycine，pH2.2
中和液：1M Tris，pH9.1
实验结果：
使用不同的抗体库，通过3轮蛋白淘选，每个淘选都观察到了回收率的显著上升(表1)，证明抗体克隆得到了有效富集。
表1蛋白淘选实验结果
Figure PCTCN2022071674-appb-000001
可以看到经过3轮淘选，不同的抗体库都得到了富集(第3轮回收率比上一轮显著提高)。
实施例2.采用酶联免疫吸附测定(ELISA)和流式细胞术(FACS)从经富集的噬菌体池筛 选特异性克隆
目的和原理：通过亲和淘选步骤富集的噬菌体池中包含各种性质的噬菌体抗体：特异克隆、非特异克隆、以及阴性克隆。为了获得特异克隆，我们需要从中分离单克隆，包装成单克隆的噬菌体，并通过酶联免疫检测(ELISA)和流式细胞术(FACS)对大量单克隆进行初筛，从中挑选到同时特异结合CD5蛋白和CD5阳性细胞系Jurkat的单克隆。特异的单克隆再进一步通过DNA测序确定其中包含的唯一的抗体序列。
在ELISA初筛中，通过链霉亲和素Streptavidin与生物素Biotin的结合，使得生物素化的靶蛋白(CD5-Fc-Bio)在反应液中更接近于天然状态的抗原构象。只结合CD5-Fc-Bio而不结合对照抗原CD19-Fc-Bio的被认定为特异克隆。FACS初筛使用CD5高表达的阳性细胞系Jurkat和CD5阴性的细胞系Raji来进行，只结合细胞Jurkat且不结合Raji细胞的被认定为特异克隆。通过ELISA和FACS两种初筛，我们可以获得既能结合重组表达的CD5蛋白，又能识别细胞表面天然状态CD5分子的候选抗体，供随后进一步筛选。
ELISA初筛实验简要步骤：
1)用深孔96孔板培养和包装单克隆噬菌体；
2)将Strepavidin用PBS稀释到2μg/mL，以100μL/孔加入到高结合酶标板中，室温结合2h；
3)弃掉包被液，每孔加入250μL封闭液，4℃封闭过夜；
4)250μL漂洗液洗板2次；
5)将带生物素标签的靶蛋白和对照蛋白用PBS稀释至2μg/mL，以100μg/孔加入预包被Strepavidin的酶标板中，室温结合1h；
6)250μL漂洗液洗板2次；
7)加入100μL步骤1)培养好的噬菌体上清到包被好靶抗原的孔，室温结合2h；
8)250μL漂洗液洗板4次；
9)加入1:2000稀释的mouse anti M13第一抗体，100μL/孔，室温孵育45min；
10)250μL漂洗液洗板4次；
11)加入1:2000稀释的HRP Donkey anti-mouse IgG，100μL/孔，室温孵育45min；
12)250μL漂洗液洗板6次；
13)加入100μL TMB显色底物，显色5至10min；
14)加入100μL 2M H 2SO 4终止反应，在酶标仪上读取结果。
FACS初筛实验简要步骤：
1)用深孔96孔板培养和包装单克隆噬菌体；
2)Raji和Jurkat细胞用PBS洗2次，用PBS重悬成1x10 7/mL浓度，以50μL分装到96孔深孔板中；
3)每孔加入50μL包装好的单克隆噬菌体，混匀后，4℃结合2h；
4)200μL PBS洗涤2次；
5)加入1:2000稀释的mouse anti M13第一抗体，100μL/孔，吹打混匀后，室温孵育45min；
6)200μL PBS洗涤2次；
7)加入1:300稀释的FITC horse anti mouse-IgG(H+L)，100μL/孔，吹打混匀后，室温孵育45min；
8)200μL PBS洗涤2次；最后用200μL PBS重悬细胞；
9)在流式细胞仪上检测样品FITC通道的荧光强度，分析结果。
主要材料和试剂：
辅助噬菌体KO7，Thermo/Invitrogen,18311019
Streptavidin,Pierce,21125
Recombinant biotinylated Human CD5 Protein,Kactus,CD5-HM401；
Recombinant biotinylated Human CD19 Protein,Kactus,CD2-HE121
High binding ELSIA plate,Costar,#3590
Corning 96 Well Clear Round Bottom TC-Treated Microplate,Costar,#3799
封闭液：PBS+3％BSA
漂洗液：PBS+0.1％Tween20
可溶型单组分TMB底物溶液,Tiangen,PA-107-02
Anti-M13 Bacteriophage Coat Protein g8p antibody,abcam,ab9225
HRP Goat anti-mouse IgG(minimal x-reactivity)Antibody,Biolegend,405306
FITC horse anti mouse-IgG(H+L),Vector,FI2000
实验结果：
从富集后的噬菌体抗体池随机挑选单克隆，包装成噬菌体后，通过噬菌体ELISA检测单克隆噬菌体与CD5-Fc-Bio蛋白、对照蛋白CD19-Fc-Bio的结合，找到CD5特异的噬菌体抗体克隆。部分克隆的ELISA结果如图2所示。从图中可知，H1，H2，H3，H4，H5，H6和H7号克隆与靶抗原CD5(CD5-Fc-Bio)结合较强，且不与对照抗原CD19-Fc-Bio结合，特异性良好。Negative phage control为阴性对照噬菌体抗体克隆，与靶抗原和对照抗原都不结合，anti-M13 phage mouse Ab/anti-mouse HRP Ab为只加第一抗体和第二抗体的阴性抗体对照，anti-mouse HRP Ab为只加第二抗体的阴性抗体对照，它们与靶抗原和对照抗原都不结合，Mouse anti human CD5 Ab/anti-mouse HRP Ab为靶抗原(CD5-Fc-Bio)的阳性抗体对照，与靶抗原结合，与对照抗原不结合。
与ELISA相对应的抗体克隆的FACS初筛结果如图3所示。其中H3，H4和H7克隆结合Jurkat，不结合Raji细胞，是特异克隆；其它克隆是阴性克隆(2种细胞都不结合)。
通过ELISA检测和FACS初筛，我们总共获得93个ELISA和FACS双阳性且特异性良好的克隆，随后我们将获得的93个双阳性且特异性良好的克隆测序，测序后得到了64种不同的单克隆序列，然后将这64个不同序列的单克隆进一步通过多细胞系的FACS鉴定和多种抗原的ELISA鉴定检测候选克隆的结合特异性。
实施例3.采用多个细胞系通过FACS鉴定单克隆特异性
实验目的和原理：用于治疗的抗体必须具有非常好的靶点特异性，只结合靶抗原，而不结合任何无关的抗原；另一方面，不同的细胞系上同一抗原的氨基酸序列会有差异(异构体或突变体)或结合的配体不一样，也需要考察我们的抗体能否与各种靶蛋白阳性的细胞都结合。为了进一步分析这些单克隆的特异性和普适性，寻找最佳的候选克隆，我们通过流式细胞术进一步评估初筛克隆的特异性。在这个实验中，我们采用多种CD5阳性的细胞系和多种CD5阴性的细胞系与这些单克隆噬菌体抗体进行反应，分析这些克隆是否可以结合不同的细胞系上的CD5抗原，以及是否与其它不表达CD5的细胞系有任何非特异的结合。通过这个实验，我们获得了若干具有优良特异性的克隆。
实验方法：与FACS初筛相同；
主要样品和试剂：
Jurkat细胞系，CD5阳性细胞系；
Raji细胞系，CD5阴性细胞系；
CCRF-CEM细胞系,CD5阳性细胞系；
NALM6细胞系,CD5阴性细胞系；
其余试剂与FACS初筛相同。
实验结果：
用于治疗的抗体必须具有非常好的靶点特异性。为了进一步分析这些单克隆抗体的特异性，我们将实施例2获得的唯一特异性克隆在更多的抗原和细胞系上应用酶联免疫吸附和流式细胞术进行了鉴定。结果显示在图4A-F中，Negative Control为阴性对照噬菌体抗体克隆。#1～2，#4～12，#14～17，#19～22，#25～27，#30～39，#42～49，#51，#54～55，#58～62，#64号克隆与2种CD5阳性细胞系Jurkat和CCRF-CEM都结合，中位荧光强度(MFI)有强有弱，与2种CD5阴性细胞系Raji和NALM6都不结合，MFI低，特异性良好；#3，#13，#18，#23，#24，#28，#29，#40，#41，#50，#57和#63号克隆与阳性细胞系Jurkat结合很弱或者不结合，与阳性细胞系CCRF-CEM不结 合，为阴性克隆；52和53号克隆只结合阳性细胞系Jurkat，不结合CCRF-CEM，说明其不能识别不同细胞系表达的不同构象或异构体的CD5抗原，不符合实验需要；56号克隆与两种阳性细胞系都能结合，但是与阴性细胞系Raji有较弱的结合，说明其结合可能为非特异性结合，不符合实验需要。
实施例4.采用不同公司的抗原通过ELISA鉴定单克隆特异性
实验目的和原理：用于治疗的抗体必须具有非常好的靶点特异性，只结合靶抗原，而不结合任何无关的抗原；另一方面，不同的公司生产的同一抗原的氨基酸序列会有差异(异构体或突变体)，也需要考察我们的抗体能否与各种靶蛋白都结合。为了进一步分析这些单克隆的特异性和普适性，寻找最佳的候选克隆，我们通过酶联免疫检测(ELISA)进一步评估初筛克隆的特异性。在这个实验中，我们采用购买自不同公司生产的CD5抗原和多种CD5不相关抗原与这些单克隆噬菌体抗体进行反应，分析这些克隆是否可以结合不同的CD5抗原，以及是否与其它CD5不相关抗原有任何非特异的结合。通过这个实验，我们获得了若干具有优良特异性的克隆。
实验方法：与ELISA初筛相同；
主要样品和试剂：
Figure PCTCN2022071674-appb-000002
其余试剂与ELISA初筛相同。
实验结果：
用于治疗的抗体必须具有非常好的靶点特异性。为了进一步分析这些单克隆抗体的特异性，我们将实施例2获得的多个克隆在多种抗原应用酶联免疫吸附(ELISA)进行了鉴定。结果显示在图5A-F中，Negative control为阴性对照噬菌体抗体克隆，与靶抗原和对照抗原都不结合，anti-M13 phage mouse Ab/anti-mouse HRP Ab为只加第一抗体和第二抗体的阴性抗体对照，anti-mouse HRP Ab为只加第二抗体的阴性抗体对照，它们与靶抗原和对照抗原都不结合，Mouse anti human CD5 Ab/anti-mouse HRP Ab为靶抗原(CD5-Fc-Bio)的阳性抗体对照，与靶抗原结合，与对照抗原不结合。anti-human IgG-HRP Ab/anti-his-HRP Ab为检测抗原标签的阳性抗体对照，与含有Fc标签或者his标签的抗原结合，说明包被的抗原已经结合到酶标板上。#1～64号克隆与3种CD5抗原都结合，与3种非相关抗原都不结合，说明能够结合不同公司的CD5抗原且特异性良好。其中#42、#60、#61和#62克隆为单域抗体，其CDR序列如下：
Figure PCTCN2022071674-appb-000003
实施例5.抗CD5 sdAbs亲和力的测定
实验目的和原理：
CD5 sdAbs与抗原间的亲和力大小可能对CAR-T在患者体内发挥杀伤作用及存续时间有着重要影响，为了确定这一重要性质，我们采用了ForteBio公司的Octet分子相互作用技术对其进行了测定。Octet系统所运用的生物膜干涉技术，是一种免标记技术，实时提供高通量的生物分子相互作用信息。该仪器发射白光到传感器表面并收集反射光，不同频率的反射光谱受到生物传感器的光膜层厚度的影响，一些频率的反射光形成了相长干涉(蓝色)，而另一些受到了相消干涉(红色)。这些干涉被光谱仪所检测到，并形成一幅干涉光谱，并以干涉光谱的相位位移强度(nm)显示。因此，结合到传感器表面的分子一旦有数量上的增减，光谱仪便会实时地检测到干涉光谱的位移，而这种位移直接反应出传感器表面生物膜的厚度，从中可以获取生物分子相互作用的高质量 的数据，从而进行生物分子间相互作用动力学参数测定(Kon,Kdis和KD)，为研发过程提供重要的信息。
简要实验步骤：
1)用上样缓冲液(1×PBS，pH 7.4，0.01％BSA和0.02％Tween 20)将抗CD5 IgG(将CD5的VHH序列与human IgG4 Fc融合构成)稀释至20μg/mL，并在生物传感器上上样，约0.8nM。
2)在60s平衡阶段后，在多种抗原浓度(100至1.563nM)下监测CD5抗原(Acro,CD5-H52H5)的结合动力学。在每个浓度下分别进行至160s结合和300s解离。
3)用10mM Glycine-HCl,pH1.5洗涤3次使芯片再生。
4)通过使用1：1结合位点模型(Biacore X-100评估软件)分析结合常数。
实验结果：
亲和力系指单个分子与其配体结合的强度，通常通过平衡解离常数(KD)进行测定和报告，平衡解离常数可用于评估两分子间相互作用的强度并对此进行排序。抗体与其抗原的结合是一个可逆的过程，结合反应的速率与反应物的浓度成正比。KD值越小，抗体对其靶标的亲和力越大。如表2所示：#42、#61、#61-42(通过linker(GGGGSGGGGSGGGGS)(SEQ ID NO：21)连接的#61和#42)、#62和#60均可与CD5抗原结合，且#61-42亲和力稍高于#42、#61、#62和#60。
表2抗CD5 IgG亲和力测定
分析物 KD(M) kon(1/Ms) kdis(1/s)
#42IgG 2.90E-09 4.80E+04 1.39E-04
#61IgG 3.96E-09 7.28E+04 2.88E-04
#61-42IgG 1.67E-09 1.13E+05 1.89E-04
#62IgG 2.74E-08 1.25E+04 3.09E-04
#60IgG 8.32E-09 2.55E+04 2.12E-04
实施例6.串联单域抗体与CD5 +靶细胞的结合研究
研究目的：考察CD5单域抗体与CD5 +靶细胞的结合能力。
研究方法：不同浓度的CD5串联单域抗体兔Fc融合蛋白61-42-rFc分别与CD5 +靶细胞孵育后，细胞用PBS洗两遍后加入荧光染料标记的兔Fc抗体标记阳性细胞，采用FCM(Flow cytometry，流式细胞术)方法检测，通过分析荧光标记的阳性CD5 +靶细胞百分比和不同浓度的CD5串联单域抗体之间的关系，由Graphpad Prism软件进行拟合计算EC50常数。本实验共进行三次独立重复实验。
研究结果：CD5串联单域抗体兔FC融合蛋白61-42rFc(通过将上述#61-42与rFc融合构成)与4株CD5阳性细胞之间均有很高的亲和力，Kd分别为：2.99±0.35nM(CCRF-CEM-Luc)；4.02±0.92nM(SUP-T1-Luc)；0.64±0.07nM(JVM-2-Luc-CD5)；1.14±0.16nM(MEC-1-CD5-Luc)。具体结果见表3和图6。
表3 61-42-rFc单域抗体与各CD5阳性细胞之间的亲和力汇总如表
Figure PCTCN2022071674-appb-000004
研究结论：CD5串联单域抗体兔Fc融合蛋白61-42-rFc与所测定的4株CD5阳性细胞之间具有稳定、良好、特异性的结合能力，EC50数值均在1～5nM。
参考文献：
1.Voisinne G,Gonzalez de Peredo A,Roncagalli R.CD5,an Undercover Regulator of TCR Signaling.Front Immunol.2018；9:2900.Published 2018 Dec 7.doi:10.3389/fimmu.2018.02900.
2.Bamberger M,Santos AM,
Figure PCTCN2022071674-appb-000005
CM,et al.A new pathway of CD5glycoprotein-mediated T cell inhibition dependent on inhibitory phosphorylation of Fyn kinase.J Biol Chem.2011；286(35):30324-30336.doi:10.1074/jbc.M111.230102.
3.Perez-Villar JJ,Whitney GS,Bowen MA,Hewgill DH,Aruffo AA,Kanner SB.CD5 negatively regulates the T-cell antigen receptor signal transduction pathway:involvement of SH2-containing phosphotyrosine phosphatase SHP-1.Mol Cell Biol.1999；19(4):2903–12.
4.Gary-Gouy H,Sainz-Perez A,Marteau JB,et al.Natural phosphorylation of CD5 in chronic lymphocytic leukemia B cells and analysis of CD5-regulated genes in a B cell  line suggest a role for CD5 in malignant phenotype.J Immunol.2007；179(7):4335-4344.doi:10.4049/jimmunol.179.7.4335
5.Freitas CMT,Johnson DK,Weber KS.T Cell Calcium Signaling Regulation by the Co-Receptor CD5.Int J Mol Sci.2018；19(5):1295.Published 2018 Apr 26.doi:10.3390/ijms19051295
6.Pui CH,Behm FG,Crist WM.Clinical and biologic relevance of immunologic marker studies in childhood acute lymphoblastic leukemia.Blood.1993；82(2):343-362.
7.Campana D,van Dongen JJ,Mehta A,et al.Stages of T-cell receptor protein expression in T-cell acute lymphoblastic leukemia.Blood.1991；77(7):1546-1554.
8.Huang H,Li Z,Huang C,et al.CD5 and CD43 Expression are Associate with Poor Prognosis in DLBCL Patients.Open Med(Wars).2018；13:605-609.Published 2018 Nov 27.doi:10.1515/med-2018-0089
9.Friedman DR,Guadalupe E,Volkheimer A,Moore JO,Weinberg JB.Clinical outcomes in chronic lymphocytic leukaemia associated with expression of CD5,a negative regulator of B-cell receptor signalling.Br J Haematol.2018；183(5):747-754.doi:10.1111/bjh.15632
10.Dillman RO,Shawler DL,Dillman JB,Royston I.Therapy of chronic lymphocytic leukemia and cutaneous T-cell lymphoma with T101 monoclonal antibody.J Clin Oncol.1984；2(8):881-891.doi:10.1200/JCO.1984.2.8.881
11.Foss FM,Raubitscheck A,Mulshine JL,et al.Phase I study of the pharmacokinetics of a radioimmunoconjugate,90Y-T101,in patients with CD5-expressing leukemia and lymphoma.Clin Cancer Res.1998；4(11):2691-2700.
12.Phage display-methods and protocols，ISSN 1064-3745 ISSN 1940-6029(electronic)Methods in Molecular Biology ISBN 978-1-4939-7446-7ISBN 978-1-4939-7447-4(eBook),DOI 10.1007/978-1-4939-7447-4.
SEQ ID NO：13#42 VH DNA序列：363bp
Figure PCTCN2022071674-appb-000006
Figure PCTCN2022071674-appb-000007
SEQ ID NO：14#60 VH DNA序列：351bp
Figure PCTCN2022071674-appb-000008
SEQ ID NO：15#61 VH DNA序列：354bp
Figure PCTCN2022071674-appb-000009
SEQ ID NO：16#62 VH DNA序列：369bp
Figure PCTCN2022071674-appb-000010
SEQ ID NO：17#42 VH氨基酸序列：121aa
Figure PCTCN2022071674-appb-000011
SEQ ID NO：18#60 VH氨基酸序列：117aa
Figure PCTCN2022071674-appb-000012
SEQ ID NO：19#61 VH氨基酸序列：118aa
Figure PCTCN2022071674-appb-000013
SEQ ID NO：20#62 VH氨基酸序列：123aa
Figure PCTCN2022071674-appb-000014

Claims (39)

  1. 靶向CD5的抗体或其抗原结合片段，其中所述抗体包括重链可变区(HCVR)，所述重链可变区包括HCDR1、HCDR2和HCDR3，所述HCDR1、HCDR2和HCDR3选自如下组合之一：
    (1)HCDR1的氨基酸序列为GFTFSHSA(SEQ ID NO：1)；
    HCDR2的氨基酸序列为IYARGGYT(SEQ ID NO：2)；
    HCDR3的氨基酸序列为ARGYHLEYMVSQDV(SEQ ID NO：3)；
    (2)HCDR1的氨基酸序列为GFTFSSYE(SEQ ID NO：4)；
    HCDR2的氨基酸序列为ISSSGSTI(SEQ ID NO：5)；
    HCDR3的氨基酸序列为ARVAQREGDV(SEQ ID NO：6)；
    (3)HCDR1的氨基酸序列为GGTFSNYA(SEQ ID NO：7)；
    HCDR2的氨基酸序列为ISAYNGDT(SEQ ID NO：8)；
    HCDR3的氨基酸序列为ARYESMSGQDI(SEQ ID NO：9)；
    (4)HCDR1的氨基酸序列为GYSFSNHW(SEQ ID NO：10)；
    HCDR2的氨基酸序列为VYPGDSDT(SEQ ID NO：11)；
    HCDR3的氨基酸序列为ARGGTIDGDYGGRQDF(SEQ ID NO：12)；或
    所述抗体包括(1)-(4)任一项中的CDR序列组合的变体，其中所述变体(1)-(4)任一项中的CDR序列相比，具有至少90％的序列一致性，或在CDR序列上共包含至少1个且不超过10，或不超过5、4、3、2或1个氨基酸改变。
  2. 如权利要求1所述的抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自如下任一项：
    (1)SEQ ID NO：17所示序列或与其有至少90％序列一致性的重链可变区序列；
    (2)SEQ ID NO：18所示序列或与其有至少90％序列一致性的重链可变区序列；
    (3)SEQ ID NO：19所示序列或与其有至少90％序列一致性的重链可变区序列；
    (4)SEQ ID NO：20所示序列或与其有至少90％序列一致性的重链可变区序列。
  3. 如权利要求1或2所述的抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自如下任一项：
    (1)SEQ ID NO：17所示的重链可变区序列；
    (2)SEQ ID NO：18所示的重链可变区序列；
    (3)SEQ ID NO：19所示的重链可变区序列；
    (4)SEQ ID NO：20所示的重链可变区序列。
  4. 如权利要求1-3任一项所述的抗体或其抗原结合片段，其中所述抗体为单域抗体。
  5. 如权利要求1-4任一项所述的抗体或其抗原结合片段，其中所述抗体为全人源抗体。
  6. 如权利要求1-5任一项所述的抗体或其抗原结合片段，其中通过生物膜干涉技术测定的所述抗体对CD5抗原的结合KD值低于10 -7M，优选低于10 -8M。
  7. 融合蛋白，包括一个或两个抗原结合功能部分，其中每个所述抗原结合功能部分包括权利要求1-6任一项所述的抗体或其抗原结合片段，优选地，所述融合蛋白还包括Fc片段。
  8. 如权利要求7所述的融合蛋白，其中所述两个抗原结合功能部分分别结合相同或不同的抗原表位。
  9. 如权利要求7或8所述的融合蛋白，包括串联连接的第一抗原结合功能部分和第二抗原结合功能部分；其中所述第一抗原结合功能部分包括第一重链可变区(HCVR)，所述第一重链可变区包括SEQ ID NO：7所示的HCDR1、SEQ ID NO：8所示的HCDR2和SEQ ID NO：9所示的HCDR3；其中所述第二抗原结合功能部分包括第二重链可变区(HCVR)，所述第二重链可变区包括SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2和SEQ ID NO：3所示的HCDR3。
  10. 如权利要求7-9任一项所述的融合蛋白，包括串联连接的SEQ ID NO：19所示的重链可变区序列和SEQ ID NO：17所示的重链可变区序列。
  11. 如权利要求7-10任一项所述的融合蛋白，其中所述抗原结合功能部分直接通过接头分子连接；优选地，所述接头分子包括SEQ ID NO：21所示的氨基酸序列。
  12. 如权利要求7-11任一项所述的融合蛋白，其中通过流式细胞术测定的所述融合蛋白与CD5阳性细胞之间结合的EC 50值在1-5nM。
  13. 编码权利要求1-6任一项的抗体或其抗原结合片段或权利要求7-12任一项所述的融合蛋白的核酸分子。
  14. 如权利要求13所述的核酸分子，其包括SEQ ID NO:13-16任一项所示的核苷酸序列。
  15. 一种表达载体，其包括权利要求13或14所述的核酸分子。
  16. 一种宿主细胞，其包括权利要求15所述的表达载体。
  17. 一种药物组合物，其包括
    1)权利要求1-6任一项的抗体或其抗原结合片段，或者权利要求7-12任一项所述的融合蛋白；以及
    2)药学上可接受的载体或稀释剂。
  18. 一种治疗疾病或病症的方法，所述方法包括通过向有需要的患者施用治疗有效量的权利要求1-6任一项所述的抗体或其抗原结合片段，权利要求7-12任一项所述的融合蛋白，权利要求16所述的宿主细胞，或权利要求17所述的药物组合物，以消除、抑制或降低CD5活性，从而预防、减轻、改善或抑制疾病或病症。
  19. 如权利要求18所述的方法，其中所述疾病或病症选自：癌症。
  20. 如权利要求19所述的方法，其中所述癌症选自：T细胞恶性肿瘤。
  21. 与权利要求1-6任一项的抗体或其抗原结合片段竞争相同表位的抗体或片段。
  22. 一种试剂盒，用于检测样品中CD5蛋白，其中，所述试剂盒包括权利要求1-6任一项所述的抗体或其抗原结合片段，或权利要求7-12任一项所述的融合蛋白。
  23. 权利要求1-6任一项所述的抗体或其抗原结合片段，权利要求7-12任一项所述的融合蛋白，或权利要求16所述的宿主细胞在制备用于消除、抑制或降低CD5活性，从而预防、减轻、改善或抑制疾病或病症的药物中的用途。
  24. 如权利要求23所述的用途，其中所述疾病或病症选自：癌症或自身免疫疾病。
  25. 如权利要求24所述的用途，其中所述癌症选自：恶性T细胞肿瘤或恶性B细胞肿瘤。
  26. 如权利要求25所述的用途，其中所述恶性T细胞肿瘤选自T细胞急性淋巴细胞白血病(T-ALL)、T细胞淋巴瘤(TCL)，所述恶性B细胞肿瘤选自慢性淋巴细胞白血病(B-CLL)或套细胞淋巴瘤(B-MCL)。
  27. 多特异性抗体分子，至少包括第一功能部分和第二功能部分，其中第一功能部分包括权利要求1-6任一项所述的抗体或其抗原结合片段；第二功能部分具有与所述第一功能部分不同的结合特异性。
  28. 如权利要求27所述的多特异性抗体分子，其中第二功能部分对免疫细胞具有结合特异性。
  29. 如权利要求27或28所述的多特异性抗体分子，其中所述第二功能部分对T细胞具有结合特异性。
  30. 如权利要求27-29任一项所述的多特异性抗体分子，其中所述第二功能部分对CD7具有结合特异性。
  31. 免疫偶联物，包括与治疗剂连接的权利要求1-6中任一项所述的抗体或其抗原结合片段。
  32. 如权利要求31所述的免疫偶联物，其中所述治疗剂是药物。
  33. 如权利要求31或32所述的免疫偶联物，其中所述治疗剂是细胞毒素。
  34. 如权利要求31-33任一项所述的免疫偶联物，其中所述治疗剂是放射性同位素。
  35. 权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求7-12任一项所述的融合蛋白、权利要求27-30任一项所述的多特异性抗体分子或权利要求31-34任一项所述的免疫偶联物在制备用于消除、抑制或降低CD5活性的药物中的用途。
  36. 如权利要求35所述的用途，其中所述药物用于预防、减轻、改善或抑制癌症或自身免疫疾病。
  37. 如权利要求36所述的用途，其中所述癌症选自：恶性T细胞肿瘤或恶性B细胞肿瘤。
  38. 如权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求7-12任一项所述的融合蛋白、权利要求27-30任一项所述的多特异性抗体分子或权利要求31-34任一项所述的免疫偶联物的用途，用于制备疫苗，较佳地为抗体疫苗，更佳地为抗独特型抗体疫苗。
  39. 一种疫苗制品，其包括如权利要求1-6任一项所述的抗体或其抗原结合片段、权利要求7-12任一项所述的融合蛋白、权利要求27-30任一项所述的多特异性抗体分子或权利要求31-34任一项所述的免疫偶联物。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115947840A (zh) * 2023-01-06 2023-04-11 南京蓬勃生物科技有限公司 抗人FcRn单域抗体及其应用
WO2024012495A1 (zh) * 2022-07-12 2024-01-18 上海驯鹿生物技术有限公司 表达靶向cd5的嵌合抗原受体(car)的细胞及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117402247B (zh) * 2023-12-14 2024-03-08 成都优赛诺生物科技有限公司 一种靶向cd5的单域抗体、嵌合抗原受体及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008121160A2 (en) * 2006-11-21 2008-10-09 Xencor, Inc. Optimized antibodies that target cd5
CN101943704A (zh) * 2010-04-30 2011-01-12 北京大学人民医院 抗cd19的抗体和抗cd5的抗体的新用途
CN102137873A (zh) * 2008-08-29 2011-07-27 西福根有限公司 抗cd5抗体
CN102786595A (zh) * 2012-08-03 2012-11-21 无锡傲锐东源生物科技有限公司 抗cd5蛋白单克隆抗体及其用途
WO2014125455A1 (fr) * 2013-02-18 2014-08-21 Universite Paris Sud Xi Un anticorps monoclonal contre le recepteur cd5 a multiples applications en medecine et en recherche anti-cancereuse
CN109266667A (zh) * 2018-11-06 2019-01-25 中国医学科学院血液病医院（血液学研究所） 靶向cd5的嵌合抗原受体及其应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4049674A1 (en) 2009-11-03 2022-08-31 City of Hope Truncated epidermal growth factor receptor (egfrt) for transduced t cell selection
NZ700362A (en) 2012-04-11 2016-09-30 Us Health Chimeric antigen receptors targeting b-cell maturation antigen
EP3172231B1 (en) 2014-07-24 2021-05-05 Bluebird Bio, Inc. Bcma chimeric antigen receptors
PL3261651T3 (pl) * 2015-02-27 2022-08-22 Icell Gene Therapeutics Llc Chimeryczne receptory antygenowe (car) ukierunkowane na nowotwory złośliwe układu krwiotwórczego, kompozycje i sposoby ich stosowania
US10786549B2 (en) * 2015-04-23 2020-09-29 Baylor College Of Medicine CD5 chimeric antigen receptor for adoptive T cell therapy
BR112020010662A2 (pt) * 2017-11-29 2020-11-10 Magenta Therapeutics, Inc. composições e métodos para a depleção de células cd5+
MX2021007547A (es) * 2018-12-19 2021-10-13 Univ Pennsylvania Uso de células t del receptor de antígeno quimérico anti-cd2/5/7 de eliminación de cd2/5/7 contra linfomas y leucemias de células t.
EP3760217A1 (en) * 2019-07-01 2021-01-06 Max-Delbrück-Centrum für Molekulare Medizin in der Helmholtz-Gemeinschaft Cd5 specific t cell receptor cell or gene therapy

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008121160A2 (en) * 2006-11-21 2008-10-09 Xencor, Inc. Optimized antibodies that target cd5
CN102137873A (zh) * 2008-08-29 2011-07-27 西福根有限公司 抗cd5抗体
CN101943704A (zh) * 2010-04-30 2011-01-12 北京大学人民医院 抗cd19的抗体和抗cd5的抗体的新用途
CN102786595A (zh) * 2012-08-03 2012-11-21 无锡傲锐东源生物科技有限公司 抗cd5蛋白单克隆抗体及其用途
WO2014125455A1 (fr) * 2013-02-18 2014-08-21 Universite Paris Sud Xi Un anticorps monoclonal contre le recepteur cd5 a multiples applications en medecine et en recherche anti-cancereuse
CN109266667A (zh) * 2018-11-06 2019-01-25 中国医学科学院血液病医院（血液学研究所） 靶向cd5的嵌合抗原受体及其应用

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
BAMBERGER MSANTOS AMGONCALVES CM ET AL.: "A new pathway of CDS glycoprotein-mediated T cell inhibition dependent on inhibitory phosphorylation of Fyn kinase", J BIOL CHEM., vol. 286, no. 35, 2011, pages 30324 - 30336, XP055077325, DOI: 10.1074/jbc.M111.230102
CAMPANA DVAN DONGEN JJMEHTA A ET AL.: "Stages of T-cell receptor protein expression in T-cell acute lymphoblastic leukemia", BLOOD, vol. 77, no. 7, 1991, pages 1546 - 1554, XP002755663
DILLMAN ROSHAWLER DLDILLMAN JBROYSTON I: "Therapy of chronic lymphocytic leukemia and cutaneous T-cell lymphoma with T101 monoclonal antibody", J CLIN ONCOL., vol. 2, no. 8, 1984, pages 881 - 891
FOSS FMRAUBITSCHECK AMULSHINE JL ET AL.: "Phase I study of the pharmacokinetics of a radioimmunoconjugate, 90Y-T101, in patients with CDS-expressing leukemia and lymphoma", CLIN CANCER RES., vol. 4, no. 11, 1998, pages 2691 - 2700
FREITAS CMTJOHNSON DKWEBER KS: "T Cell Calcium Signaling Regulation by the Co-Receptor CD5", INT J MOL SCI., vol. 19, no. 5, 26 April 2018 (2018-04-26), pages 1295
FRIEDMAN DRGUADALUPE EVOLKHEIMER AMOORE JOWEINBERG JB: "Clinical outcomes in chronic lymphocytic leukaemia associated with expression of CD5, a negative regulator of B-cell receptor signaling", BR J HAEMATOL., vol. 183, no. 5, 2018, pages 747 - 754
GARY-GOUY HSAINZ-PEREZ AMARTEAU JB ET AL.: "Natural phosphorylation of CD5 in chronic lymphocytic leukemia B cells and analysis of CDS-regulated genes in a B cell line suggest a role for CD5 in malignant phenotype.", J IMMUNOL., vol. 179, no. 7, 2007, pages 4335 - 4344
HUANG HLI ZHUANG C ET AL.: "DLBCL Patients. Open Med (Wars", vol. 13, 27 November 2018, article "CD5 and CD43 Expression are Associate with Poor Prognosis", pages: 605 - 609
PEREZ-VILLAR JJWHITNEY GSBOWEN MAHEWGILL DHARUFFO AAKANNER SB: "CD5 negatively regulates the T-cell antigen receptor signal transduction pathway: involvement of SH2-containing phosphotyrosine phosphatase SHP-1", MOL CELL BIOL., vol. 19, no. 4, 1999, pages 2903 - 12
PUI CHBEHM FGCRIST WM: "Clinical and biological relevance of immunological marker studies in childhood acute lymphoblastic leukemia", BLOOD, vol. 82, no. 2, 1993, pages 343 - 362
RAJU ET AL., BIOCHEMISTRY, vol. 40, no. 30, 2001, pages 8868 - 76
VOISINNE GGONZALEZ DE PEREDO ARONCAGALLI R: "CD5, an Undercover Regulator of TCR Signaling", FRONT IMMUNOL, vol. 9, 7 December 2018 (2018-12-07), pages 2900, XP055879490, DOI: 10.3389/fimmu.2018.02900

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024012495A1 (zh) * 2022-07-12 2024-01-18 上海驯鹿生物技术有限公司 表达靶向cd5的嵌合抗原受体(car)的细胞及其应用
CN115947840A (zh) * 2023-01-06 2023-04-11 南京蓬勃生物科技有限公司 抗人FcRn单域抗体及其应用
CN115947840B (zh) * 2023-01-06 2023-09-19 南京蓬勃生物科技有限公司 抗人FcRn单域抗体及其应用

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