CN116462763A - Antibody or binding fragment thereof for binding anti-HER 2 antibody and application thereof - Google Patents

Abstract

The invention relates to the field of biotechnology, and more particularly discloses an antibody or a binding fragment thereof for binding an anti-HER 2 antibody 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 antibody of the invention can be prepared by an in vivo abdominal water induction method or a genetic engineering method of hybridoma cells, and has high affinity and specificity with a specific anti-HER 2 antibody, so that the antibody can be effectively applied to in vivo and in vitro pharmacokinetics, pharmacodynamics and immunogenicity researches of different anti-HER 2 antibodies.

Description

Antibody or binding fragment thereof for binding anti-HER 2 antibody and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to an antibody or a binding fragment thereof for binding an anti-HER 2 antibody and application thereof, in particular to F (ab') capable of binding the anti-HER 2 antibody ₂ . Particular embodiments of the invention relate to amino acid sequences, nucleic acids and methods of making antibodies or binding fragments that bind to anti-HER 2 antibodies and uses thereof for in vivo and in vitro pharmacokinetic, pharmacodynamic and immunogenicity studies of anti-HER 2 antibodies.

Background

Human epidermal growth factor receptor-2 (Human Epidermal Growth Factor Receptor 2) is composed of 922 adenine, 1382 cytosine, 1346 guanine and 880 thymine, and is located in protooncogene of chromosome 17q21, its coded product HER2 protein is 185kD transmembrane protein with tyrosine kinase activity, p185 for short, composed of 1255 amino acids, 720-987 belongs to tyrosine kinase zone, and belongs to one of EGFR family members. The oncogenic mechanism of HER2 is that it inhibits apoptosis, promotes cancer cell proliferation, increases the invasiveness of tumor cells, causes tumor angiogenesis and lymphatic vessel neogenesis, is expressed in many tumors, and is commonly found in breast cancer and gastric cancer.

The antibody targeting HER2 can block the binding of the antibody and human epidermal growth factor by specifically binding to HER2, so as to block the growth of cancer cells and stimulate human autoimmune cells to exert tumor killing effect, thereby achieving the effect of treating cancer. Trastuzumab, pertuzumab, itumomab, ma Jituo ximab, enmtuzumab, edituzumab and detrastuzumab have been marketed worldwide since trastuzumab 1998, and with the accelerated marketing of new drugs targeting HER2, the availability of more newer drugs and biosimilar drugs has increased, and the anti-HER 2 drug market will develop rapidly in the future, estimated to reach market sizes of about 94 billions and 136 billions in 2023 and 2030, respectively.

Pharmacokinetic (PK) is a law that states the change of blood concentration over time by quantitatively studying the absorption, distribution, metabolism and excretion of drugs in an organism. Pharmacodynamics (PD) is to elucidate the quantitative relationship of drugs by studying the action and action mechanism of the drugs, adverse reactions of the drugs, factors affecting the action of the drugs, 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, simultaneously discusses the action of the organism on the medicine and the action of the medicine on the organism, and provides more scientific theoretical basis for more comprehensively and accurately knowing 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. PK/PD studies are increasingly important in drug development, and in various stages of drug development, including early preclinical studies, are critical to influence whether clinical trials continue and to support the efficient performance of the stages of development.

Based on the preclinical and clinical importance of PK/PD, the assessment and monitoring of specific antibodies for their detection is an indispensable stage of research in the drug development process. Therefore, the development of antibodies that specifically bind to antibodies that target HER2 is beneficial for safety and effectiveness guarantees in drug development. Meanwhile, due to the characteristic of being capable of specifically binding to the anti-HER 2 antibody, the kit has good application prospect in preclinical and clinical PK and PD specific detection, and can be used as positive control of ADA in immunogenicity detection.

Disclosure of Invention

In view of the importance and good application prospects of anti-drug antibodies binding to anti-HER 2 antibodies, the invention provides an antibody or binding fragment thereof binding to an anti-HER 2 antibody, which has good biological activity, high affinity and specificity with the anti-HER 2 antibody.

One aspect of the invention relates to an antibody or binding fragment thereof that binds an anti-HER 2 antibody comprising a heavy chain variable region and a light chain comprising a light chain variable region, and the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises 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. 13 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. 14 or 16.

In some embodiments of the invention, HCDR1, HCDR2 and HCDR3 are the amino acid sequences shown in SEQ ID NOS: 1, 2 and 3, respectively, and 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, HCDR1, HCDR2 and HCDR3 are the amino acid sequences shown in SEQ ID NOS: 7, 8 and 9, respectively, and LCDR1, LCDR2 and LCDR3 are the amino acid sequences shown in SEQ ID NOS: 10, 11 and 12, 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. 13, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 14.

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. 13 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. 14 or 16.

Some embodiments of the invention relate to antibodies or binding fragments thereof that bind to an anti-HER 2 antibody, which is 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 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 an anti-HER 2 antibody, 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.

Another aspect of the invention relates to an antibody that binds an anti-HER 2 antibody 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 a nucleic acid encoding an antibody or binding fragment thereof as described above that binds to an anti-HER 2 antibody, in particular said nucleic acid is capable of encoding the amino acid sequence shown in SEQ ID NOs 1-22. In some embodiments of the invention, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NOS.23-32.

The invention also relates to a vector comprising a nucleic acid as described above capable of encoding an antibody or binding fragment thereof that binds to an anti-HER 2 antibody, as described above, which is capable of protein expression in a host cell, in some embodiments of the invention.

The invention also relates to a host cell, in some embodiments of the invention, comprising a nucleic acid and/or a vector as described above, capable of expressing an antibody or binding fragment that binds an anti-HER 2 antibody. In certain embodiments of the invention, the cell is selected from the group consisting of 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 the preparation of an antibody or binding fragment thereof that binds to an anti-HER 2 antibody, comprising culturing said cell in a medium suitable for expression to express the antibody or binding fragment thereof and obtaining said antibody or binding fragment thereof.

A further aspect of the invention relates to an antibody or binding fragment thereof that binds to an anti-HER 2 antibody, for use in detecting an anti-HER 2 antibody, preferably for in vivo and in vitro pharmacokinetic, pharmacodynamic or immunogenicity studies of an anti-HER 2 antibody. In some embodiments of the invention, the anti-HER 2 antibody (drug) comprises trastuzumab, pertuzumab, itumomab, ma Jituo ximab, enmtuzumab, edituzumab, detrastuzumab.

Another aspect of the invention relates to antibodies that bind to an anti-HER 2 antibody for use in detecting an anti-HER 2 antibody, e.g., for detecting and/or measuring an anti-HER 2 antibody in a sample. Some embodiments of the invention relate to methods of administering an antibody that binds an anti-HER 2 antibody as described above to detect and/or measure the presence or amount of an anti-HER 2 antibody in a sample comprising (i) incubating a sample suspected of containing an anti-HER 2 antibody with the anti-drug antibody (i.e., an antibody that binds an anti-HER 2 antibody as described herein) under conditions that allow formation of an antibody-anti-drug antibody complex, and (ii) incubating a sample suspected of containing an anti-HER 2 antibody with the anti-drug antibody (i.e., an antibody that binds an anti-HER 2 antibody as described herein) and a secondary molecule under conditions that allow formation of an antibody-anti-drug antibody complex, and detecting the formed ternary complex, thereby determining the presence or amount of the anti-HER 2 antibody, wherein the secondary molecule is conjugated to a detectable label or molecular label, such as an antibody that specifically binds the antibody provided herein. The detectable label or reporter may be radioactive, or fluorescent or chemiluminescent. For example, the methods of detecting and/or measuring anti-HER 2 antibodies in a sample include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and fluorescence spectrophotometry.

Another aspect of the invention relates to a kit comprising an antibody or binding fragment thereof as described above that binds to an anti-HER 2 antibody for use in the detection of an anti-HER 2 antibody, or for in vivo and in vitro pharmacokinetic, pharmacodynamic and immunogenicity studies of an anti-HER 2 antibody.

Drawings

FIG. 1 is a diagram showing SDS-PAGE of antibodies (HLX 027C9D10 clone number) binding to an anti-HER 2 antibody prepared by in vivo induced abdominal water method according to the specific embodiment of the invention.

FIG. 2 is a diagram showing SDS-PAGE of antibodies (HLX 02D 3B3 clone number) binding to an anti-HER 2 antibody prepared by genetic engineering according to an embodiment of the present invention.

FIG. 3 is a diagram showing SDS-PAGE of antibodies (HLX 027C9D10 clone number) binding to an anti-HER 2 antibody prepared by genetic engineering according to an embodiment of the invention.

FIG. 4 is a SEC-HPLC profile of an antibody (HLX 02D 3B3 clone number) binding to an anti-HER 2 antibody prepared by genetic engineering methods according to embodiments of the invention.

FIG. 5 is a SEC-HPLC chromatogram of an antibody (HLX 027C9D10 clone number) binding to an anti-HER 2 antibody prepared by genetic engineering methods of specific embodiments of the invention.

FIG. 6 is a schematic representation of the binding activity of antibodies (HLX 02 2D3B3 and 7C9D10 clone numbers) that bind to anti-HER 2 antibodies according to embodiments of the invention; wherein filled circles represent the anti-HER 2 antibody of clone No. 7C9D10 (fig. 7C9D 10-NEW), filled squares represent the anti-HER 2 antibody of clone No. 2D3B3 (fig. 2D3B 3-NEW).

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 the present specification, the term "HER2" refers to human epidermal growth factor receptor-2 (Human Epidermal Growth Factor Receptor 2, HER 2), the gene of which 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, 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 by 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, unless otherwise stated, which refers to an Anti-Drug antibody of an Anti-HER 2 antibody when referred to herein as an "Anti-Drug antibody".

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 specification, the HLX02 or the HLX02 antibody is a biological analogue of anti-HER 2 antibody trastuzumab, is from Fumihong Hanzhi, and is commercially available as Hanquyou, the heavy chain amino acid sequence of which is shown as SEQ ID NO. 33, and the light chain amino acid sequence of which is shown as SEQ ID NO. 34. In the examples herein, the anti-HER 2 antibody "HLX22" used for the reverse screening was another anti-HER 2 monoclonal antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO. 35, and the light chain amino acid sequence of which is shown in SEQ ID NO. 36.

The terms "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, 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" refers to a technique in which a DNA fragment of one or more organisms is spliced and recombined with a vector DNA molecule in vitro at a 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 binds to an anti-HER 2 antibody. In some embodiments, in particular, F (ab') capable of specifically binding to an anti-HER 2 antibody ₂ A region, an antibody or binding fragment thereof that binds an anti-HER 2 antibody comprises a heavy chain comprising a heavy chain variable region, a light chain comprising a light chain variable region, a heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising complementarity determining regions LCDR1, LCDR2 and LCDR3.

In some embodiments of the invention, the antibody or binding fragment that binds to an anti-HER 2 antibody 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 light chain constant region (amino acid sequence shown as SEQ ID NO: 18).

Some embodiments of the invention provide a binding fragment that binds an anti-HER 2 antibody, 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 present invention, the heavy chain variable region of the antibody or binding fragment thereof that binds to an anti-HER 2 antibody comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region shown in SEQ ID No. 13 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. 14 or 16. In certain embodiments of the present invention, the heavy chain variable region of an antibody or binding fragment thereof that binds to an anti-HER 2 antibody comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the heavy chain variable region shown in SEQ ID No. 13, and the light chain variable region comprises complementarity determining regions LCDR, LCDR2 and LCDR3 comprised by the light chain variable region shown in SEQ ID No. 14; in still other embodiments of the present invention, the heavy chain variable region of an antibody or binding fragment thereof that binds to an anti-HER 2 antibody comprises 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 comprises 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 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, 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 is shown in SEQ ID NO. 7, the HCDR2 amino acid sequence is shown in SEQ ID NO. 8, the HCDR3 amino acid sequence is shown in SEQ ID NO. 9, and the LCDR1 amino acid sequence is shown in SEQ ID NO. 10, the LCDR2 amino acid sequence is shown in SEQ ID NO. 11, and the LCDR3 amino acid sequence is shown in SEQ ID NO. 12.

In a specific embodiment of the present invention, the heavy chain variable region of the antibody or binding fragment thereof that binds to anti-HER 2 antibody is the amino acid sequence shown in SEQ ID NO. 13, and the light chain variable region is the amino acid sequence shown in SEQ ID NO. 14; 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 an anti-HER 2 antibody comprises an amino acid sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 13 or 15, and the light chain variable region comprises an amino acid sequence that is at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No. 14 or 16, wherein "identity" refers to the extent to which two amino acid sequences have identical residues at the same position in the sequence alignment, i.e., one or more substitutions or deletions are present 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. 13 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. 14 or 16, and retain its binding activity.

Specifically, the invention provides an antibody or binding fragment thereof that binds to an anti-HER 2 antibody, which is capable of specifically binding to an anti-HER 2 antibody having heavy and light chains of SEQ ID No. 33 and SEQ ID No. 34, respectively, comprising trastuzumab and trastuzumab analogs (e.g., HLX02 antibodies in the examples, which are derived from the trade name han-kanka, in the examples), precisely, which is capable of specifically binding to F (ab') of an anti-HER 2 antibody having heavy and light chains of SEQ ID No. 33 and SEQ ID No. 34, respectively ₂ 。

Method for preparing antibody binding to HER2 antibody

The present invention provides a method for preparing the above-described antibody or binding fragment binding to an anti-HER 2 antibody, which is mainly obtained by the following 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, the antibody to the HER2 antibody is produced by means of animal immunization. The immunogens used were F (ab') ₂ The fragment is used for immunizing animals, preferably Balb/c mice. After immunization, hybridoma cell lines expressing monoclonal antibodies were selected using hybridoma technology. In a specific embodiment of the invention, the HLX02 antibody F (ab') ₂ Obtained by enzymatic cleavage methods known in the art, i.e. by cleavage of the full-length antibody and chromatographic purification.

The animal immunization method comprises the steps of immunizing a mouse four times with an immunogen at intervals of 2 weeks, injecting the immunogen subcutaneously into a peripheral immune organ through blood circulation or lymphatic circulation, stimulating corresponding B lymphocyte clones, activating and proliferating the corresponding B lymphocyte clones, and differentiating the B lymphocyte clones into sensitized B lymphocytes. A small amount of venous blood was withdrawn on day 7 after the third and fourth immunizations, and serum was isolated to examine the immune effect. When the immunodetection result shows that the immunotiter reaches the OD value of the serum dilution gradient at 1:10,000 to be more than 1.0, the hybridoma cell fusion culture can be performed with good immunoeffect, and the hybridoma cell strain expressing the 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: myeloma cells are mouse SP2/0 cells that can be immortalized in vitro. Before fusion culture, the culture solution is used for proliferation culture, when the culture solution is in the logarithmic phase, the culture solution is replaced by a new culture solution, and the cell activity rate is higher than 95% for use by counting and checking;

(2) Preparation of spleen cells of immunized mice: the method comprises the steps of (1) performing aseptic operation on a mice successfully immunized after being killed to prepare spleen cell suspension for later use;

(3) Feeder cell preparation: taking the peritoneal exudation cells from the abdomen of the sterilized mice after the sacrifice as feeder cells for standby;

(4) Cell fusion: mixing the prepared myeloma cells and the mouse spleen cells according to a certain proportion, removing the supernatant, adding PEG for fusion, adding HAT selective culture solution after reacting for a certain time to terminate the reaction, and inoculating to a 96-hole culture plate for culturing;

(5) Selective culture: after the two parent cells are treated by PEG, a mixture of 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 cloning 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 for activity detection when the cell colony is larger than 3mm or is 1/2 of the hole bottom, and screening 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: hybridoma cells secreting antibodies that bind to the predetermined anti-HER antibody are first screened, hybridoma cells having the predetermined specificity are further screened therefrom, and cell clones having stable growth and functional properties for practical use 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 techniques, namely, a recombinant expression vector containing the complete heavy chain and light chain of the antibody of the selected anti-HER 2 antibody is transfected into a host cell together, the host cell expresses and secretes the antibody into the 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 invention, the CHO cells are HEK293 cells.

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.

Nucleic acid

In some embodiments of the invention, the invention provides a nucleic acid encoding an antibody or binding fragment thereof that binds to an anti-HER 2 antibody as described above, e.g., comprising a nucleotide sequence encoding said antibody or binding fragment, in particular comprising a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NOs 1-22. The preparation method of the nucleotide 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 comprises the nucleotide sequences shown in SEQ ID NOs 23 and 24; in a further embodiment of the invention, the nucleic acid comprises the nucleotide sequences shown in SEQ ID NOS.25 and 26. IN a specific embodiment of the invention, the nucleic acid comprises the nucleotide sequences shown IN SEQ ID NOS.29 and 30. IN a specific embodiment of the invention, the nucleic acid comprises the nucleotide sequences shown IN SEQ ID NOS.31 and 32. In other embodiments of the invention, the nucleic acid may be the nucleotide sequence shown in SEQ ID NO. 23, 24, 25, 26, 29, 30, 31, 32 with one or more nucleotide substitutions, e.g.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 the above-described anti-HER 2 antibody-binding antibodies or binding fragments, respectively, e.g., comprising nucleotide sequences capable of encoding the heavy and light chains of the above-described anti-HER 2 antibody-binding antibodies or binding fragments. 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 anti-HER 2 antibody are used for detection of an anti-HER 2 antibody, e.g., in vivo or in vitro pharmacokinetic, pharmacodynamic, or immunogenicity studies. Specifically, the concentration of the anti-HER 2 antibody in human serum is detected by a bridging ELISA method by using the antibody as a capturing and detecting reagent, so that the pharmacokinetics of the drug is studied. Among them, anti-HER 2 antibodies include, but are not limited to, anti-HER 2 antibodies that are marketed or under development, such as trastuzumab, pertuzumab, itumomab, ma Jituo ximab, enmtuzumab, witnesuzumab, detrastuzumab.

Kit for detecting a substance in a sample

In a specific embodiment of the invention, the invention provides a kit for the detection of an anti-HER 2 antibody or in vivo or in vitro pharmacokinetic, pharmacodynamic and immunogenicity studies comprising an antibody or binding fragment as described above that binds an anti-HER 2 antibody. Kits will also typically include instructions for use, and/or buffers, assay consumables (e.g., ep tubes, microwell plates, etc.), materials and labels for written records, and the like.

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 anti-drug antibody corresponding to the anti-HER 2 antibody with heavy chain and light chain of SEQ ID NO. 33 and SEQ ID NO. 34, which is obtained by screening, can have higher binding affinity with the anti-HER 2 antibody, has strong specificity, and especially can specifically bind with F (ab') of the anti-HER 2 antibody ₂ The resulting anti-drug antibodies specifically bind only to the anti-HER 2 antibodies having heavy and light chains of SEQ ID No. 33 and SEQ ID No. 34, respectively, and not to other HER2 antibodies (e.g., HLX22 antibodies having heavy and light chains of SEQ ID nos. 35 and 36, respectively, in the examples). Therefore, the antibody can be used for preclinical and clinical PK/PD detection, and can be used for distinguishing detection of different drugs which are identical to HER2 targets because the specificity of the antibody can meet the sensitivity and specificity requirements of a conventional PK analysis method. Meanwhile, the antibody can also be used as a positive control for preclinical and clinical ADA detection. In addition, the antibody has good binding capacity and specificity with the corresponding antibody, and can be used for specific capture and detection of different anti-HER 2 antibodies and used in experiments such as RO, ELISA and the like.

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, and are described in detail in Sambrook et al, MOLECULARCLONING, ALABORATORYMANAUAL, second edition, coldSpringHarborLaborato ryPress,1989and edition,2001; ausubel et al,

CURRENTPROTOCOLSINMOLECULARBIOLOGY, johnWiley & Sons, newYork,1987, angeriodicicupdates; theseriesMETHODSINENZYMOLOGY, academicPress, sanDiego; wolffe, chromatins truturea and function, third, academicPress, sanDiego,1998; methodsINENZYMOLOGY, vol.304, chromatin (P.M. WassarmanandA.P. Wolffe, eds.), academic Press, san Diego,1999; and methodsinumoleclarbiligy, vol.119, chromatinProtocols (p.b. becker, ed.) humapress, totowa,1999, etc. The reagents and materials purchased in the present invention are commercially available.

Example 1 animal immunization method for preparing monoclonal antibody binding to anti-HER 2 antibody and identification

1.1 immunization of mice

F (ab') of HLX02 antibody against HER2 (anti-HER 2 antibody having heavy and light chains of SEQ ID NO:33 and SEQ ID NO:34, respectively) ₂ As an immunogen, balb/C mice were immunized 5 mice each, 50 μg each time subcutaneously, once every two weeks, and four total immunizations were performed per mouse. The animal immunization experiment is completed by Hunan Yuan Tai biotechnology Co.

1.2 immunotiter detection

1.2.1HLX02 third immunization titre assay

HLX02 antibody F (ab') was used for each at day 7 after the third immunization of mice ₂ Immunized mice were collected and their immune titers were determined by ELISA after serum separation. If the immunodetection result shows that the immunotiter reaches the serum dilution gradient of 1:10 ⁴ OD value is larger than 1.0, which indicates good immune effect. Coating with HLX02 antibody and HLX22 antibody (heavy chain and light chain are respectively anti-HER 2 antibody of SEQ ID NO:35 and SEQ ID NO: 36) as coating proteins at coating concentration of 2 μg/mL and 100 μl/well at 4deg.C overnight; the following day of washing was blocked overnight at 3% BSA 200. Mu.L/well at 4 ℃; after washing, the serum before the immunization and after the third immunization of each mouse is diluted 1:10 respectively ³ 、1:10 ⁴ 、1:10 ⁵ Then, the mixture is used as a primary antibody to be incubated for 1h at 100 mu L/Kong Changwen; after washing, 50. Mu.L/Kong Changwen of HRP-loaded anti-IgG (SIGMA; cat# A0168; lot# 097K 4831) as secondary antibody was diluted at a dilution ratio of 1:9,000 and incubated for 1h; after washing, OD was read at 450nm after development at 37℃for 10min with TMB 100. Mu.L/well. The results of the detection of the OD value of each mouse immune titer are shown in tables 1-2.

TABLE 1 results of detection of OD values of the immunotiter of coated HLX02 antibodies after the third immunization of HLX02 antibodies

TABLE 2 results of detection of OD values of the immunotiter of coated HLX22 antibodies after the third immunization with HLX02 antibodies

/>

Analysis of results: from the ELISA immunotiter assay results of the coated HLX02 antibodies of Table 1, five mice were immunized with HLX02 antibody F (ab') a third time ₂ Post serum at 1:10 ³ 、1:10 ⁴ 、1:10 ⁵ All three dilution gradients showed high OD values greater than 1.0, compared with the low OD values shown by their respective preimmune sera, indicating that five mice were better immunized and produced antibodies with high affinity to HLX02 antibodies, but at the same time, as seen from the ELISA immunotiter test results of table 2 coated HLX22 antibodies, each mouse serum also showed high OD values compared with preimmune sera, indicating that the produced antibodies also had high affinity to HLX22 antibodies; as a result of the combination of the two, the immunized mice produced antibodies against HER2 antibodies, but the antibodies present in the serum of the immunized mice were polyclonal and not strongly specific, and monoclonal screening was required in subsequent experiments to produce monoclonal antibodies that specifically bind to HLX02 antibodies.

1.2.2HLX02 antibody immunization fourth potency detection

Antibody HLX02F (ab') for each use on day 7 after the fourth immunization of mice ₂ Immunized mice were collected and their immune titers were determined by ELISA after serum separation. If the immunodetection result shows that the immunotiter reaches the serum dilution gradient of 1:10 ⁴ OD value at higher than 1.0, indicating an immune effectGood. Coating with HLX02 and HLX22 antibodies as coating proteins respectively at a coating concentration of 2 mug/mL and 100 mug/well at 4 ℃ overnight; the following day of washing was blocked overnight at 3% BSA 200. Mu.L/well at 4 ℃; after washing, the serum of each mouse before immunization and after the fourth immunization is diluted 1:10 respectively ³ 、1:10 ⁴ 、1:10 ⁵ Then, the mixture is used as a primary antibody to be incubated for 1h at 100 mu L/Kong Changwen; after washing, 50. Mu.L/Kong Changwen of HRP-loaded anti-IgG (SIGMA; cat# A0168; lot# 097K 4831) as secondary antibody was diluted at a dilution ratio of 1:9,000 and incubated for 1h; after washing, OD was read at 450nm after development at 37℃for 10min with TMB 100. Mu.L/well. The results of the detection of OD values of the immune titers of the mice are shown in tables 3-4.

TABLE 3 results of detection of OD values of the immune titers of the coated HLX02 antibodies after the fourth immunization of the HLX02 antibodies

TABLE 4 results of detection of OD values of the immune titers of the coated HLX22 antibodies after the fourth immunization of the HLX02 antibodies

Analysis of results: from the ELISA immunotiter assay results of the coated HLX02 antibodies of Table 3, five mice were immunized with the antibody HLX02F (ab') ₂ Post serum at 1:10 ⁴ 、1:10 ⁵ 、1:10 ⁶ All three dilution gradients showed an OD value greater than 1.0, compared with the low OD value shown by their respective preimmune sera, indicating that five mice were better immunized and produced antibodies with high affinity to HLX02 antibodies, but at the same time, as seen from the ELISA immunotiter test results of table 4 coated HLX22 antibodies, each mouse serum also showed a higher OD value compared with preimmune sera, indicating that the produced antibodies also had high affinity to HLX22 antibodies; as a result of the combination of the two, it was found that the antibody against HER2 was produced in the immunized mice, but the antibodies present in the serum of the immunized mice were polyclonal, and the specificity was not strong, and the later was requiredIn subsequent experiments, monoclonal antibodies that specifically bind to HLX02 antibodies were screened.

1.3 hybridoma cell screening

1.3.1HLX02 antibody hybridoma cell screening

According to the serum titer detection result of immunized mice, comprehensively considering the ELISA immune titer OD value results of the mice which reach the immune requirements for the third and fourth immunodetection and the health state of the mice, taking the mice which have high combined OD value with the HLX02 antibody and low combined OD value with the HLX22 antibody and good mouse hair color, killing the mice, taking spleen thereof, cutting the spleen into spleen cell suspension, adding a PEG fluxing agent and SP2/O mouse myeloma cells for fusion, screening the hybridoma cells successfully fused by utilizing a HAT culture medium, detecting the supernatant of each hole cell by utilizing a limiting dilution method and ELISA, and screening positive monoclonal cell strains capable of producing monoclonal antibodies after comparing the positive monoclonal cell strains with positive controls (POS: antiserum after the immunization of the mice) and negative controls (NEG: serum before the immunization of the mice). The results of the first screening are shown in Table 5; after the second screening, ten clones of HLX02 antibody-specific binding (heavy chain and light chain are respectively anti-HER 2 antibody of SEQ ID NO:33 and SEQ ID NO: 34) were selected, and ELISA detection results of each clone cell line are shown in Table 6. 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 5 ELISA detection OD value results table of first screening positive cell lines with HLX02 antibody

/>

By using the HLX02 antibody as the positive screen coating protein 1 (anti-gen 1), the HLX22 antibody (anti-HER 2 antibody with heavy and light chains of SEQ ID NO:35 and SEQ ID NO:36 respectively) as the negative screen coating protein 2 (anti-gen 2), the first screening was performed by ELISA experiments, as shown by ELISA OD results in Table 5, 10 positive clone wells of 1C9, 2D3, 2F5, 3B7, 4A11, 4C1, 5B7, 5H2, 6H3, 7C9 were first screened, and their expression supernatants all showed high OD with the HLX02 antibody, while showing low OD with the HLX22 antibody, indicating that the antibody in the supernatants of these 10 wells had high affinity and specificity with the HLX02 antibody. The results of the further second screening are shown in Table 6.

TABLE 6 ELISA detection of OD value results table for secondary screening of positive monoclonal cell lines by HLX02 antibody

As shown in Table 6, the results of ELISA detection of OD values of positive monoclonal cell strains obtained by the second screening of the first screened cell strains showed high OD values with HLX02 antibody as positive screening coating protein 1, while those obtained by screening cell strain expression supernatants of the 10 clones showed low OD values with HLX22 antibody as negative screening coating protein 2, indicating that the antibodies obtained by screening the cell strains showed high affinity and specificity with the HLX02 antibody, and were stored in a liquid nitrogen tank after performing expansion culture.

1.4 production and identification of monoclonal antibodies

1.4.1 production of monoclonal antibodies

Monoclonal antibody production was performed by in vivo ascites induction. 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 were recovered and cultured with HLX 02C 9D10 clone numbers. After 1-2 weeks, cultured hybridoma cells were inoculated into the abdominal cavity of the mice, and the monoclonal antibodies were 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 ProteinA is purified to obtain the monoclonal antibody.

1.4.2 physical and chemical identification of monoclonal antibodies (SDS-PAGE)

Reduction SDS-PAGE sample preparation: mu.g of HLX 02C 9D10 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. 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 ImageJ according to the peak area normalization method. The results of SDS-PAGE of monoclonal antibody HLX 02C 9D10 are shown in FIG. 1.Lane 1 is the gel running result after antibody reduction, and the result shows that the gel is divided into two bands of a heavy chain and a light chain after reduction, the molecular weight of the heavy chain is about 45kDa, the molecular weight of the light chain is about 30kDa, and the purity is good through gray level analysis.

1.4.3 identification of binding force of monoclonal antibodies

The affinity and specificity of the monoclonal antibody produced by the hybridoma cell strain of the HLX 02C 7D 9D10 clone number with the HLX02 antibody are detected by ELISA, the HLX02 antibody is used as a positive screening coating protein 1 (anti-gen 1), the HLX22 antibody is used as a negative screening coating protein 2 (anti-gen 2) for coating, and the OD value after color development is compared with a positive control (POS: antiserum after mouse immunization) and a negative control (NEG: serum before mouse immunization) so as to verify the specific binding force of the monoclonal antibody of the HLX 02C 9D10 clone number with the HLX02 antibody. The results of the OD values of the ELISA assays are shown in Table 7.

TABLE 7 results table of ELISA detection OD values for HLX027C9D10 monoclonal antibody

CloneNO.	7C9D10	POS	NEG
				Antigen1(HLX02)	0.821	0.811	0.091
Antigen2(HLX22)	0.117	0.789	0.087

From the OD results of the ELISA experiments of table 7, the HLX027C9D10 monoclonal antibody showed a high OD with the HLX02 antibody as positive screen coating protein 1, and a low OD with the HLX22 antibody as negative screen coating protein 2, indicating that the HLX027C9D10 monoclonal antibody had a high affinity and specificity with the HLX02 antibody.

EXAMPLE 2 sequencing of monoclonal hybridoma cells

The hybridoma cell lines with clone numbers 2D3B3 and 7C9D10 were selected for sequencing analysis (completed by Shanghai Baiying Biotechnology Co., ltd.) to obtain the CDR regions (CDRs) and the variable region amino acid sequences of the heavy chain and the light chain of the monoclonal antibodies of each clone number, as shown in Table 8-1.

Table 8-1.2D3B3, 7C9D10 clone number monoclonal antibody amino acid sequence table

EXAMPLE 3 molecular construction, production and identification of monoclonal antibodies

3.1 construction of recombinant expression vectors

The full length amino acid sequences of the complete heavy and light chains of the monoclonal antibodies that bind the anti-HER 2 antibody described above were designed and synthesized, wherein the complete heavy chain contained the heavy chain variable region (shown as SEQ ID NO:13 or 15) and the murine IgG1 heavy chain constant region (SEQ ID NO: 17), and the complete light chain contained the light chain variable region (shown as SEQ ID NO:14 or 16) and the murine Kappa light chain constant region (SEQ ID NO: 18). The heavy chain full-length sequences of the synthesized 2D3B3 and 7C9D10 monoclonal antibodies are the amino acid sequences shown in SEQ ID NO. 19 and SEQ ID NO. 21, and the light chain full-length sequences are the amino acid sequences shown in SEQ ID NO. 20 and SEQ ID NO. 22. The amino acid sequences of the heavy chain constant region and the light chain constant region and the full length of the heavy chain and the full length of the light chain of the 2D3B3, 7C9D10 monoclonal antibodies are shown in Table 8-2.

TABLE 8-2 amino acid sequences of heavy and light chain constant regions and full length of heavy and light chain of 2D2B3, 7C9D10 monoclonal antibodies

/>

Referring to Table 9, the nucleotide sequences of the heavy chain variable region of the 2D3B3 and 7C9D10 clone number monoclonal antibodies are shown as SEQ ID NO. 23 and 25, and the nucleotide sequences of the light chain variable region are shown as SEQ ID NO. 24 and 26; the nucleotide sequence of the heavy chain constant region of the monoclonal antibody of 2D3B3 and 7C9D10 is shown as SEQ ID NO. 27, and the nucleotide sequence of the light chain constant region is shown as SEQ ID NO. 28. The nucleotide sequences of the heavy chains of the monoclonal antibodies of the 2D3B3 and 7C9D10 clone numbers are respectively shown as SEQ ID NO. 29 and SEQ ID NO. 31, the nucleotide sequences of the corresponding light chains are respectively shown as SEQ ID NO. 30 and SEQ ID NO. 32, the nucleotide sequences for encoding the heavy chains and the light chains are respectively constructed on eukaryotic expression vector plasmids pcDNA3.4 by a homologous recombination method, recombinant plasmids containing complete heavy chain and light chain full-length genes are respectively obtained, and the recombinant plasmids are transformed into escherichia coli DH5 alpha to amplify and extract plasmids for eukaryotic cell system expression.

TABLE 9 nucleotide sequences

3.2 expression of antibodies

Expressed by the ExpiCHO transient expression system, the cell density was confirmed to be 7X 10 on the day of transfection ⁶ Up to 1X 10 ⁷ Cell viability about living cells/mL>98 at this time, the cells were adjusted to a final concentration of 6X 10 using fresh ExpiCHO expression medium pre-warmed at 37 ℃ ⁶ Individual cells/mL. OptiPRO pre-cooled at 4deg.C ^TM SFM dilution of plasmid of interest (1. Mu.g plasmid was added to 1mL of the medium) with OptiPRO ^TM SFM dilution of Expifectamine ^TM CHO, mixing the two materials in equal volume, and gently stirring to obtain the product ^TM The CHO/plasmid DNA mixture was incubated at room temperature for 1-5min, slowly added to the prepared cell suspension while gently shaking, and finally placed in a cell culture shaker at 37℃and 8% CO ₂ Culturing under the condition.

18-22h after transfection, expiCHO was added to the culture broth ^TM Enhance and ExpiCHO ^TM Feed, shake flask placed on a shaker at 32℃and 5% CO ₂ Culturing was continued under the conditions. On day 5 post transfection, the same volume of ExpiCHO was added ^TM Feed, slowly add while gently mix cell suspension and end the culture at day 12 of expression culture.

3.3 purification of antibodies

The cell culture supernatant was centrifuged at 15,000g for 10min, the resulting supernatant was affinity purified with NMab Protein A affinity chromatography medium, the target Protein was eluted with 100mM sodium acetate (pH 3.0), followed by neutralization with 1M Tris-HCl, and the resulting Protein was concentrated by dialysis to PBS buffer.

3.4 physical and chemical Properties identification of antibodies

3.4.1 protein concentration

The antibody concentration of the protein after liquid exchange is detected through UV280 binding extinction coefficient. The extinction coefficient is predicted from the amino acid sequence. The extinction coefficients and concentrations of antibodies for each clone number are shown in Table 10.

Table 10 extinction coefficient and concentration results of each monoclonal antibody

Clon No.	Extinction coefficient	Conc.
			2D3B3	1.522	1.360
7C9D10	1.577	0.970

3.4.2SDS-PAGE

Reduction SDS-PAGE sample preparation: mu.g of the monoclonal antibody was added to a 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. 2-3, and the purity results are shown in Table 11. As can be seen from the results of the Image J analysis combined with FIGS. 2-3, the produced antibodies of clone numbers had clear bands before (N-R) and after (R) reduction, and had good purity, and the purity was > 95%.

TABLE 11 SDS-PAGE purity results for each monoclonal antibody

Clon No.	Purity
		2D3B3	＞95.000％
7C9D10	＞95.000％

3.4.3SEC-HPLC

(1) Material preparation: mobile phase: 150mmol/L phosphate buffer, pH 7.4;

(2) Sample preparation: each clone number antibody was diluted to 0.5mg/mL with mobile phase solution. Agilent HPLC 1100 column (XBIridge BEH SEC 3.5 μm,7.8mm I.D.×30em, waters) flow rate was set at 0.8mL/min, sample volume 20. Mu.L, VWD detector wavelengths 280nm and 214nm. The percentage of high molecular aggregates, monomers and low molecular aggregates in the samples was calculated according to the area normalization method. The SEC-HPLC spectra of each monoclonal antibody are shown in FIGS. 4-5, and the purity results are shown in Table 12. According to the integral result, the purity of the antibodies of clone numbers 2D3B3 and 7C9D10 is 99.436% and 99.531%, respectively.

TABLE 12 SEC-HPLC purity results for monoclonal antibodies

Clon No.	Purity
		2D3B3	99.436％
7C9D10	99.531％

EXAMPLE 4 identification of binding Activity of monoclonal antibodies

The binding activity of each monoclonal antibody of 2D3B3 and 7C9D10 was identified by ELISA. The expressed and purified antibodies were diluted to 2. Mu.g/mL with lx PBS and coated overnight at 4℃at 30. Mu.L/well as coating protein; the following day was blocked with 60. Mu.L/Kong Shiwen of Superbrook buffer for 1h after washing with 0.05% PBST; after the washing operation, a series of concentration gradients diluted with buffer (STD 01-STD11 and Blank) 2D3B3, 7C9D10, 30. Mu.L/Kong Shiwen were added and incubated for 1h, see Table 13 for the experimental layout; after washing HRP labeled Goat anti-mouse IgG was used as detection secondary antibody at 1: incubation for 1h at 30. Mu.L/Kong Changwen after dilution at a dilution ratio of 12,000; after washing, the OD values were read at two wavelengths of 450nm and 630nm after development at 37℃for 10min at 30. Mu.L/well of TMB, and the difference between the read values at the wavelengths of 450nm and 630nm was subjected to curve fitting and calculation. Binding activity curves are shown in figure 6 and binding activity EC50 values are shown in table 14.

Table 13 monoclonal antibody binding Activity identification ELISA experiment layout template

Plate

1

2

3

4

5

6

7

8

9

10

11

12

A

STD01

STD02

STD03

STD04

STD05

STD06

STD07

STD08

STD09

STD10

STD11

Blank

B

STD01

STD02

STD03

STD04

STD05

STD06

STD07

STD08

STD09

STD10

STD11

Blank

C

STD01

STD02

S1D03

STD04

STD05

STD06

STD07

STD08

STD09

STD10

STD11

B1ank

D

STD01

STD02

STD03

STD04

STD05

STD06

STD07

STD08

STD09

STDl0

STD11

B1ank

Table 14 binding activity EC50 values for monoclonal antibodies 2D3B3, 7C9D10

Clone No.	EC50(ng/mL)
		7C9D10	14.45
2D3B3	6.97

As shown in Table 14 and FIG. 6, the 2D3B3 and 7C9D10 monoclonal antibodies have binding activity EC50 values of 6.97ng/ml and 14.45ng/ml, respectively, indicating that the antibodies have strong binding activity with the corresponding anti-HER 2 antibodies, and can be practically applied to PK/PD analysis and the like.

EXAMPLE 5 PK use of monoclonal antibodies

The monoclonal antibody with high binding activity and high specificity can be used as a capture reagent or used as a detection reagent after biotin labeling, and can be applied to PK bioanalytics of the HLX02 antibody. Monoclonal antibody 7C9D10 was used as a capture reagent and biotinylated monoclonal antibody 2D2B3 was used as a detection reagent for HLX02 concentration in serum. The 2 methods described above are all well specific, do not interfere with each other between drugs of the same target, and all perform corresponding methodological verification according to the rules, including but not limited to the following verification items: precision and accuracy, selectivity, durability, stability, specificity, etc. (method validation summary see tables 15 and 17) demonstrating that the monoclonal antibody-based method described above can be applied to detection of HLX02 in human serum in clinical settings.

5.12D3B3 and 7C9D10 antibodies applied to detection of HLX02 antibody concentration in human serum

The detection of human serum HLX02 antibody concentration adopts ELISA method, using clone 7C9D10 antibody as capture reagent and biotinylated 2D3B3 clone number antibody as detection reagent. I.e.1. Mu.g/mL of 7C9D10 antibody was coated on the plate as capture reagent. HLX02 antibodies were formulated in 100% human serum, standard curve Samples (STDs) and Quality Control Samples (QCs) were prepared, and using a Blocker ^TM Casein PBS (Casein for short) was diluted 1:200 and then placed on the plate. HLX02 antibodies in standards, quality control and samples are captured by the capture reagent. After washing, 1 μg/mL biotinylated 2D3B3 antibody was added to bind to HLX02 antibody captured in the previous step. After the plate washing operation, strepavidin-HRP was added to bind biotin. The plate was washed, TMB chromogenic solution was added, followed by stop solution, signal values at 450nm and 630nm were read using a microplate reader, and the difference between the 450nm and 630nm read was proportional to the amount of HLX02 antibody bound by the capture reagent in the initial step.

5.1.1 accuracy and precision verification

Accuracy and precision of the method (a & P) experiments were performed by 4 analysts on 3 days for 6 independent a & P analysis batches. Each of the accuracy and precision analysis lots contained at least 1 standard curve (STD 01-STD 07), one blank sample (BLK) and 3 sets of quality control samples, including 5 concentrations of upper limit of quantification (ULOQ), high concentration quality control (HQC), medium concentration quality control (MQC), low concentration quality control (LQC) and lower limit of quantification (LLOQ), each sample was subjected to a multiplex assay. The results are shown in table 15, and all quality control samples meet the acceptance criteria described in the report: the precision of the quality control samples at each concentration level was within 20% between batches, the accuracy within and between batches was within + -20% (upper and lower quantitative limits were within + -25%) and the total error between batches was within 30% (upper and lower quantitative limits were within 40%).

TABLE 15 accuracy and precision verification data sheet for detection method of HLX02 antibody concentration in human serum

/>

5.1.2 specificity verification

Specificity investigation HLX22 antibodies (final concentration up to 834 μg/mL) were added at different concentration gradients to the 2 analyte concentration levels (ULOQ and LLOQ) and 1 blank level of the validation samples, and the specificity samples were assayed in 5 replicates, each sample being multiplexed. The results of the specificity verification show that the HLX22 antibody with the content of 834 mug/mL can not interfere with the detection of the HLX02 antibody in human serum, and the results are shown in Table 16, and the specificity samples all meet the acceptance criteria: at each interferent concentration, at least 80% of the validated samples (ULOQ, LLOQ) were within ±25% of accuracy of concentration back calculated and within 25% of precision between sample wells. The blank matrix measurements without added interferents should be below the lower quantification limit (BQL).

TABLE 16 specificity verification data sheet for method of detecting HLX02 antibody concentration in human serum

/>

TABLE 17 outline of methodological verification of method for detecting concentration of HLX02 antibodies in human serum

/>

/>

Claims (18)

1. An antibody or binding fragment thereof that binds an anti-HER 2 antibody 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. 13 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. 14 or 16.

2. The antibody or binding fragment thereof that binds to an anti-HER 2 antibody 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 alternatively, the process may be performed,

the HCDR1, HCDR2 and HCDR3 are the amino acid sequences shown in SEQ ID NO 7, 8 and 9, respectively, and the LCDR1, LCDR2 and LCDR3 are the amino acid sequences shown in SEQ ID NO 10, 11 and 12, respectively.

3. The antibody or binding fragment thereof that binds to an anti-HER 2 antibody 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. 13, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO. 14; or alternatively, the process may be performed,

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 that binds to an anti-HER 2 antibody according to claim 1 or 2, wherein the antibody is a full length antibody.

5. The antibody or binding fragment thereof that binds to an anti-HER 2 antibody according to claim 1 or 2, 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 that binds to an anti-HER 2 antibody according to claim 5, wherein the heavy chain constant region comprises or consists of the amino acid sequence shown in SEQ ID No. 17 and the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID No. 18.

7. The antibody or binding fragment thereof that binds to an anti-HER 2 antibody according to claim 1 or 2, wherein 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; or alternatively, the process may be performed,

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 that binds to an anti-HER 2 antibody according to claim 1 or 2, 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. A nucleic acid encoding the antibody or binding fragment thereof of any one of claims 1-8 that binds to an anti-HER 2 antibody.

10. A nucleic acid encoding the amino acid sequence shown in SEQ ID NO. 1-22.

11. The nucleic acid according to claim 9 or 10, comprising the nucleotide sequence shown in SEQ ID NOs 23-26 or 29-32.

12. A vector comprising the nucleic acid of any one of claims 9-11.

13. A host cell comprising a nucleic acid according to any one of claims 9-11 and/or a vector according to claim 12.

14. The host cell of claim 13, wherein the cell is selected from 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.

15. A method of producing an antibody or binding fragment thereof that binds an anti-HER 2 antibody, comprising: culturing the cell according to claim 13 or 14 in a medium suitable for expression to express the antibody or binding fragment thereof and obtaining the antibody or binding fragment thereof.

16. The antibody or binding fragment thereof that binds to an anti-HER 2 antibody according to any one of claims 1-8 for use in detecting an anti-HER 2 antibody.

17. The use of an antibody or binding fragment thereof that binds to an anti-HER 2 antibody according to claim 16 for in vivo and in vitro pharmacokinetic, pharmacodynamic or immunogenicity studies of an anti-HER 2 antibody.

18. A kit comprising the antibody or binding fragment thereof of any one of claims 1-8 that binds to an anti-HER 2 antibody.