WO2016162505A1 - Thérapies d'agent de liaison à her2 - Google Patents

Thérapies d'agent de liaison à her2 Download PDF

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WO2016162505A1
WO2016162505A1 PCT/EP2016/057800 EP2016057800W WO2016162505A1 WO 2016162505 A1 WO2016162505 A1 WO 2016162505A1 EP 2016057800 W EP2016057800 W EP 2016057800W WO 2016162505 A1 WO2016162505 A1 WO 2016162505A1
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her2
specific binding
binding member
cancer
copy number
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PCT/EP2016/057800
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Sarah Batey
Haijun Sun
Kinmei LEUNG
Robert Rowlands
Francis Y. Lee
Matthew Anthony MAURER
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F-Star Biotechnology Limited
F-Star Biotechnologische Forschungs- Und Entwicklungsges.M.B.H
Bristol-Myers Squibb Company
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Publication of WO2016162505A1 publication Critical patent/WO2016162505A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to combination treatments for cancer, comprising the combined administration of (i) a Epidermal Growth Factor Receptor 2 (HER2) binding agent, e.g., a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain, e.g. CH3 domain, of the specific binding member, 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.
  • HER2 Epidermal Growth Factor Receptor 2
  • the present invention also relates to the use of Human HER2 gene copy number (GCN) and HER2 mRNA levels as biomarkers.
  • GCN Human HER2 gene copy number
  • HER2 mRNA levels as biomarkers.
  • the present invention relates to the use of HER2 gene copy number and HER2 mRNA levels as biomarkers 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, e.g. CH3 domain, of the specific binding member, and specific binding members which compete with such a binding member for binding to HER2.
  • the present invention 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.
  • Human Epidermal Growth Factor Receptor 2 (also referred to as HER2, HER2/neu or ErbB-2) is an 185kDa 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 normally regulates cell growth and survival, as well as adhesion, migration, differentiation, and other cellular responses (Hudis, C, 2007).
  • Overexpression and amplification of HER2 is observed in the development of a variety of solid cancers including breast (Yarden, Y, 2001 ), gastric (Gravalos ef al., 2008), stomach (Ruschoff et al., 2010), colorectal (Ochs er a/., 2004), ovarian (Lanitis et al., 2012), pancreatic (Lei er a/., 1995), endometrial (Berchuk ef al., 1991 ) and non-small cell lung cancers (Brabender et al., 2001 ).
  • HER2 targeting therapies have been approved for treatment of HER2 positive tumours.
  • HerceptinTM is approved for the treatment of metastatic breast cancer in combination with TaxolTM (paclitaxel) and alone for the treatment of HER2 positive breast cancer in patients who have received one or more chemotherapy courses for metastatic disease.
  • trastuzumab also enhances the efficacy of adjuvant chemotherapy (paclitaxel, docetaxel and vinorelbine) in operable or locally advanced HER2 positive tumours, it is considered standard of care for patients with early or advanced stages of HER2-overexpressing breast cancer.
  • Trastuzumab has also been approved for treatment of HER2 positive metastatic cancer of the stomach or gastroesophageal junction cancer, in combination with chemotherapy (cisplatin and either capecitabine or 5-fluorouracil) in patients who have not received prior treatment for their metastatic disease (Genentech, About Herceptin. [online] Available at: http://www.herceptin.com/about [accessed on 17 September 2013]).
  • PerjetaTM has also been approved for the treatment of HER2 positive metastatic breast cancer in combination with trastuzumab and docetaxel (Genentech, PERJETA Can Help Strengthen Your Treatment, [online] Available at: http://www.perjeta.com/patient/about [accessed on 17
  • Pertuzumab targets a different domain of HER2 and has a different mechanism of action than trastuzumab. Specifically, pertuzumab is a HER2 dimerisation inhibitor, which prevents HER2 from pairing with other HER receptors (EGFR/HER1 , HER3 and HER4).
  • KadcylaTM (ado- trastuzumab emtansine, T-DM1 ) is an antibody-drug conjugate, which comprises trastuzumab linked to the cytotoxic agent mertansine (DM1 ), which disrupts the assembly of microtubules in dividing cells resulting in cell death, and is approved for the treatment of metastatic breast cancer in patients who have received prior treatment with trastuzumab and a taxane chemotherapy (Genentech, How KadcylaTM (ado-trastuzumab emtansine) is Believed to Work (Proposed Mechanism of Action),
  • TykerbTM (lapatinib) is a small molecule kinase inhibitor that blocks the catalytic action of both HER2 and EGFR. It has been approved in combination with FemaraTM (letrozole) for treatment of HER2 positive, hormone receptor positive, metastatic breast cancer in postmenopausal women, and in combination with XelodaTM (capecitabine) for the treatment of advanced or metastatic HER2 positive breast cancer in patients who have received prior therapy including an anthracycline, a taxane, and HerceptinTM (U.S. Food and Drug Administration, Highlights of Prescribing Information, [online] Available at: http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/022059s007lbl.pdf
  • trastuzumab standard of care status for HER2 positive breast cancer, 20-50% of patients from adjuvant settings and around 70% of patients from monotherapy settings go on to develop resistance to trastuzumab (Wolff et al., 2007; and Harris et ai, 2007).
  • therascreenTM KRAS test is an EGFR immunohistochemistry test which identifies patients having EGFR positive metastatic colorectal cancer with wild-type KRAS genes to be treated with ErbituxTM (cetuximab).
  • the DAKO C-kit PharmDx immunohistochemistry test identifies patients with c-kit positive gastrointestinal stromal tumours susceptible to treatment with Gleevec (imatinib).
  • a number of diagnostic tests have also been approved for identification of HER2 positive tumours for treatment with HerceptinTM (trastuzumab) (Hamburg and Collins, 2010), such as the
  • kits for immunohistochemistry of HER2 positive tumours include Oracle (Leica Biosystems) and Pathway (Ventana).
  • Preclinical and clinical research efforts to identify biomarkers predictive of the clinical response to treatment also have the potential to identify additional patients with "non-traditional" HER2+ cancers, including colorectal cancer, ovarian cancer and others, which are likely to benefit from HER2 targeting therapies (Gun et a/., 2013).
  • HER2 positive tumours which may be susceptible to treatment with a HER2 specific biological therapeutic are usually identified in the first instance by using
  • IHC immunohistochemistry
  • the HER2 gene copy number is compared to an internal control such as CEP17, where the FISH score is less than two, in other words the HER2/CEP17 ratio is less than 2, the HER2 gene status is said to be non-amplified and the tumour is considered HER2 negative.
  • the FISH score is equal to or greater than 2
  • the HER2 gene status is said to be amplified, and the tumour is considered HER2 positive and is reported to a clinician for possible treatment with a HER2 specific therapy.
  • Antigen-binding Fc fragments (also referred to as FcabTM [Fc fragment with Antigen Binding]) comprising e.g., a modified lgG1 Fc domain which binds to HER2 with high affinity, are described in WO 2009/132876 A1 and WO 2009/000006 A1 which are hereby incorporated by reference in their entirety.
  • Specific binding members described herein include antigen binding Fc fragments described herein which each has one or more amino acid modifications in at least one structural loop region, wherein the modified structural loop region specifically binds to an epitope of an antigen, e.g., Her2, to which an unmodified Fc fragment does not significantly bind.
  • the loops which are not CDR-loops in a native immunoglobulin do not have antigen binding or epitope binding specificity but contribute to the correct folding of the entire immunoglobulin molecule and/or its effector or other functions and are therefore called structural loops for the purpose of this invention.
  • a "structural loop" or “non-CDR-loop” according to the present invention is to be understood in the following manner: immunoglobulins are made of domains with a so called immunoglobulin fold. In essence, anti-parallel beta sheets are connected by loops to form a compressed antiparallel beta barrel. In the variable region, some of the loops of the domains contribute essentially to the specificity of the antibody, i.e., the binding to an antigen. These loops are called CDR-loops. All other loops of antibody domains are rather contributing to the structure of the molecule and/or the effector function. These loops are defined herein as structural loops or non- CDR-loops.
  • Fcabs have been shown to have favourable properties, such as half-lives of approximately 60 hours in mice.
  • Figure 1 shows an alignment of the antigen-binding Fc fragment H561-4 amino acid sequence (H561-4) with the wild-type lgG1 amino acid sequence (WT) in the same region.
  • the amino acids are numbered according to Kabat - Kabat numbering is shown above the aligned sequences (Note: Kabat numbering does not have residues 293-294, 297-298, 315-316, 356,362, 380, 403-404, 409, 412-413, 429, 431-431 in lgG1 ).
  • Part of the AB loop is shown in bold, and part of the EF loop is shown in bold and doubly underlined.
  • Figure 2 demonstrates that H561-4 binds to a different epitope on HER2 than trastuzumab or pertuzumab.
  • Figure 2A shows a surface plasmon resonance (SPR) analysis of epitope competition of trastuzumab and H561-4 using BIAcore.
  • SPR surface plasmon resonance
  • trastuzumab after the first injection (dotted black line).
  • a CM5 BIAcore chip coated with 1000 RU of HER2 ECD was saturated with trastuzumab by injection of 10 pg/ml of trastuzumab for 3 min followed by a second injection of a mixture of H561-4 (10 pg/ml) and trastuzumab (10 pg/ml) showing when a HER2 chip is saturated with trastuzumab H561 -4 is still able to bind (solid black line).
  • CM5 BIAcore chip coated with 000 RU of HER2 ECD was saturated with H561-4 by injection of 10 pg/ml of H561- 4 for 3 min followed by a second injection of H561-4 (10 Mg/ml) showing that the HER2 chip is saturated with H561-4 after the first injection (dashed black line).
  • FIG. 2B shows a SPR analysis of epitope competition of pertuzumab (PE) and H561-4 using Octet.
  • PE pertuzumab
  • FIG. 2B shows a SPR analysis of epitope competition of pertuzumab (PE) and H561-4 using Octet.
  • a streptavidin coated tip was loaded by incubation in biot-HER2 for 30 min and then washed for 1 minute in buffer.
  • HER2 was saturated with pertuzumab by incubation of 325 nM pertuzumab for 30 min followed by a second incubation of pertuzumab at 325 nM showing that the HER2 coated tip is saturated with pertuzumab after the first incubation (grey line).
  • a streptavidin coated tip was loaded by incubation in biot-HER2 for 30 min, and then washed for 1 minute in buffer.
  • HER2 was saturated with pertuzumab by incubation of 325 nM pertuzumab for 30 min followed by a second incubation of H561-4 at 325 nM and pertuzumab at 325 nM showing that a HER2 coated tip saturated with pertuzumab is still able to bind H561-4 (black line).
  • Figure 2C shows the predicted binding region of H561 -4 (black labelling spanning domain 1 and 3) on the extracellular domain of HER2.
  • the binding epitope of H561-4 was predicted using Pepscan CLIPS (Chemical Linkage of Peptides onto Scaffolds) technology.
  • HER2 domains are indicated by roman numerals.
  • the table in Figure 2C indicates the predicted linear sequences for each of the binding epitopes of H561 -4, that together form the binding pocket spanning domains 1 and 3.
  • Figure 3 demonstrates that H561 -4 induces apoptosis and leads to HER2 internalization and degradation.
  • Figure 3A shows that H561-4 treatment of SKBr3 cells caused a reduction in the total number of cells and in the percentage of viable cells. PI and Annexin V staining showed an increase in late apoptotic and necrotic cells after H561 -4 treatment.
  • Figure 3B shows that H561 -4 treatment of SKBr3 cells caused a reduction in total HER2 and in phosphorylated HER2.
  • Figure 3C shows that H561 -4 treatment of SKBr3 cells caused a reduction in cell surface HER2 and total HER2.
  • FIG. 3D shows that caspase 3/7 activity is induced by H561 -4 treatment of SKBr3 cells but not by treatment of SKBr3 cells with trastuzumab, wt antigen-binding Fc fragment, or an lgG1 control (ctrl).
  • Figure 4 demonstrates efficacy of H561 -4 treatment human patient derived tumour xenograft (PDX) models with HER2 gene copy numbers greater than or equal to 10.
  • the error bars in Figures 4A-H represent the standard error of the mean (SEM).
  • Mice were treated intravenously (i.v.) with compounds listed in the legend ( " separates the dosing compounds of the first treatment cycle from those of the second cycle). Arrows indicate the dosing schedule.
  • After implanted tumours reached the mean volume of 100 mm 3 they were treated with 4 weekly doses of TR+PE combination therapy (or 4 weekly doses of H561 -4 as a control).
  • the TR+PE treated tumours continued to grow albeit at a slower rate.
  • a 33-day interval was implemented to allow the clearance of these antibodies before the second dosing cycle.
  • mice were re-dosed weekly with TR+PE or with H561 -4.
  • the error bars represent the standard error of the mean (SEM).
  • Figure 6 shows a scatter plot of the T/C values of H561 -4 and trastuzumab treatment in PDX models with HER2 gene copy numbers (GCN) greater than or equal to 10, compared with PDX models with GCN of less than 10. P values were calculated by unpaired T-test using Graphpad prism software.
  • Figure 7 shows a scatter plot of the T/C values of H561 -4 and trastuzumab treatment in PDX models with HER2 mRNA levels (mRNA) greater than or equal to 200, compared with PDX models with mRNA levels of less than 200. P values were calculated by unpaired T-test using Graphpad prism software.
  • Figure 8 shows a scatter plot of the T/C values of H561-4 and trastuzumab treatment in PDX models with HER2 GCN greater than 10 compared to PDX models with GCN less than 10.
  • the T/C values were calculated using the updated T/C equation.
  • P values were calculated by unpaired T-test using Graphpad prism software.
  • Figure 9 shows a scatter plot of the T/C values of H561 -4 and trastuzumab treatment in PDX models with HER2 mRNA level greater than or equal to 200 compared to PDX models with HER2 mRNA level of less than 200.
  • the T/C values were calculated using the updated T/C equation.
  • P values were calculated by unpaired T-test using Graphpad prism software.
  • Figure 10 shows a scatter plot of the T/C values of H561 -4 and trastuzumab treatment in PDX models with HER2 GCN greater than 18 as determined by FISH compared to PDX models with GCN less than 18. P values were calculated by unpaired T-test using Graphpad prism software.
  • Figure 11 shows a scatter plot of the gene copy number determined by FISH and qPCR in each of 17 PDX tumour models.
  • Figure 12 shows a scatter plot of the T/C values of H561 -4 and trastuzumab treatment in PDX models with HER2 protein overexpression positive compared to PDX models with HER2 protein overexpression negative. P values were calculated by unpaired T-test using Graphpad prism software. Detailed description of the invention
  • a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain, e.g. CH3 domain, of the specific binding member can be used to treat cancers with a HER2 gene copy number of greater than or equal to 10 per tumour cell.
  • the present inventors have also discovered that such specific binding members can be used to treat cancers with a high HER2 mRNA levels.
  • such specific binding members can be used to treat cancers resistant to treatment with known HER2 specific treatments, such as trastuzumab monotherapy, or treatment with trastuzumab in combination with pertuzumab.
  • Specific binding members comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain, e.g., CH3 domain, of the specific binding member can be used in combination with another treatment, e.g., surgery, radiation therapy or chemotherapy, such as immunotherapy.
  • another treatment e.g., surgery, radiation therapy or chemotherapy, such as immunotherapy.
  • HER2 is known to be expressed in a number of different cancers. More data is available in breast cancer than in other cancers known to express HER2, as this is perhaps the most well studied HER2 positive cancer. Most data is available for tumours having an IHC score of 3+. Hoff et al. (2002) determined that 8% of all breast cancer patients had gene copy number greater than or equal to 10. In the largest adjuvant breast cancer trial of trastuzumab (the HERA trial), gene copy number of the tumours was assessed in 2071 (61 %) patients by FISH. Of those patients, a gene copy number of greater than or equal to 10 was identified in about 65% of the patients. This represents about 13% of all breast cancer patients (Dowsett et al., 2009).
  • a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain, e.g., CH3 domain, of the specific binding member, as well as a specific binding member which competes with such a specific binding member for binding to HER2, for use in a method of treating cancer in a patient, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, wherein the cancer has a HER2 gene copy number of greater than or equal to 10 per tumour cell.
  • a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain, e.g. CH3 domain, of the specific binding member, as well as a specific binding member which competes with such a specific binding member for binding to HER2, for use in a method of treating cancer in a patient, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, wherein said cancer has a high HER2 mRNA level.
  • immunotherapy e.g., therapy with an immuno-oncology agent
  • a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain, e.g. CH3 domain, of the specific binding member, as well as a specific binding member which competes with such a specific binding member for binding to HER2, for use in a method of treating cancer in a patient, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, wherein said cancer is resistant or refractory for treatment with trastuzumab and/or trastuzumab plus pertuzumab (e.g., is intrinsically resistant or refractory or has acquired resistance or refractory status).
  • immunotherapy e.g., therapy with an immuno-oncology agent
  • a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain, e.g. CH3 domain, of the specific binding member, as well as a specific binding member which competes with such a specific binding member for binding to HER2, for use in a method of treating cancer in a patient, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, wherein said cancer (i) is resistant or refractory for treatment with trastuzumab and/or trastuzumab plus pertuzumab (e.g., is intrinsically resistant or refractory or has acquired resistance or refractory status) and (ii) has a HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level.
  • immunotherapy e.g., therapy with an immuno-oncology agent
  • the present invention provides a specific binding member which binds to HER2 for use in a method of treating cancer in a patient, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, wherein said cancer has a HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell, and wherein the specific binding member is:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member which competes with a specific binding member according to (a) for binding to HER2.
  • a patient as referred to herein in one embodiment, is a human patient.
  • a tumour sample obtained from said patient may have been determined to have a HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell.
  • the present invention provides a specific binding member which binds to HER2 for use in a method of treating cancer in a patient, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, wherein a tumour sample obtained from said patient has been determined to have an average HER2 gene copy number of greater than or equal to 10 per tumour cell, and wherein the specific binding member is:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the method may comprise:
  • the present invention therefore also provides a specific binding member which binds to HER2 for use in a method of treating cancer in a patient, wherein the method comprises:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the term "HER2 gene copy number”, as used herein may refer to the average HER2 gene copy number per tumour/cancer cell.
  • the HER2 gene copy number may be greater than or equal to 10.
  • the HER2 gene copy number 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.
  • the HER2 gene copy number is greater than 10. In another example, the HER2 gene copy number is greater than or equal to 11. In another example, the HER2 gene copy number is greater than or equal to 12. In another example, the HER2 gene copy number is greater than or equal to 13. In another example, the HER2 gene copy number is greater than or equal to 14. In a further example, the HER2 gene copy number is greater than or equal to 15. In another example, the HER2 gene copy number is greater than or equal to 16. In another example, the HER2 gene copy number is greater than or equal to 17. In another example, the HER2 gene copy number is greater than or equal to 18. In another example, the HER2 gene copy number is greater than or equal to 19.
  • the HER2 gene copy number is greater than or equal to 20. In a further example, the HER2 gene copy number is greater than or equal to 23. In a yet further example, the HER2 gene copy number is greater than or equal to 25. Preferably, the HER2 gene copy number, e.g. the average HER2 gene copy number, is greater than or equal to 15.
  • the HER2 gene copy number e.g., the average HER2 gene copy number, is greater than or equal to 10, wherein the gene copy number is determined by quantitative polymerase chain reaction (qPCR).
  • qPCR quantitative polymerase chain reaction
  • the HER2 gene copy number e.g., the average HER2 gene copy number, is greater than or equal to 18, wherein the gene copy number is determined by FISH.
  • the present invention provides a specific binding member which binds to HER2 for use in a method of treating cancer in a patient, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, wherein said cancer has a high HER2 mRNA level, and wherein the specific binding member is.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent, wherein said cancer has a high HER2 mRNA level, and wherein the specific binding member is.
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • a tumour sample obtained from said patient may have been determined to have a high HER2 mRNA level.
  • the present invention provides a specific binding member which binds to HER2 for use in a method of treating cancer in a patient, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, wherein a tumour sample obtained from said patient has been determined to have a high HER2 mRNA level, and wherein the specific binding member is:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the method may comprise:
  • the present invention therefore also provides a specific binding member which binds to HER2 for use in a method of treating cancer in a patient, wherein the method comprises:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • Levels of mRNA encoding a specific protein in a sample may be measured indirectly through quantifying, by quantitative PCR, the copies of cDNA derived from the mRNA in the sample by reverse transcription PCR (RT-PCR), relative to the cDNA copies of a reference, preferably a housekeeping gene.
  • the reference gene has a normal profile across diverse tissue/sample types and is used as a standard.
  • the HER2 mRNA level in a tumour/cancer or tumour sample as referred to herein, preferably refers to the HER2 mRNA level as reflected by the HER2 cDNA copy number in the tumour/cancer or tumour sample, determined by RT-PCR followed by quantitative PCR, relative to the mRNA level of a reference (e.g.
  • cDNA copy number may be determined from quantitative PCR data using CopyCallerTM Software version 2.0 (Life Technologies), or another equivalent software which would be known to the skilled person.
  • CopyCallerTM Software performs a comparative cycle threshold relative quantitation analysis of the number of copies of the HER2 cDNA relative to the number of copies of the cDNA of a housekeeping gene.
  • the cDNA copy number may be normalised by reference to a reference cDNA sample, such as a cDNA sample obtained from a pool of 10 human cell lines which have or are expected to have two copies of the HER2 gene per cell (LifeTechnologies, CopyCaller Software - User Manual).
  • a reference cDNA sample such as a cDNA sample obtained from a pool of 10 human cell lines which have or are expected to have two copies of the HER2 gene per cell (LifeTechnologies, CopyCaller Software - User Manual).
  • a high HER2 mRNA level in a tumour/cancer or tumour sample thus preferably refers to a high HER2 mRNA level as reflected by a high HER2 cDNA copy number in the
  • tumour/cancer or tumour sample as determined by RT-PCR followed by quantitative PCR, relative to the mRNA level of a reference gene, as reflected by the number of cDNA copies of the reference gene, as determined by RT-PCR followed by quantitative PCR, in the tumour/cancer or tumour sample, respectively.
  • reverse transcription PCR is used to transcribe the HER2 or reference gene mRNA in a tumour sample to cDNA, which is then quantified using qPCR.
  • the reference gene is preferably a housekeeping gene.
  • the reference gene is TATA-binding protein (TBP) but other suitable reference genes are also known in the art.
  • the HER2 mRNA level in tumour/cancer or tumour sample may be normalized relative to the HER2 mRNA level in a control cell sample known to have two copies of the HER2 gene per cell.
  • the HER2 mRNA level in tumour/cancer or tumour sample as reflected by the HER2 cDNA copy number in the tumour/cancer or tumour sample, determined by RT-PCR followed by quantitative PCR, may be normalized relative to the HER2 mRNA level as reflected by the HER2 cDNA copy number, determined by RT-PCR followed by quantitative PCR, in a control cell sample known to have two copies of the HER2 gene per cell.
  • the HER2 mRNA level of the control sample is preferably the HER2 mRNA level as reflected by the cDNA copy number in the control sample, as determined by RT-PCR followed by quantitative PCR, relative to the mRNA level of a reference gene, as reflected by the number of cDNA copies of the reference gene, as determined by RT-PCR followed by quantitative PCR, in the control sample.
  • the reference gene is preferably the same reference gene as used to determine the HER2 mRNA level in the tumour/cancer or tumour sample, e.g., TBP.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample thus preferably refers to a high HER2 mRNA level as reflected by a high HER2 cDNA copy number in the
  • tumour/cancer or tumour sample determined by RT-PCR followed by quantitative PCR, relative to the mRNA level of a reference gene, as reflected by the number of cDNA copies of the reference gene determined by RT-PCR followed by quantitative PCR, in the tumour/cancer or tumour sample, respectively.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample may refer or correspond to a HER2 cDNA copy number 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 in a sample of said cancer/tumour relative to the cDNA copy number of a reference gene in said sample, wherein the cDNA copy number of HER2 and the reference gene in the sample is determined by RT-PCR followed by quantitative PCR.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample may refer or correspond to a HER2 cDNA copy number greater than or equal to 168 in a sample of said cancer/tumour relative to the cDNA copy number of a reference gene in said sample, wherein the cDNA copy number of HER2 and the reference gene in the sample is determined by RT-PCR followed by quantitative PCR.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample may refer or correspond to a HER2 cDNA copy number greater than or equal to 248 in a sample of said cancer/tumour relative to the cDNA copy number of a reference gene in said sample, wherein the cDNA copy number of HER2 and the reference gene in the sample is determined by RT-PCR followed by quantitative PCR.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample may refer or correspond to a HER2 cDNA copy number greater than or equal to 200 in a sample of said cancer/tumour relative to the cDNA copy number of a reference gene in said sample, wherein the cDNA copy number of HER2 and the reference gene in the sample is determined by RT- PCR followed by quantitative PCR.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample may refer or correspond to a HER2 cDNA copy number greater than or equal to 229 in a sample of said cancer/tumour relative to the cDNA copy number of a reference gene in said sample, wherein the cDNA copy number of HER2 and the reference gene in the sample is determined by RT- PCR followed by quantitative PCR.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample may refer or correspond to a HER2 cDNA copy number of 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 1 00, 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 in a sample of said cancer/tumour relative to the cDNA copy number of a reference gene in said sample, wherein the cDNA copy number of HER2 and the reference gene in the sample is determined by RT-PCR followed by quantitative PCR.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample may refer or correspond to a HER2 cDNA copy number of less than or equal to 820 in a sample of said cancer/tumour relative to the cDNA copy number of a reference gene in said sample, wherein the cDNA copy number of HER2 and the reference gene in the sample is determined by RT-PCR followed by quantitative PCR.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample may refer or correspond to a HER2 cDNA copy number greater than or equal to 200 and less than or equal to 820 in a sample of said cancer/tumour relative to the cDNA copy number of a reference gene in said sample, wherein the cDNA copy number of HER2 and the reference gene in the sample is determined by RT-PCR followed by quantitative PCR.
  • a high HER2 mRNA level in a tumour/cancer or tumour sample may refer or correspond to a HER2 cDNA copy number greater than or equal to 229 and less than or equal to 820 in a sample of said cancer/tumour relative to the cDNA copy number of a reference gene in said sample, wherein the cDNA copy number of HER2 and the reference gene in the sample is determined by RT-PCR followed by quantitative PCR.
  • a cancer as referred to herein may be a gastric cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer (for example, non-small cell lung cancer), stomach cancer, or endometrial cancer. All of these cancers have been shown to overexpress HER2.
  • the cancer is gastric cancer, breast cancer, or colorectal cancer. More preferably, the cancer is gastric cancer or breast cancer.
  • the cancer is gastric cancer.
  • Gastric cancer, as referred to herein includes oesophageal cancer.
  • the cancer is breast cancer.
  • the HER2 gene copy number of the cancer is as set out above.
  • HER2 positive HER2+
  • overexpressing HER2 HER2
  • a cancer as referred to herein, may be HER2 positive.
  • a cancer as referred to herein may overexpress HER2. Whether a cancer is HER2 positive or overexpresses HER2 may, for example, be determined initially using immunohistochemistry (IHC), optionally followed by methods such as qPCR as outlined above.
  • IHC immunohistochemistry
  • a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain, e.g. CH3 domain, of the specific binding member can be used to treat cancers which are resistant or refractory for treatment with trastuzumab and/or trastuzumab plus pertuzumab.
  • the cancer may be intrinsically resistant or refractory for treatment with trastuzumab and/or trastuzumab plus pertuzumab, or may have acquired resistance or refractoriness to treatment with trastuzumab and/or trastuzumab plus pertuzumab.
  • the cancer may be a gastric cancer which is resistant or refractory for treatment with trastuzumab and/or trastuzumab plus pertuzumab.
  • the cancer may be a breast cancer which is resistant or refractory for treatment with trastuzumab and/or trastuzumab plus pertuzumab.
  • Methods for determining whether a cancer is resistant or refractory for treatment with trastuzumab and/or trastuzumab plus pertuzumab are well known in the art and would be apparent to the skilled person.
  • a breast cancer which is resistant to treatment with trastuzumab may show progression at the first radiological reassessment at 8-12 weeks or within 3 months after first-line trastuzumab treatment with or without chemotherapy in the metastatic setting or new recurrences diagnosed during or within 12 months after adjuvant trastuzumab.
  • a breast cancer which is refractory to trastuzumab may show disease progression after two or more lines of trastuzumab-containing regimens that initially achieved disease response or stabilization at the first radiological assessment (Wong ef a/., 2011).
  • a patient as referred to herein may have exhibited an inadequate response to trastuzumab and/or trastuzumab plus pertuzumab treatment.
  • An inadequate response to trastuzumab and/or trastuzumab plus pertuzumab may refer to a lack of tumour growth retardation, or insufficient tumour growth retardation, when the patient was treated with trastuzumab and/or trastuzumab plus pertuzumab.
  • a patient who exhibited an inadequate response to trastuzumab and/or trastuzumab plus pertuzumab may refer to a patient who had to discontinue treatment with trastuzumab and/or trastuzumab plus pertuzumab due to adverse effects or adverse events resulting from the treatment with trastuzumab and/or trastuzumab plus pertuzumab, such as side effects, in particular serious side effects.
  • an inadequate response to trastuzumab and/or trastuzumab plus pertuzumab refers to a lack of tumour growth retardation, or insufficient tumour growth retardation, when the patient was treated with trastuzumab and/or trastuzumab plus pertuzumab.
  • a medical practitioner will have no difficulty in determining whether a given patient has exhibited an inadequate response to trastuzumab and/or trastuzumab plus pertuzumab treatment, as medical practitioners are experienced in judging whether a given cancer treatment was or was not successful in the case of a particular patient, and hence should or should not be continued.
  • a long period of stable disease may be considered a favourable clinical state if a patient's disease burden and clinical symptoms are light at the start of treatment, whereas if the disease burden is high and clinical symptoms are significant at the start of treatment, then stable disease may be considered an inadequate response.
  • kits for treating patients having cancer that (i) exhibit an inadequate response to trastuzumab and/or trastuzumab plus pertuzumab; (ii) wherein the cancer is intrinsically resistant or refractory to trastuzumab and/or trastuzumab plus pertuzumab; or (iii) wherein the cancer has acquired resistance to trastuzumab and/or trastuzumab plus pertuzumab, the method comprising administering to said patient a specific binding member and optionally another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent,
  • immunotherapy e.g., therapy with an immuno-oncology agent
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • a “specific binding member” is a molecule, e.g., a protein that binds specifically to another molecule, e.g., a protein, e.g., HER2 (e.g., human HER2).
  • a specific binding member may be natural or partly or wholly synthetically produced.
  • a specific binding member normally comprises a molecule having an antigen-binding site.
  • a specific binding member may be an antibody molecule, or fragment thereof, that comprises an antigen-binding site.
  • a specific binding member may also be a non-antibody protein scaffold that binds specifically to an antigen, such as a molecule comprising a modified immunoglobulin like fold, e.g., a fibronectin domain ( 10 Fn3 domain).
  • An antibody may be a monoclonal antibody, especially a human monoclonal antibody, which can be obtained according to the standard methods well known to the person skilled in the art.
  • a specific binding member may be a protein, e.g., an antibody constant region comprising a HE 2 antigen binding site engineered into a structural loop region of a CH3 domain of the specific binding member and containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14)
  • Antibody fragments that comprise an antigen-binding site include, but are not limited to, Fab fragments (consisting of VL, VH, CL and CH1 domains); F(ab')2 fragments (bivalent fragment comprising two linked Fab fragments); single chain Fv molecules (scFv) (consisting of a VH domain and a VL domain linked by a peptide linker which allows the two domains to associate to form an antigen binding site; Bird er a/. [1988] Science, 242, 423-426; Huston er a/.
  • antigen-binding immunoglobulin e.g., IgG
  • heavy chain constant region fragments such as antigen-binding Fc fragments.
  • Antigen-binding Fc fragments may comprise an antigen-binding site engineered into one or more structural loop regions of a constant domain of the Fc fragment, e.g. the CH2 or CH3 domain.
  • the preparation of antigen-binding Fc fragments is described in WO 2006/072620 and WO2009/132876.
  • a specific binding member for use in the present invention preferably is, or comprises, an antigen- binding Fc fragment, also referred to as FcabTM. More preferably, a specific binding member for use in the present invention is an antigen-binding Fc fragment.
  • the specific binding member may be an lgA1 , lgA2, IgD, IgE, lgG1 , lgG2, lgG3, lgG4 or IgM antigen-binding Fc fragment.
  • a specific binding member as referred to herein is an lgG1 (e.g., human lgG1 ) antigen-binding Fc fragment.
  • a specific binding member is an lgG1 antigen-binding Fc fragment comprising a hinge or portion thereof, a CH2 domain and a CH3 domain.
  • Antigen-binding Fc fragments may be incorporated into immunoglobulin molecules.
  • a specific binding member for use in the present invention may be an lgA1 , lgA2, IgD, IgE, lgG1 , lgG2, lgG3, lgG4 or IgM molecule comprising an antigen-binding site in the Fc region.
  • Such molecules may comprise a second CDR-based binding site in the variable region of the molecule, and hence are bispecific.
  • the specific binding member for use in the present invention may be an lgG1 molecule comprising an antigen-binding site in the Fc region.
  • a specific binding member as referred to herein preferably has a molecular weight (MW) of 60 kD or less, more preferably of 55 kD or less, 54 kD or less, or 53 kD or less.
  • Specific binding member H561 -4 disclosed herein has a MW of approximately 53 kD.
  • Specific binding member H561 -4 is also known as FS102.
  • a specific binding member, as referred to herein, may comprise a HER2 antigen binding site engineered into a structural loop region of a CH3 domain of the specific binding member, wherein the HER2 antigen binding site comprises or contains the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14).
  • a specific binding member may comprise a HER2 antigen binding site engineered into a structural loop region of a CH3 domain of a specific binding member, wherein the HER2 antigen binding site contains the amino acid sequences FFTYW (SEQ ID NO: 12), NGQPE (SEQ ID NO: 13), and DRRRWTA (SEQ ID NO: 14).
  • a specific binding member may comprise a HER2 antigen binding site engineered into a structural loop region of a CH3 domain of the specific binding member, wherein the HER2 antigen binding site comprises the amino acid sequence FFTYW (SEQ ID NO: 12) and the amino acid sequence DRRRWTA (SEQ ID NO: 14).
  • a specific binding member may comprise a HER2 antigen binding site engineered into a structural loop region of a CH3 domain of the specific binding member, wherein the HER2 antigen binding site comprises the amino acid sequence FFTYW (SEQ ID NO: 12) in the AB loop, and amino acid sequence DRRRWTA (SEQ ID NO: 14) in the EF loop of the CH3 domain.
  • a specific binding member comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain of the specific binding member, wherein the HER2 antigen binding site comprises the amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), wherein SEQ ID NO: 12 is located in residues 14-18 of the AB loop of the CH3 domain, and SEQ ID NO: 14 is located in residues 92-98 of the EF loop of the CH3 domain, and wherein the residues are numbered according to the IMGT (ImMunoGeneTics) numbering scheme.
  • IMGT ImMunoGeneTics
  • a specific binding member comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain of the specific binding member, wherein the HER2 antigen binding site comprises the amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), wherein SEQ ID NO: 12 is located in residues 381 -385, and SEQ ID NO: 14 is located in residues 440-450 of the EF loop of the CH3 domain, and wherein the residues are numbered according to KABAT (Kabat et al., 1987, Sequences of Proteins of Immunological Interest. 4 th Edition.
  • the backbone of 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, wherein the HER2 antigen binding site comprises the amino acid sequence FFTYW (SEQ ID NO: 12) and the amino acid sequence DRRRWTA (SEQ ID NO: 14) may be an lgG1 , e.g., human lgG1 , heavy chain constant region.
  • Human lgG1 may be of any allotype, e.g., G1 m, G1 m1 (a), G1 m2(x), G1 m3(f), G1 m17(z) (see, e.g., Jefferis et al. (2009) mAbs 1 :1 ).
  • a specific binding member preferably comprises the CH3 domain of SEQ ID NO: 11.
  • a specific binding member may further comprise the CH2 domain of SEQ ID NO: 10.
  • a specific binding member as referred to herein preferably comprises the sequence of SEQ ID NO: 8.
  • a specific binding member as referred to herein is a dimer of a polypeptide of SEQ ID NO: 8.
  • the specific binding member may be a dimer formed by two polypeptides of SEQ ID NO: 8, such as a dimer consisting of two polypeptides, wherein each polypeptide consists of the sequence shown in SEQ ID NO: 8.
  • a dimer comprises 1 or 2 disulfide bridges in the hinge region.
  • a specific binding member is a protein comprising two polypeptides, wherein each polypeptide comprises or consists of SEQ ID NO: 8, wherein the two polypeptides are linked through 1 or 2 disulfide bridges formed through cysteines that are located in the hinge region (the second and fifth cysteines in SEQ ID NO: 8) (such an specific binding member is also referred to as H561-4 herein).
  • a specific binding member may comprise the CH3 domain of SEQ ID NO: 11 , or the CH3 domain of SEQ ID NO: 11 minus one or two C-terminal residues (e.g. the C-terminal lysine or C-terminal lysine and glycine residue).
  • a specific binding member, as referred herein, may comprise the sequence of SEQ ID NO: 8, or the sequence of SEQ ID NO: 8 minus one or two C-terminal residues (e.g. the C-terminal lysine or lysine and glycine residues).
  • any reference to SEQ ID NOs 8 and 11 as used herein should be interpreted as also applying to these sequences lacking one or two of the C-terminal amino acid residues (e.g. the C- terminal lysine or lysine and glycine residues).
  • Reference to a CH3 domain as referred herein encompasses a CH3 domain which is missing one or two C-terminal amino acids residues (e.g. the C- terminal lysine or lysine and glycine residues).
  • a specific binding member is a protein comprising two polypeptides, wherein each polypeptide comprises or consists of SEQ ID NO: 8, wherein the two polypeptides are linked through 1 or 2 disulfide bridges formed through cysteines that are located in the hinge region (the second and fifth cysteines in SEQ ID NO: 8), and wherein one or both of the polypeptides is lacking the C-terminal lysine ( ) or the C-terminal lysine and adjacent glycine (GK) residues.
  • a specific binding member may comprise a HER2 antigen binding site engineered into a structural loop region of a CH3 domain of the specific binding member, wherein the HER2 antigen binding site comprises or contains the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14) with 3, 2 or 1 amino acid substitutions, deletions or additions.
  • An amino acid substitution may be a conservative amino acid substitution.
  • a specific binding member may comprise a HER2 antigen binding site engineered into a structural loop region of a CH3 domain of a specific binding member, wherein the HER2 antigen binding site contains the amino acid sequences FFTYW (SEQ ID NO: 12), NGQPE (SEQ ID NO: 13), and DRRRWTA (SEQ ID NO: 14) with 3, 2 or 1 amino acid substitutions, deletions or additions.
  • Variants also include those comprising an amino acid sequence that differs from SEQ ID NO: 8 in at most 1 , 2, 3, 4, 5, 1-3, 1-5 or 1-10 amino acid substitutions (e.g., conservative substitutions), deletions or additions) and/or those that are at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, wherein the variants bind specifically to human HER2, preferably at the same or overlapping epitope, and optionally induce apoptosis of HER2 positive cancer cells.
  • amino acid sequence that differs from SEQ ID NO: 8 in at most 1 , 2, 3, 4, 5, 1-3, 1-5 or 1-10 amino acid substitutions (e.g., conservative substitutions), deletions or additions) and/or those that are at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, wherein the variants bind specifically to human HER2, preferably at the same or overlapping epitope, and optionally induce apopto
  • Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the referenced amino acid sequence sequence, after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • a specific binding member comprising a thus-altered amino acid sequence may retain the ability to bind HER2.
  • it may bind HER2 with same affinity as a specific binding member in which the substitution is not made.
  • the binding affinity of an antibody is usually characterized in terms of the concentration of the antibody, at which half of the antigen binding sites are occupied, known as the dissociation constant (Kd).
  • Specific binding members preferably have the HER2 binding property of a binding affinity Kd ⁇ 10 ⁇ 8 M and/or a potency of ⁇ 50 ⁇ 10 ⁇ 8 M, or a Kd or IC50 of less than 10 9 M, preferably less than 10 10 M or even less than 10" 11 M, most preferred in the picomolar range.
  • the IC50 also called EC50 or 50% saturation concentration
  • the potency of a binder is usually defined by its IC50 (hereby understood as an EC50 value). This can be calculated for a given binder by determining the concentration of binder needed to elicit half saturation of the maximum binding. Elucidating an IC50 or EC50 value is useful for comparing the potency of specific binding member or specific binding member variants with similar efficacies, in particular when determined in saturation binding assays, not in competition assays. In this case it is considered as the concentration, which determines the plasma concentration to obtain a half-maximal (50%) effect in vivo. The lower the IC50 or EC50, the greater the potency of the specific binding member, and the lower the concentration of the EC50 value
  • An isolated protein that binds specifically to human HER2 may comprise two polypeptides, wherein each polypeptide comprises a human lgG1 heavy chain fragment comprising a CH2 domain and a CH3 domain, wherein the CH3 domain comprises an AB loop comprising the amino acid sequence of SEQ ID NO: 191 , a CD loop comprising the amino acid sequence of SEQ ID NO: 241 and an EF loop comprising the amino acid sequence 370.
  • the CH3 domain may comprise the amino acid sequence SEQ ID NO: 11.
  • the CH2 domain may comprise the amino acid sequence SEQ ID NO: 10.
  • the isolated protein may comprise a CH2 domain comprising SEQ ID NO: 10 and a CH3 domain comprising SEQ ID NO: 11.
  • the human lgG1 heavy chain fragment may comprise a hinge.
  • the two polypeptides may be connected by a disulfide bond.
  • the two polypeptides may be connected by 2 disulfide bonds.
  • the isolated protein may bind to human Her2 with a binding affinity of Kd ⁇ 10" 8 M.
  • the isolated protein may be cytotoxic.
  • the isolated protein may 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 may have a molecular weight of up to 60 kD.
  • an isolated protein that specifically binds to human HER2 has a binding affinity of Kd ⁇ 10 8 M, is cytotoxic, and has a molecular weight of up to 60 kD.
  • an isolated protein that specifically binds to human Her2 comprises two polypeptides, wherein each polypeptide comprises a human lgG1 heavy chain fragment comprising a CH2 domain and a CH3 domain, wherein the CH3 domain comprises an AB loop comprising the amino acid sequence of SEQ ID NO: 191 , a CD loop comprising the amino acid sequence of SEQ ID NO: 241 and an EF loop comprising the amino acid sequence 370; and wherein the isolated protein has a molecular weight of up to 60 kD, a binding affinity of Kd ⁇ 10 "8 M and is cytotoxic.
  • pharmaceutical compositions comprising any of the isolated proteins described herein and a pharmaceutically acceptable carrier.
  • a binding agent that competes for binding to HER2 with, or binds to the same epitope as, a HER2 binding agent may be an antibody (e.g., full length antibody) or an antigen binding fragment thereof, such as an antibody or antigen binding fragment that binds to HER2 through CDRs.
  • a specific binding member which competes with 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14), or a specific binding member as set out above, for binding to HER2, preferably comprises a HER2 antigen binding site engineered into one or more, preferably two, structural loop regions of a constant domain of the specific binding member. More preferably, such a specific binding member comprises a HER2 antigen binding site engineered into one or more, preferably two, structural loop regions of a CH3 domain of the specific binding member.
  • the structural loops in the CH3 domain are located at residues 7-21 (AB loop), 25-39 (BC loop), 41-81 (CD loop), 83-85 (DE loop), 89-103 (EF loop) and 106-1 17 (FG loop), wherein the residues are numbered according to IMGT (ImMunoGeneTics) numbering scheme (WO 2006/072620 A1 ).
  • a specific binding member comprises a HER2 antigen binding site engineered into structural loop regions AB and EF of a CH3 domain of the specific binding member.
  • such a specific binding member may comprise a HER2 antigen binding site engineered into residues 14-18 and 92- 98 of the CH3 domain of the specific binding member, wherein the residues are numbered according to the IMGT numbering scheme. Residues 14-18 are located in the AB loop and residues 92-98 are located in the EF loop of the CH3 domain.
  • Such a specific binding member may comprise a HER2 antigen binding site engineered into residues 381-385 and 440-450 of the specific binding member, wherein the residues are numbered according to KABAT. Residues 381-385 are located in the AB loop and residues 440-450 are located in the EF loop of the CH3 domain.
  • such a specific binding member may comprise a HER2 antigen binding site engineered into structural loop regions AB, CD, and EF of a CH3 domain of the specific binding member.
  • the 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14), with which the specific binding member competes for binding to HER2, is preferably a dimer of a polypeptide of SEQ ID NO: 8, e.g., a protein comprising two polypeptides, each of which comprises SEQ ID NO: 8.
  • HER2 e.g., human HER2
  • HER2 e.g., human HER2
  • Such methods include competition methods using surface plasmon resonance (SPR), enzyme-linked immunosorbent assays (ELISA), fluorescence activated cell sorting (FACS), or immunocytochemistry.
  • SPR surface plasmon resonance
  • ELISA enzyme-linked immunosorbent assays
  • FACS fluorescence activated cell sorting
  • SPR surface plasmon resonance
  • BIAcore surface plasmon resonance
  • BIAcore an enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS), or immunocytochemistry.
  • a specific binding member which competes with a second specific binding member for binding to HER2 competes with the second binding member for binding to HER2, as determined using surface plasmon resonance (SPR), e.g. BIAcore.
  • SPR surface plasmon resonance
  • trastuzumab and pertuzumab do not compete with specific binding member H561-4, which comprises a HER2 antigen binding site engineered into a structural loop region of its CH3 domain containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14), for binding to HER2.
  • the HER2 is immobilised on a chip surface (BIAcore), on a plate (ELISA), or displayed on a cell surface (FACS and immunohistochemistry).
  • BiAcore chip surface
  • ELISA ELISA
  • FACS and immunohistochemistry One of the two specific binding members to be compared is then incubated with the immobilized HER2 for a time and at a concentration that leads to saturation of the HER2 antigen, thereby blocking all of the HER2 epitopes to which this specific binding member binds.
  • the second specific binding member is then incubated with the immobilized HER2.
  • Binding of the second specific binding member to the HER2 indicates that the specific binding members bind to different epitopes on HER2, whereas if the second binding member does not bind to the immobilized HER2, this indicates that the second specific binding member competes with the first specific binding member for binding to HER2, and that the two specific binding members bind to the same (or overlapping) epitopes on HER2.
  • a binding member inhibits the binding of H561-4 to HER2 (e.g., human HER2) by at least 50%, 60%, 70%, 80%, 90%, 95% or more, as determined, e.g., by Biacore, ELISA or FACS.
  • H561-4 inhibits the binding of the binding member to HER2 by at least 50%, 60%, 70%, 80%, 90%, 95% or more, as determined, e.g., by Biacore, ELISA or FACS.
  • a binding member inhibits the binding of H561-4 to HER2 by at least 50%, 60%, 70%, 80%, 90%, 95% or more
  • H561 -4 inhibits the binding of the binding member to HER2 by at least 50%, 60%, 70%, 80%, 90%, 95% or more, as determined, e.g., by Biacore, ELISA or FACS (i.e., the competition is both ways).
  • a binding member competes for binding to HER2 with H561-4, but not with trastuzumab or pertuzumab. In certain embodiments, a binding member does not inhibit the binding of trastuzumab or pertuzumab to HER2 by more than 50%, 40%, 30%, 20%, 10% or less, as determined, e.g., by Biacore, ELISA or FACS. In certain embodiments, trastuzumab or pertuzumab does not inhibit the binding of H561-4 to HER2 by more than 50%, 40%, 30%, 20%, 10% or less, as determined, e.g., by Biacore, ELISA or FACS.
  • a binding member inhibits the binding of H561-4 to HER2 and/or H561-4 inhibits the binding of the binding member by at least 50%, 60%, 70%, 80%, 90%, 95% or more, but does not inhibit the binding of trastuzumab or pertuzumab and/or the to HER2 and/or trastuzumab or pertuzumab does not inhibit the binding of H561-4 to HER2 by more than 50%, 40%, 30%, 20%, 10% or less, as determined, e.g., by Biacore, ELISA or FACS.
  • a binding member inhibits the binding of H561-4 to HER2 and/or H561-4 inhibits the binding of the binding member by at least 50%, but does not inhibit the binding of trastuzumab or pertuzumab to HER2 and/or trastuzumab or pertuzumab does not inhibit the binding of H561-4 to HER2 by more than 50%, as determined, e.g., by Biacore, ELISA or FACS.
  • a binding member inhibits the binding of H561-4 to HER2 and/or H561-4 inhibits the binding of the binding member by at least 90%, but does not inhibit the binding of trastuzumab or pertuzumab to HER2 and/or trastuzumab or pertuzumab does not inhibit the binding of H561-4 to HER2 by more than 10%, as determined, e.g., by Biacore, ELISA or FACS.
  • a specific binding member as referred to herein may bind dimeric HER2.
  • the specific binding member may bind dimeric HER2 with an affinity of 1 nM, or an affinity that is higher.
  • the specific binding member may preferentially bind dimeric HER2 compared with monomeric HER2.
  • the specific binding member binds to the same epitope on the human HER2 extracellular domain as a protein comprising two polypeptides, each comprising SEQ ID NO: 8.
  • the epitope may be a conformational, i.e., non-linear, epitope.
  • the epitope may span domains 1 and 3 of HER2.
  • the epitope may comprise, or consist of, SEQ ID NOs 15, 16 and 17.
  • the epitope may comprise, or consist of, amino acids 13 to 27, 31 to 45, and 420 to 475 of the HER2 extracellular domain.
  • the epitope may be located within SEQ ID NOs 15, 16 and 17, or amino acids 13 to 27, 31 to 45, and 420 to 475 of the HER2 extracellular domain.
  • the sequence of the HER2 extracellular domain is shown in SEQ ID NO: 18.
  • an antigen in this case HER2
  • HDX-MS hydrogen/deuterium exchange mass spectrometry
  • X-ray Crystallography X-ray Crystallography
  • Residue mutations which cause a loss in binding are indicated to be part of the epitope bound by the specific binding member. However care must be taken to ensure that a mutation does not cause a general loss in structure of the HER2.
  • the antigen, here HER2 and the specific binding member are crystallised as a complex. The crystal data is then used to determine the epitope bound by the specific binding member. The HER2 epitopes bound by two different specific binding members can then be compared.
  • HER2 gene copy number may be determined using fluorescence in situ hybridization (FISH), chromogenic in situ hybridisation (CISH), or quantitative polymerase chain reaction (qPCR).
  • FISH fluorescence in situ hybridization
  • CISH chromogenic in situ hybridisation
  • qPCR quantitative polymerase chain reaction
  • HER2 gene copy number is determined using FISH.
  • Methods for determining HER2 mRNA levels are similarly known in the art and would be apparent to the skilled person.
  • HER2 mRNA level may be determined using reverse transcription PCR (RT-PCR) followed by quantitative PCR (qPCR).
  • Overexpression of the HER2 gene can be measured by immunohistochemistry (IHC) to determine the amount of HER2 expressed on the cell surface.
  • IHC immunohistochemistry
  • FISH employs fluorescence-tagged probes to detect particular DNA sequences in tissue samples by fluorescence microscopy.
  • FISH is used to quantitatively determine HER2 gene amplification in formalin-fixed, paraffin-embedded (FFPE) cancer tissue specimens using specific probes which hybridise with HER2 sequences.
  • FFPE formalin-fixed, paraffin-embedded
  • Different FDA approved methods exist for performing FISH to determine HER2 GCN Press et al, 2002). In one method, both HER2 and chromosome 17 numbers are measured and compared, to determine the average HER2 GCN per tumour cell in the samples.
  • CEP17 probes (chromosome enumeration probes, which are directly labelled fluorescent DNA probes specific to the alpha satellite DNA sequence at the centromeric region of chromosome 17) may be used in this test as an internal control for chromosomal aneuploidy.
  • the HER2 gene and CEP17 are labelled with different fluorophores and can be viewed using specific filters for the microscope.
  • Measurement of HER2 gene amplification is based on the ratio between the number of HER2 copies to CEP17 copies counted by microscopy, thereby eliminating aneuploidy of chromosome 17 as a source of increased HER2 GCN. Where, for example, the ratio is equal to or greater than 10, the average HER2 GCN per tumour/cancer cell is greater than or equal to 10.
  • a HER2, or average HER2, gene copy number per tumour/cancer cell, as referred to herein, may therefore exclude any increase in HER2 gene copy number due to chromosomal aneuploidy, in particular aneuploidy of chromosome 17.
  • overall HER2 gene copy number may be determined by direct counting of the HER2 signals without normalisation to CEP17 signal. This method is preferred because counting overall HER2 gene copy number can avoid false-positive or false-negative results due to loss or gain of the pericentromeric region of chromosome 17 which is more commonly observed than true polysomy (duplication of the entire chromosome) that can result in alterations in the HER2/CEP17 ratio (Wolff et al., 2013; Hanna et al., 2014).
  • FISH may be performed using only a HER2 probe on FFPE as an indirect method for localization of HER2 gene (Press et al, 2002).
  • the gene copy number obtained by FISH and qPCR for a particular tumour has been found to be highly correlative (see Example 9). Thus, conversion between GCN values obtained by different methods (e.g., FISH and qPCR) is possible.
  • a specific binding member comprising a HER2 antigen binding site engineered into a structural loop region of a constant domain, e.g., CH3 domain, of the specific binding member, as well as a specific binding member which competes with such a specific binding member for binding to HER2, in the manufacture of a medicament for treating cancer in a patient, wherein the cancer has a HER2 gene copy number, e.g., an average HER2 gene copy number, of greater than or equal to 10 per tumour cell, and/or wherein the cancer has a high HER2 mRNA level.
  • the present invention further provides the use of a specific binding member which binds to HER2, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, in the manufacture of a medicament for treating cancer in a patient, wherein said cancer has a HER2 gene copy number, e.g., an average HER2 gene copy number, of greater than or equal to 10 per tumour cell, and/or the cancer has a high HER2 mRNA level, and wherein the specific binding member is:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • a specific binding member which binds to HER2 optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, in the manufacture of a medicament for treating cancer in a patient, wherein a tumour sample obtained from said patient has been determined to have an average HER2 gene copy number of greater than or equal to 10 per tumour cell, and/or wherein a tumour sample obtained from said patient has been determined to have a high HER2 mRNA level, and wherein the specific binding member is:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • a specific binding member which binds to HER2 optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, in the manufacture of a medicament for the treatment of cancer in a patient, the treatment 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • a specific binding member which binds to HER2 optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, in the manufacture of a medicament for the treatment of cancer in a patient, the treatment 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member which competes with a specific binding member according to
  • the present invention provides a method of treating cancer in a patient, wherein said cancer has HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell, and wherein the method comprises administering to the patient a therapeutically effective amount of:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • a tumour sample obtained from said patient may have been determined to have a HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell.
  • the present invention provides a method of treating cancer in a patient, wherein a tumour sample obtained from said patient has been determined to have an average HER2 gene copy number of greater than or equal to 10 per tumour cell, and wherein the method comprises administering to the patient a therapeutically effective amount of:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the method may comprise:
  • treating said patient with said specific binding member optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, if the HER2 gene copy number, e.g. the average HER2 gene copy number, is greater than or equal to 10 per tumour cell.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent, if the HER2 gene copy number, e.g. the average HER2 gene copy number, is greater than or equal to 10 per tumour cell.
  • the present invention therefore also provides a method of treating cancer in a patient, wherein the method comprises:
  • the gene copy number e.g. the average gene copy number
  • administering a therapeutically effective amount of the specific binding member to the patient, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, if the HER2 gene copy number, e.g. the average gene copy number, is greater than or equal to 10 per tumour cell, wherein the specific binding member is:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the present invention provides a method of treating cancer in a patient, wherein said cancer has a high HER2 mRNA level, and wherein the method comprises administering to the patient a therapeutically effective amount of:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the present invention provides a specific binding member which binds to HER2, optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, for use in a method of treating cancer in a patient, wherein said cancer has a high HER2 mRNA level, and wherein the specific binding member is:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • Tumour samples obtained from said patient may have been determined to have high HER2 mRNA levels.
  • the present invention provides a method of treating cancer in a patient, wherein a tumour sample obtained from said patient has been determined to have a high HER2 mRNA level, 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 engineered into a structural loop region of a CH3 domain of the specific binding member and containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the method may comprise:
  • the present invention therefore also provides a method of treating cancer in a patient, wherein the method comprises:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the specific binding members described herein, as single agents or in combination with 1, 2, 3 or more agents are designed to be used in methods of treatment of patients, preferably human patients.
  • Specific binding members, as single agents or in combination with another agent will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member.
  • pharmaceutical compositions described herein, and for use in accordance with the present invention may comprise, in addition to the active ingredient(s), a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient(s).
  • the precise nature of the carrier or other material will depend on the route of administration, which may be by injection, e.g. intravenous or subcutaneous.
  • the specific binding member as a single agent or in combination with another agent (e.g., immuno-oncology agent), may be administered intravenously, or subcutaneously, preferably, but also by any type of administration including, but not limited to, orally, intranasally, intraotically, transdermally, mucosal, topically (e.g., gels, salves, lotions, creams, etc.), intraperitoneal, intramuscularly, intrapulmonary (e.g., AERxTM inhalable technology commercially available from Aradigm, or InhanceTM pulmonary delivery system commercially available from Inhale Therapeutics), vaginally, parenterally, rectally, or intraocularly.
  • AERxTM inhalable technology commercially available from Aradigm, or InhanceTM pulmonary delivery system commercially available from Inhale Therapeutics
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient(s) will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed, as required.
  • Many methods for the preparation of pharmaceutical formulations are known to those skilled in the art. See e.g. Robinson ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978.
  • Administration may be in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom.
  • treatment refers to amelioration of at least one symptom.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular patient being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the type of conjugate, the method of
  • Specific dosages indicated herein, or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered, may be used.
  • a therapeutically effective amount or suitable dose of a specific binding member can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the size and location of the area to be treated, and the precise nature of the specific binding member.
  • Treatments may be repeated at daily, twice-weekly, weekly, biweekly, every three weeks or monthly intervals, at the discretion of the physician. Treatment may be given before, and/or after surgery, and may be administered or applied directly at the anatomical site of surgical treatment. Treatment may be given before and/or after another therapy, e.g., radiation therapy or chemotherapy.
  • the present invention provides a method of identifying a cancer in a patient which is susceptible to treatment with: (a) 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 1 ); or
  • HER2 gene per tumour cell in a tumour sample obtained from the patient, wherein a gene copy number, e.g. an average gene copy number, of greater than or equal to 10 per tumour cell indicates that the cancer is susceptible to treatment with the specific binding member.
  • a method of identifying a cancer in a patient which is susceptible to treatment may further comprise (ii) selecting said patient for treatment with the specific binding member if the
  • HER2 gene copy number e.g. the average HER2 gene copy number, is greater than or equal to 10 per tumour cell.
  • the present invention provides a method of predicting the response of a cancer to treatment with:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • determining the HER2 gene copy number, e.g. the average HER2 gene copy number, of the HER2 gene per tumour cell in a tumour sample obtained from a patient wherein a gene copy number, e.g. the average HER2 gene copy number, of greater than or equal to 10 per tumour cell indicates that the cancer is susceptible to treatment with the specific binding member, and wherein a gene copy number, e.g. the average HER2 gene copy number, of less than 10 per tumour cell indicates that the cancer is not susceptible to treatment with the specific binding member.
  • a method of predicting the response of a cancer to treatment may further comprise:
  • the present invention provides a method of identifying a cancer in a patient which is susceptible to treatment with:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • a method of identifying a cancer in a patient which is susceptible to treatment may further comprise (ii) selecting said patient for treatment with the specific binding member if the HER2 mRNA level of the tumour sample is high.
  • the present invention provides a method of predicting the response of a cancer to treatment with:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • a method of predicting the response of a cancer to treatment may further comprise:
  • the present invention provides a specific binding member which binds to HER2, wherein the specific binding member is a dimer of a polypeptide of SEQ ID NO: 8.
  • the specific binding member may be a dimer formed by two polypeptides of SEQ ID NO: 8, such as a dimer consisting of two polypeptides, wherein each polypeptide consists of the sequence shown in SEQ ID NO: 8 (such a specific binding member is also referred to as H561-4 herein).
  • a pharmaceutical composition comprising the specific binding member of the invention.
  • the pharmaceutical composition may comprise the specific binding member of the invention and a pharmaceutically acceptable excipient.
  • a nucleic acid encoding the specific binding member of the invention is also provided.
  • the nucleic acid comprises or consists of the sequence of SEQ ID NO: 1. This nucleotide sequence has been optimized for expression in Chinese Hamster Ovary (CHO) cells but the skilled person would have no difficulty in designing other nucelotide sequences encoding the specific binding member of the invention.
  • a vector comprising a nucleic acid encoding the specific binding member of the invention, as is a host cell comprising a vector or nucleic acid of the invention.
  • a HER2 binding agent is administered with another treatment, either simultaneously, or consecutively, to a subject, e.g., a subject having cancer.
  • a HER2 binding agent may be administered with one of more of: radiotherapy, surgery, or chemotherapy, e.g., targeted chemotherapy or immunotherapy.
  • Immunotherapy e.g., cancer immuntherapy includes cancer vaccines and immuno-oncology agents.
  • a HER2 binding agent may be, e.g., a protein, an antibody, antibody fragment or a small molecule, that binds to HER2.
  • a HER2 binding agent may be a protein comprising a heavy chain constant region fragment comprising a CH2 and CH3 domains, wherein the CH3 domain comprises a HER2 binding site, and maybe, e.g., H561-4 or an analog or derivative thereof.
  • a subject having cancer that (i) has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory to treatment with trastuzumab and/or pertuzumab (e.g., intrinsically refractory or resistant or having acquired refractory status or resistance) and/or (ii) has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level is treated by administration of a HER2 binding agent and another therapy (such as radiotherapy, surgery or chemotherapy), wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises
  • a method of treatment of a subject having cancer that (i) has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory to treatment with trastuzumab and/or pertuzumab (e.g., intrinsically refractory or resistant or having acquired refractory status or resistance) and/or (ii) has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level comprises administering to the subject having the cancer a HER2 binding agent, e.g., H561-4, and one or more immuno-oncology agents.
  • a HER2 binding agent e.g., H561-4
  • Immunotherapy e.g., therapy with an immuno-oncology agent
  • the administration of a HER2 binding agent with an immuno-oncology agent has a synergic effect in the treatment of cancer, e.g., in inhibiting tumour growth.
  • a HER2 binding agent is sequentially administered prior to administration of the immuno-oncology agent. In one aspect, a HER2 binding agent is administered concurrently with the immunology-oncology agent. In yet one aspect, a HER2 binding agent is sequentially administered after administration of the immuno-oncology agent.
  • the administration of the two agents may start at times that are, e.g., 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 more weeks apart, or administration of the second agent may start, e.g., 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 more weeks after the first agent has been administered.
  • a HER2 binding agent and an immuno-oncology agent are administered simultaneously, e.g., are infused simultaneously, e.g., over a period of 30 or 60 minutes to a patient.
  • a HER2 binding agent may be co-formulated with an immuno-oncology agent.
  • Immuno-oncology agents include, for example, a small molecule drug, antibody or fragment thereof, or other biologic or small molecule.
  • biologic immuno-oncology agents include, but are not limited to, antibodies, antibody fragments, vaccines and cytokines.
  • the antibody is a monoclonal antibody. In certain aspects, the monoclonal antibody is humanized or human.
  • the immuno-oncology agent is (i) an agonist of a stimulatory (including a co- stimulatory) molecule (e.g., receptor or ligand) or (ii) an antagonist of an inhibitory (including a co- inhibitory) molecule (e.g., receptor or ligand) on immune cells, e.g., T cells, both of which result in amplifying antigen-specific T cell responses.
  • a stimulatory including a co- stimulatory
  • an antagonist of an inhibitory (including a co- inhibitory) molecule e.g., receptor or ligand
  • an immuno-oncology agent is (i) an agonist of a stimulatory (including a co-stimulatory) molecule (e.g., receptor or ligand) or (ii) an antagonist of an inhibitory (including a co-inhibitory) molecule (e.g., receptor or ligand) on cells involved in innate immunity, e.g., NK cells, and wherein the immuno-oncology agent enhances innate immunity.
  • Such immuno-oncology agents are often referred to as immune checkpoint regulators, e.g., immune checkpoint inhibitor or immune checkpoint stimulator.
  • an immuno-oncology agent targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin super family (IgSF).
  • an immuno-oncology agent may be an agent that targets (or binds specifically to) a member of the B7 family of membrane-bound ligands, which includes B7-1 , B7-2, B7-H1 (PD-L1 ), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6, or a co-stimulatory or co-inhibitory receptor binding specifically to a B7 family member.
  • An immuno-oncology agent may be an agent that targets a member of the TNF family of membrane bound ligands or a co-stimulatory or co-inhibitory receptor binding specifically thereto, e.g., a TNF receptor family member.
  • exemplary TNF and TNFR family members that may be targeted by immuno-oncology agents include CD40 and CD40L, OX-40, OX-40L, GITR, GITRL, CD70, CD27L, CD30, CD30L, 4-1 BBL, CD137 (4-1 BB), TRAIL/Apo2-L, TRAILR1/DR4,
  • TRAILR2/DR5, TRAILR3, TRAILR4, OPG RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LT ⁇ R, LIGHT, DcR3, HVEM, VEGI TL1A, TRAMP/DR3, EDAR, EDA1 , XEDAR, EDA2, TNFR1 , Lymphotoxin a/TNF , TNFR2, TNFa, LT R, Lymphotoxin a 1 ⁇ 2, FAS, FASL, RELT, DR6, TROY and NGFR.
  • An immuno-oncology agent that may be used in combination with a HER2 agent for treating cancer may be an agent, e.g., an antibody, targeting an IgSF member, such as a B7 family member, a B7 receptor family member, a TNF family member or a TNFR family member, such as those described above.
  • an agent e.g., an antibody, targeting an IgSF member, such as a B7 family member, a B7 receptor family member, a TNF family member or a TNFR family member, such as those described above.
  • a HER2 binding agent is administered with one or more of (i) an antagonist of a protein that inhibits T cell activation (e.g., immune checkpoint inhibitor) 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-H4, 2B4, CD48, GARP, PD1 H, LAIR1 , TIM-1 , and TIM-4, and (ii) an agonist of a protein that stimulates T cell activation such as B7-1 , B7-2, CD28, 4-1 BB (CD137), 4-1 BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, CD40L, DR3 and CD28H.
  • an immuno-oncology agent is an agent that inhibits (i.e., an antagonist of) a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF- ⁇ , VEGF, and other immunosuppressive cytokines) or is an agonist of a cytokine, such as IL-2, IL-7, IL-12, IL-15, IL-21 and IFNa (e.g., the cytokine itself) that stimulates T cell activation, and stimulates an immune response.
  • a cytokine that inhibits T cell activation e.g., IL-6, IL-10, TGF- ⁇ , VEGF, and other immunosuppressive cytokines
  • Anti-HER2 binding agent can be combined with an antagonist of KIR.
  • agents for combination therapies include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1 R antagonists such as CSF-1 R antagonist antibodies including RG7155 (W011/70024, W011/107553, WO11/131407, W013/87699, W013/119716, W013/132044) or FPA-008 (W011/140249; W013169264; WO14/036357).
  • CSF-1 R antagonists such as CSF-1 R antagonist antibodies including RG7155 (W011/70024, W011/107553, WO11/131407, W013/87699, W013/119716, W013/132044) or FPA-008 (W011/140249; W013169264; WO14/036357).
  • Immuno-oncology agents also include agents that inhibit TGF- ⁇ signaling.
  • Additional agents that may be combined with a HER2 binding agent include agents that enhance tumour antigen presentation, e.g., dendritic cell vaccines, GM-CSF secreting cellular vaccines, CpG oligonucleotides, and imiquimod, or therapies that enhance the immunogenicity of tumour cells (e.g., anthracyclines).
  • agents that enhance tumour antigen presentation e.g., dendritic cell vaccines, GM-CSF secreting cellular vaccines, CpG oligonucleotides, and imiquimod
  • therapies that enhance the immunogenicity of tumour cells e.g., anthracyclines.
  • therapies that may be combined with a HER2 binding agent include therapies that deplete or block Treg cells, e.g., an agent that specifically binds to CD25.
  • Another therapy that may be combined with a HER2 binding agent is a therapy that inhibits a metabolic enzyme such as indoleamine dioxigenase (IDO), dioxigenase, arginase, or nitric oxide synthetase.
  • IDO indoleamine dioxigenase
  • dioxigenase dioxigenase
  • arginase arginase
  • nitric oxide synthetase nitric oxide synthetase
  • Another class of agents that may be used includes agents that inhibit the formation of adenosine or inhibit the adenosine A2A receptor.
  • Other therapies that may be combined with a HER2 binding agent for treating cancer include therapies that reverse/prevent T cell anergy or exhaustion and therapies that trigger an innate immune activation and/or inflammation at a tumour site.
  • a HER2 binding agent may be combined with more than one immuno-oncology agent, and may be, e.g., combined with a combinatorial approach that targets multiple elements of the immune pathway, such as one or more of the following: a therapy that enhances tumour antigen presentation (e.g., dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpG oligonucleotides, imiquimod); a therapy that inhibits negative immune regulation e.g., by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking Treg or other immune suppressing cells; a therapy that stimulates positive immune regulation, e.g., with agonists that stimulate the CD-137, OX-40 and/or GITR pathway and/or stimulate T cell effector function; a therapy that increases systemically the frequency of anti-tumour T cells; a therapy that depletes or inhibits Tregs, such as Tregs in the tumour, e.g.,
  • a HER2 binding agent can be used with one or more agonistic agents that ligate positive costimulatory receptors; one or more antagonists (blocking agents) that attenuate signaling through inhibitory receptors, such as antagonists that overcome distinct immune suppressive pathways within the tumour microenvironment (e.g., block PD-L1/PD-1/PD-L2 interactions); one or more agents that increase systemically the frequency of anti-tumour immune cells, such as T cells, deplete or inhibit Tregs (e.g., by inhibiting CD25); one or more agents that inhibit metabolic enzymes such as IDO; one or more agents that reverse/prevent T cell anergy or exhaustion; and one or more agents that trigger innate immune activation and/or inflammation at tumour sites.
  • agonistic agents that ligate positive costimulatory receptors
  • antagonists blocking agents that attenuate signaling through inhibitory receptors, such as antagonists that overcome distinct immune suppressive pathways within the tumour microenvironment (e.g., block PD-L1/PD
  • a subject having cancer that (i) has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory to treatment with trastuzumab and/or pertuzumab and/or (ii) has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or high HER2 mRNA levels is treated by administration of a HER2 binding agent and an immuno-oncology agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C-terminal amino acids.
  • the immuno-oncology agent is a CTLA-4 antagonist, such as an antagonistic CTLA-4 antibody.
  • Suitable CTLA-4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.
  • the immuno-oncology agent is a PD-1 antagonist, such as an antagonistic PD-1 antibody.
  • Suitable PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA
  • the immuno-oncology agent may also include pidilizumab (CT-011 ), though its specificity for PD-1 binding has been questioned.
  • Another approach to target the PD-1 receptor is the recombinant protein composed of the
  • AMP-224 extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of lgG1 , called AMP-224.
  • the immuno-oncology agent is a PD-L1 antagonist, such as an antagonistic PD-L1 antibody.
  • Suitable PD-L1 antibodies include, for example, MPDL3280A (RG7446; WO2010/077634), durvalumab (MEDI4736), BMS-936559 (WO2007/005874), MSB0010718C (WO2013/79174) or rHigM12B7.
  • the immuno-oncology agent is a LAG-3 antagonist, such as an antagonistic LAG-3 antibody.
  • LAG 3 antibodies include, for example, BMS-986016 (W010/19570, W014/08218), or lMP-731 or IMP-32 (WO08/132601 , WO09/44273).
  • the immuno-oncology agent is a CD137 (4-1 BB) agonist, such as an agonistic CD137 antibody.
  • Suitable CD137 antibodies include, for example, urelumab or PF-05082566 (W012/32433).
  • the immuno-oncology agent is a GITR agonist, such as an agonistic GITR antibody.
  • GITR antibodies include, for example, TRX-518 (WO06/105021 , WO09/009116), MK-4166 (W011/028683) or a GITR antibody disclosed in WO2015/031667 or WO2015/187835
  • the immuno-oncology agent is an OX40 agonist, such as an agonistic OX40 antibody.
  • OX40 antibodies include, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888; WO06/029879).
  • the immuno-oncology agent is a CD40 agonist, such as an agonistic CD40 antibody.
  • the immuno-oncology agent is a CD40 antagonist, such as an antagonistic CD40 antibody.
  • Suitable CD40 antibodies include, for example, lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893 or Chi Lob 7/4.
  • the immuno-oncology agent is a CD27 agonist, such as an agonistic CD27 antibody.
  • Suitable CD27 antibodies include, for example, varlilumab (CDX-1127).
  • the immuno-oncology agent is MGA271 (to B7H3) (W011/109400).
  • the immuno-oncology agent is a KIR antagonist, such as lirilumab.
  • the immuno-oncology agent is an IDO antagonist.
  • IDO antagonists include, for example, INCB-024360 (WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919 (WO09/73620, WO09/1156652, W011/56652, W012/142237) or F001287.
  • the immuno-oncology agent is a Toll-like receptor agonist, e.g., a TLR2/4 agonist (e.g., Bacillus Calmette-Guerin); a TLR7 agonist (e.g., Hiltonol or Imiquimod); a TLR7/8 agonist (e.g., Resiquimod); or a TLR9 agonist (e.g., CpG7909).
  • a TLR2/4 agonist e.g., Bacillus Calmette-Guerin
  • TLR7 agonist e.g., Hiltonol or Imiquimod
  • TLR7/8 agonist e.g., Resiquimod
  • a TLR9 agonist e.g., CpG7909
  • the immuno-oncology agent is a TGF- ⁇ inhibitor, e.g., GC1008, LY2157299, TEW7197 or lMC-TRL
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab, is treated by administration of a HER2 binding agent and a CTLA-4 antagonist agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C- terminal amino acids.
  • the CTLA-4 antagonist may be ipilimumab.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level
  • a HER2 binding agent and a CTLA-4 antagonist agent wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C-terminal amino acids.
  • the CTLA-4 antagonist may be ipilimumab.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that (i) has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab and (ii) has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level, is treated by administration of a HER2 binding agent and a CTLA-4 antagonist agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab, is treated by administration of a HER2 binding agent and an antagonist PD-1 pathway agent (e.g., a PD- 1 , PD-L1 or PD-L2 antagonist), wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level
  • a HER2 binding agent and an antagonist PD-1 pathway agent e.g., a PD-1 , PD-L1 or PD-L2 antagonist
  • the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 4), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C-terminal amino acids.
  • the antagonist PD-1 pathway agent may be nivolumab.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that (i) has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab and (ii) has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level, is treated by administration of a HER2 binding agent and an antagonist PD-1 pathway agent (e.g., a PD-1 , PD-L1 or PD-L2 antagonist), wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab, is treated by administration of a HER2 binding agent and a LAG-3 antagonist agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C- terminal amino acids.
  • the LAG-3 antagonist may be BMS-986016.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level
  • a HER2 binding agent and a LAG-3 antagonist agent wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C-terminal amino acids.
  • the LAG-3 antagonist may be BMS-986016.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that (i) has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab and (ii) has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level, is treated by administration of a HER2 binding agent and a LAG-3 antagonist agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab
  • a HER2 binding agent and a CD137 agonist agent wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C- terminal amino acids.
  • the CD137 agonist may be urelumab.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level, is treated by administration of a HER2 binding agent and a CD137 agonist agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and D RWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C-terminal amino acids.
  • the CD 37 agonist may be urelumab.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that (i) has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab and (ii) has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level, is treated by administration of a HER2 binding agent and a CD137 agonist agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab, is treated by administration of a HER2 binding agent and IDO antagonist agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C- terminal amino acids.
  • the IDO antagonist may be F001287.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level
  • a HER2 binding agent and IDO antagonist agent wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C-terminal amino acids.
  • the IDO antagonist may be F001287.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that (i) has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab and (ii) has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level, is treated by administration of a HER2 binding agent and IDO antagonist agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO:
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab
  • a HER2 binding agent and KIR antagonist agent wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C- terminal amino acids.
  • the KIR antagonist may be lirilumab.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or has a high HER2 mRNA level
  • a HER2 binding agent and KIR antagonist agent wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO: 8 with or without the last 1 or 2 C-terminal amino acids.
  • the KIR antagonist may be lirilumab.
  • a subject having cancer e.g., gastric cancer, colorectal cancer or breast cancer, that (i) has exhibited an inadequate response to trastuzumab and/or pertuzumab or a cancer that is refractory or resistant (intrinsically or acquired) to treatment with trastuzumab and/or pertuzumab and (ii) has an average HER2 gene copy number of greater than or equal to 10 per tumour cell and/or a high HER2 mRNA level, is treated by administration of a HER2 binding agent and KIR antagonist agent, wherein the HER2 binding agent comprises a HER2 antigen binding site engineered into a structural loop region of a CH3 domain, and comprising, e.g., amino acid sequences FFTYW (SEQ ID NO: 12) and DRRRWTA (SEQ ID NO: 14), or a HER2 binding agent that competes therewith for binding to HER2.
  • the HER2 binding agent comprises SEQ ID NO:
  • a composition comprising a specific binding agent according to the present invention may also be administered in combination with an existing therapeutic agent, e.g., abitaterone acetate (Zytiga), afatinib, aflibercept, anastrozole, bevacizumab, bicalutamide, BRAF inhibitors, carboplatin, capecitabine, cetuximab, cisplatin, crizotinib, cyclophosphoamide, docetaxel, pegylated liposomal doxorubicin, enzalutamide (XTANDI), epirubicin, eribulin mesylate, erlotinib, etoposide, everolimus, exemestane, FOLFIRI, FOLFOX, fluoracil, 5-fluorouracil, flutamide, fulvestrant, gefitinib, gemcitabine, goserelin, hedgehog pathway inhibitors, irenotecan, ixabepilone,
  • two therapeutic agents are coformulated.
  • a specific binding member is formulated together with an immuno-oncology agent.
  • the combination of active ingredients form a fixed dose combination.
  • the present invention further provides a method of producing the specific binding memberof the invention, comprising culturing the recombinant host cell of the invention under conditions for production of the specific binding member.
  • the method may further comprise isolating and/or purifying the specific binding member.
  • the method may also comprise formulating the specific binding member into a pharmaceutical composition.
  • a HER2 binding agent e.g., a HER2 Fcab
  • a subject having cancer such as a subject with relapsed or refractory solid tumours that overexpress HER2, at 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.
  • a HER2 binding agent may be administered at 0.1-0.5 mg/kg, 0.1-1 mg/kg, 0.5-1 mg/kg, 1-3 mg/kg, 1-6 mg/kg, 6-10 mg/kg, 2-4 mg/kg, 5-7 mg/kg or 9-10 mg/kg.
  • Subjects may be subjects with locally un-resectable and/or metastatic solid cancers that over express human HER2 by standard clinical pathology criteria.
  • subjects are subjects who have no standard treatment options.
  • subjects have histologically or cytologically confirmed solid tumour malignancy that is unresectable/locally advanced and/or metastatic and for which standard curative or palliative measures are not available or are no longer effective.
  • HER2 status in subjects with breast cancer may follow the 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) criteria (Wolff et al, 2013) as practicable.
  • Assessment of HER2 status in subjects with gastric and gastroesophageal junction adenocarcinoma may follow the criteria published by Ruschoff et al (2012) as practicable.
  • HER2 status in subjects with non-breast/non-gastric cancers may follow local institutional criteria.
  • Subjects with breast and gastric/gastroesophageal junction cancers may have HER2 testing performed using an assay kit/methodology specifically FDA-approved for their cancer type as practicable.
  • the clinical pathology report includes IHC as 3+.
  • subjects with breast cancer have been treated with at least one or at least two FDA-approved anti-HER2 directed therapies, wherein the subjects have refractory or
  • subjects with gastric and gastroesophageal junction cancers have been treated with at least one FDA-approved anti-HER2 directed therapy; wherein the subjects have refractory or relapsed/progressive disease during or following their last prior anti-HER2 directed therapy.
  • subjects who have non-breast, non-gastric, non-gastroesophageal junction cancers did not have any prior treatment with anti-HER2 therapy (e.g., if there is no FDA-approved anti-HER2 agent for their specific cancer type), but had refractory or relapsed/progressive disease during or following their last prior anti-cancer therapy.
  • a subject treated with an a HER2 binding agent has a prior cumulative doxorubicin dose ⁇ 360 mg/m2 and/or a prior cumulative epirubicin dose ⁇ 720 mg/m2.
  • a subject treated with an HER2 binding agent has adequate organ function as defined below:
  • LVEF Left ventricular ejection fraction
  • 2D Echo 2 dimensional echocardiogram
  • MUGA multi-gated acquisition scan
  • LN local institutional lower limit normal
  • subjects that are treated with a HER2 binding agent do not have one or more or all of the following conditions; symptoms or criteria:
  • immunoconjugates include biological therapies (including monoclonal antibodies or other engineered proteins), targeted small molecules (including but not limited to kinase inhibitors), hormonal therapies (except for gonadotropin releasing hormone agonists/antagonists for prostate cancer which may be continued while on study) within 3 weeks of scheduled dosing day 1 ;
  • trastuzumab Receipt of trastuzumab, pertuzumab, or ado-trastuzumab emtansine within 4 weeks of scheduled dosing day 1 ;
  • b Myocardial infarction, unstable angina, coronary artery bypass graft, coronary artery angioplasty or stent placement within 12 months before scheduled dosing day 1 ; c. History of congestive heart failure;
  • HIV human immunodeficiency virus
  • j Non-malignant interstitial lung disease
  • k Dyspnea of any cause requiring supplemental oxygen therapy
  • Hypersensitivity/infusion reaction to monoclonal antibodies, other therapeutic proteins, or allergy to any component/excipient of the HER2 binding agent finished drug product arginine, glycine, phosphoric acid, or polysorbate 80
  • standard therapies such as anti-histamines, 5-HT3 antagonists, or corticosteroids.
  • a subject may receive weekly ( ⁇ 1 day) or less frequent (e.g., once every two weeks or once every 3 weeks) intravenous (IV) infusions of a HER2 binding agent during an initial 21 day (3-week) or 28-day (4-week) treatment cycle (Cycle 1 ).
  • IV intravenous
  • subjects may be assessed for safety, tolerability, dose limiting toxicity, PK, immunogenicity, and clinical disease response.
  • Subjects e.g., subjects without clinical disease progression and/or without unacceptable toxicity, may continue receiving a Her2 binding agent for up to six 21 (Q3W) to 28-day (weekly or Q2W) cycles.
  • Subjects may continue to receive a HER2 binding agent at the same dose they were originally assigned, unless modified downward for earlier toxicity.
  • a subject having cancer such as a cancer having a higher than normal HER2 gene copy number, e.g., a solid tumour that overexpresses HER2, comprising administering to the subject 0.1-10 mg/kg, 0.1-1 mg/kg or 1-10 mg/kg of a HER2 binding agent weekly, every two weeks or every three weeks.
  • the method comprises administering to the patient once every week, once every two weeks, once every three weeks 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or (b) a specific binding member which competes with a specific binding member according to
  • the method optionally comprises administering another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • the present invention provides a method of treating cancer in a patient, wherein said cancer has HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks an amount of 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 4); or
  • the method optionally comprises administering another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • a tumour sample obtained from said patient may have been determined to have a HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell.
  • the present invention provides a method of treating cancer in a patient, wherein a tumour sample obtained from said patient has been determined to have an average HER2 gene copy number of greater than or equal to 10 per tumour cell, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks an amount of 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the method optionally comprises administering another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • the method may comprise:
  • treating said patient with said specific binding member optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, if the HER2 gene copy number, e.g. the average HER2 gene copy number, is greater than or equal to 10 per tumour cell.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent, if the HER2 gene copy number, e.g. the average HER2 gene copy number, is greater than or equal to 10 per tumour cell.
  • the present invention therefore also provides a method of treating cancer in a patient, wherein the method comprises:
  • 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the method optionally comprises administering another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • the present invention provides a method of treating cancer in a patient, wherein said cancer has a high HER2 mRNA level, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks an amount of 0.1-10 mg/kg, 0.1- 0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the method optionally comprises administering another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • Tumour samples obtained from said patient may have been determined to have high HER2 mRNA levels.
  • the present invention provides a method of treating cancer in a patient, wherein a tumour sample obtained from said patient has been determined to have a high HER2 mRNA level, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks an amount of 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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) 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO
  • the method optionally comprises administering another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • the method may comprise:
  • the present invention therefore also provides a method of treating cancer in a patient, wherein the method comprises:
  • the specific binding member is: a) 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 containing the amino acid sequences FFTYW (SEQ ID NO: 12), and DRRRWTA (SEQ ID NO: 14); or
  • the method optionally comprises administering another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • the method comprises administering to the patient once very week, once every two weeks, once every three weeks 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 specifically binds to human HER2 and comprises two polypeptides, wherein each polypeptide comprises a human lgG1 heavy chain fragment comprising a CH2 domain and a CH3 domain, wherein the CH3 domain comprises an AB loop comprising the amino acid sequence of SEQ ID NO: 191 , a CD loop comprising the amino acid sequence of SEQ ID NO: 241 and an EF loop comprising the amino acid sequence 370; and wherein the HER2 binding agent has a molecular weight of up
  • the present invention provides a method of treating cancer in a patient, wherein said cancer has a HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks a pharmaceutical composition comprising 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 specifically binds to human HER2 and comprises two polypeptides, wherein each polypeptide comprises a human lgG1 heavy chain fragment comprising a CH2 domain and a CH3 domain, wherein the CH3 domain comprises an AB loop comprising the amino acid sequence of S
  • a tumour sample obtained from said patient may have been determined to have a HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell.
  • the present invention provides a method of treating cancer in a patient, wherein a tumour sample obtained from said patient has been determined to have an average HER2 gene copy number of greater than or equal to 10 per tumour cell, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks a pharmaceutical composition comprising 0.1 -10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.gO.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 specifically binds to human HER2 and comprises two polypeptides, wherein each polypeptide comprises a human
  • the method may comprise:
  • the present invention therefore also provides a method of treating cancer in a patient, wherein the method comprises:
  • a pharmaceutical composition comprising 0.1 -10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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, if the HER2 gene copy number, e.g.
  • the average gene copy number is greater than or equal to 10 per tumour cell
  • the HER2 binding agent specifically binds to human HER2 and comprises two polypeptides, wherein each polypeptide comprises a human lgG1 heavy chain fragment comprising a CH2 domain and a CH3 domain, wherein the CH3 domain comprises an AB loop comprising the amino acid sequence of SEQ ID NO: 191 , a CD loop comprising the amino acid sequence of SEQ ID NO: 241 and an EF loop comprising the amino acid sequence 370; and wherein the HER2 binding agent has a molecular weight of up to 60 kD, a binding affinity of Kd ⁇ 10 ⁇ 8 M and is cytotoxic, and wherein the method optionally comprises administering another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • the present invention provides a method of treating cancer in a patient, wherein said cancer has a high HER2 mRNA level, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks a pharmaceutical composition comprising 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 specifically binds to human HER2 and comprises two polypeptides, wherein each polypeptide comprises a human lgG1 heavy chain fragment comprising a CH2 domain and a CH3 domain, wherein the CH3 domain comprises an AB loop comprising the amino acid sequence of SEQ ID NO: 191 , a CD loop comprising the amino acid sequence of SEQ ID NO: 191
  • Tumour samples obtained from said patient may have been determined to have high HER2 mRNA levels.
  • the present invention provides a method of treating cancer in a patient, wherein a tumour sample obtained from said patient has been determined to have a high HER2 mRNA level, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks a pharmaceutical composition comprising 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1 -10 mg/kg (e.g., 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 specifically binds to human HER2 and comprises two polypeptides, wherein each polypeptide comprises a human lgG1 heavy chain fragment comprising a CH2 domain and a CH3 domain, wherein the CH3 domain comprises an
  • the method may comprise:
  • the present invention therefore also provides a method of treating cancer in a patient, wherein the method comprises:
  • a pharmaceutical composition comprising 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 , if the HER2 mRNA levels are high, wherein the HER2 binding agent specifically binds to human HER2 and comprises two polypeptides, wherein each polypeptide comprises a human lgG1 heavy chain fragment comprising a CH2 domain and a CH3 domain, wherein the CH3 domain comprises an AB loop comprising the amino acid sequence of SEQ ID NO: 191, a CD loop comprising the amino acid sequence of SEQ ID NO: 241 and an EF loop comprising the amino acid sequence 370;
  • the present invention provides a method of treating cancer in a patient, wherein said cancer has HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks a pharmaceutical composition comprising 0.1 -10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 specifically binds to human HER2 and comprises two polypeptides, each of which comprises an amino acid sequence consisting of SEQ ID NO: 8, or a sequence that differs therefrom in at most 5, 4, 3, 2 or 1 amino acid deletions, additions or substitutions, wherein the HER2 gene copy number
  • a tumour sample obtained from said patient may have been determined to have a HER2 gene copy number, e.g. an average HER2 gene copy number, of greater than or equal to 10 per tumour cell.
  • the present invention provides a method of treating cancer in a patient, wherein a tumour sample obtained from said patient has been determined to have an average HER2 gene copy number of greater than or equal to 10 per tumour cell, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks a pharmaceutical composition comprising 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 specifically binds to human HER2 and comprises two polypeptides, each of which comprises an amino acid sequence consisting
  • the method may comprise:
  • treating said patient with said HER2 binding agent optionally in combination with another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent, if the HER2 gene copy number, e.g. the average HER2 gene copy number, is greater than or equal to 10 per tumour cell.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent, if the HER2 gene copy number, e.g. the average HER2 gene copy number, is greater than or equal to 10 per tumour cell.
  • the present invention therefore also provides a method of treating cancer in a patient, wherein the method comprises:
  • a pharmaceutical composition comprising 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 if the HER2 gene copy number, e.g.
  • the average HER2 gene copy number is greater than or equal to 10 per tumour cell, wherein the HER2 binding agent specifically binds to human HER2 and comprises two polypeptides, each of which comprises an amino acid sequence consisting of SEQ ID NO: 8, or a sequence that differs therefrom in at most 5, 4, 3, 2 or 1 amino acid deletions, additions or substitutions, wherein the HER2 binding agent has a molecular weight of up to 60 kD, a binding affinity of Kd ⁇ 10 8 M and is cytotoxic, and wherein the method optionally comprises administering another therapy, such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • another therapy such as immunotherapy, e.g., therapy with an immuno-oncology agent.
  • the present invention provides a method of treating cancer in a patient, wherein said cancer has a high HER2 mRNA level, and wherein the method comprises administering to the patient once every week, once every two weeks or once every three weeks a pharmaceutical composition comprising 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 specifically binds to human HER2 and comprises two polypeptides, each of which comprises an amino acid sequence consisting of SEQ ID NO: 8, or a sequence that differs therefrom in at most 5, 4, 3, 2 or 1 amino acid deletions, additions or substitutions, wherein the HER2 binding agent has a molecular weight of up to 60 kD, a binding affinity of
  • Tumour samples obtained from said patient may have been determined to have high HER2 mRNA levels.
  • the present invention provides a method of treating cancer in a patient, wherein a tumour sample obtained from said patient has been determined to have a high HER2 mRNA level, and wherein the method comprises administering to patient once every week, once every two weeks or once every three weeks a pharmaceutical composition comprising 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 specifically binds to human HER2 and comprises two polypeptides, each of which comprises an amino acid sequence consisting of SEQ ID NO: 8, or a sequence that differs therefrom in at most 5, 4, 3, 2 or 1 amino acid deletions, additions or
  • the method may comprise:
  • the present invention therefore also provides a method of treating cancer in a patient, wherein the method comprises:
  • a pharmaceutical composition comprising 0.1-10 mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, or 1-10 mg/kg (e.g., 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 if the HER2 imRNA levels are high, wherein the HER2 binding agent specifically binds to human HER2 and comprises two polypeptides, each of which comprises an amino acid sequence consisting of SEQ ID NO: 8, or a sequence that differs therefrom in at most 5, 4, 3, 2 or 1 amino acid deletions, additions or substitutions, wherein the HER2 binding agent has a molecular weight of up to 60 kD, a binding affinity of Kd ⁇ 10 8 M and is cytotoxic, and wherein the method
  • Example 1 - H561-4 binds to a different epitope on HER2 than trastuzumab or pertuzumab
  • SPR Surface Plasmon resonance
  • a BIAcore 3000 (GE healthcare) was used to determine if H561-4 could bind to a human HER2 coated chip that was saturated with trastuzumab (TR) and vice versa.
  • TR trastuzumab
  • a CM5 chip was coated with 1000 RU of extracellular domain (ECD) of human HER2 (BenderMed Systems) using standard amine coupling, experiments were carried out using a flow rate of 20 ⁇ /min in HBS-P buffer (GE
  • the HER2 surface was regenerated using 4 M MgC .
  • the first HER2 binding compound H561-4 or trastuzumab
  • the second HER2 binding compound Trastuzumab or H561-4
  • the second injection was performed in the presence of 10 pg/ml of the first compound in order to eliminate the dissociation of compound 1 during the second injection
  • dissociation in buffer Figure 2A
  • injections 1 and 2 were the same compound (trastuzumab followed by trastuzumab or H561-4 followed by H561-4) little or no binding was observed at the second injection, showing that the HER2 binding surface was saturated in both cases.
  • Table 1 below shows the response (RU) observed with the different combinations of injections:
  • Table 1 shows that the binding response of H561-4 on a HER2 chip saturated with trastuzumab (246 RU) is similar to that of H561-4 binding on a naked HER2 surface (253-254 RU), indicating that H561- 4 does not compete with trastuzumab for binding to HER2.
  • the same result was seen when the injection series was reversed in that the binding response of trastuzumab on a H561-4 saturated surface (746 RU) is similar to that when binding to a naked HER2 surface (771-794 RU).
  • a Streptavidin coated tip (ForteBio) was coated by incubation in 50 g/ml biot-HER2 for 30 mins, the tip was then washed in buffer for 1 min then incubated in 325 nM pertuzumab for 30 mins to saturate all the pertuzumab binding sites then immediately transferred into a mixture of H561-4 (325 nM) and pertuzumab (325 nM) and incubated for 30 mins before dissociation in buffer for 10 mins.
  • Table 2 shows that the binding response of H561-4 on a HER2 chip saturated with pertuzumab (0.41 RU) is significantly greater than the additional binding response of pertuzumab (0.04 RU), indicating that H561-4 does not compete with pertuzumab for binding to HER2.
  • Example 2 Investigating binding of H561 -4 to HER2 Comparison ofH561-4 binding to monomeric versus dimeric HER2
  • H561 -4 was found to preferentially bind to dimeric HER2 compared with monomeric HER2.
  • ELISA based and SPR based methods were used to compare binding of H561 -4 to monomeric HER2 (extracellular domain [ECD] of HER2 without tag) versus dimeric HER2 (ECD of HER2 with Fc tag, R&D biological).
  • ECD extracellular domain
  • HER2 with Fc tag, R&D biological
  • the HER2 was coated onto a plate surface and was therefore immobilised HER2
  • the H561 -4 was captured and the HER2 was injected as the analyte and was therefore soluble HER2.
  • HER2 ECD untagged Monomeric HER2 (HER2 ECD untagged) and dimeric HER2 (HER2 ECD Fc-tag) were immobilised at 2 pg/ml on a 96-well maxisorp plate (Nunc) in PBS overnight at 4°C. The plates were blocked for 1 hour with 200 ⁇ of 5% BSA in PBS.
  • the blocking solution was removed and 100 ⁇ of the dilution series of H561 -4 and trastuzumab were added to the plate and incubated at RT for 1 hour.
  • the dilution series was removed and the plate was washed 3x with phosphate buffered saline with Tween 20 (PBST).
  • a 1 :5000 dilution of Strep-HRP (Abeam ab) was prepared in 5% BSA and 100 ⁇ was added to the plate and incubated for 1 hour at RT. The plate was then washed 3x with PBST before 100 ⁇ of TMB (Roche) were added, and the plate incubated at RT for 2 mins. The reaction was stopped using 100 ⁇ of 2 M H2SO4 and the plate was read at 450 nM.
  • the trastuzumab control had the same half maximal effective concentration (EC50) (1.3 - 1 .5 nM) on monomeric and dimeric HER2, whereas H561 -4 had a 10-fold preference for the dimeric HER2 compared with monomeric HER2, as shown by the EC50 values of 1 .7 nM and 20.6 nM, respectively.
  • EC50 refers to the concentration of the binding member which induces a response halfway between the baseline and maximum after a specified exposure time.
  • an anti- EGFR-H561 -4 mAb 2 (EGFR/H561 -4) was used.
  • This molecule is an anti-EGFR antibody with the Fc region modified to contain the binding loops of H561 -4 in the CH3 domain.
  • This EGFR HER2 bispecifc molecule was then captured on a EGFR coated BIAcore chip via the EGFR binding specificity, which is in the Fab arms, thereby leaving the HER2 binding CH3 domain exposed to the analyte.
  • trastuzumab-EGFR antigen-binding Fc fragment (TR/EGFR).
  • This bispecific comprises the HER2 mAb trastuzumab with the Fc region modified to contain the binding loops 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).
  • the TR EGFR was captured on the EGFR chip via the EGFR antigen-binding Fc fragment Fc region thereby leaving the HER2 binding specificity in the Fab arms exposed to the analyte.
  • a BIAcore streptavidin chip was immobilised with 1000 RU of biot-EGFR. Experiments were carried out using a flow rate of 20 ⁇ /min in HBS-P buffer (GE Healthcare). The EGFR/HER2 bispecific molecules were captured by injecting 60 ⁇ of 100 mM of the mAb 2 , and the EGFR surface was regenerated using 50 mM NaOH. Dilution series of monomeric (untagged HER2 ECD, in house) and dimeric (Fc-tagged HER2 ECD, R&D) were injected as the analyte. The data were fit using the Langmuir 1 :1 binding model.
  • Table 3 below shows the KD values obtained by the 1 :1 fits of the data.
  • H561 -4 shows only weak binding to soluble monomeric HER2 even at concentrations up to 1000 nM, whereas the affinity to dimeric HER2 is 1 nM.
  • Trastuzumab binds to monomeric HER2 with an affinity of 1 nM and to dimeric HER2 with a sub-nM affinity.
  • H561 -4 was determined to bind across two domains of extracellular HER2. These domains were domains 1 and 3 of HER2, with binding epitopes mapped to amino acids 13 through to 27 (LPASPETHLDMLRHL) and amino acids 31 through to 45
  • the linear and CLIPS (Chemical Linkage of Peptides onto Scaffolds) peptides were synthesized based on the amino acid sequence of extracellular HER2 using standard FMOC-chemistry and de- protected using trifluoric acid with scavengers.
  • the constrained peptides were synthesized on chemical scaffolds in order to reconstruct conformational epitopes, using CLIPS technology
  • the peptides were incubated with a 1 :1000 dilution of antibody peroxidase conjugate for one hour at 25° C. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 microlitres of 3 percent 1 1202 were added. After one hour, the colour development was measured. The colour development was quantified with a charge coupled device (CCD)— camera and an image processing system (as firstly described in Slootstra et al., 1996, Structural aspects of antibody-antigen interaction revealed through small random peptide libraries, Molecular Diversity, 1 , 87-96).
  • CCD charge coupled device
  • the raw data were therefore optical values obtained by a CCD-camera.
  • the values mostly range from 0 to 3000, a log scale similar to 1 to 3 of a standard 96-well plate ELISA-reader. In this experiment, the values obtained ranged from 0-380.
  • the CCD-camera first took a picture of the card before peroxidase colouring and then another picture after peroxidase colouring. These two pictures were subtracted from each other, which resulted in the raw-data. This was copied into the Peplabtm database. Then the values were copied to excel and this file was labelled as a raw-data file. One follow-up manipulation was allowed. Sometimes a well contains an air-bubble resulting in a false-positive value, the cards were manually inspected and any values caused by an air-bubble were scored as 0. Positive values were taken as a binding signal (the epitope map) and mapped to the sequence of HER2. Computer generated figures of the extracellular HER2 protein were generated and the positive binding signals mapped, indicating the areas of antigen-binding Fc fragment binding (Figure 2C).
  • Example 3 - H561 -4 induces apoptosis and leads to unique and profound HER2 internalization and degradation
  • the HER2 overexpressing breast cancer cell line SKBr3 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 was cultured in McCoy's 5a + GlutaMAX Medium (Invitrogen) containing 10% fetal bovine serum (FBS). Trastuzumab (Roche) was used as a control in all mechanism of action studies.
  • H561-4 has anti-proliferative activity in SKBr3 cells and induces apoptosis
  • SKBr3 cells were seeded at 7.5 x 10 3 cells per well in 96 well tissue culture plates and incubated overnight at 37°C, 5% CO2 with 100 ⁇ per well. The next day 20 ⁇ media containing the treatments (H561 -4 or trastuzumab) were added to give a concentration dilution series of H561-4 or trastuzumab. The cells were incubated with the treatments for 5 days at 37°C, 5% CC .
  • the cells were then stained with a mixture of 1 pg/ml final Hoechst stain (Sigma Aldrich, H6024), 2.5 pg/ml Propidium Iodide (PI) and 1 :100 Alexa-488 Annexin V (Invitrogen, V13245) in Annexin V Binding Buffer (12.5 mM CaCI 2 , 140 mM NaCI, 10 mM Hepes, pH 7.4) and incubated at room temperature (RT) for 2 hours. The cells were then read on the ImageXpress Micro (Molecular Devices). The cell numbers in each well were counted using the Hoechst staining at 4x magnification.
  • Annexin V and PI staining was used to determine the percentage of viable cells (cells not stained with PI), early apoptotic cells (cells stained with Annexin V only), late apoptotic cells (cells stained with Annexin V and PI) and necrotic cells (cells stained with PI only) with a 40x magnification.
  • H561-4 caused a 37% reduction in cell number compared to a 22% reduction by trastuzumab.
  • trastuzumab the remaining H561-4 treated cells only 43% were viable compared with 79% for trastuzumab. This reduction in total cells and viable cells was associated with an increase in late apoptotic and necrotic cells (18% and 35%, respectively) for the H561-4 treated cells only.
  • the results of this experiment are shown in Figure 3A.
  • H561-4 causes internalisation and degradation ofHER2
  • SKBr3 cells were seeded at 3.3 x 10 5 cells/ml with 3 ml/well in a 6 well plate and incubated overnight at 37°C, 5% CO2. The next day the media were changed to media containing 0.1 % FBS and incubated for at least 2 hours at 37°C, 5% CO2 before the media were removed and replaced with media containing 0.1 % FBS and the treatments (H561-4 or trastuzumab) at 200 nM. The cells were incubated at 37°C, 5% CO2 for 24 h, 48 h or 72 h.
  • the lysates were then collected on ice: the media were removed and 250 ⁇ of the lysis buffer (10mM Tris pH7.5, 150mM NaCI, 1 mM EDTA, 1 :100 protease cocktail [Calbiochem, 539131], 1 :100 phosphatase cocktails [Calbiochem, 524625]), was added.
  • the cells were then scraped from the well surface and 250 ⁇ were incubated on ice for 15 mins, centrifuged at 14,000 rpm for 15 min at 4°C and the supernatant was collected for analysis.
  • the samples were mixed with NuPAGE loading buffer containing ⁇ -mercaptoethanol, incubated at 95°C for 10 mins then stored at -20°C until use. 20 ⁇ of the samples were run on a 4-12 % Bis-Tris SDS PAGE and transferred to a nitrocellulose membrane, the membrane was blocked with 5 %
  • H561-4 treatment caused a reduction in total HER2 at 24 h, with a complete reduction at 48 h and 72 h (Figure 3B).
  • trastuzumab treatment did not detectably reduce HER2 levels at the time points studied ( Figure 3B).
  • H561-4 treatment also caused a concomitant reduction in pHER2. This reduction was evident at 24 h, with a further reduction at 48 h and no detectable pHER2 remaining at 72 h.
  • Trastuzumab treatment only caused a slight reduction in pHER2 at the 72 h time point (Figure 3B).
  • Immunofluorescence imaging was then used to quantify the degradation of HER2.
  • S Br3 cells were seeded in a 96-well plate at 1x10 4 cells/well and incubated overnight at 37°C, 5% CO2. The next day, treatments (H561-4; trastuzumab [TR]) or the controls, (wild-type [WT] antigen-binding Fc fragment; or lgG1 isotype control, Sigma 15154 [lgG/lgG1 ctrl]) were added to the media to give a final concentration of 200 nM.
  • the cells were incubated for 24 h, 48 h or 72 h at 37°C, 5% C02, the supernatant removed, and the cells washed with PBS before being fixed with 4% formaldehyde in PBS for 20 mins.
  • half of the cells were then permeabilised in 0.1 % TritonX100/Glucose 0.1 mg/ml in PBS for total protein imaging.
  • the cells were then washed and blocked in 5% FBS/PBS for 1 hour at RT, before being incubated for 1 hour at RT with the primary antibody diluted :100 in PBS (Mouse Anti- Her2 MGR2, Enzo Life Sciences).
  • the cells were then washed twice and before being incubated for 1 h at RT in the dark with the 5 g/ml of secondary antibody (anti-mouse alexa647, Invitrogen A21237) and 1 pg/ml of Hoechst (Invitrogen, 33342).
  • the cells were then washed and stored in 0.05 % NaN3 in PBS until they were read on the ImageXpress Micro (Molecular Devices).
  • the analysis was performed with 40x imaging, the multiwave scoring function was used to count the cell nuclei and the number of cells with HER2 positive staining, and then the per cent of HER2 positive cells was calculated.
  • H561-4 treatment of SKBr3 cells at 200 nM resulted in a decrease in both cell surface and total HER2.
  • the reduction in HER2 levels was detectable after 24 hours treatment in increased at increased time points (48 hours and 72 hours) ( Figure 3C).
  • H561-4 treatment causes caspase-dependent apoptosis
  • SKBr3 cells were seeded at 7.5 x 10 4 cells/ml in a 96 well plate with 100 ⁇ per well and incubated overnight at 37°C and 5% CO2.
  • the next day treatments (H561-4, trastuzumab, wild-type (WT) antigen-binding Fc fragment or lgG1 isotype control) were added to the wells in media to give a final concentrations of 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM and 0.01 nM, plus untreated controls.
  • the cells were incubated for 5 days at 37°C and 5% CO2 before the caspase 3/7 activity was measured using the Promega Caspase-Glo 3/7 assay. 50 ⁇ of Caspase 3/7 Glo reagent was added into each well, mixed and then the plate was incubated at RT for 2 hours before luminescence was measured using the Synergy 4 microplate reader (BIOTEK).
  • Example 4 In vivo efficacy studies: H561 -4 treatment of human patient derived tumour xenograft (PDX) models with HER2 gene copy numbers greater than or equal to 10
  • mice bearing human patient derived xenograft tumours were evaluated using mice bearing human patient derived xenograft tumours. Immunodeficient mice were used enabling the xenotransplantation and growth of human tumours. Mice were implanted with tumours at approximately 5-7 weeks old apart from the GA0060 PDX model where mice were implanted at approximately 6-8 weeks. The GA0060 work was carried out by Crown Bioscience which uses a slightly different experimental procedure to the clinical research
  • tumour xenograft used as a test tumour in this study was derived from a surgical specimen from a patient which was directly implanted subcutaneously into nude mice, hence the designation "patient derived tumour xenograft" (PDX). Tumour fragments were obtained from xenografts in serial passage in nude mice.
  • PDX patient derived tumour xenograft
  • tumours were cut into fragments (2-5 mm diameter or 2-4 mm in diameter for GA0060) and then used for unilateral subcutaneous implantation in to the flank of mice.
  • tumour-bearing animals were stratified into the various groups according to tumour volume. Only mice carrying a tumour of appropriate size (50 mm 3 to 250 mm 3 , or 100 mm 3 to 300 mm 3 for GA0060) were considered for randomization.
  • mice were randomized when the required number of mice qualified for randomization. The day of randomization was designated as day 0. The first day of dosing was also day 0. Animals were sacrificed if their tumour exceeded a volume of 2000 mm 3 . If an animal was sacrificed due to tumour load, then the last observation carried forward (LOCF) was used for subsequent tumour volume values.
  • LOCF last observation carried forward
  • the absolute tumour volume (ATV) was determined by two-dimensional measurement with a caliper one day after tumour implantation and then twice weekly (i. e. on the same days on which mice were weighed).
  • Tumour volumes were calculated according to the formula: (a x b 2 ) x 0.5, where a represents the largest and b the perpendicular tumour diameter.
  • Tumour inhibition for a particular day was calculated from the ratio of the mean absolute tumour volume values (i.e mean growth of tumours) of test versus control groups multiplied by 100%.
  • Day x denotes any day where the minimum (optimal) T/C was observed. Day 0 was the first day of dosing.
  • T/C% For all PDX models apart from the GA0060 PDX model, the minimum (or optimum) T/C% value recorded for a particular test group during an experiment represents the maximum anti-tumour efficacy for the respective treatment. T/C values were calculated if at least 50% of the randomized animals in a group were alive on the day in question.
  • the dosing regimen was twice weekly depending on the study.
  • the absolute tumour volume (ATV) was determined by two-dimensional measurement with a caliper one day after tumour implantation and then twice weekly. Tumour volumes were calculated according to the formula: (a x b 2 ) x 0.5 where a represents the largest and b the perpendicular tumour diameter. Tumour inhibition for a particular day (T/C in %) was calculated from the ratio of the mean absolute tumour volume values of test versus control groups multiplied by 100%. The difference between the two T/C equations is explained in Example 7.
  • GXF281 was isolated from a primary adenocarcinoma of the stomach from a 46 year old male patient and was studied in NMRI nude mice by Oncotest (Freiburg, Germany).
  • H561 -4 (10 mg/kg), trastuzumab (10 mg/kg) and PBS as a vehicle control (10ml/kg) were injected once a week for 5 weeks. Each group contained 6 mice.
  • the tumour volume was measured twice a week for up to 81 days and the mean absolute tumour volume was plotted (Figure 4A). Five weekly treatments with H561-4 resulted in complete tumour regression (minimum T/C value of less than 0 %), whereas five weekly treatments with trastuzumab resulted in only moderate efficacy (minimum T/C of 30 %).
  • GXA3039 was isolated from a primary adenocarcinoma of the stomach from a 65 year old male patient and was studied in NMRI nude mice by Oncotest (Freiburg, Germany). H561-4 (10 mg/kg), trastuzumab (10 mg/kg) and PBS as a vehicle control (10ml/kg) were injected once a week for 4 weeks. Each group contained 5 mice. The tumour volume was measured twice a week for up to 91 days and the mean absolute tumour volume was plotted (Figure 4B).
  • CXF1991 was isolated from a primary adenocarcinoma of the colon from a 59 year old female patient and was studied in NMRI nude mice by Oncotest (Freiburg, Germany).
  • H561-4 (10 mg/kg), trastuzumab (10 mg/kg) and PBS as a vehicle control (10ml/kg) were injected once a week for 6 weeks.
  • Each group contained 5 mice.
  • the tumour volume was measured twice a week for up to 62 days and the mean absolute tumour volume was plotted (Figure 4C).
  • CXF2102 was isolated from a liver metastasis of an adenocarcinoma of the colon from patient and was studied in NMRI nude mice by Oncotest (Freiburg, Germany), the age and gender of this patient are not known.
  • H561-4 (10 mg/kg), trastuzumab (10 mg/kg) and PBS as a vehicle control (10ml/kg) were injected once a week for 4 weeks. Each group contained 5 mice. The tumour volume was measured twice a week for up to 58 days and the mean absolute tumour volume was plotted (Figure 4D).
  • GXA3054 was isolated from a primary tumour of an adenocarcinoma of the stomach from 65 year old male patient and was studied in NMRI nude mice by Oncotest (Freiburg, Germany). H561-4 (10 mg/kg), and PBS as a vehicle control (10ml/kg) were injected once a week for 5 weeks. There was a break in dosing for 4 weeks, then weekly dosing was resumed for 4 weeks. Each group contained 5 mice, a trastuzumab only group was not included in this model. The tumour volume was measured twice a week for up to 100 days and the mean absolute tumour volume was plotted (Figure 4E). The weekly treatments with H561-4 resulted in tumour regression (minimum T/C value of 17 %).
  • GXA3038 was isolated from a primary tumour of an adenocarcinoma of the stomach from 63 year old male patient and was studied in NMRI nude mice by Oncotest (Freiburg, Germany). H561-4 (10 mg/kg), and PBS as a vehicle control (10ml/kg) were injected once a week for 5 weeks. There was a break in dosing for 1 week then weekly dosing was resumed for 4 weeks. Each group contained 5 mice, a trastuzumab only group was not included in this model. The tumour volume was measured twice a week for up to 77 days and the mean absolute tumour volume was plotted (Figure 4F). The weekly treatments with H561-4 resulted in minor tumour regression (minimum T/C value of 55 %).
  • HBCx-13B was isolated from a lymphoid metastasis carcinoma and was studied in athymic nude mice (Hsd;Athymic Nude-Fox1nu) by Xentech (Evry, France).
  • H561-4 (10 mg/kg), trastuzumab (10 mg/kg) and PBS as a vehicle control (10ml/kg) were injected twice a week for 8 weeks. Each group contained 9 mice.
  • the tumour volume was measured twice a week for up to 56 days and the mean absolute tumour volume was plotted (Figure 4G). Twice weekly treatments with H561-4 resulted in significant tumour regression (minimum T/C value of 7 %), whereas six weekly treatments with trastuzumab resulted in only minor efficacy (minimum T/C of 48 %).
  • GA0060 was isolated from human gastric cancer and was studied in BALB/c nude mice by Crown Bioscience (Beijing, China). H561-4 (30 and 60 mg/kg), trastuzumab (30 mg/kg) and PBS as a vehicle control (10ml/kg) were injected twice a week for 6 weeks each group contained 10 mice. The tumour volume was measured twice a week for up to 41 days and the mean absolute tumour volume was plotted (Figure 4H). The T/C values were calculated using the updated T/C equation. Six biweekly treatments with H561-4 resulted in minor tumour regression (T/C value of 49% and 60%, at 60 and 30 mg/kg respectively), whereas six bi-weekly treatment with trastuzumab resulted in tumour regression (T/C value of 25%). 60 mg/kg trastuzumab was not tested because it already caused tumour regression at 30 mg/kg.
  • Example 5 - H561-4 shows efficacy in a trastuzumab and pertuzumab resistance model
  • H561-4 has an anti-tumour effect in patient derived xenograft (PDX) models with HER2 gene copy numbers greater than or equal to 10. It was also observed the H561-4 has a significantly superior anti-tumour effect to trastuzumab in these PDX models.
  • PDX patient derived xenograft
  • trastuzumab is often used in combination with pertuzumab in the clinic to treat breast and gastric cancer.
  • GXF281 gastric PDX model GXF281
  • trastuzumab and pertuzumab combination treatment resulted in a reduction in tumour growth.
  • the tumour eventually progressed.
  • H561-4 treatment of tumours that had progressed on trastuzumab and pertuzumab combination treatment resulted in complete tumour regression.
  • tumour fragments from the patient derived tumour GXF281 were implanted subcutaneously in anesthetised NMRI nu/nu mice. Mice with suitable tumour growth (50 mm 3 -250 mm 3 tumours) were randomised into 3 groups as follows:
  • mice received 4 weekly i.v. injections of PBS at 10 ml/kg.
  • Group 2 20 mice received 4 weekly i.v. injections of trastuzumab at 10 mg/kg and pertuzumab at 10 mg/kg.
  • mice received 4 weekly i.v. injections of H561-4 at 10 mg/kg
  • mice from group 2 were randomised into 4 groups as follows:
  • Group 5 5 mice received 7 weekly i.v. injections of trastuzumab at 10 mg/kg and pertuzumab at 10 mg/kg.
  • mice received 7 weekly i.v. injections of H561-4 at 10 mg/kg
  • mice received 7 weekly i.v. injections of H561-4 at 3.6 mg/kg
  • tumour volumes were monitored twice a week for 105 days (Figure 5).
  • Group 3 showed complete tumour regression with no mice having a detectable tumour after day 46 of the study and no regrowth occurred up to the end of the study (day 105).
  • Group 2 had slower tumour growth than the control group (group 1 ).
  • Repeated dosing of mice with trastuzumab and pertuzumab did not slow tumour growth further (group 4 and group 5 had similar tumour growth).
  • Example 6 - H561 -4 selectively inhibits tumour growth in PDX models with HER2 gene copy numbers greater than or equal to 10
  • H561 -4 The in vivo activity of H561 -4 was studied in 23 different patient derived xenograft (PDX) models, all of which were classified as HER2 positive by IHC according to the criteria used by Oncotest.
  • PDX patient derived xenograft
  • the T/C value was calculated using the following equation:
  • Day x denotes any day where the minimum (optimal) T/C was observed. Day 0 was the first day of dosing.
  • Table 4 shows the efficacy criteria as defined by the minimum T/C values (in %):
  • DNA from each of the tumours was provided at an initial concentration of 100ng/pl and subsequently diluted to Sng/ ⁇ in molecular grade water (SIGMA #W4502-1 L).
  • a master mix for the PCR reaction was created consisting of 2x Taqman Genotyping Master Mix (Invitrogen, #371355), 1 ⁇ HER2 taqman probe and primers set (Invitrogen, Gene Assay ID: Hs00817646 Cat No:4400291 ) and 1 ⁇ housekeeping RNAseP taqman probe and primers set (Invitrogen, #4403326). 20ng of sample DNA was added in dH20 resulting in a final reaction volume of 20ul.
  • the quantitative PCR conditions were as follows: Hold at 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and then 60°C for 60 seconds. After acquisition of raw CT (Cycle Threshold) data, a manual CT of 0.2 and an auto- baseline were applied to the results. All data obtained were normalized by a housekeeping gene (RNAseP taqman probe and primers set, Invitrogen, #4403326) of which it was known that there were 2 copies per cell. The results of the quantitative PCR were expressed in arbitrary units (AU, related to the copy number per cell). First, for each sample the difference between the CT value (Cycle Threshold) of a housekeeping gene and the HER2 gene was calculated, as follows:
  • Target gene expression 2 (Cy housekeeping gene (RNAse P)- C T HER2 gene)
  • Results were analyzed for linear amplification and exported to Copy CallerTM Software version 2.0 (Invitrogen) for copy number determination of the HER2 gene.
  • a DNA reference sample (Roche, #11691112001 ) found to have 2 copies of HER2 (CT values of HER2 gene and housekeeping gene were equal, indicating 2 copies per cell of HER2) was included in the assay to enable exact copy number determination and to allow for assay running adjustment. 2 AU was considered to be the normal value for HER2 gene in the reference genomic DNA. Where the copy number obtained for the human standard genomic DNA was not exactly 2 AU, a multiplier was applied to convert it to 2 AU. This multiplier was also applied to the results obtained for the tumour samples. The gene copy number for each sample is listed in Table 5.
  • Table 5 shows the efficacy as defined by the minimum T/C values (in %) of H561-4 and trastuzumab treated PDX models as well as gene copy number (GCN) in each of the PDX tumour samples tested:
  • H561-4 had efficacy in 6 out of 7 PDX models with a HER2 gene copy number (GCN) greater than or equal to 10, whereas there was only borderline activity in 2 of the PDX models with a HER2 GCN of less than 10.
  • Trastuzumab had moderate activity in 2 PDX models with a GCN greater than or equal to 10 and borderline activity in another.
  • trastuzumab had borderline activity in 3 PDX models and moderate activity in another 3 models.
  • Figure 6 shows a scatter plot of the T/C values of H561-4 and trastuzumab treatment in PDX models with a HER2 GCN greater than or equal to 10, compared with PDX models with GCN of less than 10.
  • the scatter plot shows that H561-4 has statistically greater efficacy in cancers with GCN greater than or equal to 10 compared with cancers with GCN of less than 10.
  • Trastuzumab activity is independent of HER2 gene copy number value. Determination ofmRNA levels in the PDX tumour models by Reverse-Transcription (RT) PCR followed by quantitative PCR: mRNA from each of the tumours was provided at a concentration of 250 ⁇ / ⁇ .
  • RT Reverse-Transcription
  • mRNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Invitrogen, #N8080234) as per the manufacturer's instructions. Transcribed cDNA was then quantified using a Nanodrop 2000 (Thermo Scientific) to determine cDNA concentration in ng/ ⁇ . The cDNA obtained was diluted to 25ng/pl in molecular grade water (SIGMA #W4502-1 L).
  • a master mix for the quantitative PCR reaction was created consisting of 2x Taqman Gene Expression Master Mix (Invitrogen, # 4369016), 1.25 ⁇ HER2 taqman probe and primers set (Invitrogen, Hs01001580_m1 #4331182) and 1.25 ⁇ housekeeping human TBP taqman probe and primers set (Invitrogen, Hs00427620_m1 #4331182). 50ng of sample cDNA was added along with dhbO to produce a final reaction volume of 25 ⁇ .
  • the quantitative PCR conditions were as follows: Hold at 95°C for 10 minutes followed by 50 cycles of 95°C for 15 seconds and then 61 °C for 30 seconds. After acquisition of raw CT (Cycle Threshold) data, a manual CT of 0.2 and an auto-baseline were applied to the results.
  • a "human standard cDNA” reverse-transcribed from a "universal human reference RNA” (Agilent technologies, #740000) made by pooling 10 human cell lines, which are expected to have two copies of the HER2 gene per cell (as the cell lines are considered to have a normal profile) was used as a reference at the same concentration as the tumour sample cDNA.
  • This human standard cDNA is considered to have a normal mRNA profile.
  • HER2 ex ression 2 (Cy housekeeping gene(TBP) - C-. HER2 gene)
  • Results were analyzed for linear amplification and exported to Copy CallerTM Software version 2.0 (Invitrogen). This software was used to determine the cDNA copy number of HER2 relative to the cDNA copy number of the housekeeping gene in the tumour samples, normalised to the HER2 cDNA copy number in the human standard cDNA sample.
  • HER2 expression in the tumour sample x constant cDNA copy number
  • Table 6 shows the efficacy as defined by the minimum T/C values (in %) of H561 -4 and trastuzumab treated PDX models as well as the HER2 mRNA levels for each of the PDX tumour samples.
  • PAXF2005 H561-4 had efficacy in 6 out of 7 PDX models with a HER2 mRNA level >200 in the tumour, and efficacy in all PDX models with a HER2 mRNA level ⁇ 200 and ⁇ 820 in the tumour, whereas there was only borderline activity in 2 of the PDX models with a HER2 m NA level ⁇ 200 in the tumour.
  • the HER2 mRNA level in those models which had mRNA levels ⁇ 820 was 168, whereas for all models tested the HER2 mRNA level was 248.
  • Trastuzumab had moderate activity in 2 PDX models with a HER2 mRNA level >200 and borderline activity in another.
  • FIG. 7 shows a scatter plot of the T/C values of H561-4 and trastuzumab treatment in PDX models with a HER2 mRNA level greater than or equal to 200 in the tumour, compared with PDX models with an mRNA level of less than 200 in the tumour.
  • H561-4 has statistically greater efficacy in cancers with a HER2 mRNA levels ⁇ 200, compared with cancers with a HER2 mRNA level ⁇ 200 .
  • the data show that H561-4 has statistically greater efficacy in cancers with HER2 mRNA levels >200 and ⁇ 820, compared with PDX models with a HER2 mRNA levels ⁇ 200 and >820.
  • Trastuzumab activity is independent of the HER2 mRNA level in the range tested.
  • Example 7 - H561-4 selectively inhibits tumour growth in PDX models with HER2 gene copy numbers greater than or equal to 10 using an updated measure of T/C values.
  • T/C values were calculated using the following equation: where x is approximately one dose period after the final dose if at least 50% of the animals in a group were alive.
  • this equation is referred to as the updated T/C equation.
  • This updated equation was designed to facilitate the comparison of data collected from different clinical research institutions.
  • Day x denoted any day where the minimum (optimal) T/C observed. Therefore x could be any day over the entire period of each experiment.
  • Day x becomes a fixed time point of approximately one dose period after the final dose (if at least 50% of the animals in a group were alive). Therefore the updated T/C formula has the advantage of standardising Day x, which allows us to compare the effect of treatments from different studies.
  • the basal subtraction step was not included in the updated T/C equation, this was compensated by the ranked distribution of inoculated mice evenly to obtain equal mean tumour sizes across different test groups. Values from mice removed due to tumour load were considered beyond the day of sacrifice using the Last Observation Carried Forward methodology if this increased the group mean.
  • T/C efficacy values were determined as shown in Table 4.
  • Table 7 shows the efficacy as defined by the T/C values (%)(using the updated T/C formula) of H561- 4 and trastuzumab treated PDX models.
  • H561 -4 had efficacy in 7 out of 8 PDX models with HER2 gene copy number of greater than 10, 6 of which had high activity, whereas there was no activity in all of the PDX models with HER2 GCN less than 10.
  • Trastuzumab had moderate activity in 3 PDX models with GCN greater than 10 and borderline activity in another 3, but none of them showed high activity.
  • trastuzumab had borderline activity in 1 model.
  • Figure 8 shows a scatter plot of the T/C values of H561-4 and trastuzumab treatment in PDX models with HER2 GCN greater than 10 compared to PDX models with GCN less than 10.
  • the scatter plot shows that H561-4 has statistically greater efficacy in PDX models with GCN greater than 10 compared to PDX models with GCN less than 10.
  • the mean activities of trastuzumab in PDX models with GCN greater than or less than 10 are both higher the 50%, supporting the notion that HER2 gene copy number value are not predictive to the trastuzumab efficacy.
  • Table 8 shows the efficacy as defined by the T/C values (%) (using the updated T/C formula) of H561- 4 and trastuzumab treated PDX models.
  • HER2 mRNA TIC (Treatment/Control tumour volume number %)
  • H561 -4 had efficacy in 7 out of 8 PDX models with a HER2 mRNA level > 200 in the tumour, and efficacy in all PDX models with a HER2 mRNA level > 200 and ⁇ 820 in the tumour, whereas there was no activity in all of the PDX models with a HER2 mRNA level ⁇ 200 in the tumour.
  • Trastuzumab had moderate activity in 3 PDX models with a HER2 mRNA level ⁇ 200 and borderline activity in another 3, but none of them showed high activity. In the PDX models with an mRNA level ⁇ 200, trastuzumab had borderline activity in 1 model.
  • Figure 9 shows a scatter plot of the T/C values of H561 -4 and trastuzumab treatment in PDX models with a HER2 mRNA level greater than or equal to 200 in the tumour, compared with PDX models with an mRNA level of less than 200 in the tumour.
  • the scatter plot shows that H561-4 has statistically greater efficacy in PDX models with a HER2 mRNA level > 200 and ⁇ 820 compared to PDX models with a HER2 mRNA levels ⁇ 200 and > 820.
  • trastuzumab in PDX models in the groups of HER2 mRNA ⁇ 200 and the group ⁇ 200 are both higher than 50%, supporting the notion that HER2 mRNA levels are not predictive to the trastuzumab efficacy.
  • Example 8 - H561 -4 selectively inhibits PDX models with HER2 gene copy numbers of greater than 18 where the gene copy number is determined by FISH
  • the HER2 gene copy numbers determined by FISH in relation to the in vivo activity of H561 -4 was studied in 17 different patient derived xenograft (PDX) models, including breast, gastric and colon models.
  • PDX patient derived xenograft
  • the efficacy of the H561 -4 treatment and trastuzumab treatment was assessed based on the T/C value using the updated T/C equation:
  • FFPE paraffin-embedded
  • the slides were heated to 73 °C for 5 minutes for DNA denaturation, followed by probe hybridisation at 37 °C for 14-18 hours. Unspecific hybridization events were washed away in the post-hybridization buffer at 72 °C for 2 minutes. The slides were then incubated with DAPI counterstain for 15 minutes in the dark until viewing using fluorescence microscopy for signal enumeration. Enumeration was evaluated based on 20-60 tumour cells per sample and an average of the HER2 gene copy number was taken. The HER2 gene copy number determined by FISH for each sample is listed in Table 9. Table 9 shows the efficacy as defined by the T/C values (in %) of H561 -4 and trastuzumab treated PDX models as well as gene copy number (GCN) in each PDX tumour tested:
  • H561 -4 had efficacy in 7 out of 8 PDX models with HER2 gene copy number of greater than 18 as determined by FISH, 6 of which had high activity, whereas there was no activity in all of the PDX models with HER2 GCN less than 18.
  • Trastuzumab had moderate activity in 3 PDX models with GCN greater than 18 and borderline activity in another 3, but none of them showed high activity.
  • trastuzumab had borderline activity in 1 model.
  • Figure 10 shows a scatter plot of the T/C values of H561 -4 and trastuzumab treatment in PDX models with HER2 GCN greater than 18 compared to PDX models with GCN less than 18.
  • the scatter plot shows that H561-4 has statistically greater efficacy in PDX models with GCN greater than 18 compared to PDX models with GCN less than 18.
  • Trastuzumab activity is independent of HER2 gene copy number value.
  • the mean activities of trastuzumab in PDX models in the group with GCN greater than 18 or in the group with less than 18 are both higher than 50%, supporting the notion that HER2 gene copy number value are not predictive to the trastuzumab efficacy.
  • the HER2 gene copy numbers were determined by qPCR and FISH.
  • HER2 GCN in PDX tumours determined by the qPCR varied from 1 to 76 copies, while GCN in these tumours as determined by FISH varied from 2 to 40 copies.
  • Tumours with high HER2 gene amplification determined by either method are statistically more likely to respond to H561 -4 treatment.
  • the relationship of the GCN determined by the two methods was assessed in 17 PDX tumours where data collected by both methods are available.
  • the HER2 gene copy numbers determined by both methods are listed in Table 10.
  • Example 10 Investigating variability of HER2 IHC scoring determined by different protocols.
  • Immunohistochemicai (IHC) assay is a commonly used method for evaluating HER2 protein expression in tissue samples by antibodies that bind specifically to the antigen.
  • IHC immunohistochemicai
  • the semi-quantitative nature of IHC means that it can be inexact and does not always provide an accurate reading.
  • HER2 antibodies and scoring systems are in use, variability in HER2 IHC results between providers of IHC assays could be a concern.
  • HercepTest has been approved by the FDA as a standardised IHC assay for determination of HER2 protein overexpression in breast and gastric cancers, it has been reported in some studies that most HercepTest HER2 positive samples lacked HER2 gene amplification as determined by FISH assay (Jacobs et al., 1999). On the other hand, breast cancers may exhibit HER2 protein overexpression in the absence of gene amplification, however this phenomenon previously was observed only in 7% of cases (Persons et al., 997). These discrepancies contradict the current clinical practices to deem the HercepTest 3+ positive population to be HER2 amplified.
  • IHC assays including HercepTest and the immunohistochemicai assay developed by Oncotest were used to identify overexpression of HER2 protein in 22 formalin-fixed, paraffin-embedded PDX tumours.
  • the aim was to investigate the reliability of IHC as a H561 -4 biomarker.
  • HercepTest IHC assay the manufacture protocol of HercepTest was used. Briefly, the formalin-fixed, paraffin-embedded cancer tissues were deparaffinised in xylene three times for 5 minutes, followed by rehydration in industrial methylated spirits (IMS) two times for 5 minutes. The slides were then incubated in Epitope Retrieval Solution for 40 minutes at 97 °C and socked in wash buffer for 20 minutes prior to staining. The slides were incubated with the primary rabbit anti-human HER2 antibody for 30 minutes followed by incubation with secondary goat anti-rabbit antibody linked to horseradish peroxidase for 30 minutes. DAB chromogen was applied for enzymatic conversion for signal visualisation.
  • IMS industrial methylated spirits
  • the immunohistochemicai assay developed by Oncotest was carried out by first deparaffinising and rehydration of the tissue.
  • the slides were incubated in 10 mM Tri-Sodium Citrate for antigen retrieval for 20 minutes at 720 watt microwave.
  • the samples were permeabilised by 0.2% triton X100 and blocked by 3% H2O2 to inactivate endogenous peroxidase.
  • the slides were blocked for unspecific binding in 10% BSA and incubated with polyclonal rabbit anti-human HER2 antibody (Dako Cat# A0485) overnight at 4 °C.
  • the slides were incubated the next day with biotinylated goat anti-rabbit IgG (Jackson Cat# 1 11 -065-045) for 60 minutes at room temperature.
  • the biotinylated secondary antibody was detect by incubation with an avidin/biotinyiated enzyme complex (ABC complex, Vector Lab Cat#PK-4000) followed by signal development with DAB chromogen.
  • HER2 protein overexpression is graded on a scale of 0 to 3+.
  • the level of IHC staining of tumour tissue sections determined by the percentage of tumour expressing HER2 protein and the intensity of the HER2 stain is used to score the HER2 protein expression.
  • the guidelines for the scoring of HER2 by IHC are shown in Table 11 below (in 2013, the assessment of IHC 2+ was updated from 'weakly positive' (as used in the 2007 guidelines) to 'equivocal')
  • the HER2 IHC overexpression scores determined by HercepTest and by the IHC method developed by Oncotest for each PDX tumour tested in efficacy studies are listed in Table 12.
  • the HER2 GCN determined by either FISH or qPCR methods or the HER2 mRNA are much more predictive in selecting a biomarker positive population to be the responsive patient population to H561-4 (p ⁇ 0.0001 ; unpaired f test).
  • Figure 12 shows a scatter plot of the T/C values of H561-4 and trastuzumab treatment in PDX models with HER2 protein overexpression positive compared to PDX models with HER2 protein
  • trastuzumab overexpression negative determined by HercepTest.
  • the mean activities of trastuzumab in PDX models in both HER2 protein overexpression positive or negative are higher than 50%, suggesting the notion that HER2 protein overexpression is not robustly predictive to the trastuzumab efficacy.
  • Nucleotide sequence of antigen-binding Fc fragment H561 -4 (SEQ ID NO: 1 )
  • Sequence "ACCTGCCCCCCTTGTCCT" at the start of the H561-4 nucleotide sequence encodes part of the lgG1 hinge region.
  • the sequence encoding the CH2 domain of H561-4 is underlined.
  • the sequence encoding the CH3 domain of H561-4 is shown in italics.
  • the sequence encoding the part of the AB structural loop of the CH3 domain believed to be involved in antigen binding is shown in bold, and the part of the sequence encoding the EF structural loop of the CH3 domain believed to be involved in antigen binding is shown in bold and doubly underlined.
  • Nucleotide sequence of the antigen-binding Fc fragment H561-4 CH2 domain (SEQ ID NO: 3)
  • Nucleotide sequence of the antigen-binding Fc fragment H561-4 CH3 domain (SEQ ID NO: 4)
  • Nucleotide sequence encoding part of the H561 -4 CD structural loop region (SEQ ID NO: 6)
  • Nucleotide sequence encoding the part of the H561 -4 EF structural loop region believed to be involved in antigen binding (SEQ ID NO: 7)
  • Amino acid sequence of antigen-binding Fc fragment H561-4 (SEQ ID NO: 8)
  • TCPPCP at the start of the H561 -4 amino acid sequence represents part of the lgG1 hinge region.
  • the CH2 domain of H561 -4 is underlined.
  • the CH3 domain of H561-4 is shown in italics.
  • Part of the AB structural loop of the CH3 domain is shown in bold and part of the EF structural loop of the CH3 domain is shown in bold and doubly underlined.
  • Cleavage of the C-terminal Lysine (K) or C-terminal lysine and the adjacent glycine (GK) of the CH3 domain has been reported.
  • the C- terminal lysine and the adjacent glycine (also present in SEQ ID NO: 11 , below) are shown boxed in the above sequence.
  • HER2 binding members for use herein may lack the C-terminal lysine or the C- terminal lysine and glycine.
  • Amino acid sequence of the part of the H561 -4 AB structural loop region believed to be involved in antigen binding (SEQ ID NO: 12)

Abstract

La présente invention concerne une combinaison de traitements pour le cancer, comprenant l'administration combinée de (i) un agent de liaison de Récepteur 2 du Facteur de Croissance Épidermique (HER2), et des éléments de liaison spécifiques qui sont en compétition avec un tel élément de liaison pour la liaison à HER2, et (ii) d'une radiothérapie, d'une chirurgie et/ou d'une chimiothérapie telle qu'une immunothérapie. La présente invention concerne également l'utilisation du nombre de copies du gène de HER2 humain et des niveaux de ARNm de HER2 en tant que biomarqueurs, par exemple pour identifier des cancers qui répondront à un traitement avec un élément de liaison spécifique comprenant un site de liaison à l'antigène HER2 mis au point dans une région de boucle structurale d'un domaine constant de l'élément de liaison spécifique, et des éléments de liaison spécifiques qui sont en compétition avec un tel élément de liaison pour la liaison à HER2. La présente invention concerne également des compositions comprenant un élément de liaison spécifique comprenant un site de liaison à l'antigène HER2 mis au point dans une région de boucle structurale d'un domaine CH3 de l'élément de liaison spécifique et un autre agent, par exemple un produit thérapeutique, tel qu'un agent immuno-oncologique et des utilisations de celui-ci.
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