CN117069853B

CN117069853B - Antibody targeting trastuzumab and application thereof

Info

Publication number: CN117069853B
Application number: CN202311314926.3A
Authority: CN
Inventors: 欧伦; 王雪莉; 王变珍; 谢新遥
Original assignee: United Power Pharma Tech Co ltd
Current assignee: United Power Pharma Tech Co ltd
Priority date: 2023-10-11
Filing date: 2023-10-11
Publication date: 2024-01-05
Anticipated expiration: 2043-10-11
Also published as: CN117069853A

Abstract

The present application provides an antibody targeting trastuzumab and uses thereof. The application also provides nucleic acids encoding the antibodies, expression vectors comprising the nucleic acids, host cells comprising the nucleic acids or the expression vectors, detection reagents or kits for detecting trastuzumab, and the like. The antibody specifically combined with trastuzumab prepared by the application not only can provide technical support for monitoring the concentration of the drug in blood, but also can be used as a positive reference for ADA detection.

Description

Antibody targeting trastuzumab and application thereof

Technical Field

The present application relates generally to the field of antibodies. More specifically, the present application relates to the preparation and use of an antibody targeting trastuzumab.

Background

The epidermal growth factor receptor family (epidermal growth factor receptor family, erbB family) includes four family members, erbB1 (EGFR, HER 1), erbB2 (HER 2), erbB3 (HER 3) and ErbB4 (HER 4). HER2 protein has tyrosine protein kinase activity and heterodimerizes with other members of the EGFR family to perform signaling functions. The overexpression of HER2 causes autophosphorylation of receptor cytoplasmic tyrosine residues, starts a signal path, causes abnormal proliferation of cells, plays an important role in the occurrence and development processes of tumors such as breast cancer, gastric cancer, colorectal cancer, lung cancer and the like, and HER2 becomes an ideal tumor treatment target.

Trastuzumab was the first anti-HER 2 monoclonal antibody developed in 1990 to interfere with the HER2 signaling pathway by inhibiting multiple mechanisms such as receptor dimerization, receptor internalization and degradation, and inhibition of the PI3K-AKT signaling pathway. Clinical trials demonstrate that chemotherapy in combination with trastuzumab can increase the overall survival of HER2 positive metastatic breast cancer females, thus trastuzumab becomes the first-line therapeutic for metastatic and early HER2 positive breast cancer.

Along with the continuous expansion of trastuzumab in clinical tumor application, the content of antibodies in blood needs to be detected, and the pharmacokinetic analysis of the medicine is carried out; monitoring the production of Anti-drug antibody (ADA) directs the clinical rational administration, so it is necessary to prepare an antibody capable of Anti-trastuzumab. An antibody against trastuzumab recognizes the variable region of trastuzumab and produces an antibody that specifically binds. Antibodies against trastuzumab can be classified into two types, competitive (antigen-blocking) and non-competitive (antigen-non-blocking).

Disclosure of Invention

To meet the detection needs of trastuzumab in clinical patients, provided herein is an idiotype antibody targeting trastuzumab. Specifically, the present application provides the following technical solutions.

In a first aspect, the present application provides an antibody or antigen binding portion thereof that specifically binds trastuzumab comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, wherein according to the Kabat numbering system the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6; or according to an IMGT numbering system, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 11, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 13.

In a second aspect, the present application provides a nucleic acid molecule encoding an antibody or antigen binding portion thereof of the first aspect.

In a third aspect, the present application provides an expression vector comprising a nucleic acid molecule according to the second aspect.

In a fourth aspect, the present application provides a host cell comprising the nucleic acid molecule of the second aspect or the expression vector of the third aspect.

In a fifth aspect, the present application provides a method of preparing an antibody or antigen binding portion thereof of the first aspect, comprising:

a) Culturing the host cell of the fourth aspect; and

b) Recovering the antibody or antigen binding portion thereof from the host cell or from a supernatant of a culture of the host cell.

In a sixth aspect, the present application provides a detection reagent or kit comprising an antibody or antigen-binding portion thereof according to the first aspect.

In a seventh aspect, the present application provides the use of an antibody or antigen binding portion thereof of the first aspect for detecting trastuzumab in a biological sample of a subject.

In an eighth aspect, the present application provides the use of an antibody or antigen binding portion thereof of the first aspect for determining the content of trastuzumab in a biological sample of a patient administered with trastuzumab.

The antibody specifically combined with trastuzumab prepared by the application not only can provide technical support for monitoring the concentration of the drug in blood, but also can be used as a positive reference for ADA detection.

Drawings

FIG. 1 shows SDS-PAGE patterns of trastuzumab before and after Ide S cleavage, wherein M is protein Marker;1,2: trastuzumab under non-reducing and reducing conditions; 3,4: trastuzumab F (ab') 2 fragments under non-reducing and reducing conditions;

FIG. 2 shows the results of a flow cytometry assay for Her2 positive breast cancer cells SKBR3 and trastuzumab binding by anti-trastuzumab antibody 9G6 prepared in the present application, wherein trastuzumab binds to SKBR3 cells, the peak pattern shifts right, the peak pattern shifts left after adding a seropositive control to immunized mice, the results of 9G6 clone antibody are similar to immune serum, the peak pattern shifts left;

FIG. 3 shows SDS-PAGE electrophoresis and affinity assay of anti-trastuzumab antibody 9G6 expressed by HEK293 cells. A: SDS-PAGE electrophoresis of anti-trastuzumab antibody 9G6, wherein M: protein markers; 1, non-reducing condition; 2, reducing conditions; b: binding and dissociation curves for Fortebio detection of anti-trastuzumab antibody 9G6 with trastuzumab F (ab') 2 fragment;

FIG. 4 shows a fitted curve of anti-trastuzumab antibody 9G6 binding to trastuzumab;

figure 5 shows a fitted curve of 9G6 competition trastuzumab with anti-trastuzumab antibodies bound to SKBR3 cells.

Detailed description of the invention

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, the terms of the present application are understood according to conventional usage by those of ordinary skill in the relevant art.

Definition of the definition

The term "about" as used herein refers to + -10% of the recited figures, e.g., about 1% refers to a range of 0.9% to 1.1%.

The term "antibody" as used herein refers to any form of antibody or fragment thereof that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

The term "specific binding" as used herein is a term well known in the art, and methods for determining such specific binding of an antibody to an antigen are also well known in the art. For example, in some embodiments, "specific binding" refers to binding of an antibody to a desired target, but not significantly to other targets. Antibodies bind the intended target epitope with significantly increased affinity and/or for a longer duration than other epitopes.

The term "antigen binding portion" as used herein includes fragments or derivatives of antibodies that substantially retain their binding activity. Thus, the term "antigen binding portion" refers to a portion of a full-length antibody, typically an antigen binding or variable region thereof. Examples of antigen binding moieties include, but are not limited to: fab fragments, fab 'fragments, F (ab') 2 fragments, fv fragments, diabodies, single chain antibody molecules such as sc-Fv and multispecific antibodies formed from antibody fragments. It is also contemplated that the antigen binding portion may comprise conservative amino acid substitutions that do not substantially alter its binding activity.

The term "Fab fragment" as used herein comprises a light chain and a heavy chain CH1 and variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

The term "Fab ' fragment" as used herein contains a portion or fragment of one light chain and one heavy chain, said portion or fragment containing a VH domain and a CH1 domain and a region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of 2 Fab ' fragments to form a F (ab ') 2 molecule.

The term "F (ab') 2 fragment" as used herein contains two light chains and two heavy chains, the heavy chains containing a portion of the constant region between the CH1 and CH2 domains such that an interchain disulfide bond is formed between the two heavy chains. The F (ab ') 2 fragment thus consists of two Fab' fragments, which are linked together by a disulfide bond between the two heavy chains.

The term "Fv fragment" as used herein comprises variable regions from the heavy and light chains, but lacks constant regions.

The term "single chain Fv" or "scFv" as used herein refers to an antibody fragment comprising the VH domain and the VL domain of an antibody, wherein these domains are present as a single polypeptide chain. Generally, fv polypeptides also comprise a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding.

The term "diabody" as used herein refers to a small antibody fragment having two antigen-binding sites, said fragment comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) (VH-VL or VL-VH) linked thereto in the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same strand, each domain is forced to pair with the complementary domain of the other strand, thereby creating two antigen binding sites.

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining region" or "CDR" (e.g., residues 24-34 (LCDR-1), 50-56 (LCDR-2) and 89-97 (LCDR-3) in the light chain variable domain and residues 31-35 (HCDR-1), 50-65 (HCDR-2) and 95-102 (HCDR-3) in the heavy chain variable domain); kabat et al, (1991) Sequences of Proteins of Immunological Interest, version 5, public Health Service, national Institutes of Health, bethesda, md., and/or amino acid residues from the "hypervariable loop" (i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and residues 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain); chothia and Lesk, (1987) J. Mol biol 196: a frame region "is defined herein as residues outside the framework region of the variable domain.

The term "CDR" as used herein may be labeled and defined in a manner known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system, using tool websites including but not limited to AbRSA websites (http:// cao.labshare. Cn/AbRSA/CDRs. Php), abYsis websites (www.abysis.org/analysis/sequence_input/key_analysis. Cgi) and IMGT websites (http:// www.imgt.org/3 Dstructure-DB/cgi/DonGapAlig. Cgi#rest). "CDR" as used herein is numbered according to the Kabat numbering system or the IMGT numbering system.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations in a small number of individuals.

The term "EC" as used herein ₅₀ "means half maximum effective concentration (concentration for% of maximal effect, EC) ₅₀ ) Refers to the concentration that causes 50% of the maximum effect.

The term "affinity" as used herein refers to the binding force between molecules, which is essentially a non-covalent force. Methods for determining affinity between molecules, which demonstrate the ability to bind between molecules (e.g., between an antibody and an antigen, between a receptor and a ligand), are well known in the art and include, but are not limited to, biological membrane interferometry (BLI), solid phase radioimmunoassay (SP-RIA), equilibrium dialysis, binding antigen precipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), surface Plasmon Resonance (SPR), and the like. The magnitude of the affinity can be determined by an affinity constant K _D Representing the affinity constant K _D The higher the two, the greater the ability to bond.

Detailed Description

Along with the continuous expansion of trastuzumab in clinical tumor application, the content of antibodies in blood needs to be detected, and the pharmacokinetic analysis of the medicine is carried out; monitoring the production of anti-drug antibodies, guiding the clinical rational administration, it is therefore necessary to prepare antibodies capable of anti-trastuzumab. The inventor of the application prepares an anti-trastuzumab antibody by immunizing a mouse with a part of trastuzumab, and tests show that the anti-trastuzumab antibody can bind to trastuzumab with high affinity, so that support is provided for accurately detecting the content of trastuzumab in blood and carrying out pharmacokinetic analysis of medicines.

EGFR (epidermal growth factor receptor, abbreviated as EGFR, erbB-1 or HER 1) is one of the members of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB 1, EGFR), HER2 (erbB 2, NEU), HER3 (erbB 3), and HER4 (erbB 4). The HER family plays an important regulatory role in cellular physiology. EGFR is widely distributed on the surfaces of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes and the like, and EGFR signaling pathways play an important role in physiological processes such as growth, proliferation, differentiation and the like of cells. EGFR is divided into three regions: an extracellular ligand binding domain, a transmembrane domain and an intracellular kinase domain.

Specifically, the application provides the following technical scheme:

in a first aspect, the present application provides an antibody or antigen binding portion thereof that specifically binds trastuzumab comprising a heavy chain variable region comprising any one or more of HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID No. 1, the amino acid sequence of HCDR2 is shown in SEQ ID No. 2, and the amino acid sequence of HCDR3 is shown in SEQ ID No. 3 according to the Kabat numbering system; or according to the IMGT numbering system, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 10, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 11.

In a preferred embodiment, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 according to the numbering system of Kabat, wherein the amino acid sequence of HCDR1 is shown in SEQ ID No. 1, the amino acid sequence of HCDR2 is shown in SEQ ID No. 2, and the amino acid sequence of HCDR3 is shown in SEQ ID No. 3; or according to the IMGT numbering system, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 10, and the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 11.

In some embodiments, the antibody or antigen binding portion thereof further comprises a light chain variable region comprising any one or more of LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID No. 4, the amino acid sequence of LCDR2 is shown in SEQ ID No. 5, and the amino acid sequence of LCDR3 is shown in SEQ ID No. 6 according to the Kabat numbering system; or according to the IMGT numbering system, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 13.

In a preferred embodiment, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO. 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO. 5 and the amino acid sequence of LCDR3 is shown in SEQ ID NO. 6 according to the Kabat numbering system; or according to the IMGT numbering system, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 13.

In a preferred embodiment, the antibody or antigen binding portion thereof comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID No. 1, the amino acid sequence of HCDR2 is shown in SEQ ID No. 2, the amino acid sequence of HCDR3 is shown in SEQ ID No. 3, the amino acid sequence of LCDR1 is shown in SEQ ID No. 4, the amino acid sequence of LCDR2 is shown in SEQ ID No. 5 and the amino acid sequence of LCDR3 is shown in SEQ ID No. 6 according to the Kabat numbering system; or according to an IMGT numbering system, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 11, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 13.

In specific embodiments, the antigen binding portion is selected from the group consisting of: fab fragment, fab 'fragment, F (ab') ₂ Fragments, fv fragments, scFv fragments, fd fragments or single domain antibodies.

In some embodiments, the antibody is a murine antibody.

In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7 and/or the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

In preferred embodiments, the nucleic acids described herein may be codon optimized nucleic acids suitable for expression in a host cell. For example, according to the degeneracy of the codons, they still encode the same protein. Methods for codon optimization according to the host cell used are well known to those skilled in the art.

Any suitable expression vector may be used. For example, prokaryotic cloning vectors include plasmids derived from E.coli, such as colEl, pCRl, pBR322, pMB9, pUC, pKSM and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages. An example of a vector that can be used in yeast is a 2. Mu. Plasmid. Suitable vectors for expression in mammalian cells include the following well known derivatives: SV-40, adenovirus, retrovirus-derived DNA sequences, and shuttle vectors derived from functional mammalian vectors (such as those described above) and combinations of functional plasmid and phage DNA.

Additional eukaryotic expression vectors are known in the art (e.g., P J. Southern & P. Berg, J. Mol. Appl. Genet, 1:327-341 (1982); subramanni et al, mol. Cell. Biol, 1:854-864 (1981); kaufmann & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene); J. Mol. Biol, 159:601-621 (1982); kaufhiann & Sharp, mol. Cell. Biol, 159:601-664 (1982); scahill et al," Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells, "Proc. Nat 'l Acad. Sci USA, 80:4654-4659 (1983); urlaub & Chasin, proc. Nat' l Acad. 4216-4220, 1980), incorporated herein by reference in its entirety.

Expression vectors useful in the present application contain at least one expression control sequence operably linked to a DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate expression of the cloned DNA sequences. Examples of useful expression control sequences are the lac system, trp system, tac system, trc system, the major operator and promoter regions of phage lambda, the control regions of fd coat proteins, the glycolytic promoters of yeast, such as the 3-phosphoglycerate kinase promoter, the promoters of yeast acid phosphatase, such as the Pho5, the promoters of yeast alpha-mating factors, and promoters derived from polyomaviruses, adenoviruses, retroviruses and simian viruses, such as the early and late promoters of SV40 and other sequences known to control gene expression in prokaryotic or eukaryotic cells and viruses thereof or combinations thereof.

In some embodiments, the host cell described herein is a mammalian cell. Mammalian cells may include, but are not limited to, CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, and per.c6 cells. One skilled in the art can select an appropriate host cell as desired.

a) Culturing the host cell of the fourth aspect; and

The methods of preparing anti-trastuzumab monoclonal antibodies disclosed herein can comprise: culturing the host cell under expression conditions to express the anti-trastuzumab monoclonal antibody; isolation and purification of the expressed anti-trastuzumab monoclonal antibody. By using the method, a crude anti-trastuzumab monoclonal antibody can be obtained. The anti-trastuzumab monoclonal antibody is then purified to a substantially homogeneous material, e.g., as a single band on SDS-PAGE electrophoresis, by purification methods including trastuzumab-based affinity purification, non-denaturing gel purification, HPLC or RP-HPLC, size exclusion, purification on a protein a column, or any combination of these techniques.

In some embodiments, the kit further comprises instructions for how to perform the assay.

The antibodies or antigen binding portions thereof described herein can be conjugated to a detectable moiety. Exemplary detectable moieties include, but are not limited to, radioisotopes such as iodine 125, iodine-131, cesium-137, iridium 192 and cobalt 60, horseradish peroxidase, fluorescein isothiocyanate, biotin, alkaline phosphatase, chemiluminescent agents such as luminols, and the like. One skilled in the art can select the appropriate detectable moiety to bind to the antibodies or antigen binding portions thereof of the present application as desired to achieve different detection objectives.

The term "subject" as used herein refers to mammals, including but not limited to primates, cows, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or human. Particularly preferred mammals are humans. As used herein, "patient" and "subject" may be used interchangeably.

In some embodiments of the seventh and eighth aspects, a "biological sample" refers to any sample taken from a subject (e.g., a human (or other animal), such as a human with cancer, or a human suspected of having cancer, and containing trastuzumab, the biological sample may be a bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, fluid from the scrotum (e.g., ascites in the testes), vaginal rinse, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage, drainage fluid from the nipple, aspiration fluid from a different part of the body (e.g., thyroid, breast), intraocular fluid (e.g., aqueous humor), and the like.

In this specification and claims, the words "comprise", "comprising" and "include" mean "including but not limited to", and are not intended to exclude other moieties, additives, components or steps.

It should be understood that features, characteristics, components or steps described in particular aspects, embodiments or examples of the present application may be applied to any other aspects, embodiments or examples described herein unless contradicted by context.

The foregoing disclosure generally describes the present application, examples are further illustrative of the present application and are not to be construed as limiting the present application. Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, see for example Sambrook, j., fritsch, EF. and maniis, t. (1989) Molecular Cloning: a Laboratory Manual, second edition, cold spring Harbor Laboratory Press.

Example 1: preparation of trastuzumab F (ab') 2 fragments

1mg trastuzumab (available from Roche) and 1000U Ide S protease (available from Acro) were added to digestion buffer (10 mM sodium phosphate, 10mM NaCl, pH 6.5), mixed and incubated at 37℃for 60 minutes. And (3) combining the enzyme-digested product with the Fc fragment by using Protein A affinity chromatography, collecting the flow-through, removing Ide S protease by using a nickel column, and collecting the flow-through to obtain the trastuzumab F (ab') 2 fragment. The harvested protein shows a molecular weight of 100kD in a non-reduced state by SDS-PAGE gel electrophoresis, and a molecular weight of 25kD after reduction, and the result is shown in FIG. 1.

Example 2: preparation of antibodies against trastuzumab

2.1 Trastuzumab F (ab') 2 antigen immunized Balb/C mice

Healthy female Balb/C mice of 6-8 weeks of age were selected as subjects for immunization. 400 mg trastuzumab F (ab ') 2 protein was adjusted to 0.5 ml in volume with physiological saline, mixed with 0.5 ml complete Freund's adjuvant (Sigma), placed in a 2 ml pre-chilled syringe, and emulsified with a tissue high speed disperser on ice for 20 min, and the solution turned milky. Taking a proper amount of emulsified liquid drops, placing the emulsified liquid drops in ice water for 10 min, and keeping the liquid drops not to scatter, so that the emulsified liquid drops show that the emulsified liquid drops are completely emulsified, and the protein and the immunoadjuvant form a water-in-oil state; emulsified antigen was injected at 3 points under the skin of Balb/C mice, 100 per point ml. Once every two weeks, a total of 3 subcutaneous immunizations were performed, followed by two subcutaneous immunizations adjuvanted to incomplete Freund's adjuvant (Sigma). The antigen was injected intraperitoneally with 50mg, once daily, 3 times in total.

2.2 Fusion of spleen cells and myeloma cells of immunized mice

After the immunized mice were killed by cervical removal, 75% alcohol was used for soaking and sterilizing, the abdominal cavity was opened, the spleen was removed and placed in a petri dish, 3 ml of prim1640 basal medium was added, the spleen of the mice was placed on a 70mm filter screen, the spleen of the mice was lightly ground with the needle of a 10ml syringe, and a spleen single cell suspension was collected. Spleen cells and mouse myeloma cells SP2/0 were cell fused with PEG 1500 (Roche, cat.10783641001). The fused cells were centrifuged at 300g for 5 minutes, resuspended in 100 ml HAT medium, and cultured in a cell incubator for 10-15 days by adding 10 96-well plates with feeder cells.

2.3 ELISA detection of hybridoma secreted antibodies

Clone growth was seen in the well plate on day 7 after hybridoma fusion, and hybridoma supernatant 50 ml was taken for ELISA detection. ELISA plates were coated with trastuzumab F (ab') 2 at a concentration of 1mg/ml and 50 ml hybridoma supernatant was added thereto. Immune serum was set as a positive control and culture medium blank. The secondary antibody selected HRP-labeled goat anti-mouse IgG (Boobolone Biotechnology Co., ltd., 1:5000). After incubation was completed, the optical density was measured in a microplate reader at a wavelength of 450 nm. ELISA screening was performed again 2 days after HAT medium exchange, and clones positive for both were detected by flow cytometry (FACS).

Example 3: identification of antibodies against trastuzumab

3.1 Flow cytometry detection of hybridoma antibodies and cell binding Activity

Resuscitated and subcultured SKBR3 cells (ATCC number: HTB-30) for at least 3 passages were blown uniformly and the cells were collected, centrifuged at 250 g/min for 5min, counted in resuspension, and cell density was adjusted to 2.5X106 cells/mL. The groups were set as blank, APC-labeled rat anti-human IgG Fc (BioLegend, clone number: M1310G 05) secondary antibody, trastuzumab, immune seropositive control and experimental (trastuzumab+hybridoma supernatant 50. Mu.l). 50 μl of hybridoma supernatant, 10 μl of trastuzumab (final concentration 1 μg/ml), was added to the tube of the experimental group, incubated at room temperature for 10 min after mixing, then 40 μl of SKBR3 cell suspension was added, incubated at room temperature for 30 min, washed with 3 mL PBS buffer containing 2% FBS, centrifuged at 250 g/min for 5min, and resuspended in 50 μl PBS buffer containing 2% FBS; the blank was added with 0.5 μl of water; trastuzumab groups were added with 0.5 μl of APC-labeled rat anti-human IgG Fc (BioLegend, clone number: M1310G 05), and both experimental and positive control groups were added with 0.5 μl of APC-labeled rat anti-human IgG Fc antibody; after mixing, incubate for 30 min at room temperature in the dark. After the incubation was completed, the test tubes of each group were washed with PBS buffer containing 2% FBS, centrifuged at 250. 250 g/min for 5min, resuspended in 100. Mu.l of PBS buffer containing 2% FBS, and detected by an upflow cytometer (Aisen, instrument model: novoCyte). The hybridoma antibodies were competing (antigen-blocking) antibodies, and FACS results showed that the APC pathway was shifted to the left compared to the trastuzumab panel, see specifically fig. 2, from which hybridoma cells secreting antibodies against trastuzumab were selected, clone No. 9G6.

3.2. Preparation of hybridoma antibodies

Hybridoma cell 9G6 clones were cultured in RPIM 1640 medium containing 10% low IgG serum, and the cultured cell cultures were harvested, centrifuged at 3000×g for 20 min, and the supernatants were collected and filtered with 0.45 μm filters. 5 mL Protein A (Thermo Scientific) affinity chromatography column was equilibrated with a mixed buffer (pH 7.4) of 20 mM PB and 150 mM NaCl, flow rate 5 mL/min, and volume greater than 5CV. The filtered sample solution was loaded at a flow rate of 5 mL/min. After loading was completed, the Protein A affinity column was washed with a mixed buffer (pH 7.4) of 20 mM PB and 150 mM NaCl at a flow rate of 5 mL/min. The whole elution peak was collected by eluting with 50 mM citric acid (pH 3.0) buffer at a flow rate of 5 mL/min, while the pH of the collected eluate was adjusted to about 7.0 with 1M Tris HCl (pH 9.0) buffer. The protein concentration was measured by Nanodrop micro-spectrophotometer and the resulting antibody was designated 9G6.

3.3 antibody variable region Gene sequencing

The purified antibody is detected by ELISA kit (Boolon, BF 06001) for identifying Ig class/subclass of the mouse monoclonal antibody, and the subtype of the antibody is determined to be IgG1, and the light chain is determined to be subtype k. The total RNA of hybridoma cell 9G6 was extracted by TRIZOL, and the heavy and light chains of the obtained antibody were reverse transcribed and amplified by the nested PCR method according to the instructions of the 5' -RACE kit (Bio, B605102-0010). The PCR product was about 800bp, and the DNA fragment was cut and recovered, and cloned into pClone007 Vector (Optimum, pClone007 Vector Kit). The resulting vector was transformed into TSC-C01 competent cells (Optimus Praeparata) and 5 clones were selected for sequencing, and the amino acid sequences of the variable regions of the light and heavy chains of the antibodies were shown in SEQ ID NO. 9 and SEQ ID NO. 10.

Example 4: expression and identification of antibodies against trastuzumab

4.1 Expression of antibodies

Synthesizing the heavy chain and light chain nucleic acid sequences (Jiangsu Saikovia Boc, technology Co., ltd.) obtained in the above manner, cloning the heavy chain sequences into a heavy chain vector pQKXM13 (Beijing immune Aristolochia medical technology Co., ltd., number: pQKXM 13) of a eukaryotic expression vector of a mouse IgG1 skeleton by using a homologous recombination technique to obtain a heavy chain expression vector pQK G6-H; the light chain sequence was cloned into the eukaryotic expression vector kappa light chain vector pQKXM16 (Beijing immune ark medical science and technology Co., ltd.; no.: pQKXM 16) of the mouse IgG1 framework to obtain the light chain expression vector pQK G6-L. Heavy chain expressionVector pQK G6-H and light chain expression vector pQK G6-L were co-transfected into HEK293 cells (ATCC, accession No. CRL-1573) for expression. HEK293 cells were cultured in OPM-293 CD05 serum-free medium (o Pu Mai, cat: 81075-001), 36.5℃and 7.5% CO ₂ Suspension culture at 120 rpm. At the time of transfection, the recombinant plasmids pQK G6-H and pQK G6-L were mixed in a weight ratio of 1:1 (total DNA 100 mg) in 10mL OPM-293 CD05 medium, followed by addition of 100 mL PEI (3 mg/mL concentration), vortexing rapidly, and stationary incubation at room temperature for 15 min. The mixture is then added to the cell culture described above. Cells at 36.5℃and 7.5% CO ₂ The expressed antibodies were harvested by continued culture at 120 rpm/min for 7 days.

4.2 Purification of antibodies

The harvested cell cultures were centrifuged at 3000 Xg for 20 min, and the supernatants were collected and filtered with 0.45 μm filters. The whole elution peak was collected by eluting with 5 mL Protein A affinity chromatography (GE) antibody, 50 mM citric acid (pH 3.0) buffer at a flow rate of 5 mL/min, and the pH of the collected eluate was adjusted to about 7.0 with 1M Tris HCl (pH 9.0) buffer. The obtained proteins were detected by SDS-PAGE and Coomassie blue staining (FIG. 3A).

4.3 Identification of antibodies

4.3.1 Fortebio assay of antibody affinity

The affinity constant KD of the purified antibody was measured by using a molecular interaction device Fortebio Octet QK (Molecular Devices). Murine antibody was immobilized by a sensor (sensor) of Protein a at a concentration of 0.25 μm. Trastuzumab F (ab') 2 fragments were loaded at concentrations of 600 nM,300 nM,150 nM and 75 nM. The resulting antibody was hardly dissociated from trastuzumab (fig. 3B). The KD value of the measurement result of the affinity constant is smaller than <1.0E-12.

4.3.2 ELISA detection of binding of anti-trastuzumab antibodies to trastuzumab

ELISA plates were coated with trastuzumab F (ab') 2 at a protein concentration of 1G/ml, antibody 9G6 at an initial concentration of 40. Mu.g/ml, diluted in a double ratio, added to ELISA plates at 100. Mu.l per well, and PBS solution was used as a blank. The secondary antibody selected HRP-labeled goat anti-mouse IgG (Boobolone Biotechnology Co., ltd., 1:5000) was measured for optical density in an enzyme-labeled instrument at a wavelength of 450 nm, and the result is shown in FIG. 4, which shows that the GraphPad Prims software calculated its EC50 to be 0.0613. Mu.g/ml.

4.3.3 Flow cytometry to detect binding of anti-trastuzumab antibodies to trastuzumab

The SKBR3 cells after pancreatin digestion were blown up uniformly and the cells were collected, centrifuged at 250 g/min for 5min, counted in resuspended, and the cell density was adjusted to 2.0X106 cells/mL. The groups were set as blank, secondary, trastuzumab and experimental (trastuzumab+antibody 9G 6). Trastuzumab 10 μg/ml was added to the tubes of the experimental group, 9G6 antibody concentration was diluted sequentially in multiple ratios from 100 μg/ml for 9 concentrations, mixed well and incubated at room temperature for 10 min, then 80 μl of SKBR3 cell suspension was added, incubated at room temperature for 30 min, washed with 3 mL PBS buffer containing 2% FBS, centrifuged at 250G/min for 5min, resuspended in 50 μl PBS buffer containing 2% FBS; the trastuzumab and experimental groups were added with 0.5 μl of APC-labeled rat anti-human IgG Fc (BioLegend, clone No. M1310G 05), mixed well and incubated at room temperature in the dark for 30 min. After completion of incubation, the cells were washed with PBS buffer containing 2% FBS, resuspended in 100. Mu.l of PBS buffer containing 2% FBS, and detected by an upflow cytometer (Aisen, apparatus model: novoCyte), and the results are shown in FIG. 5. It can be seen that the fluorescence intensity of trastuzumab gradually decreases with increasing concentration of antibody 9G6.

While the foregoing has been with a general description and specific embodiments, it will be apparent to those skilled in the art that various modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the application and are intended to be within the scope of the invention as claimed.

Sequence listing

SEQ ID NO:1 (amino acid sequence of HCDR1 of 9G6 antibody according to Kabat numbering system)

DYYIY

SEQ ID NO:2 (amino acid sequence of HCDR2 of 9G6 antibody according to Kabat numbering system)

RINPYNGATNYNQNFKD

SEQ ID NO:3 (amino acid sequence of HCDR3 of 9G6 antibody according to Kabat numbering system)

GGTGAWFAY

SEQ ID NO:4 (amino acid sequence of LCDR1 of 9G6 antibody according to Kabat numbering system)

SASSSVSYMH

SEQ ID NO:5 (amino acid sequence of LCDR2 of 9G6 antibody according to Kabat numbering System)

DTSKLAS

SEQ ID NO:6 (amino acid sequence of LCDR3 of 9G6 antibody according to Kabat numbering system)

QQWSSNPPT

SEQ ID NO:7 (amino acid sequence of VH of 9G6 antibody)

EVQLLESGPEL VKPGASVKIS CKASTYSFTD YYIYWVKQSH VKSLEWIGRI NPYNGATNYN QNFKDKAYLT VDKASSTAYM VLHSLTSEDS AVYYCSRGGT GAWFAYWGQG TLVTVSA

SEQ ID NO:8 (amino acid sequence of VL of 9G6 antibody)

QIVLTQS PAIMSASPGE KVTMTCSASS SVSYMHWYQQ KSGTSPKRWI YDTSKLASGV PARFSGSGSG TSYSLTISSM EAEDAATYYC QQWSSNPPTF GGGTKLEIK

SEQ ID NO:9 (9G 6 antibody according to the amino acid sequence of HCDR1 of IMGT numbering system)

TYSFTDYY

SEQ ID NO:10 (9G 6 antibody according to the amino acid sequence of HCDR2 of the IMGT numbering system)

INPYNGAT

SEQ ID NO:11 (9G 6 antibody according to the amino acid sequence of HCDR3 of the IMGT numbering system)

SRGGTGAWFAY

SEQ ID NO:12 (9G 6 antibody according to the amino acid sequence of LCDR1 of the IMGT numbering System)

SSVSY

SEQ ID NO:13 (9G 6 antibody according to the amino acid sequence of LCDR3 of the IMGT numbering system)

QQWSSNPPT

SEQ ID NO:14 (9G 6 antibody according to the amino acid sequence of LCDR2 of the IMGT numbering System)

DT。

Claims

1. An antibody or antigen binding portion thereof that specifically binds trastuzumab comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID No. 1, the amino acid sequence of HCDR2 is shown in SEQ ID No. 2, the amino acid sequence of HCDR3 is shown in SEQ ID No. 3, the amino acid sequence of LCDR1 is shown in SEQ ID No. 4, the amino acid sequence of LCDR2 is shown in SEQ ID No. 5 and the amino acid sequence of LCDR3 is shown in SEQ ID No. 6 according to the Kabat numbering system; or according to an IMGT numbering system, the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 10, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 11, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 12, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 14, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 13.

2. The antibody or antigen-binding portion thereof of claim 1, wherein the antigen-binding portion is selected from the group consisting of: fab fragment, fab 'fragment, F (ab') ₂ Fragments, fv fragments, scFv fragments or Fd fragments.

3. The antibody or antigen-binding portion thereof according to claim 1 or 2, wherein the antibody is a murine antibody and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 7 and/or the amino acid sequence of the light chain variable region is shown in SEQ ID No. 8.

4. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-3.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. A method of making the antibody or antigen-binding portion thereof of any one of claims 1-3, comprising:

a) Culturing the host cell of claim 6; and

8. A detection reagent or kit comprising the antibody or antigen-binding portion thereof of any one of claims 1-3.

9. Use of the antibody or antigen-binding portion thereof of any one of claims 1-3 for detecting trastuzumab in a biological sample of a subject.

10. Use of the antibody or antigen binding portion thereof of any one of claims 1-3 for determining the content of trastuzumab in a biological sample of a patient administered with trastuzumab.