ANTIBODY FOR HER2 CONCOMITANT DIAGNOSIS IMMUNOHISTOCHEMICAL DETECTION AND APPLICATION THEREOF

Abstract

The invention provides an antibody for human epidermal growth factor receptor 2 (Her2) concomitant diagnostic immunohistochemical detection (IHC). When the antibody is used as a primary antibody for immunohistochemical detection of Her2 expression, false positives in detection results caused by the absence of an extracellular region can be avoided. Moreover, the antibody can still recognize and bind to the corresponding epitope in the detection sample when the patient's immunohistochemical detection sample is not repaired by an antigen (or epitope), thereby reducing the issue of a false negative caused by the difference in repairing methods of the antigen (or epitope) in an IHC test.

Claims

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

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

3. The antibody according to claim 1, wherein the antibody competitively binds to the same or a similar epitope with a CDR-defined antibody, and the CDRs 1-3 of a heavy chain variable region of the CDR-defined antibody have the amino acid sequences shown in SEQ ID NOs: 1-3, respectively; and the CDRs 1-3 of a light chain variable region of the CDR-defined antibody have the amino acid sequences shown in SEQ ID NOs: 4-6, respectively.

4. The antibody according to claim 1, wherein, in the antibody: (i) the CDR 1 of the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence obtained after 1 or 2 amino acids substitution on SEQ ID NO: 1; or/and the CDR 2 of the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence obtained after 1, 2, 3, 4 or 5 amino acids substitution on SEQ ID NO: 2; or the CDR 3 of the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence obtained after 1, 2 or 3 amino acids substitution on SEQ ID NO: 3; and (ii) the CDR 1 of the light chain variable region has an amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence obtained after 1, 2, 3, or 4 amino acids substitution on SEQ ID NO: 4; or the CDR 2 of the light chain variable region has an amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence obtained after 1 or 2 amino acids substitution on SEQ ID NO: 5; or/and the CDR 3 of the light chain variable region has an amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence obtained after 1 or 2 amino acids substitution on SEQ ID NO: 6; and (iii) the immunoglobulin Fc fragment is a murine-derived IgG Fc fragment, or further is a murine-derived IgG1 Fc fragment.

5. The antibody according to claim 4, wherein, in the antibody: (i) the CDRs 1-3 of the heavy chain variable region have an amino acid sequences shown in SEQ ID NOs: 1-3, respectively; and (ii) the CDRs 1-3 of the light chain variable region have an amino acid sequences shown in SEQ ID NOs: 4-6, respectively.

6. The antibody according to claim 1, wherein: (i) the heavy chain of the antibody comprises an amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% sequence identity thereto; and (ii) the light chain of the antibody comprises an amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% sequence identity thereto; and (iii) the CDRs 1-3 of the heavy chain variable region of the antibody have an amino acid sequences shown in SEQ ID NOs: 1-3, respectively; the CDRs 1-3 of the light chain variable region of the antibody have an amino acid sequences shown in SEQ ID NOs: 4-6, respectively.

7. The antibody according to claim 6, wherein (i) the heavy chain of the antibody has an amino acid sequence shown in SEQ ID NO: 7; and (ii) the light chain of the antibody has an amino acid sequence shown in SEQ ID NO:8.

8. A nucleic acid molecule encoding the antibody according to claim 1.

9. A vector comprising the nucleic acid molecule according to claim 7.

10. A method for immunohistochemical detection, comprising detecting binding of the antibody according to claim 1 to the extracellular domain IV region of Her2 protein, wherein the method does not require a step of antigen or epitope retrieval during the immunohistochemical detection.

11. A Her2 companion diagnostic immunohistochemical detection kit comprising the antibody according to claim 1, wherein the kit has the function of not requiring a step of the antigen or epitope retrieval during the immunohistochemical detection.

12. The antibody according to claim 1, wherein, in the antibody, the CDR 1 of the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence obtained after 1 or 2 amino acids substitution on SEQ ID NO: 1; or/and the CDR 2 of the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence obtained after 1, 2, 3, 4 or 5 amino acids substitution on SEQ ID NO: 2; or/and the CDR 3 of the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence obtained after 1, 2 or 3 amino acids substitution on SEQ ID NO: 3.

13. The antibody according to claim 1, wherein, in the antibody, the CDR 1 of the light chain variable region has an amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence obtained after 1, 2, 3, or 4 amino acids substitution on SEQ ID NO: 4; or/and the CDR 2 of the light chain variable region has an amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence obtained after 1 or 2 amino acids substitution on SEQ ID NO: 5; or/and the CDR 3 of the light chain variable region has an amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence obtained after 1 or 2 amino acids substitution on SEQ ID NO: 6.

14. The antibody according to claim 1, wherein, in the antibody, the immunoglobulin Fc fragment is a murine-derived IgG Fc fragment, or further is a murine-derived IgG1 Fc fragment.

15. The antibody according to claim 1, wherein: (i) the heavy chain of the antibody comprises an amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 80% sequence identity thereto; and (ii) (ii) the CDRs 1-3 of the heavy chain variable region of the antibody have an amino acid sequences shown in SEQ ID NOs: 1-3, respectively; the CDRs 1-3 of the light chain variable region of the antibody have an amino acid sequences shown in SEQ ID NOs: 4-6, respectively.

16. The antibody according to claim 1, wherein: (i) the light chain of the antibody comprises an amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 80% sequence identity thereto; and (ii) the CDRs 1-3 of the heavy chain variable region of the antibody have an amino acid sequences shown in SEQ ID NOs: 1-3, respectively; the CDRs 1-3 of the light chain variable region of the antibody have an amino acid sequences shown in SEQ ID NOs: 4-6, respectively.

17. The method according to claim 10, wherein the Her2 companion diagnostic immunohistochemical detection product is a matching one used before RC48-ADC targeted drug therapy.

18. The method according to claim 10, wherein an indication targeted by the Her2 companion diagnostic immunohistochemical detection product is a Her2-related cancer.

19. The method according to claim 10, wherein the Her2-related cancer is selected from the group consisting of breast carcinoma, gastric carcinoma, gastroesophageal carcinoma, oesophageal carcinoma, ovarian carcinoma, endometrial carcinoma, lung carcinoma, urothelial carcinoma, and bladder carcinoma.

20. The Her2 companion diagnostic immunohistochemical detection kit comprising the antibody according to claim 1, wherein the Her2 companion diagnostic immunohistochemical detection product is a matching one used before RC48-ADC targeted drug therapy.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0032] FIG. 1. Guidelines for Her2 detection using a verified immunohistochemistry method;

[0033] FIG. 2A shows the results of immunohistochemical staining of the case of serial number 1 in Table 7 with RC48M antibody as the primary antibody;

[0034] FIG. 2B shows the results of immunohistochemical staining of the case of serial number 1 in Table 7 with 4B5 antibody as the primary antibody;

[0035] FIG. 3A shows the results of immunohistochemical staining of the case of serial number 2 in Table 7 with RC48M antibody as the primary antibody;

[0036] FIG. 3B shows the results of immunohistochemical staining of the case of serial number 2 in Table 7 with 4B5 antibody as the primary antibody;

[0037] FIG. 4A shows the results of immunohistochemical staining of the case of serial number 3 in Table 7 with RC48M antibody as the primary antibody;

[0038] FIG. 4B shows the results of immunohistochemical staining of the case of serial number 3 in Table 7 with 4B5 antibody as the primary antibody;

[0039] FIG. 5A shows the results of immunohistochemical staining of the case of serial number 4 in Table 7 with RC48M antibody as the primary antibody,

[0040] FIG. 5B shows the results of immunohistochemical staining of the case of serial number 4 in Table 7 with 4B5 antibody as the primary antibody;

[0041] FIG. 6A shows the results of immunohistochemical staining of the case of serial number 5 in Table 7 with RC48M antibody as the primary antibody;

[0042] FIG. 6B shows the results of immunohistochemical staining of the case of serial number 5 in Table 7 with 4B5 antibody as the primary antibody;

[0043] FIG. 7A shows the results of immunohistochemical staining of the case of serial number 6 in Table 7 with RC48M antibody as the primary antibody;

[0044] FIG. 7B shows the results of immunohistochemical staining of the case of serial number 6 in Table 7 with 4B5 antibody as the primary antibody;

[0045] FIG. 8A shows the results of immunohistochemical staining of the case of serial number 7 in Table 7 with RC48M antibody as the primary antibody;

[0046] FIG. 8B shows the results of immunohistochemical staining of the case of serial number 7 in Table 7 with 4B5 antibody as the primary antibody;

[0047] FIG. 9 shows the structural formula of an RC48-ADC drug, wherein RC48 represents the RC48 antibody.

DETAILED DESCRIPTION

Definition

[0048] Unless otherwise defined, all terms used herein have the same meaning as understood by those of ordinary skill in the art. For definitions and terms in the art, professionals can in particular refer to Current Protocols in Molecular Biology (Ausubel). The abbreviation for amino acid residues is a standard 3-letter and/or 1-letter code used in the art to refer to one of the 20 commonly used L-amino acids.

[0049] As used herein, “antibody” is used in the broadest scope and encompasses various antibody structures including, but not limited to, a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., bispecific antibody), and an antibody fragment. In particular, “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 (one-fifth or one-quarter region on the heavy chain near the N-terminal) and a heavy chain constant region (three-quarters or four-fifths region on the heavy chain near the C-terminal). Each light chain comprises a light chain variable region (a half region on the light chain near the N-terminal) and a light chain constant region (a half region on the light chain near the C-terminal). The variable region of the heavy chain and the variable region of the light chain can be further subdivided into multiple regions with high variability, which is called a complementary determining region (CDR). The “CDR” refers to the hypervariable regions of the heavy and light chains of immunoglobulins, including those determined by the Kabat, Chothia, or IMGT systems. There are three heavy chain CDRs and three light chain CDRs per antibody. Depending on the circumstances, the term CDR as used herein is intended to indicate one or several or even all of these regions, which comprise most of the amino acid residues responsible for binding through the affinity of an antibody to an antigen or a recognition epitope thereof.

[0050] The term “Her2 detection antibody” as used in the present disclosure refers to an antibody capable of detecting the expression state of Her2 in a case and capable of binding to Her2. In immunohistochemistry, Her2 detection antibody is used as a primary antibody to detect the expression state of Her2. The “Her2 detection antibody” involved in the present disclosure is capable of targeting the extracellular domain IV region of Her2 protein.

[0051] The term “RC48M antibody” as used in the present disclosure refers to an antibody in which 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.

[0052] The term “RC48-ADC” or “RC48-ADC drug” or “RC48-ADC targeted drug” as used in the present disclosure refers to an antibody-drug conjugate (i.e. antibody-drug conjugate, ADC) comprising an antibody capable of specifically binding Her2 disclosed in patents WO2015074528A1 and CN105008398A, wherein the antibody is called “RC48 antibody”, and (i) the amino acid sequences of the CDRs 1-3 of its heavy chain region are DYYIH, RVNPDHGDSYYNQKFKD and ARNYLFDHW, respectively; and (ii) the amino acid sequences of the CDRs 1-3 of its light chain are KASQDVGTAVA, WASIRHT and HQFATYT, respectively. Or further, the antibody is derived from an antibody secreted by the hybridomas deposited in the China General Microbiological Culture Collection Center with the depositary number CGMCC No. 8102 on Aug. 22, 2013. Or further, the antibody is derived from an antibody secreted by the CHO cells deposited in the China Center for Type Culture Collection with the depositary number CCTCC C2013170 on Nov. 6, 2013. The structure of an RC48-ADC drug is shown in FIG. 9.

EXAMPLES

[0053] The present disclosure will be further described from examples in the following. It should be noted that the following examples are further illustrations and explanations of the present disclosure, and should not be considered as limiting the present disclosure.

[0054] In the following examples, the RC48M antibody was used as an example of an IHC detection antibody targeting the extracellular domain IV region of Her2 protein. The amino acid sequence of the heavy chain variable region of the RC48M antibody is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region of the RC48M antibody is shown in SEQ ID NO: 8. The detection of breast carcinoma samples was used as an example.

Example 1 Comparison of the Overall Agreement Rate Between the IHC Detection of RC48M Antibody (Primary Antibody) and the IHC Detection of PATHWAY—Anti-Her2/Neu (4B5) Rabbit Monoclonal Primary Antibody (Hereinafter Referred to as 4B5 Antibody) and the Problem of False Negative

[0055] Comparative detection and analysis of 49 invasive breast carcinoma samples.

[0056] 1) For 4B5 antibody, follow the product instruction method;

[0057] 2) Main procedure steps of RC48M IHC detection

TABLE-US-00005 TABLE 3 Main procedure steps of RC48M IHC detection Step Procedure and Parameter Description Primary antibody (dilution ratio) RC48M (1:500) Incubation conditions and time Room temperature (18~20° C.), for the primary antibody 20 min Antigen retrieval conditions without retrieval Detection system for the Polymer Refine Detection secondary antibody Color development time of DAB 5 min

[0058] See Table 4 for a list of detection results.

[0059] Table 4 List of detection results from 49 samples

TABLE-US-00006 Pathology 4B5 RC48M Number Tissue Type Number Antibody Antibody 1 breast carcinoma 18-04155-1 3+ 3+ 2 breast carcinoma 18-05735-1 3+ 3+ 3 breast carcinoma 18-05736-2 3+ 3+ 4 breast carcinoma 18-06464-1 3+ 3+ 5 breast carcinoma 18-07814-1 3+ 3+ 6 breast carcinoma 18-08167-1 3+ 3+ 7 breast carcinoma 18-08709-1 3+ 3+ 8 breast carcinoma 18-09022-1 3+ 3+ 9 breast carcinoma 18-07600-2 3+ 2+ 10 breast carcinoma 18-09839-1 3+ 2+ 11 breast carcinoma 18-09840-1 3+ 2+ 12 breast carcinoma 18-07150-1 2+ 2+ 13 breast carcinoma 18-07607-5 1+ 2+ 14 breast carcinoma 18-08555-1 1+ 2+ 15 breast carcinoma 18-08929-1 1+ 2+ 16 breast carcinoma 18-09503-1 1+ 2+ 17 breast carcinoma 18-05833-1 1+ 1+ 18 breast carcinoma 18-05899-1 1+ 1+ 19 breast carcinoma 18-07234-1 1+ 1+ 20 breast carcinoma 18-07384-4 1+ 1+ 21 breast carcinoma 18-07817-1 1+ 1+ 22 breast carcinoma 18-10298-2 1+ 1+ 23 breast carcinoma 18-06079-2 1+ 0  24 breast carcinoma 18-07486-1 1+ 0  25 breast carcinoma 18-07728-2 1+ 0  26 breast carcinoma 18-08047-1 1+ 0  27 breast carcinoma 18-08459-1 1+ 0  28 breast carcinoma 18-09312-1 1+ 0  29 breast carcinoma 18-10091-1 1+ 0  30 breast carcinoma 18-08952-1 0  2+ 31 breast carcinoma 18-08580-7 0  1+ 32 breast carcinoma 18-05734-1 0  0  33 breast carcinoma 18-05827-1 0  0  34 breast carcinoma 18-05829-1 0  0  35 breast carcinoma 18-06431-1 0  0  36 breast carcinoma 18-06551-3 0  0  37 breast carcinoma 18-06714 0  0  38 breast carcinoma 18-06715-3 0  0  39 breast carcinoma 18-06856-1 0  0  40 breast carcinoma 18-07599-1 0  0  41 breast carcinoma 18-07822-1 0  0  42 breast carcinoma 18-08233-1 0  0  43 breast carcinoma 18-08345-1 0  0  44 breast carcinoma 18-08930-1 0  0  45 breast carcinoma 18-09161-1 0  0  46 breast carcinoma 18-09361-3 0  0  47 breast carcinoma 18-09437-1 0  0  48 breast carcinoma 18-09673-1 0  0  49 breast carcinoma 18-09674-1 0  0  Note: the grading judgment of the staining results was referred to the rules in the “Guideline for HER2 Detection of Breast carcinoma (2014 Edition)”, the same below. The above results were summarized, and the results were shown in Table 5.

TABLE-US-00007 TABLE 5 Statistical analysis table of detection results Number of Cases RC48M Antibody 4B5 antibody 0 1+ 2+ 3+ Total 0  18 1 1 0 20 1+ 7 6 4 0 17 2+ 0 0 1 0 1 3+ 0 0 3 8 11 Total 25 7 9 8 49

[0060] From the above statistical results, there were 33 cases (18+6+1+8) with the same detection grade, accounting for 67.3% (33/49). From the judgment guidance of the current medical decision, and 1+ were determined as negative, 2+ was suspicious, and 3+ was positive, then a total of 26 cases (18+7+1)+6 cases case+8 cases=41 cases (accounting for 83.7%). The use of both will lead to the same medical decision. The cases with differences indecisions are summarized in Table 6.

TABLE-US-00008 TABLE 6 List of samples with differences in medical decisions Pathology 4B5 RC48M Number Tissue Type Number Antibody Antibody 9 breast carcinoma 18-07600-2 3+ 2+ 10 breast carcinoma 18-09839-1 3+ 2+ 11 breast carcinoma 18-09840-1 3+ 2+ 13 breast carcinoma 18-07607-5 1+ 2+ 14 breast carcinoma 18-08555-1 1+ 2+ 15 breast carcinoma 18-08929-1 1+ 2+ 16 breast carcinoma 18-09503-1 1+ 2+ 30 breast carcinoma 18-08952-1 0  2+

[0061] As mentioned above, Seema Jabbar et al. (Reference 6: Comparison of Two FDA-Approved Her2 Immunohistochemical Assays for Breast Carcinoma: HercepTest and Pathway Her2 (4B5), Am J Clin Pathol 2018; 149: S93-S94) conducted HercepTest and Pathway Her2 (4B5) (referred to as 4B5) detections on a total of 95 breast carcinoma samples. The detection results of HercepTest were 72 suspicious+23 positive; wherein the 72 suspicious samples were detected by 4B5, and 52 (52/72=72%) were negative. Of the 95 samples detected by 4B5, 52 (55%) were negative, 25 (26%) were suspicious, and 18 (19%) were positive. Confirmation of the results by FISH detection shows that 4B5 has a higher consistency with the results of FISH detection compared to HercepTest detection. In addition, among the 52 negative detected by 4B5, 3 cases were verified as positive by FISH detection, which indicates that there was a certain false negative result in the 4B5 detection. Combining with the results in Table 6, part of the results of the 4B5 detection were interpreted as 2+ for the results of the R48M detection and became suspicious cases, which required a process of the FISH or CISH detection (ISH detection) to determine. Although the use of the RC48M detection resulted in the addition of further ISH confirmation detection steps in some cases, this to a certain extent avoids the problem of partial false negatives in the results of 4B5 detection, thereby avoiding the problem of missing the better treatment period and missed the use of targeted therapeutic drugs to develop targeted therapy due to the problem of false negative detection in some patients. In addition, combining the results of Reference 6 and the above detection data, the consistency of the results of the RC48M detection and the results of the FISH detection should also be higher than the results of the HercepTest detection.

Example 2 Analysis of False Positive Problem

[0062] A total of 7 phase I clinical samples of RC48-ADC drug were detected in parallel with RC48M antibody and 4B5 for comparison. Seven paraffin sections of breast carcinoma tissue were used in each group. The primary antibody in Group 1 was RC48M antibody and in Group 2 was 4B5 antibody; and the two groups of experiments both used the rabbit and mouse universal immunohistochemical secondary antibody kit (purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.). The specific experimental process is as follows:

[0063] (1) Dewaxing and hydration: the slices were baked at 65° C. for 2 h, then soaked sequentially as follows: in xylene for 20 min, in absolute ethanol for 5 min, in 95% ethanol for 5 min, and in 75% ethanol for 5 min; and then the slices treated as above were transferred into 0.01 M PBST for use;

[0064] (2) Antigen retrieval: the slices of Group 2 were transferred into the sodium citrate buffer and heated at 98° C. for 20 min; and the slices of Group 1 did not require antigen retrieval.

[0065] (3) Blocking: the slices of Group 1 and Group 2 were blocked with 3% hydrogen peroxide for 10 min, and then soaked twice with 0.01 M PBST for 3 min at a time;

[0066] (4) Primary antibody: the slices of Group 1 and Group 2 were placed in a wet box, the RC48M antibody was diluted to 1 μg/ml, and then the diluted RC48M antibody and 4B5 antibody were added dropwise onto the corresponding slices of Group 1 and Group 2, respectively, incubated at 37° C. for 60 min, and soaked twice with 0.01 M PBST for 3 min each time after returned to room temperature;

[0067] (5) Secondary antibody: the rabbit and mouse universal secondary antibody in the rabbit and mouse universal immunohistochemistry secondary antibody kit was added dropwise onto the slices of Group 1 and Group 2 in an amount of 100 μl/piece, respectively, incubated at 37° C. for 20 min, and soaked twice with PBST (0.01 M) for 3 min each time; then the reaction amplifying agent in the above kit was added dropwise onto the slices of Group 1 and Group 2 in an amount of 100 μl/piece, incubated at 37° C. for 20 min, and soaked twice with PBST (0.01 M) for 3 min each time;

[0068] (6) DAB color development: DAB color development kit (purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.) was employed. 50 μl each of the dilution buffer, DAB chromogen and substrate in 1 mL of the kit were taken out and mixed well, then added onto the slices, color developed at room temperature for 1-3 min, and washed with distilled water;

[0069] (7) Hematoxylin counterstaining: 100 μL of hematoxylin staining solution (purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.) was added dropwise to each slice, stained at room temperature for 2-5 min, and washed with tap water for 5 min until return of blue.

[0070] (8) Dehydration: the slices of Group 1 and Group 2 were soaked sequentially as follows: in 75% ethanol for 5 min, 95% ethanol for 5 min, in 100% ethanol for 5 min, and in xylene for 10 min; and then the slices were taken out and xylene was volatilized.

[0071] (9) Mounting.

[0072] Then observed under the microscope and pictures were taken, with the objective lens 10×, the eyepiece 20×, and magnification 200 times. The staining results are shown in FIGS. 2-8, where FIGS. 2A, 3A, 4A, 5A, 6A, 7A, and 8A show the results of immunohistochemical staining with RC48M antibody as the primary antibody, and FIGS. 2B, 3B, 4B, 5B, 6B, 7B, and 8B show the results of immunohistochemical staining with 4B5 antibody as the primary antibody. The RC48M antibody and 4B5 antibody had similar staining in the cases of serial numbers 2, 3, 4, 5, 6, and 7, but showed a difference in the case of serial number 1, where RC48M staining was negative and 4B5 staining was positive. The above staining results were interpreted according to the rules in the “Guideline for HER2 Detection of Breast carcinoma (2014 Edition)”. See Table 7 for the interpretation results. The staining results of the RC48M antibody and the 4B5 antibody were 3+ in the cases of serial numbers 3, 4, 5, and 6; were 2+ in the case of serial number 2; showed a difference of 1 grade in the case of serial number 7; and showed a difference of negative and 3+ in the case of serial number 1. According to the clinical trial drug efficacy information corresponding to the cases represented by each serial number, the disease of the case represented by serial number 1 continued to progress after clinical medication. That is, for the patient who was positive for the detection, the use of targeted drugs was ineffective, which means that the case of serial number 1 had false positives when detected with 4B5 antibody. The results show that the RC48M antibody can effectively reduce the false positive problems in the detection caused by mutations in the extracellular region of Her2, so that it can more accurately determine the follow-up treatment regimen for such patients and avoid ineffective targeted drug treatment, and thus greatly saves the ineffective treatment investment for such patients and provide more accurate detection reference for the subsequent selection of other treatment regimens.

TABLE-US-00009 TABLE 7 Detection results and clinical efficacy Serial Number RC48M 4B5 Clinical Efficacy 1 0  3+ PD 2 2+ 2+ PD 3 3+ 3+ PR 4 3+ 3+ SD 5 3+ 3+ PR 6 3+ 3+ PR 7 2+ 3+ SD Note: PD stands for progressive disease; PR stands for partial remission; and SD stands for stable disease.

[0073] The present disclosure has been exemplified by various specific embodiments. However, those of ordinary skill in the art can understand that the present disclosure is not limited to specific embodiments. Those of ordinary skill in the art can make various modifications and variations within the scope of the present disclosure, and various technical features mentioned in various places of this specification can be combined with each other without departing from the spirit and scope of the present disclosure. Such modifications and variations are all within the scope of the present disclosure.