TW201642897A - HER2 binding agent therapies - Google Patents

Abstract

The present application relates to combination treatments for cancer, comprising the combined administration of (i) a Epidermal Growth Factor Receptor 2 (HER2) binding agent, and specific binding members which compete with such a binding member for binding to HER2, and (ii) radiotherapy, surgery and/or chemotherapy, such as immunotherapy. The present application also relates to the use of human HER2 gene copy number and HER2 mRNA levels as biomarkers, e.g. to identify cancers which will respond to treatment with a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain of the specific binding member, and specific binding members which compete with such a binding member for binding to HER2. The present application also relates to compositions comprising a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a CH3 domain of the specific binding member and another agent, e.g., a therapeutic, such as an immuno-oncology agent and uses thereof.

Description

HER2 binding agent treatment Field of invention

The present invention relates to a combination therapy for cancer comprising the combined administration of (i) a second type of epidermal growth factor receptor (HER2) binding agent, such as a specific binding member, comprising a design comprising one of the binding members. A fixed domain, such as a HER2 antigen binding site of a structural loop region of the CH3 domain, and a specific binding member that competes with the binding member for HER2 binding, and (ii) radiation therapy, surgery, and/or chemotherapy, Such as immunotherapy. The invention also relates to the use of human HER2 gene sets (GCN) and HER2 mRNA levels as biomarkers. In particular, the present invention relates to the use of HER2 gene sets and HER2 mRNA levels as biomarkers to identify cancers that are responsive to treatment with a particular binding member, the specific binding member comprising a design that is designed to be fixed. A domain, such as a HER2 antigen binding site of a structural loop region of the CH3 domain, and a specific binding member that competes with the binding member for HER2 binding. The invention also relates to a composition comprising a specific binding member designed to comprise a HER2 antigen binding site in a structural loop region of its CH3 domain; and comprising another agent, such as a therapeutic agent, Such as an immunological antitumor agent; and the use of the composition.

Background of the invention

The human type 2 epidermal growth factor receptor (also known as HER2, HER2/neu or ErbB-2) is a 185 kDa cytoplasmic transmembrane tyrosine kinase receptor. It is encoded by the c-erbB-2 gene located on the long arm of chromosome 17q and is a member of the HER family (Ross et al. 2003). The HER family typically regulates cell growth and survival, as well as its attachment, migration, differentiation, and other cellular responses (Hudis, C, 2007).

Overexpression and amplification of HER2 have been observed in the development of various solid cancers, including breast cancer (Yarden, Y, 2001), stomach cancer (Gravalos et al., 2008), and gastric cancer (Rüschoff et al.) 2010 B), colorectal cancer (Ochs et al. 2004), ovarian cancer (Lanitis et al. 2012), pancreatic cancer (Lei et al. 1995), intrauterine Membrane cancer (Berchuk et al. 1991) and non-small cell lung cancer (Brabender et al. 2001).

In 2008, breast cancer, colorectal cancer and stomach cancer accounted for 30% of all cancer-confirmed cases and 24% of all cancer deaths (CRUK and World Health Organization (WHO) World Cancer Report (World Cancer Report) )). In particular, breast cancer is the leading cause of cancer death in women. Overexpression or expansion of HER2 in 15 to 30% of breast cancers, accompanied by poor prognosis, short period of disease-free and short-term survival, and aggressiveness of cancer phenotypes (Vinatzer et al. 2005) ). About 20% of breast cancer patients will develop tumors that contain genomic alterations involving amplification of an amplicon on chromosome 17 containing the HER2 proto-oncogene. Role (Ross, J's 2009 B; and Hicks et al. 2005). These tumors represent a more aggressive breast cancer subtype and contribute significantly to the death of the disease (Hudis C, 2007).

Several HER2 target therapies have been approved for the treatment of HER2-positive tumors.

Herceptin (Herceptin) ^TM (monoclonal antibody Trastuzumab (of trastuzumab)) has been approved and Taxol (Taxol ^TM) (paclitaxel (paclitaxel)) were combined for the treatment of metastatic breast cancer, and approved for separate Treatment of HER2-positive breast cancer in patients who have undergone chemotherapy for one or more metastatic disease. Because trastuzumab antibody also enhances the efficacy of adjuvant chemotherapy (pacific paclitaxel, docetaxel, and vinorelbine) for surgical or locally advanced HER2-positive tumors, Standard care for early or late HER2 over-expression breast cancer patients. In patients who have not been treated for metastatic disease, trastuzumab antibody has also been approved for treatment with the chemotherapy (cisplatin and capecitabine or 5-fluorouracil) for the treatment of the stomach or HER2-positive metastatic cancer of the gastroesophageal junction cancer (Genentech, Herceptin) can be found online at http://www.Herceptin.com/about[September 2013 17th browsing]).

Perjeta ^TM (pertuzumab) has also been approved for the treatment of HER2-positive metastatic breast cancer with the combination of trastuzumab antibody and European paclitaxel (Genentech) The company can refer to "PERJETA can help strengthen your treatment" at http://www.Perjeta.com/patient/about. [Browse on September 17, 2013]). Perezetab antibodies target another distinct domain of HER2 and its mechanism of action is different from that of trastuzumab antibodies. Specifically, the inhibitor of Pertitudin alone against HER2 dimerization of the system prevents HER2 from pairing with other HER receptors (EGFR/HER1, HER3 and HER4). HE Aining (Kadcyla ^TM) (Trastuzumab monoclonal antibody Ai Tanxin (emtansine) of conjugate, T-DM1) based an antibody - drug complex, which comprises a cytotoxic agent Mo Tanxin (mertansine) (DM1) a linked trastuzumab antibody that disrupts microtubule assembly in dividing cells resulting in cell death and is approved in patients who have received trastuzumab antibody and taxane chemotherapy for the treatment of metastatic breast cancer (gene Technology (Genentech) company, can be found on the website http://www.gene.com/media/product-information/kadcyla-moa "why believe Herceptin Ning (Kadcyla) ^TM (Qu Is there a curative effect of the conjugate of the antibody and emtansine (the proposed mechanism of action)? B. [Browse on September 17, 2013]).

Techno ingot (Tykerb ^TM) (lapatinib (of lapatinib)) based, a small molecule kinase inhibitors, which simultaneously block the catalytic action of HER2 with EGFR. Milk which have been approved and the complex admittance (Femara ^TM) (letrozole (the letrozole)) for the combined treatment of HER2-positive women after menopause, hormone receptor-positive metastatic breast cancer; previously approved and accepted anthracycline comprises Su patients, taxanes and Herceptin (Herceptin) ^TM) treatment, the tumor was cut (Xeloda ^TM) (capecitabine (capecitabine)) for the combined treatment of advanced or metastatic HER2-positive breast cancer (which may See the US Food and Drug Administration's "Highlights of Prescribing Information" on http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/022059s007lbl.pdf. View on the 28th of the month]). There are other drugs in the clinical development stage.

Although approved HER2-specific therapies have improved standard care for HER2-positive breast cancer and gastric cancer, significant medical needs remain unmet due to innate or acquired resistance to these drugs. For HER2-positive breast cancer, despite the standard care status of trastuzumab antibodies, approximately 70% of patients in the adjuvant regimen and approximately 70% of patients in the single-drug regimen still develop trastrol alone Resistance to antibodies (Wolff et al., 2007; and Harris et al., 2007).

Emerging trends in cancer treatment are based on the assessment of the underlying genetic or molecular mechanisms of cancer and the use of it as a biomarker to screen out patients for treatment. The US Food and Drug Administration has approved diagnostic tests based on biomarkers with specific cancer-related properties to identify patients for treatment with specific cancer therapies. As of May 2013, the US Food and Drug Administration has approved 15 diagnostic tests, which are also known as companion diagnostics ("Genetic Engineering and Biotechnology News" in 2013" Diagnostics: 52 items were selected". Available at http://www.genengnews.com/insight-and-intelligenceand153/companion-diagnostics-52-pick-up/77899813/ [Browse on September 28, 2013] ]), there are various other diagnostic tests being developed. For example, therascreen ^TM KRAS chemical detection system detecting one EGFR immunohistochemistry, which identify EGFR-positive patients suffering from metastatic colorectal cancer with the wild-type KRAS gene, and such patients available Erbitux (Erbitux ^TM) (West Treatment with a single antibody (cetuximab). The DAKO C-kit PharmDx immunohistochemistry test identifies patients with c-kit-positive gastrointestinal stromal tumors that are suitable for treatment with gleevec (imatinib). Several diagnostic tests are also approved for identifying suitable for treatment with Herceptin (Herceptin) ^TM (monoclonal antibody trastuzumab) of HER2-positive tumors (Hamburg, 2010 and the Collins B above), such as immunohistochemistry Herceptest ^TM and Her2 FISH pharmDx Kit ^TM, which is generally combined the like. Other commercially available immunohistochemistry kits for HER2-positive tumors include Oracle (Leica Biosystems) and Pathway (Ventana). Preclinical and clinical research work to identify biomarkers that are predictive of clinical response to treatment, and has the potential to identify "non-traditional" HER2-positive cancers including colorectal cancer, ovarian cancer, and other cancers. In other patients, these patients are likely to benefit from HER2 target treatment (Gun et al. 2013).

Due to the aggressive nature of tumors and their overexpression of HER2 or the amplification of the HER2 gene, HER2 amplification has become an increasingly important predictor of disease prognosis and is widely tested. HER2-positive tumors that may be suitable for treatment with a HER2-specific biotherapeutic agent are typically first identified by using immunohistochemistry (IHC). Samples from patients were evaluated in a semi-quantitative manner using a scoring system that reflects the intensity of staining and the percentage of stained tumor cells in the sample. The level of performance or overexpression of HER2 is measured and scored to give a strong and complete membrane staining score of 3+ and is considered strongly positive or clear. Weak to moderate end observed in more than 10% of tumor cells The whole membrane staining is given a score of 2+ and is considered weakly positive or uncertain. When the membrane staining was weak or incomplete and occurred in less than 10% of tumor cells, the sample scored 1+/0 and was considered HER2 negative. Those tumors with a score of 3+ in the IHC analysis, ie those who overexpress the HER2 protein on the surface of tumor cells, will be notified to the clinician to seek the possibility of treatment with HER2-specific therapies.

Because IHC is only semi-quantitative, and the measurement of HER2 protein overexpression on the cell surface may be inaccurate, and the readings provided are not always accurate. In particular, it may be difficult to correctly identify a tumor with a weak positive HER2 expression by IHC, that is, a tumor that achieves a 2+ score in the analysis. To clarify the HER2 status of such tumors, the sample is further detected, for example, by fluorescence in situ hybridization (FISH) to determine the HER2 gene amplification level in the tumor sample. Different methods for performing FISH are known. In comparing the HER2 gene sets with an internal control group such as CEP17, when the FISH score is less than 2, that is, when the HER2/CEP17 ratio is less than 2, the HER2 gene status is regarded as non-amplified, and the tumor is treated as Negative for HER2. When the FISH score is equal to or greater than 2, the HER2 gene status is considered to be amplifying and the tumor is considered HER2 positive, and the clinician is notified to seek the possibility of treatment with HER2-specific therapy.

Although these multi-layer tests were performed on HER2-positive tumors, not all tumors that were identified as HER2-positive were responsive to current HER2-specific therapies. Thus, there remains a need in the art for a therapy for a HER2-positive tumor patient, as well as a means for identifying a patient who may benefit from such therapy. In addition, as described above, for the needle Known therapies for HER2, such as the acquired resistance of trastuzumab antibody, indicate that there is a need in the art for a therapy suitable for treating such drug resistant HER2-positive cancers.

WO 2009/132876 A1 and WO 2009/000006 A1 mentioned antigen-binding Fc fragment (also referred Fcab ^TM [Fc fragment having the antigen-binding]), which contains, for example with high affinity binding of HER2 modified type domain IgG1Fc , etc., which is hereby incorporated by reference in its entirety into this application. The specific binding member groups described herein include the antigen-binding Fc fragments described herein, wherein each has one or more amino acid modifications in at least one structural loop region, wherein the modified structural loop region Binding to an antigen, such as an epitope of Her2, in a specific manner, while the unmodified Fc fragment does not significantly bind to it.

In natural immunoglobulins, these loops are not specific for CDR loops and do not have antigen binding or epitope binding, but contribute to proper folding or other functions of immunoglobulin molecules and/or their effectors. Therefore, it is referred to as a structural ring for the purpose of the present invention. Thus, a "structural loop" or "non-CDR loop" as in the present invention should be understood in the following manner: The immunoglobulin system consists of a domain having a so-called immunoglobulin fold. In essence, the anti-parallel beta flaps are joined by a loop to form a compressed anti-parallel beta bucket. In the variable region, some of the loop systems of the domains substantially contribute to antibody specificity, i.e., binding to an antigen. These loops are referred to as CDR loops. All other loops of the antibody domain are more conducive to the structure and/or effector function of the molecule. These loops are defined herein as structural loops or non-CDR loops.

These Fcabs have been shown to have advantageous properties, such as in mice. The half life is about 60 hours.

According to an embodiment of the present invention, a specific binding member that binds to a human type II epidermal growth factor receptor (HER2) is provided, which is useful in a therapeutic method for cancer in a patient, which is selectively In combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent, wherein each tumor cell of the cancer has an average number of HER2 gene sets greater than or equal to 10, and wherein the specific binding member is: (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) And DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with HER2-specific binding members as one of (a).

Figure 1 shows the ratio of the antigen-binding Fc fragment H561-4 amino acid sequence (H561-4) to the wild-type IgG1 amino acid sequence (WT) of the same region. The amino acid is numbered according to Kabat, and the Bate number is shown above the alignment sequence (Note: Bet's number does not have 293 to 294, 297 to 298 of IgG1, 315 to 316, 356, 362, 380, 403 to 404, 409, 412 to 413, 429, 431 to 431 residues). The part of the AB ring is shown in bold. The part of the EF ring is shown in bold with a double bottom line.

Figure 2 is a graph showing that the epitope-binding line to which H561-4 binds to HER2 is different from that of the trastuzumab antibody or the Pertuzumab antibody. Figure 2A shows surface plasmon resonance (SPR) analysis using BIAcore's trastuzumab antibody to compete with the epitope of H561-4. A CM5 BIAcore wafer coated with 1000 RU of HER2 ECD was prepared by injecting 10 μg/ml of trastuzumab antibody for 3 minutes followed by a second injection of 10 μg/ml of trastuzumab antibody. The trastuzumab antibody (TR) was saturated, indicating that after the first injection, the HER2 wafer was saturated with the trastuzumab antibody (black dotted line). By injecting 10 μg/ml of trastuzumab antibody for 3 minutes, followed by a second injection of H561-4 (10 μg/ml) with a mixture of trastuzumab antibody (10 μg/ml). A CM5 BIAcore wafer coated with 1000 RU of HER2 ECD was saturated with trastuzumab antibody, which showed that when the HER2 wafer was saturated with trastuzumab antibody H561-4, it was still able to bind (black solid line) . A CM5 BIAcore wafer coated with 1000 RU of HER2 ECD was presented on H561-4 by injecting 10 μg/ml of H561-4 for 3 minutes followed by a second injection of H561-4 (10 μg/ml). Saturated, it shows that after the first injection, the HER2 wafer has been saturated with H561-4 (black dashed line). The coating was carried out by injecting 10 μg/ml of H561-4 for 3 minutes, followed by a second injection of a mixture of trastuzumab antibody (10 μg/ml) and H561-4 (10 μg/ml). A CM5 BIAcore wafer with 1000 RU of HER2 ECD is saturated with H561-4, which shows that when the HER2 wafer is saturated with H561-4 trastuzole antibody, it is still able to bind (wide Black dash). Figure 2B shows SPR analysis of the epitope-targeting effect of Octetibole antibody (PE) with H561-4 using Octet. The streptavidin coated tip was loaded by incubation in biot-HER2 for 30 minutes and then in buffer for 1 minute. By culturing with 325 nM Pertamine monoclonal antibody for 30 minutes, followed by incubation with 325 nM per bead monoclonal antibody for a second time, HER2 was saturated with Perezetim antibody, which was shown after the first incubation. The HER2 coated tip has been saturated with the Pertamine monoclonal antibody (grey line). The streptavidin coated tip was loaded by incubation in biot-HER2 for 30 minutes and then in buffer for 1 minute. By culturing with 325 nM Pertamine monoclonal antibody for 30 minutes, followed by incubation with 325 nM of H561-4 and 325 nM Peroxida monoclonal antibody for a second time, HER2 was saturated with Pertatum monoclonal antibody. The HER2 coated tip of the Pertitux monoclonal antibody was still able to bind to H561-4 (black line). Figure 2C shows predicted H561-4 binding regions on the HER2 extracellular domain (cross-domains 1 and 3 of black markers). The binding epitope of H561-4 was predicted using Pepscan CLIPS (using a chemical bond to link the peptide to the scaffold) technique. The HER2 domain is shown in Roman numerals. The table in Figure 2C shows the predicted linear sequences for each of the binding epitopes of H561-4, which together form the binding pocket spans 1 and 3.

Figure 3 shows that H561-4 triggers apoptosis and leads to HER2 internalization and degradation. Figure 3A shows the reduction in the total number of cells and the percentage of viable cells when SKBr3 cells were treated with H561-4. The staining of PI with type V annexin showed that late apoptosis and necrotic cells increased after H561-4 treatment. Figure 3B shows when SKBr3 is treated with H561-4 When cells are present, this results in a decrease in total HER2 and phosphorylated HER2. Figure 3C shows the reduction in cell surface HER2 and total HER2 when SKBr3 cells were treated with H561-4. When treated with trastuzumab antibody, wild-type (WT) antigen-binding Fc fragment (SEQ ID NO: 19) or IgG1 control (IgG), no such decrease in Her2 was observed in SKBr3 cells. Not seen in untreated cells. Figure 3D shows the induction of apoptotic protease 3/7 activity when treated with H561-4 for SKBr3 cells; when SKBr3 cells were treated with trastuzumab antibody, wild-type antigen-binding Fc fragment or IgG1 control group (ctrl) otherwise.

Figure 4 shows the efficacy of H561-4 treatment in a tumor xenograft (PDX) model derived from a human patient with a HER2 gene set of greater than or equal to 10. Figure 4A shows in vivo tumor response data from gastric tumors (GXF281) with a high number of HER2 gene sets (15 sets of HER2 gene sets per cell). The arrow indicates the number of days of treatment. Figure 4B shows in vivo tumor response data from gastric tumors (GXA 3039) with a high number of HER2 gene sets (25 sets of HER2 gene sets per cell). The arrow indicates the number of days of treatment. Figure 4C shows in vivo tumor response data from colorectal tumors (CXF1991) with a high number of HER2 gene sets (32 sets of HER2 gene sets per cell). The arrow indicates the number of days of treatment. Figure 4D shows in vivo tumor response data from colorectal tumors (CXF2102) with a high number of HER2 gene sets (35 sets of HER2 genes per cell). The arrow indicates the number of days of treatment. Figure 4E shows in vivo tumor response data from gastric tumors (GXA3054) with a high number of HER2 gene sets (76 sets of HER2 gene sets per cell). The arrow indicates the number of days of treatment. Figure 4F shows that the number of sets from the HER2 gene is high (per cell In vivo tumor response data for gastric tumors (GXA 3038) with a HER2 gene set of 29 sets. The arrow indicates the number of days of treatment. Figure 4G shows in vivo tumor response data from breast tumors (HBCx-13B) with a high number of HER2 gene sets (23 sets of HER2 gene sets per cell). The arrow indicates the number of days of treatment. Figure 4H shows in vivo tumor response data from gastric tumors (GA0060) with a high number of HER2 gene sets (43 sets of HER2 gene sets per cell). The arrow indicates the number of days of treatment. The error bars in Figures 4A-H represent the standard error (SEM) of the mean.

Figure 5 shows that in GXF281 gastric PDX mode (25 sets of HER2 gene per cell), H561-4 overcomes the resistance of trastuzumab antibody plus peroxidase monoclonal antibody (TR+PE) . Mice were treated intravenously (i.v.) with the compounds listed in the legend ("/" separated from the compound administered in the first treatment cycle and the second cycle administered). The arrow indicates the administration schedule. After the average volume of the transplanted tumor reached 100 cubic millimeters, the four-week weekly dose of TR+PE combined therapy (or four weeks of H561-4 for the weekly dose) was used for treatment. Although the growth rate of TR+PE-treated tumors slowed down, it continued to grow. The behavioral period was 33 days apart to allow the antibodies to be cleared prior to the second dosing cycle. After the interval, if the average tumor volume exceeds 500 cubic millimeters, the mice are again given TR+PE or H561-4 every week. The error bars represent the standard error (SEM) of the mean.

Figure 6 is a graph showing the spread of therapeutic T/C values of H561-4 and trastuzumab antibodies in a PDX mode with a HER2 gene set number greater than or equal to 10 in a PDX mode with a set number of genes (GCN) of less than 10. Figure. use The Graphpad prism software calculates the P value by an unpaired T-check.

Figure 7 is a graph showing the treatment of H561-4 and trastuzumab antibody in PDX mode with HER2 mRNA level (mRNA) greater than or equal to 200 in PDX mode with mRNA level below 200. A scatter plot of C values. P values were calculated by unpaired T-test using the Graphpad prism software.

Figure 8 is a scatter plot showing the therapeutic T/C values of H561-4 and trastuzumab antibodies in PDX mode with HER2 gene sets greater than 10 compared to PDX mode with GCN less than 10. Calculate the T/C value using the updated T/C formula. P values were calculated by unpaired T-test using the Graphpad prism software.

Figure 9 is a graph showing the therapeutic T/C of H561-4 and trastuzumab antibody in PDX mode with HER2 mRNA level higher than or equal to 200 in PDX mode with HER2 mRNA level below 200. The scatter plot of values. Calculate the T/C value using the updated T/C formula. P values were calculated by unpaired T-test using the Graphpad prism software.

Figure 10 is a graph showing the therapeutic T/C values of H561-4 and trastuzumab antibodies in a PDX mode with a set of genes greater than 18 compared to a PDX model with a HER2 gene set of less than 18 as determined by FISH. The scatter map. P values were calculated by unpaired T-test using the Graphpad prism software.

Figure 11 is a scatter plot showing the number of sets of genes determined by FISH and qPCR in each of the 17 PDX tumor models. Based on the correlation analysis, the number of sets of genes determined by the two methods was highly correlated (Pearson r = 0.90; p <0.0001; 95% CI = 0.74 - 0.96).

Figure 12 shows that compared to the overexpression of HER2 protein Negative PDX mode, a scatter plot of therapeutic T/C values for H561-4 and trastuzumab antibodies in PDX mode positive for HER2 protein overexpression. P values were calculated by unpaired T-test using the Graphpad prism software.

Detailed description of the invention

The inventors of the present application have discovered a specific binding member comprising a HER2 antigen binding site designed into a structural loop region of a fixed domain such as the CH3 domain, and which is useful for treating each Cancer cells with a HER2 gene set greater than or equal to 10 cancers. The inventors of the present application have also discovered that such specific binding members can be used to treat cancers with high HER2 mRNA levels. In particular, such specific binding members can be used to treat cancers that are resistant to treatment with known HER2-specific therapies, known single-drug therapies for HER2-specific therapies such as trastuzumab antibodies, or combined use The treatment of the bead monoclonal antibody and the pertamine monoclonal antibody. A specific binding member comprises a HER2 antigen binding site designed to be placed in a structural domain of a fixed domain, such as the CH3 domain, which can be used in combination with another treatment, such as surgery, radiation therapy or chemotherapy, such as immunization. therapy.

As explained above, HER2 is known to behave in several different cancers. The available breast cancer data is more than other cancers known to exhibit HER2, as breast cancer research may be the most abundant of HER2-positive cancers. Most of the data available is for tumors with an IHC score of 3+. Hoff et al. (June 2002) measured 8% of all breast cancer patients with a set of genes greater than or equal to 10. Auxiliary of monoclonal antibodies to the largest scale In the breast cancer test (HERA test), the number of tumor genes in 2071 (61%) patients was evaluated by FISH. In these patients, it was found that about 65% of patients had a set of genes greater than or equal to 10. This represents approximately 13% of all breast cancer patients (Dowsett et al., 2009). Although some clinical trials have studied the number of sets of HER2 genes, there is very little information about the corresponding HER2 mRNA levels in these cancers. However, it is known that an increase in the number of sets of genes in a cell usually results in an increase in the expression of the gene and thus contributes to an increase in the level of mRNA encoded by the gene. Therefore, a patient with a high HER2 gene set (i.e., equal to or greater than 10) per tumor cell of the cancer is expected to have a high HER2 mRNA level.

Thus, for a significant number of patients with cancer, each tumor cell has a HER2 gene set number greater than or equal to 10, and such patients will benefit from treatment with a specific binding member comprising A HER2 antigen binding site in a structural loop region of a fixed domain such as the CH3 domain is designed; or a treatment that benefits from binding to one of the specific binding members of the specific binding member in HER2 binding. The same applies to patients with high levels of HER2 mRNA in cancers.

The inventors of the present application have shown that the number of sets of genes can be used to predict the treatment of a specific binding member as disclosed in the present application, and cannot be used to predict the use of previously approved HER-2 specific trastuzumab. Treatment of antibodies.

In the present application, a specific binding member is provided, the specific binding member comprising a knot designed to be placed in a fixed domain such as the CH3 domain a HER2 antigen binding site of the loop region, and a therapeutic method for providing a specific binding member that competes with the one specific binding member for HER2 binding for use in a cancer of a patient, optionally In combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent, wherein each tumor cell of the cancer has a HER2 gene set number greater than or equal to 10.

Also provided in the present application is a specific binding member comprising a HER2 antigen binding site designed into a structural loop region of a fixed domain such as the CH3 domain, and providing for HER2 binding. One of the specific binding members that compete with the one specific binding member for use in a treatment for cancer in a patient, which is selectively used in combination with another therapy such as immunotherapy, such as the use of an immunological antitumor agent Therapy wherein the cancer has a high HER2 mRNA level.

Further provided is a specific binding member comprising a HER2 antigen binding site designed to be placed in a structural domain of a fixed domain, such as the CH3 domain, and to provide a specificity in HER2 binding A sexually binding member competes with one of the specific binding members for use in a treatment for cancer in a patient, optionally in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent, wherein Cancer is resistant or refractory to a treatment system of trastuzumab antibody and/or trastuzumab antibody plus pertituzil antibody (eg, is resistant or refractory in nature) , or for acquired or refractory conditions).

a specific binding member is also provided, the specific binding member a HER2 antigen binding site comprising a structural loop region designed to be placed in a fixed domain thereof, such as the CH3 domain, and a specific binding member that competes with the specific binding member for binding to HER2 for use In a treatment for cancer, it is selectively used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent, wherein the cancer (i) is for trastuzumab antibody and / Or a treatment with a trastuzumab antibody plus a pertamine monoclonal antibody that is resistant or refractory (eg, is resistant or refractory in nature, or is acquired or refractory); (ii) Each tumor cell has a HER2 gene set number greater than or equal to 10 and/or a high HER2 mRNA level.

In one aspect, the invention provides a method of specifically binding to one of HER2 binding for use in a method of treatment for cancer in a patient, optionally in combination with another therapy such as immunotherapy, such as using one A therapy for immunizing an antitumor agent, wherein each tumor cell of the cancer has a HER2 gene set, such as an average number of HER2 genes, greater than or equal to 10, and wherein the specific binding member is: (a) a specific binding A member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with HER2-specific binding members as one of (a).

A patient as referred to in an embodiment is a human patient.

Measuring a tumor sample obtained from the patient, wherein each tumor is swollen The number of sets of HER2 genes possessed by tumor cells, such as the average number of sets of HER2 genes, is greater than or equal to 10. Thus, the present invention provides for the specific binding of one of the bindings to HER2 to a member for use in a method of treatment for cancer in a patient, optionally in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent The therapy, wherein a tumor sample obtained from the patient is measured, and wherein each tumor cell has an average number of HER2 gene sets greater than or equal to 10, and wherein the specific binding member is: (a) a specific A sex binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ IDENTIFICATION Number: 14); or (b) a specific binding member that competes with HER2-specific binding members as one of (a).

The method may comprise: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; (ii) if the number of sets of HER2 genes per tumor cell, such as an average The HER2 gene set number system is greater than or equal to 10, the specific binding member is used to treat the patient; and (iii) the patient is selectively treated with another therapy, such as immunotherapy, such as the use of an immunological antineoplastic agent. .

Accordingly, the present invention also provides a specific binding member that binds to HER2 for use in a method of treatment for cancer in a patient, wherein the method comprises: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; (ii) if the number of sets of HER2 genes per tumor cell, such as the average number of sets of genes, is greater than or equal to 10, treating the patient with the specific binding member; and (iii) selectively treating the patient with another therapy, such as immunotherapy, such as the use of an immunotherapeutic agent, wherein the specific binding member And: (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) with DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with HER2-specific binding members as one of (a).

As noted above, the term "HER2 gene sets" as used in this application may refer to the average number of HER2 genes per tumor/cancer cell. The HER2 gene set can be greater than or equal to 10. Optionally, the HER2 gene set may be greater than or equal to 11, greater than or equal to 12, greater than or equal to 13, greater than or equal to 14, greater than or equal to 15, greater than or equal to 16, greater than or equal to 17, greater than or equal to 18, greater than or Equal to 19, greater than or equal to 20, greater than or equal to 21, greater than or equal to 22, greater than or equal to 23, greater than or equal to 24, or greater than or equal to 25. In one example, the HER2 gene set number is greater than 10. In another example, the HER2 gene set number is greater than or equal to 11. In another example, the HER2 gene set number is greater than or equal to 12. In another example, the HER2 gene set number is greater than or equal to 13. In another example, the HER2 base Because the number of sets is greater than or equal to 14. In yet another example, the HER2 gene set number is greater than or equal to 15. In another example, the HER2 gene set number is greater than or equal to 16. In another example, the HER2 gene set number is greater than or equal to 17. In another example, the HER2 gene set number is greater than or equal to 18. In another example, the HER2 gene set number is greater than or equal to 19. In another example, the HER2 gene set number is greater than or equal to 20. In another example, the HER2 gene set number is greater than or equal to 23. In yet another example, the HER2 gene set number is greater than or equal to 25. The HER2 gene set number, such as the average HER2 gene set number, is preferably greater than or equal to 15.

The HER2 gene set number, such as the average HER2 gene set number, is preferably greater than or equal to 10, wherein the set of genes is determined by quantitative polymerase chain reaction (qPCR).

The HER2 gene set number, such as the average HER2 gene set number, is preferably greater than or equal to 18, wherein the set of genes is determined by FISH.

In one aspect, the invention provides a method of specifically binding to one of HER2 binding for use in a method of treatment for cancer in a patient, optionally in combination with another therapy such as immunotherapy, such as using one Therapeutic of an anti-tumor agent, wherein the cancer has a high HER2 mRNA level, and wherein the specific binding member is: (a) a specific binding member comprising a CH3 domain engineered to be placed in the specific binding member a HER2 antigen binding site of a structural loop region, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, which is HER2 binding effect and (a) One of the specific binding members competes.

A tumor sample obtained from the patient has been determined to have a high HER2 mRNA level by measurement. Thus, the present invention provides for the specific binding of one of the bindings to HER2 to a member for use in a method of treatment for cancer in a patient, optionally in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent Therapy wherein a tumor sample obtained from the patient is determined and has a high HER2 mRNA level, wherein the specific binding member is: (a) a specific binding member comprising a designed insert The HER2 antigen binding site of a structural loop region of the CH3 domain of the specific binding member, and the amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) A specific binding member that competes for HER2 binding with a specific binding member such as (a).

The method can comprise: (i) determining a HER2 mRNA level of a tumor sample obtained from the patient; (ii) treating the patient with the specific binding member if the level of the HER2 mRNA is high; Iii) selectively treating the patient with another therapy, such as immunotherapy, such as the use of an immunotherapeutic agent.

Accordingly, the present invention also provides a specific binding member that binds to HER2 for use in a method of treatment for cancer in a patient, wherein the method comprises: (i) determining the HER2 mRNA level of a tumor sample obtained from the patient; (ii) treating the patient with the specific binding member if the level of HER2 mRNA is high; and (iii) selectively The patient is treated with another therapy, such as immunotherapy, such as the use of an immunotherapeutic agent, wherein the specific binding member is: (a) a specific binding member comprising a designed specific binding. a HER2 antigen binding site of a structural loop region of the CH3 domain of the member, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member It competes with HER2-specific binding members in one of (a).

The mRNA level encoding a specific protein can be indirectly measured by the quantitative action of quantitative PCR; the cDNA copy relative to a reference and preferably a housekeeping gene, and by reverse transcription PCR (RT- PCR) measures cDNA copies derived from mRNA as in this example. The reference gene has a normal profile in a variety of tissue/sample types and is used as a standard. The HER2 mRNA level in a tumor/cancer or tumor sample as referred to in the present application preferably refers to the number of HER2 cDNA sets in the tumor/cancer or tumor sample determined by RT-PCR and quantitative PCR. Reflecting the HER2 mRNA level, and the mRNA level relative to a reference gene (such as a housekeeping gene); and the mRNA level of the reference gene refers to one of the tumors determined by RT-PCR and quantitative PCR. / The number of cDNA sets of reference genes in a cancer or tumor sample is reflected. The knowledge can be used CopyCaller ^TM software, Version 2.0 (Life Technologies (Life Technologies)) or, for those skilled in the art skilled other equivalent software, data from quantitative PCR assay cDNA copy number. CopyCaller ^TM system soft copy number relative to a housekeeping gene cDNA, comparing the threshold cycle number of copies of HER2 cDNA of relative quantitative analysis. Alternatively, a cDNA sample (Life Technologies' CopyCaller software from Life Technologies may be referenced by reference to a reference cDNA sample, such as from a collection of 10 human cell lines each cell has or is expected to have two sets of HER2 genes. The user manual), while standardizing the number of sets of cDNA.

Thus, a high HER2 mRNA level in a tumor/cancer or tumor sample as referred to in this application preferably refers to a high HER2 mRNA position as reflected by the high number of HER2 cDNA sets in a tumor/cancer or tumor sample. The sequence is determined by RT-PCR and quantitative PCR, and the mRNA level relative to a reference gene; and the mRNA level of the reference gene is determined by RT-PCR and quantitative PCR. The number of cDNA sets of reference genes in the tumor/cancer or tumor sample is reflected. Specifically, HER2 or a reference gene in a tumor sample is transcribed into cDNA using reverse transcription PCR, and then quantified using qPCR. The reference gene is preferably a housekeeping gene. In a preferred embodiment, the reference gene is a TATA binding protein (TBP), but other suitable reference genes are also known in the art. The HER2 mRNA level in a tumor/cancer or tumor sample can be normalized relative to the HER2 mRNA level in a control cell sample that is known to have two sets of HER2 genes per cell. Especially, tumor/cancer Or the HER2 mRNA level in the tumor sample, which is reflected by the number of HER2 cDNA sets in the tumor/cancer or tumor sample determined by RT-PCR and quantitative PCR, which can be relative to each cell known to have two The HER2 mRNA level reflected in the HER2 cDNA set in a control cell sample of the HER2 gene was normalized, and the HER2 cDNA sets were sequentially determined by RT-PCR and quantitative PCR. The HER2 mRNA level of the control sample is preferably the HER2 mRNA level reflected by the number of cDNA sets in the control sample determined by RT-PCR and quantitative PCR, which is one of the samples in the control group. The mRNA level of the reference gene is expressed; and the mRNA level of the reference gene is reflected by the number of cDNA sets of the reference gene determined by RT-PCR and quantitative PCR. Preferably, the reference gene is the same reference gene, such as TBP, used to determine HER2 mRNA levels in tumor/cancer or tumor samples.

Thus, a high HER2 mRNA level in a tumor/cancer or tumor sample as referred to in this application preferably refers to a high HER2 mRNA position as reflected by the high number of HER2 cDNA sets in a tumor/cancer or tumor sample. The quasi- and its lines are relative to the mRNA level of a reference gene; the HER2 cDNA sets are determined by RT-PCR and quantitative PCR, and the mRNA levels of the reference genes are sequentially sequenced by RT-PCR and quantification. The number of cDNA sets of the reference gene in the tumor/cancer or tumor sample determined by PCR is reflected. The high HER2 mRNA level in a tumor/cancer or tumor sample may refer to or correspond to the number of HER2 cDNA sets in the same cancer/tumor, and the system is greater than or equal to 75, greater than or equal to 80, greater than or equal to 90. , greater than or equal to 100, greater than or equal to 110, greater than or equal to 120, greater than or equal to 130, greater than or equal to 140, greater than or equal to 150, greater than or equal to 160, greater than or equal to 168, greater than or equal to 170, greater than or equal to 180, greater than or equal to 190, greater than or equal to 200, greater than or equal to 210, greater than or equal to 220, greater than or equal to 229 or greater than or equal to 248, which is relative to the number of cDNA sets of a reference gene in the sample, wherein the cDNA sets of the HER2 and the reference gene in the sample are sequentially sequenced by RT-PCR Quantitative PCR assay. The high HER2 mRNA level in a tumor/cancer or tumor sample may refer to or correspond to the number of HER2 cDNA sets in the same cancer/tumor, and the line is greater than or equal to 168, which is relative to a reference in the sample. The cDNA set of the gene, wherein the cDNA sets of the HER2 and the reference gene in the sample are sequentially determined by RT-PCR and quantitative PCR. The high HER2 mRNA level in a tumor/cancer or tumor sample may refer to or correspond to the number of HER2 cDNA sets in the same cancer/tumor, and the line is greater than or equal to 248, relative to one of the samples For the cDNA set of the reference gene, the cDNA sets of the HER2 and the reference gene in the sample are determined by RT-PCR and quantitative PCR in sequence. Preferably, the high HER2 mRNA level in a tumor/cancer or tumor sample may or may correspond to the number of HER2 cDNA sets in the same cancer/tumor, and the line is greater than or equal to 200, relative to the sample In the case of the cDNA set of a reference gene, the cDNA sets of the HER2 and the reference gene in the sample are sequentially determined by RT-PCR and quantitative PCR. More preferably, the high HER2 mRNA level in a tumor/cancer or tumor sample may or may correspond to the number of HER2 cDNA sets in the same cancer/tumor, and the line is greater than or equal to 229, relative to the sample a reference gene In terms of the number of sets of cDNA, the cDNA sets of HER2 and the reference gene in the sample were determined by RT-PCR and quantitative PCR in sequence.

In addition to the lower limit described above, a high HER2 mRNA level in a tumor/cancer or tumor sample may refer to or correspond to the number of HER2 cDNA sets in the same cancer/tumor, and the system is less than or equal to 820. , less than or equal to 850, less than or equal to 900, less than or equal to 950, less than or equal to 1000, less than or equal to 1100, less than or equal to 1200, less than or equal to 1300, less than or equal to 1400, less than or equal to 1500, less than or equal to 1600. , less than or equal to 1700, less than or equal to 1800 or less than or equal to 1900, which is relative to the number of cDNA sets of a reference gene in the sample, wherein the cDNA sets of the HER2 and the reference gene in the sample are sequentially RT-PCR and quantitative PCR assays. Preferably, the high HER2 mRNA level in a tumor/cancer or tumor sample may or may correspond to the number of HER2 cDNA sets in the same cancer/tumor, and the system is less than or equal to 820, relative to the sample In the case of the cDNA set of a reference gene, the cDNA sets of the HER2 and the reference gene in the sample are sequentially determined by RT-PCR and quantitative PCR.

Preferably, the high HER2 mRNA level in a tumor/cancer or tumor sample may or may correspond to the number of HER2 cDNA sets in the same cancer/tumor, and the system is greater than or equal to 200 and less than or equal to 820, It is based on the number of cDNA sets of a reference gene in the sample, wherein the cDNA sets of the HER2 and the reference gene in the sample are sequentially determined by RT-PCR and quantitative PCR. More preferably, a high HER2 mRNA level in a tumor/cancer or tumor sample may or may correspond to the cancer/tumor The number of HER2 cDNA sets in a sample, and the system is greater than or equal to 229 and less than or equal to 820, which is relative to the number of cDNA sets of a reference gene in the sample, wherein the cDNA sets of the HER2 and the reference gene in the sample are It was determined by RT-PCR and quantitative PCR in sequence.

The cancer as referred to in the present application may be stomach cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer (for example, non-small cell lung cancer), gastric cancer or endometrial cancer. The owners of these cancers all show excessive expression of HER2. Preferably, the cancer is gastric cancer, breast cancer or colorectal cancer. More preferably, the cancer is stomach cancer or breast cancer. In a preferred embodiment, the cancer is a stomach cancer. The stomach cancer system as referred to in the present application includes esophageal cancer. In another preferred embodiment, the cancer is breast cancer. The HER2 gene set number of the cancer is as described above. The cancer can be referred to as HER2 positive (HER2+) or as overexpressing HER2. Thus, one of the cancers as mentioned in this application can be HER2 positive. Additionally or alternatively, a cancer as referred to in this application may overexpress HER2. Whether a cancer is HER2 positive or overexpresses HER2 is initially determined, for example, using immunohistochemistry (IHC), and is then selectively determined by methods as outlined above, such as qPCR.

The inventors of the present application have discovered a specific binding member comprising a HER2 antigen binding site designed to be placed in a structural loop region of a fixed domain such as the CH3 domain, and useful for treating a The treatment with the bead monoclonal antibody and/or the trastuzumab antibody plus the betobeta monoclonal antibody is resistant or refractory to cancer. The cancer may be essentially a mixture of trastuzumab antibody and/or trastuzumab antibody plus perindopril. Treatment with antibodies is either resistant or refractory, or may result in acquired resistance or non-reactivity to treatment with trastuzumab antibody and/or trastuzumab antibody plus pertamine monoclonal antibody. The cancer may be a stomach cancer that is resistant or refractory to the treatment of trastuzumab antibody and/or trastuzumab antibody plus pertamine monoclonal antibody. Optionally, the cancer can be breast cancer, which is resistant or refractory to treatment with trastuzumab antibody and/or trastuzumab antibody plus pertituzil antibody. A method for determining whether a cancer is resistant or refractory to the treatment of a trastuzumab antibody and/or a trastuzumab antibody plus a perttomycin monoclonal antibody is well known in the art and is Skilled by the skilled person. For example, breast cancer that is resistant to treatment with trastuzumab antibody may be re-evaluated for the first time in weeks 8 to 12 or worse within three months after first-line trastuzumab antibody treatment. , whether or not chemotherapy is performed for the metastatic condition, or whether a new relapse is diagnosed during the treatment of the adjuvant trastuzumab antibody or within 12 months after the treatment. For a refractory breast cancer with a trastuzumab antibody, the disease may be worsened during the first radiological assessment after a treatment regimen containing trastuzumab antibodies that may contribute to the disease response or stabilize two or more lines. (Wong et al., 2011).

Additionally or alternatively, a patient as referred to in the present application may have demonstrated insufficient response to treatment with trastuzumab antibody and/or trastuzumab antibody plus perttomycin monoclonal antibody ( Inadequate response). Insufficient response to trastuzumab antibody and/or trastuzumab antibody plus pertamine monoclonal antibody may mean that the patient receives trastuzumab When the antibody of the strain and/or the antibody of trastuzumab and the antibody of pertituzide were treated, no tumor growth retardation occurred, or the degree of tumor growth retardation was insufficient. Optionally, a patient who is less responsive to trastuzumab antibody and/or trastuzumab antibody plus perindopril monoclonal antibody may be referred to as trastuzumab antibody and/or trastone The adverse reactions or adverse events caused by the treatment of the bead monoclonal antibody plus the pertamine monoclonal antibody, such as side effects and especially severe side effects, stop the trastuzumab antibody and/or the trastuzumab antibody plus One of the treatments for the treatment of the antibody to Perdole beads. Insufficient response to trastuzumab antibody and/or trastuzumab antibody plus pertamine monoclonal antibody, preferably when the patient receives trastuzumab antibody and/or trastuzide When the antibody was treated with the polyclonal antibody of Pertamine, no tumor growth retardation occurred, or the degree of tumor growth retardation was insufficient. The medical practitioner will have no difficulty in determining whether a particular patient has insufficient response to the treatment of trastuzumab antibody and/or trastuzumab antibody plus pertamine monoclonal antibody, as the medical practitioner is in the judgment Experience in the success of cancer treatment for specific patients and whether they should continue treatment. For example, if a patient's disease load and clinical symptoms are mild at the beginning of treatment, the long-term stability of the disease may be considered a favorable clinical state; if the disease load is large at the beginning of treatment and the clinical symptoms are significant, the disease may be stable. It is considered to be insufficiently reactive.

In the present application, there is provided a method for treating a cancer patient, (i) exhibiting insufficient response to trastuzumab antibody and/or trastuzumab antibody plus pertituzil antibody (ii) wherein the cancer is inherently resistant or refractory to the trastuzumab antibody and/or the trastuzumab antibody plus the pertallazine monoclonal antibody; or (iii) wherein the cancer has There is acquired resistance to trastuzumab antibody and/or trastuzumab antibody plus permethate monoclonal antibody, the method comprising administering a specific binding member to the patient, and selectively administering Another therapy, such as immunotherapy, such as the use of an immunotherapeutic agent, wherein the specific binding member is: (a) a specific binding member comprising a CH3 domain engineered to be placed in the specific binding member a HER2 antigen binding site of a structural loop region, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, which is HER2 binds to compete with a specific binding member such as (a).

A "specific binding member" is a molecule, such as a protein that binds to another molecule in a specific manner, such as a protein, such as HER2 (such as human HER2). A specific binding member can be native, or produced in part or in whole synthetically. A specific binding member typically comprises a molecule having one antigen binding site. For example, a specific binding member can be an antibody molecule or a fragment thereof comprising one of the antigen binding sites. A specific binding member can also be a non-antibody protein scaffold that binds to an antigen in a specific manner, such as a molecule comprising a modified immunoglobulin-like fold, such as a fibronectin domain ( ¹⁰ Fn3 domain) ). In the art, various methods are available for obtaining antibodies and antibody fragments against a target antigen. The antibody can be a monoclonal antibody, particularly a human monoclonal antibody, which can be obtained according to standard methods well known to those skilled in the art. A specific binding member can be a protein, such as an antibody immobilization region, and a HER2 antigen binding site thereof comprising a structural loop region designed to be inserted into the CH3 domain of the specific binding member, and comprising an amino acid Sequence FFTYW (sequence identification number: 12) and DRRRWTA (sequence identification number: 14).

Antibody fragments comprising an antigen binding site include, but are not limited to, Fab fragments (consisting of VL, VH, CL and CH1 domains); F(ab')2 fragments (containing bivalent fragments of two linked Fab fragments); a single-chain Fv molecule (scFv) consisting of a VH domain joined by a peptide linker and a VL domain that associates the two domains to form an antigen binding site; Bird et al., 1988 "Science" No. 242, pp. 423-426; B. Huston et al., 1988, "PNAS USA", No. 85, 5879-5883, B); Bispecific single-chain Fv dimer (PCT/US92) /09965); Fv fragment (consisting of the VL domain and VH domain of a single antibody); dAb fragment (consisting of a VH domain or a VL domain; Ward et al., 1989, "Nature", No. 341, 544- 546 pages in B; McCafferty et al., 1990, "Nature", No. 348, pp. 552-554; Holt et al., 2003, "Trends in Biotechnology," No. 21, pp. 484-490, B); Fd Fragments (consisting of the VH domain and the CH1 domain); double-stranded antibodies (multivalent or multispecific fragments constructed by gene fusion; WO94/13804; Holliger, 1993) a journal "Proc.Natl.Acad.Sci.USA" 6444-6448 on page 90 of the text B); and antigen-binding immunoglobulin (e.g. IgG) heavy chain fragment fixation, such as antigen-binding Fc fragment.

The antigen-binding Fc fragment may comprise an antigen binding site that is designed to be placed into a fixed domain of the Fc fragment, such as one of the CH2 or CH3 domains or a plurality of structural loop regions. The preparation of antigen-binding Fc fragments is described in WO 2006/072620 and WO 2009/132876. For use in the present invention, one specific binding member is preferably an antigen-binding Fc fragment, or Fc comprising an antigen-binding fragment thereof, antigen-binding Fc fragment also referred Fcab ^TM. One of the specific binding members for use in the present invention is a better one-antigen-binding Fc fragment. The specific binding member can be an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or lgM antigen binding Fc fragment. Most preferably, one of the specific binding members as referred to in this application is an IgGl (e.g., human IgGl) antigen binding Fc fragment. In a specific embodiment, a specific binding member is an IgGl antigen binding Fc fragment comprising a hub or portion thereof, a CH2 domain, and a CH3 domain.

An antigen-binding Fc fragment can be incorporated into an immunoglobulin molecule. Thus, one of the specific binding members for use in the present invention may be an IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM molecule comprising an antigen binding site in the Fc region. The molecules may comprise a second CDR-type binding site in the variable region of the molecule to have bispecificity. In particular, a specific binding member for use in the present invention may be an IgG1 molecule comprising an antigen binding site in the Fc region.

The molecular weight (MW) of a specific binding member as mentioned in the present application is preferably 60 kD or less, more preferably 55 kD or less, 54 kD or less or 53 kD or less. The specific binding member H561-4 disclosed in the present application has a molecular weight of about 53 kD. The specific binding member H561-4 is also known as FS102.

A specific binding member as referred to in the application, which may comprise a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, wherein the HER2 antigen binding site is Contains or contains the amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14). Optionally, a specific binding member may comprise a HER2 antigen binding site designed to be inserted into a structural loop region of a CH3 domain of a specific binding member, wherein the HER2 antigen binding site comprises an amino acid sequence FFTYW ( Sequence identification number: 12), NGQPE (sequence identification number: 13) and DRRRWTA (sequence identification number: 14).

A specific binding member as referred to in the application, which may comprise a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, wherein the HER2 antigen binding site is The amino acid sequence FFTYW (SEQ ID NO: 12) and the amino acid sequence DRRRWTA (SEQ ID NO: 14) are included. In particular, a specific binding member as referred to in the present application, which may comprise a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, wherein the HER2 antigen binding site The dot contains the amino acid sequence FFTYW (SEQ ID NO: 12) located in the AB loop, and the amino acid sequence DRRRWTA (SEQ ID NO: 14) located in the EF loop of the CH3 domain. A specific binding member, as referred to in this application, preferably comprises a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, wherein the HER2 antigen binding site is Contains amino acid sequence FFTYW (sequence identification number: 12) and DRRRWTA (sequence identification No.: 14), wherein the sequence identification number: 12 is located in the 14th to 18th residues of the AB loop of the CH3 domain, and the sequence identification number: 14 is located at the 92nd to 98th residues of the EF loop of the CH3 domain, and The residues are numbered according to the IMGT (ImMunoGeneTics) numbering method. In another preferred embodiment, a specific binding member, as referred to in the present application, comprises a HER2 antigen binding site of a structural loop region designed to be inserted into the CH3 domain of the specific binding member, wherein The HER2 antigen binding site comprises an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), wherein the sequence identification number: 12 is located in the 381 to 385 residues of the EF ring in the CH3 domain. Base, and sequence identification number: 14 is located in the 440th to 450th residues, and the residues are numbered according to KABAT (Kabat et al., published by the US Department of Health and Human Services in 1987) The fourth edition of the "Sequences of Proteins of Immunological Interest" and its updated version are currently available at the website immuno.bme.nwu.edu or using any kind of Internet search. The engine searches for "Kabat"). The backbone of a specific binding member may comprise a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, wherein the HER2 antigen binding site comprises an amino acid sequence FFTYW ( The sequence identification number: 12) and the amino acid sequence DRRRWTA (SEQ ID NO: 14) can be a heavy chain immobilization region of IgG1 such as human IgG1. Human IgG1 can be of any isotype, such as G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z) (see, for example, Jefferis et al., 2009 issuer "mAbs", page 1, page 1B Text).

One of the specific binding member lines as referred to in the present application preferably comprises a CH3 domain of sequence identification number: 11. A specific binding member may further comprise a CH2 domain of sequence identification number: 10. One of the specific binding member lines as referred to in the present application preferably comprises the sequence of Sequence Identification Number: 8. In a particular embodiment, one of the polypeptides, as specifically mentioned in the present application, specifically binds to a member of the sequence identification number: 8 as a dimer of a polypeptide. For example, the specific binding member can be a dimer formed by two polypeptides of sequence identification number: 8, such as a dimer composed of two polypeptides, wherein each polypeptide is identified by sequence identification number: The sequence shown consists of the sequence. In a particular embodiment, the dimerization system comprises one or two disulfide bonds located in the hinge zone. For example, in a particular embodiment, a specific binding member is a protein comprising two polypeptides, wherein each polypeptide comprises a sequence ID: 8 or consists of a sequence ID: 8 wherein the two polypeptides are located at a hub The region is linked by one or two disulfide bonds formed by cysteine (sequence identification number: the second and fifth cysteine in 8) (this specific binding member is in this application) Also known as H561-4).

It has previously been reported that the loss of the C-terminus of the CH3 domain from the amino acid (K) or the C-terminal lysine and the adjacent glycine (GK) residue occurs during the manufacture of the immunoglobulin. This so-called 'C-terminal shearing action' is believed to be a general phenomenon and does not affect the function of such micro-variants (Beck et al., 2013). It is expected that a C-terminal truncation may occur during the manufacturing of H561-4. The H561-4 CH3 domain includes a C-terminal lysine and a glycine residue which may be c-terminally cleaved, and is shown in Sequence ID: 11 and is also present, for example, in Sequence Identification Number: 8, as follows The official sequence table is shown.

Thus, it can be seen that one of the specific binding members as referred to in the present application may comprise a CH3 domain of sequence identification number: 11 or a subtraction of one or two C-terminal residues (eg, C-terminal lysine or C). Sequence identification number: 11 from the CH3 domain of the amine acid and the glycine residue). A specific binding member as referred to in this application may comprise a sequence of sequence number: 8 or a subtraction of one or two C-terminal residues (eg, C-terminal amide or lysine and glycine) Sequence of sequence identification number: 8). When referring to the sequence identification numbers 8 and 11 used in the present application, it should be construed as being also applicable to the absence of one or two C-terminal amino acid residues (such as C-terminal amino acid or lysine and glycine). These sequences of residues). When referring to a CH3 domain as described in this application, it encompasses a CH3 domain that loses one or two C-terminal amino acid residues (such as a C-terminal amino acid or an lysine and a glycine residue). In a particular embodiment, a specific binding member comprises a protein comprising two polypeptides, wherein each polypeptide comprises a sequence ID: 8 or consists of a sequence ID: 8 wherein the two polypeptides are located in a hub Linked by one or two disulfide bonds formed by cysteine (sequence identification number: the second and fifth cysteine in 8), and one or both of the polypeptides are absent The C-terminus is separated from the amino acid (K) or the C-terminal acid and the adjacent glycine (GK) residue.

Variants of such specific binding members can also be used in the present invention. For example, a specific binding member as referred to in the present application, which may comprise a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, wherein the HER2 antigen binding site Dot contains or contains amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14) and has 3, 2 or 1 amines Substituted, deleted or added. The amino acid substitution can be a retention amino acid substitution. Optionally, a specific binding member may comprise a HER2 antigen binding site designed to be inserted into a structural loop region of a CH3 domain of a specific binding member, wherein the HER2 antigen binding site comprises an amino acid sequence FFTYW ( Sequence identification number: 12), NGQPE (sequence identification number: 13) and DRRRWTA (sequence identification number: 14) and their substitution, deletion or addition with 3, 2 or 1 amino acids. Also included are the variants and the amino acid sequence contained therein at up to 1, 2, 3, 4, 5, 1 to 3, 1 to 5 or 1 to 10 amine groups. Acid substitution (eg, retention substitution), deletion or addition) differs from sequence identification number: 8, and/or includes agreement with sequence identification number: 8 of at least 90%, 95%, 96%, 97%, 98. % or 99% of such variants, wherein the variants bind to human HER2 in a specific manner, preferably at the same or overlapping epitopes, and selectively elicit apoptosis in HER2-positive cancer cells. For example, the percent amino acid sequence identity (%) is defined as the amino acid residue in a candidate sequence after the ratio of the sequence and, as appropriate, the gap is included to achieve the maximum sequence identity percentage. The percentage of amino acid residues in the reference amino acid sequence is consistent. The alignment can be performed for the purpose of determining the percent identity of the amino acid sequence in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine the appropriate parameters for measuring the alignment, including any algorithm required to achieve maximum alignment over the full length of the sequence being compared. These changes usually do not result in loss of function and therefore include such changes One of the amino acid sequence-specific binding members retains the ability to bind to HER2. For example, its HER2 binding affinity may be the same as a specific binding member that does not have this substitution.

Characterization of the binding affinity of antibodies is generally performed on antibody concentrations that occupy half of the antigen binding sites, and is referred to as dissociation constant (Kd).

With respect to the HER2 binding properties of the specific binding member, the binding affinity Kd is preferably less than 10 ^-8 M and/or its IC50 potency is less than 10 ^-8 M, or its Kd or IC50 system is less than 10 ^-9 M, preferably less than 10 ^-10 M or even less than 10 ^-11 M, and best in the range of Pimol.

IC50, also known as EC50 or 50% saturation concentration, is a measure of the binding potency of a specific binding member. It is the molar concentration of a binding agent that reaches 50% of the maximum possible binding in equilibrium or saturation. The effectiveness of a binding agent is generally defined by its IC50 (here understood as the EC50 value). The IC50 of a particular binding agent can be calculated by determining the concentration of binding agent required to initiate the maximum saturation of the semi-saturated state. The elucidation of IC50 or EC50 values is useful for comparing the efficacy of specific binding members or specific binding member variants with similar efficacy, especially when assayed in a saturation binding assay rather than a competition assay. In this case, the IC50 or EC50 value is regarded as the concentration, which measures the plasma concentration which achieves half of the maximum effect (50%) in vivo. The lower the IC50 or EC50, the greater the potency of specific binding members and the lower the concentration of specific binding members required to inhibit maximal biological responses such as effector function or cytotoxic activity.

An isolated protein (a HER2 binding agent) that binds to human HER2 in a specific manner may comprise two polypeptides, wherein each human IgG1 heavy chain fragment contained in the polypeptide comprises a CH2 domain and a CH3 domain, wherein the CH3 domain The included AB loop system comprises the amino acid sequence of SEQ ID NO: 191, and comprises a CD loop system comprising the amino acid sequence of SEQ ID NO: 241 and an EF loop system comprising an amino acid sequence. 370. The CH3 domain may comprise an amino acid sequence of sequence identification number: 11. The CH2 domain may comprise an amino acid sequence of sequence identification number: 10. The CH2 domain comprised by the isolated protein may comprise a sequence identification number: 10, and the CH3 domain contained therein may comprise a sequence identification number: 11. The human IgG1 heavy chain fragment can comprise a hub. The two polypeptides can be linked by a disulfide bond. The two polypeptides can be linked by two disulfide bonds. The isolated protein binds to human Her2 and its binding affinity Kd is less than 10 ^-8 M. The isolated protein can be cytotoxic. The isolated protein can trigger at least one of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDC), or apoptotic activity. The isolated protein can have a molecular weight of up to 60 kD. In a particular embodiment, the binding affinity Kd of an isolated protein that binds to human Her2 in a specific manner is less than 10 ^-8 M, which is cytotoxic, and its molecular weight is up to 60 kD. In a particular embodiment, an isolated protein line that binds to human Her2 in a specific manner comprises two polypeptides, wherein each human IgG1 heavy chain fragment comprised comprises a CH2 domain and a CH3 domain, wherein the CH3 domain The included AB loop system comprises the amino acid sequence of SEQ ID NO: 191, and comprises a CD loop system comprising the amino acid sequence of SEQ ID NO: 241 and an EF loop system comprising an amino acid sequence. 370; and the molecular weight of the isolated protein is up to 60 kD, the binding affinity is less than 10 ^-8 M, and is cytotoxic. A pharmaceutical composition comprising any of the isolated proteins described in the application and a pharmaceutically acceptable carrier is provided in the present application.

A binding agent that competes with a HER2 binding agent for binding to a HER2 binding agent or that binds to one of the HER2 binding agents, and may be an antibody (such as a full length antibody) or an antigen binding fragment thereof, such as via a CDR. HER2 binds to one of the antibodies or antigen-binding fragments. In a particular embodiment, the HER2 binding competes with a specific binding member and the specific binding member comprises a HER2 antigen binding site designed to be inserted into a structural loop region of its CH3 domain and comprises an amino acid sequence FFTYW (SEQ ID NO: 12) specifically binds to one of DRRRWTA (SEQ ID NO: 14), or competes with a specific binding member as described above for one specific binding member, preferably comprising One of the one or more and preferably two structural loop regions of one of the specific binding members is placed in one of the HER2 antigen binding sites. Preferably, the one specific binding member comprises one of the one or more and preferably two structural loop regions of the CH3 domain of the specific binding member. The structural loops in the CH3 domain are located at 7th to 21th residues (AB loop), 25th to 39th residues (BC loop), 41st to 81th residues (CD loop), 83rd to 85th residues a base (DE loop), a 89th to 103th residue (EF loop), and a 106th to 117th residue (FG loop), wherein the residues are based on the IMGT (ImMunoGeneTics) numbering method (WO 2006/072620 A1) Numbering. More preferably, the one specific binding member comprises a HER2 antigen binding site of the structural loop region AB and EF designed to be inserted into the CH3 domain of the specific binding member. Especially, the one The specific binding member may comprise a 14th to 18th residue of the CH3 domain of the specific binding member and a HER2 antigen binding site of the 92nd to 98th residues, wherein the residues are based on IMGT Numbering is numbered. The 14th to 18th residues are located in the AB loop, while the 92nd to 98th residues are located in the EF loop of the CH3 domain. The specific binding member may comprise a HER2 antigen binding site of 381 to 385 residues and 440 to 450 residues designed to be inserted into the specific binding member, wherein the residues are based on KABAT Numbering. The 381th to 385th residues are located in the AB loop, while the 440th to 450th residues are located in the EF loop of the CH3 domain. Optionally, the specific binding member can comprise a HER2 antigen binding site of structural loop regions AB, CD and EF that are designed to be placed into the CH3 domain of the specific binding member. The specific binding member may comprise a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and the amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (sequence) Identification number: 14), the specific binding member competes with HER2 binding, which is preferably a dimer of a polypeptide of sequence identification number: 8, such as a protein comprising two polypeptides, in the two polypeptides Each of them contains a sequence identification number: 8.

Suitable methods for determining whether two specific binding members compete for binding by the same target are well known in the art and will be apparent to those skilled in the art, and in this case the HER2 (e.g., human HER2) ). Such methods include competitive methods using surface plasma resonance (SPR), enzyme linked immunosorbent assay (ELISA), fluorescent activated cell sorting (FACS) or immunocytochemistry. Surface electricity The slurry resonance (SPR) method includes BIAcore. Thus, one of the specific binding members that competes with the second specific binding member in HER2 binding can be, for example, surface plasma resonance (SPR) (eg, BIAcore), enzyme-linked immunosorbent assay (ELISA), and fluorescent-activated type. As measured by cell sorting technique (FACS) or immunocytochemistry, it competes with the second binding member for HER2 binding. One of the specific binding members that compete with the second specific binding member for HER2 binding is preferably competitive with the second binding member in HER2 binding as measured using surface plasma resonance (SPR) as BIAcore. As shown in Example 1 of the present application, the trastuzumab antibody and the Pertuxim mono antibody did not compete with the specific binding member H561-4 for HER2 binding; H561-4 was designed to be placed into its CH3 A HER2 antigen binding site of a structural loop region of the domain, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14).

For the competition methods listed above, HER2 is immobilized on a wafer surface (BIAcore method), either on a plate (ELISA) or on a cell surface (FACS and immunohistochemistry). Then, one of the two specific binding members to be compared is incubated with the immobilized HER2 for a period of time to cause saturation of the HER2 antigen, thereby blocking all HER2 epitopes bound by the specific binding member. . The second specific binding member is then incubated with the immobilized HER2. Binding of the second specific binding member to HER2 indicates that the specific binding member lines bind to different epitopes on HER2; however, if the second binding member does not bind to the immobilized HER2, then the second specific binding is indicated The member line competes with the first specific binding member for HER2 binding, And the bispecific binding member line binds to the same (or overlapping) epitope on HER2.

In a particular embodiment, the inhibition of binding of H561-4 to HER2 (eg, human HER2) by a binding member is at least 50%, 60%, 70%, 80%, 90%, 95% or higher, such as Measured by Biacore, ELISA or FACS. In a particular embodiment, H561-4 inhibits binding of the binding member to HER2 by at least 50%, 60%, 70%, 80%, 90%, 95% or higher, such as by Biacore, ELISA Or measured by FACS. In a particular embodiment, the inhibition of binding of H561-4 to HER2 by a binding member is at least 50%, 60%, 70%, 80%, 90%, 95% or higher, and H561-4 for The inhibition of binding of the binding member to HER2 is at least 50%, 60%, 70%, 80%, 90%, 95% or higher, as determined by Biacore, ELISA or FACS (ie, the competition system) Two-way).

In a particular embodiment, a binding member competes with H561-4 for HER2 binding, but does not compete with a trastuzumab antibody or a pertolecular antibody. In a particular embodiment, the binding of a binding member to the binding of the trastuzumab antibody or the Pertuzumab antibody to HER2 is no more than 50%, 40%, 30%, 20%, 10% or less. , as measured by Biacore, ELISA or FACS. In a particular embodiment, the inhibition of binding of H561-4 to HER2 by a trastuzumab antibody or a pertolecular antibody is no more than 50%, 40%, 30%, 20%, 10% or less. , as measured by Biacore, ELISA or FACS. In a particular embodiment, inhibition of binding of H561-4 to HER2 by a binding member and/or H561-4 The inhibition of the binding of the binding member is at least 50%, 60%, 70%, 80%, 90%, 95% or higher, but for the binding of the trastuzumab antibody or the Pertuzumab antibody Inhibition of action and/or inhibition of binding to HER2 and/or inhibition of binding of H561-4 to HER2 by trastuzumab antibody or pertolecular monoclonal antibody is not more than 50%, 40%, 30 %, 20%, 10% or lower, as measured by Biacore, ELISA or FACS. For example, inhibition of binding of H561-4 to HER2 by a binding member and/or inhibition of binding of H561-4 to the binding member is at least 50%, but for trastuzumab antibody or pertustidine Inhibition of binding of the strain antibody to HER2 and/or inhibition of binding of H561-4 to HER2 by trastuzumab antibody or Pertamine monoclonal antibody, such as by Biacore, ELISA or FACS Measured. In another example, inhibition of binding of H561-4 to HER2 by a binding member and/or inhibition of binding of H561-4 to the binding member is at least 90%, but for trastuzumab antibodies or Inhibition of binding of Pertitux monoclonal antibody to HER2 and/or inhibition of binding of H561-4 to HER2 by trastuzumab antibody or Pertoxaz antibody alone, such as by Biacore Measured by ELISA or FACS.

One of the specific binding members as mentioned in the present application can bind to the dimeric HER2. The binding affinity of the specific binding member to the dimeric HER2 can be 1 nM or higher. This specific binding member may preferentially bind to the dimeric HER2 compared to the haplotype HER2. Preferably, the specific binding member binds to the same epitope on the human HER2 extracellular domain, the specific binding member comprising one of the two polypeptides, Each polypeptide line contains a sequence identification number: 8. The epitope can have a configuration, that is, a non-linear epitope. This epitope can span domain 1 and domain 3 of HER2. The epitope may comprise, or consist of, sequence identification numbers 15, 16 and 17. The epitope may comprise, or consist of, 13th to 27th, 31st to 45th, and 420th to 475th amino acids of the HER2 extracellular domain. Optionally, the epitope can be located within sequence IDs 15, 16 and 17, or within the 13th to 27th, 31st to 45th and 420th to 475th amino acids of the HER2 extracellular domain. The sequence of the HER2 extracellular domain is shown in Sequence ID: 18.

Methods for determining whether the two specific binding members bind to the same epitope are well known in the art and will be apparent to those skilled in the art. Such methods include peptide scanning techniques (Pepscan), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and X-ray crystal diffraction. In the localization study of epitopes, an antigen and, in this case, HER2, can be divided into small regions, ie peptides or domains, and then tested for binding to specific binding members. The epitope to which the specific binding member binds can be inferred by showing the region of binding. Antigenic epitopes can be compared by comparison to regions of two different specific binding members. Alternatively, mutations can be made in the antigen and its HER2 in the present application, and then tested for binding to a specific binding member. If a residue mutation results in a loss of binding, it indicates that it is part of the epitope that the specific binding member binds to. Care must be taken, however, to ensure that the mutation does not result in a complete loss of HER2 structure. In the X-ray crystal diffraction method, the antigen and its HER2 system and the special in the present application The heterosexual bonding member crystallizes into a complex. The crystal data is then used to determine the epitope that the specific binding member binds to. The HER2 epitopes to which the two different specific binding members bind can then be compared.

Methods for determining the number of sets of HER2 genes are well known in the art and will be apparent to those skilled in the art. For example, the HER2 gene sets can be determined using fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), or quantitative polymerase chain reaction (qPCR). Previous studies have reported that, as shown by linear regression analysis, the HER2 gene sets are highly correlated by CISH and FISH (Garcia-Caballero et al. 2010). Further studies confirmed that the HER2 amplification state measured by the two methods is also strongly correlated (Arnould et al., 2012; B, Mollerup et al., 2012). Therefore, it is expected that the number of sets of HER2 genes measured by CISH is similar to the number of sets of genes measured by FISH. The number of HER2 gene sets is preferably determined using FISH. Likewise, methods for determining HER2 mRNA levels are known in the art and will be apparent to those skilled in the art. For example, HER2 mRNA levels can be determined using reverse transcription PCR (RT-PCR) followed by quantitative PCR (qPCR). Excessive expression of the HER2 gene can be measured by immunohistochemistry (IHC) to determine the amount of HER2 expressed on the cell surface.

FISH uses fluorescently labeled probes to detect specific DNA sequences in tissue samples by fluorescence microscopy. The HER2 gene expansion in cancer-fixed and embedded paraffin-embedded (FFPE) cancer tissue samples was determined quantitatively by the FISH method using a specific probe heterozygous for the HER2 sequence. Increase the effect. The US Food and Drug Administration has approved different methods for determining the number of HER2 gene sets by FISH (Press et al., 2002). In one method, the average number of HER2 genes per tumor cell in a sample is determined by measuring and comparing the number of HER2 and chromosome 17. In this assay, a CEP17 probe (a chromosomal counting probe and its direct-labeled fluorescent DNA probe specific for the alpha satellite DNA sequence in the region of chromosome 17) can be used as a chromosome aneuploidy. Internal control group. The HER2 gene and CEP17 are labeled with different fluorophores and can be viewed using a specific filter of the microscope. The measurement of HER2 gene amplification is based on the ratio of the number of HER2 copies counted by microscopy to the number of copies of CEP17, thereby eliminating the possibility that the HER2 gene set is increased by the aneuploidy of chromosome 17. For example, when the ratio is greater than or equal to 10, the average number of HER2 gene sets per tumor/cancer cell is greater than or equal to 10. With regard to the number of HER2 gene sets or the average number of HER2 genes per tumor/cancer cell referred to in this application, any increase in the number of HER2 genes attributed to chromosomal aneuploidy can be excluded, especially due to Aneuploidy of chromosome 17. Optionally, in a preferred method, the overall HER2 gene set can be determined by directly counting the HER2 signal and without standardizing the CEP17 signal. This is the preferred method because counting the overall HER2 gene set avoids false positive or false negative results attributed to loss or increase in the near mid-segment of chromosome 17, which is more universal than in true multichromosomal (replicating whole chromosomes) The observed in the middle is higher and can result in a change in the HER2/CEP17 ratio (Wolff et al., 2013, B; Hanna et al., 2014). Alternatively, on FFPE Only one HER2 probe was used for FISH as an indirect method for mapping the HER2 gene (Press et al., 2002).

It has been found that the gene set number of a particular tumor obtained by FISH and qPCR is highly correlated (see Example 9). Thus, conversion between GCN values obtained by different methods, such as FISH and qPCR, is possible.

Also provided is the use of a specific binding member for the manufacture of a therapeutic drug for a cancer comprising a HER2 antigen binding site designed to be placed in a structural loop region of a fixed domain such as the CH3 domain. And providing a specific binding member that competes with the specific binding member for binding to HER2, wherein each tumor cell of the cancer has a HER2 gene set, such as an average number of HER2 genes, greater than or equal to 10, And/or wherein the cancer has a high HER2 mRNA level.

The invention further provides the use of a specific binding member that binds to HER2 in the manufacture of a therapeutic drug for a cancer, which is optionally used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent, Wherein each tumor cell of the cancer has a HER2 gene set, such as an average number of HER2 genes, greater than or equal to 10, and/or the cancer has a high HER2 mRNA level, and wherein the specific binding member is: a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that is associated with HER2 binding (a) One of the specific binding members competes.

Also provided is the use of a specific binding member that binds to HER2 in the manufacture of a therapeutic drug for a disease, which is optionally used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent; a tumor sample obtained from the patient, and measuring that each of the tumor cells has an average number of HER2 gene sets greater than or equal to 10; and/or wherein a tumor sample obtained from the patient is measured and measured Having a high HER2 mRNA level, wherein the specific binding member is: (a) a specific binding member comprising a HER2 antigen binding site of a structural loop region designed to be inserted into the CH3 domain of the specific binding member Point, and containing amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, which is in HER2 binding and one of (a) Specific binding members compete.

Further provided is the use of a specific binding member that binds to HER2 in the manufacture of a therapeutic drug for a cancer, which is optionally used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent, the treatment Including: (i) determining the number of sets of HER2 genes for each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; and (ii) if the number of HER2 genes per tumor cell, such as the average HER2 gene If the number of sets is greater than or equal to 10, the specific binding member is administered to the patient, optionally in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent; Wherein the specific binding member is: (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with HER2-specific binding members as one of (a).

Also provided is the use of a specific binding member that binds to HER2 in the manufacture of a therapeutic drug for a cancer, which is optionally used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent, the treatment The method comprises: (i) determining a HER2 mRNA level of a tumor sample obtained from the patient; and (ii) treating the patient with the specific binding member if the HER2 mRNA level is high, optionally In combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent; wherein the specific binding member is: (a) a specific binding member comprising a CH3 designed to be placed in the specific binding member a HER2 antigen binding site of a structural loop region of the domain, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, It competes with a specific binding member such as (a) in HER2 binding.

In one aspect, the invention provides for treating a patient with cancer In one method, wherein the number of HER2 genes per tumor cell of the cancer, such as the average number of HER2 genes, is greater than or equal to 10, and wherein the method comprises administering to the patient a therapeutically effective amount of the following: a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with a specific binding member such as (a) for HER2 binding; or (c) as (a) or (b) Any of the specific binding members, which are used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

A tumor sample obtained from the patient is determined, wherein each tumor cell has a HER2 gene set, such as an average number of HER2 genes, which is greater than or equal to 10. Accordingly, the present invention provides a method for treating cancer in a patient, wherein a tumor sample obtained from the patient is measured, and wherein each tumor cell has an average number of HER2 gene sets greater than or equal to 10, and wherein The method comprises administering to the patient a therapeutically effective amount of: (a) a specific binding member comprising a HER2 antigen designed into a structural loop region of the CH3 domain of the specific binding member. Binding site, and containing amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, which is in HER2 binding and (a) one of the specific binding members competes; or (c) any of the specific binding members of (a) or (b), which is used in combination with another therapy such as immunotherapy, such as the use of an immunization Anti-tumor therapy.

The method may comprise: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; and (ii) if the number of sets of HER2 genes per tumor cell, such as Where the average HER2 gene set number is greater than or equal to 10, the patient is treated with the specific binding member, optionally in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

Accordingly, the present invention also provides a method of treating cancer in a patient, wherein the method comprises: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; And (ii) if the number of sets of HER2 genes per tumor cell, such as the average number of sets of genes, is greater than or equal to 10, then the patient is administered a therapeutically effective amount of a specific binding member, optionally with another such as immunotherapy A combination of therapies, such as the use of an immunotherapeutic agent, wherein the specific binding member is: (a) a specific binding member comprising a structural loop region designed to be inserted into the CH3 domain of the specific binding member a HER2 antigen binding site, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with HER2-specific binding members as one of (a).

In one aspect, the invention provides a method of treating cancer in a patient, wherein the cancer has a high HER2 mRNA level, and wherein the method comprises administering to the patient a therapeutically effective amount of the following: (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) And DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with HER2-specific binding members as one of (a); or (c) as (a) or b) Any of the specific binding members, which are used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

In one aspect, the invention provides a specific binding member that binds to HER2, which is optionally used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent, for use in a diseased cancer In a method of treatment wherein the cancer has a high HER2 mRNA level, and wherein the specific binding member is: (a) a specific binding member comprising a CH3 domain engineered to be placed in the specific binding member a HER2 antigen binding site of a structural loop region, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); (b) a specific binding member that competes with HER2-specific binding members as one of (a).

Tumor samples obtained from this patient may have been found to have high HER2 mRNA levels by assay. Accordingly, the present invention provides a method of treating cancer in a patient, wherein a tumor sample obtained from the patient has been determined to have a high HER2 mRNA level by assay, and wherein the method comprises administering to the patient A therapeutically effective amount of: (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid Sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with HER2 for binding to a specific binding member such as (a); (c) Any of the specific binding members of (a) or (b), which are used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

The method can comprise: (i) determining a HER2 mRNA level of a tumor sample obtained from the patient; and (ii) treating the patient with the specific binding member if the HER2 mRNA level is high, It is optionally used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

Accordingly, the present invention also provides a method of treating cancer in a patient, wherein the method comprises: (i) determining the HER2 mRNA level of a tumor sample obtained from the patient; and (ii) administering a therapeutically effective amount of a specific binding member to the patient if the patient has a high HER2 mRNA level. Optionally, in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent, wherein the specific binding member is: (a) a specific binding member comprising a designed specific binding a HER2 antigen binding site of a structural loop region of the CH3 domain of the member, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member It competes with HER2-specific binding members in one of (a).

The specific binding members described herein as a single agent or in combination with one, two, three or more agents (eg, immuno-anti-tumor agents) are designed to treat patients and preferably human patients. In the method. A specific binding member for use as a single agent or in combination with another agent (e.g., an immuno-antitumor agent) is usually administered in the form of a pharmaceutical composition which may comprise at least one component other than the specific binding member. Thus, in addition to the active ingredient, the pharmaceutical compositions described herein and as used herein may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or are well known to those skilled in the art. Other substances. These substances should not be toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance will depend on the route of administration, which may be via injection, such as intravenous or subcutaneous injection. The specific binding member used as a single agent or in combination with another agent (such as an immuno-antitumor agent) may preferably be administered intravenously or subcutaneously, but may also be administered by any means including but not limited to oral or nasal. Internal, intra-oral, transdermal, mucosal, topical (eg gel, ointment, lotion, cream, etc.), intraperitoneal, intramuscular, intrapulmonary (eg AERx ^TM inhalability available from Aradigm ⁾ The technology is either Inhance ^(TM) pulmonary delivery system available from Inhale Therapeutics, vaginal, parenteral, rectal or intraocular administration.

Liquid pharmaceutical compositions typically comprise a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil or synthetic oil. Physiological saline solutions, dextrose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous injection or injection into a site of pain, the active ingredient is in the form of a parenterally acceptable aqueous solution which is pyrogen free and has suitable pH, isotonicity and stability. One skilled in the art will be able to prepare a suitable solution using, for example, an isotonic emollient carrier such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be used as needed. Those skilled in the art are aware of numerous methods for preparing pharmaceutical formulations. See, for example, Marcel Dekker Co., New York, USA, published in 1978 and edited by Robinson, "Sustained and Controlled Release Drug Delivery Systems".

Administration can be administered in a "therapeutically effective amount" sufficient to show one of the benefits to a patient. This benefit can at least improve at least one symptom. Thus, "treatment" A designated disease means improving at least one symptom. The actual amount administered and the rate and duration of administration will depend on the nature and severity of the condition being treated, the particular condition being treated, the clinical condition of the individual patient, the cause of the condition, and the delivery site of the composition. , the type of compound, the method of administration, the schedule of administration, and other factors known to the practitioner. It is within the responsibility of the general practitioner and other physicians to open treatment prescriptions, such as determining the dosage, and may be subject to the severity and/or severity of the symptoms of the disease being treated. Suitable antibody dosages are well known in the art (Ledermann et al. (1991) in the journal "Int. J. Cancer", 47th, pp. 659-664; and Bagshawe et al. (1991) in the journal "Antibody," Immunoconjugates and Radiopharmaceuticals, No. 4, pp. 915-922, B). Specific dosages suitable for the type of drug administered, as indicated in this application or in the Physicians' Desk Reference (2003), may also be used. A therapeutically effective amount or a suitable dose of a specific binding member can be determined by comparing its in vitro activity with in vivo activity in an animal mode. Methods for extrapolating effective doses in mice to other test animals to humans are known. The exact dose will depend on several factors, including the size and location of the region to be treated and the exact nature of the specific binding member. Treatment can be performed daily, twice a week, once a week, once every two weeks, once every three weeks or once a month, at the discretion of the physician. Treatment can be administered before and/or after surgery and can be administered or administered directly to the anatomical site of the surgical treatment. Treatment can be administered before and/or after another therapy such as radiation therapy or chemotherapy.

In one aspect, the invention provides a method for identifying whether a cancer of a patient is susceptible to treatment by: (a) a specific binding member comprising a designer designed to be placed in the specific binding member a HER2 antigen binding site of a structural loop region of the CH3 domain, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, Is in competition with HER2 binding to a specific binding member such as (a); the method comprises: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as an average The number of sets of genes, wherein if the number of sets of genes per tumor cell, such as the average number of sets of genes, is greater than or equal to 10, it indicates that the cancer line is sensitive to the treatment of the specific binding member.

A method for identifying whether a cancer of a patient is sensitive to treatment may further comprise (ii) if the number of HER2 genes per tumor cell, such as the average number of HER2 gene sets, is greater than or equal to 10, the patient is selected for the Treatment of specific binding members.

In one aspect, the invention provides a method for predicting a response of a cancer to treatment of: (a) a specific binding member comprising a CH3 domain engineered to be placed in the specific binding member a HER2 antigen binding site in the structural loop region, and an amino acid sequence containing FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that binds to HER2 Functionally competing with a specific binding member such as (a); the method comprises: (i) determining the HER2 gene set of the HER2 gene of each tumor cell of a tumor sample obtained from a patient, such as the average number of HER2 genes, wherein if the number of sets of genes per tumor cell, such as the average number of HER2 genes, is greater than or equal to 10, indicating that the cancer line is sensitive to the treatment of the specific binding member, and if the number of sets of genes per tumor cell, such as the average number of HER2 gene sets is less than 10, it indicates that the cancer system is not treated for the specific binding member. sensitive.

A method for predicting a response of a cancer to treatment, which may further comprise: (ii) if the number of HER2 genes per tumor cell, such as the average number of HER2 gene sets, is greater than or equal to 10, the cancer is selected for the specific binding. Treatment of members.

In one aspect, the invention provides a method for identifying whether a cancer of a patient is susceptible to treatment by: (a) a specific binding member comprising a designer designed to be placed in the specific binding member a HER2 antigen binding site of a structural loop region of the CH3 domain, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, Is a member that competes with a specific binding member such as (a) in HER2 binding; or (c) a member that specifically binds to one of (a) or (b), which is used in combination with another therapy such as immunotherapy, Such as the use of an immunotherapeutic agent; the method comprises: (i) determining the HER2 mRNA position of a tumor sample obtained from the patient Quasi, where a high HER2 mRNA level is present, indicates that the cancer line is sensitive to the treatment of the specific binding member.

A method for identifying whether a cancer of a patient is sensitive to treatment may further comprise (ii) if the tumor sample has a high HER2 mRNA level, the patient is selected for treatment with the specific binding member.

In one aspect, the invention provides a method for predicting a response of a cancer to treatment of: (a) a specific binding member comprising a CH3 domain engineered to be placed in the specific binding member a HER2 antigen binding site in the structural loop region, and an amino acid sequence containing FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that binds to HER2 Functionally competes with a member of a specific binding member such as (a); or (c) a member that specifically binds to one of (a) or (b), which is used in combination with another therapy such as immunotherapy, such as the use of an immune Therapeutic effect of an antitumor agent; the method comprises: (i) determining a HER2 mRNA level of a tumor sample obtained from a patient, wherein if the tumor sample has a high HER2 mRNA level, indicating that the cancer system is specific for the tumor Sensitive members are sensitive to treatment.

A method for predicting a response of a cancer to treatment, which may further comprise: (i) if the tumor sample has a high HER2 mRNA level, the cancer is selected for treatment of the specific binding member.

In one aspect, the invention provides a specific binding member that binds to HER2, wherein the specific binding member is a dimer of a polypeptide of sequence number: 8. For example, the specific binding member can be a dimer formed by two polypeptides of sequence identification number: 8, such as a dimer composed of two polypeptides, wherein each polypeptide is identified by sequence identification number: The sequence shown is composed (this specific binding member is also referred to as H561-4 in this application).

A pharmaceutical composition comprising one of the specific binding members of the invention is also provided. The pharmaceutical composition can comprise a specific binding member of the invention and a pharmaceutically acceptable excipient. A nucleic acid encoding one of the specific binding members of the invention is also provided. Preferably, the nucleic acid comprises or consists of a sequence of sequence identification number: 1. The nucleotide sequence has been optimized in Chinese hamster ovary (CHO) cells, and the skilled artisan should have no difficulty designing other nucleotide sequences encoding the specific binding members of the present invention. Also provided is a vector comprising a nucleic acid comprising one of the specific binding members of the invention, and a host cell comprising a vector or a nucleic acid of the invention.

Combined therapy

In a particular embodiment, a HER2 binding agent is administered to a subject simultaneously or sequentially with another therapy, such as an individual suffering from a cancer. For example, a HER2 binding agent can be administered in combination with one or more of the following: radiation therapy, surgery, or chemotherapy, such as targeted chemotherapy or immunotherapy. Immunotherapy, such as cancer immuntherapy, includes cancer vaccines and immunosuppressive agents. A HER2 binding agent can be, for example, HER2 binds to a protein, an antibody, an antibody fragment or a small molecule. The HER2 binding agent can be a protein comprising a heavy chain immobilization fragment comprising a CH2 and CH3 domain, wherein the CH3 domain comprises a HER2 binding site and can be, for example, H561-4 or an analog or derivative thereof. Things.

For example, when a body is suffering from a cancer system (i) insufficient response to trastuzumab antibody and/or pertamine monoclonal antibody, or cancer of the disease for trastuzumab antibody and/or perindopril Treatment of monoclonal antibodies is refractory (eg, refractory or resistant in nature, or refractory or resistant in nature); and/or (ii) average number of HER2 genes per tumor cell When the system is greater than or equal to 10 and/or has a high HER2 mRNA level, the treatment is carried out by administering a HER2 binding agent with another therapy (such as radiation therapy, surgery or chemotherapy), wherein the HER2 binding agent system comprises A HER2 antigen binding site designed into a structural loop region of the CH3 domain, and comprising, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or administered in combination with HER2 A HER2 binding agent that competes with it. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids.

In a particular embodiment, a method for treating a subject suffering from cancer comprises administering to a subject afflicted with a cancer a HER2 binding agent, such as H561-4, and one or more immune anti-tumor agents, wherein the cancer system (i) Insufficient response to trastuzumab antibody and/or pertituzate antibody, or refractory to treatment with trastuzumab antibody and/or pertabamycin antibody (eg essentially refractory or Resistant, or difficult to appear The condition or resistance); and/or (ii) the average HER2 gene set per tumor cell is greater than or equal to 10 and/or has a high HER2 mRNA level. Immunotherapy, such as the use of an immunotherapeutic agent, is effective in enhancing, stimulating, and/or upregulating an immune response in a body. In one aspect, administration of a HER2 binding agent along with an immunological anti-tumor agent produces a synergistic effect in cancer treatment, such as in inhibiting tumor growth.

In one aspect, a HER2 binding agent is administered sequentially prior to administration of the immunotherapeutic agent. In one aspect, a HER2 binding agent is administered concurrently with an immunotherapeutic agent. In another aspect, a HER2 binding agent is administered sequentially following administration of an immunotherapeutic agent. The time at which the two agents are started to be administered may be, for example, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one. Or multiple weeks; or in the first drug administration such as 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days Or after one or more weeks, the second drug system begins to be administered.

In a particular aspect, a HER2 binding agent is administered to a patient simultaneously with an immuno-anti-tumor agent, such as at the same time, for a period of 30 or 60 minutes, such as simultaneous infusion to the patient. A HER2 binding agent can be formulated with an immunological antitumor agent. Immunological anti-tumor agents include, for example, small molecule drugs, antibodies or fragments thereof or other biomolecules or small molecules. Examples of biological immunological antitumor agents include, but are not limited to, antibodies, antibody fragments, vaccines, and interleukins. In one aspect, the anti-system is a monoclonal antibody. In a particular aspect, the monoclonal antibody is resistant to humanized or human monoclonal antibodies.

In one aspect, the immuno-antitumor agent acts on one of (i) an irritating (including a co-stimulatory) molecule (eg, a receptor or a ligand) agonist of an immune cell, such as a T cell, or (ii) an inhibitory (including a total inhibitory) molecule (such as a receptor or a ligand) antagonist, both of which result in amplification of an antigen-specific T cell response. In a particular aspect, an immuno-anti-tumor agent acts on one of (i) a stimulatory (including a co-stimulatory) molecule (such as a receptor or a ligand) that is involved in innate immunity, such as NK cells. Or an antagonist of (ii) an inhibitory (including a total inhibitory) molecule (such as a receptor or a ligand), and wherein the immunological antitumor agent enhances innate immunity. Such immunological antineoplastic agents are commonly referred to as immunological check stimulators, such as immunosuppressive stimulators or immunosuppressive stimulators.

In a particular embodiment, an immuno-anti-tumor agent is characterized by a stimulatory or inhibitory molecule that is a member of one of the immunoglobulin superfamilies (IgSF). For example, an immuno-antitumor agent can include B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, a member of the B7 family of membrane-bound ligands such as B7-H4, B7-H5 (VISTA) and B7-H6 or a co-stimulatory or co-inhibitory receptor specifically binding to a member of a B7 family One of the agents (or in combination with it in a specific manner). An immunoanti-tumor agent can be one of the members of the TNF family of membrane-bound ligands or one of the co-stimulatory or co-inhibitory receptors, such as a TNF receptor family member, specifically binding to it. Exemplary TNF and TNFR family members that can be targeted by immunotherapeutic agents include CD40 and CD40L, OX-40, OX-40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR , TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS , FASL, RELT, DR6, TROY and NGFR. An immunotherapeutic agent which can be used in combination with a HER2 agent for treating cancer, and can be one of the agents of an IgSF member such as the B7 family member, the B7 receptor family member, the TNF family member or the TNFR family member described above. , such as an antibody.

In one aspect, a HER2 binding agent can be administered with one or more of the following: (i) a protein that inhibits T cell activation (eg, immunosuppressive stimulators) such as CTLA-4, PD-1, PD -L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7- One of H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1 and TIM-4 antagonists, and (ii) a protein that stimulates T cell activation such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, CD40L, DR3 and CD28H one of the agonists. In one aspect, the immuno-antitumor agent inhibits an interleukin (ie, as an antagonist thereof) and the interleukin inhibits T Cell activation (eg IL-6, IL-10, TGF-ß, VEGF and other immunosuppressive interleukins); or an interleukin agonist such as IL-2, IL-7, IL- 12. IL-15, IL-21 and IFNα (such as interleukin itself), which stimulate T cell activation and stimulate an immune response.

Other agents that can be combined with a HER2 binding agent for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, an anti-HER2 binding agent can be used in combination with an antagonist of KIR.

Other agents for use in combination therapy include agents that inhibit or deplete macrophages or mononuclear leukocytes, including, but not limited to, CSF-1R antagonists, such as CSF-1R antagonist antibodies, and including RG7155 (WO11/70024, WO11) /107553, WO11/131407, WO13/87699, WO13/119716, WO13/132044) or FPA-008 (WO11/140249; WO 13169264; WO14/036357).

Immunological antitumor agents also include agents that inhibit TGF-beta signaling.

Other agents that can be used in combination with a HER2 binding agent include agents that enhance the presentation of tumor antigens, such as dendritic cell vaccines, GM-CSF secreted cell vaccines, CpG oligonucleotides, and imiquimod, or include tumor enhancement Immunotherapeutic therapy of cells (eg, anthracycline).

Other therapies that can be used in combination with a HER2 binding agent include therapies that deplete or block Treg cells, such as one that binds to CD25 in a specific manner.

Another treatment that can be combined with a HER2 binding agent One of the metabolic enzymes such as guanamine dioxygenase (IDO), dioxygenase, arginase or nitric oxide synthase.

Another type of agent that can be used includes agents that inhibit adenosine formation or inhibit adenosine A2A receptors.

Other therapies that can be combined with a HER2 binding agent for the treatment of cancer include therapies that reverse/prevent T cell anergy or failure, and include an innate immune activation and/or inflammatory therapy that triggers a tumor site.

A HER2 binding agent can be used in combination with more than one immuno-antitumor agent, and for example, in combination with one of a plurality of parts of the immune pathway, such as one or more of the following: a therapy for enhancing tumor antigen presentation (eg dendritic cell vaccine, GM-CSF secreted cell vaccine, CpG oligonucleotide, imiquimod); one of the therapies that inhibit negative immunomodulation, such as by inhibiting CTLA-4 and/or PD1 /PD-L1/PD-L2 pathway and / or depletion or blocking of Treg or other immunosuppressive cells; one of the treatments that stimulate positive immunomodulation, such as by stimulating CD-137, OX-40 and / or GITR pathways And/or an agonist that stimulates T cell effector function; one that increases systemic frequency of anti-tumor T cells; one that depletes or inhibits Treg, such as Treg in tumors, such as using a CD25 antagonist (such as Dali Beads) Monoclonal antibody (daclizumab) or depletion by ex vivo anti-CD25 beads; one of the functions affecting the function of inhibitory bone marrow cells in tumors; one of the therapies that enhance the immunogenicity of tumor cells (such as anthracycline); Adoptive T cell or NK cell metastasis, Including genetically modified cells, such as cell by transformation of the chimeric antigen receptor (CAR-T therapy); inhibition of a metabolic enzyme such as indazol One of the treatments of indole dioxygenase (IDO), dioxygenase, arginase or nitric oxide synthase; one of the treatments to reverse/prevent T cell anergy or failure; triggering a congenital part of a tumor One of immuno-activating and/or inflammatory treatments; the administration of immunostimulatory interleukins or the blocking of immunosuppressive interleukins.

For example, a HER2 binding agent can be used in combination with one or more agonistic agents that bind to a positive costimulatory receptor; one or more antagonists (blockers) that attenuate signaling via an inhibitory receptor. ), such as overcoming antagonists of different immunosuppressive pathways within the tumor microenvironment (eg, blocking PD-L1/PD-1/PD-L2 interactions); systemically increasing the frequency and depletion of anti-tumor immune cells such as T cells Or inhibiting one or more agents of Treg (eg, by inhibiting CD25); inhibiting one or more agents of a metabolic enzyme such as IDO; reversing/preventing one or more agents of T cell anergy or failure; and inducing innate immunity at the tumor site One or more agents that activate and/or inflammatory. In one embodiment, the individual (i) who is afflicted with cancer has insufficient response to the trastuzumab antibody and/or the pertamine monoclonal antibody, or the cancer that is afflicted with the trastuzumab antibody and/or culture Treatment with a bead monoclonal antibody is refractory and/or (ii) the average HER2 gene set per tumor cell is greater than or equal to 10 and/or has a high HER2 mRNA level, and by administering a HER2 binding agent with An immunotherapeutic agent, wherein the HER2 binding agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) And DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for its binding. In special In certain embodiments, the HER2 binding agent comprises the sequence number: 8 and its presence or absence of the last 1 or 2 C-terminal amino acids.

In one aspect, the immuno-antitumor agent is a CTLA-4 antagonist, such as an antagonist CTLA-4 antibody. Suitable CTLA-4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.

In one aspect, the immuno-antitumor agent is a PD-1 antagonist, such as an antagonist PD-1 antibody. Suitable PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab) or MEDI-0680 (AMP-514; WO2012/145493). Immuno-anti-tumor agents may also include pidilizumab (CT-011), although its specificity for PD-1 binding has been questioned. Another method of targeting the PD-1 receptor is a recombinant protein consisting of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, which is called AMP-224.

In one aspect, the immuno-antitumor agent is a PD-L1 antagonist, such as an antagonist PD-L1 antibody. Suitable PD-L1 antibodies include, for example, MPDL3280A (RG7446; WO2010/077634), duvaluumab (MEDI4736), BMS-936559 (WO2007/005874), MSB0010718C (WO2013/79174) or rHigM12B7.

In one aspect, the immuno-antitumor agent is a LAG-3 antagonist, such as an antagonist LAG-3 antibody. Suitable LAG3 antibodies include, for example, BMS-986016 (WO 10/19570, WO 14/08218); or IMP-731 or IMP-321 (WO 08/132601, WO 09/44273).

In one aspect, the immuno-antitumor agent is a CD137 (4-1BB) agonist, such as a potent CD137 antibody. Suitable CD137 antibodies include, for example, urelumab or PF-05082566 (WO 12/32433).

In one aspect, the immuno-antitumor agent is a GITR agonist, such as an agonistic GITR antibody. Suitable GITR antibodies include, for example, a GITR antibody as disclosed in TRX-518 (WO06/105021, WO09/009116), MK-4166 (WO11/028683) or WO2015/031667 or WO2015/187835.

In one aspect, the immuno-antitumor agent is an OX40 agonist, such as an agonistic OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888; WO06/029879).

In one aspect, the immuno-antitumor agent is a CD40 agonist, such as a agonistic CD40 antibody. In a particular embodiment, the immuno-anti-tumor agent is a CD40 antagonist, such as an antagonist CD40 antibody. Suitable CD40 antibodies include, for example, lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870, 893 or Chi Lob 7/4.

In one aspect, the immuno-antitumor agent is a CD27 agonist, such as a potent CD27 antibody. Suitable CD27 antibodies include, for example, Vallilumab (CDX-1127).

In one aspect, the immuno-antitumor agent is MGA271 (to B7H3) (WO 11/109400).

In one aspect, the immuno-antitumor agent is a KIR antagonist, such as a lililumab antibody.

In one aspect, the immuno-anti-tumor agent is an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919 (WO09/73620, WO09/1156652, WO11) /56652, WO12/142237) or F001287. In one aspect, the immuno-antitumor agent is a Toll-like receptor agonist, such as a TLR2/4 agonist (such as Bacillus Calmette-Guerin); a TLR7 agonist (eg, Hiltonol or imiquimod; a TLR7/8 agonist (such as Resiquimod); or a TLR9 agonist (such as CpG7909).

In one aspect, the immuno-antitumor agent is a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197 or IMC-TR1.

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is insufficiently responding to an antibody to trastuzumab and/or a pertamine monoclonal antibody, or a cancer suffering from a pertussis When the treatment of monoclonal antibodies and/or perphenezate antibodies is refractory or (essential or acquired), it is treated by administering a HER2 binding agent with a CTLA-4 antagonist, wherein the HER2 binding The agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or administered A HER2 binding agent that competes with HER2 binding. In a particular embodiment, the HER2 binding agent comprises a sequence Column identification number: 8 and its presence or absence of the last 1 or 2 C-terminal amino acids. The CTLA-4 antagonist can be an Ipti monoclonal antibody (ipilimumab).

In one embodiment, an individual having cancer, such as stomach cancer, colorectal cancer, or breast cancer, and each of the tumor cells having an average HER2 gene set of greater than or equal to 10 and/or having a high HER2 mRNA level is by Administration of a HER2 binding agent to a CTLA-4 antagonist, wherein the HER2 binding agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprising, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for its binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The CTLA-4 antagonist can be an Ipti monoclonal antibody (ipilimumab).

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is (i) underreacting to an antibody to trastuzumab and/or percetomycin, or a cancer Treatment with trastuzumab monobody and/or pertitubeta antibody is refractory or (essential or acquired), and (ii) the average number of HER2 genes per tumor cell is greater than or equal to 10 and / Or having a high HER2 mRNA level, treated by administering a HER2 binding agent comprising a HER2 antigen designed to be placed in a structural loop region of the CH3 domain, by administering a HER2 binding agent. The binding site, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for binding. In special In certain embodiments, the HER2 binding agent comprises the sequence number: 8 and its presence or absence of the last 1 or 2 C-terminal amino acids. The CTLA-4 antagonist can be an Ipti monoclonal antibody (ipilimumab).

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is insufficiently responding to an antibody to trastuzumab and/or a pertamine monoclonal antibody, or a cancer suffering from a pertussis Treatment of monoclonal antibodies and/or perphenezam monoclonal antibodies is refractory or (essential or acquired) by administering a HER2 binding agent with an agent that antagonizes the PD-1 pathway (eg, PD- 1. A PD-L1 or PD-L2 antagonist), wherein the HER2 binding agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprises, for example, an amino acid sequence FFTYW ( Sequence ID: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for its binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The agent that antagonizes the PD-1 pathway may be a nivolumab antibody.

In one embodiment, an individual having cancer, such as stomach cancer, colorectal cancer, or breast cancer, and each of the tumor cells having an average HER2 gene set of greater than or equal to 10 and/or having a high HER2 mRNA level is by Administration of a HER2 binding agent with an agent that antagonizes the PD-1 pathway, such as a PD-1, PD-L1 or PD-L2 antagonist, wherein the HER2 binding agent comprises a structure designed to be placed into the CH3 domain a HER2 antigen binding site in the loop region, and includes, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or administered at HER2 binds to a HER2 binding agent that competes with it. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The agent that antagonizes the PD-1 pathway may be a nivolumab antibody.

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is (i) underreacting to an antibody to trastuzumab and/or percetomycin, or a cancer Treatment with trastuzumab monobody and/or pertitubeta antibody is refractory or (essential or acquired), and (ii) the average number of HER2 genes per tumor cell is greater than or equal to 10 and / Or having a high HER2 mRNA level, by administering a HER2 binding agent with an agent that antagonizes the PD-1 pathway (such as PD-1, PD-L1 or PD-L2 antagonist), wherein the HER2 binds The agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or administered A HER2 binding agent that competes with HER2 binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The agent that antagonizes the PD-1 pathway may be a nivolumab antibody.

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is insufficiently responding to an antibody to trastuzumab and/or a pertamine monoclonal antibody, or a cancer suffering from a pertussis When the treatment of monoclonal antibodies and/or pertamine monoclonal antibodies is refractory or (essential or acquired), it is antagonized by administration of a HER2 binding agent with a LAG-3. The agent is treated, wherein the HER2 binding agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (sequence recognition) No.: 14), or a HER2 binding agent that competes with HER2 for its binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The LAG-3 antagonist can be BMS-986016.

In one embodiment, an individual having cancer, such as stomach cancer, colorectal cancer, or breast cancer, and each of the tumor cells having an average HER2 gene set of greater than or equal to 10 and/or having a high HER2 mRNA level is by Administration of a HER2 binding agent to a LAG-3 antagonist comprising a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprising, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for its binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The LAG-3 antagonist can be BMS-986016.

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is (i) underreacting to an antibody to trastuzumab and/or percetomycin, or a cancer Treatment with trastuzumab monobody and/or pertitubeta antibody is refractory or (essential or acquired), and (ii) the average number of HER2 genes per tumor cell is greater than or equal to 10 and / Or by having a high HER2 mRNA level, by administering A HER2 binding agent is treated with a LAG-3 antagonist, wherein the HER2 binding agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprises, for example, an amino acid sequence FFTYW (sequence) Identification number: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for its binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The LAG-3 antagonist can be BMS-986016.

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is insufficiently responding to an antibody to trastuzumab and/or a pertamine monoclonal antibody, or a cancer suffering from a pertussis When the treatment of monoclonal antibodies and/or pertamine monoclonal antibodies is refractory or (essential or acquired), it is treated by administering a HER2 binding agent with a CD137 agonist, wherein the HER2 binding The agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or administered A HER2 binding agent that competes with HER2 binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The CD137 agonist can be a urelumab antibody.

In one embodiment, an individual having cancer, such as stomach cancer, colorectal cancer, or breast cancer, and each of the tumor cells having an average HER2 gene set of greater than or equal to 10 and/or having a high HER2 mRNA level is by Administering a HER2 binding agent with a CD137 agonist for treatment, Wherein the HER2 binding agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14) Or, a HER2 binding agent that competes with HER2 binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The CD137 agonist can be a urelumab antibody.

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is (i) underreacting to an antibody to trastuzumab and/or percetomycin, or a cancer Treatment with trastuzumab monobody and/or pertitubeta antibody is refractory or (essential or acquired), and (ii) the average number of HER2 genes per tumor cell is greater than or equal to 10 and / Or having a high HER2 mRNA level, by administering a HER2 binding agent comprising a HER2 antigen binding designed to be placed in a structural loop region of the CH3 domain, by administering a HER2 binding agent. The site, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for its binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The CD137 agonist can be a urelumab antibody.

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is insufficiently responding to an antibody to trastuzumab and/or a pertamine monoclonal antibody, or a cancer suffering from a pertussis Monoclonal resistance When the treatment of the body and/or the betulin monoclonal antibody is refractory or (essential or acquired), it is treated by administering a HER2 binding agent and an IDO antagonist, wherein the HER2 binding agent comprises Designing a HER2 antigen binding site in a structural loop region of the CH3 domain, and including, for example, the amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or administration of HER2 binding A HER2 binding agent that competes with it. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The IDO antagonist can be F001287.

In one embodiment, an individual having cancer, such as stomach cancer, colorectal cancer, or breast cancer, and each of the tumor cells having an average HER2 gene set of greater than or equal to 10 and/or having a high HER2 mRNA level is by Administration of a HER2 binding agent comprising an HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain and comprising an amino acid sequence FFTYW (sequence recognition) is administered in combination with an IDO antagonist. ID: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for its binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The IDO antagonist can be F001287.

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is (i) underreacting to an antibody to trastuzumab and/or percetomycin, or a cancer Treatment with trastuzumab antibody and/or pertamine monoclonal antibody is refractory or has (essential or post- And (ii) when the average HER2 gene set per tumor cell is greater than or equal to 10 and/or has a high HER2 mRNA level, by administering a HER2 binding agent with an IDO antagonist, Wherein the HER2 binding agent comprises a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14) Or, a HER2 binding agent that competes with HER2 binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The IDO antagonist can be F001287.

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is insufficiently responding to an antibody to trastuzumab and/or a pertamine monoclonal antibody, or a cancer suffering from a pertussis When the treatment of monoclonal antibodies and/or pertamine monoclonal antibodies is refractory or (essential or acquired), it is treated by administering a HER2 binding agent with a KIR antagonist, wherein the HER2 binding agent is A HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain, and comprising, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or administered at HER2 A HER2 binding agent that binds to its competition. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The KIR antagonist can be a lililu monoclonal antibody (lirilumab).

In one embodiment, the average number of HER2 genes in a cancer, such as a stomach cancer, colorectal cancer, or breast cancer, and each of its tumor cells is greater than An individual equal to 10 and/or having a high HER2 mRNA level is treated by administering a HER2 binding agent comprising a structural loop that is designed to be placed in the CH3 domain, by administering a HER2 binding agent. A HER2 antigen binding site of the region, and comprises, for example, an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for its binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The KIR antagonist can be a lililu monoclonal antibody (lirilumab).

In one embodiment, an individual suffering from a cancer, such as a stomach cancer, a colorectal cancer, or a breast cancer, is (i) underreacting to an antibody to trastuzumab and/or percetomycin, or a cancer Treatment with trastuzumab monobody and/or pertitubeta antibody is refractory or (essential or acquired), and (ii) the average number of HER2 genes per tumor cell is greater than or equal to 10 and / Or having a high HER2 mRNA level, which is treated by administering a HER2 binding agent comprising a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain, by administering a HER2 binding agent. And include, for example, the amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes with HER2 for its binding. In a particular embodiment, the HER2 binding agent comprises the sequence ID: 8 and with or without the last 1 or 2 C-terminal amino acids. The KIR antagonist can be a lililu monoclonal antibody (lirilumab).

Composition comprising a specific binding agent as in the present invention It can also be administered in combination with an existing therapeutic agent, such as abitaterone acetate (Zytiga), afatinib, aflibercept, anastrozole, Bevacizumab, bicalutamide, BRAF inhibitor, carboplatin, capecitabine, cetuximab, cisplatin ), crizotinib, cyclophosphamide, oligoerythritol, PEGylated doxorubicin liposome, enzalutamide (XTANDI), epirubicin (epirubicin), eribulin mesylate, erlotinib, etoposide, everolimus, exemestane, FOLFIRI, FOLFOX, Fluoril, 5-fluorouracil, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hedgehog pathway inhibitor, Irenotecan, ixabepilone, lapatinib, letrozole (letroz) Ole), formazan tetrahydrofolate, leuprorelin, amine formazan, mitoxantrone, oxaliplatin, paclitaxel, albumin-bound Pacific paclitaxel nanoparticles, Pa Monoclonal antibody (panitumumab), pemetrexed (pemetrexed), perphenazine monoclonal antibody, prednisone, radium dichloride (radio 223) (radio-treated bone (Xofigo) injection), thunder Moss monoclonal antibody (ramucirumab), regorafenib, S-1, tamoxifen, topotecan, trastuzumab antibody, T-DM1, triptor Ruilin (triptorelin) and vinorelbine (vinorelbine).

In a particular embodiment, the two therapeutic agents (active ingredients) are co-formulated. For example, a specific binding member is formulated with an immunoanti-tumor agent. In a particular embodiment, the compositions of the active ingredients form a fixed dose of the composition.

The invention further provides a method of producing a specific binding member of the invention comprising culturing a recombinant host cell of the invention under conditions which produce a specific binding member. The method can further comprise isolating and/or purifying the specific binding member. The method can also include formulating the specific binding member to a pharmaceutical composition.

Exemplary mode of administration

For example, a HER2 binding agent such as HER2 Fcab can be administered to an individual suffering from cancer by intravenous injection (IV), such as an individual having a relapsed or refractory overexpressing HER2 solid tumor, at a dose of 0.1. To 10 or 20 mg/kg, such as 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 Mg/kg or 10 mg/kg. For example, a HER2 binding agent administered may be 0.1 to 0.5 mg/kg, 0.1 to 1 mg/kg, 0.5 to 1 mg/kg, 1 to 3 mg/kg, 1 to 6 mg/kg, 6 to 10 Mg/kg, 2 to 4 mg/kg, 5 to 7 mg/kg or 9 to 10 mg/kg. Such individuals may be individuals who have been unable to locally excise and/or metastatic solid cancers that overexpress human HER2 on a standard clinical pathology basis. In a particular embodiment, the systems have no individual to choose from for the standard treatment option. In a particular embodiment The individuals have unremovable/locally advanced and/or metastatic histologically or cytologically confirmed solid tumor malignancy, and no standard therapeutic or palliative measures are available for the tumor or have Efficacy.

The HER2 status of individuals with breast cancer can be assessed following the practical use of the 2013 American Society of Clinical Oncology (ASCO)/American College of Pathologists (CAP) benchmark (Wolff et al., 2013). The HER2 status of individuals with adenocarcinoma at the junction of the stomach and the gastroesophageal tract can be assessed in accordance with the published guidelines of Rüschoff et al. (2012). The HER2 status of a non-breast/non-gastric cancer body can be assessed against the baseline set by the local agency. Individuals with breast and stomach/gastroesophageal junction cancers can be tested for HER2 using a practical set of assays/methods approved by the FDA for their cancer types. The clinical pathology report for a particular individual includes 3+ IHC.

In a particular embodiment, a system for breast cancer has been treated with at least one or at least two FDA-approved anti-HER2 targeting therapies, wherein during or after the last last anti-HER2 directed therapy, the individuals are refractory Or disease recurrence/deterioration. In a particular embodiment, a system for cancer of the stomach and gastroesophageal junction has been treated with at least one FDA approved anti-HER2 directed therapy; wherein during or after its last last anti-HER2 directed therapy, Individuals are refractory or relapsed/deteriorated. In a particular embodiment, an individual suffering from non-breast cancer, non-gastric cancer, non-gastric esophageal junction cancer has not previously received any treatment for anti-HER2 therapy (eg, an anti-HER2 agent that is not approved by the FDA for its particular cancer type), but In its last last anticancer therapy During or after these individuals are refractory or relapsed/deteriorated. In a particular embodiment, the individual has previously been administered adjuvant anti-HER2 therapy after surgery.

In a particular embodiment, the previous cumulative dose of doxorubicin in one of the individual treated with a HER2 binding agent is less than or equal to 360 mg/m2, and/or the previous epirubicin The cumulative dose is less than or equal to 720 mg/m2.

In a particular embodiment, a body treated with a HER2 binding agent has sufficient organ function as defined below: (i) serum aspartate transaminase (AST) and alanine transaminase (ALT) system Less than or equal to 2.5 times the upper limit of normal (ULN); if liver dysfunction is due to a potential malignancy, AST and ALT must be less than or equal to 5 times the upper limit of normal; (ii) unless due to JB (Gilbert) syndrome, otherwise the total serum bilirubin system is less than or equal to the upper limit of normal; (iii) serum creatinine is less than or equal to 1.25 times the upper limit of normal; or if serum creatinine is greater than 1.25 times the upper limit of normal, then The calculated (Cockcroft-Gault formula) has a renal glomerular filtration rate (GFR) greater than or equal to 60 ml/min; (iv) a heme system greater than or equal to 9.0 g/dl And the number of red blood cells required to be infused per month is not more than 1 unit; (v) the absolute neutrophil white blood cell number is greater than or equal to 1,500/cubic millimeter (supported by growth factors in the previous 21 days); (vi) number of platelets The system is greater than or equal to 100,000 / cubic millimeter (before 7 days without platelet transfusion and not supported by growth factors); (vii) activated partial clotting time (aPTT) is less than or equal to 1.25 times the upper limit of normal and international normalized ratio (INR) is less than or equal to 1.3 (unless the The individual is receiving a therapeutic anticoagulant); (viii) the left ventricular ejection rate (LVEF) or multi-gate detection scan (MUGA) is greater than or equal to 50 as determined by 2D Echo. %, or greater than or equal to the normal lower limit (LLN) set by the local authority, and whichever is higher; and/or (ix) the magnesium, calcium and phosphorus in the serum are in the normal reference range for local testing Inside.

In a particular embodiment, the individual treated with a HER2 binding agent does not have one or more of the following conditions, symptoms or criteria:

1. Primary brain or other central nervous system malignancy;

2. History of any meningeal metastasis;

3. Current brain metastases or treatments for brain metastases within 1 month of the first day of scheduled dosing; a. For corticosteroids, for brain tolerance in terms of glucose tolerance The tolerated dose of the transferred corticosteroid may be less than or equal to the second grade (symptomatic; indicates that there is a modified diet or oral medication) and hyperglycemia is lower than or equal to the second grade (fasting blood glucose values above 160 to 250 mg / dl [higher than 8.9 to 13.9 mm / liter]).

4. History of second or other primary cancer other than: 1) therapeutically treated non-melanoma skin cancer; 2) therapeutic treatment Treatment of cervical or breast carcinoma in situ; or 3) other primary solid tumors that have been treated for the purpose of healing, and no known current disease, and no treatment during the past three years;

5. Receive any trial-type treatment within 4 weeks from the first day of scheduled dosing;

6. Receive cytotoxic chemotherapeutic treatment within 3 weeks from the first day of scheduled dosing (within nitrosourea and mitomycin C for 6 weeks);

7. Receiving radiation therapy within 3 weeks from the first day of scheduled dosing, unless the radiation contains a limited field for non-visceral structures such as limb bone metastases;

8. Receive the following treatments within 3 weeks from the first day of scheduled dosing: immunotherapy (including interferon, interleukin, immune complex), biological therapy (including monoclonal antibodies or other genetically engineered proteins) Targeting small molecules (including but not limited to kinase inhibitors), hormone therapy (except for gonadotropin-releasing agonists/antagonists for prostate cancer, which may continue during the study);

9. Receiving a conjugate of trastuzumab antibody, pertatumizole antibody, or trastuzumab antibody and emtansine within 4 weeks from the first day of scheduled administration;

10. Receiving lapatinib within 7 days of the first day of scheduled dosing;

11. Exclude concurrent medical conditions: a. Uncontrolled hypertension at a systolic blood pressure below 160 mm Hg and a diastolic blood pressure below 90 mm Hg; b. Myocardium within 12 months prior to the first day of dosing Infarction, instability Stereotype angina pectoris, coronary bypass surgery, coronary angioplasty or stent placement; c. history of septic heart failure; d. absolute reduction in left ventricular ejection rate (LVEF) greater than or equal to 16 absolute percent or greater than or A medical history equal to 10% of the total score, and in the previous anti-HER2 therapy, from above the lower limit of normal to below the lower limit of normal, even if the asymptomatic and LVEF reduction is restored; e. The researchers determined a clinically significant 12-lead electrocardiogram (ECG) abnormality; f. hemorrhagic, embolic or thrombotic stroke within 6 months from the first day of scheduled dosing; g. previous bone marrow or stem cell transplantation; h. previously known to be infected with human immunodeficiency virus (HIV); or infection with type B or hepatitis C requiring treatment; i. need to use parenteral antimicrobial agents or any current infection above level 2; Non-malignant interstitial lung disease; k. dyspnea caused by any cause and need to supplement oxygen therapy; l significant trauma or major surgery within 21 days from the first day of scheduled dosing (major surgery refers to opening the body cavity) Such as open chest Surgery, laparotomy, laparoscopic resection and major orthopedic surgery, such as replacement joints, open reduction and internal fixation); m. may increase the risk associated with participating in the study or any other acute that may interfere with the interpretation of the findings Or chronic disease or mental condition (package) Including alcohol and illegal drug abuse) or laboratory test abnormalities, and will prompt the researcher or medical supervisor to determine that the individual is not suitable for participation in the study.

12. Not yet recovered from previous adverse effects of anticancer therapy to pre-treatment baseline or first grade, except for hair loss, anemia (heme must meet the participation criteria of this study) and peripheral neuropathy (which must have been restored to low) Beyond or equal to the second level).

13. Allergic/infusion reactions for monoclonal antibodies, other therapeutic proteins, or any component/excipient (spermine acid, glycine, phosphoric acid or polysorbate 80) for the HER2 binding agent drug product. Allergies, and subsequent infusions with standard therapies such as antihistamines, 5-HT3 antagonists, or corticosteroids, remain uncontrollable or preventive.

During the initial 21 (3 weeks) or 28 (4 weeks) treatment cycles (first cycle), one body can be once or less per week (±1 day) (eg every two weeks) One or three times a week, receiving an intravenous (IV) infusion of a HER2 binding agent. During the first cycle, the individual can be assessed for safety, tolerability, dose-limiting toxicity, PK, immunogenicity, and clinical disease response. Individuals who have not experienced clinical disease progression and/or have not experienced unacceptable toxicity may continue to receive a HER2 binding agent for up to six rounds for six days (every three weeks) to 28 days (once or per week) Two weeks) cycle. An individual may continue to receive a HER2 binding agent at the same dose as it was originally assigned, unless it is adjusted downward due to previous toxicity.

The present application provides a method for treating a body suffering from cancer, such as a cancer with a higher number of HER2 genes than normal, such as an over-expression One of the HER2 solid tumors, the method comprising administering to the individual 0.1 to 10 mg/kg, 0.1 to 1 mg/kg or 1 to 10 mg/kg of HER2 per week, every two weeks or every three weeks. Binding agent once. In one aspect, the method comprises administering to the patient a frequency of 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, once a week, once every two weeks, once every three weeks. , 0.5 to 1 mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg) , 3 mg/kg, 6 mg/kg or 10 mg/kg): (a) a specific binding member comprising a structural loop region designed to be inserted into the CH3 domain of the specific binding member a HER2 antigen binding site, and an amino acid sequence containing FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, which is linked to HER2 binding (a) One of the specific binding members competes, wherein the method optionally comprises administering another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

In one aspect, the invention provides a method of treating cancer in a patient, wherein the number of HER2 genes per tumor cell of the cancer, such as the average number of HER2 genes, is greater than or equal to 10; and wherein the method comprises The patient is administered once a week, every two weeks or every three weeks and the amount is 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/kg): (a) a specific binding member comprising a designed specific binding member a HER2 antigen binding site of a structural loop region of the CH3 domain, and an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, It competes for HER2 binding with a specific binding member such as (a), wherein the method optionally includes administration of another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

A tumor sample obtained from the patient is determined, wherein each tumor cell has a HER2 gene set, such as an average number of HER2 genes, which is greater than or equal to 10. Accordingly, the present invention provides a method of treating cancer in a patient, wherein a tumor sample obtained from the patient is measured, and wherein each tumor cell has an average number of HER2 gene sets greater than or equal to 10; The method comprises administering to the patient once a week, every two weeks or every three weeks and the amount is 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg. /kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10) Each of the following: (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid Sequence FFTYW (sequence identification number: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with a specific binding member such as (a) for HER2 binding, wherein the method optionally includes administration such as immunization Another treatment for therapy, such as the use of an immunotherapeutic agent.

The method may comprise: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; and (ii) if the number of sets of HER2 genes per tumor cell, such as The average HER2 gene set is greater than or equal to 10, and the patient is treated with the specific binding member, optionally in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

Accordingly, the present invention also provides a method of treating cancer in a patient, wherein the method comprises: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; And (ii) if the number of HER2 gene sets per tumor cell, such as the average number of sets of genes, is greater than or equal to 10, the patient is administered once a week, every two weeks, or every three weeks, and the amount is 0.1 to 10 mg. /kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, A specific binding member of 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/kg, wherein the specific binding member is: (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) And DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes for HER2 binding with a specific binding member such as (a), wherein the method optionally includes administration such as Another treatment for immunotherapy, such as the use of an immunotherapeutic agent.

In one aspect, the invention provides a method of treating cancer in a patient, wherein the cancer has a high HER2 mRNA level, and wherein the method comprises the disease for each week, every two weeks or every three weeks Once administered, the amount is 0.1 to 10 mg / kg, 0.1 to 0.5 mg / kg, 0.5 to 1 mg / kg or 1 to 10 mg / kg (such as 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg , 0.4 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg or 10 mg / kg) of the following: (a) a specific binding member, It comprises a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and contains an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14) Or (b) a specific binding member that competes with a specific binding member such as (a) for HER2 binding, wherein the method optionally includes administering another therapy such as immunotherapy, such as An immunotherapeutic agent.

Tumor samples obtained from this patient may have been found to have high HER2 mRNA levels by assay. Accordingly, the present invention provides a method of treating cancer in a patient, wherein a tumor sample obtained from the patient has been determined to have a high HER2 mRNA level by assay, and wherein the method includes, per week, every two The patient is administered once a week or every three weeks in an amount of 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, The following: 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg or 10 mg / kg): (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) And DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes for HER2 binding with a specific binding member such as (a), wherein the method optionally includes administration such as Another treatment for immunotherapy, such as the use of an immune anti-tumor Tumor therapy.

The method can comprise: (i) determining a HER2 mRNA level of a tumor sample obtained from the patient; and (ii) treating the patient with the specific binding member if the HER2 mRNA level is high, It is optionally used in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

Accordingly, the present invention also provides a method of treating cancer in a patient, wherein the method comprises: (i) determining a HER2 mRNA level of a tumor sample obtained from the patient; and (ii) if having a high HER2 mRNA position For the patient, the patient is administered once a week, every two weeks or every three weeks and the amount is 0.1 to 10 mg / kg, 0.1 to 0.5 mg / kg, 0.5 to 1 mg / kg or 1 to 10 mg. /kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10) a specific binding member of mg/kg), wherein the specific binding member is: (a) a specific binding member comprising a HER2 antigen of a structural loop region designed to be inserted into the CH3 domain of the specific binding member Binding site, and containing amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, which is linked to HER2 and (a) One of the specific binding members competes, wherein the method optionally includes administration The immunotherapy of another therapy, such as an immune therapy is the use of anti-tumor agents.

In one aspect, the method comprises administering 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg, or the patient once a week, every two weeks, every three weeks. 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg) /kg or 10 mg/kg) of a pharmaceutical composition comprising a HER2 binding agent that binds to human HER2 in a specific manner and comprising two polypeptides, wherein a human IgG1 heavy chain fragment comprising each polypeptide comprises a CH2 domain and a CH3 domain, wherein the AB loop system included in the CH3 domain comprises an amino acid sequence of sequence identification number: 191, and a CD loop system comprises a sequence identification number: 241 amino acid sequence and The included EF ring system comprises an amino acid sequence 370; and wherein the HER2 binding agent has a molecular weight of up to 60 kD, a binding affinity of less than 10 ^-8 M, and is cytotoxic; and wherein the method selectively comprises Give another treatment such as immunotherapy, A therapy using anti-tumor agents of the immunity.

In one aspect, the invention provides a method of treating cancer in a patient, wherein each tumor cell of the cancer has a HER2 gene set, such as an average number of HER2 genes, greater than or equal to 10; and the method The method comprises administering to the patient once a week, every two weeks, every three weeks a pharmaceutical composition comprising 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/ Kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/kg) of a HER2 binding agent that binds to human HER2 in a specific manner and comprises two polypeptides, wherein a human IgG1 heavy chain fragment contained in each polypeptide comprises a a CH2 domain and a CH3 domain, wherein the AB loop system included in the CH3 domain comprises an amino acid sequence of sequence identification number: 191, and a CD loop system comprises a sequence identification number: 241 amino acid sequence and included One EF ring system contains an amine Acid sequence 370; HER2 binding agent and wherein the molecular weight of up to 60kD system, which system is less than the binding affinity of 10 ^-8 M, and is cytotoxic; and wherein the method optionally comprises administering another therapy, such as immunotherapy, the Such as the use of an immunotherapeutic agent.

A tumor sample obtained from the patient is determined, wherein each tumor cell has a HER2 gene set, such as an average number of HER2 genes, which is greater than or equal to 10. Accordingly, the present invention provides a method of treating cancer in a patient, wherein a tumor sample obtained from the patient is measured, and wherein each tumor cell has an average number of HER2 gene sets greater than or equal to 10; The method comprises administering to the patient a pharmaceutical composition once a week, every two weeks, every three weeks, the pharmaceutical composition comprising 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 Mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/ a HER2 binding agent of kilograms, 6 mg/kg or 10 mg/kg, which binds to human HER2 in a specific manner and comprises two polypeptides, wherein each polypeptide comprises a human IgG1 heavy chain fragment The invention comprises a CH2 domain and a CH3 domain, wherein the AB loop system included in the CH3 domain comprises an amino acid sequence of sequence identification number: 191, and a CD ring system comprises a sequence identification number: 241 amino acid sequence and An EF ring system Included in amino acid sequence 370; and wherein the HER2 binding agent has a molecular weight of up to 60 kD, a binding affinity of less than 10 ^-8 M, and is cytotoxic; and wherein the method optionally comprises administering another such as immunotherapy A therapy such as the use of an immunotherapeutic agent.

The method may comprise: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; and (ii) if the number of sets of HER2 genes per tumor cell, such as The average HER2 gene set is greater than or equal to 10, and the HER2 binding agent is used to treat the patient, and optionally in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

Accordingly, the present invention also provides a method of treating cancer in a patient, wherein the method comprises: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; And (ii) if the number of HER2 gene sets per tumor cell is greater than or equal to 10, the pharmaceutical composition is administered once a week, every two weeks, every three weeks for the patient. The composition comprises 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg) a HER2 binding agent of /kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/kg, wherein the HER2 binding agent is in a specific manner Human HER2 binds to and comprises two polypeptides, wherein a human IgG1 heavy chain fragment contained in each polypeptide comprises a CH2 domain and a CH3 domain, wherein the AB3 system contained in the CH3 domain comprises an amino acid sequence number: 191 Acid sequence, a CD included The ring system comprises the amino acid sequence of SEQ ID NO: 241 and the included EF ring system comprises an amino acid sequence 370; and wherein the HER2 binding agent has a molecular weight of up to 60 kD and a binding affinity of less than 10 ^-8 M And having cytotoxicity; and wherein the method optionally comprises administering another therapy, such as immunotherapy, such as the use of an immunotherapeutic agent.

In one aspect, the invention provides a method of treating cancer in a patient, wherein the cancer has a high HER2 mRNA level, and wherein the method comprises treating the disease every week, every two weeks, every three weeks Suffering from administration of a pharmaceutical composition comprising 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2) a HER2 binding agent of mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/kg, The HER2 binding agent binds to human HER2 in a specific manner and comprises two polypeptides, wherein a human IgG1 heavy chain fragment contained in each polypeptide comprises a CH2 domain and a CH3 domain, wherein the CH3 domain comprises an AB loop Is an amino acid sequence comprising SEQ ID NO: 191, comprising a CD loop system comprising the amino acid sequence of SEQ ID NO: 241 and an EF loop system comprising an amino acid sequence 370; and wherein the HER2 The molecular weight of the binder is up to 60kD, Based binding affinity less than 10 ^-8 M, and is cytotoxic; and wherein the method optionally comprises administering another therapy, such as immunotherapy, the therapy such as the use of an immune anti-tumor agents.

Tumor samples obtained from this patient may have been found to have high HER2 mRNA levels by assay. Accordingly, the present invention provides a method of treating cancer in a patient, wherein a tumor sample obtained from the patient has been determined to have a high HER2 mRNA level by assay, and wherein the method includes, per week, every two The patient is administered a pharmaceutical composition once a week, every three weeks, and the pharmaceutical composition comprises 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/ Kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg) /kg) of a HER2 binding agent that binds to human HER2 in a specific manner and comprises two polypeptides, wherein a human IgG1 heavy chain fragment contained in each polypeptide comprises a CH2 domain and a CH3 domain, wherein The AB loop system included in the CH3 domain comprises the amino acid sequence of sequence identification number: 191, and the CD loop system comprises a sequence identification number: 241 amino acid sequence and an EF ring system comprising an amine. Acid sequence 370; and the HER2 junction therein Agent-based molecular weight up to 60kD, which is less than the binding affinity-based 10 ^-8 M, and is cytotoxic; and wherein the method optionally comprises administering another therapy, such as immunotherapy, the therapy such as the use of an immune anti-tumor agents .

The method can comprise: (i) determining a HER2 mRNA level of a tumor sample obtained from the patient; and (ii) treating the patient with the HER2 mRNA level if the patient has a high HER2 mRNA level, and selecting Sexually with another therapy such as immunotherapy Use, for example, the use of an immunotherapeutic agent.

Accordingly, the present invention also provides a method of treating cancer in a patient, wherein the method comprises: (i) determining a HER2 mRNA level of a tumor sample obtained from the patient; and (ii) if having a high HER2 mRNA position The patient is administered a pharmaceutical composition once a week, every two weeks, every three weeks, and the pharmaceutical composition comprises 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 Mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/ a HER2 binding agent of kilograms, 6 mg/kg or 10 mg/kg, wherein the HER2 binding agent binds to human HER2 in a specific manner and comprises two polypeptides, wherein each polypeptide comprises a human IgG1 heavy chain fragment The system comprises a CH2 domain and a CH3 domain, wherein the AB3 system contained in the CH3 domain comprises an amino acid sequence of sequence identification number: 191, and a CD ring system comprises a sequence identification number: 241 amino acid sequence And the included EF ring system comprises an amino acid sequence 370 HER2 binding agent and wherein the molecular weight of up to 60kD system, which system is less than the binding affinity of 10 ^-8 M, and is cytotoxic; and wherein the method optionally comprises administering another therapy, such as immunotherapy, the use as an immune Anti-tumor therapy.

In one aspect, the invention provides a method of treating cancer in a patient, wherein the number of HER2 genes per tumor cell of the cancer, such as the average number of HER2 genes, is greater than or equal to 10; and wherein the method comprises The patient is administered a pharmaceutical composition once a week, every two weeks, every three weeks, and the pharmaceutical composition comprises 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg) /kg or 10 mg/kg) of a HER2 binding agent that binds to human HER2 in a specific manner and comprises two polypeptides, each of which contains a sequence identification number: 8 or An amino acid sequence consisting of a sequence of at least 5, 4, 3, 2 or 1 amino acid deletions, additions or substitutions, wherein the molecular weight of the HER2 binding agent is up to 60 kD, the combination thereof Affinity is less than 10 ^-8 M, and has Cytotoxicity; and wherein the method optionally comprises administering another therapy, such as immunotherapy, such as the use of an immunotherapeutic agent.

A tumor sample obtained from the patient is determined, wherein each tumor cell has a HER2 gene set, such as an average number of HER2 genes, which is greater than or equal to 10. Accordingly, the present invention provides a method of treating cancer in a patient, wherein a tumor sample obtained from the patient is measured, and wherein each tumor cell has an average number of HER2 gene sets greater than or equal to 10; The method comprises administering to the patient a pharmaceutical composition once a week, every two weeks, every three weeks, the pharmaceutical composition comprising 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 Mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/ a HER2 binding agent of kilograms, 6 mg/kg or 10 mg/kg) that binds to human HER2 in a specific manner and comprises two polypeptides, each of which comprises sequence recognition Number: 8 or an amino acid sequence consisting of a sequence of at least 5, 4, 3, 2 or 1 amino acid deletions, additions or substitutions, wherein the molecular weight of the HER2 binding agent is up to 60kD, its binding affinity is small 10 ^-8 M, and is cytotoxic; and wherein the method optionally comprises administering another therapy, such as immunotherapy, the therapy such as the use of an immune anti-tumor agents.

The method may comprise: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; and (ii) if the number of sets of HER2 genes per tumor cell, such as The average HER2 gene set is greater than or equal to 10, and the HER2 binding agent is used to treat the patient, and optionally in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

Accordingly, the present invention also provides a method of treating cancer in a patient, wherein the method comprises: (i) determining the number of sets of genes for the HER2 gene of each tumor cell of a tumor sample obtained from the patient, such as the average number of sets of genes; And (ii) if the number of HER2 genes per tumor cell, such as the average number of HER2 genes, is greater than or equal to 10, the patient is administered a pharmaceutical composition once a week, every two weeks, every three weeks. The pharmaceutical composition comprises 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/kg (eg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg) a HER2 binding agent of 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/kg, wherein the HER2 binding agent is specific Sexually binds to human HER2 and comprises two polypeptides, each of which comprises a sequence identification number: 8 or a difference of up to 5, 4, 3, 2 or 1 amino acid deletion, An amino group consisting of a sequence added or substituted Sequence, wherein the molecular weight of the HER2 binding agent is based up to 60kD, which is less than the binding affinity-based 10 ^-8 M, and is cytotoxic; and wherein the method optionally comprises administering another therapy, such as immunotherapy, the use of such a Therapy for immunizing anti-tumor agents.

In one aspect, the invention provides a method of treating cancer in a patient, wherein the cancer has a high HER2 mRNA level, and wherein the method comprises treating the disease every week, every two weeks, every three weeks Suffering from administration of a pharmaceutical composition comprising 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2) a HER2 binding agent of mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/kg, The HER2 binding agent binds to human HER2 in a specific manner and comprises two polypeptides, each of which comprises a sequence identification number: 8 or a difference of up to 5, 4, 3, 2 or An amino acid sequence consisting of a sequence of amino acid deletions, additions or substitutions, wherein the HER2 binding agent has a molecular weight of up to 60 kD, a binding affinity of less than 10 ^-8 M, and cytotoxicity; Wherein the method optionally includes administering such as Phytophthora Therapies another therapy, such as an immune therapy is the use of anti-tumor agents.

Tumor samples obtained from this patient may have been found to have high HER2 mRNA levels by assay. Accordingly, the present invention provides a method of treating cancer in a patient, wherein a tumor sample obtained from the patient has been determined to have a high HER2 mRNA level by assay, and wherein the method includes, per week, every two A pharmaceutical composition is administered to the patient once every three weeks, and the pharmaceutical composition comprises 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 mg/kg or 1 to 10 mg/kg. (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg or 10 mg/ a kilogram of a HER2 binding agent that binds to human HER2 in a specific manner and comprises two polypeptides, each of which comprises a sequence identification number of 8 or at most An amino acid sequence consisting of a sequence of 5, 4, 3, 2 or 1 amino acid deleted, added or substituted, wherein the HER2 binding agent has a molecular weight of up to 60 kD and a binding affinity of less than 10 ^-8 M, and has cytotoxicity; and the party Optionally comprise administering another therapy, such as immunotherapy, the therapy such as the use of an immune anti-tumor agents.

The method can comprise: (i) determining a HER2 mRNA level of a tumor sample obtained from the patient; (ii) If the HER2 mRNA level is high, the patient is treated with the HER2 binding agent, and optionally in combination with another therapy such as immunotherapy, such as the use of an immunotherapeutic agent.

Accordingly, the present invention also provides a method of treating cancer in a patient, wherein the method comprises: (i) determining a HER2 mRNA level of a tumor sample obtained from the patient; and (ii) if having a high HER2 mRNA position The patient is administered a pharmaceutical composition once a week, every two weeks, every three weeks, and the pharmaceutical composition comprises 0.1 to 10 mg/kg, 0.1 to 0.5 mg/kg, 0.5 to 1 Mg/kg or 1 to 10 mg/kg (eg 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.7 mg/kg, 1 mg/kg, 3 mg/ a HER2 binding agent of kilograms, 6 mg/kg or 10 mg/kg, wherein the HER2 binding agent binds to human HER2 in a specific manner and comprises two polypeptides, each of which comprises a sequence Identification number: 8 or an amino acid sequence consisting of a sequence of at least 5, 4, 3, 2 or 1 amino acid deletions, additions or substitutions, wherein the molecular weight of the HER2 binding agent is Up to 60kD, with a binding affinity of less than 10 ^-8 M, and with cells Toxicity; and wherein the method optionally comprises administering another therapy, such as immunotherapy, such as the use of an immunotherapeutic agent.

Other aspects and embodiments of the present invention will be apparent to those skilled in the art in view of this disclosure.

&quot;and/or&quot; as used in this application is intended to be an explicit disclosure of each of the two specified features or components, with or without the other. For example, "A and/or B" is considered to be an explicit disclosure of each of (i) A, (ii) B, and (iii) A and B, as each is individually recited in this application.

The descriptions and definitions of the listed features are not limited to any particular aspect and embodiment of the invention, and are equally applicable to all aspects and embodiments described, unless the context dictates otherwise.

Particular aspects and embodiments of the present invention will now be described by way of example and with reference to the accompanying drawings.

Instance Example 1-H561-4 binds to an epitope on HER2 that is different from a combination of a trastuzumab antibody or a pertolecular antibody

Surface Plasma Resonance (SPR) was used to determine whether H561-4 competes with the known anti-HER2 antibody, trastuzumab antibody and Pertuzumab antibody, in HER2 binding.

Competition with trastuzumab antibody

BIAcore 3000 (GE Healthcare) was used to determine whether H561-4 can bind to one of the human HER2 coated wafers saturated with trastuzumab antibody (TR) and vice versa. CM5 wafers were coated with 1000 RU of human HER2 extracellular domain (ECD) (BenderMed Systems) using standard amine coupling, and the experimental system was 20 μl in HBS-P buffer (GE Healthcare) The flow rate was carried out at /min, and regeneration of the HER2 surface was carried out using 4M magnesium chloride. The first HER2 binding compound (H561-4 or trastuzumab antibody) was injected at 10 μg/ml for 3 minutes, and then the second HER2 binding compound (trastuzole antibody or H561-4) was injected for 3 minutes ( The second injection was carried out in the presence of 10 μg/ml of the first compound to eliminate the dissociation of Compound 1 during the second injection), followed by dissociation in the buffer (Fig. 2A). In the case of the first injection and the second injection of the same compound (in the order of trastuzumab antibody and trastuzumab antibody or sequentially H561-4 and H561-4), observed in the second injection Too little or no binding, indicating that the HER2 binding surface is saturated in both cases. Table 1 below shows the reactions (RU) observed in different injection combinations:

Table 1 shows that the binding reaction of H561-4 on a HER2 wafer saturated with trastuzumab antibody (246RU) is similar to the binding reaction of H561-4 on a naked HER2 surface (253 to 254 RU), indicating H561-4 does not compete with the trastuzumab antibody for HER2 binding. The same result was observed when the injection series was reversed, in which the binding reaction of the trastuzumab antibody on a surface saturated with H561-4 (746 RU) and the binding reaction on a naked HER2 surface (771 to 794 RU) )similar.

Competition with Pertamine monoclonal antibody

Octet (ForteBio) was used to determine whether H561-4 binds to one of the human HER2 coated tip of Perepol Monoclonal Antibody (PE) and vice versa. All steps were performed in a power buffer (ForteBio) at 1000 rpm. A streptavidin-coated tip (ForteBio) was applied by incubation in 50 μg/ml of biot-HER2 for 30 minutes, and then the tip was washed in buffer for 1 minute. Then, it was cultured in 325 nM Pertactone monoclonal antibody for 30 minutes to saturate all of the Pertulin monoclonal antibody binding sites, and then immediately transferred to H561-4 (325 nM) and Perosite monoclonal antibody (325 nM). The mixture was incubated for 30 minutes and then dissociated in buffer for 10 minutes. The experiment was repeated using a new tip to serve as a control group to ensure that all of the Pertulin monoclonal antibody binding sites were occupied. In the first step, Pertatum monoclonal antibody (325 nM) was used. Only Peptomycin monoclonal antibody (325 nM) was used in the second step (Fig. 2B). In the case where the two steps were all Peptomycin monoclonal antibodies, little or no additional binding was observed in the second step, indicating that the HER2 binding surface has been saturated with Perepolizut antibody. Table 2 below shows the reactions (RU) observed in different injection combinations:

Table 2 shows that the binding reaction (0.41 RU) of H561-4 on the HER2 wafer saturated with Perotuztab antibody was significantly greater than the additional binding reaction (0.04 RU) of the Pertatum monoclonal antibody, indicating that H561-4 was HER2 binding does not compete with Pertuzumab antibodies.

Example 2 - Studying the binding of H561-4 to HER2 Comparison of the binding of H561-4 to haplotype HER2 relative to dimeric HER2

Compared to the haplotype HER2, it was found that H561-4 preferentially binds to the dimeric HER2. ELISA and SPR methods were used to compare H561-4 with haplotype HER2 (extracellular domain of HER2 [ECD] without labeling) relative to dimeric HER2 (ECD for HER2 and Fc-tag, R&D Bio Inc.) The combination. In the ELISA format, HER2 was coated on a plate surface and thus stationary HER2; in the BIAcore experiment, H561-4 was captured and injected into HER2 as an analyte, and thus soluble HER2.

ELISA-based comparison of H561-4 binding to haplotype HER2 and dimeric HER2

Monomeric HER2 (unlabeled HER2 ECD) and dimeric HER2 (with Fc-tagged HER2 ECD) were immobilized on a 96-well Maxisorp plate (Nunc) at 2 μg/ml and placed on a 96-well Maxisorp plate (Nunc) 4 ° C in PBS overnight. Blocking of the plates was performed for 1 hour using 200 microliters of 5% BSA in PBS. Follow Hna Biosciences (Innova Bioscience) biotinylation kit (#704-0010), H561-4 and trastuzumab antibody (Roche) biotin according to the manufacturer's instructions Chemical. A dilution series of biotinylated H561-4 or biotinylated trastuzumab antibody was prepared in 5% BSA. The blocking solution was removed, and 100 μl of a dilution series of H561-4 and trastuzumab antibody was added to the plate and incubated for 1 hour at room temperature. Remove the dilution series and use phosphorus with Tween 20 The plate was washed three times with acid buffered saline (PBST).

A 1:5000 dilution of Strep-HRP (Abeam antibody company) was prepared in 5% BSA, 100 microliters was added to the plate and incubated for 1 hour at room temperature. The plate was then washed three times with PBST, followed by the addition of 100 microliters of TMB (Roche) and the plate was incubated for 2 minutes at room temperature. The reaction was stopped using 100 microliters of 2M sulfuric acid and the plate was read at 450 nM.

Trastuzumab monoclonal antibody control group with the same half maximal effective concentration (EC ₅₀₎ (1.3 to 1.5 nM) in a monomeric and dimeric type of HER2; haplotype Compared to the HER2, H561-4 for The dimer type HER2 has a 10-fold preference, as indicated by EC ₅₀ values of 1.7 nM and 20.6 nM, respectively. EC ₅₀ means after a specified exposure time, half of the binding reaction between the initiator concentration between the base member and a maximum value.

Comparison of BIAcore expression of H561-4 binding to haplotype HER2 and dimeric HER2

To capture H561-4 as directed to allow binding to soluble HER2, anti-EGFR-H561-4 mAb ² (EGFR/H561-4) was used. This molecule is an anti-EGFR antibody whose Fc region has been engineered to contain a binding loop of H561-4 in the CH3 domain. The EGFR/HER2 bispecific molecule is then captured on the EGFR coated BIAcore wafer via the EGFR binding specificity at the Fab arm, leaving the HER2 binding CH3 domain exposed to the analyte. To allow comparison with the HER2 mAb trastuzumab antibody, a similar bispecificity, the trastuzumab antibody-EGFR antigen binding Fc fragment (TR/EGFR), was constructed. The bispecific line comprises a HER2 mAb trastuzumab antibody, the Fc region of which is engineered to contain a binding loop of an anti-EGFR antigen binding Fc fragment in the CH3 domain. Both the EGFR mAb and the EGFR antigen binding Fc fragment have similar binding affinities for EGFR (1 nM). TR/EGFR was captured on the EGFR wafer via the EGFR antigen binding Fc fragment Fc region, leaving the HER2 binding specificity of the Fab arm exposed to the analyte.

BIAcore streptavidin wafers were fixed with 1000 RU of biot-EGFR. The experiment was carried out in a HBS-P buffer (GE Healthcare) at a flow rate of 20 μl/min. The EGFR/HER2 bispecific molecule was captured by injecting 60 microliters of 100 mM mAb ² and the surface of EGFR was regenerated using 50 mM sodium hydroxide. A dilution series of haplotype (unlabeled HER2 ECD, internal) and dimeric (plus Fc-tagged HER2 ECD, Research and Development) was injected as an analyte. Data fitting was performed using Langmuir's 1:1 binding mode.

The following Table 3 data lines showed by 1: 1 K _D values obtained from fitting. H561-4 showed weak binding to soluble haplotype HER2, even at concentrations up to 1000 nM; and affinity to dimeric HER2 was 1 nM. The binding affinity of the trastuzumab antibody to the haplotype HER2 was 1 nM, and its binding affinity to the dimeric HER2 was sub-nM grade.

Prediction of H561-4 epitope on HER2 using peptide scanning technology

Using the CLIPS technique (peptide scanning technique), it was determined that the binding of H561-4 spans two domains of extracellular HER2. These domains are Domain 1 and Domain 3 of HER2, and their binding epitopes are located in the 13th to 27th amino acids (LPASPETHLDMLRHL) and 31st to 45th amino acids (CQVVQGNLELTYLPT) of Domain 1. . The 420th to 475th amino acid (SLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTA) in domain 3 has a larger binding site. In terms of linear structure, these three different epitopes appear to be random, however when viewing the configuration format, a unique binding pocket spanning one of the two domains is revealed (Fig. 2C).

Deprotection using standard FMOC chemistry and trifluoro acid with scavengers, based on the amino acid sequence of extracellular HER2, synthesize linear and CLIPS (linking peptides to scaffolds using chemical bonds) peptides. CLIPS used techniques, synthetic peptides prepared by chemically beam holder, to reconstruct the bit configuration epitope (Timmerman et al., 2007 Journal "J MoL Recognit."^"(TM) technology using CLIPS 20 pp 283-99 of "Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS ^TM technology" (in Chinese). 0.5 mM solution with CLIPS template on a credit card format polypropylene PEPSCAN card (455 peptide format/card) (1,3-bis(bromomethyl)benzene/acetonitrile in ammonium bicarbonate (20 mM vs. pH 7.9) (1:1 [vol/vol) reaction, coupling the side chains of the numerous cysteine acids in the peptide to the CLIPS template. When the solution completely covers the card, the card is gently shaken in solution for 30 to 60 minutes. Finally, the cards were extensively washed with excess water and sonicated for 30 minutes in 70 °C interrupt buffer containing 1% SDS/0.1% β-mercaptoethanol in PBS (pH 7.2). The sonication treatment was carried out in water for 45 minutes. The binding of the antibody to each peptide was tested in a PEPSCAN-type ELISA. A 455-well credit card format polypropylene card containing a covalently linked peptide was incubated with a primary antibody solution. The antibody solution consists, for example, of 1 μg/ml of primary antibody diluted in blocking solution, 5% horse serum, and 5% ovalbumin (weight/volume) in PBS/1% Tween 80.

After washing, the peptides were incubated with a 1:1000 diluted antibody peroxidase complex for 1 hour at 25 °C. After washing, the peroxidase was added with 2,2'- azo-di-3-ethylbenzothiazoline sulfonate (ABTS) and 2 μl of 3% 11202. After 1 hour, measurement Color development. The color rendering is quantified using a charge coupled device (CCD), a camera and an image processing system (as originally described by Slootstra et al., 1996, "Molecular Diversity", No. 1, pp. 87-96" via a small random peptide library. "Structural aspects of antibody-antigen interaction revealed through small pulp libraries" (described in B).

Therefore, the raw data is the optical value obtained by the CCD-camera. These values are mostly in the range of 0 to 3000 with a logarithmic scale similar to the logarithmic scale of 1 to 3 of a standard 96-well plate ELISA-reader. The values obtained in this experiment ranged from 0 to 380.

The CCD-camera first takes a picture of the card before the peroxidase is colored, and then takes another picture after the peroxidase is colored. The two photos are subtracted from each other to produce raw data. Copy it to the Peplabm database. The values are then copied to Excel and the file is marked as the original data file. A follow-up operation can be tolerated. Sometimes, the holes contain bubbles that cause false positives, manually check the cards, and record any value due to bubbles as zero. Positive values will be considered as binding signals (antigenic epitope maps) and sequences mapped to HER2. A computer generated map of the extracellular HER2 protein was output, the location of the positive binding signal was mapped, and the region of the antigen binding Fc fragment binding was indicated (Fig. 2C).

Example 3 -H561-4 triggers apoptosis and leads to unique and far-reaching HER2 internalization and degradation

The breast cancer cell line SKBr3 overexpressing HER2 was used as a model system to determine the mechanism of action of H561-4. The SKBr3 cell line was obtained from ATCC (HTB-30) and cultured in McCoy 5a+GlutaMAX medium (Invitrogen) containing 10% fetal bovine serum (FBS). In all studies of the mechanism of action, trastuzumab antibody (Roche) was used as a control group.

H561-4 has antiproliferative activity and induces apoptosis in SKBr3 cells

SKBr3 cells were seeded into 96-well tissue culture dishes at 7.5 x 10 ³ cells per well, and cultured overnight at 37 ° C, 5% carbon dioxide at 100 μL per well. On the next day, 20 μl of the medium containing the therapeutic agent (H561-4 or trastuzumab antibody) was added to obtain a concentration dilution series of one of H561-4 or trastuzumab antibody. The cells were incubated with the therapeutic agent for 5 days at 37 ° C, 5% carbon dioxide. Then stained with 1 μg/ml of final Heechst (Sigma Aldrich H6024), 2.5 μg/ml propidium iodide (PI) and 1:100 Alexa -488 Type V Annexin (Invitrogen V13245) in Type V Annexin Binding Buffer (12.5 mM calcium chloride, 140 mM sodium chloride, 10 mM 4-hydroxyethylperidine) A mixture of one of ethanesulfonic acid (Hepes), pH 7.4) was stained with cells and incubated at room temperature (RT) for 2 hours. The cells were then read on an ImageXpress Micro (Molecular Devices). The number of cells in each well was counted using Hoechst staining and magnification 4x. Using a type V annexin and propidium iodide staining to measure live cells (cells not stained with propidium iodide) and early apoptotic cells (only subjected to type V membrane association) at 40-fold magnification Percentage of late apoptotic cells (cells stained with type V annexin and propidium iodide) and necrotic cells (cells stained only with propidium iodide). H561-4 resulted in a 37% reduction in cell number at the highest concentration tested (300 nM) and a 22% reduction in cell number compared to trastuzumab antibody. In addition, only 43% of the cells remaining after treatment with H561-4 survived, compared to 79% of the cells in the case of trastuzumab antibodies. In the case of cells treated with H561-4 only, the decrease in total cells and viable cells was associated with an increase in late apoptotic cells and necrotic cells (18% and 35%, respectively). The results of this experiment are shown in Figure 3A.

Internalization and degradation of HER2 induced by H561-4

The SKBr3 cell line was inoculated to a 6-well plate at 3.3 x ^{10 5} cells/ml and 3 ml/well, and cultured overnight at 37 ° C, 5% carbon dioxide. The next day, the medium was changed to medium containing 0.1% fetal bovine serum, and cultured at 37 ° C, 5% carbon dioxide for at least 2 hours, then the medium was removed, and replaced with 0.1% fetal bovine serum and 200 nM therapeutic agent ( Medium for H561-4 or trastuzumab antibody). The cell line was incubated at 37 ° C, 5% carbon dioxide for 24 hours, 48 hours or 72 hours. The lysate was then collected on ice: the medium was removed, and 250 μl of lysis buffer (pH 7.5 of 10 mM Tris, 150 mM sodium chloride, 1 mM EDTA, 1:100 protease cocktail [Kyle Biochemical (Kyle Biochemical ( Calbiochem) 539131], 1:100 phosphatase cocktail [Kell Biochemical Company 524625]). The cells were then scraped from the surface of the wells and 250 μl was placed on ice for 15 minutes, centrifuged at 14,000 rpm for 15 minutes at 4 ° C and the supernatant was collected for analysis. The sample was mixed with NuPAGE loading buffer containing β-mercaptoethanol, incubated at 95 ° C for 10 minutes, and then stored at -20 ° C until use. Allow 20 μl of the sample to run on 4 to 12% Bis-Tris SDS PAGE and transfer to a nitrocellulose membrane, block the membrane with 5% Marvel/TBST, and then probe HER2 (Cell Signalling Company Catalog No. 2248), Phosphor-HER2 (Saitong Corporation Catalog No. 2243) or α-β-actin (Sigma) as a loading control A1978). These membranes are then washed and a suitable HRP complex secondary antibody (an anti-mouse antibody from Jackson Immunoresearch, or an anti-rabbit antibody from Jackson Immunology) is added. These films were developed and imaged using ECL reagent (GE Healthcare, Inc., RPN 2105).

Treatment with H561-4 for 24 hours resulted in a decrease in total HER2, resulting in a complete decrease at 48 hours and 72 hours (Fig. 3B). Conversely, trastuzum monosaccharide Treatment of the antibody did not result in a detectable decrease in HER2 level at the time point studied (Fig. 3B). Treatment with H561-4 also resulted in a decrease in pHER2. The decrease was evident at 24 hours, further decreased at 48 hours, and no pHER2 residue was detected at 72 hours. At 72 hours, treatment with trastuzumab antibody only resulted in a slight decrease in pHER2 (Fig. 3B).

Immunofluorescence imaging was used to quantify the degradation of HER2. The SKBr3 cell line was seeded at 1 x 10 ⁴ cells/well into 96-well plates and incubated overnight at 37 ° C in 5% carbon dioxide. The next day, a therapeutic agent (H561-4; trastuzumab antibody [TR]) or a control group (wild type [WT] antigen-binding Fc fragment; or IgG1 isotype control group, ie, Sikma (" Sigma) I5154 [IgG/IgG1 control]), resulting in a final concentration of 200 nM.

The cell lines were incubated at 37 ° C, 5% carbon dioxide for 24 hours, 48 hours or 72 hours, the supernatant was removed, the cells were washed with PBS, and then fixed with 4% formaldehyde in PBS for 20 minutes. For cell surface imaging, half of the cells were permeabilized in 0.1% Triton X100/0.1 mg/ml glucose in PBS for imaging of total protein. The cells were then washed and blocked for 1 hour at room temperature with 5% FBS/PBS and then diluted 1:100 with primary antibody in PBS at room temperature (Enzo Life Sciences) The mice were incubated with Her2 MGR2) for 1 hour. The cells were then washed twice, then at room temperature and in the dark with 5 μg/ml of secondary antibody (Invitrogen A21237 anti-mouse alexa647) and 1 μg/ml of Hoechst (Invitrogen ( Invitrogen) company 33342) cultured for 1 hour. Then wash the cells, and Prior to reading on ImageXpress Micro (Molecular Devices), it was stored in 0.05% sodium azide in PBS. The analysis was performed with a 40-fold imaging effect, and the multi-wave scoring function was used to count the number of cells and cells having HER2 positive staining, and then the percentage of HER2-positive cells was calculated.

Treatment of SKBr3 cells with 200 nM H561-4 resulted in a decrease in cell surface and total HER2. A decrease in the HER2 level was detected after 24 hours of treatment, and the degree of decrease increased with time (48 hours and 72 hours) (Fig. 3C). Treatment with the control antibody, trastuzumab antibody, did not result in a detectable decrease in HER2 levels (Fig. 3C).

Treatment with H561-4 triggers apoptosis-dependent apoptosis

The SKBr3 cell line was seeded into 96-well plates at 7.5 x 10 ⁴ cells/ml and 100 microliters per well, and cultured overnight at 37 ° C with 5% carbon dioxide. On the next day, the therapeutic agent (H561-4, trastuzumab antibody, wild type (WT) antigen-binding Fc fragment or IgG1 isotype control group) in the medium was added to the well to obtain 1000 nM, 100 nM, 10 nM, Final concentrations of 1 nM, 0.1 nM, and 0.01 nM were added to the untreated control group. The cell line was cultured for five days at 37 ° C with 5% carbon dioxide, and then the apoptosis protease 3/7 activity was measured using Promega's apoptosis protease-Glo 3/7 assay. Add 50 μl of the Apoptotic protease 3/7 Glo reagent to each well, mix and incubate the plate for 2 hours at room temperature, then measure the luminescence using a Synergy Type 4 microplate reader (BIOTEK). .

Treatment of SKBr3 cells with H561-4 resulted in a significant dose-dependent manner of eliciting protease 3/7 activity (Fig. 3D); Treatment with the monoclonal antibody did not result in a detectable increase in the activity of the apoptotic protease 3/7, as was the treatment with the wild-type antigen-binding Fc fragment or the IgG1 isotype control group.

Taken together, the above results demonstrate that when SK56 cells are treated with H561-4, apoptosis is induced and internalization and degradation of HER2 are caused.

Example 4 - In vivo efficacy study: Treatment of tumor xenograft (PDX) patterns in human patients with H561-4 treatment of HER2 gene sets greater than or equal to 10

The in vivo efficacy of H561-4 in a tumor pattern expressing HER2 with a set of genes greater than or equal to 10 was evaluated using mice bearing xenograft tumors derived from human patients. The use of immunocompromised mice allows xenografts and growth of human tumors. Tumors were implanted in mice about 5 to 7 weeks old, which is different from the GA0060 PDX mode in which tumors were implanted in mice about 6 to 8 weeks old. The research work of GA0060 was carried out by Crown BioScience, and the experimental procedures used were slightly different from other PDX modes used by other clinical research institutions. Any differences in the experimental procedures of Crown Biotech are shown below. All experiments were approved by the local authorities and conducted in accordance with all applicable international, national and local regulations and guidelines. Animals screened for participation in the testing procedure have an undisputed health condition. Mice received at least 7 days of environmental acclimatization after arrival and prior to the start of any experiment. The tumor xenograft system used as the test tumor in this study was derived from a surgical specimen of a patient and implanted directly into the skin of a nude mouse, hence the term "patient-derived tumor xenograft" (PDX). Obtained from xenografts subcultured in nude mice Tumor fragment. After the tumor was taken out from the donor mouse, it was cut into fragments (2 to 5 mm in diameter or 2 to 4 mm in diameter in the GA0060 mode), and then transplanted subcutaneously into the flank of the unilateral mouse. Tumor-bearing animals were randomly stratified into different groups based on tumor volume. Only mice with appropriate tumor sizes (50 to 250 mm, or 100 to 300 mm in the GA0060 mode) were considered for inclusion in the randomization group.

When the number of mice required for randomization was reached, the mice were randomized into groups. The random group will be set to the 0th day. The first day of administration was also day 0. Animals are sacrificed when the tumor volume exceeds 2000 cubic millimeters. If an animal is sacrificed due to tumor burden, the final observation (LOCF) is used as the subsequent tumor volume value.

For all PDX modes except the GA0060 PDX mode, antibody samples were diluted to 1 mg/ml in PBS and administered at 10 mg/kg. Depending on the study, the dosing regimen is administered once a week or twice a week. Absolute tumor volume (ATV) was determined using a caliper for two-dimensional measurements on the next day after tumor transplantation and twice a week thereafter (i.e., on the same day the mice were weighed). The tumor volume was calculated according to the following formula: (axb ² ) x 0.5, where a represents the largest tumor diameter and b represents the vertical tumor diameter. Tumor inhibition (T/C in %) was calculated from the ratio of the mean absolute tumor volume value (i.e., mean tumor growth) of the test group to the control group multiplied by 100%.

Day x is any day at which the lowest (best) T/C is observed. Day 0 was the first day of dosing.

For all PDX modes other than the GA0060 PDX mode, the lowest (or optimal) T/C% value recorded for a particular test during a study represents the maximum anti-tumor efficacy of each treatment. The T/C value is calculated if at least 50% of the randomized animals in the group survive on that day in question.

For the GA0060 PDX mode, antibody samples were diluted to 6 mg/ml in PBS and administered at 60 mg/kg. The dosing regimen is administered twice a week, depending on the study. Absolute tumor volume (ATV) was determined using a caliper for two-dimensional measurements on the next day after tumor transplantation and twice a week thereafter. The tumor volume was calculated according to the following formula: (axb ² ) x 0.5, where a represents the largest tumor diameter and b represents the vertical tumor diameter. The tumor inhibition (T/C in %) was calculated from the ratio of the mean absolute tumor volume values of the test group to the control group multiplied by 100%. The difference between the two T/C formulas is illustrated in Example 7.

Where at least 50% of the animals in the group survive, x is about one dose period after the final dose.

For the sake of simplicity, this formula is referred to as the updated T/C formula.

Efficacy of H561-4 in PDX mode GXF281 in the stomach

GXF281 was isolated from primary adenocarcinoma in the stomach of a 46-year-old male patient and was performed by Oncotest (Freiburg, Germany) Studies were performed in NMRI nude mice. H561-4 (10 mg/kg), trastuzumab antibody (10 mg/kg) and PBS (10 ml/kg) as a vehicle control group were injected once a week for a total of 5 weeks. Each group included 6 mice. Tumor volume was measured twice a week and for up to 81 days, and the mean absolute tumor volume was plotted (Figure 4A). A total of five rounds of H561-4 treatment per week resulted in complete tumor regression (minimum T/C values were less than 0%), while a total of five rounds of trastuzumab antibody treatment per week produced only moderate efficacy (minimum T/ C is 30%).

Therapeutic effect of H561-4 in gastric PDX mode GXA3039

GXA3039 was isolated from primary adenocarcinoma of the stomach of a 65 year old male patient and was studied by NMRI nude mice by Oncotest (Freiburg, Germany). H561-4 (10 mg/kg), trastuzumab antibody (10 mg/kg) and PBS (10 ml/kg) as a vehicle control group were injected once a week for a total of 4 weeks. Each group included 5 mice. Tumor volume was measured twice a week and lasted for up to 91 days, and the mean absolute tumor volume was plotted (Figure 4B). A total of four rounds of H561-4 treatment per week resulted in complete tumor regression (minimum T/C values were less than 0%), while a total of four rounds of trastuzumab antibody treatment per week produced only mild efficacy (minimum T /C is 88%).

Therapeutic effect of H561-4 in colorectal PDX mode CXF1991

CXF1991 was isolated from a primary adenocarcinoma of the colon of a 59-year-old female patient and was studied by NMRI nude mice by Oncotest (Freiburg, Germany). H561-4 (10 mg/kg), trastuzumab antibody (10 mg/kg) and PBS as vehicle control (10 ml/kg) Jin) is injected once a week for a total of 6 weeks. Each group included 5 mice. Tumor volume was measured twice a week and for up to 62 days, and the mean absolute tumor volume was plotted (Figure 4C). A total of six rounds of H561-4 treatment per week resulted in complete tumor regression (minimum T/C values were less than 0%), while a total of six rounds of trastuzumab antibody treatment per week produced only mild efficacy (minimum T /C is 57%).

Therapeutic effect of H561-4 in colorectal PDX mode CXF2102

CXF2102 was isolated from liver metastases of colonic adenocarcinoma of patients and was studied by NMRI nude mice by Oncotest (Freiburg, Germany), whose age and sex were unknown. H561-4 (10 mg/kg), trastuzumab antibody (10 mg/kg) and PBS (10 ml/kg) as a vehicle control group were injected once a week for a total of 4 weeks. Each group included 5 mice. Tumor volume was measured twice a week and for up to 58 days, and the mean absolute tumor volume was plotted (Figure 4D). A total of four rounds of weekly H561-4 treatment resulted in significant tumor regression (minimum T/C values were less than 0%), while a total of six rounds of weekly trastuzumab antibody treatment did not elicit significant effects ( The minimum T/C is 76%).

Efficacy of H561-4 in PDX mode GXA3054 in stomach

GXA3054 was isolated from a primary tumor of a 65-year-old male patient with gastric adenocarcinoma and was studied by NMRI nude mice by Oncotest (Freiburg, Germany). H561-4 (10 mg/kg) and PBS (10 ml/kg) as a vehicle control group were injected once a week for a total of 5 weeks. Suspension of administration for 4 weeks, then resumed weekly dosing Once for 4 weeks. Each group included 5 mice, and one group that received only the antibody to trastuzumab was not included in this model. Tumor volume was measured twice a week and for up to 100 days per week, and the mean absolute tumor volume was plotted (Figure 4E). H561-4 treatment per week caused tumor regression (lowest T/C value of 17%).

Therapeutic effect of H561-4 in PDX mode GXA3038 in the stomach

GXA3038 was isolated from a primary tumor of a 63-year-old male patient with gastric adenocarcinoma and was studied by NMRI nude mice by Oncotest (Freiburg, Germany). H561-4 (10 mg/kg) and PBS (10 ml/kg) as a vehicle control group were injected once a week for a total of 5 weeks. The administration was suspended for 1 week and then resumed once a week for 4 weeks. Each group included 5 mice, and one group that received only the antibody to trastuzumab was not included in this model. Tumor volume was measured twice a week and for up to 77 days, and the mean absolute tumor volume was plotted (Figure 4F). H561-4 treatment per week caused a slight tumor regression (minimum T/C value of 55%).

Therapeutic effect of H561-4 in breast PDX mode HBCx-13B

HBCx-13B was isolated from a lymphatic metastatic malignancy and was studied by Xentech (Evry, France) in athymic nude mice (Hsd; athymic nude mice-Fox1nu). H561-4 (10 mg/kg), trastuzumab antibody (10 mg/kg), and PBS (10 ml/kg) as a vehicle control group were injected twice a week for a total of 8 weeks. Each group included 9 mice. Tumor volume was measured twice a week for up to 56 days and the mean absolute tumor volume was plotted (Figure 4G). Twice a week Treatment with H561-4 caused significant tumor regression (lowest T/C value of 7%), while a total of six rounds of trastuzumab antibody treatment per week produced only mild efficacy (minimum T/C of 48%).

Therapeutic effect of H561-4 in gastric PDX mode GA0060

GA0060 was isolated from human gastric cancer, and was studied in BALB/c nude mice by Guanke Biotechnology Co., Ltd. (Beijing, China). H561-4 (30 and 60 mg/kg), trastuzumab antibody (30 mg/kg) and PBS (10 ml/kg) as a vehicle control group were injected twice a week, and a total of 6 injections were given. week. Each group included 10 mice. Tumor volume was measured twice a week for up to 41 days, and the mean absolute tumor volume was plotted (Figure 4H). Calculate the T/C value using the updated T/C formula. A total of six rounds of H561-4 treatment per week resulted in mild tumor regression (60% vs. 30 mg/kg T/C values of 49% and 60%, respectively), and a total of six rounds of trastone per week. Single antibody treatment resulted in tumor regression (T/C value of 25%). A 60 mg/kg trastuzumab antibody was not tested because at 30 mg/kg it has caused tumor regression.

Example 5-H561-4 shows efficacy in the resistance pattern of trastuzumab antibody and pertituzate antibody

As shown in Example 4 above, H561-4 has an anti-tumor effect in a patient-derived xenograft (PDX) mode in which the HER2 gene set is greater than or equal to 10. It was also observed that the anti-tumor effect of H561-4 in these PDX modes was significantly better than that of the trastuzumab antibody.

Clinically, trastuzumab antibody is often used in combination with pertitubeta monoclonal antibody for the treatment of breast cancer and gastric cancer. PX mode GXF281 in the stomach Among them, the combined therapy of trastuzumab antibody and pertoxazide antibody caused a slowing of tumor growth. However, the tumor eventually deteriorated. H561-4 caused complete regression of the tumor when used to treat tumors that were still aggravated by the combined therapy of the trastuzumab antibody and the pertallazine antibody. These results are shown in Figure 5.

Tumor fragments obtained from the patient-derived tumor GXF281 were implanted subcutaneously in NMRI nu/nu mice under anesthesia. Mice with suitable tumor growth (50 mm to 250 mm mm tumors) were randomly divided into the following three groups:

‧Group 1: 5 mice, which received a four-week intravenous injection of 10 ml/kg PBS once a week.

‧ Group 2: 20 mice, which received a four-week intravenous injection of 10 mg/kg of trastuzumab antibody and 10 mg/kg of Pertoxagen monoclonal antibody once a week.

‧Group 3: 5 mice, which received a four-week intravenous injection of 10 mg/kg of H561-4 once a week.

On the 53rd day, when the average tumor volume of the mice of the second group reached 500 mm 3 , the mice of the second group (Trastuzole monoclonal antibody and Pertuxone monoclonal antibody combined treatment group) were randomly divided into the following groups. Four groups:

‧Group 4: 5 mice, which did not receive further treatment.

‧Group 5: 5 mice received an intravenous injection of 10 mg/kg of trastuzumab antibody and 10 mg/kg of Pertuzumab antibody once a week for seven rounds.

‧Group 6: 5 mice, which received an intravenous injection of 10 mg/kg of H561-4 once a week for seven rounds.

‧Group 7: 5 mice, which received an intravenous injection of 3.6 mg/kg H561-4 once a week for seven rounds.

Tumor volume was monitored twice a week for 105 days (Figure 5). Group 3 showed complete regression of the tumor, no mice had detectable tumors after day 46 of the study, and no tumor re-growth did not occur until the end of the study (day 105). The tumor growth of the second group was slower than that of the control group (group 1). Repeated administration of trastuzumab antibody and pertituzate antibody in mice did not further slow tumor growth (the tumor growth of Group 4 and Group 5 was similar). H561-4 (dose of 10 mg/kg or 3.6 mg/kg) caused significant tumor regression when used to treat mice that were still aggravated by combination therapy with trastuzumab antibody and pertituzil antibody The mean tumor size of group 6 on day 105 was 19 cubic millimeters, compared to the average tumor size of 716 cubic millimeters on day 53 before H561-4 treatment; the average tumor size of group 7 on day 105. The average tumor size was 3595 cubic millimeters compared to the 53rd day before H561-4 treatment was 659 cubic millimeters.

Example 6-H561-4 selectively inhibits tumor growth in a PDX mode with a HER2 gene set greater than or equal to 10

The in vivo activity of H561-4 was studied in 23 different patient-derived xenograft (PDX) models, which were classified as HER2 positive by IHC according to the criteria used by Oncotest. The efficacy of H561-4 treatment and trastuzumab antibody treatment was evaluated based on the lowest T/C value expressed as a percentage. Calculate the T/C value using the following formula: Mean tumor volume of the control group on day x - average tumor volume of the control group on day 0

Day x is any day at which the lowest (best) T/C is observed. Day 0 was the first day of dosing.

Table 4 shows the efficacy criteria defined by the lowest T/C value (in %):

The number of HER2 gene sets in the PDX tumor pattern was determined by quantitative PCR:

DNA from each tumor was supplied at an initial concentration of 100 ng/μl, and then diluted to 5 ng/μl in water at a molecular grade (SIGMA) #W4502-1L. Prepare a premix for the PCR reaction, and its line is a 2x Taigman genotype analysis premix (Invitrogen #371355), 1 microliter of HER2 Tagman probe and The primer set (Invitrogen's genetic analysis identification number: Hs00817646 and catalog number 4400291) and 1 microliter of housekeeping RNAseP Taqman probe and primer set (Invitrogen's #4403326) Composed of. A 20 ng DNA sample was added to deionized water such that the final reaction volume was 20 microliters. The conditions used for quantitative PCR were as follows: maintaining at 95 ° C for 10 minutes, followed by 40 cycles each at 95 ° C for 15 seconds, and then at 60 ° C for 60 seconds. After obtaining the original C _T (cycle threshold) data, the C _T value of the manual 0.2 and an automatic baseline are applied to the results.

All data obtained were normalized by a housekeeping gene (RNAseP Taqman probe and primer set, Invitrogen #4403326), which is known to have 2 of the genes in each cell. copy. The results of quantitative PCR are shown in arbitrary units (AU and its lines are related to the number of sets per cell). First, the difference between the CT gene value (circulation threshold) of one of the housekeeping genes and the HER2 gene was calculated as follows: the target gene expression effect = 2 ( ^C _T ^{housekeeping gene (RNAse P)-C} _T ^{HER2 gene)}

Analysis of the results of linear amplification, and transmission to the Copy Caller ^TM software, Version 2.0 (Invitrogen (Invitrogen) Corporation), to determine the copy number of the HER2 gene. Included in the analysis were DNA reference samples found to have two sets of HER2 (Roche Pharmaceuticals #11691112001) (since the HER2 gene and the housekeeping gene have equal C _T values, indicating that each cell has two sets of HER2), The determination is carried out by determining the exact number of sets and allowing adjustments. The 2AU is considered to be a normal value of the HER2 gene in the reference genome DNA. When the number of sets obtained from human standard genomic DNA is not exactly 2 AU, a multiplier is applied to convert it to 2 AU. This multiplier also applies to the results obtained from tumor samples. The number of sets of genes for each sample is shown in Table 5.

Table 5 shows the efficacy as defined by the lowest T/C value (in %) for the PDX pattern treated with H561-4 and trastuzumab antibody, and the number of sets of genes for each PDX tumor sample tested (GCN) ):

A PDX mode with a HER2 gene set number (GCN) greater than or equal to 10. In general, H561-4 has efficacy in six of the seven modes; whereas for PDX mode with a HER2 gene set of less than 10, H561-4 has marginal activity in only two of these modes. In the PDX mode in which the number of sets of genes is greater than or equal to 10, the trastuzumab antibody has moderate activity in two of the modes and edge activity in the other mode. In the PDX mode with a set number of genes less than 10, the trastuzumab antibody has marginal activity in three of the PDX modes, and the other three modes have moderate activity. Figure 6 shows a scatter plot of the therapeutic T/C values of H561-4 and trastuzumab antibodies in the PDX mode with a HER2 gene set greater than or equal to 10, and compared to the PDX pattern with a set of genes less than 10. The scatter plot shows the efficacy of H561-4 in cancers with a set number of genes greater than or equal to 10, which is statistically greater than the efficacy in cancers with less than 10 sets of genes. The activity of the trastuzumab antibody was independent of the number of HER2 gene sets.

The mRNA levels in the PDX tumor model were determined by reverse transcription (RT) PCR and quantitative PCR in sequence:

mRNA from each tumor was provided at a concentration of 250 Ng/μl. mRNA reverse transcription was performed using a high capacity cDNA reverse transcription kit (Invitrogen #N8080234) following the manufacturer's instructions. The transcribed cDNA was then quantified using a Nanodrop 2000 (Thermo Scientific) to determine the cDNA concentration and its line in nanograms per microliter. The resulting cDNA was diluted to 25 Ng/μl in water at a molecular grade (Sigmama (SIGMA) #W4502-1L). Preparation of a master mix for quantitative PCR reactions, and its 2x Tegman gene performance premix (Invitrogen #4369016), 1.25 microliters of HER2 Tagman probe and primer The group (Invitrogen's Hs01001580_m1 #4331182) and 1.25 microliters of the housekeeping human TBP Tagman probe and the introduction group (Invitrogen's Hs00427620_m1 #4331182). A 50 ng cDNA sample was added along with deionized water such that the final reaction volume became 25 microliters. The conditions used for quantitative PCR were as follows: maintained at 95 ° C for 10 minutes, followed by 50 cycles each at 95 ° C for 15 seconds, and then at 61 ° C for 30 seconds. After obtaining the original C _T (cycle threshold) data, the CT value of the manual 0.2 and an automatic baseline are applied to the results.

The "Human Standard cDNA" was used as a reference and its concentration was the same as that of the tumor cDNA sample, which was reverse transcribed from "Universal Human Reference RNA" (Agilent Technologies, Inc. #740000). "Universal human reference RNA" is formed by pooling 10 human cell lines that are expected to have two sets of HER2 genes per cell (as the cell line is considered to have a normal profile). This human standard cDNA is considered to have a normal mRNA profile.

All data obtained were normalized by a housekeeping gene (the housekeeping human TBP Tagman probe and the introduction group (Invitrogen's Hs00427620_m1 #4331182)), which is known to be in many tissue types. Has a constant performance level. The results of quantitative PCR are shown in arbitrary units (AU). First, the difference between the CT gene value (circulation threshold) of one of the housekeeping genes and the HER2 gene was calculated as follows: HER2 expression effect = 2 ( ^C _T ^{housekeeping gene (TBP)-C} _T ^{HER2 gene)}

Analysis results on linear amplification, and transmission to the Copy Caller ^TM software, Version 2.0 (Invitrogen (Invitrogen) Company). Using this software, the number of sets of HER2 cDNA in the tumor sample relative to the cDNA set of the housekeeping gene was determined, and the number of HER2 cDNA sets in the human standard cDNA sample was normalized.

Standardization was carried out by setting the normal value of the number of sets of HER2 cDNA relative to the number of cDNA sets of the housekeeping gene in the human standard cDNA to 2 AU. A multiplier (constant) is applied to convert it to 2 AU. The multiplier (constant) is also applicable to the results obtained from the tumor sample, and the resulting cDNA sets are listed in Table 6 in the form of mRNA levels.

That is, the HER2 expression of standard cDNA is x constant = 2

HER2 expression in tumor samples x constant = number of cDNA sets

The constant is used to normalize the HER2 expression effect in the standard cDNA to a multiplier of the value 2.

Table 6 shows the efficacy as defined by the lowest T/C value (in %) for the PDX pattern treated with H561-4 and trastuzumab antibody, and the HER2 mRNA level for each PDX tumor sample.

In the PDX mode in which the HER2 mRNA level in the tumor is higher than or equal to 200, H561-4 has efficacy in six of the seven modes; and the HER2 mRNA level in the tumor is higher than or equal to 200 and lower. Either or all of the PDX modes have a therapeutic effect; whereas in the PDX mode in which the HER2 mRNA level in the tumor is below 200, H561-4 has marginal activity only in two of the modes. The HER2 mRNA level in these patterns with mRNA levels below or equal to 820 was 168, while the HER2 mRNA level for all modes tested was 248. The HER2 mRNA level is higher than or In the PDX mode equal to 200, the trastuzumab antibody has moderate activity in two of the modes and edge activity in the other mode. In the PDX mode where the mRNA level is below 200, the trastuzumab antibody has marginal activity in three of the PDX modes, and the other three modes have moderate activity. Figure 7 shows a scatter plot of the therapeutic T/C values of H561-4 and trastuzumab antibodies in the PDX mode with a HER2 mRNA level above 200 in the tumor, and a lower level of mRNA in the tumor. Compare with the PDX mode of 200. This shows that the efficacy of H561-4 in cancers with HER2 mRNA levels above or equal to 200 is statistically greater than in cancers with HER2 mRNA levels below 200. In particular, the data show that the efficacy of H561-4 in cancers with HER2 mRNA levels above or equal to 200 and below or equal to 820 is statistically greater than in PDX mode where HER2 mRNA levels are below 200 and greater than 820. Efficacy. Within the range tested, the activity of the trastuzumab antibody was independent of the HER2 mRNA level.

Example 7 - Using updated T/C values measurements, H561-4 was found to selectively inhibit tumor growth in a PDX mode with a HER2 gene set greater than or equal to 10.

A different T/C value calculation formula was used to evaluate the efficacy of H561-4 treatment with trastuzumab antibody treatment.

Calculate the T/C value using the following formula: Where at least 50% of the animals in the group survive, x is about one dose period after the final dose.

As shown in Example 4 above, this formula is referred to as an updated T/C formula. The updated formula is designed to facilitate comparison of data collected by different clinical research institutions.

Compared to the T/C formula used in Example 6, the updated T/C formula incorporates two changes: the xth day is defined as the time point of comparison and the base deduction is removed.

In the T/C formula previously used, day x refers to any day at which the lowest (best) T/C value is observed. Thus, day x can be any day of the entire period of each experiment. In the updated version of the T/C formula, day x becomes a fixed time point for about one dose period after the final dose (if at least 50% of the animals in the group survive). Therefore, the updated T/C formula has the advantage of standardizing day x, allowing us to compare the therapeutic effects of different studies. Although the basic deduction step was not included in the updated T/C formula, this was compensated by the uniform ordering distribution of the vaccinated mice, while equal mean tumor sizes were obtained in different experimental groups. Using the final observation methodology, the values from mice that were removed due to tumor burden were taken into account after the sacrifice date, if this practice increased the mean of the group.

Using this updated version of the formula, we re-evaluated the T/C values of the 23 different patient-derived xenograft (PDX) patterns listed in Tables 5 and 6, together with the HER2 gene sets (GCN). Table 7, and together with the HER2 mRNA level, are summarized in Table 8. The T/C efficacy values were determined as shown in Table 4.

Treatment with trastuzumab antibody in GXA 3054 in the stomach and GXA3038 PDX in the stomach. This information is also included in Tables 7 and 8. in.

Table 7 shows the efficacy as defined by the T/C value (%) (using the updated T/C formula) for the PDX mode treated with H561-4 and trastuzumab antibody.

* ⁺ These groups were treated with 30* and 60 ⁺ mg/kg H561-4 to achieve the maximum efficacy observed in in vivo studies.

For PDX patterns with a HER2 gene set greater than 10, H561-4 is effective in seven of the eight modes, with high activity in six of the modes; and in all PDX modes with a HER2 gene set of less than 10 Not active. In the PDX mode in which the number of sets of genes is greater than 10, the trastuzumab antibody has moderate activity in three of the modes, and has marginal activity in the other three modes, but does not show high activity. In the PDX mode with a gene set of less than 10, the trastuzumab antibody has marginal activity in one of the modes.

Figure 8 shows a scatter plot of the therapeutic T/C values of H561-4 and trastuzumab antibodies in the PDX mode with a HER2 gene set greater than 10, and compared to the PDX pattern with a set of genes less than 10. The scatter plot shows the efficacy of H561-4 in the PDX mode with a set of genes greater than 10, which is statistically greater than the efficacy in the PDX mode with a set of genes less than 10. The average activity of the trastuzumab antibody in the PDX mode with a set number of genes greater than 10 or less than 10 is higher than 50%, which supports the notion that the value of the HER2 gene set cannot be used to predict the efficacy of the antibody to trastuzumab.

In summary, the T/C value derived from the updated T/C formula The same conclusion as before (which is described in Example 6).

Table 8 shows the efficacy as defined by the T/C value (%) (using the updated T/C formula) for the PDX mode treated with H561-4 and trastuzumab antibody.

* ⁺ These groups were treated with 30* and 60 ⁺ mg/kg H561-4 to achieve the maximum efficacy observed in in vivo studies.

In the PDX mode in which the HER2 mRNA level in the tumor is higher than or equal to 200, H561-4 has efficacy in seven of the eight modes; and the HER2 mRNA level in the tumor is higher than or equal to 200 and lower. Either in all PDX modes with or equal to 820; and no activity in all PDX modes where the HER2 mRNA level in the tumor is below 200. In the PDX mode in which the HER2 mRNA level is higher than or equal to 200, the trastuzumab antibody has moderate activity in three of the modes, and the other three modes have marginal activity, but no high activity is shown therein. By. In the PDX mode where the mRNA level is below 200, the trastuzumab antibody has marginal activity in one of the modes.

Figure 9 shows a scatter plot of the therapeutic T/C values of H561-4 and trastuzumab antibodies in the PDX mode with a HER2 mRNA level above 200 in the tumor, and a lower level of mRNA in the tumor. Compare with the PDX mode of 200. The scatter plot shows the efficacy of H561-4 in the PDX mode with HER2 mRNA levels above or equal to 200 and below or equal to 820, which is statistically greater than the PDX mode with HER2 mRNA levels below 200 and greater than 820. The efficacy of the disease. In the group with HER2 mRNA higher than or equal to 200 and the group with HER2 mRNA lower than 200, the average activity of trastuzumab antibody in PDX mode was higher than 50%, which supported the HER2 mRNA level. The concept used to predict the efficacy of trastuzumab antibodies.

The T/C values obtained using the updated T/C formula derive the same conclusions as before.

Example 8-H561-4 selectively inhibits the PDX pattern of HER2 gene sets greater than 18 and the number of sets of genes in it is measured by FISH

The relationship between the number of HER2 gene sets measured by FISH and the in vivo activity of H561-4 was investigated in 17 different patient-derived xenograft (PDX) patterns including breast, stomach, and colon patterns. Evaluate the efficacy of H561-4 treatment with trastuzumab antibody treatment based on the T/C value of the updated T/C formula:

The efficacy criteria defined by the T/C values are summarized in Table 4. The number of HER2 gene sets in the PDX tumor model was determined by fluorescence in situ hybridization (FISH): a tissue sample of formaldehyde fixed and embedded in paraffin (FFPE) was obtained from the PDX model. FISH analysis was performed using PathVysion HER-2 DNA Probe Kit II (Abbott Molecular Corporation #06N46-030). The analysis uses a direct count of the HER2 signal without standardizing the CEP17 signal. Deparaffinated in xylene for 5 minutes A total of three times, followed by rehydration in industrial methanol-containing alcohol (IMS) for 5 minutes for a total of two times, and incubation in citrate buffer at pH 6.0 to 95 ° C for 30 minutes for pretreatment of tissue sections. . After pretreatment, the sections were incubated in pepsin buffer at 37 ° C for 20 minutes for protease digestion. To effectively label the HER2 DNA sequence, the sections were heated to 73 °C for 5 minutes to denature the DNA; then probe hybridization was carried out at 37 °C for 14 to 18 hours. The non-specific hybrid item was removed by washing in a post-hybrid buffer at 72 °C for 2 minutes. Prior to signal counting using fluorescence microscopy, the sections were stained in the dark for 15 minutes in contrast to DAPI. Cell counts were performed on the basis of 20 to 60 tumor cells per sample, and the average number of HER2 gene sets was obtained. The HER2 gene sets for each sample measured by FISH are listed in Table 9.

Table 9 shows the efficacy defined by the T/C value (in %) of the PDX pattern treated with H561-4 and trastuzumab antibody, and the number of sets of genes (GCN) for each PDX tumor tested: * ⁺ These groups were treated with 30* and 60 ⁺ mg/kg H561-4 to achieve the maximum efficacy observed in in vivo studies.

For PDX patterns with a HER2 gene set greater than 18, H561-4 is effective in seven of the eight modes and is highly active in six of the modes; and in all PDX modes with a HER2 gene set of less than 18 Not active. In the PDX mode in which the number of sets of genes is greater than 18, the trastuzumab antibody has moderate activity in three of the modes, and has marginal activity in the other three modes, but does not show high activity. In the PDX mode with a set of genes less than 18, the trastuzumab antibody has marginal activity in one of the modes.

Figure 10 shows a scatter plot of the therapeutic T/C values of H561-4 and trastuzumab antibodies in the PDX mode with a HER2 gene set greater than 18, and compared to the PDX pattern with a set of genes less than 18. The scatter plot shows the efficacy of H561-4 in the PDX mode with a set of genes greater than 18, which is statistically greater than The efficacy of the PDX model with a set number of genes less than 18. The trastuzumab antibody activity was independent of the number of HER2 gene sets. In groups with more than 18 gene sets or groups with less than 18 gene sets, the average activity of trastuzumab antibodies in PDX mode was higher than 50%, which supported the value of HER2 gene sets not used for prediction. The concept of the efficacy of trastrol monoclonal antibody.

Example 9 - Determination of the number of HER2 gene sets and their relationship by FISH and qPCR

The HER2 gene sets were determined by qPCR and FISH as shown in Examples 6 and 8 above. The number of HER2 gene sets in PDX tumors measured by qPCR ranged from 1 to 76 sets, and the number of sets of genes in these tumors varied from 2 to 40 sets as measured by FISH. The tumors with high HER2 gene amplification measured by either method (the number of sets of genes measured by qPCR is greater than or equal to 10 or the number of sets of genes measured by FISH is greater than or equal to 18) are statistically more May respond to H561-4 treatment. Among the 17 PDX tumors in which the data collected by the two methods were available, the relationship between the number of sets of genes measured by the two methods was evaluated. The series of HER2 gene sets measured by the two methods are shown in Table 10.

Table 10. Summary of HER2 gene sets in PDX tumors as measured by qPCR and FISH

Correlation analysis was performed to define the relationship between the number of sets of HER2 genes measured by qPCR and measured by FISH. The scatter plot in Figure 11 shows the relationship between the number of sets of genes measured by FISH and by qPCR in each PDX tumor sample, while the point system adjacent to the diagonal indicates a 1:1 correlation. Based on the correlation analysis, the number of sets of genes measured by the two methods was highly correlated (Pearson r = 0.90; p was less than 0.0001; 95% CI = 0.74 - 0.96).

Example 10 - Study of the variability of the HER2 immunohistochemical score determined by different procedures

The HER2 amplification state has been used as a prognostic factor. Immunohistochemistry (IHC) analysis is a common method for assessing the expression of HER2 proteins in tissue samples by antibodies that bind to antigens in a specific manner. As mentioned earlier, the semi-quantitative nature of IHC indicates that it may be inaccurate and that the readings provided are not always accurate. In addition, given the different analysis The variability of the results of HER2 immunohistochemical analysis between the different IHC analysis suppliers, the procedure, the HER2 antibody used and the scoring system may be of concern.

Although the US Food and Drug Administration (FDA) has approved HercepTest as a standardized IHC assay for the determination of HER2 protein overexpression in breast and gastric cancer, several studies have indicated that most HercepTest HER2 positive samples are not as measured by FISH analysis. Amplification of the HER2 gene (Jacobs et al. 1999). On the other hand, although breast cancer may exhibit excessive expression of HER2 protein in the absence of gene amplification, this phenomenon was previously observed in only 7% of cases (Persons et al., 1997). These differences are inconsistent with the current clinical practice of treating the HercepTest 3+ positive population as a HER2 amplification.

In this study, different IHC analyses, including immunohistochemical analysis developed by HercepTest and Oncotest, were used to identify HER2 protein overexpression in 22 PDX tumors fixed in formaldehyde and embedded in paraffin. Its purpose was to investigate the credibility of IHC as a H561-4 biomarker.

For the HercepTest immunohistochemical analysis, the HercepTest manufacturer's operating procedures were used. Briefly, cancer tissues fixed in formaldehyde and embedded in paraffin were deparaffinized in xylene for three minutes for a total of three times, followed by rehydration in industrial methanol-containing alcohol (IMS) for a total of five minutes. The sections were then incubated for 40 minutes in an epitope-repairing solution at 97 ° C and the wash buffer was placed for 20 minutes prior to staining. Slice system and Primary rabbit anti-human HER2 antibody was incubated for 30 minutes and then incubated with a secondary goat anti-rabbit antibody linked to wasabi peroxidase for 30 minutes. Apply DAB chromogen to visualize the signal in an enzyme-based manner.

The immunohistochemical analysis developed by Oncotest first deparaffinized and rehydrated the tissue. In a 720 watt microwave oven, the sections were incubated with 10 mM trisodium citrate for antigen retrieval for 20 minutes. Sample permeabilization was carried out by 0.2% Triton X100 and blocked by 3% hydrogen peroxide to deactivate endogenous hydrogen peroxide. Non-specific binding of the sections was blocked in 10% BSA and cultured overnight at 4 °C with multiple rabbit anti-human HER2 antibodies (Dako Company Catalog No. A0485). The next day, the sections were incubated with biotinylated goat anti-rabbit IgG (Jackson Corporation catalog number 111-065-045) for 60 minutes at room temperature. By culturing with avidin/biotinylase complex (ABC complex, Vector Lab, catalog number PK-4000), followed by signal coloring with DAB chromogen, the assay was performed. Biotinylated secondary antibody.

Both methods use the American Society of Clinical Oncology (ASCO) scoring guidelines. Briefly, HER2 protein overexpression was scored on a scale of 0 to 3+. The IHC staining level of tumor tissue sections determined by the percentage of tumors expressing HER2 protein and the intensity of HER2 staining was used to assess the score of HER2 protein expression. The HER2 scoring guidelines by IHC are shown in Table 11 below (in 2013, the IHC 2+ rating was updated from “weak positive” (as used in the 2007 edition of the guidelines) to “unsure”).

Table 11: American Society of Clinical Oncology (ASCO) guidelines for scoring HER2 protein overexpression

In the efficacy study, the HER2 IHC overexpression scores for each of the PDX tumors tested by HercepTest and by the IHC method developed by Oncotest were summarized in Table 12. Although the HercepTest and Oncotest methods use the same scoring criteria, the HER2 IHC results obtained by two different staining methods show great differences, and in the case of tumors with a HER2 gene set of less than or equal to 10 measured by qPCR. This is the case. In addition, there is an inconsistency even between the same method repeated by Oncotest (n=1 versus n=2). Thus, there is a significant difference between the IHC showing multiple analyses between different suppliers and between the same suppliers.

The number of HER2-negative cases detected by HercepTest was much higher than that of the IHC test performed by Oncotest (8 cases vs. 2 cases for the tumors tested by the two methods). The other side In fact, the positive rate detected by HercepTest is reported to be high (Jacobs et al. 1999). Taken together, these results point to the variability of the HER2 IHC results. Previous studies have pointed out different topics that may lead to variability when IHC is used for HER2 proteins, including variability in tissue fixation and processing, and variability in antibody sensitivity and specificity. However, in the current study, the reason is unlikely to be the primary antibody specificity, since both methods use the same antibody.

Comparing the overall HER2 IHC results with the HER2 gene sets and the results of HER2 mRNA (Examples 7 and 8), it was found that the screening would be a patient population that responded to H561-4 ( p less than 0.0001; unpaired t - Assay) One of the biomarker positive populations, the HER2 gene set or HER2 mRNA is more predictive as determined by the FISH method or the qPCR method.

Figure 12 shows a scatter plot of therapeutic T/C values of H561-4 and trastuzumab antibodies in PDX mode positive for HER2 protein overexpression, as compared to HER2 protein overexpression as determined by HercepTest The PDX pattern presented negative is compared. Although HercepTest was approved by the FDA for the screening of concomitant test kits for patients receiving Herceptin treatment, trastuzumab antibody activity was not associated with HER2 protein overexpression status in the 16 samples tested. (p=0.09; unpaired t-test). In the PDX mode in which the HER2 protein was positive or negative, the average activity of the trastuzumab antibody was higher than 50%, indicating that the overexpression of the HER2 protein could not predict the efficacy of the trastuzumab antibody alone. Concept.

Intriguingly, the scatter plot shows when IHC is used The efficacy of H561-4 in the PDX mode, which is positive for HER2 protein overexpression, measured by HercepTest is statistically greater than that in the PDX mode, which is negative for HER2 protein overexpression. These results demonstrate that the HER2 IHC status is more predictive of H561-4 than for the trastuzumab antibody in screening for reactive populations.

Table 12. Efficacy as defined by T/C values (in %) for the PDX pattern of H561-4 and trastuzumab antibody treated in each PDX tumor tested and by HercepTest and Oncotest The HER2 protein over-performance score measured by the company's IHC method: * ⁺ These groups were treated with 30* and 60 ⁺ mg/kg H561-4 to achieve the maximum efficacy observed in in vivo studies.

Informal sequence listing

Nucleotide sequence of antigen-binding Fc fragment H561-4 (SEQ ID NO: 1)

The "ACCTGCCCCCCTTGTCCT" sequence at the beginning of the H561-4 nucleotide sequence encodes a portion of the IgG1 hub. The bottom line is drawn below the sequence of the CH2 domain coded H561-4. The sequence encoding the CH3 domain of H561-4 is shown in italics. The sequence encoding the AB structural loop of the CH3 domain involved in antigen binding is shown in bold, while the sequence encoding the EF structural loop of the CH3 domain believed to be involved in antigen binding is shown in bold and double bottom lines.

Nucleotide sequence of the antigen-binding Fc fragment H561-4 pivot region (SEQ ID NO: 2) ACCTGCCCCCCTTGTCCT

Nucleotide sequence of the antigen-binding Fc fragment H561-4 CH2 domain (SEQ ID NO: 3)

Nucleotide sequence of antigen-binding Fc fragment H561-4 CH3 domain (SEQ ID NO: 4)

Nucleotide sequence encoding a portion of the H561-4 AB structural loop region believed to be involved in antigen binding (SEQ ID NO: 5) TTCTTCACCTACTGG

Nucleotide sequence encoding a portion of the H561-4 CD structural loop region (sequence recognition) No.: 6) AACGGCCAGCCCGAG

Nucleotide sequence encoding a portion of the H561-4 EF structural loop region involved in antigen binding (SEQ ID NO: 7) GACCGGCGGAGATGGACCGCC

Amino acid sequence of antigen-binding Fc fragment H561-4 (SEQ ID NO: 8)

The "TCPPCP" sequence at the beginning of the H561-4 amino acid sequence represents a portion of the IgGl hub. A bottom line is drawn below the CH2 field of H561-4. The CH3 domain of H561-4 is shown in italics. One part of the AB structural ring of the CH3 domain is shown in bold, while one part of the EF structural ring of the CH3 domain is shown in bold and double bottom lines. It has been reported that the C-terminus of the CH3 domain is cleaved from the amine acid (K) or the C-terminal amide acid and the adjacent glycine acid (GK). In the above sequence, the C-terminal amino acid and the adjacent glycine (also present in the following sequence number: 11) are shown in a box. The HER2 binding member for use in this application may lack the C-terminal lysine or C-terminal amic acid and glycine.

Amino acid sequence of H561-4 hub (sequence identification number: 9) TCPPCP

Amino acid sequence of H561-4 CH2 domain (SEQ ID NO: 10)

Amino acid sequence of H561-4 CH3 domain (SEQ ID NO: 11)

Amino acid sequence of a portion of the H561-4 AB structural loop involved in antigen binding (SEQ ID NO: 12) FFTYW

Amino acid sequence of a part of H561-4 CD structural loop region (SEQ ID NO: 13) NGQPE

Amino acid sequence of a portion of the H561-4 EF structural loop involved in antigen binding (SEQ ID NO: 14) DRRRWTA

The HER2 amino acid sequence bound by H561-4 as determined by peptide scanning technique (Pepscan) (SEQ ID NO: 15) LPASPETHLDMLRHL

The HER2 amino acid sequence bound by H561-4 as determined by peptide scanning technique (Pepscan) (SEQ ID NO: 16) CQVVQGNLELTYLPT

The HER2 amino acid sequence bound by H561-4 as determined by peptide scanning technique (Pepscan) (SEQ ID NO: 17)

Amino acid sequence of the HER2 extracellular domain (SEQ ID NO: 18)

The amino acid sequence of the wild-type Fc antigen-binding fragment used as a control in the experiment (SEQ ID NO: 19)

references

All documents mentioned in this specification are incorporated herein by reference in their entirety.

Arnould et al ., Accuracy of HER2 status determination on breast core-needle biopsies (immunohistochemistry, FISH, CISH and SISH vs FISH). Mod Path. 2012. 25: 675-682

Beck et al . Characterization of Therapeutic Antibodies and Related Products. Anal. Chem. 2013, 85, 715-736

Berchuck, A. et al., Overexprerssion of HER2/neu in endometrial cancer is associated with advanced stage disease. AJOG; 164 (1): 15-21, 1991.

Brabender, J et al ., Epidermal Growth Factor Receptor and HER2-neu mRNA Expression in Non-Small Cell Lung Cancer is Correlated with Survival. Clin Cancer Res; 7: 1850, 2001.

CRUK and WHO world cancer report. World Cancer Report 2008, Editor Boyle P, et al., WHO International Agency for Research on Cancer, ISBN 9789283204237

Dowsett, M et al., Disease-free survival according to degree of HER2 amplification for patients treated with adjuvant chemotherapy with or without 1 year of trastuzumab, the HERA trial, J Clin Oncol, 27(18): 2962-9, 2009.

Garcia-Caballero et al., Determination of HER2 amplification in primary breast cancer using dual-color chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens. Histopathology. 2010. 56: 472-480

Gravalos, C and Jimeno, A. Her2 in gastric cancer: a new prognostic factor and a novel therapeutic target. Ann Oncol; 19 (9): 1523-1529, 2008.

Gun, S et al., Molecular cytogenetics as a clinical test for prognostic and predictive biomarkers in newly diagnosed ovarian cancer. J Ovarian Res 6:2, 2013.

Hamburg M.A and Collins, F.S. The path to personalised medicine, 10.1056/NEJMp1006304, 15 June 2010.

Hanna et al ., HER2 in situ hybridization in breast cancer: Clinical implications of polysomy 17 and genetic heterogeneity. Mod Pathol: 27: 4-18 (2014)

Harris L et al., Predictors of Resistance to Preoperative Trastuzumab and Vinorelbine for HER-2 Positive Early Breast Cancer. Clin Cancer Res, 13:1198-1207, 2007.

Hicks D, et al., Assessment of the HER2 status in breast cancer by Fluorescence In Situ Hybridisation: a technical review with interpretive guidelines. Hum Pathol, 36:250-261, 2005.

Hoff, E et al., HER2/neu amplification in breast cancer. Am J Clin Pathol, 117:916-921, 2002.

Hudis, C. Trastuzumab-Mechanism of Action and Use in Clinical Practice. N Engl J Med, 357:39-51, 2007

Jacobs, TW et al.-Specificity of HercepTest in Determining HER-2/neu Status of Breast Cancers Using the United States Food and Drug Administration-Approved Scoring System. Journal of Clinical Oncology, Vol 17, No 7 (July), 1999: Pp 1983-1987.

Lanitis et al., Primary Human Ovarian Epithelial Cancer Cells Broadly Express HER2 at Immunologically-Detectable Levels. PLOSone;7 (11):e49829, 2012.

Lei et al., Overexpression of HER2/neu oncogene in pancreatic cancer correlated with shortened survival. International Journal of Pancreatology; 17 (1), 15-21, 1995

Mollerup et al ., Dual color chromogenic in situ hybridization for determination of HER2 status in breast cancer: a large comparative study to current state of the art fluorescence in situ hybridization. BMC Clinical Path. 2012. 12:3

Ochs et al ., Expression of Vascular Endothelial Growth Factor and HER2/neu in Stage II Colon Cancer and Correlation with Survival. Clinical Colorectal Cancer; 4 (4): 262-267, 2004.

Persons et al., Quantitation of HER-2/neu and c-myc gene amplification in breast carcinoma using fluorescence in situ hybridization. Mod Pathol 10:720-727, 1997

Press et al., Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. JCO, 20 (14): 3095-3105, 2002.

Rüschoff et al., HER2 diagnostics in gastric cancer-guideline validation and development of standardized immunohistochemical testing. Vichows Arch; 457:299-307, 2010.

Ross J. Breast cancer biomarkers and HER2 testing after 10 years of anti-HER2 therapy. Drug News Perspect; 22:93-106, 2009

Ross J et al ., The HER2/neu Gene and Protein in Breast Cancer 2003: Biomarker and Target of Therapy. The Oncologist, 8:307-325, 2003.

Timmerman et al . (2007). Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS ^TM technology. J Mol Recognit; 20:283-99.

Slootstra et al (1996) Structural aspects of antibody-antigen interaction revealed through small random peptide libraries Molecular Diversity, 1:... 87-96.

Vinatzer et al ., Expression of HER2 and the Coamplified Genes GRB7 and MLN64 in Human Breast Cancer: Quantatative Real-time Reverse Transcription PCR as a Diagnostic Alternative to Immunohistochemistry and Fluorescence In situ Hybridisation. Clin Cancer Res; 11 (23):8348- 8357, 2005.

Wolff A et al., American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factors Receptor 2 Testing in Breast Cancer. J Clin Oncol; 25:118/145, 2007

Wolff et al., Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update. J Clin Onco; 31(31): 3997-4013, 2013.

Wong et al . (2011) Intergrating molecular and clinical evidence in the management of trastuzumab resistant or refractory HER-2+ Metastatic breast cancer, The Oncologist; 16:1535-1546.

Yarden, Y. Biology of HER2 and Its Importance in Breast Cancer. Oncology; 61 (2): 1-13, 2001.

Equivalent part:

Many equivalents of the specific embodiments disclosed herein will be understood or appreciated by those skilled in the <RTIgt; These equivalents are intended to be included in the scope of the following claims.

<110> F-Star Biotechnology Co., Ltd. F-Star Biotechnology R&D Co., Ltd.

<120> HER2 binding agent treatment

<130> TEK/CP7196157

<140> TW105111069

<141> 2016-04-08

<150> US62/288,618

<151> 2016-01-29

<150> US62/190,610

<151> 2015-07-09

<150> US62/144498

<151> 2015-04-08

<160> 19

<170> Patent Application Software Version 3.3

<210> 1

<211> 681

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence: Nucleotide sequence of antigen-binding Fc fragment H561-4

<400> 1

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence: nucleotide sequence of the H561-4 hub of the binding Fc fragment

<400> 2

<210> 3

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence: Nucleotide sequence of the antigen-binding Fc fragment H561-4 CH2 domain

<400> 3

<210> 4

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence: Nucleotide sequence of the antigen-binding Fc fragment H561-4 CH3 domain

<400> 4

<210> 5

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence: encoding a H561-4 AB structural loop region believed to be involved in antigen binding Part of the nucleotide sequence

<400> 5

<210> 6

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence: a nucleotide sequence encoding a portion of the H561-4 CD structural loop region

<400> 6

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic sequence: encoding the H561-4 EF structural loop region believed to be involved in antigen binding Part of the nucleotide sequence

<400> 7

<210> 8

<211> 223

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: amino acid sequence of antigen-binding Fc fragment H561-4

<400> 8

<210> 9

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: amino acid sequence of the H561-4 pivot region

<400> 9

<210> 10

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: amino acid sequence of H561-4 CH2 domain

<400> 10

<210> 11

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: amino acid sequence of H561-4 CH3 domain

<400> 11

<210> 12

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: an amino acid sequence of a portion of the H561-4AB structural loop region believed to be involved in antigen binding

<400> 12

<210> 13

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: amino acid sequence of a portion of the H561-4 CD structural loop region

<400> 13

<210> 14

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic sequence: an amino acid sequence of a portion of the H561-4 EF structural loop region believed to be involved in antigen binding

<400> 14

<210> 15

<211> 15

<212> PRT

<213> Homo sapiens

<400> 15

<210> 16

<211> 15

<212> PRT

<213> Homo sapiens

<400> 16

<210> 17

<211> 52

<212> PRT

<213> Homo sapiens

<400> 17

<210> 18

<211> 630

<212> PRT

<213> Homo sapiens

<400> 18

<210> 19

<211> 223

<212> PRT

<213> Homo sapiens

<400> 19

Claims (70)

A specific binding member that binds to human type 2 epidermal growth factor receptor (HER2) for use in a method of treating cancer in a patient, optionally in combination with another therapy such as immunotherapy, the immunization Therapy is such as the use of an immunotherapeutic agent, wherein each tumor cell of the cancer has an average number of HER2 gene sets greater than or equal to 10, and wherein the specific binding member is: (a) a specific binding member , comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14 Or (b) a specific binding member that competes with HER2-specific binding members as one of (a). A specific binding member of claim 1, wherein a tumor sample obtained from the patient is determined to have a mean HER2 gene set number greater than or equal to 10 per tumor cell. A specific binding member according to claim 1 or 2, wherein the cancer line has or is determined to have a high HER2 mRNA level, wherein a high HER2 mRNA level corresponds to a tumor sample obtained from the patient greater than or The number of HER2 cDNA sets equal to 200, which is relative to the number of cDNA sets of a reference gene in the sample, wherein the cDNA sets of the HER2 and the reference gene in the sample are sequentially determined by RT-PCR and quantitative PCR. A specific binding member according to claim 1 or 2, wherein the cancer line has or is determined to have a high HER2 mRNA level, wherein a high HER2 mRNA level corresponds to a tumor sample obtained from the patient greater than or The number of HER2 cDNA sets equal to 200 and less than or equal to 820, which is relative to the number of cDNA sets of a reference gene in the sample, wherein the cDNA sets of the HER2 and the reference gene in the sample are sequentially sequenced by RT-PCR Quantitative PCR assay. The specific binding member of claim 1, wherein the method comprises: (i) determining an average number of sets of the HER2 gene per tumor cell in a tumor sample obtained from the patient; and (ii) if each tumor If the average HER2 gene set number of the cells is greater than or equal to 10, the patient is treated with the specific binding member. The specific binding member according to any one of claims 1 to 5, wherein the HER2 gene set number is greater than or equal to 11, greater than or equal to 12, greater than or equal to 13, greater than or equal to 14, greater than or equal to 15, greater than or equal to 16. Greater than or equal to 17, or greater than or equal to 18. The specific binding member of claim 6, wherein the HER2 gene set number is greater than or equal to 18. The specific binding member according to any one of claims 1 to 7, wherein the cancer is gastric cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, gastric cancer or endometrial cancer. The specific binding member of claim 8, wherein the cancer is gastric cancer, breast cancer or colorectal cancer. The specific binding member of claim 8, wherein the cancer is a stomach cancer. The specific binding member of claim 8, wherein the cancer is breast cancer. The specific binding member according to any one of claims 1 to 11, wherein the patient exhibits an antibody to trastuzumab and/or trastuzumab plus pertamine monoclonal antibody ( Pertuzumab) has an inadequate response. The specific binding member according to any one of claims 1 to 12, wherein the cancer is refractory to the treatment of a trastuzumab antibody or a trastuzumab antibody plus a pertituzil antibody. The specific binding member of any one of claims 1 to 13, wherein the specific binding member of part (b) of claim 1 comprises a structural loop region designed to be inserted into the CH3 domain of the specific binding member One of the HER2 antigen binding sites. The specific binding member of claim 14, wherein the specific binding member comprises a HER2 antigen binding site of structural loop regions AB and EF that are designed to be inserted into the CH3 domain of the specific binding member. The specific binding member according to any one of claims 1 to 15, wherein the specific binding member-antigen-binding Fc fragment according to part (a) of claim 1 and/or part (b) of claim 1 Or contain an antigen-binding Fc fragment. The specific binding member of any one of claims 1 to 16, wherein the specific binding member of part (a) of claim 1 and/or part (b) of claim 1 comprises a sequence identification number: The CH3 domain of 11 or the sequence number: 11 of the CH3 domain minus one or two C-terminal amino acids. As the specific binding member of claim 17, wherein part (a) of claim 1 The specific binding member of the sub-portion and/or part (b) of claim 1 further comprises a CH2 domain of sequence identification number: 10. The specific binding member according to any one of claims 1 to 18, wherein the specific binding member of part (a) of claim 1 is a dimer of a polypeptide of sequence identification number: 8, or is a sequence Identification of a polypeptide of number 8 minus one or two dimers of a C-terminal amino acid. The specific binding member according to any one of claims 1 to 19, wherein the specific binding member of the part (b) of claim 1 is specific for HER2 binding and one of the parts (a) of claim 1 Sexual binding member competition, as determined by surface plasma resonance (SPR), competitive enzyme linked immunosorbent assay (ELISA), fluorescent activated cell sorting (FACS) or competitive immunocytochemistry. The specific binding member according to any one of claims 1 to 20, wherein the specific binding member of the part (b) of claim 1 is bound to the epitope at HER2, and is related to (a) of claim 1 One of the partial binding members is identical; and wherein the specific binding member of part (a) of claim 1 is a dimer of a polypeptide of sequence number: 8 or a polypeptide of sequence number: 8. One or two dimers of the C-terminal amino acid are subtracted. The specific binding member of claim 2 or claim 5, wherein the HER2 gene set uses fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or quantitative polymerase Chain reaction (qPCR) assay. The specific binding member of claim 22, wherein the HER2 gene set The FISH assay was used. The specific binding member according to any one of claims 3 to 19, wherein the method comprises determining a HER2 mRNA level of a tumor sample obtained from the patient, wherein a high HER2 mRNA level corresponds to the tumor sample The number of HER2 cDNA sets greater than or equal to 200, relative to the number of cDNA sets of a reference gene in the sample, and wherein the high HER2 mRNA level indicates that the average HER2 gene set number per tumor cell is greater than or equal to 10 . The specific binding member of claim 24, wherein the cDNA set of the HER2 and the reference gene is sequentially determined by reverse transcription PCR and quantitative PCR. A method of treating cancer in a patient, wherein each tumor cell of the cancer has an average number of HER2 gene sets greater than or equal to 10, and wherein the method comprises administering to the patient a therapeutically effective amount of each of the following : (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12 And DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with HER2 for binding to a specific binding member such as (a); or (c) as (a) or A specific binding member of (b) which is used in combination with an immunological antitumor agent. The method of claim 26, wherein a tumor sample obtained from the patient is Each tumor cell was determined to have an average number of HER2 gene sets greater than or equal to 10. The method of claim 26 or 27, wherein the cancer has or is determined to have a high HER2 mRNA level, wherein the high HER2 mRNA level corresponds to greater than or equal to 200 in a tumor sample obtained from the patient. The number of HER2 cDNA sets is relative to the number of cDNA sets of a reference gene in the sample, wherein the cDNA sets of the HER2 and the reference gene in the sample are sequentially determined by RT-PCR and quantitative PCR. The method of claim 26 or 27, wherein the cancer has or is determined to have a high HER2 mRNA level, wherein the high HER2 mRNA level corresponds to greater than or equal to 200 in a tumor sample obtained from the patient. And a HER2 cDNA set of less than or equal to 820, wherein the number of cDNA sets of the HER2 and the reference gene in the sample is sequentially determined by RT-PCR with respect to the number of cDNA sets of a reference gene in the sample. Quantitative PCR assay. The method of claim 26, wherein the method comprises: (i) determining an average number of sets of HER2 genes per tumor cell in a tumor sample obtained from the patient; and (ii) an average HER2 per tumor cell If the number of sets of genes is greater than or equal to 10, the patient is administered a therapeutically effective amount of a specific binding member that is selectively used in combination with an immunoantitumor agent. A method of identifying whether a patient's cancer is sensitive to the treatment of each of the following: (a) a specific binding member comprising a HER2 antigen binding site designed to be inserted into a structural loop region of the CH3 domain of the specific binding member, and comprising an amino acid sequence FFTYW (SEQ ID NO: 12) And DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member that competes with HER2-specific binding members as one of (a); or (c) as (a) or b) one of the specific binding members, which is used in combination with an immunological antitumor agent; the method comprising: (i) determining the average number of sets of genes of the HER2 gene per tumor cell in a tumor sample obtained from the patient, wherein If the average number of sets of genes per tumor cell is greater than or equal to 10, it indicates that the cancer is sensitive to the treatment of the specific binding member. The method of claim 31, wherein the method further comprises: (ii) if the average number of HER2 genes per tumor cell is greater than or equal to 10, then selecting the patient for treatment of the specific binding member, optionally Used in combination with an immunological antitumor agent. A method of predicting the response of a cancer to treatment of: (a) a specific binding member comprising a HER2 antigen binding site designed to be placed in a structural loop region of the CH3 domain of the specific binding member, And containing amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member, which is specific for HER2 binding and one of (a) Combine member competition; or (c) a specific binding member according to (a) or (b), which is used in combination with an immunological antitumor agent; the method comprising: (i) determining each tumor cell in a tumor sample obtained from a patient The average number of sets of genes for the HER2 gene, wherein the number of sets of genes per tumor cell is greater than or equal to 10 indicates that the cancer is sensitive to the treatment of the specific binding member, and the number of sets of genes per tumor cell is less than 10 indicating that the cancer has specificity for the cancer The treatment of the combined members is not sensitive. The method of claim 33, wherein the method further comprises: (ii) if the number of HER2 gene sets per tumor cell is greater than or equal to 10, then selecting the patient for treatment of the specific binding member, optionally with An immunological antitumor agent is used in combination. The method of any one of clauses 26 to 34, wherein the HER2 gene set number is greater than or equal to 11, greater than or equal to 12, greater than or equal to 13, greater than or equal to 14, greater than or equal to 15, greater than or equal to 16, greater than Or equal to 17, or greater than or equal to 18. The method of claim 35, wherein the HER2 gene set number is greater than or equal to 18. The method of any one of claims 26 to 36, wherein the cancer is gastric cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, gastric cancer or endometrial cancer. The method of claim 37, wherein the cancer is gastric cancer, breast cancer or colorectal cancer. The method of claim 38, wherein the cancer is a stomach cancer. The method of claim 38, wherein the cancer is breast cancer. The method of any one of claims 26 to 40, wherein the patient exhibits insufficient response to the trastuzumab antibody and/or the trastuzumab antibody plus the pertallazine antibody. One of the claims 26 to 41 uses one of the specific binding members, wherein the cancer is refractory to the treatment of the trastuzumab antibody and/or the trastuzumab antibody plus the pertallazine antibody. of. The method of any one of claims 26 to 42, wherein the specific binding member of part (b) of claim 26, part (b) of claim item 31, or part (b) of claim item 33 is designed to comprise A HER2 antigen binding site of a structural loop region of the CH3 domain of the specific binding member is placed. The method of claim 43, wherein the specific binding member comprises a HER2 antigen binding site of the structural loop region AB and EF of the CH3 domain designed to be inserted into the specific binding member. The method of any one of claims 26 to 44, wherein (a) and/or (b) of claim 26, (a) and/or (b) of claim 31, or (a) of claim 33 And/or part (b) of the specific binding member is an antigen-binding Fc fragment or comprises an antigen-binding Fc fragment. The method of any one of claims 26 to 45, wherein (a) and/or (b) of claim 26, (a) and/or (b) of claim 31 or (a) of claim 33 And/or the specific binding member of part (b) comprising the CH3 domain of sequence identification number: 11, or the CH3 domain of sequence identification number: 11 minus one or two C-terminal amino acids. The method of claim 46, wherein part (a) and/or (b) of claim 26, The specific binding member of part (a) and/or (b) of claim 31 or part (a) and/or (b) of claim 33 further comprises a CH2 domain of sequence identification number: 10. The method of any one of claims 26 to 47, wherein the specific binding member sequence identification number of part (a) of claim 26, part (a) of claim 31, or part (a) of claim 33 A dimer of a polypeptide of 8 or a polypeptide of sequence number: 8 minus one or two dimers of a C-terminal amino acid. The method of any one of claims 26 to 48, wherein the specific binding member of part (b) of claim 26, part (b) of claim item 31, or part (b) of claim item 33 is in HER2 The binding is in cooperation with a specific binding member such as part (a) of claim 26, part (a) of claim 29, or part (a) of claim 33, respectively, by surface plasma resonance (SPR) Or a competitive enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) or competitive immunocytochemistry. The method of any one of claims 26 to 49, wherein the specific binding member of part (b) of claim 26, part (b) of claim item 31, or part (b) of claim item 33 is on HER2 The combined epitope is the same as the specific binding member of part (a) of claim 26, part (a) of claim 29, or part (a) of claim 33, respectively, and wherein The specific binding member of part (a), part (a) of claim 29 or part (a) of claim 33, which comprises a protein of two polypeptides having the sequence number: 8 or Sequence identification number: 8 of 2 One polypeptide of one or two C-terminal amino acids is subtracted from the polypeptide. The method of any one of claims 26 to 50, wherein the HER2 gene set is determined using fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) or quantitative polymerase chain reaction (qPCR). The method of claim 51, wherein the HER2 gene set is determined using FISH. The method of any one of clauses 26 to 52, wherein the method comprises determining a HER2 mRNA level in a tumor sample obtained from the patient, wherein a high HER2 mRNA level corresponds to greater than or equal to the tumor sample The HER2 cDNA set of 200 is relative to the cDNA set of a reference gene in the sample, and its high HER2 mRNA level indicates that the average HER2 gene set number per tumor cell is greater than or equal to 10. The method of claim 53, wherein the HER2 and the cDNA set of the reference gene are sequentially determined using reverse transcription PCR and quantitative PCR. The specific binding member according to any one of claims 1 to 25, which is for use in a therapeutic method for cancer of a patient, which is used in combination with an immunological antitumor agent. One of the claims 55 specifically binds to a member, wherein the immuno-antitumor agent is a CTLA-4 antagonist, a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a LAG-3 antagonist, a CD- 137 agonist or KIR antagonist. The method of any one of claims 26 to 54, wherein the specific binding member is administered in combination with an immunological antitumor agent. The method of claim 57, wherein the immunological antitumor agent is CTLA-4 antagonist An anti-agent, a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a LAG-3 antagonist, a CD-137 agonist or a KIR antagonist. A composition comprising a HER2 binding agent and an immunopotent anti-tumor agent. The composition of claim 59, wherein the HER2 binding agent competes with a specific binding member for binding to a specific binding member in the binding of human HER2, the specific binding member comprising CH3 designed to be inserted into the specific binding member A HER2 antigen binding site of a structural loop region of the domain, and the specific binding member contains the amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14). The composition of claim 59 or 60, wherein the epitope to which the HER2 binding agent specifically binds is identical to the binding of a specific binding member comprising a specific binding member designed to be inserted A HER2 antigen binding site of a structural loop region of the CH3 domain, and the specific binding member contains an amino acid sequence FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14). The composition of any one of claims 59 to 61, wherein the immunological antitumor agent is a CTLA-4 antagonist, a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a LAG-3 antagonist , CD-137 agonist or KIR antagonist. The composition of any one of claims 59 to 62 which is a fixed dose combination of the HER2 binding agent and one of the immunological antitumor agents. A specific binding member for use in a method of treating a cancer of a patient according to any one of claims 1 to 59, wherein the specific binding member is administered once a week at 0.1 to 10 mg/kg Every two weeks Once, every three weeks or once every four weeks. A specific binding member of claim 64 for use in a method of treating cancer in a patient, wherein the specific binding member is administered once every two weeks, once every two weeks, at a dose of 0.1 to 5 mg/kg Once every three weeks or once every four weeks. A specific binding member according to claim 65, wherein the specific binding member is administered once every week, once every two weeks, every 0.5 to 5 mg/kg. Once every three weeks or once every four weeks. A specific binding member of claim 64 for use in a method of treating cancer in a patient, wherein the specific binding member is administered once every two weeks, once every two weeks, at a dose of 0.1 to 1 mg/kg. Once every three weeks or once every four weeks. A specific binding member of a therapeutic method of claim 67 for use in a cancer of a patient, wherein the specific binding member is 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 04 mg/kg. Or 0.5 mg/kg for administration once a week, once every two weeks, once every three weeks or once every four weeks. The specific binding member of any one of the treatment methods of any one of claims 64 to 68, wherein the specific binding member is administered in combination with an immunological antitumor agent. A specific binding member of a therapeutic method according to claim 69, wherein the immunological antitumor agent is a CTLA-4 antagonist, a PD-1 antagonist, a PD-L1 antagonist, and a PD-L2. Antagonist, LAG-3 An antagonist, a CD-137 agonist or a KIR antagonist.