CN117467013A - Antibodies or binding fragments thereof specifically binding to IgG kappa light chain and uses thereof - Google Patents
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- CN117467013A CN117467013A CN202310788177.1A CN202310788177A CN117467013A CN 117467013 A CN117467013 A CN 117467013A CN 202310788177 A CN202310788177 A CN 202310788177A CN 117467013 A CN117467013 A CN 117467013A
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Abstract
The invention relates to the technical field of biology, and in particular discloses an antibody specifically binding to an IgG kappa type light chain or a binding fragment and application thereof. The antibody comprises a heavy chain comprising a heavy chain variable region and a constant region and a light chain comprising a light chain variable region and a constant region. The antibodies of the present invention can be prepared by in vivo abdominal water induction method or genetic engineering method of hybridoma cells, and all show high affinity and specificity with IgG kappa type light chains, so that the antibodies can be effectively applied to in vivo and in vitro pharmacokinetic, pharmacodynamic and immunogenicity studies of different IgG kappa type light chains or IgG comprising kappa type light chains.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an antibody specifically binding to an IgG kappa type light chain or a binding fragment and application thereof. Particular embodiments of the invention relate to amino acid sequences, nucleic acids and methods of preparation of antibodies or binding fragments thereof that specifically bind to kappa-type light chains of human IgG, and uses thereof for in vivo and in vitro pharmacokinetic, pharmacodynamic and immunogenicity studies of human IgG comprising kappa-type light chains.
Background
IgG is the most abundant and predominant immunoglobulin in humans, accounting for 70% -85% of total immunoglobulin, has a molecular weight of about 150KD, and contains 2-3% of sugar. Each IgG molecule consists of two heavy chains and two light chains. Two heavy chains are linked to each other by disulfide bonds, each heavy chain being linked to one light chain by disulfide bonds. The light chain constant region amino acid sequence and antigenicity are classified into kappa (kappa) type and lambda (lambda) type, and the corresponding IgG is named kappa type and lambda type IgG, respectively. IgG can be divided into 4 subtypes, depending on the composition of heavy chain amino acids, number of interchain disulfide bonds, and position: igG 1, igG 2, igG 3 and IgG 4.
Therapeutic antibodies (Abs) are tailored biological agents for the diagnosis and treatment of various diseases, such as cancer, inflammatory diseases and autoimmune diseases, that can activate or inhibit biological processes associated with a particular disease by binding to a particular antigen (Ag), including can be used to prevent proliferation of cancer cells. Most antibodies approved by the market are derived from IgG as a carrier, and mainly comprise the following four types: murine, chimeric, humanized and fully human. Since 2003, the approval rates of humanized and fully human antibodies have exceeded those of chimeric and murine origin, becoming the major class of therapeutic antibodies. In most of the humanized antibodies on the market, the heavy chain subtype used was IgG 1, accounting for 72.4% of the antibody drug counted, and in addition was IgG 2 heavy chain and a few IgG 4 heavy chains in sequence. Therefore, it is very important to develop a generic antibody capable of binding to various subtypes of human IgG. Since kappa light chains represent the major (about 90%) subtype of antibody therapeutics, the invention has great application value for Pharmacokinetic (PK) and immunogenicity studies of antibody therapeutics in preclinical or clinical studies.
Pharmacokinetic (PK) is a discipline that states the law of change of blood concentration over time by quantitatively studying drug absorption, distribution, metabolism and excretion in an organism. Pharmacodynamics (PD) is a discipline for elucidating the relationship between the drug and the dose-effect by studying the action and mechanism of action of the drug, adverse reactions of the drug, factors affecting the action of the drug, and the like. To describe more precisely the relationship between drug dose and drug effect, sheiner is incorporated into the effector compartment in classical pharmacokinetics, known as pharmacokinetic-pharmacodynamic model (PK/PD modeling). The PK/PD model organically combines two interrelated dynamic processes of PK and PD, and simultaneously discusses the action of the organism on the medicine and the action of the medicine on the organism so as to more comprehensively and accurately know the rule of the effect of the medicine changing along with the dosage (or concentration) and time, thereby providing more scientific theoretical basis for the safety and effectiveness of clinical medication. In various stages of drug development, including early preclinical studies, PK/PD studies are increasingly important in the drug development process, which is a key to influencing whether clinical trials continue and supporting the efficient performance of the various stages of development. Immunogenicity of a drug refers to the ability of the drug and/or its metabolite to elicit an immune response or immune related event to itself or related proteins. Anti-drug antibodies (ADA) are the primary means of evaluating the immunogenicity of antibody drugs. If a clinically relevant immune response is observed, its underlying mechanism should be investigated and key influencing factors determined. These studies help control and ease the formulation and implementation of immune response strategies, including modifying product prescriptions or screening high risk patient populations, and the like.
Based on preclinical and clinical importance, PK/PD and ADA are indispensable in the development of antibody therapeutics. Therefore, the development of antibodies that bind human kappa IgG (including the different subclasses of IgG1 to 4) for use in PK/PD and ADA assays would be of great interest in the biomedical field.
Disclosure of Invention
In view of the importance and good application prospects of antibody-binding anti-drug antibodies, the invention provides an antibody or a binding fragment thereof specifically binding to an IgG kappa-type light chain, which can have high affinity and specificity with the IgG kappa-type light chain.
One aspect of the invention relates to an antibody or binding fragment thereof that specifically binds to an IgG kappa-type light chain comprising a heavy chain variable region and a light chain comprising a light chain variable region, and the heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3.
In some embodiments of the invention, the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 comprised by the heavy chain variable region set forth in SEQ ID NO. 7 or 15, and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2, and LCDR3 comprised by the light chain variable region set forth in SEQ ID NO. 8 or 16.
In some embodiments of the invention, the HCDR1, HCDR2 and HCDR3 are the amino acid sequences shown in SEQ ID NOs 1, 2 and 3, respectively, and the LCDR1, LCDR2 and LCDR3 are the amino acid sequences shown in SEQ ID NOs 4, 5 and 6, respectively.
In some embodiments of the invention, the HCDR1, HCDR2 and HCDR3 are the amino acid sequences shown in SEQ ID NOs 9, 10 and 11, respectively, and the LCDR1, LCDR2 and LCDR3 are the amino acid sequences shown in SEQ ID NOs 12, 13 and 14, respectively.
In some embodiments of the invention, more specifically, the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 7 and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 8.
In some embodiments of the invention, more specifically, the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 15 and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 16.
In some embodiments of the invention, the heavy chain variable region comprises an amino acid sequence having at least 70%, 80%, 90% or 95% identity to SEQ ID NO. 7 or 15, and the light chain variable region comprises an amino acid sequence having at least 70%, 80%, 90% or 95% identity to SEQ ID NO. 8 or 16.
Some embodiments of the invention relate to antibodies or binding fragments thereof that specifically bind to IgG kappa-type light chains, which antibodies are full length antibodies, comprising constant region sequences. In some embodiments, the constant region sequence is murine or human, wherein the heavy chain constant region comprises a constant region of human or murine IgA, igG, igM, igE or IgD and the light chain constant region comprises a constant region of human or murine kappa or lambda. In some embodiments of the invention, the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO. 17 and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID NO. 18.
Some embodiments of the invention relate to antibodies or binding fragments thereof that bind to IgG kappa-type light chains, wherein the binding fragments are Fab, fab ', F (ab') 2 Single chain Fv (scFv) fragments, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2 Fv-Fc fusion, scFv-Fv fusion or VHH.
Another aspect of the invention relates to an antibody that specifically binds to an IgG kappa-type light chain, comprising a heavy chain and a light chain; in some embodiments of the invention, the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 19, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 20; in other embodiments of the invention, the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 21 and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 22.
The invention also relates to an antibody-label conjugate, wherein the antibody or binding fragment thereof that specifically binds to an IgG kappa light chain is linked to a label; wherein the label may be a fluorescent dye, radionuclide, chemiluminescent molecule, colloidal gold, biotin, avidin, or enzyme.
The invention also relates to a nucleic acid encoding an antibody or binding fragment thereof as described above that specifically binds to an IgG kappa-type light chain, in some embodiments of the invention, in particular said nucleic acid is capable of encoding the amino acid sequence shown in SEQ ID NOs 1-22.
The invention also relates to a vector comprising, in some embodiments of the invention, a nucleic acid as described above capable of encoding an antibody or binding fragment thereof as described above that specifically binds to an IgG kappa-type light chain, which is capable of protein expression in a host cell.
The invention also relates to a host cell comprising a nucleic acid and/or vector as described above and capable of expressing an antibody or binding fragment thereof that binds to an IgG kappa-type light chain in some embodiments of the invention. In certain embodiments of the invention, the host cell is selected from COS-7 (monkey kidney cell 7), NSO cell, SP2/0 cell, CHO (Chinese hamster ovary) cell, W138, BHK (baby hamster kidney) cell, MDCK, myeloma cell line, huT78 cell, HEK293 cell, E.coli, B.subtilis, streptomyces, pseudomonas, proteus mirabilis, staphylococcus, fungi (e.g., aspergillus, pichia, saccharomyces cerevisiae, schizosaccharomyces, and Neurospora crassa).
Another aspect of the invention relates to a method for preparing an antibody or binding fragment thereof that specifically binds to an IgG kappa-type light chain, comprising culturing the cells in a medium suitable for expression to express the antibody or binding fragment thereof and obtaining the antibody or binding fragment thereof.
Another aspect of the invention also relates to a method of assaying IgG kappa light chains or IgG comprising kappa light chains in a biological sample, comprising:
(a) Contacting a biological sample with a capture reagent, said capture reagent being a target protein to which said IgG is directed, thereby forming an immune complex;
(b) Contacting the immunocomplex from step (a) with an antibody or binding fragment thereof that specifically binds to an IgG kappa-type light chain as described above and a label secondary molecule attached thereto, or contacting the immunocomplex from step (a) with an antibody-label conjugate as described above,
(c) Determining the presence or absence or amount of IgG kappa light chains or IgG comprising kappa light chains bound to the antibody or binding fragment thereof by detecting the label.
Wherein the label is a fluorescent dye, a chemiluminescent molecule, a radionuclide, colloidal gold, biotin, avidin, or an enzyme.
Another aspect of the invention relates to an antibody or binding fragment thereof that specifically binds to an IgG kappa-type light chain and the use of an antibody-label conjugate as described above for detecting IgG of an IgG kappa-type light chain and/or an IgG comprising a kappa-type light chain, preferably for in vivo and in vitro pharmacokinetic, pharmacodynamic or immunogenicity studies of a human IgG kappa-type light chain and/or a human IgG comprising a kappa-type light chain.
Another aspect of the invention relates to antibodies or binding fragments thereof that specifically bind to kappa-type light chains of IgG and antibody-tag conjugates as described above for use in detecting kappa-type light chains of IgG and/or IgG comprising kappa-type light chains, e.g., human IgG for detecting and/or measuring kappa-type light chains in a sample.
Another aspect of the invention relates to a kit comprising an antibody or binding fragment thereof as described above that specifically binds to a human IgG kappa-type light chain for detection of human IgG of the human IgG kappa-type light chain and/or comprising a kappa-type light chain, or for in vivo and in vitro pharmacokinetic, pharmacodynamic and immunogenicity studies of human IgG kappa-type light chain and/or comprising a kappa-type light chain, and instructions for use.
Drawings
FIG. 1 is a SDS-PAGE result of antibodies (clone No. 2D2B 1) binding to IgG kappa type light chains prepared by in vivo induced abdominal water method according to an embodiment of the present invention.
FIG. 2 is a SDS-PAGE result of antibodies (clone No. 2B5H 4) binding to IgG kappa type light chains prepared by in vivo induced abdominal water method according to an embodiment of the invention.
FIG. 3 is a SDS-PAGE result of antibodies (clone No. 2B5F 7) binding to IgG kappa type light chains prepared by in vivo induced abdominal water method according to an embodiment of the invention.
FIG. 4 is a SDS-PAGE result of antibodies (clone No. 3D1G 4) binding to IgG kappa type light chains prepared by in vivo induced abdominal water method according to an embodiment of the invention.
FIG. 5 is a SDS-PAGE result of antibodies (clone number 3D1C 3) binding to IgG kappa type light chains prepared by in vivo induced abdominal water method according to an embodiment of the invention.
In fig. 1 to 5, lane1 is the result after antibody reduction, and Lane2 is the band before antibody reduction; the results show that the antibodies after reduction split into heavy and light chain bands.
FIG. 6 is a graph showing a curve fit of the binding of 2D2B1, 2B5H4, 2B5F7, 3D1G4, 3D1C3 monoclonal antibodies and commercial monoclonal antibodies R10Z8E9 to anti-C-MET antibodies according to embodiments of the present invention.
FIG. 7 is a graph showing a curve fit of the binding of 2D2B1, 2B5H4, 2B5F7, 3D1G4, 3D1C3 monoclonal antibodies and commercial monoclonal antibodies R10Z8E9 to an anti-DR 4 antibody according to an embodiment of the invention.
FIG. 8 is a graph showing a curve fit of the binding of 2D2B1, 2B5H4, 2B5F7, 3D1G4, 3D1C3 monoclonal antibodies and commercial monoclonal antibodies R10Z8E9 to lambda-chain human IgG drug anti-PD-L1 antibodies in an embodiment of the invention.
FIG. 9 is a graph showing the binding of a kappa light chain human IgG drug anti-HER 2 antibody (IgG 1, trastuzumab) using 2D2B1 as the capture antibody and 2B5F7-HRP as the detection antibody in an embodiment of the invention.
Detailed Description
Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Unless defined otherwise, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used in this specification is well known and commonly employed in the art.
Definition of the definition
In this specification, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies but also antigen-binding fragments of antibodies.
In the present specification, "Anti-Drug Antibodies (ADA)" refers to an immunoglobulin produced in vivo as a result of the use of a certain antibody Drug that is capable of specifically binding to the antibody Drug, and is also an antibody in nature, but is herein distinguished from an antibody, herein referred to as an Anti-Drug antibody, which, unless otherwise stated, when referred to herein, refers to an Anti-human IgG kappa light chain (kappa type light chain) and/or an Anti-human IgG antibody comprising kappa light chains (kappa type light chain).
An "antibody fragment" or "binding fragment" refers to a molecule that is different from an intact antibody, which comprises a portion of the intact antibody and binds to an antigen to which the intact antibody binds. For example, antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') 2 The method comprises the steps of carrying out a first treatment on the surface of the A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.
In the present specification, a "light chain" is a peptide chain of a relatively small molecular weight in an immunoglobulin (IgG, antibody), generally comprising about 214 amino acids, and is classified into a "kappa light chain (kappa type light chain)" and a "lambda light chain (lambda type light chain)" in terms of their structure and antigenicity of a constant region.
In the present specification, "human IgG" includes a fully human antibody, a fully humanized antibody or a partially humanized antibody comprising a kappa-type light chain of human IgG, and a human chimeric antibody.
In the present specification, "full length antibody", "intact antibody" and "intact antibody" are used interchangeably herein to refer to antibodies that are substantially similar in structure to the native antibody structure or have a heavy chain comprising an Fc region as defined herein.
In this specification, anti-c-MET antibodies are kappa-type light chain, igG 2-type monoclonal antibodies targeting c-MET and comprising the heavy and light chains shown in SEQ ID NOs 23 and 24. Wherein, c-MET is a cell mesenchymal epithelial transforming factor (cell-mesenchymal epithelial transition factor) which is a receptor tyrosine kinase family member; the tumor c-MET signaling pathway can be frequently activated by tumor cells, promoting tumor formation, invasive growth, and metastasis. anti-c-MET monoclonal antibodies bind to c-MET, inhibit phosphorylation of intracellular tyrosine kinase, and block downstream signaling.
In the present specification, an anti-death receptor 4 antibody (anti-DR 4 antibody) is a kappa-type light chain, igG 1-type monoclonal antibody targeting death receptor 4, comprising the heavy and light chains shown in SEQ ID NOS 25 and 26. Wherein death receptor 4 (DR 4) belongs to tumor necrosis factor receptor superfamily member (TNFRSF 10A), cytoplasmic region contains death domain, and is distributed in tumor cells, lymphoid tissue and activated T cells, and TRAIL binds with cell surface DR4 to induce apoptosis. Wherein the amino acid sequence of the death domain in DR4 is relatively conserved, expressed in normal tissues such as spleen, peripheral blood leukocytes, small intestine, thyroid, activated T cells, etc., and in tumor tissues. DR4 on the surface of tumor cells in Fab region of anti-death receptor 4 monoclonal antibody multimerizes, enhancing pro-apoptotic signal, and causing apoptosis of tumor cells.
In this specification, an anti-PD-L1 antibody is a lambda type light chain, igG1 type antibody targeting PD-L1, comprising the heavy and light chains shown in SEQ ID NOs 27 and 28. Among them, PD-L1 is a ligand-1 of programmed death molecule, which is an important immunosuppressive factor expressed on the surface of tumor cells, and can interact with PD-1 receptor to make tumor cells escape T cell recognition.
In the present specification, the anti-HER 2 antibody is an antibody of kappa type light chain targeting HER2, and in a specific embodiment of the present invention, the anti-HER 2 antibody is trastuzumab. Wherein HER2 refers to human EGFR-2 (Human Epidermal Growth Factor Receptor 2, HER 2), and the gene is a found breast cancer proto-oncogene. HER2 protein is a transmembrane protein with tyrosine kinase activity, belongs to one of EGFR family members, and is one of the important targets for developing medicaments for treating tumors at present.
In this specification, the term "Pharmacokinetics (PK)" is a discipline for quantitatively studying the absorption, distribution, metabolism and excretion processes of drugs in living bodies and describing the dynamic laws of drugs in the bodies using mathematical principles and methods. The concentration of the medicine at the action part is dynamically changed under the influence of the in-vivo process of the medicine, and is an important component of preclinical research and clinical research of the medicine.
In the present specification, the term "Pharmacodynamics (PD)" is a discipline for researching the action of a drug on a body and its mechanism, that is, the change rule of the physiological functions of organs and the metabolic activities of cells of the body under the action of the drug, and is also an important component of preclinical and clinical research of the drug.
In this specification, the term "complementarity determining region" or "CDR" is a region of an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and/or contains antigen-contacting residues ("antigen-contacting points"). The CDRs are mainly responsible for binding to the epitope, and sequentially comprise a CDR1, a CDR2 and a CDR3 from the N-terminal, wherein the CDRs of the heavy chain variable region are sequentially HCDR1, HCDR2 and HCDR3, and the CDRs of the light chain variable region sequentially comprise LCDR1, LCDR2 and LCDR3. In a given heavy chain variable region amino acid sequence, the exact amino acid sequence boundaries of each CDR can be determined using any one of a number of well-known antibody CDR assignment systems, or a combination thereof. It is well known to those skilled in the art that CDRs of antibodies can be defined in a variety of ways, such as Chothia (Chothia et al (1989) Nature 342:877-883, al-Lazikani et al, journal of Molecular Biology,273,927-948 (1997)), kabat (Kabat et al, U.S. device of Health and Human Services, national Institutes of Health (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT) (world Wide Web IMGT. Cis. Fr /), based on topology of the antibody and North CDR definitions based on neighbor-transmitted clusters (affinity propagation clustering) using a large number of crystal structures. It will be appreciated by those skilled in the art that unless otherwise specified, the term "complementarity determining region" or "CDR" will be understood to encompass complementarity determining regions defined by any of the above known schemes as described by the present invention.
In the present specification, the term "host cell" refers to a cell that can be used to introduce exogenous nucleic acid (mainly, vector), and includes, but is not limited to, a prokaryotic cell such as E.coli, a fungal cell such as a yeast cell, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, heLa cell, BHK cell, HEK293 cell or human cell.
In the present specification, the term "ELISA" refers to an ELISA assay, which is a test method commonly used in immunology, by binding an antigen or antibody to a solid support, and performing qualitative and quantitative experiments of immune reaction using specific binding of a drug-resistant antibody.
In the present specification, the term "genetic engineering technique" refers to a technique in which a DNA fragment of one or more organisms is subjected to splice recombination with a vector DNA molecule in vitro at the molecular level to produce a new recombinant DNA molecule, which is then transferred into another organism to inherit and express a new protein.
In the present specification, the term "in vivo induced abdominal water method" refers to inoculating hybridoma cells corresponding to a monoclonal antibody into the abdominal cavity of a mouse, which produce and secrete the monoclonal antibody after proliferation in the abdominal cavity of the mouse.
In this specification, the term "a & P Run" refers to precision and accuracy.
Antibodies and binding fragments
The present invention provides an antibody or binding fragment thereof that specifically binds to a human IgG kappa-type light chain. Wherein the antibody or binding fragment thereof that specifically binds to a human IgG kappa-type light chain comprises a heavy chain comprising a heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3 and a light chain comprising complementarity determining regions LCDR1, LCDR2 and LCDR3.
In some embodiments of the invention, an antibody or binding fragment thereof that specifically binds to a human IgG kappa-type light chain may be a full-length antibody comprising a constant region sequence; in some embodiments, the constant region sequence is a murine or human constant region, wherein the heavy chain constant region comprises a human or murine IgA, igG, igM, igE or IgD constant region and the light chain constant region comprises a human or murine kappa or lambda constant region. In a specific embodiment of the invention, the heavy chain constant region is a murine IgG1 heavy chain constant region (amino acid sequence shown as SEQ ID NO: 17) and the light chain constant region is a murine kappa-type light chain constant region (amino acid sequence shown as SEQ ID NO: 18). It will be appreciated that one skilled in the art may refer to the heavy chain variable region or HCDR1, HCDR2, HCDR3, and/or the light chain variable region or LCDR1, LCDR2 or LCDR3 according to the disclosure of antibodies or binding fragments thereof that bind to human IgG kappa light chains.
Some embodiments of the invention provide an antibody or binding fragment thereof that specifically binds to a human IgG kappa-type light chain, wherein the binding fragment may be Fab, fab ', F (ab') 2 Single chain Fv (scFv) fragments, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2 Fv-Fc fusion, scFv-Fv fusion.
In some embodiments of the invention, the heavy chain variable region of the above-described antibody or binding fragment thereof that specifically binds to a human IgG kappa-type light chain comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region shown in SEQ ID No. 7 or 15, and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2 and LCDR3 comprised by the light chain variable region shown in SEQ ID No. 8 or 16. In certain embodiments of the invention, the heavy chain variable region of an antibody or binding fragment thereof that binds to a human IgG kappa-type light chain comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region shown in SEQ ID No. 7, and the light chain variable region thereof comprises complementarity determining regions LCDR, LCDR2 and LCDR3 comprised by the light chain variable region shown in SEQ ID No. 8; in still other embodiments of the present invention, the heavy chain variable region of an antibody or binding fragment thereof that binds to a human IgG kappa-type light chain comprises the complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region shown in SEQ ID No. 15, and the light chain variable region thereof comprises the complementarity determining regions LCDR1, LCDR2 and LCDR3 comprised by the heavy chain variable region shown in SEQ ID No. 16.
In some embodiments of the invention, the HCDR1 amino acid sequence of the antibody or binding fragment thereof that specifically binds human IgG kappa light chain is shown in SEQ ID NO. 1, the HCDR2 amino acid sequence is shown in SEQ ID NO. 2, the HCDR3 amino acid sequence is shown in SEQ ID NO. 3, and the LCDR1 amino acid sequence is shown in SEQ ID NO. 4, the LCDR2 amino acid sequence is shown in SEQ ID NO. 5, and the LCDR3 amino acid sequence is shown in SEQ ID NO. 6. In another embodiment of the present invention, the HCDR1 amino acid sequence of the antibody or binding fragment thereof that specifically binds human IgG kappa-type light chain is shown as SEQ ID NO. 9, the HCDR2 amino acid sequence is shown as SEQ ID NO. 10, the HCDR3 amino acid sequence is shown as SEQ ID NO. 11, and the LCDR1 amino acid sequence is shown as SEQ ID NO. 12, the LCDR2 amino acid sequence is shown as SEQ ID NO. 13, and the LCDR3 amino acid sequence is shown as SEQ ID NO. 14.
In a specific embodiment of the present invention, the heavy chain variable region of the above-mentioned antibody or binding fragment thereof binding to human IgG kappa-type light chain is an amino acid sequence shown as SEQ ID NO. 7, and the light chain variable region is an amino acid sequence shown as SEQ ID NO. 8; in another embodiment of the present invention, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 15 and the light chain variable region has the amino acid sequence shown in SEQ ID NO. 16. In some embodiments of the invention, the heavy chain variable region of an antibody or binding fragment thereof that binds a human IgG kappa-type light chain comprises an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 7 or 15, and the light chain variable region comprises an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 8 or 16, wherein "identity" refers to the extent to which two amino acid sequences have identical residues at identical positions in the sequence alignment, i.e. there are one or more substitutions or deletions in the full-length amino acid sequence, and retains its binding activity. In some embodiments of the invention, the heavy chain variable region may comprise an amino acid sequence having one or more amino acid substitutions or deletions from the amino acid sequence set forth in SEQ ID NO. 7 or 15, and retains its binding activity; accordingly, the light chain variable region may comprise an amino acid sequence having one or more amino acid substitutions or deletions from the amino acid sequence set forth in SEQ ID NO. 8 or 16, and retain its binding activity.
Binding to IgG kappa light chainsMethod for producing antibodies or binding fragments thereof
The present invention provides a method for binding an antibody or binding fragment thereof to an IgG kappa light chain, which is essentially obtained by two methods: (1) Obtained by an in vivo ascites induction method of hybridoma cells of the monoclonal antibody; (2) obtained by genetic engineering techniques.
In some embodiments of the invention, antibodies or binding fragments thereof that bind to IgG kappa-type light chains are produced by means of animal immunization. The immunogens used are antibodies comprising kappa-type light chains and/or kappa-type light chains, respectively, and the immunized animals used are preferably Balb/c mice. After immunization, hybridoma cell lines expressing monoclonal antibodies were selected using hybridoma technology. In certain embodiments of the invention, the immunogen used is an IgG1, igG 2 humanized antibody drug mixture and the immunized animal used is a Balb/c mouse. After immunization, monoclonal antibodies with high affinity for kappa human IgG were screened using hybridoma technology.
The animal was immunized by dividing the immunogen four times with the immunization interval of 0, 21, 35, 49 days. Multiple injections are performed subcutaneously in mice, and immunogens enter peripheral immune organs through the blood circulation or lymphatic circulation, stimulating the corresponding B lymphocyte clones to activate, proliferate, and differentiate into sensitized B lymphocytes. A small amount of venous blood was withdrawn on day 10 after the fourth immunization, and serum was isolated to examine the immune effect. The result of the immunoassay shows that the immunological titer reaches a serum dilution gradient of 1:10 4 When the OD value is more than 1.0, the method shows that the immune effect is good, hybridoma cell fusion culture can be carried out, and hybridoma cell strains expressing monoclonal antibody can be screened by utilizing the hybridoma technology.
The fusion culture process of the hybridoma cells comprises the following steps:
(1) Preparation of myeloma cells: selecting SP2/0 myeloma cells of a mouse, carrying out proliferation culture by using a culture solution before carrying out fusion culture, and changing the SP2/0 myeloma cells into a new culture solution when the SP2/0 myeloma cells are in a logarithmic growth phase, and counting and checking that the cell viability is higher than 95% for use;
(2) Preparation of spleen cells of immunized mice: killing mice successfully immunized, and taking the spleen of the mice to prepare spleen cell suspension for later use in a sterile operation;
(3) Feeder cell preparation: sterilizing the killed mice, and taking the peritoneal exudation cells from the abdomen as feeder cells for later use;
(4) Cell fusion: mixing the prepared myeloma cells and the mouse spleen cells according to a certain proportion, removing supernatant, adding PEG for fusion, adding HAT selective culture solution for stopping reaction after reacting for a certain time, and then inoculating to a 96-well culture plate for culturing;
(5) Selective culture: after the two parent cells are treated by PEG, a mixture containing various cell components can be formed, including unfused free parent cells, fusion between myeloma cells, fusion between immune B cells and heterokaryotic cells fused between myeloma cells and immune B cells, only heterokaryotic cells fused between myeloma cells and immune B cells can form hybridomas, and clone culture is needed to be selected. HAT medium is commonly used and contains Huang Dieling (H), aminopterin (a), thymidine (T). In HAT medium, unfused myeloma cells lack hypoxanthine-guanine-phosphoribosyl transferase and cannot synthesize DNA by salvage pathways to die. Unfused B lymphocytes have hypoxanthine-guanine-phosphoribosyl transferase, but do not themselves survive in vitro for long periods and die. Only fused hybridoma cells survive and proliferate in HAT medium due to the hypoxanthine-guanine-phosphoribosyl transferase obtained from spleen cells and the unlimited proliferation of myeloma cells. And inoculating the 96-well plate to replace HAT culture solution at two continuous weeks, and taking supernatant to perform activity detection when the cell colony is larger than 3mm or is 1/2 of the hole bottom, and screening out fusion cells which are successfully fused.
After hybridoma cells are grown in HAT medium to form clones, only a few of them secrete antibodies of predetermined specificity, and a plurality of clones grow in a large number of culture wells, and the secreted antibodies may be different, so that screening and cloning are necessary. The hybridoma technology screening process for hybridoma cell strains expressing monoclonal antibodies comprises the following steps:
(1) Screening of hybridoma-positive clones: firstly, hybridoma cells which can secrete anti-drug antibodies combined with a preset kappa type light chain IgG antibody are screened out, then hybridoma cells with preset specificity are screened out, and then cell clones which can be used for practical application and have stable growth and functional characteristics are selected. As a general method, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, radioimmunoassay, indirect hemagglutination assay, hemolysis plaque assay, etc., are mentioned, and ELISA is preferred for screening in the present invention.
(2) Cloning: in order to prevent overgrowth of irrelevant clones, it is necessary to clone the hybrid clones positive for the detection antibody as early as possible, otherwise the cells secreted by the antibody will be inhibited by the cells not secreted, since the growth rate of the cells not secreted by the antibody will be faster than that of the cells secreted, and the cells secreted will be lost as a result of competition between the two. Even cloned hybridoma cells require periodic recloning to prevent mutation or chromosome loss of the hybridoma cells, thereby losing the ability to produce antibodies. Cloning methods include limiting dilution, soft agar plates, micromanipulations and fluorescence activated cell sorting, and the present invention preferably uses limiting dilution for cloning culture. The cell strain to be cloned is counted and diluted to make each hole contain 0.5-1 cells, and then is cultured until the visible cell clone is subjected to combination detection, and a positive hole grown by a single clone is selected for clone culture and combination detection is performed again.
(3) Preservation of hybridoma cells after screening: counting the screened positive monoclonal cell strains, centrifuging according to the counting result, adding a certain amount of cell freezing solution, uniformly mixing, subpackaging the cell suspension into a freezing tube, placing the freezing tube in a program cooling box, and transferring the freezing tube into liquid nitrogen for long-term storage after overnight at-80 ℃.
The selected hybridoma cell suspension cultured after resuscitating was injected into the abdominal cavity of mice that had been injected with incomplete adjuvant one week in advance. Collecting ascites after the abdominal cavity of the mice is swelled after 1-2 weeks, centrifuging to obtain supernatant, and purifying to obtain the corresponding antibody. Wherein, the incomplete adjuvant can be liquid paraffin or Freund's incomplete adjuvant.
In another specific embodiment of the invention, the antibody is prepared by genetic engineering technology, namely, a recombinant expression vector containing the complete heavy chain and the light chain of the antibody which specifically binds to the kappa-type light chain of human IgG is transfected into a host cell together, the host cell expresses and secretes the antibody into a supernatant, the expression is finished after a certain period of culture, the supernatant is centrifugally taken, and the corresponding antibody is obtained after purification. Among them, the host cell may be COS-7 (monkey kidney cell 7), NSO cell, SP2/0 cell, CHO (Chinese hamster ovary) cell, W138, BHK (baby hamster kidney) cell, MDCK, myeloma cell line, huT78 cell, HEK293 cell, escherichia coli, bacillus subtilis, streptomyces, pseudomonas, proteus mirabilis, staphylococcus, fungi (such as Aspergillus, pichia, saccharomyces cerevisiae, schizosaccharomyces, and Neurospora crassa). Preferably, in a specific embodiment of the present invention, CHO cells are used as host cells. More preferably, in a specific embodiment of the invention, the host cell is a HEK293 cell.
The purification can be performed by using Protein A or Protein G filler, both of which can specifically bind to IgG, preferably Protein A affinity filler.
Antibody-label conjugates
In a further aspect the invention relates to an antibody-label conjugate, i.e. an antibody or binding fragment thereof specifically binding to an IgG kappa light chain of the invention is linked to a label.
Wherein the label may be radioactive, or fluorescent or chemiluminescent. For example, the label is a fluorescent dye (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dot, or cyanine dye derivative (e.g., cy7, alexa 750)), a chemiluminescent molecule (e.g., an acridine ester compound), a radionuclide (e.g., 3 H、 125 I、 35 S、 14 c or 32 P), colloidal gold, biotin, avidin (e.g., streptavidin), and enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.).
Method for determining IgG kappa light chain or kappa light chain-containing IgG in biological sample
In another aspect the invention also relates to a method for determining IgG kappa-type light chains or IgG comprising kappa-type light chains in a biological sample; in one embodiment of the invention, a method for determining kappa light chain IgG or kappa light chain IgG comprising the same in a biological sample comprises: (a) Contacting the biological sample with a capture reagent, the capture reagent being a target protein to which the IgG is directed, thereby forming an immune complex; (b) Contacting the immune complex from step (a) with an IgG kappa-type light chain antibody or binding fragment thereof as described above that specifically binds to a marker secondary molecule attached thereto; (c) Determining the presence or absence or amount of IgG kappa light chains or IgG comprising kappa light chains bound to the antibody or binding fragment thereof by detecting the label. The secondary molecule may specifically bind to an IgG kappa-type light chain antibody or binding fragment thereof, e.g., specifically bind to an Fc fragment of an IgG kappa-type light chain antibody.
In another embodiment of the invention, a method for determining kappa light chain IgG or kappa light chain IgG comprising in a biological sample, comprises: (a) Contacting the biological sample with a capture reagent, the capture reagent being a target protein to which the IgG is directed, thereby forming an immune complex; (b) Contacting the immunocomplex from step (a) with an antibody-label conjugate as described above; (c) Determining the presence or absence or amount of IgG kappa light chains or IgG comprising kappa light chains bound to the antibody or binding fragment thereof by detecting the label.
Wherein the biological sample may be derived from blood tissue or solid organ tissue. For example, but not by way of limitation, the biological sample may be human or mammalian blood, plasma or serum.
The label may be radioactive, or fluorescent or chemiluminescent. For example, the label is a fluorescent dye (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dot, or cyanine dye derivative (e.g., cy7, alexa 750)), chemiluminescentMolecules (e.g. acridinium esters), radionuclides (e.g., 3 H、 125 I、 35 S、 14 C or 32 P), colloidal gold, biotin, avidin (e.g., streptavidin), and enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.).
The methods of detecting and/or measuring human IgG kappa-type light chains and/or human IgG comprising kappa-type light chains in a sample include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and fluorescence spectrophotometry.
Nucleic acid
In some embodiments of the invention, a nucleic acid is provided that encodes an antibody or binding fragment thereof that binds to a human IgG kappa-type light chain as described above, and in particular comprises a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NOs 1-22. The preparation method of the nucleic acid is a conventional preparation method in the field, including but not limited to the following preparation methods: obtained by a gene cloning technique such as a PCR method or the like, or by a method of artificial total sequence synthesis. In a specific embodiment of the invention, the nucleic acid is a nucleotide sequence encoding a polypeptide comprising the amino acid sequences shown in SEQ ID NOS 1-3 and the amino acid sequences shown in SEQ ID NOS 4-6; in other embodiments of the invention, the nucleic acid is a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequences shown in SEQ ID NOs 7 and 8. In other embodiments of the invention, the nucleic acid is a nucleotide sequence encoding a polypeptide comprising the amino acid sequences set forth in SEQ ID NOS 9-11 and the amino acid sequences set forth in SEQ ID NOS 12-14; in other embodiments of the invention, the nucleic acid is a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequences shown in SEQ ID NOs 15 and 16. In a specific embodiment of the invention, the nucleic acid is a nucleotide sequence encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 19 and the amino acid sequence shown in SEQ ID NO. 20; in other embodiments of the invention, the nucleic acid is a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequences shown in SEQ ID NOS.21 and 22. In other embodiments of the invention, the nucleic acid has a nucleotide sequence that retains 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the above sequence.
Carrier body
In some embodiments of the invention, a vector is provided comprising nucleic acids encoding antibodies or binding fragments thereof that bind human IgG kappa light chains, such as comprising nucleotide sequences encoding heavy and light chains (SEQ ID NOS: 19 and 20, or SEQ ID NOS: 21 and 22) of the antibodies or binding fragments thereof, respectively, that bind human IgG kappa light chains. In some embodiments of the invention, the vector is constructed after codon optimization according to the expression system, e.g., in particular embodiments, CHO cells or 293 cell expression systems are used. In a specific embodiment of the invention, the vector is pcDNA3.4.
Host cells
The invention provides a host cell which can be used for transfection of a recombinant expression vector. For example, the host cell is COS-7 (monkey kidney cell 7), NSO cell, SP2/0 cell, CHO (Chinese hamster ovary) cell, W138, BHK (baby hamster kidney) cell, MDCK, myeloma cell line, huT78 cell, HEK293 cell, escherichia coli, bacillus subtilis, streptomyces, pseudomonas, proteus mirabilis, staphylococcus, fungus (e.g., aspergillus, pichia, saccharomyces cerevisiae, schizosaccharomyces, or Neurospora crassa). In a specific embodiment of the invention, the recombinant expression vector is obtained by carrying out transformation amplification before transfection and then extracting, and the transformed prokaryotic cells are DH5 alpha escherichia coli, wherein DH5 alpha escherichia coli is competent cells.
Use of the same
In embodiments of the invention, antibodies or binding fragments thereof that bind to an IgG kappa-type light chain are used to detect IgG kappa-type light chains and/or IgG comprising kappa-type light chains, e.g., in vivo and in vitro pharmacokinetic, pharmacodynamic, or immunogenicity studies. Specifically, the antibody is used as a capturing and detecting reagent, and the concentration of human IgG of the kappa type light chain and/or the kappa type light chain of the human IgG is detected in human serum by a bridging ELISA, so that the pharmacokinetics of the drug is studied.
Kit for detecting a substance in a sample
In a specific embodiment of the invention, the invention provides a kit for detection of IgG kappa-type light chains and/or IgG comprising kappa-type light chains or in vivo and in vitro pharmacokinetic, pharmacodynamic and immunogenicity studies comprising an antibody or binding fragment as described above that binds IgG kappa-type light chains and/or IgG comprising kappa-type light chains. The kit generally further comprises instructions for use, and/or buffers, experimental consumables (e.g., ep tubes, microwell plates, etc.), materials for written records, labels, etc., and may further comprise standards, quality controls, coating fluids, sealing fluids, color-developing fluids.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The invention has the positive progress effects that:
the antibody (monoclonal antibody) which can be combined with the kappa-type light chain of the IgG and is screened by the invention can be specifically combined with kappa-type IgG, has the characteristics of high sensitivity, high specificity and universality on the IgG, and can be widely applied to content detection of human IgG, such as in-vivo and in-vitro pharmacokinetics, pharmacodynamics and immunogenicity research in clinic and clinic. Since kappa light chains represent the major (about 90%) subtype of antibody therapeutics, and the monoclonal antibodies of the invention have very high specificity, they are highly versatile; in addition, the high sensitivity of the monoclonal antibody can also meet the requirements of pharmacokinetics, pharmacodynamics and immunogenicity research in the development of antibody medicaments.
The invention will be further illustrated by means of examples which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. The room temperature described in the examples is room temperature conventional in the art, typically 10-30 ℃.
The technical features mentioned in the different embodiments described throughout the present invention can be implemented in combination with each other.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the present invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art, see in particular Sambrook et al MOLECULAR CLONING: A LABORATORY MANUAL, second edition, cold Spring Harbor Laboratory Press,1989 and Third edition,2001; ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987 and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; METHODS IN ENZYMOLOGY, vol.304, chromatin (p.m. wassman and a.p. wolffe, eds.), academic Press, sanDiego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatin Protocols (p.b. becker, ed.) Humana Press, totowa,1999, etc. The reagents and materials purchased in the present invention are commercially available.
Sequence listing
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Examples
Example 1 animal immunization method for preparing monoclonal antibody specifically binding to IgG kappa light chain and identification
1.1 immunization of mice
A mixture of kappa-type light chain human IgG drug anti-c-MET antibodies (IgG 2, heavy and light chain as shown in SEQ ID nos. 23 and 24) and anti-DR 4 antibodies (IgG 1, heavy and light chain as shown in SEQ ID nos. 25 and 26) were immunized 5 mice, 50 μg each time subcutaneously, and once on days 0, 21, 35, 49, for a total of 4 immunizations; mice were drawn a small amount of venous blood on day 59 and serum was isolated to test for immune effects.
1.2 immunotiter detection
1.2.1 fourth immunotiter detection of anti-c-MET antibody (IgG 2)/anti-DR 4 antibody
On day 10 after the fourth immunization of mice, 5 mice immunized with an anti-c-MET antibody (IgG 2), a mixture of anti-DR 4 antibodies (IgG 1) were venous blood drawn, and serum was isolated; the immune titer of the antibody in the serum is measured by ELISA, if the immune titer reaches the serum dilution gradient of 1:10 as shown by the immune detection result 4 The OD value of more than 1.0 indicates that the immunization effect is good. Respectively with anti-c-METAntibodies, anti-DR 4 antibodies and monkey IgG (as controls) as coating proteins were coated at a coating concentration of 2. Mu.g/mL, 100. Mu.L/well at 4deg.C overnight; the following day of washing was followed by blocking overnight at 4℃with 200. Mu.L/well of 1% BSA; serum from each mouse before immunization and after the fourth immunization was diluted 1:10 after washing 3 、1:10 4 、1:10 5 As primary antibody, 100 μl/well incubated for 1h at room temperature; after washing, HRP-loaded anti-IgG (SIGMA; cat#: A0168; lot NO: #068M 4764V) was used as secondary antibody at 1: dilution ratio of 9,000, 50. Mu.L/well, incubation at room temperature for 1h; after washing, the OD was read at 450nm after development at 37℃for 10min at 100. Mu.L/well of TMB. The results of the detection of the OD value of each mouse immune titer are shown in tables 1-3.
TABLE 1 results of detection of OD values of the immune titers of the anti-c-MET antibody/anti-DR 4 antibody coated with the anti-c-MET antibody after the fourth immunization
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TABLE 2 results of detection of OD values of the immune titers of the anti-c-MET antibody/anti-DR 4 antibody coated with the anti-DR 4 antibody after the fourth immunization
TABLE 3 results of detection of OD values of the immune titers of coated monkey IgG after the fourth immunization with anti-c-MET antibody/anti-DR 4 antibody
Analysis of results:
from the ELISA immunotiter measurements of the coated anti-c-MET antibodies shown in Table 1, it was found that the serum after the fourth immunization of the anti-c-MET antibody/anti-DR 4 antibody mixture was 1:10 4 Dilution ofNext, 1, 3, 4, 5 mice showed high OD values greater than 1.0. And, 5 th mouse was at 1:10 5 Still shows a high OD value of greater than 1.0 at the dilution gradient, indicating that the immunization of mice 5 was the best, resulting in antibodies with high affinity for anti-c-MET antibodies.
From the ELISA immunotiter measurements of the coated anti-DR 4 antibodies shown in Table 2, the sera of five mice after the fourth immunization of the anti-c-MET antibody/anti-DR 4 antibody mixture were 1:10 4 Only the 5 th mouse showed a high OD value of greater than 1.0 upon dilution, indicating that the fifth mouse was immunized well and produced an antibody with high affinity to the anti-DR 4 antibody.
From the ELISA immunotiter measurements of coated monkey IgG shown in Table 3, the sera of five mice after the fourth immunization of the anti-c-MET antibody/anti-DR 4 antibody mixture were 1:10 4 The OD values of mice detected at dilution were all less than 1.0, indicating that none of the 5 mice produced antibodies with high affinity for non-human IgG such as monkey IgG.
In summary, the fifth mouse immunized produced antibodies with high affinity to kappa-type light chain human IgG against the c-MET/anti-DR 4 antibodies, but the antibodies present in the serum of the immunized mice were polyclonal and were present at 1:10 3 Can bind to monkey IgG even under dilution, and monoclonal antibodies capable of specifically binding to kappa-type human IgG need to be screened in subsequent experiments.
1.3 hybridoma cell screening
1.3.1 anti-c-MET antibody/anti-DR 4 antibody hybridoma cell screening
According to the serum titer detection result of the immunized mice, the 5 th mouse is sacrificed, spleen is sheared to prepare spleen cell suspension, then PEG fluxing agent and SP2/O mouse myeloma cells are added for fusion, and HAT culture medium is utilized for screening hybridoma cells which are successfully fused. The OD value of the supernatants of each well was measured by using an anti-c-MET antibody/anti-DR 4 antibody mixture, an anti-c-MET antibody, an anti-DR 4 antibody as a positive screening coating, monkey IgG, monkey serum as an anti-screening protein, and anti-c-MET antibody/anti-DR 4 antibody antiserum as a positive control (POS) after immunization of the mice, and serum as a negative control (NEG) before immunization of the mice. Screening out monoclonal cell strains capable of producing positive results on the positive screening coating protein and producing negative results on the negative screening coating protein. Evaluation criteria: the signal to noise ratio (S/N) is more than or equal to 2.1, and the result is positive, otherwise, the result is negative. And (3) performing expansion culture on the screened monoclonal cell strain, and then freezing and storing the monoclonal cell strain in a liquid nitrogen tank for seed preservation.
TABLE 4 ELISA detection of positive cell lines screened for anti-c-MET antibody/anti-DR 4 antibody hybridoma cells results table
Analysis of results: as shown in Table 4, the selected 2D2B1, 2B5H4, 2B5F7, 3D1G4, 3D1C3 5 clones, the cell culture supernatants of which all showed high OD values with kappa-type light chain human IgG such as anti-c-MET antibody/anti-DR 4 antibody mixture, anti-c-MET antibody, anti-DR 4 antibody, etc., and S/N > 2.1. While with monkey IgG and monkey serum shows low OD values, S/N < 2.1. The 5 monoclonal antibodies are positive cell strains, and have high affinity and specificity with kappa-type light chain human IgG.
1.4 determination of monoclonal antibody subtype
Diluting the Goat Anti-Mouse IgG AM concentrate (50X) in subtype assay kit (Biodragon; cat#BF 16001) with coating solution to working solution, adding ELISA plate, 50 μl/well, standing at 4deg.C overnight or 37 deg.C for 1 hr; washing the antigen plate with distilled water or PBS for 3 times, rinsing for 3min each time, throwing away the washing liquid, and reversely wiping on clean paper; 200. Mu.L/well of 1% BSA was added, and BSA was thrown off overnight at 4℃or at 37℃for 1 hour; respectively adding the antibodies to be detected into an antigen coating plate according to the sequence of the vertical rows A-H of the ELISA plate, 50 mu L/hole, and standing at 4 ℃ overnight or 37 ℃ for 1H; washing with distilled water or PBS for 3 times, throwing away the washing liquid, and wiping the washing liquid with a back-off button on clean paper; sequentially adding Rabbit Anti-Mouse IgG 1, igG 2a, igG 2b, igG 3, igA, igM, kappa, gamma subtype typing reagents from 1-12 wells in row-H, respectively, and allowing 50 mu L of each well to stand at 4 ℃ overnight or 37 ℃ for 1H; washing with distilled water or PBS for 3 times, throwing away the washing liquid, and wiping the washing liquid with a back-off button on clean paper; HRP-coat Anti-Rabbit IgG (H+L), 50. Mu.L/well, overnight at 4℃or 1H at 37 ℃; washing with distilled water or PBS for 3 times, throwing away the washing liquid, reversely wiping on clean paper, adding ABTS,50 μl/hole, and standing at 37deg.C for 10min; the subclass type was judged from the reading at wavelength 405nm and the results are shown in Table 5.
TABLE 5 monoclonal antibody subtype determination results Table
| Clone number | Subtype type |
| 2D2B1 | IgG 2a,κ |
| 2B5H4 | IgG 2a,κ |
| 2B5F7 | IgG 2a,κ |
| 3D1G4 | IgG 1,κ |
| 3D1C3 | IgG 1,κ |
1.5 preparation and identification of monoclonal antibodies
1.5.1 preparation of monoclonal antibodies
The monoclonal antibody is produced by adopting an in-vivo ascites induction method. Balb/C mice were first pre-treated with 0.5mL of incomplete adjuvant such as liquid paraffin or pristane by intraperitoneal injection, and simultaneously, the frozen hybridoma cell lines of clone numbers 2D2B1, 2B5H4, 2B5F7, 3D1G4, and 3D1C3 were recovered for culture. After 1-2 weeks, cultured hybridoma cells were inoculated into the abdominal cavity of the mice, and the monoclonal antibody was produced and secreted after proliferation of the hybridoma cells in the abdominal cavity of the mice. After 1-2 weeks, when the abdomen of the mouse expands, the ascites of the abdominal cavity of the mouse is extracted by a syringe, and the Protein A is purified to obtain the monoclonal antibody.
1.5.2 physical and chemical identification of monoclonal antibodies (SDS-PAGE)
Reduction SDS-PAGE sample preparation: mu.g of the monoclonal antibody was added to 5 XSDS loading buffer (containing DTT), heated in a dry bath at 70℃for 10min, cooled to room temperature and centrifuged for use. Non-reducing SDS-PAGE sample preparation: mu.g of the monoclonal antibody was added to 5 XSDS loading buffer (without DTT) and the mixture was centrifuged for later use. The treated samples were gel-electrophoresed and protein bands were visualized by coomassie blue staining. Protein gels with chromogenic protein bands were scanned using a gel imager, the molecular weights of the light and heavy chains were known by comparison with the protein Marker bands, and the band purity was calculated by Image J according to the peak area normalization method. The SDS-PAGE results of each monoclonal antibody are shown in FIGS. 1-5, and the purity results are shown in Table 6. In fig. 1-5, M is a Marker band, lane 1 is a running result of monoclonal antibody reduction, and the result shows that after the monoclonal antibodies are reduced, the monoclonal antibodies are equally divided into a heavy chain band and a light chain band, and Lane 2 is a band before reduction. As shown by the combination of Image J analysis results, the produced antibodies with clone numbers have clear bands before and after reduction, and have good purity which is more than 90 percent.
TABLE 6 SDS-PAGE purity results for each monoclonal antibody
| Clone No. | Purity |
| 2D2B1 | >90.0％ |
| 2B5H4 | >90.0％ |
| 2B5F7 | >90.0％ |
| 3D1G4 | >90.0％ |
| 3D1C3 | >90.0％ |
1.5.3 identification of the binding Capacity of monoclonal antibodies
Monoclonal antibodies produced by hybridoma cell lines 2D2B1, 2B5H4, 2B5F7, 3D1G4, and 3D1C3 were tested for affinity and specificity with anti-C-MET antibodies and anti-DR 4 antibodies, respectively, by ELISA. The anti-C-MET antibody and the anti-DR 4 antibody are used as positive screening coating proteins, monkey IgG and monkey serum are used as negative screening coating proteins for coating, and the OD value after color development is compared with positive control POS and negative control NEG, so that the specific binding force between the 2D2B1, 2B5H4, 2B5F7, 3D1G4 and 3D1C3 monoclonal antibodies and the anti-C-MET antibody and the anti-DR 4 antibody human IgG of kappa type light chains is verified. Signal to noise ratio (S/N)
And the result is more than or equal to 2.1, and the result is negative if the result is more than or equal to 2.1. The results of the OD values of the ELISA assays are shown in Table 7.
Tables 7.2D2B1, 2B5H4, 2B5F7, 3D1G4, 3D1C3 monoclonal antibody ELISA detection OD value results tables
| Clone NO. | 2D2B1 | 2B5H4 | 2B5F7 | 3D1C3 | 3D1G4 | POS | NEG |
| anti-c-MET antibodies | 3.066 | 1.907 | 2.230 | 1.719 | 0.862 | 2.067 | 0.188 |
| anti-DR 4 antibodies | 1.318 | 0.595 | 2.091 | 1.487 | 0.834 | 2.544 | 0.138 |
| Monkey IgG | 0.247 | 0.285 | 0.256 | 0.226 | 0.232 | 2.712 | 0.297 |
| Monkey serum | 0.141 | 0.192 | 0.183 | 0.100 | 0.168 | 2.443 | 0.140 |
Analysis of results: as shown in Table 7, these 5 monoclonal antibodies 2D2B1, 2B5H4, 2B5F7, 3D1G4, 3D1C3, etc. showed high OD values with both the anti-C-MET antibody and the anti-DR 4 antibody of the positive screening coating protein, and S/N >2.1, while showing low OD values with the anti-screening coating protein monkey IgG and monkey serum, S/N < 2.1, indicating that 2D2B1, 2B5H4, 2B5F7, 3D1G4, 3D1C3 have high affinity with kappa human IgG such as anti-C-MET antibody, anti-DR 4 antibody, etc.
EXAMPLE 2 identification of the binding Capacity of monoclonal antibodies to different human IgG drugs
Different subtypes of kappa-type light chain human IgG drug anti-c-MET antibody, anti-DR 4 antibody, and lambda-type light chain human IgG drug anti-PD-L1 antibody (IgG 1, which comprises the heavy and light chains shown in SEQ ID NOS: 27 and 28) were diluted to 2 μg/mL with 1 XPBS. They were coated as coating proteins at 30. Mu.L/well at 4℃overnight, respectively; the following day was blocked with Casein buffer 60. Mu.L/Kong Shiwen for 1h after washing with 0.05% PBST; 2D2B1, 2B5H4, 2B5F7, 3D1G4, 3D1C3 and commercial monoclonal antibody R10Z8E9 (control) diluted with buffer were added after washing operation and incubated for 1H at 30. Mu.L/Kong Shiwen; after washing, using HRP labeled Goat anti-mouse IgG as a detection secondary antibody, and after dilution at a dilution ratio of 1:10,000, incubating for 1h at 30 mu L/Kong Shiwen, and referring to the experimental layout chart as shown in Table 8; after washing, the OD value is read at 450nm and 630nm after the color development is carried out for 10min at 37 ℃ with TMB of 30 mu L/hole, and the difference of the read values of the two wavelengths is subjected to curve fitting and calculation, and the combined curves are shown in fig. 6-8, and according to the results of fig. 6-8, the effect that 2D2B1, 2B5H4, 2B5F7, 3D1G4 and 3D1C3 are combined with only different subtypes of kappa-type light chain human IgG drugs and are not combined with lambda-type light chain human IgG drugs is demonstrated, so that the monoclonal antibodies can specifically identify kappa-type light chain human IgG drugs and are not influenced by lambda-type light chain human IgG drugs.
TABLE 8 identification of binding Capacity of monoclonal antibodies to different human IgG drugs ELISA experiment layout templates
Hybridoma cell lines with monoclonal numbers 2D2B1 and 2B5F7 were selected for sequencing analysis (Shanghai Baiying Biotech Co., ltd.) and the specific sequences thereof are shown in the sequence table.
PK applications of examples 3.2D2B1 and 2B5F7
4.1 Suitability verification of 2D2B1 Capture reagent and 2B5F7-HRP detection reagent
2D2B1 was used as capture antibody, diluted to 2. Mu.g/mL with 1XPBS, 30. Mu.L/well coated overnight at 4deg.C; the following day was blocked with Casein buffer 60. Mu.L/Kong Shiwen for 1h after washing with 0.05% PBST; after the washing procedure, a series of concentration gradients (STD 01-STD 07 and Blank) of human IgG drug anti-HER 2 antibody (IgG 1, a commercial trastuzumab) diluted with Casein buffer was added and incubated for 1h at 30. Mu.L/Kong Shiwen; washing, diluting with 2B5F7-HRP (obtained by reacting activated amine reactive aldehyde group formed by oxidizing sugar molecule of horseradish peroxide with amino group on 2B5F 7) at a dilution ratio of 1:8000, incubating for 1h at 30 μl/Kong Shiwen, and referring to experimental layout chart as shown in table 9; after washing, after developing color for 10min at 37 ℃ with TMB 30 mu L/hole, OD values are read at 450nm and 630nm, and the difference value of the two-wavelength reading values is subjected to curve fitting and calculation, and the combination curve is shown in FIG. 9, and according to the result of FIG. 9, the 2D2B1 serving as a capture reagent and the 2B5F7-HRP serving as a detection reagent can be used for successfully detecting kappa-type light chain human IgG drugs of different subtypes.
Table 9.2D2B1 ELISA test layout template for verifying the suitability of Capture reagent and 2B5F7-HRP detection reagent
Application of 4.2.2D2B1 and 2B5F7-HRP to PK detection of preclinical anti-c-MET antibody actual sample
2D2B1/2B5F7-HRP and a commercial polyclonal Sheep anti-human/Goat anti-human-HRP were used for PK detection of the anti-c-MET antibodies (IgG 2, kappa light chain, heavy and light chain as shown in SEQ ID NOS: 23 and 24) in rat samples. Randomly selecting blood samples of 16 rat samples, diluting the blood samples with rat mixed serum for 2 times, and coating the blood samples with 2D2B1 or Sheep anti-human serving as a capture antibody at a concentration of 2 mug/mL at 4 ℃ overnight; the following day was blocked with Casein buffer 60. Mu.L/Kong Shiwen for 1h after washing with 0.05% PBST; after washing, 16 diluted rat samples were added and MRD100, 30. Mu.L/Kong Shiwen were incubated for 1h; after washing, 2B5F7-HRP or Goat anti-human-HRP is used as detection antibody, and 1 is diluted according to the dilution ratio: after 8000, 30. Mu.L/Kong Shiwen was incubated for 1h; after washing, the OD value is read at the dual wavelength of 450nm and 630nm after the color development is carried out for 10min at the temperature of 37 ℃ by using TMB of 30 mu L/hole, and the difference value of the read value at the dual wavelength is subjected to curve fitting and calculation, and the detection result and the back calculation result are shown in Table 10. Comparing the PK detection results of the actual samples of the pre-clinical anti-c-MET antibody with that of the 2D2B1/2B5F7-HRP and a commercial multi-antibody system Sheep anti-human/Goat anti-human-HRP.
Table 10.2D2B1/2B5F7-HRP and commercial polyclonal antibody System PK detection comparison Table of preclinical anti-c-MET antibody actual samples
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From Table 10, it can be seen that the concentration of anti-c-MET antibody in the rat serum detected by 2D2B1/2B5F7-HRP is relatively close to the detection result of the commercial multi-antibody system shaep anti-human/coat anti-human-HRP, which indicates that 2D2B1 and 2B5F7-HRP can be used for PK detection of actual samples of preclinical kappa type light chain human IgG drugs.
The embodiments described above are intended to provide those skilled in the art with a full range of modifications and variations to the embodiments described above without departing from the spirit of the invention, and therefore the scope of the invention is not limited to the embodiments described above, but is to be accorded the broadest scope consistent with the novel features set forth in the claims.
Claims (21)
1. An antibody or binding fragment thereof that specifically binds to an IgG kappa-type light chain comprising a heavy chain variable region and a light chain comprising a light chain variable region;
the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region shown in SEQ ID NO. 7 or 15, and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2 and LCDR3 comprised by the light chain variable region shown in SEQ ID NO. 8 or 16.
2. The antibody or binding fragment thereof according to claim 1, wherein,
the HCDR1, HCDR2 and HCDR3 are respectively the amino acid sequences shown in SEQ ID NO. 1, 2 and 3, and the LCDR1, LCDR2 and LCDR3 are respectively the amino acid sequences shown in SEQ ID NO. 4, 5 and 6; or,
the HCDR1, HCDR2 and HCDR3 are the amino acid sequences shown in SEQ ID NO. 9, 10 and 11, respectively, and the LCDR1, LCDR2 and LCDR3 are the amino acid sequences shown in SEQ ID NO. 12, 13 and 14, respectively.
3. The antibody or binding fragment thereof according to claim 1 or 2, wherein,
the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 7, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 8; or,
the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 15, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 16.
4. The antibody or binding fragment thereof of any one of claims 1 to 3, wherein the antibody is a full length antibody.
5. The antibody or binding fragment thereof of any one of claims 1 to 4, wherein the antibody comprises a constant region sequence; the constant region sequence is a murine or human constant region, wherein the heavy chain constant region comprises a human or murine IgA, igG, igM, igE or IgD constant region and the light chain constant region comprises a human or murine kappa or lambda constant region.
6. The antibody or binding fragment thereof of claim 5, wherein the heavy chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID No. 17 and the light chain constant region comprises or consists of the amino acid sequence set forth in SEQ ID No. 18.
7. The antibody or binding fragment thereof of any one of claims 1-6, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID No. 19 and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID No. 20; or,
the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 21, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 22.
8. The antibody or binding fragment thereof of any one of claims 1-7, wherein the binding fragment is Fab, fab ', F (ab') 2 Single chain Fv (scFv) fragments, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2 Fv-Fc fusion, scFv-Fv fusion or VHH.
9. The antibody or binding fragment thereof of any one of claims 1-8, which does not bind to an IgG lambda type light chain.
10. An antibody-label conjugate, wherein the antibody or binding fragment thereof according to any one of claims 1-9 is linked to a label.
11. The antibody-label conjugate of claim 10, wherein the label is a fluorescent dye, a chemiluminescent molecule, a radionuclide, colloidal gold, biotin, avidin, or an enzyme.
12. A nucleic acid encoding the antibody or binding fragment thereof according to any one of claims 1-9.
13. A vector comprising the nucleic acid of claim 12.
14. A host cell comprising the nucleic acid of claim 12 and/or the vector of claim 13.
15. The host cell of claim 14, wherein the host cell is selected from the group consisting of COS-7 (monkey kidney cell 7), NSO cells, SP2/0 cells, CHO (chinese hamster ovary) cells, W138, BHK (baby hamster kidney) cells, MDCK, myeloma cell lines, huT78 cells, HEK293 cells, escherichia coli, bacillus subtilis, streptomyces, pseudomonas, proteus mirabilis, staphylococcus, aspergillus, pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, and neurospora crassa.
16. A method of producing an antibody or binding fragment thereof that specifically binds to an IgG kappa-type light chain, comprising: culturing the host cell according to claim 14 or 15 in a medium suitable for expression to express the antibody or binding fragment thereof, and recovering the antibody or binding fragment thereof from the medium.
17. A method for assaying IgG kappa-type light chains or IgG comprising kappa-type light chains in a biological sample, comprising:
(a) Contacting a biological sample with a capture reagent, said capture reagent being a target protein to which said IgG is directed, thereby forming an immune complex;
(b) Contacting the immunocomplex from step (a) with an antibody or binding fragment thereof that specifically binds to an IgG kappa-type light chain, as defined in any of claims 1-9, and a label secondary molecule attached, or contacting the immunocomplex from step (a) with an antibody-label conjugate as defined in any of claims 10-11,
(c) Determining the presence or absence or amount of IgG kappa-type light chains or IgG comprising kappa-type light chains bound to the antibody or binding fragment thereof by detecting the label.
18. The method of claim 17, wherein the label is a fluorescent dye, a chemiluminescent molecule, a radionuclide, colloidal gold, biotin, avidin, or an enzyme.
19. Use of an antibody or binding fragment thereof that binds to an IgG kappa light chain according to any one of claims 1-9 and/or an antibody-label conjugate according to any one of claims 10-11 for detecting IgG kappa light chains and/or IgG comprising kappa light chains.
20. The use according to claim 19 for the detection of IgG kappa-type light chains and/or in vivo and in vitro pharmacokinetic, pharmacodynamic or immunogenicity studies of IgG comprising kappa-type light chains.
21. A kit comprising the antibody or binding fragment thereof of any one of claims 1-9 that specifically binds to an IgG kappa light chain, and/or the antibody-label conjugate of any one of claims 10-11 that binds to an IgG kappa light chain, and instructions for use.
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