阅读说明：本技术 一种Her2伴随诊断免疫组化检测抗体及其应用 (Her2 concomitant diagnosis immunohistochemical detection antibody and application thereof ) 是由 于占娇 李元浩 黄长江 梁旺 毛海燕 房健民 于 2020-02-25 设计创作，主要内容包括：本发明提供了一种人表皮生长因子受体2(Her2)伴随诊断免疫组化检测(IHC)用抗体,该抗体作为一抗对样品的Her2表达情况进行免疫组化检测时,能够避免因胞外区缺失导致的检测结果的假阳性。并且,所述抗体在患者免疫组化检测样本不经抗原(或表位)修复处理的情况下,仍然能够识别并结合检测样本中的相应表位,从而降低了IHC检测中因抗原(或表位)修复处理方法的差异所产生的假阴性问题。(The invention provides an antibody for human epidermal growth factor receptor 2(Her2) concomitant diagnostic immunohistochemical detection (IHC), which is used as a primary antibody to avoid false positive of a detection result caused by extracellular region deletion when the antibody is used for performing immunohistochemical detection on the Her2 expression condition of a sample. Moreover, the antibody can still recognize and combine with the corresponding epitope in the detection sample under the condition that the patient immunohistochemical detection sample is not subjected to antigen (or epitope) repair treatment, so that the false negative problem caused by the difference of the antigen (or epitope) repair treatment method in IHC detection is reduced.)

1. An antibody for human epidermal growth factor receptor 2(Her2) companion diagnostic immunohistochemical assay (IHC), wherein the antibody specifically binds to the extracellular domain IV region of Her2 protein, and the antibody has the property of effectively detecting IHC samples without antigen or epitope retrieval treatment.

2. The antibody of claim 1, wherein the antibody Fc-fragment is a non-human mammalian antibody Fc-fragment, preferably a murine Fc-fragment or a rabbit Fc-fragment.

3. The antibody of claim 1 or 2, wherein the antibody competes for binding to the same or similar epitope with an antibody defined by the following CDRs of which CDR1-3 of the heavy chain variable region are set forth in SEQ ID NOs: 1-3; the CDR1-3 of the light chain variable region of the antibody defined by the CDR are respectively shown in SEQ ID NO: 4-6.

4. The antibody of any one of claims 1-3, wherein in said antibody:

(i) CDR1 of the heavy chain variable region has the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 amino acid sequence after 1 or 2 amino acid substitutions; or/and CDR2 has the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 by 1, 2, 3, 4 or 5 amino acid substitutions; or/and CDR3 has the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3 amino acid sequence after 1, 2 or 3 amino acid substitutions; and/or

(ii) CDR1 of the light chain variable region has the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4 amino acid sequence after 1, 2, 3 or 4 amino acid substitutions; or/and CDR2 has the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5 amino acid sequence after 1 or 2 amino acid substitutions; or/and CDR3 has the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6 amino acid sequence after 1 or 2 amino acid substitutions;

and/or

(iii) The immunoglobulin Fc fragment is a murine IgGFc fragment, or further a murine IgG1Fc fragment.

5. The antibody of claim 4, wherein in said antibody:

(i) CDR1-3 of the heavy chain variable region is SEQ ID NO: 1-3; and

(ii) CDR1-3 of the light chain variable region is SEQ ID NO: 4-6.

6. The antibody according to any one of claims 1 to 5,

(i) the heavy chain of the antibody comprises SEQ ID NO:7 or a sequence having at least 80% sequence identity thereto; and/or

(ii) The light chain of the antibody comprises SEQ id no:8 or a sequence having at least 80% sequence identity thereto; and is

(iii) CDR1-3 of the heavy chain variable region of the antibody is respectively shown in SEQ ID NO: 1-3; CDR1-3 of the variable region of the light chain of the antibody is respectively shown in SEQ ID NO: 4-6.

7. The antibody of claim 6,

(i) the heavy chain of the antibody is shown as SEQ ID NO: 7; and

(ii) the light chain of the antibody is shown as SEQ ID NO: 8.

8. A nucleic acid molecule encoding the antibody of any one of claims 1 to 7.

9. A vector comprising the nucleic acid molecule of claim 7.

10. Use of the antibody of any one of claims 1-7 in the manufacture of Her2 in a companion diagnostic immunohistochemical test product that functions without an antigen or epitope retrieval step during immunohistochemical testing; preferably, the Her2 companion diagnostic immunohistochemical detection product is a Her2 companion diagnostic immunohistochemical detection product matched before RC48-ADC targeting drug treatment; more preferably, the Her2 companion diagnostic immunohistochemical test product is directed to Her2 related cancer, more preferably breast cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, ovarian cancer, endometrial cancer, lung cancer, urothelial cancer or bladder cancer.

11. A Her2 companion diagnostic immunohistochemical test kit comprising the antibody of any one of claims 1-7, the kit functioning without an antigen or epitope retrieval step during the immunohistochemical test; preferably, the Her2 companion diagnostic immunohistochemical detection kit is a Her2 companion diagnostic immunohistochemical detection kit matched before RC48-ADC targeting drug treatment; more preferably, the Her2 companion diagnostic immunohistochemical detection kit is directed to Her2 related cancer, more preferably breast cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, ovarian cancer, endometrial cancer, lung cancer, urothelial cancer or bladder cancer.

Technical Field

The invention relates to the field of biological medicine companion diagnosis, in particular to a detection antibody for companion diagnosis of a targeted Her2(human epidermal growth factor receptor 2) medicine, and a detection application and a detection method thereof.

Background

Cancer is today the major disease endangering human health. In china, the incidence of cancer is in a rapid rise. There is increasing evidence that cancer is a complex and diverse disease, and patients may exhibit similar symptoms and have the same pathological changes, but may be caused by completely different genetic changes. Because of this heterogeneity, the response rates of pathologically homogeneous cancer patients to currently available drugs vary widely. Often only a fraction of patients with a particular tumor respond to a particular treatment, and many patients often suffer from unnecessary and/or damaging (side effects) treatments because the susceptibility and resistance of different tumor individuals to the drug cannot be judged prior to treatment. The theoretical and practical basis for supporting individualized or precise medical care is the difference in molecular genetics between individuals, which is considered to be a determinant of human disease susceptibility and drug response. The early examples fully demonstrate the relationship between genetics and precise treatment of tumors: when targeted anticancer drugs gefitinib (Iressa) and erlotinib (Tarceva) are initially on the market, the side effects of drugs applied to lung cancer patients are greatly improved compared with chemotherapy, but the drug effect is not very obvious, and the drugs are only used as second-line drugs for treating lung cancer. However, it was discovered that gefitinib and erlotinib are excellent in therapeutic effect in lung cancer patients carrying EGFR specific gene mutation, and have become the first-line standard therapeutic drugs for clinical treatment of such patients. This demonstrates the relevance of tumor gene mutation to drug and therapeutic response, and the decision of the patient's targeted treatment regimen based on the information and combination of gene mutations is the major direction of precision therapy (document 1: precision therapy of tumors: concept, technology and prospect, Hangzhou Bohai, etc., science and technology guide, Vol 33, No 15, pp 14-21, 2015).

The companion diagnosis (CDx) is an in vitro diagnosis technology related to targeted drugs, and mainly obtains the therapeutic response of different patients to specific drugs by measuring the expression levels of proteins and variant genes in human bodies, screens the most appropriate drug-taking population and performs personalized treatment in a targeted manner, thereby improving the treatment prognosis and reducing the health care expenditure. The FDA issued concomitant diagnostic guidelines on 6/8 of 2014 in the united states. Companion diagnostics help to identify the patient population most likely to respond to a therapeutic drug, facilitate the use of the drug in a relatively limited market, and improve the effectiveness and safety of the drug. CDx facilitates the design of small sample clinical trial protocols in drug development, with less investment in the development process to achieve clearer and more definite results. The advantages of the concomitant diagnosis are that an effective treatment scheme can be screened for the patient, the time and cost of ineffective treatment are saved, the medication compliance of the patient is improved, the incidence rate of adverse reactions is reduced, and the safety and the curative effect of the medicine are ensured (reference 2: concomitant diagnosis-individualized treatment booster, Toxon road penetration, pharmaceutical progress, volume 39, phase 6, page 463-477, 2015).

Her2, also known as ErbB2, is a second member of the EGFR family that causes activation of the EGFR signaling pathway by heterodimerization with the other three members of the EGFR family, which is often associated with abnormal cell proliferation and tumor formation, and thus Her2 becomes one of the Therapeutic targets for a variety of Cancers (such as breast, gastric, gastroesophageal, esophageal, ovarian, endometrial, lung, urothelial, bladder, etc.; see document 16: Human epilame Growth factor receptor 2(HER2) in Cancers: Overexpression and Therapeutic implants, Nidaiqbal and naved Iqbal, Molecular Biology International, Volume 2014, Article ID852748), and there are currently a variety of Therapeutic drugs including trastuzumab and trastuzumab 2 as targets or in clinical stage. The increased Her-2 activity is generally thought to be associated with Her2 gene amplification, up-regulation of Her2 protein expression, and mutation of Her-2 protein. Among these, the HER2 gene is most commonly amplified and thus causes the HER-2 protein expression to be upregulated. Among 34 accompanying diagnostic reagents approved by the FDA in the United states (by 2019, 2 months later) (reference 3: https:// www.fda.gov/medical devices/products and medical procedures/InVitroDiacoustics/ucm301431. htm). It was shown that since 1998 there are 9 approved companion diagnostic kit products related to targeted Her2 drugs (document 4: Her2 testing: Current status and future directives, edition a. perez et al, cancer treatment Reviews, vol. 40, p. 276-. This suggests a continuing market demand for concomitant diagnostic agents for this targeted drug.

TABLE 1 FDA approved companion diagnostic kit before HER-2 targeted therapy (ref. 4)

Note: CISH, chromogenic in situ hybridization;

FISH, fluorescence in situ hybridization;

HER2, human epidermal growth factor receptor 2;

IHC, immunohistochemistry.

Currently, there are four diagnostic kits for use with U.S. FDA-approved targeted Her2 drugs based on immunohistochemical detection (2 semi-quantitative IHCs, 2 IHCs) and five based on in situ hybridization (two FISH, three CISH). Immunohistochemistry (IHC) detects Her2 protein expression on cell membranes, and In Situ Hybridization (ISH) detects Her2 gene amplification. The principles and characteristics of the three methods for detection are shown in table 2.

TABLE 2 IHC, CISH and FISH detection principles and characteristics

Although the results of CISH and FISH detection are more accurate than the results of IHC detection, the IHC method is economical (approximate price Comparison using IHC and FISH is given in a statement of evaluation made in 2006 according to McGill University Health Centre (MUHC), the price of IHC is about $ 85 for a single test and the price of FISH is about $ 381 for a single test using IHC and FISH (document 5: https:// mucc. ca/sites/default/files/micro/m-TAU/HER2reportdredgedraft4 fine May2006.pdf, page 15), Seema Jaar et al (document 6: composition of two-microwave 2 microbial Assays for Imeast investment of Imeast city: pTceest herthrest 2 (FDA) and the price of FISH is about $ 32 for a single test and the price of FISH for a single test of China-60 (FISH-5072 for a total cost of FISH) is about $ 3 for a single test and the price of FISH (document 6: US 120: US) and the cost of FISH general test for a total cost of FISH 2 for a single test of China hospital and a general test of FISH 32, respectively (document 3: US) is about $ 3, the expense of the price for a total cost of a total test of China hospital and FISH 2 for a total test of the IHC method of the IHC, the IHC method of The detection cost is about 1200 yuan for FISH and about 150 yuan for IHC, the FISH cost reaches 8 times of the IHC detection cost), the method is quick, the film reading has morphological basis, and the method is easy to operate on a large number of samples, has higher sensitivity and relatively high repeatability between the interior of a laboratory and the laboratory, so the IHC method as a primary screening tool is superior to the CISH method. Pathologists often use the IHC method as the first test method for condition assessment. However, based on the results of false positive in cases judged to be positive by IHC method 2+, the Her2 detection protocol for breast cancer recommended by ASCO/CAP (American society of clinical oncology/College of American Patholoists) is as follows: first, the detection is usually performed by the IHC method, the FISH detection is positive in the case of 3+ result, and the FISH detection is negative in the case of 1+ or less IHC result. Cases with IHC detection result of 2+ require to be confirmed again by FISH method (see specifically FIG. 1, reference 7: Human Epidermal Growth factory receptor 2Testing in Breast Cancer, American Society of Clinical sciences Clinical guidelines for Clinical findings in Clinical application, Antonio C. wolff, etc., Arch Pathol Lab Med, Vol.142, p.1364 + 1382, 11 months in 2018).

Disclosure of Invention

In order to solve the problems, the invention provides a detection antibody targeting the extracellular region of Her2, and the detection antibody specifically binds to the extracellular domain IV region of Her2 protein.

An exemplary amino acid sequence for Her2 is as NCBI GenBank ID: AAA75493.1 shows that the corresponding protein extracellular domain IV region is located at amino acid 511-643. Johan Rockberg et al (document 14: Discovery of epitopes for targeting the human epidermal growth factor receptor 2(HER2) with antibiotics, Johan Rockberg et al, MOLECULAR ONCOLOGY, Vol.3, p.238-247, 2009) and "HER 2/ERBB2 Protein" marketed by Beijing Yinqi Kangshen science and technology Co., Ltd. (Sino Biological), the Human body is a Human body, recombinant (ECD, domain IV, His Tag) "(polyhistidine-tagged Human ERBB2(AAA75493.1) (Pro489-Cys630) region) (Cat:10004-H08H4, https:// www.sinobiological.com/Human-HER2-ErbB2-Protein-ECD-domain-I V-His-Tag-p223119.html) (characteristically, its binding ability was examined by functional ELISA; and EC50 values for binding to herceptin, herceptin were 10-40ng/mL) demonstrated that the extracellular domain IV region of the HER2 Protein could serve as an independent immunogen and still retain the stereo configuration of the corresponding region when in intact HER 2.

Antibodies targeting the Her2 protein extracellular domain IV with the following CDR combinations are disclosed in the prior art: (i) the heavy chain variable region CDR1-3 is: GFNIKDTYIH, RIYPTNGYTRYADSVKG, WGGDGFYAMDV, respectively; and light chain variable region CDR1-3 is: RASQDVNTAVAW, SASFLES, QQHYTTPPT (WO9222653A 1); (ii) the heavy chain variable region CDR1-3 is: GFNIKDTYIH, RIYPTNGYTRYADSVKG, WGGDGFYAMDY, respectively; the light chain variable region CDR1-3 is: RASQDVNTAVA, SASFLES, QQHYTTPPT, respectively; (document 15: Molecular dynamics of Trastuzumab F (ab') 2structure in registration with HER2 as a thermal agent of Breast cancer, S Hermanto et al, Journal of Physics: Conference Series, volume 835, No. 1, pages 1-11, 2017). Such antibody uses are all therapeutic antibodies.

The present invention surprisingly found that antibodies specifically binding to the IV region of the extracellular domain of Her2 protein can be used as primary antibodies (primary antibodies) in immunohistochemical detection, and can be specifically and effectively recognized and detected by the antibodies in the patient immunohistochemical detection sample without antigen (or epitope) repair treatment. Therefore, the false negative problem caused by the difference of the antigen (or epitope) repairing treatment method in IHC detection is effectively reduced. Meanwhile, the repairing step is not needed, so that the working intensity can be effectively reduced, the detection time is shortened, and the IHC detection efficiency is improved.

Furthermore, it was also surprisingly found that antibodies (amino acid sequences of heavy chain CDR1-3 region are DYYIH, RVNPDHGDSYYNQKFKD, ARNYLLFW, and amino acid sequences of () light chain CDR1-3 region are KASQDVGTAVA, WASIRHT, HQFATYT) binding to the same epitope (located in the extracellular domain IV region of Her2 protein) or antibodies with the same light chain and heavy chain CDR as above, in the drug conjugate (RC48) of the Her2 targeting antibody (RC 48-ADC) disclosed in WO2015074528A1 and CN105008398A, can be used as concomitant diagnosis IHC antibodies for determining Her2 positivity, and since the epitope is located in the extracellular domain IV region of Her2 protein, the detection of corresponding IHC antibody avoids the possibility that a mutant form in which the extracellular region of Her2 is deleted can also be detected positively, thus effectively solving the problem of false intracellular region detection of primary antibody (mary antibody) against Her2 positive. Meanwhile, as the CDR region of the antibody in the targeted drug is the same, the epitope combined in the detection process is completely the same as the targeted epitope of the therapeutic drug, the high correspondence of the detection antibody and the medicinal antibody combined epitope highly ensures the pertinence and the effectiveness of the targeted drug in use.

The technical scheme of the invention is as follows:

the invention provides an antibody for a human epidermal growth factor receptor 2(Her2) companion diagnostic immunohistochemical detection (IHC), wherein the antibody specifically binds to an extracellular domain IV region of a Her2 protein, and the antibody has the performance of effectively detecting an IHC sample which is not subjected to antigen or epitope repair treatment.

Further, the antibody Fc segment is a non-human mammal antibody Fc segment, preferably a murine Fc segment or a rabbit Fc segment.

Further, the antibody competes for binding to the same or similar epitope with an antibody defined by the following CDRs of which CDRs 1-3 of the heavy chain variable region are set forth in SEQ ID NOs: 1-3; CDR1-3 of the light chain variable region of the antibody defined by the CDR are respectively shown in SEQ ID NO: 4-6.

Further, in the antibody:

(i) CDR1 of the heavy chain variable region has the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 amino acid sequence after 1 or 2 amino acid substitutions; or/and CDR2 has the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 by 1, 2, 3, 4 or 5 amino acid substitutions; or/and CDR3 has the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3 amino acid sequence after 1, 2 or 3 amino acid substitutions; and/or

(ii) CDR1 of the light chain variable region has the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence shown in SEQ ID NO: 4 amino acid sequence after 1, 2, 3 or 4 amino acid substitutions; or/and CDR2 has the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5 amino acid sequence after 1 or 2 amino acid substitutions; or/and CDR3 has the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6 amino acid sequence after 1 or 2 amino acid substitutions;

and/or

(iii) The immunoglobulin Fc fragment is a murine IgG Fc fragment, or further a murine IgG1Fc fragment.

Further, in the antibody:

(i) CDR1-3 of the heavy chain variable region is SEQ ID NO: 1-3; and

(ii) CDR1-3 of the light chain variable region is SEQ ID NO: 4-6.

Further, the antibody:

(i) the heavy chain of the antibody comprises SEQ ID NO:7 or a sequence having at least 80% sequence identity thereto; and/or

(ii) The light chain of the antibody comprises SEQ ID NO:8 or a sequence having at least 80% sequence identity thereto; and is

(iii) CDR1-3 of the heavy chain variable region of the antibody is respectively shown in SEQ ID NO: 1-3; CDR1-3 of the variable region of the light chain of the antibody is respectively shown in SEQ ID NO: 4-6.

Further, there is provided an antibody R48M having the following amino acid sequence information:

1) heavy chain (Hc)

EVQLVQSGAEVKKPGATVKISCKVSGYTFTDYYIHWVQQAPGKGLEWMGRVNPDHGDSYYNQKFKDKATITADKSTDTAYMELSSLRSEDTAVYFCARNYLFDHWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGI(SEQ ID NO：7)

2) Light chain (Lc)

DIQMTQSPSSVSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASIRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQFATYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO：8)

The invention further provides a nucleic acid molecule encoding the antibody described above.

The invention further provides a vector comprising the nucleic acid molecule described above.

The invention further provides application of the antibody in preparing a Her2 companion diagnostic immunohistochemical detection product, wherein the immunohistochemical detection product has a function of avoiding an antigen or epitope repairing step in an immunohistochemical detection process.

The invention further provides a Her2 companion diagnostic immunohistochemical detection kit, which comprises the antibody, and the kit has the function of not needing an antigen or epitope repair step in the immunohistochemical detection process.

The invention further provides a Her2 companion diagnostic immunohistochemical detection method using the above antibody as a detection primary antibody. Or further no antigen or epitope repair step during said immunohistochemical detection.

The indication of the Her2 concomitant diagnosis is Her2 related cancer, and more preferably breast cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, ovarian cancer, endometrial cancer, lung cancer, urothelial cancer or bladder cancer. (see document 16)

The technical scheme of the invention has the following beneficial effects: the invention provides an antibody capable of targeting an extracellular domain IV region of a Her2 protein, and when the antibody is used as a primary antibody for carrying out immunohistochemical detection on the Her2 expression condition of a sample, false positive of a detection result caused by extracellular region deletion can be effectively avoided. In addition, in the immunohistochemical detection process of the Her2 detection antibody provided by the invention, the repair treatment step is not required, so that the false negative problem caused by the difference of the antigen (or epitope) repair treatment method in IHC detection is effectively reduced. Meanwhile, the working strength can be effectively reduced, and the detection time is shortened, so that the IHC detection efficiency is improved.

Drawings

FIG. 1 Her 2test guidelines using a validated immunohistochemical method;

FIG. 2A shows the immunohistochemical staining results of the case number 1 in Table 7, the primary antibody being RC48M antibody;

FIG. 2B shows the immunohistochemical staining results of the primary antibody 4B5 for case number 1 in Table 7;

FIG. 3A shows the immunohistochemical staining results of the case number 2 in Table 7, the primary antibody being RC48M antibody;

FIG. 3B shows immunohistochemical staining results for case number 2 in Table 7, primary antibody 4B 5;

FIG. 4A shows the immunohistochemical staining results of the case No. 3 in Table 7, the primary antibody being RC48M antibody;

FIG. 4B shows immunohistochemical staining results for case number 3 in Table 7, primary antibody 4B5 antibody;

FIG. 5A shows the immunohistochemical staining results of the case No. 4 in Table 7, the primary antibody being RC48M antibody;

FIG. 5B shows immunohistochemical staining results for case number 4 in Table 7, primary antibody 4B 5;

FIG. 6A shows the immunohistochemical staining results of the case No. 5 in Table 7, the primary antibody being RC48M antibody;

FIG. 6B shows immunohistochemical staining results for case number 5 in Table 7, primary antibody 4B 5;

FIG. 7A shows the immunohistochemical staining results of the case No. 6 in Table 7, the primary antibody being RC48M antibody;

FIG. 7B shows immunohistochemical staining results for case number 6 in Table 7, primary antibody 4B 5;

FIG. 8A shows the immunohistochemical staining results of the case No. 7 in Table 7, the primary antibody being RC48M antibody;

FIG. 8B shows immunohistochemical staining results for case number 7 in Table 7, primary antibody 4B 5;

FIG. 9 shows the structural formula of an RC48-ADC drug, wherein RC48 represents RC48 antibody.

Detailed Description

Definition of

Unless otherwise defined, all terms used herein have the same meaning as understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in molecular biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

As used herein, "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. In particular, an "antibody" as used herein refers to a protein comprising at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (the region of the heavy chain near one fifth or one fourth of the N terminus) and a heavy chain constant region (the region of the heavy chain near three quarters or four fifths of the C terminus). Each light chain comprises a light chain variable region (the region of the light chain near one-half of the N-terminus) and a light chain constant region (the region of the light chain near one-half of the C-terminus). The heavy and light chain variable region regions can also be subdivided into regions of high variability, referred to as Complementarity Determining Regions (CDRs). The "CDR" refers to the hypervariable region of the heavy and light chains of an immunoglobulin, and is determined by the Kabat, Chothia or IMGT systems, etc. There are three heavy chain CDRs and three light chain CDRs per antibody. The term CDR is used herein to indicate one of these regions, or several or even all of these regions, as the case may be, which comprise the majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope.

The term "Her 2 detection antibody" used herein refers to an antibody capable of detecting the expression state of Her2 in a case, which is capable of binding to Her2, and the Her2 detection antibody is used as a primary antibody in immunohistochemical chemistry to detect the expression state of Her 2. The invention relates to a Her2 detection antibody which can target the extracellular domain IV region of a Her2 protein.

The term "RC 48M antibody" as used herein refers to an antibody having the amino acid sequence shown in SEQ ID NO. 7 for the heavy chain variable region and SEQ ID NO.8 for the light chain variable region.

The term "RC 48-ADC" or "RC 48-ADC drug" or "RC 48-ADC targeting drug" as used herein refers to an antibody drug conjugate (i.e. antibody drug conjugate, ADC) comprising a moiety capable of specifically binding to Her2 as disclosed in patents WO2015074528a1 and CN105008398A, wherein said antibody is referred to as "RC 48 antibody", wherein (i) the amino acid sequences of the CDR1-3 regions of the heavy chain are DYYIH, rvnpdhgdsyqkfkd, arny lfdhw, respectively; and (ii) the light chain CDR1-3 have amino acid sequences of KASQDVGTAVA, WASIRHT, HQFATYT, respectively; or further the antibody is derived from the antibody secreted by the hybridoma preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.8102 at 22/08.2013. Or further the antibody is derived from an antibody secreted by CHO cells deposited at the China center for type culture Collection with the collection number CCTCCC2013170 on day 06 of 11 months 2013. The structure of one RC48-ADC drug is shown in FIG. 9.