Combination of ErbB-2 / ErbB-3 bispecific antibodies and endocrine therapy for breast cancer

Abstract

The present invention relates to a method for treating a subject having or at risk of developing breast cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an ErbB-2 / ErbB-3 bispecific antibody in combination with a therapeutically effective amount of an endocrine therapy drug, wherein the bispecific antibody has an antigen binding site that binds to the extracellular portion of ErbB-2 and an antigen binding site that binds to the extracellular portion of ErbB-3; and a device for use in the method.

Description

[0001] This application claims priority to US application No. 62 / 507,675, filed May 17, 2017, the contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of antibodies. Specifically, it relates to the field of therapeutic (human) antibodies for treating diseases involving abnormal cells. More specifically, it relates to antibodies that can bind to ErbB-2 and ErbB-3, and the use of these antibodies in combination with endocrine therapy drugs to treat subjects with breast cancer.

[0003] Some types of breast cancer are affected by hormones in the blood. Estrogen receptor (ER)-positive and progesterone receptor (PR)-positive breast cancer cells have receptors that attach to hormones, which help the cancer cells grow. About two-thirds of breast cancers are hormone receptor-positive. Their cells have receptors that attach to the hormones estrogen (ER-positive cancers) and / or progesterone (PR-positive cancers). For these cancers, high estrogen levels help the cancer cells grow and spread.

[0004] There are various drugs that interfere with this mechanism.

[0005] Drugs that block estrogen receptors

[0006] These drugs play a role by preventing estrogen from affecting breast cancer cells. An example of such a drug is tamoxifen. The drug blocks the estrogen receptors in breast cancer cells. It prevents estrogen from binding to cancer cells and prevents it from commanding cancer cells to grow and divide. Although tamoxifen acts as an anti-estrogen in breast cells, it acts as estrogen in other tissues (such as uterus and bone). Therefore, it is referred to as selective estrogen receptor modulators (SERM). Tamoxifen is one of the most famous SERMs. Other SERMs approved for medical use include bazedoxifene (Duavee), broparestrol (Acnestrol), clomifene (Clomid), cyclofenil (Sexovid), lasofoxifene (Fablyn), ormeloxifene (Centron, Novex, Novex-DS, Sevista), ospemifene (Osphena), raloxifene (Evista), tamoxifen (Nolvadex) and toremifene (Fareston), some of which have been approved for the treatment of hormone receptor-positive breast cancer. More SERMs have not yet been approved, but they are also functional.

[0007] Tamoxifen may be started after surgery (adjuvant therapy) or before surgery (neoadjuvant therapy), and it is usually taken for 5 to 10 years. After menopause, aromatase inhibitors may be used.

[0008] In women at high risk of breast cancer, tamoxifen may be used to help reduce the risk of developing breast cancer.

[0009] Toremifene (Fareston) is another SERM currently approved for the treatment of metastatic breast cancer. Most of these drugs are taken orally, most often as a pill.

[0010] Fulvestrant (Faslodex)

[0011] Fulvestrant is a drug that blocks estrogen receptors and also temporarily eliminates them. Fulvestrant is a selective estrogen receptor degrader (SERD). Other SERDs include Brilanestrant and Elacestrant. Fulvestrant is used to treat metastatic breast cancer, including after other hormonal drugs (such as tamoxifen and often aromatase inhibitors) have stopped working.

[0012] It can be administered by intramuscular injection.Fulvestrant is currently approved for use in postmenopausal women.

[0013] Aromatase inhibitor (AI)

[0014] Aromatase inhibitors (AIs) are medications that block the production of estrogen. Before menopause, most estrogen is produced by the ovaries. However, for women whose ovaries no longer function due to menopause or medical treatment, enzymes in fat tissue, breasts, or skin (called aromatase) still produce small amounts of estrogen. AIs work by blocking aromatase from producing estrogen.

[0015] These drugs are used in postmenopausal women, but they can also be used in premenopausal women if combined with ovarian ablation.

[0016] There are many AIs. Exemplary AIs include: Letrozole (Femara), Anastrozole (Arimidex), and Exemestane (Aromasin).

[0017] These medicines come as pills that you take every day.

[0018] For postmenopausal women whose cancer is hormone receptor-positive, doctors may recommend taking an AI during adjuvant therapy.

[0019] Luteinizing hormone-releasing hormone (LHRH) analogs: These medications are more commonly used than oophorectomy. They block the body's signals to the ovaries to produce estrogen, which can cause temporary menopause. Common LHRH medications include goserelin (Zoladex) and leuprolide (Lupron). They can be used alone or with other hormone medications (tamoxifen, aromatase inhibitors, fulvestrant) as hormone therapy in premenopausal women.

[0020] Chemotherapy drugs: Some chemotherapy drugs can damage the ovaries in premenopausal women, so they no longer make estrogen. In some women, ovarian function returns after months or years, but in others, the damage to the ovaries is permanent and leads to menopause.

[0021] The drugs mentioned above are collectively referred to as endocrine therapy drugs for breast cancer. Recently, Lumachi et al. (Lumachi, F., et al., "Endocrine therapy of breast cancer." Current medicinal chemistry 18.4 (2011): 513-522) reviewed endocrine therapy and concomitant drugs. In the context of the present invention, endocrine therapy drugs refer to drugs used for endocrine therapy of breast cancer.

[0022] In the context of the invention described herein, the term endocrine therapy includes therapeutic drugs that interfere with the action of hormones, typically estrogen or progesterone hormones, in cancer cells. This can be done directly through the action of the drug in cancer cells, or indirectly, for example by reducing the amount of estrogen that can reach cancer cells, including by directly or indirectly interfering with the action of estrogen on tumors. Summary of the Invention

[0023] The present invention provides a method for treating a subject having or at risk of breast cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an ErbB-2 / ErbB-3 bispecific antibody in combination with a therapeutically effective amount of an endocrine therapy drug, wherein the bispecific antibody has an antigen binding site that binds to the extracellular portion of ErbB-2 and an antigen binding site that binds to the extracellular portion of ErbB-3.

[0024] The present invention provides a combination of an ErbB-2 / ErbB-3 bispecific antibody and an endocrine therapy drug for treating a subject having or at risk of having breast cancer, wherein the bispecific antibody has an antigen binding site that binds to the extracellular portion of ErbB-2 and an antigen binding site that binds to the extracellular portion of ErbB-3.

[0025] The present invention also provides the use of an ErbB-2 / ErbB-3 bispecific antibody and an endocrine therapy drug in the preparation of a medicament for treating a subject having breast cancer or at risk of having such a cancer, wherein the bispecific antibody has an antigen binding site that can bind to the extracellular portion of ErbB-2 and an antigen binding site that can bind to the extracellular portion of ErbB-3.

[0026] Also provided are products comprising an ErbB-2 / ErbB-3 bispecific antibody and an endocrine therapy drug for simultaneous, separate, or sequential use in treating a subject having or at risk of breast cancer, wherein the bispecific antibody has an antigen binding site that binds to the extracellular portion of ErbB-2 and an antigen binding site that binds to the extracellular portion of ErbB-3.

[0027] The present invention also provides a method of treating a subject having or at risk of having breast cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that binds to the extracellular portion of ErbB-2 and inhibits ErbB-2 / ErbB-3 dimerization on the cancer cells, wherein the cancer is a hormone receptor-positive cancer.

[0028] Also provided are antibodies that bind to the extracellular portion of ErbB-2 and inhibit ErbB-2 / ErbB-3 dimerization on cancer cells for use in treating a subject having or at risk of having breast cancer, wherein the cancer is a hormone receptor-positive cancer.

[0029] Also provided are combinations of ErbB-2 and / or ErbB-3 antibodies and an endocrine therapy agent for treating a subject having or at risk of having breast cancer, wherein the breast cancer is hormone receptor-positive breast cancer, and wherein the antibody inhibits ErbB-2, ErbB-3 dimerization.

[0030] The present invention also provides the use of ErbB-2 and / or ErbB-3 antibodies and endocrine therapy drugs in the preparation of a medicament for treating a subject having breast cancer or at risk of having such cancer, wherein the breast cancer is hormone receptor-positive breast cancer, and wherein the antibodies inhibit ErbB-2 and ErbB-3 dimerization.

[0031] Also provided are products comprising an ErbB-2 and / or ErbB-3 antibody and an endocrine therapy for simultaneous, separate, or sequential use in treating a subject having or at risk of breast cancer, wherein the cancer is a hormone receptor-positive cancer and wherein the antibody inhibits ErbB-2, ErbB-3 dimerization.

[0032] The antibody is preferably a bispecific antibody having an antigen binding site that binds to the extracellular portion of ErbB-2 and an antigen binding site that binds to the extracellular portion of ErbB-3. In one embodiment, the method further comprises administering to a subject in need thereof a therapeutically effective amount of an endocrine therapy drug.

[0033] The antibody may be MCLA-128.

[0034] Endocrine therapy drugs are preferably drugs that interfere with the effects of estrogen or progesterone hormones in cancer cells. Endocrine therapy drugs preferably include aromatase inhibitors; selective estrogen receptor modulators (SERMs); or selective estrogen receptor downregulators (SERDs). Endocrine therapy drugs may include selective estrogen receptor modulators (SERMs) selected from tamoxifen (Nolvadex), bromostriene (Acnestrol), cyclofenil (Sexovid), raloxifene (Evista) and toremifene (Fareston). Endocrine therapy drugs preferably include tamoxifen, fulvestrant or its equivalent. In one embodiment, endocrine therapy drugs include letrozole or its equivalent.

[0035] In one embodiment, the cancer is an immunohistochemically ErbB-2+ cancer or an immunohistochemically ErbB-2++ cancer without ErbB-2 gene amplification.

[0036] In one embodiment, the breast cancer is a metastatic breast cancer MBC that is ER positive with low HER2 expression by IHC 1+ or IHC 2+ in combination with negative FISH.

[0037] In one embodiment, the method further comprises administering to the patient a cyclin-dependent kinase 4 / 6 inhibitor. The cyclin-dependent kinase 4 / 6 inhibitor can be, for example, Palbociclib, Ribociclib, or Abemaciclib.

[0038] In one embodiment, the subject having breast cancer or at risk of such cancer (including subjects at risk of recurrence) has been treated with one, preferably two, endocrine therapies prior to starting treatment with an ErbB-2 / ErbB-3 bispecific antibody as described herein. The subject has preferably received these prior therapies to treat metastases. Additionally, the subject has preferably received a cyclin-dependent kinase inhibitor prior to starting treatment with an ErbB-2 / ErbB-3 bispecific antibody as described herein. Detailed Description of the Invention

[0040] ErbB-2 / ErbB-3 bispecific antibodies are described in PCT / NL2015 / 050125, published as WO2015 / 130173. This application is incorporated herein by reference. In particular, the present invention relates to nucleic acid molecules, amino acid molecules, and sequences encoding such bispecific antibodies or their constant or variable portions. Also particularly, the present invention relates to methods for producing such bispecific antibodies (and references therein).

[0041] EP17164292; EP17164382 and US 15 / 476,260 also describe ErbB-2 / ErbB-3 bispecific antibodies and their uses. EP17164292; EP17164382 and US 15 / 476,260 are incorporated herein by reference.

[0042] In one embodiment, the breast cancer is a hormone receptor positive breast cancer. In one embodiment, the hormone positive breast cancer is an estrogen receptor positive breast cancer. In one embodiment, the hormone positive breast cancer is a progesterone receptor positive breast cancer. Breast cancer is routinely tested for the presence of the above hormone receptors, and the skilled person can use recognized classifications and tests. Reference is made to Hammond et al. (2010: J. of Clinical Oncology Vol 28: pp 2784-2794), which describes suitable tests and thus provides guidance. For example, a patient suitable for treatment according to the present invention is one in which at least 1% of the tumor nuclei in a tumor biopsy are immunoreactive, as determined by immunohistochemistry; positive for estrogen receptors and / or progesterone receptors.

[0043] Furthermore, another example of a suitable patient for treatment according to the present invention is a patient having a cancer with a documented hormone receptor positive status (estrogen receptor positive [ER+] and / or progesterone receptor positive [PR+]) by local criteria comprising ≥1% of positively stained cells, based on local analysis of a recent tumor biopsy.

[0044] Furthermore, another example of a suitable patient for treatment according to the present invention is a patient having a cancer with documented hormone receptor positive status (estrogen receptor positive [ER+] and / or progesterone receptor positive [PR+]) as determined by immunohistochemistry of ≥1% positive cells on a tumor biopsy.

[0045] Breast cancer can be ErbB-2 negative or ErbB-2 positive. Wolff et al. (2013: J. of Clinical Oncology Vol 31: pp 3997-4013) describe such a test and provide recommendations. The generally accepted stratification of breast cancer based on ErbB-2 expression is: ErbB-2-; ErbB-2+; ErbB-2++ without ErbB-2 gene amplification; ErbB-2++ with ErbB-2 gene amplification, and ErbB-2+++. In one embodiment, the breast cancer is ErbB-2+ or ErbB-2++ without ErbB-2 gene amplification, including the absence of gene amplification at the level of detection. Fluorescence in situ hybridization (FISH) can be used to determine the presence or absence of gene amplification. Therefore, patients suitable for treatment according to the present invention may be patients with cancer that does not show ErbB-2 gene amplification according to FISH analysis, which is understood by those of ordinary skill in the art to be FISH negative.

[0046] Suitable patients for treatment according to the present invention may be patients with ER-positive metastatic breast cancer (MBC) with low HER2 expression by immunohistochemistry (IHC) 1+ or IHC 2+ combined with negative fluorescence in situ hybridization (FISH).

[0047] In one embodiment, the breast cancer is ErbB-3 positive breast cancer.

[0048] In one embodiment, the bispecific antibody can reduce ligand-induced receptor function of ErbB-3 on ErbB-2 and ErbB-3 positive cells.

[0049] As used herein, the term "antigen binding site" refers to a site derived from and preferably present on an antibody that is capable of binding to an antigen. Unmodified antigen binding sites are typically formed by and present in the variable domains of an antibody. The variable domains comprise the antigen binding site. An antigen-binding variable domain is a variable domain that comprises an antigen-binding site that binds to an antigen.

[0050] In one embodiment, the antibody variable domains of the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL). The antigen binding site may be present in the combined VH / VL variable domains, or may be present only in the VH region or only in the VL region. When the antigen binding site is present in only one of the two regions of the variable domain, the corresponding variable region may promote the folding and / or stability of the bound variable region, but will not significantly promote the binding of the antigen itself.

[0051] Antigen binding as used herein refers to the typical binding ability of an antibody to its antigen. Antibodies comprising an antigen binding site that binds to ErbB-3 bind to ErbB-3 and, other things being equal, do not bind to the cognate receptors ErbB-1 and ErbB-4 of the same species. Considering that the ErbB family is a family of cell surface receptors, the binding is typically assessed on cells expressing the receptors. The antibodies of the present invention preferably bind to human ErbB-2, human ErbB-3, or a combination thereof.

[0052] In contrast to the random, nonspecific sticking of antibodies, antigen binding by antibodies is typically mediated by the complementary regions of the antibodies and the specific three-dimensional structure of both the antigen and the variable domain, allowing the two structures to fit together precisely (similar to the interaction of a lock and key). Since antibodies typically recognize epitopes of antigens, and since these epitopes may also be present in other compounds, antibodies that bind to ErbB-2 or ErbB-3 according to the present invention may also recognize other proteins if such other compounds contain the same epitope. Therefore, the term "binding" does not exclude the binding of antibodies to other proteins containing the same epitope. Such other proteins are preferably not human proteins. The ErbB-2 antigen binding site and the ErbB-3 antigen binding site as defined in the present invention do not typically bind to other proteins on the cell membranes of postnatal (preferably adult) humans. As outlined in more detail below, the bispecific antibodies according to the present invention are typically capable of binding to ErbB-2 or ErbB-3 with a binding affinity of at least 1×10e-6 M.

[0053] As used herein, the term "interfering with binding" means that the antibody is directed against an epitope on ErbB-3 and that the antibody competes with the ligand for binding to ErbB-3. The antibody may reduce ligand binding, displace the ligand when it is already bound to ErbB-3, or at least partially prevent the ligand from binding to ErbB-3 (e.g., by steric hindrance).

[0054] As used herein, the term "antibody" means a protein molecule preferably belonging to the immunoglobulin class of proteins, which comprises one or more variable domains that bind to an epitope on an antigen, wherein such domains are derived from the variable domains of an antibody or share sequence homology therewith. Antibodies for therapeutic use are preferably as close as possible to the natural antibodies of the subject to be treated (e.g., human antibodies for human subjects). Antibody binding can be expressed in terms of specificity and affinity. Specificity determines which antigen or epitope is specifically bound by the binding domain. Affinity is a measure of the strength of binding to a specific antigen or epitope. Specific binding is defined as binding with an affinity (KD) of at least 1×10e-6M, more preferably 1×10e-7M, and more preferably greater than 1×10e-9M. Generally speaking, the affinity of antibodies for therapeutic applications is as high as 1×10e-10M or higher. Antibodies (e.g., bispecific antibodies of the present invention) may comprise the constant domains (Fc portions) of natural antibodies. The antibodies of the present invention are typically bispecific full-length antibodies, preferably human IgG subclasses. Preferably, the antibodies of the present invention are human IgG1 subclasses. Such antibodies of the present invention have good ADCC properties, have a favorable half-life when administered to humans in vivo, and have CH3 engineering technology that can provide modified heavy chains that preferentially form heterodimers over homodimers when co-expressed in clonal cells.

[0055] The antibodies of the present invention are preferably "full-length" antibodies. The term "full-length" according to the present invention is defined as comprising a substantially complete antibody, however, the antibody does not necessarily have all the functions of a complete antibody. For the avoidance of doubt, a full-length antibody comprises two heavy chains and two light chains. Each chain comprises a constant (C) region and a variable (V) region, which can be decomposed into domains referred to as CH1, CH2, CH3, VH and CL, VL. The antibody binds to the antigen through the variable domains contained in the Fab portion, and after binding, can interact with the molecules and cells of the immune system through the constant domains (primarily through the Fc portion). The terms "variable domain," "VH / VL pair," and "VH / VL" are used interchangeably herein. Full-length antibodies according to the present invention include antibodies in which mutations that provide desired properties may be present. Such mutations should not be deletions of substantial portions of any region. However, antibodies in which one or more amino acid residues are deleted without substantially changing the binding properties of the resulting antibody are included within the term "full-length antibody." For example, an IgG antibody may have insertions, deletions, or combinations thereof of 1 to 20 amino acid residues in the constant region. For example, when an antibody itself has low ADCC activity, the ADCC activity of the antibody can be improved by slightly modifying the constant region of the antibody (Junttila, TT, K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Arnplified Breast Cancer." Cancer Research 70(11): 4481-4489).

[0056] Because of the favorable half-life of full-length IgG antibodies and the need to maintain close to completely autologous (human) molecules for reasons of immunogenicity, it is preferred. The antibody of the present invention is preferably a bispecific IgG antibody, preferably a bispecific full-length IgG1 antibody. Based on the long circulation half-life of IgG1 in humans, it is advantageous. In order to prevent any immunogenicity in humans, it is preferred that the bispecific IgG antibody according to the present invention is a human IgG1.

[0057] The term "bispecific" (bispecific, bs) means that a portion of an antibody (as defined above) binds to an epitope on an antigen, while a second portion binds to a different epitope. Different epitopes are generally present on different antigens. According to the present invention, the first and second antigens are actually two different proteins. A preferred bispecific antibody is an antibody that comprises parts of two different monoclonal antibodies and therefore binds to two different types of antigens. One arm of a bispecific antibody generally comprises the variable domains of one antibody, while the other arm comprises the variable domains of another antibody. The heavy chain variable regions of the bispecific antibodies of the present invention are generally different from each other, while in the bispecific antibodies of the present invention, the light chain variable regions are preferably the same. Bispecific antibodies in which different heavy chain variable regions are associated with the same or common light chain are also referred to as bispecific antibodies with a common light chain. Therefore, a bispecific antibody according to the present invention is also provided, wherein the two arms comprise a common light chain.

[0058] Preferred bispecific antibodies can be obtained by co-expressing two different heavy chains and a common light chain in a single cell. When using a wild-type CH3 domain, co-expression of two different heavy chains and a common light chain will result in three different species: AA, AB, and BB. To increase the percentage of the desired bispecific product (AB), CH3 modification can be used, or in other words, heavy chains with compatible heterodimerization domains as defined below can be used.

[0059] As used herein, the term "compatible heterodimerization domain" refers to a protein domain that has been engineered such that engineered domain A' will preferentially form heterodimers with engineered domain B', and vice versa, while homodimerization between A'-A' and B'-B' is reduced.

[0060] The term "common light chain" according to the present invention refers to a light chain that can be identical or have some amino acid sequence differences without affecting the binding specificity of the full-length antibody. For example, within the definition of a common light chain used herein, it is possible to prepare or discover different but still functionally equivalent light chains, such as by introducing and testing conservative amino acid changes, not promoting or only partially promoting changes in amino acids in regions of binding specificity when paired with a heavy chain. With or without the term "rearrangement", the terms 'common light chain', 'common VL', 'single light chain', 'single VL' are all used interchangeably herein. One aspect of the present invention is to use a human light chain that can be combined with different heavy chains to form an antibody with a functional antigen-binding domain as a common light chain (WO2004 / 009618, WO2009 / 157771, Merchant et al., 1998 and Nissim et al., 1994). Preferably, the common light chain has a germline sequence. A preferred germline sequence is a light chain variable region that is frequently used in the human repertoire and has good thermodynamic stability, yield, and solubility. A preferred germline light chain is O12, preferably a rearranged germline human κ light chain IgVκ1-39*01 / IGJκ1*01 or a fragment or functional equivalent thereof (i.e., the same IgVκ1-39 gene segment, but a different IGJκ gene segment) (according to the nomenclature of the IMGT database on the World Wide Web imgt.org). Thus, a bispecific antibody according to the invention is also provided, wherein the common light chain is a germline light chain, preferably a rearranged germline human κ light chain comprising the IgVK1-39 gene segment, most preferably a rearranged germline human κ light chain IgVK1-39*01 / IGJK1*01. The terms rearranged germline human κ light chain IgVκ1-39*01 / IGJκ1*01, IGKV1-39 / IGKJ1, huVκ1-39 light chain or abbreviated huVκ1-39 are used interchangeably throughout the application. Obviously, those skilled in the art will recognize that "common" also refers to functional equivalents of light chains that are not identical in amino acid sequence. There are many variants of the light chain, in which there are mutations (deletions, substitutions, additions) that do not substantially affect the formation of the functional binding region. The light chain of the present invention may also be a light chain as described above having an insertion, deletion, substitution, or combination thereof of 1 to 5 amino acids.

[0061] Antibodies in which the VH is capable of specifically recognizing a first antigen and the VL paired with the VH in the immunoglobulin variable domain is capable of specifically recognizing a second antigen are also contemplated. The resulting VH / VL pair will bind to either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008 / 027236, WO 2010 / 108127, and Schaefer et al. (Cancer Cell 20, 472-486, October 2011), which are different from the bispecific antibodies of the present invention and are also referred to as "two-in-one" antibodies.

[0062] As used herein, the term "ErbB-2" refers to the protein encoded by the ERBB-2 gene in humans. Alternative names for the gene or protein include: CD340; HER-2; HER-2 / neu; MLN 19; NEU; NGL; TKR1. The ERBB-2 gene is often referred to as HER2 (from human epidermal growth factor receptor 2). When ErbB-2 is mentioned herein, the reference is to human ErbB-2. Antibodies comprising an antigen binding site that binds to ErbB-2 bind to human ErbB-2. Due to the sequence and tertiary structure similarities between human and other mammalian orthologs, the ErbB-2 antigen binding site may also bind to such orthologs, but not necessarily. The database accession numbers for the human ErbB-2 protein and its encoding gene are (NP_001005862.1; NP_004439.2; NC_000017.10; NT_010783.15; NC_018928.2). The accession numbers are given primarily to provide an additional means of identifying ErbB-2 as a target, the actual sequence of the ErbB-2 protein to which the antibody binds may vary, for example due to mutations in the encoding gene, such as those that occur in some cancers, etc. The ErbB-2 antigen binding site binds to ErbB-2 and its various variants, such as those expressed by some ErbB-2-positive tumor cells.

[0063] As used herein, the term "ErbB-3" refers to the protein encoded by the ERBB-3 gene in humans. Alternative names for the gene or protein are: HER3; LCCS2; MDA-BF-1; c-ErbB-3; c-erbb-3; erbb-3-S; p180-Erbb-3; p45-sErbb-3; and p85-sErbb-3. When ErbB-3 is referred to herein, the reference is to human ErbB-3. Antibodies comprising an antigen binding site that binds ErbB-3 bind to human ErbB-3. Due to the sequence and tertiary structure similarities between human and other mammalian orthologs, the ErbB-3 antigen binding site may also bind to such orthologs, but not necessarily. The database accession numbers for the human ErbB-3 protein and its encoding gene are (NP_001005915.1; NP_001973.2; NC_000012.11; NC_018923.2; NT_029419.12). The accession numbers are primarily given to provide additional methods for identifying ErbB-3 as a target. The actual sequence of the ErbB-3 protein bound by the antibody may vary, for example due to mutations in the encoding gene, such as those that occur in some cancers. The ErbB-3 antigen binding site binds to ErbB-3 and its various variants, such as those expressed by some ErbB-2-positive tumor cells.

[0064] The bispecific antibodies of the present invention, comprising a first antigen binding site that binds ErbB-2 and a second antigen binding site that binds ErbB-3, can reduce the ligand-induced receptor function of ErbB-3 on ErbB-2 and ErbB-3 positive cells. In the presence of an excess of ErbB-2, in the absence of a ligand for a detectable ErbB-3 chain in the heterodimer, the ErbB-2 / ErbB-3 heterodimer can provide a growth signal to the expressing cell. This ErbB-3 receptor function is referred to herein as the ligand-independent receptor function of ErbB-3. In the presence of an ErbB-3 ligand, the ErbB-2 / ErbB-3 heterodimer also provides a growth signal to the expressing cell. This ErbB-3 receptor function is referred to herein as the ligand-induced receptor function of ErbB-3.

[0065] As used herein, the term "ErbB-3 ligand" refers to a polypeptide that binds to and activates ErbB-3. Some examples of ErbB-3 ligands include, but are not limited to, neuregulin (NRG) 1 and neuregulin 2, betacellulin, heparin-binding epidermal growth factor, and epiregulin. The term includes biologically active fragments and / or variants of naturally occurring polypeptides.

[0066] In a preferred embodiment of the present invention, the ligand-induced receptor function of ErbB-3 is the ErbB-3 ligand-induced growth of ErbB-2 and ErbB-3 positive cells. In a preferred embodiment, the cells are MCF-7 cells ( HTB-22 TM );SKBR3( HTB-30 TM ) cells; NCI-87( CRL-5822 TM ) cells; BxPC-3-luc2 cells (Perkin Elmer 125058), BT-474 cells ( HTB-20 TM ) or JIMT1 cells (DSMZ no.: ACC 589).

[0067] In a preferred embodiment, ErbB-2 and ErbB-3 positive cells contain at least 50,000 ErbB-2 receptors on the cell surface. In a preferred embodiment, at least 100,000 ErbB-2 receptors. In a preferred embodiment, ErbB-2 and ErbB-3 positive cells contain at least 1,000,000 ErbB-2 receptors on the cell surface. In another preferred embodiment, ErbB-2 and ErbB-3 positive cells contain no more than 1,000,000 ErbB-2 receptors on the cell surface. Currently used treatments, such as trastuzumab (Herceptin) and pertuzumab, are only prescribed to patients with malignant ErbB-2 positive cells that have more than 1,000,000 ErbB-2 receptors on their cell surface to achieve a clinical response. Patients with ErbB-2 positive tumor cells that have more than 1,000,000 ErbB-2 receptors on their cell surface are typically classified as ErbB-2 [+++]. For example, using the HercepTest™ and / or HER2 FISH sold by Dako Denmark A / S (pharm Dx TM ), and / or using the The assay is used to categorize patients. Trastuzumab and Pertuzumab are prescribed only for ErbB-2[+++] patients because patients with lower ErbB-2 concentrations generally do not show an adequate clinical response when treated with trastuzumab and Pertuzumab. However, the present invention provides bispecific antibodies that have improved binding affinity for cells with lower ErbB-2 receptor concentrations compared to trastuzumab. As shown in the Examples, the proliferation of such cells with low ErbB2 expression is effectively counteracted by the antibodies according to the present invention. Such low ErbB-2 receptor concentrations are present in malignant cells of patients categorized as ErbB-2[++] or ErbB-2[+]. Similarly, recurrent ErbB-2-positive tumors typically have ErbB-2 receptor concentrations below 1,000,000 receptors / cell. Therefore, such ErbB-2[++] or ErbB-2[+] patients, as well as patients with recurrent ErbB-2-positive tumors, are preferably treated with the bispecific antibodies according to the present invention. Thus, also provided are bispecific antibodies comprising a first antigen binding site that binds ErbB-2 and a second antigen binding site that binds ErbB-3, wherein the antibody can reduce ligand-induced growth of ErbB-2 and ErbB-3-positive cells that have fewer than 1,000,000 ErbB-2 cell surface receptors. Also provided are methods for treating a subject having or at risk of having an ErbB-2, ErbB-3, or ErbB-2 / ErbB-3-positive tumor, wherein the tumor has fewer than 1,000,000 ErbB-2 cell surface receptors per cell, the method comprising administering to the subject a bispecific antibody or pharmaceutical composition according to the invention. Also provided herein are bispecific antibodies according to the present invention for use in treating a subject having, or at risk for, an ErbB-2, ErbB-3, or ErbB-2 / ErbB-3 positive tumor, wherein the tumor has fewer than 1,000,000 ErbB-2 cell surface receptors per cell. The antibodies according to the present invention are generally capable of reducing ligand-induced receptor function, preferably ligand-induced growth, of ErbB-3 on ErbB-2 and ErbB-3 positive cells. The antibodies according to the present invention preferably comprise a first antigen binding site that binds to domain I of ErbB-2 and a second antigen binding site that binds to domain III of ErbB-3. In a preferred embodiment, as explained in more detail below, the affinity of the second antigen binding site for ErbB-3 positive cells is equal to or greater than the affinity of the first antigen binding site for ErbB-2 positive cells.The affinity of the second antigen binding site for ErbB-3 positive cells is preferably less than or equal to 2.0 nM, more preferably less than or equal to 1.39 nM, more preferably less than or equal to 0.99 nM. The affinity of the first antigen binding site for ErbB-2 positive cells is preferably less than or equal to 5.0 nM, preferably less than or equal to 4.5 nM, preferably less than or equal to 4.0 nM.

[0068] In a preferred embodiment, the antibody according to the invention comprises an antigen binding site that binds to at least one amino acid of domain I of ErbB-2 selected from the group consisting of T144, T164, R166, P172, G179, S180 and R181, and a surface-exposed amino acid residue located within about 5 amino acid positions of T144, T164, R166, P172, G179, S180 or R181.

[0069] In a preferred embodiment, the antibody according to the present invention preferably comprises an antigen binding site that binds to at least one amino acid of domain III of ErbB-3 selected from R426 and located at a position 1000 ft from R426 in the native ErbB-3 protein. surface-exposed amino acid residues within.

[0070] To determine whether a tumor is ErbB-3 positive, a technician can, for example, determine ErbB-3 gene amplification and / or staining by immunohistochemistry. At least 10% of the tumor cells in the biopsy should be positive. The biopsy may also contain 20%, 30%, 40%, 50%, 60%, 70% or more positive cells.

[0071] The ligand-induced receptor function used herein is reduced by at least 20%, preferably at least 30%, 40%, 50%, 60% or at least 70%. In a particularly preferred embodiment, the ligand-induced receptor function is reduced by 80%, more preferably by 90%. Preferably, the reduction is determined by determining the ligand-induced receptor function in the presence of the bispecific antibody of the present invention and comparing it with the same function in the absence of the antibody under other conditions being equal. The conditions comprise at least the presence of an ErbB-3 ligand. The amount of the ligand present is preferably an amount that induces half the maximum growth of ErbB-2 and ErbB-3 positive cell lines. The ErbB-2 and ErbB-3 positive cell lines used for this test are preferably MCF-7 cell lines ( HTB-22 TM ), SKBR3 cell line ( HTB-30 TM ) cells, JIMT 1 cell line (DSMZ ACC589) or NCI-87 cell line ( CRL-5822TM The assay and / or ligand used to determine ErbB-3 ligand-induced receptor function is preferably an assay for ErbB-3 ligand-induced growth reduction as specified in the examples.

[0072] The ErbB-2 protein contains several domains (see Landgraf, R Breast Cancer Res. 2007; 9(1): 202- Figure 1 For reference). The extracellular domains are referred to as domains I to IV. The positions of the corresponding domains of the antigen binding sites of the antibodies described herein have been mapped (see Examples). The bispecific antibodies of the present invention having an antigen binding site (first antigen binding site) that binds to domain I or domain IV (first antigen binding site) of ErbB-2 comprise heavy chain variable regions that maintain significant binding specificity and affinity for ErbB-2 when combined with a variety of light chains. It was found that bispecific antibodies having an antigen binding site (first antigen binding site) that binds to domain I or domain IV (first antigen binding site) of ErbB-2 and an antigen binding site (second antigen binding site) for ErbB-3 were more effective in reducing the ligand-induced receptor function of ErbB-3 when compared to bispecific antibodies comprising an antigen binding site (first antigen binding site) that binds to other extracellular domains of ErbB-2. Bispecific antibodies comprising an antigen binding site (first antigen binding site) that binds to ErbB-2, wherein the antigen binding site binds to domain I or domain IV of ErbB-2 are preferred. Preferably, the antigen binding site binds to domain IV of ErbB-2. It has been found that bispecific antibodies having an antigen binding site (first antigen binding site) that binds to ErbB-2 and further comprising ADCC are more effective than other ErbB-2 binding antibodies that do not have significant ADCC activity, particularly in vivo. Therefore, bispecific antibodies according to the present invention that exhibit ADCC are preferred. It has been found that antibodies in which the first antigen binding site binds to domain IV of ErbB-2 have inherent ADCC activity. ErbB-2 binding antibodies that bind to domain I with low inherent ADCC activity can be modified to enhance ADCC activity, and the Fc region mediates antibody function by binding to different receptors on immune effector cells (e.g., macrophages, natural killer cells, B cells, and neutrophils). Some of these receptors (e.g., CD16A (FcγRIIIA) and CD32A (FcγRIIA)) activate cells to mount a response against antigens. Other receptors (e.g., CD32B) inhibit the activation of immune cells. By engineering the Fc region (by introducing amino acid substitutions) to bind to activating receptors with greater selectivity, antibodies with greater cytotoxic activity, which is desirable for anti-cancer mAbs, can be generated.

[0073] One technique for enhancing ADCC of antibodies is afucosylation. (See, e.g., Junttila, TT, K. Parsons, et al. (2010). "Superior In vivo Efficacy of Afucosylated Trastuzumab in the Treatment of HER2-Amplified Breast Cancer." Cancer Research 70(11): 4481-4489). Therefore, afucosylated bispecific antibodies according to the present invention are also provided. Alternatively or additionally, ADCC enhancement can be achieved using a variety of other strategies, including, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics), all of which seek to improve Fc binding to the low-affinity activating FcγRIIIa and / or reduce binding to the low-affinity inhibitory FcγRIIb.

[0074] There are several in vitro methods for determining the effectiveness of antibodies or effector cells in eliciting ADCC. These include chromium 51 [Cr51] release assays, europium [Eu] release assays, and sulfur 35 [S35] release assays. Generally, a labeled target cell line expressing a surface-exposed antigen is incubated with an antibody specific for the antigen. After washing, effector cells expressing the Fc receptor CD16 are typically co-incubated with antibody-labeled target cells. Target cell lysis is then typically measured by release of intracellular markers (e.g., by scintillation counter or spectrophotometry). A preferred test is described in detail in the Examples.

[0075] One advantage of the present invention is the fact that binding of antibodies according to the invention (e.g., PB4188) to ErbB-2 and ErbB-3 positive cells results in internalization to the same extent as trastuzumab. If a combination of trastuzumab and pertuzumab is used, the internalization of these antibodies is enhanced. However, this enhanced internalization results in reduced ADCC. Therefore, antibodies according to the invention that result in essentially the same degree of internalization as trastuzumab are preferred over a combination of trastuzumab and pertuzumab, because ADCC activity is better maintained with such antibodies.

[0076] Antibodies comprising an antigen binding site that binds to ErbB-3 of the present invention interfere with the binding of ErbB-3 ligands to ErbB-3. Such antibodies are more effective in reducing ErbB-3 ligand-induced receptor function on ErbB-2 and ErbB-3 positive cell lines, particularly in the case of bispecific antibodies that also comprise an antigen binding site that binds to ErbB-2.

[0077] Some preferred embodiments of the present invention provide bispecific antibodies comprising a first antigen binding site that binds to ErbB-2 and a second antigen binding site that binds to ErbB-3, wherein the first antigen binding site binds to domain I of ErbB-2. As shown in the Examples, bispecific antibodies with these characteristics are able to bind well to ErbB-2 and ErbB-3 positive cells and counteract their activities (e.g., ligand-induced receptor function of ErbB-3 and ligand-induced growth of ErbB-2 and ErbB-3 positive cells). In addition, bispecific antibodies according to the present invention comprising a first antigen binding site that binds to domain I of ErbB-2 are particularly suitable for use in combination with existing anti-ErbB-2 treatments (e.g., trastuzumab and pertuzumab) because trastuzumab and pertuzumab bind to different domains of ErbB-2. Trastuzumab binds to domain IV of ErbB-2 and pertuzumab binds to domain II of ErbB-2. Therefore, the bispecific antibody according to the present invention that binds to domain I of ErbB-2 is preferred because it does not compete with trastuzumab and pertuzumab for the same epitope.

[0078] Another preferred embodiment provides a bispecific antibody comprising a first antigen binding site that binds ErbB-2 and a second antigen binding site that binds ErbB-3, wherein the second antigen binding site binds to domain III of ErbB-3. Such antibodies according to the present invention are particularly suitable for combination therapy with currently used anti-ErbB-3 binding molecules that do not bind to domain III of ErbB-3 (e.g., MM-121 (Merrimack Pharmaceuticals; also known as #Ab6) and RG7116 (Roche) that bind to domain I of ErbB-3), because the different binding molecules do not compete with each other for the same epitope.

[0079] Preferably, bispecific antibodies are provided that comprise a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein the first antigen-binding site binds to domain I of ErbB-2 and the second antigen-binding site binds to domain III of ErbB-3. Such antibodies are particularly suitable for combination therapy with anti-ErbB-2 binding molecules that do not bind to domain I of ErbB-2 (e.g., trastuzumab and pertuzumab) and with anti-ErbB-3 binding molecules that do not bind to domain III of ErbB-3 (e.g., MM-121 (#Ab6) and RG7116).

[0080] A preferred embodiment provides a bispecific antibody comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein the first antigen-binding site binds to domain I of ErbB-2 and the second antigen-binding site binds to domain III of ErbB-3, and wherein the antibody can reduce ligand-induced receptor function of ErbB-3 on ErbB-2 and ErbB-3 positive cells. The antibody can preferably reduce ligand-induced growth of ErbB-2 and ErbB-3 positive cells.

[0081] Other embodiments of the present invention provide bispecific antibodies comprising a first antigen binding site that binds ErbB-2 and a second antigen binding site that binds ErbB-3, wherein the affinity (KD) of the second antigen binding site for ErbB-3-positive cells is equal to or higher than the affinity of the first antigen binding site for ErbB-2-positive cells. In contrast to bispecific compounds that have a higher affinity for ErbB-2 than for ErbB-3 (e.g., MM111 from Merrimack Pharmaceuticals), the present invention provides bispecific antibodies with ErbB-3-specific arms that have a higher affinity for ErbB-3 on cells than the affinity of the ErbB-2-specific arms for ErbB-2 on cells. Despite the low cell surface concentration of ErbB-3, such bispecific antibodies are still able to better bind to ErbB-3. This provides the advantage of enhanced functional activity against ErbB-3 compared to the compounds of the prior art, which means that these bispecific antibodies according to the present invention are better able to counteract ErbB-3 activity (e.g., ligand-induced growth).

[0082] As used herein, the term "affinity" refers to the KD value.

[0083] The affinity (KD) of the second antigen-binding site for ErbB-3-positive cells is preferably less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.39 nM, and more preferably less than or equal to 0.99 nM. In a preferred embodiment, the affinity of the second antigen-binding site for ErbB-3 on SK BR 3 cells is less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.39 nM, and preferably less than or equal to 0.99 nM. In one embodiment, the affinity is in the range of 1.39 to 0.59 nM. In a preferred embodiment, the affinity of the second antigen-binding site for ErbB-3 on BT 474 cells is less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.0 nM, more preferably less than 0.5 nM, more preferably less than or equal to 0.31 nM, and more preferably less than or equal to 0.23 nM. In one embodiment, the affinity is in the range of 0.31 to 0.15 nM. The above affinity is preferably measured as using steady state cellular affinity measurements, wherein cells are incubated with radiolabeled antibodies at 4°C and cell-bound radioactivity is measured as described in the Examples.

[0084] The affinity (KD) of the first antigen binding site for ErbB-2 positive cells is preferably less than or equal to 5.0 nM, more preferably less than or equal to 4.5 nM, more preferably less than or equal to 3.9 nM. In a preferred embodiment, the affinity of the first antigen binding site for ErbB-2 on SK BR 3 cells is less than or equal to 5.0 nM, preferably less than or equal to 4.5 nM, more preferably less than or equal to 4.0 nM, more preferably less than or equal to 3.5 nM, more preferably less than or equal to 3.0 nM, more preferably less than or equal to 2.3 nM. In one embodiment, the affinity is in the range of 3.0 to 1.6 nM. In a preferred embodiment, the affinity of the first antigen binding site for ErbB-2 on BT 474 cells is less than or equal to 5.0 nM, preferably less than or equal to 4.5 nM, more preferably less than or equal to 3.9 nM. In one embodiment, the affinity is in the range of 4.5 to 3.3 nM. The above affinity is preferably measured as using steady-state cellular affinity measurements, wherein cells are incubated with radiolabeled antibodies at 4°C and the cell-bound radioactivity is measured as described in the Examples.

[0085] In a preferred embodiment, a bispecific antibody according to the present invention is provided, wherein the affinity (KD) of the bispecific antibody for BT 474 cells is less than or equal to 5.0 nM, preferably less than or equal to 4.5 nM, more preferably less than or equal to 4.0 nM, more preferably less than or equal to 3.5 nM, more preferably less than or equal to 3.7 nM, preferably less than or equal to 3.2 nM. In one embodiment, the affinity is in the range of 3.7 to 2.7 nM. In a preferred embodiment, a bispecific antibody according to the present invention is provided, wherein the affinity of the bispecific antibody for SK BR 3 cells is less than or equal to 5.0 nM, preferably less than or equal to 4.5 nM, more preferably less than or equal to 4.0 nM, more preferably less than or equal to 3.5 nM, more preferably less than or equal to 3.0 nM, preferably less than or equal to 2.5 nM, more preferably less than or equal to 2.0 nM. In one embodiment, the affinity is in the range of 2.4 to 1.6 nM. Likewise, the above affinities are preferably measured as using steady-state cellular affinity measurements, wherein cells are incubated with radiolabeled antibodies at 4°C and cell-bound radioactivity is measured as described in the Examples.

[0086] Other preferred embodiments of the present invention provide bispecific antibodies comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein the affinity (KD) of the second antigen-binding site for ErbB-3-positive cells is equal to or greater than the affinity of the first antigen-binding site for ErbB-2-positive cells, and wherein the antibody can reduce ligand-induced receptor function of ErbB-3 on both ErbB-2 and ErbB-3-positive cells. The antibody can preferably reduce ligand-induced growth of ErbB-2 and ErbB-3-positive cells.

[0087] The above-mentioned antibodies according to the present invention that have a high affinity for ErbB-3 preferably bind to domain I of ErbB2 and / or domain III of ErbB-3. Therefore, a bispecific antibody according to the present invention is also provided, which comprises a first antigen binding site that binds to domain I of ErbB-2 and a second antigen binding site that binds to ErbB-3, wherein the affinity (KD) of the second antigen binding site for ErbB-3-positive cells is equal to or higher than the affinity of the first antigen binding site for ErbB-2-positive cells. A bispecific antibody according to the present invention is also provided, which comprises a first antigen binding site that binds to domain I of ErbB-2 and a second antigen binding site that binds to domain III of ErbB-3, wherein the affinity of the second antigen binding site for ErbB-3-positive cells is equal to or higher than the affinity of the first antigen binding site for ErbB-2-positive cells. In a particularly preferred embodiment, a bispecific antibody according to the present invention is provided, which comprises a first antigen binding site that binds to domain I of ErbB-2 and a second antigen binding site that binds to domain III of ErbB-3, wherein the affinity of the second antigen binding site for ErbB-3-positive cells is equal to or higher than the affinity of the first antigen binding site for ErbB-2-positive cells.

[0088] The second antigen binding site preferably binds to domain III of ErbB-3 with an affinity (KD) for ErbB-3-positive cells of less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, preferably less than or equal to 1.39 nM, more preferably less than or equal to 0.99 nM. In a preferred embodiment, the second antigen binding site binds to domain III of ErbB-3 with an affinity for ErbB-3 on SK BR 3 cells of less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, preferably less than or equal to 1.39 nM, more preferably less than or equal to 0.99 nM. In one embodiment, the affinity is in the range of 1.39 to 0.59 nM. In a preferred embodiment, the second antigen-binding site binds to domain III of ErbB-3 with an affinity for ErbB-3 on BT 474 cells of less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.0 nM, more preferably less than or equal to 0.5 nM, more preferably less than or equal to 0.31 nM, more preferably less than or equal to 0.23 nM. In one embodiment, the affinity is in the range of 0.31 to 0.15 nM.

[0089] The first antigen binding site preferably binds to domain I of ErbB-2 with an affinity (KD) for ErbB-2-positive cells of less than or equal to 5.0 nM, more preferably less than or equal to 4.5 nM, more preferably less than or equal to 3.9 nM. In a preferred embodiment, the first antigen binding site binds to domain I of ErbB-2 with an affinity for ErbB-2 on SK BR 3 cells of less than or equal to 5.0 nM, more preferably less than or equal to 4.5 nM, more preferably less than or equal to 4.0 nM, more preferably less than or equal to 3.5 nM, more preferably less than or equal to 3.0 nM, more preferably less than or equal to 2.5 nM, more preferably less than or equal to 2.3 nM. In one embodiment, the affinity is in the range of 3.0 to 1.6 nM. The affinity of the bispecific antibody for SK BR 3 cells is preferably less than or equal to 5.0 nM, more preferably less than or equal to 4.5 nM, more preferably less than or equal to 4.0 nM, more preferably less than or equal to 3.5 nM, more preferably less than or equal to 3.0 nM, more preferably less than or equal to 2.5 nM, more preferably less than or equal to 2.4 nM, more preferably less than or equal to 2.0 nM. In one embodiment, the affinity is in the range of 2.4 to 1.6 nM.

[0090] In a preferred embodiment, the first antigen binding site binds to domain I of ErbB-2 and has an affinity (KD) for ErbB-2 on BT 474 cells of less than or equal to 5.0 nM, more preferably less than or equal to 4.5 nM, preferably less than or equal to 3.9 nM. In one embodiment, the affinity is in the range of 4.5 to 3.3 nM. The affinity of the bispecific antibody for BT 474 cells is preferably less than or equal to 5.0 nM, more preferably less than or equal to 4.5 nM, more preferably less than or equal to 4.0 nM, more preferably less than or equal to 3.7 nM, more preferably less than or equal to 3.2 nM. In one embodiment, the affinity is in the range of 3.7 to 2.7 nM.

[0091] Likewise, the above affinities are preferably measured as using steady-state cellular affinity measurements, wherein cells are incubated with radiolabeled antibodies at 4°C and cell-bound radioactivity is measured as described in the Examples.

[0092] Another preferred embodiment provides a bispecific antibody according to the present invention, comprising a first antigen binding site that binds to ErbB-2 and a second antigen binding site that binds to ErbB-3, wherein the antibody can reduce ligand-induced receptor function of ErbB-3 on ErbB-2 and ErbB-3 positive cells, wherein the bispecific antibody does not significantly affect the survival of cardiomyocytes. Cardiotoxicity is a known risk factor in ErbB-2 targeted therapy, and the frequency of complications increases when trastuzumab is used in combination with anthracyclines, thereby inducing cardiac stress. For example, the combination of doxycycline (DOX) and trastuzumab induces severe cardiac side effects. Clinical studies estimate that 5% to 10% of patients receiving trastuzumab in the adjuvant setting for breast cancer develop cardiac dysfunction (Guarneri et al., J Clin Oncol., 1985, 3: 818-26; Ewer MS et al., Nat Rev Cardiol 2010; 7: 564-75). However, a retrospective study showed that when trastuzumab is used with DOX in the adjuvant setting, the risk of developing silent cardiac dysfunction is actually as high as about 25% (Wadhwa et al., Breast Cancer Res Treat 2009; 117: 357-64). As shown in the Examples, the present invention provides antibodies that target ErbB-2 and do not affect, or affect to a significantly lesser extent, the survival of cardiomyocytes compared to trastuzumab and pertuzumab. This offers a significant advantage due to reduced cardiotoxicity. This is already advantageous for people who do not have impaired cardiac function, and is even more so for people who do have or are at risk of impaired cardiac function (e.g., subjects with congestive heart failure (CHF), left ventricular dysfunction (LVD) and / or a decrease in left ventricular ejection fraction (LVEF) ≥ 10%, and / or subjects who have had a myocardial infarction). Therefore, antibodies according to the present invention that do not significantly affect cardiomyocyte survival are preferred. In vitro, cardiomyocyte function is measured, for example, by determining cardiomyocyte viability, by determining BNP (B-type natriuretic peptide, which is a cardiac biomarker), by determining QT prolongation and / or by determining mitochondrial membrane potential.

[0093] The antibody according to the present invention preferably comprises a first antigen binding site that binds to domain I of ErbB-2 and a second antigen binding site that binds to domain III of ErbB-3. One embodiment provides an antibody according to the present invention that does not significantly affect cardiomyocyte survival, comprising a first antigen binding site that binds to ErbB-2 and a second antigen binding site that binds to ErbB-3, wherein the affinity of the second antigen binding site for ErbB-3-positive cells is equal to or greater than the affinity of the first antigen binding site for ErbB-2-positive cells. The affinity of the second antigen binding site for ErbB-3-positive cells is preferably less than or equal to 2.0 nM, more preferably less than or equal to 1.39 nM, more preferably less than or equal to 0.99 nM. The affinity of the first antigen binding site for ErbB-2-positive cells is preferably less than or equal to 5.0 nM, preferably less than or equal to 4.5 nM, preferably less than or equal to 4.0 nM.

[0094] In a preferred embodiment, the antibody that does not significantly affect cardiomyocyte survival comprises:

[0095] - at least the CDR3 sequence (preferably at least the CDR1, CDR2 and CDR3 sequences) of the ErbB-2-specific heavy chain variable region or at least the heavy chain variable region sequence selected from the group consisting of: Figure 10A or Figure 10E , MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 shown in, or a heavy chain variable region sequence that differs from said heavy chain variable region sequence at at most 15 amino acids, preferably at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably at most 1, 2, 3, 4 or 5 amino acids; and / or at least the CDR3 sequence (preferably at least CDR1, CDR2 and CDR3 sequence) of the ErbB-3 specific heavy chain variable region or at least a heavy chain variable region sequence selected from the group consisting of: Figure 10B or Figure 10E or Figure 11or a heavy chain variable region sequence that differs from said heavy chain variable region sequence at at most 15 amino acids, preferably at at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably at at most 1, 2, 3, 4 or 5 amino acids. In a preferred embodiment, the antibody is PB4188.

[0096] Another aspect of the present invention provides an antibody according to the present invention comprising a first antigen binding site that binds ErbB-2 and a second antigen binding site that binds ErbB-3, wherein the antibody comprises an antigen binding site that binds at least one amino acid residue of ErbB-2 domain I selected from T144, T164, R166, P172, G179, S180, and R181, and a surface-exposed amino acid residue located within about 5 amino acid positions of T144, T164, R166, P172, G179, S180, or R181. The amino acid residues are numbered according to Protein Data Bank (PDB) ID #1S78. As shown in the Examples, antibodies that bind to this region of domain I of ErbB-2 exhibit particularly good binding characteristics and are able to counteract the activity of ErbB-2-positive cells (e.g., ligand-induced receptor function of ErbB-3 on ErbB-2 and ErbB-3-positive cells, and / or ligand-induced growth of such cells). In addition, such antibodies are particularly suitable for combination therapy with currently known anti-ErbB-2 monoclonal antibodies, such as trastuzumab (which binds to domain IV of ErbB-2) and pertuzumab (which binds to domain II of ErbB-2), because they bind to different domains of ErbB-2. Therefore, these antibodies can be used simultaneously without competing for the same epitope. The term "surface-exposed amino acid residue located at about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181" refers to an amino acid residue that is in the primary amino acid sequence within about the first five amino acid residues adjacent to the residue and is at least partially exposed to the outside of the protein so that it can be bound by the antibody (see, for example, Figure 21B of WO2015 / 130173). Preferably, the amino acid residue located within about 5 amino acid positions of T144, T164, R166, P172, G179, S180 or R181 is selected from the group consisting of L139, C140, Y141, Q142, D143, 1145, L146, W147, K148, D149, L159, T160, L161, 1162, D163, N165, S167, R168, A169, C170, H171, C173, S174, P175, M176, C177, K178, C182, W183, G184, E185 and S186.Preferably, the antibody comprises an antigen binding site that binds to at least 2 or at least 3 amino acid residues of domain I of ErbB-2 selected from T144, T164, R166, P172, G179, S180 and R181, and a surface-exposed amino acid residue located within 5 amino acid positions of T144, T164, R166, P172, G179, S180 or R181.

[0097] In a preferred embodiment, a bispecific antibody according to the invention is provided, wherein the antibody comprises an antigen binding site that binds to at least T144, R166, and R181 of domain I of ErbB-2. Another embodiment provides a bispecific antibody according to the invention, wherein the antibody comprises an antigen binding site that binds to at least T144, R166, P172, G179, and R181 of domain I of ErbB-2. Another embodiment provides a bispecific antibody according to the invention, wherein the antibody comprises an antigen binding site that binds to at least T144, T164, R166, P172, G179, S180, and R181 of domain I of ErbB-2.

[0098] Another aspect of the present invention provides an antibody comprising a first antigen binding site that binds to ErbB-2 and a second antigen binding site that binds to ErbB-3, wherein the antibody comprises an antigen binding site that binds to at least one amino acid of domain III of ErbB-3 selected from R426 and located 100% from R426 in a native ErbB-3 protein. The amino acid residues are numbered as those in Protein Data Bank (PDB) ID# 4P59. As shown in the Examples, antibodies that bind to this region of domain III of ErbB-3 exhibit particularly good binding characteristics and are able to counteract the activity of ErbB-3-positive cells (e.g., ligand-induced receptor function of ErbB-3 on ErbB-2 and ErbB-3-positive cells, and / or ligand-induced growth of such cells). The term "located within 100 nm of the native ErbB-3 protein" refers to a region of the ErbB-3 domain that is located 100 nm from the native ErbB-3 protein. The term "surface exposed amino acid residue within" refers to an amino acid residue that is spatially located within a certain distance from R426. The tertiary structure of the ErbB-3 protein is located within the tertiary structure of the ErbB-3 protein and is at least partially exposed to the outside of the protein so that it can be bound by an antibody. The amino acid residues within are selected from L423, Y424, N425, G427, G452, R453, Y455, E480, R481, L482, D483 and K485 (see, for example, Figure 21C and Table 15 of WO2015 / 130173). In a preferred embodiment, a bispecific antibody according to the invention is provided, wherein the antibody comprises an antigen binding site that binds to at least R426 of domain III of ErbB-3. Preferably, the antibody comprises an antigen binding site that binds to at least R426 of domain III of ErbB-3.

[0099] The bispecific antibodies of the present invention are preferably afucosylated to enhance ADCC activity. When compared to the same antibody produced in normal CHO cells, the bispecific antibodies of the present invention preferably contain a reduced amount of fucosylated N-linked carbohydrate structures in the Fc region.

[0100] The bispecific antibodies of the present invention are preferably used in humans. To this end, the bispecific antibodies of the present invention are preferably human antibodies or humanized antibodies.

[0101] Human tolerance to polypeptides is controlled by many different aspects. Whether it is T cell-mediated, B cell-mediated, or other immunity, it is one of the variables encompassed in human tolerance to polypeptides. The constant region of the bispecific antibodies of the present invention is preferably a human constant region. The constant region may contain one or more, preferably no more than 10, preferably no more than 5 amino acid differences relative to the constant region of a naturally occurring human antibody. Preferably, the constant portion is entirely derived from naturally occurring human antibodies. The various antibodies produced herein are derived from a library of human antibody variable domains. Therefore, these variable domains are human. The unique CDR region may be derived from a human, synthetic, or derived from other organisms. When a variable region has an amino acid sequence identical to the amino acid sequence of a naturally occurring human antibody variable region, it is considered a human variable region, but with respect to the CDR region. The variable region of the ErbB-2-binding VH, ErbB-3-binding VH, or light chain in the antibodies of the present invention may contain one or more, preferably no more than 10, preferably no more than 5 amino acid differences compared to the variable region of a naturally occurring human antibody, without calculating possible differences in the amino acid sequence of the CDR region. Such mutations also occur in nature in the case of somatic hypermutation.

[0102] At least with respect to the heavy chain variable region, antibodies can be derived from a variety of animal species. It is common practice to humanize, for example, a murine heavy chain variable region. There are a variety of methods for achieving this, including: grafting the CDRs into a human heavy chain variable region having a 3D structure that matches the 3D structure of the murine heavy chain variable region; deimmunizing the murine heavy chain variable region, preferably by removing known or suspected T or B cell epitopes from the murine heavy chain variable region. Removal is typically performed by replacing one or more amino acids in the epitope with other (usually conservative) amino acids so that the sequence of the epitope is modified so that it is no longer a T cell epitope or a B cell epitope.

[0103] Such deimmunized murine heavy chain variable regions have lower immunogenicity in humans than the original murine heavy chain variable regions. Preferably, the variable regions or domains of the present invention are further humanized, for example, as vennered. By using venner technology, external residues that are easily encountered by the immune system are selectively replaced with human residues to provide hybrid molecules containing a vennered surface that is weakly immunogenic or substantially non-immunogenic. The animal used in the present invention is preferably a mammal, more preferably a primate, and most preferably a human.

[0104] The bispecific antibody according to the present invention preferably comprises the constant region of human antibody.According to the difference in its heavy chain constant domain, antibody can be divided into five categories or isotypes: IgG, IgA, IgM, IgD and IgE.These categories or isotypes comprise at least one described heavy chain named with corresponding Greek letters.In a preferred embodiment, the invention provides antibody according to the present invention, wherein the constant region is selected from IgG, IgA, IgM, IgD and IgE constant region, more preferably, the constant region comprises IgG constant region, more preferably IgG1 constant region, preferably mutated IgG1 constant region.Some variations in IgG1 constant region naturally occur, for example, such as allotype G1m1, 17 and G1m3, and / or it is allowed when the immunological properties of the obtained antibody are not changed.Usually, insertion, deletion, replacement or its combination between about 1 to 10 amino acids are allowed in the constant region.

[0105] In one embodiment, the present invention provides an antibody comprising a variable domain that binds ErbB-2, wherein the antibody comprises at least a CDR3 sequence of an ErbB-2-specific heavy chain variable region selected from the group consisting of: Figure 10A or Figure 10EMF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 as shown in, or wherein the antibody comprises a heavy chain CDR3 sequence that differs in at most three, preferably at most two, preferably not more than one amino acid from a CDR3 sequence of a VH selected from the group consisting of: Figure 10A or Figure 10E MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 shown in. The antibody preferably comprises at least the CDR3 sequence of MF1849, MF2971, MF3958, MF3004 or MF3991, most preferably at least the CDR3 sequence of MF3958.

[0106] The antibody preferably comprises at least CDR1, CDR2 and CDR3 sequences of the ErbB-2 specific heavy chain variable region selected from the group consisting of: Figure 10A or Figure 10E MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 shown in, or with MF2926, MF2930, MF1849; MF2973 , MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898. The antibody preferably comprises at least the CDR1, CDR2 and CDR3 sequences of MF1849, MF2971, MF3958, MF3004 or MF3991, most preferably at least the CDR1, CDR2 and CDR3 sequences of MF3958.

[0107] The present invention also provides an antibody comprising a variable domain that binds to ErbB-3, wherein the antibody comprises at least a CDR3 sequence of an ErbB-3 specific heavy chain variable region selected from the group consisting of: Figure 10B or Figure 10E or Figure 11 or wherein said antibody comprises a heavy chain CDR3 sequence that differs in at most three, preferably at most two, and preferably not more than one amino acid from a CDR3 sequence of a VH selected from the group consisting of: Figure 10B or Figure 10E or Figure 11 MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074. The antibody preferably comprises at least the CDR3 sequence of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065, most preferably at least the CDR3 sequence of MF3178.

[0108] The antibody preferably comprises at least CDR1, CDR2 and CDR3 sequences of the ErbB-3 specific heavy chain variable region selected from the group consisting of: Figure 10B or Figure 10E or Figure 11MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 shown in, or with at most three, preferably at most, of the following CDR1, CDR2 and CDR3 sequences: The heavy chain CDR1, CDR2 and CDR3 sequences differ in at most two, preferably at at most one, amino acid sequence: MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074. The antibody preferably comprises at least the CDR1, CDR2 and CDR3 sequences of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065, most preferably at least the CDR1, CDR2 and CDR3 sequences of MF3178.

[0109] In one embodiment, the present invention provides a bispecific antibody comprising a first antigen binding site that binds ErbB-2 and a second antigen binding site that binds ErbB-3, wherein the first antigen binding site comprises at least a CDR3 sequence of an ErbB-2-specific heavy chain variable region selected from the group consisting of: Figure 10A or Figure 10E MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 shown in, or a heavy chain CDR3 sequence that differs in at most three, preferably at most two, and preferably not more than one amino acid from a CDR3 sequence of a VH selected from the group consisting of: Figure 10A or Figure 10EMF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 as shown in FIG, and wherein the second antigen binding site comprises at least a CDR3 sequence of an ErbB-3 specific heavy chain variable region selected from the group consisting of: Figure 10B or Figure 10E or Figure 11 or a heavy chain CDR3 sequence that differs in at most three, preferably at most two, and preferably not more than one amino acid from a CDR3 sequence of a VH selected from the group consisting of: Figure 10B or Figure 10E or Figure 11 MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 shown in. The first antigen binding site preferably comprises at least the CDR3 sequence of MF1849, MF2971, MF3958, MF3004 or MF3991, most preferably at least the CDR3 sequence of MF3958, and the second antigen binding site preferably comprises at least the CDR3 sequence of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065, most preferably at least the CDR3 sequence of MF3178.

[0110] The first antigen binding site preferably comprises at least CDR1, CDR2 and CDR3 sequences of the ErbB-2 specific heavy chain variable region selected from the group consisting of: Figure 10A or Figure 10EMF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 shown in, or heavy chain CDR1, CDR2 that differ from the following CDR1, CDR2 and CDR3 sequences in at most three, preferably at most two, preferably at most one amino acid sequence: and CDR3 sequences: MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898, and the second antigen-binding site preferably comprises at least the CDR1, CDR2 and CDR3 sequences of the ErbB-3 specific heavy chain variable region selected from the group consisting of: Figure 10B or Figure 10E or Figure 11 or heavy chain CDR1, CDR2 and CDR3 sequences that differ from the following CDR1, CDR2 and CDR3 sequences at at most three, preferably at at most two, and preferably at at most one amino acid position: Figure 10B or Figure 10E or Figure 11MF3178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059;MF6060;MF6061;MF6062;MF6063;MF6064;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 or MF6074 shown in. The first antigen binding site preferably comprises at least the CDR1, CDR2 and CDR3 sequences of MF1849, MF2971, MF3958, MF3004 or MF3991, most preferably at least the CDR1, CDR2 and CDR3 sequences of MF3958, and the second antigen binding site preferably comprises at least the CDR1, CDR2 and CDR3 sequences of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065, most preferably at least the CDR1, CDR2 and CDR3 sequences of MF3178.

[0111] A preferred embodiment provides a bispecific antibody comprising a first antigen binding site that binds to ErbB-2 and a second antigen binding site that binds to ErbB-3, wherein the first antigen binding site comprises at least the CDR3 sequence of MF3958, or a CDR3 sequence that differs from the CDR3 sequence of MF3958 at at most three, preferably at most two, and preferably at no more than one amino acid, and wherein the second antigen binding site comprises at least the CDR3 sequence of MF3178, or a CDR3 sequence that differs from the CDR3 sequence of MF3178 at at most three, preferably at most two, and preferably at no more than one amino acid.

[0112] In one embodiment the invention provides a bispecific antibody comprising a first antigen binding site that binds to ErbB-2 and a second antigen binding site that binds to ErbB-3, wherein the first antigen binding site comprises at least the CDR1, CDR2 and CDR3 sequence of MF3958, or CDR1, CDR2 and CDR3 sequences that differ from the CDR1, CDR2 and CDR3 sequences of MF3958 at at most three, preferably at most two, preferably at most one amino acid, and wherein the second antigen binding site comprises at least the CDR1, CDR2 and CDR3 sequence of MF3178, or CDR1, CDR2 and CDR3 sequences that differ from the CDR1, CDR2 and CDR3 sequences of MF3178 at at most three, preferably at most two, preferably at most one amino acid.

[0113] In one embodiment the invention provides a bispecific antibody comprising a first antigen binding site that binds ErbB-2 and a second antigen binding site that binds ErbB-3, wherein the first antigen binding site comprises at least the CDR3 sequence of MF3958 and wherein the second antigen binding site comprises at least the CDR3 sequence of MF3178.

[0114] In one embodiment, the invention provides a bispecific antibody comprising a first antigen binding site that binds ErbB-2 and a second antigen binding site that binds ErbB-3, wherein the first antigen binding site comprises at least the CDR1, CDR2, and CDR3 sequences of MF3958, and wherein the second antigen binding site comprises at least the CDR1, CDR2, and CDR3 sequences of MF3178.

[0115] For example, the CDR sequence can be changed for optimization purposes, preferably in order to improve the binding efficacy or stability of the antibody. For example, optimization can be performed by a mutagenesis program, and then the stability and / or binding affinity of the resulting antibody can be preferably tested, and the improved ErbB-2 or ErbB-3 specific CDR sequence can be preferably selected. The technician can well produce antibody variants comprising the CDR sequence of at least one change according to the present invention. For example, conservative amino acid replacements can be used. Some examples of conservative amino acid replacements include replacing another hydrophobic residue with a hydrophobic residue (for example, isoleucine, valine, leucine or methionine), and replacing another polar residue with a polar residue, for example, replacing lysine with arginine, replacing aspartic acid with glutamic acid, or replacing asparagine with glutamine.

[0116] In one embodiment, the present invention provides an antibody comprising a variable domain that binds to ErbB-2, wherein the VH chain of the variable domain comprises the following VH chain amino acid sequence: Figure 10A or Figure 10E MF2926; MF2930; MF1849; MF2973; MF3004; MF3958 (which is a humanized MF2971); MF2971; MF3025; MF2916; MF3991 (which is a humanized MF3004); MF3031; MF2889; MF2913; MF1847; MF3001, MF3003 or MF1898; or comprising a molecule relative to Figure 10A or Figure 10E The above VH chain sequence has at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, of the following VH chain amino acid sequence: Figure 10A or Figure 10EMF2926; MF2930; MF1849; MF2973; MF3004; MF3958 (which is a humanized MF2971); MF2971; MF3025; MF2916; MF3991 (which is a humanized MF3004); MF3031; MF2889; MF2913; MF1847; MF3001, MF3003 or MF1898 shown in FIG. The VH chain of the variable domain that binds to ErbB-2 preferably comprises the following amino acid sequence:

[0117] -like Figure 10A MF1849 as shown in ; or

[0118] - MF2971 or a humanized form thereof, wherein said humanized form preferably comprises the amino acid sequence of MF3958; or

[0119] -MF3004 or a humanized form thereof, wherein the humanized form preferably comprises the amino acid sequence of MF3991. In one embodiment, the VH chain of the variable domain that binds to ErbB-2 comprises the amino acid sequence of the following VH chain: MF1849; or MF2971 or a humanized form thereof, wherein the humanized form preferably comprises the amino acid sequence of the following VH chain: MF3958; or MF3004 or a humanized form thereof, wherein the humanized form preferably comprises the amino acid sequence of MF3991, wherein the VH sequence is Figure 10A The corresponding sequence shown in has at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof. In a preferred embodiment, the VH chain of the variable domain that binds to ErbB-2 comprises the amino acid sequence of MF3958; or comprises a VH chain sequence having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH chain sequence. Figure 10A The amino acid sequence of MF3958 is shown in . The antibody comprising a variable domain that binds to ErbB-2 is preferably a bispecific antibody, which preferably also comprises a variable domain that binds to ErbB-3. The VH chain of the variable domain that binds to Erb-B3 preferably comprises the following VH chain amino acid sequence: Figure 10B or Figure 10E or Figure 11MF3178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059;MF6060;MF6061;MF6062;MF6063;MF6064;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 or MF6074; or containing reference to Figure 10B or Figure 10E or Figure 11 The VH chain sequence of the following VH chain has at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof: Figure 10B or Figure 10E or Figure 11 The VH chain of the variable domain that binds to Erb-B3 preferably comprises the amino acid sequence of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065; or comprises the amino acid sequence relative to MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065; or comprises the amino acid sequence relative to MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065; Figure 10B or Figure 11 The amino acid sequence of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065 has at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the corresponding VH chain sequence. In a preferred embodiment, the VH chain of the variable domain that binds to ErbB-3 comprises the amino acid sequence of MF3178; or comprises a VH chain sequence having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the corresponding VH chain sequence. Figure 10B The amino acid sequence of MF3178 is shown in . Preferably, the above amino acid insertions, deletions, and substitutions do not exist in the CDR3 region. The above amino acid insertions, deletions, and substitutions also preferably do not exist in the CDR1 and CDR2 regions. The above amino acid insertions, deletions, and substitutions also preferably do not exist in the FR4 region.

[0120] The present invention also provides an antibody comprising a variable domain that binds to ErbB-3, wherein the VH chain of the variable region comprises the following VH chain amino acid sequence: Figure 10B or Figure 10E or Figure 11 or MF6074; or a VH chain comprising the amino acid sequence of at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof relative to said VH chain sequence: Figure 10B or Figure 10E or Figure 11 MF3178;MF3176;MF3163;MF3099;MF3307;MF6055;MF6056;MF6057;MF6058;MF6059;MF6060;MF6061;MF6062;MF6063;MF6064;MF6065;MF6066;MF6067;MF6068;MF6069;MF6070;MF6071;MF6072;MF6073 or MF6074 shown in. The VH chain of the variable domain that binds to ErbB3 preferably comprises the amino acid sequence of the following VH chain: MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065; or comprises the amino acid sequence of the VH chain of MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065 having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof relative to the VH chain sequence. In a preferred embodiment, the VH chain of the variable domain that binds to ErbB-3 comprises Figure 10B or comprising at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH chain sequence; Figure 10BThe amino acid sequence of the VH chain MF3178 is shown in . The antibody comprising a variable domain that binds to ErbB-3 is preferably a bispecific antibody, which preferably also comprises a variable domain that binds to ErbB-2. The VH chain of the variable domain that binds to ErbB-2 preferably comprises Figure 10A or Figure 10E The VH chain of the variable domain that binds to ErbB-2 preferably comprises the following amino acid sequence: MF1849; or MF2971 or a humanized form thereof, wherein the humanized form preferably comprises the following amino acid sequence: MF3958; or MF3004 or a humanized form thereof, wherein the humanized form preferably comprises Figure 10A In one embodiment, the VH sequence of the Erb-B2 binding protein is relative to Figure 10A The corresponding sequence described in has at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof. In a preferred embodiment, Figure 10A The ErbB-2-binding VH chain comprises the amino acid sequence of MF3958; or comprises the amino acid sequence of MF3958 having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and more preferably up to 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or a combination thereof relative to the VH chain sequence. Preferably, these amino acid insertions, deletions, and substitutions are not present in the CDR3 region. These amino acid insertions, deletions, and substitutions are also preferably not present in the CDR1 and CDR2 regions. These amino acid insertions, deletions, and substitutions are also preferably not present in the FR4 region.

[0121] Also provided is an antibody according to the invention, wherein the antibody comprises an ErbB-2 specific heavy chain variable region sequence selected from the group consisting of: Figure 10A or Figure 10EMF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 shown in, or wherein the antibody comprises the heavy chain variable region sequence of MF2926, MF2930, MF1849; MF2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 and MF1898 The heavy chain variable region sequences of F2973, MF3004, MF3958, MF2971, MF3025, MF2916, MF3991, MF3031, MF2889, MF2913, MF1847, MF3001, MF3003 or MF1898 differ in at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acids.

[0122] Also provided is an antibody according to the invention, wherein the antibody comprises an ErbB-3 specific heavy chain variable region sequence selected from the group consisting of: Figure 10B or Figure 10E or Figure 11 and MF6074, or wherein the antibody comprises the heavy chain variable region sequences of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 and MF6074 as shown in The heavy chain variable region sequences of F3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074 differ in the heavy chain variable region sequences at at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at at most 1, 2, 3, 4 or 5 amino acids.

[0123] In one embodiment, the present invention provides an antibody comprising two antigen binding sites that bind to ErbB-2, wherein at least one of the antigen binding sites binds to domain I of ErbB-2. Preferably, both antigen binding sites bind to domain I of ErbB-2. Such antibodies according to the present invention are particularly suitable for combination therapy with currently used anti-ErbB-2 binding molecules that do not bind to domain I of ErbB-2 (e.g., trastuzumab, which binds to domain IV of ErbB-2, and pertuzumab, which binds to domain II of ErbB-2), because the different binding molecules do not compete with each other for the same epitope.

[0124] Also provided are antibodies comprising two antigen-binding sites that bind to ErbB-2, wherein at least one of the antigen-binding sites binds to domain I of ErbB-2, and wherein the affinity (KD) of the at least one antigen-binding site for ErbB-2-positive cells is less than or equal to 5.0 nM, preferably less than or equal to 4.5 nM, more preferably less than or equal to 3.9 nM. Preferably, both antigen-binding sites bind to domain I of ErbB-2. In a preferred embodiment, the affinity of the at least one antigen-binding site for ErbB-2 on SK BR 3 cells is less than or equal to 5.0 nM, preferably less than or equal to 4.5 nM, more preferably less than or equal to 4.0 nM, more preferably less than or equal to 3.5 nM, more preferably less than or equal to 3.0 nM, more preferably less than or equal to 2.3 nM. In one embodiment, the affinity is in the range of 3.0 to 1.6 nM. In a preferred embodiment, the affinity of the at least one antigen binding site for ErbB-2 on BT 474 cells is lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 3.9 nM. In one embodiment, the affinity is in the range of 4.5 to 3.3 nM.

[0125] The above affinity is preferably measured as using steady-state cellular affinity measurements, wherein cells are incubated with radiolabeled antibodies at 4°C and the cell-bound radioactivity is measured as described in the Examples.

[0126] The present invention also provides an antibody comprising two variable domains that bind to ErbB-2, wherein the VH chain of the variable domain comprises the following VH chain amino acid sequence: Figure 10A or Figure 10EMF2926; MF2930; MF1849; MF2973; MF3004; MF3958 (which is a humanized MF2971); MF2971; MF3025; MF2916; MF3991 (which is a humanized MF3004); MF3031; MF2889; MF2913; MF1847; MF3001, MF3003 or MF1898; or relative to Figure 10A or Figure 10E The amino acid sequence of the VH chain shown in the corresponding sequence has up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably up to 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof: Figure 10A or Figure 10E MF2926; MF2930; MF1849; MF2973; MF3004; MF3958 (which is a humanized MF2971); MF2971; MF3025; MF2916; MF3991 (which is a humanized MF3004); MF3031; MF2889; MF2913; MF1847; MF3001, MF3003 or MF1898 shown in . The VH preferably comprises the amino acid sequence of the following VH chain: MF1849; or MF2971 or a humanized form thereof, wherein the humanized form preferably comprises the following amino acid sequence: MF3958; or MF3004 or a humanized form thereof, wherein the humanized form preferably comprises as Figure 10A or MF3991 or a humanized form thereof, wherein the humanized form preferably comprises the amino acid sequence of the VH chain of MF1849; or MF2971 or a humanized form thereof, wherein the humanized form preferably comprises the amino acid sequence of MF3958; or MF3004 or a humanized form thereof, wherein the humanized form preferably comprises the amino acid sequence of MF3958 relative to Figure 10A The corresponding sequence shown in has up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably up to 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof. Figure 10A The variable domains of the antibodies preferably comprise the same VH chain, preferably having Figure 10A or Figure 10E The sequences shown in . Antibodies with variable domains that have the same VH chain are not bispecific antibodies. Figure 10A or Figure 10E or Figure 11 The same VH chain sequence as shown in, or relative to Figure 10A or Figure 10E or Figure 11Corresponding sequences shown are identical for the purposes of the present invention if they have the same VH chain sequence with 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof.

[0127] In one embodiment, the present invention provides antibodies comprising two antigen binding sites that bind to ErbB-3, wherein at least one of the antigen binding sites binds to domain III of ErbB-3. Preferably, both antigen binding sites bind to domain III of ErbB-3. Such antibodies according to the present invention are particularly suitable for combination therapy with currently used anti-ErbB-3 binding molecules that do not bind to domain III of ErbB-3 (e.g., MM-121 (#Ab6) and RG7116, which bind to domain I of ErbB-3), because the different binding molecules do not compete with each other for the same epitope.

[0128] Also provided are antibodies comprising two antigen-binding sites that bind to ErbB-3, wherein at least one of the antigen-binding sites binds to domain III of ErbB-3, and wherein the affinity (KD) of the at least one antigen-binding site for ErbB-3-positive cells is less than or equal to 2.0 nM, preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.39 nM, and more preferably less than or equal to 0.99 nM. Preferably, both antigen-binding sites bind to domain III of ErbB-3. In a preferred embodiment, the affinity of the at least one antigen-binding site for ErbB-3 on SK BR 3 cells is less than or equal to 2.0 nM, preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.39 nM, and more preferably less than or equal to 0.99 nM. In one embodiment, the affinity is in the range of 1.39 to 0.59 nM. In a preferred embodiment, the at least one antigen binding site has an affinity for ErbB-3 on BT 474 cells of less than or equal to 2.0 nM, more preferably less than or equal to 1.5 nM, more preferably less than or equal to 1.0 nM, more preferably less than or equal to 0.5 nM, more preferably less than or equal to 0.31 nM, more preferably less than or equal to 0.23 nM. In one embodiment, the affinity is in the range of 0.31 to 0.15 nM.

[0129] Likewise, the above affinities are preferably measured as using steady-state cellular affinity measurements, wherein cells are incubated with radiolabeled antibodies at 4°C and cell-bound radioactivity is measured as described in the Examples.

[0130] The present invention also provides an antibody comprising two variable domains, each of which binds to ErbB3, wherein the VH of the variable domain comprises the following VH chain amino acid sequence: Figure 10B or Figure 10E or Figure 11 or MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074; or comprising up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or more, relative to any of said VH chain sequences 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, of the amino acid sequence of the VH chain of MF3178; MF3176; MF3163; MF3099; MF3307; MF6055; MF6056; MF6057; MF6058; MF6059; MF6060; MF6061; MF6062; MF6063; MF6064; MF6065; MF6066; MF6067; MF6068; MF6069; MF6070; MF6071; MF6072; MF6073 or MF6074. The VH preferably comprises the amino acid sequence of the VH chain MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065; or comprises the amino acid sequence of the VH chain MF3178, MF3176, MF3163, MF6058, MF6061 or MF6065 having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to any of the VH chain sequences. The VH preferably comprises the amino acid sequence of the VH chain MF3178; or comprises the amino acid sequence of the VH chain MF3178 having at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably at most 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or combinations thereof relative to the VH chain sequence of MF3178. Figure 10B The amino acid sequence of the VH chain MF3178 is shown in FIG. The variable domain of the antibody preferably comprises the same VH chain, which preferably has Figure 10B or Figure 10E or Figure 11 The sequences shown in . Antibodies with variable domains that have the same VH chain are not bispecific antibodies. Figure 10B or Figure 10E or Figure 11 The same VH chain sequence as shown in, or relative to Figure 10B or Figure 10E or Figure 11If the VH chain sequences of the sequences of the proteins are identical to the VH chain sequences of the proteins, they are identical if the sequences of the proteins have 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof.

[0131] The ErbB-2 / ErbB-3 specific antibodies disclosed herein are preferably bispecific antibodies. The antibodies preferably comprise a variable domain having a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of the ErbB-2 specific heavy chain variable region of MF3958 as shown in FIG10 , or CDR sequences that differ from the CDR1, CDR2, and CDR3 sequences of MF3958 at no more than 3 amino acids, preferably at no more than 2 amino acids, preferably at no more than 1 amino acid, and a variable domain having a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of the ErbB-3 specific heavy chain variable region of MF3178 as shown in FIG10 , or CDR sequences that differ from the CDR1, CDR2, and CDR3 sequences of MF3178 at no more than 3 amino acids, preferably at no more than 2 amino acids, preferably at no more than 1 amino acid.

[0132] The ErbB-2 / ErbB-3 specific antibodies preferably comprise: an ErbB-2 specific variable domain having a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region of MF3958 as shown in FIG10 with 0 to 10, preferably 0 to 5, preferably 0, 1 or 2 amino acid substitutions; and an ErbB-3 specific variable domain having a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region of MF3178 as shown in FIG10 with 0 to 10, preferably 0 to 5, preferably 0, 1 or 2 amino acid substitutions. The light chains in these antibodies are preferably Figure 10C of light chain.

[0133] The method of treating a subject having or at risk of having breast cancer preferably further comprises determining the expression level of estrogen receptor, ErbB-2, ErbB-3, or a combination thereof on cells of the cancer.

[0134] The object that suffers from breast cancer or is in the risk of suffering from breast cancer is preferably people.If a people once suffered from breast cancer in the past, but it is in remission and makes it impossible to find cancer by routine examination, then this people is in the risk of suffering from breast cancer.Can think that such people have been cured, but when compared with the normal healthy individual of the same age, this people suffers from the risk of breast cancer higher.Breast cancer can be the recurrence cancer at the primary cancer position that is in remission or normally is the metastasis of breast cancer at the position different from the position of the primary cancer position.

[0135] The present invention also provides a method for treating a subject having breast cancer or at risk of having such a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that binds to the extracellular portion of ErbB-2 and inhibits ErbB-2 / ErbB-3 dimerization on cancer cells, wherein the cancer is a hormone receptor-positive cancer. The antibody is preferably a bispecific antibody having an antigen binding site that binds to the extracellular portion of ErbB-2 and an antigen binding site that binds to the extracellular portion of ErbB-3. The antibody is preferably a bispecific ErbB-2 / ErbB-3-specific antibody disclosed herein. The method preferably further comprises administering to a subject in need thereof a therapeutically effective amount of an endocrine therapeutic drug. The cancer is preferably an immunohistochemically ErbB-2+ cancer or an immunohistochemically ErbB-2++ cancer without ErbB-2 gene amplification.

[0136] Typically, the bispecific antibody and endocrine therapy will be administered repeatedly during the course of treatment. For example, in certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of endocrine therapy and multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of the bispecific antibody are administered to a subject in need of treatment.

[0137] In some embodiments, the administration of endocrine therapy can be given daily, several days per week, weekly, every two weeks, every 3 or 4 weeks, or at longer intervals, and the administration of bispecific antibodies can be given weekly, every two weeks, every 3 or 4 weeks, or at longer intervals. The bispecific antibodies and endocrine therapy can be, but not necessarily, administered according to the same administration regimen. Thus, endocrine therapy and bispecific antibodies can be administered on the same day or on different days in different orders (first endocrine, second bispecific, or vice versa). Endocrine therapy can be administered 1 or more days before or after the bispecific antibody, or vice versa.

[0138] In some embodiments, the dosage of bispecific antibody and / or endocrine therapy varies over time. Throughout the treatment process, the dosage of endocrine therapy and / or bispecific therapy may be identical. Alternatively, the dosage of endocrine therapy and / or bispecific therapy may be higher at the beginning, such as loading a higher dosage (which may be a unique dosage or several dosages), followed by a maintenance dose. Alternatively, the dosage of endocrine therapy and / or bispecific therapy may be lower at the beginning (which may be a unique dosage or several dosages), followed by a maintenance dose. Additionally or alternatively, the endocrine therapy of the medicament starting from a weekly regimen or the initial administration regimen of the two may be changed to a biweekly regimen or other. Clinicians can use the preferred dosage and / or dosage regimen ensured by the situation of the treated patient.

[0139] Treatment with endocrine therapy can be initiated with a smaller dose than the optimal dose of the compound. Thereafter, the dose can be increased in small amounts until the optimal effect is achieved under the circumstances. For convenience, if necessary, the total daily dose can be divided and administered in batches throughout the day. Intermittent treatment (e.g., one week for three weeks or three weeks for four weeks) can also be used.

[0140] In certain embodiments, the bispecific antibody is administered at a flat dose of about 750 mg every 3 weeks, but alternatively, it may be given at a dosage range of 300 mg to 900 mg flat dose in a dosing schedule of weekly, biweekly, every 3 weeks, every 4 weeks, or longer intervals.

[0141] When a range is given herein between the numbers 1 and 2, the range includes the numbers 1 and 2. For example, a range between 2 and 5 includes the numbers 2 and 5.

[0142] When referring to an affinity that is higher than another affinity herein, Kd = lower than another Kd. For the avoidance of doubt, a Kd of 10e-9 M is lower than a Kd of 10e-8 M. An antibody with a Kd of 10e-9 M for a target has a higher affinity than when the Kd is 10e-8 M.

[0143] For purposes of clarity and concise description, features are described herein as part of the same or different embodiments; however, it should be understood that the scope of the invention may include embodiments having combinations of all or some of the described features. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 Mean and individual tumor growth changes in human HBCx-34 breast tumor xenografts. Treatment began 36 days after HBCx-34 implantation. MCLA-128 vehicle and 25 mg / kg of MCLA-128 were administered on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, and D56, and letrozole vehicle and 2.5 mg / kg of letrozole were administered daily for 57 days. Initial group size: 9 to 10 animals.

[0146] Figure 2T / C% in human HBCx-34 breast tumor xenografts. Treatment began 36 days after HBCx-34 implantation. MCLA-128 vehicle and 25 mg / kg of MCLA-128 were administered on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, and D56, and letrozole vehicle and 2.5 mg / kg of letrozole were administered daily for 57 days. Initial group size: 9 to 10 animals.

[0147] Figure 3 Mean and individual relative body weight changes in human HBCx-34 breast tumor xenografts. Treatment began 36 days after HBCx-34 implantation. MCLA-128 vehicle and 25 mg / kg of MCLA-128 were administered on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, and D56, and letrozole vehicle and 2.5 mg / kg of letrozole were administered daily for 57 days. Initial group size: 9 to 10 animals.

[0148] Figure 4. In vivo growth characteristics of the HBCx-34 PDX model. A: IHC characteristics of HBCx-34 PDX tumors. B: Athymic nude mice were sc-implanted with HBCx-34 tumors. Estrogen-supplemented animals were treated with fulvestrant vehicle, and estrogen-naive animals were treated with vehicle or letrozole. Estrogen supplementation induced faster tumor growth, and letrozole significantly reduced the formation of estrogen-independent tumors. T-tests were performed to compare tumor volumes of estrogen-independent tumors treated with vehicle or letrozole (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

[0149] Figure 5 Tumor volume in MCF-7 xenograft mice on day 25. Treatment started

[0150] MCLA-128 vehicle, 25 mg / kg of MCLA-128 were administered on D0, D1, D4, D8, D11, D15, D22, and D25, and hormone therapy vehicle and tamoxifen were administered every other day (D1, D3, D5, etc.). Fulvestrant was administered once a week. Statistical significance compared to the vehicle group for KruskalWallisDunn's test or Mann-Whimey U test: ns indicates not significant, * indicates 0.01 < P < 0.05, ** indicates 0.001 < P < 0.01, *** indicates P < 0.001. TGI > 60% indicates potential therapeutic activity. MTV: mean tumor volume, G1 MTV: Group 1 (vehicle) MTV, TGI: tumor growth inhibition.

[0151] Figure 6 Tumor volumes in MCF-7 xenograft mice treated with MCLA-128, fulvestrant, or tamoxifen as single agents or in combination. For each time point, a statistical t-test compared fulvestrant with fulvestrant plus MCLA-128 and tamoxifen with tamoxifen plus MCLA-128. ns indicates not significant; * indicates P < 0.05; ** indicates P < 0.01.

[0152] Figure 7 Inter-assay correlation between two independent VeraTag assays. Assay scores obtained in rounds 1 and 2 were plotted in an XY graph and compared using x=y linear regression. All assays showed good correlation between the results at the two time points, with only the "Group 7 Assay 3" sample exhibiting a coefficient of variation (CV%) exceeding 20%.

[0153] Figure 8 Modulation of total HER2, total HER3, and HER2:HER3 dimer levels in MCF-7 tumors after an 8-day treatment period. Xenograft mice were treated for 8 days with a total of 3 doses of MCLA-128 or 2 doses of fulvestrant, or the combination. 24 hours after the last dose, tumors were harvested, processed for FFPE, and subjected to VeraTag analysis for the indicated assays. Five independent tumors from each treatment group were used for the final analysis. Statistical t-tests were used to compare all groups independently. ** indicates P < 0.01.

[0154] Figure 9Reverse phase protein array (RPPA) in MCF-7 tumor xenografts treated with vehicle, MCLA-128, fulvestrant, tamoxifen, MCLA-128 + fulvestrant, or MCLA-128 + tamoxifen. Proteins detected in this assay included (phospho)-Akt and (phospho)-HER3 (full data provided in Appendix 4). One-way ANOVA compared levels in all tumors from the treatment groups with those from the PBS group. ** indicates P < 0.01.

[0155] Figure 10. Nucleic acid and amino acid sequences of the VH chains, common light chains and heavy chains of the antibodies of the invention. Where a leader sequence is indicated in the figure, it is not part of the VH chain or antibody but is typically cleaved during protein processing in cells producing the protein.

[0156] Figure 11 Amino acid and nucleotide alignment of MF3178 variants. CDR regions are indicated.

[0157] Figure 12 .Study design of combination therapy: MCLA-128 + endocrine therapy.

[0158] Figure 13 Treatment administration for combination therapy studies: MCLA-128 + endocrine therapy - all cycles. Example

[0159] As used herein, "MFXXXX" (wherein X is independently a number 0 to 9) refers to a Fab comprising a variable domain wherein VH has an amino acid sequence identified by four numbers. Unless otherwise indicated, the light chain variable region of the variable domain typically has Figure 10C The light chain is usually Figure 10C . "MFXXXX VH" refers to the amino acid sequence of the VH identified by four numbers. MF also contains the constant region of the light chain and the constant region of the heavy chain that typically interacts with the constant region of the light chain. PG refers to a monospecific antibody comprising the same heavy and light chains. PB refers to a bispecific antibody having two different heavy chains. The VH variable regions of the heavy chains are different, and usually the CH3 regions are also different, with one heavy chain having a KK mutation in its CH3 domain and the other having a complementary DE mutation in its CH3 domain (see PCT / NL2013 / 050294 (published as WO2013 / 157954) for reference).

[0160] Example 1

[0161] The anti-tumor efficacy of HER2 / HER3-targeting antibodies was determined in combination therapy with aromatase inhibitors. MCLA-128 was used as a preferred example of a bispecific HER2 / HER3-targeting antibody. It was used as a single agent and in combination with the aromatase inhibitor letrozole. The hormone-dependent HBCx-34 patient-derived breast cancer xenograft model established in immunodeficient mice was used as an example of breast cancer.

[0162] Human tumor xenograft models

[0163] With the informed consent of patients treated at the cancer center, human tumor samples of various histological origins were obtained and established as transplantable xenografts in immunodeficient mice. The transplanted samples were residual material of primary tumors or metastases obtained before or after treatment. These patient-derived xenograft (PDX) models can be established without prior in vitro culture and have been studied for histological, cytogenetic, genetic and other biomarkers and for their response to standard-of-care (SOC) treatment.

[0164] The HBCx-34 PDX model is derived from untreated Primary invasive ductal breast carcinoma. The HBCx-34 PDX model has mutant ATM (a gene encoding a protein involved in double-strand DNA repair) and wt p53, is ER+ / PR+, and is a responder to docetaxel, capecitabine, tamoxifen, and the doxorubicin / cyclophosphamide combination and a low responder to letrozole.

[0165] The HBCx-34 tumor model takes approximately 35 days from the day of implantation (with estrogen supplementation) to achieve a tumor size of 60 to 200 mm. 3 The largest tumor in the range takes about 80 days to reach 2000mm 3 In mice bearing HBCx-34, HBCx-34 did not exhibit overt cachectic properties, but no weight gain was observed.

[0166] MCLA-128 is an IgG1 bispecific antibody with enhanced ADCC targeting the HER2:HER3 dimer. MCLA-128 demonstrated superior in vitro potency compared to other anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of heregulin (HRG), thereby overcoming one of the resistance mechanisms associated with current HER2 therapies.

[0167] Tumor-bearing mice received estrogen (β-estradiol, 8.5 mg / l) diluted in drinking water from the day of tumor implantation until the date of inclusion. During the treatment period, mice did not receive estrogen supplementation.

[0168] Animals and maintenance conditions

[0169] Outbred athymic (nu / nu) female mice (HSD: Athymic Nude-Foxn1) weighing 18 to 25 g were used. nu 》) (Harlan Laboratories, Gannat, France) were assigned to the animal facility for acclimatization and had ad libitum access to food and water for at least 6 days before the operation (Protocol 4-1).

[0170] Scheme 4-1 Animal Characteristics

[0171]

[0172] Test compounds and preparations

[0173] MCLA-128 vehicle was readily available and used for dosing from day 0 to day 38 and stored at +4°C. 0.9% NaCl (CDM Lavoisier batch 6F134) was then used as the vehicle for dosing and MCLA-128 formulation from day 42 to day 56 (end of study). 0.9% NaCl aliquots (CDM Lavoisier batch 6F134) were prepared weekly from day 42 to day 56 and stored at +4°C. Letrozole vehicle (CDM Lavoisier batch 6F134): 0.9% NaCl was readily available. 0.9% NaCl aliquots (CDM Lavoisier batch 6F134) were prepared weekly and stored at +4°C.

[0174] MCLA-128 was readily available and used for injections from day 0 to day 42. Thereafter, an MCLA-128 aliquot was diluted in 0.9% NaCl to obtain a 2.5 mg / ml working solution, which was prepared prior to each injection. This was used for dosing from day 42 to day 56 (end of study).

[0175] Letrozole tablets (Actavis) were dissolved in 0.9% NaCl under magnetic stirring to form a final solution of 0.25 mg / ml. The dosing solution was stable for 7 days and stored at +4°C protected from light.

[0176] Tumor transplantation model induction

[0177] Tumors of the same generation were transplanted subcutaneously into 6 to 24 mice (donor mice, generation (n-1)). When these tumors reached 1000 to 2000 mm 3 At 4 hr, the donor mice were sacrificed by cervical dislocation and the tumors were aseptically excised and dissected. After removing the necrotic area, the tumors were cut into pieces measuring approximately 20 mm. 3 fragments and transferred to culture medium before grafting.

[0178] 95 mice were anesthetized with 100 mg / kg ketamine hydrochloride (batch 5D92, expiration date: 2017 / 03, Virbac) and 10 mg / kg xylazine (batch KP0AX9X, expiration date: 2017 / 08, Bayer), and then the skin was sterilized with chlorhexidine solution, incised at the level of the interscapular region, and 20 mm 3 The tumor fragment was placed in the subcutaneous tissue. The skin was closed with clips.

[0179] All mice from the same experiment were implanted on the same day.

[0180] Treatment phase

[0181] 40 mice with subcutaneous growth ranging from 62.5 to 256 mm 3 Mice with HBCx-34 tumors were randomly assigned based on their tumor volume to give homogeneous mean and median tumor volumes in each treatment arm. Treatment was randomly assigned to boxes housing up to 5 mice and started 36 days after tumor implantation (42% enrollment rate). The study was terminated 57 days after treatment began.

[0182] Tumor measurement and animal observation

[0183] Tumor volume was assessed by measuring tumor diameter with calipers every two weeks during the experimental period.

[0184] Use the formula TV(mm 3 )=[length (mm)×width (mm)2] / 2, where length and width are the longest and shortest diameters of the tumor, respectively.

[0185] All animals were weighed every two weeks during the experimental period.

[0186] Toxicity of different treatments was determined as: percent body weight loss (%BWL) = 100 - (mean BWx / mean BW0 x 100), where BWx is the mean BW on any day during treatment and BW0 is the mean BW on the first day of treatment.

[0187] As outlined in the protocol below, a total of 4 groups were used. Each group initially contained 10 mice.

[0188]

[0189] In group 1, MCLA-128 vehicle and letrozole vehicle were administered at 10 ml / kg ip on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, and D56 and po qd×57, respectively;

[0190] In groups 2 and 4, MCLA-128 was administered ip at 25 mg / kg on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, and D56;

[0191] In Groups 3 and 4, letrozole was administered at 2.5 mg / kg, po, qd×57.

[0192] All treatment doses were adjusted based on body weight at each injection.

[0193]

[0194] result

[0195] The average percentage change in body weight during treatment was as follows Figure 3 shown.

[0196] In Group 1, MCLA-128 vehicle was administered ip on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, and D56, and letrozole vehicle was administered po qd×57, both at 10 ml / kg, and were well tolerated, with a maximum mean body weight loss of 0.4% on day 4 and a maximum individual body weight loss of 3.7% on day 49.

[0197] In Group 2, MCLA-128 was administered ip at 25 mg / kg on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, and D56, administered at 10 ml / kg and was well tolerated with a maximum mean body weight loss of 0.4% on Day 25 and a maximum individual body weight loss of 8.3% on Day 39.

[0198] In Group 3, letrozole 2.5 mg / kg po was administered in 10 ml / kg qd x 57 and was well tolerated with no mean weight loss on day 4 and a maximum individual weight loss of 4.1%.

[0199] In Group 4, MCLA-128 was administered at 25 mg / kg ip on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, and D56, and letrozole was administered at 2.5 rng / kg po qd x 57, both administered at 10 ml / kg. These were well tolerated, with a maximum mean body weight loss of 0.7% on day 4 and a maximum individual body weight loss of 4.2% on day 4.

[0200] Tumor growth curves (average tumor volume over time) are shown in Figure 1 The T / C percentage values for each treatment group are shown in Table 1 and are Figure 2 The definitions of partial and complete responses are defined in Table 2. Statistical analyses are shown in Tables 3 and 4.

[0201] In this study, tumors were measured every two weeks during the experimental period.

[0202] In Group 2, MCLA-128 was administered at 25 mg / kg ip on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, and D56, and administered at 10 ml / kg ip. This did not induce statistically significant tumor growth inhibition, with a TGDi of 0.93 on day 28, a best T / C% of 91.14%, a T / C% of 101.56% (at the end of the control group), and a best TGI% of 31.59% at the end of the control group. However, stabilization of 1 / 9 tumors and partial regression of 1 / 9 tumors were observed.

[0203] In Group 3, letrozole at 2.5 mg / kg, administered at 10 ml / kg po, qd x 57, induced statistically significant tumor growth inhibition (Mann Whitney test: P < 0.001 compared to the vehicle group; Dunn's test, p < 0.05 compared to the control group), with TGDi > 1.1 on day 56 (end of the control group), the best T / C% = 18.68%, and the best TGI% = 145.70% on day 35. In addition, partial tumor regression was observed in 7 / 10 and complete tumor regression was observed in 1 / 10.

[0204] In Group 4, the combination of MCLA-128 and letrozole (wherein MCLA-128 was administered at 25 mg / kg ip on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56 and letrozole was administered at 2.5 mg / kg po qd x 57, both administered at 10 ml / kg) induced statistically significant tumor growth inhibition (Mann Whitney test and Dunn test: P < 0.001 compared with the vehicle group), with TGDi > 1.1 on day 56 (end of control group), best T / C% = 9.64%, and TGI% = 169.03% on day 32. Additionally, 5 / 10 partial tumor regressions and 5 / 10 complete tumor regressions were observed, with 5 / 10 tumor-free survivors at the end of the study.

[0205] discuss

[0206] Based on body weight data and clinical observations, all test compounds, as single agents or in combination, were well tolerated at the doses and schedules tested.

[0207] In the HBCx-34 model, MCLA-128 alone did not induce tumor growth inhibition, while letrozole alone induced statistically significant tumor growth inhibition. The combination of letrozole and MCLA-128 induced statistically significant synergistic tumor growth inhibition.

[0208] Treatment options for breast cancer patients are guided by three biomarkers: HER2, estrogen (ER) and progesterone (PR) receptors. Patients with HER2 overexpression are eligible for HER2-targeted therapies, such as trastuzumab and pertuzumab. ER / PR-positive patients will receive anti-estrogen therapy or aromatase inhibitors. Anti-estrogen drugs (such as tamoxifen and fulvestrant) regulate ER activity, while aromatase inhibitors (AIs, such as letrozole or exemestane) inhibit the conversion of androgens to estrogens, thereby depleting the level of ER stimulation in patients. Anti-estrogen and aromatase inhibitors are used in different ER+ breast cancer patient groups, for example, AIs are used as first-line treatment for postmenopausal patients. In Example 2, it was demonstrated that the addition of MCLA-128 to tamoxifen or fulvestrant enhanced the activity of anti-estrogen therapy.

[0209] Example 2

[0210] Evaluation of MCLA-128 as monotherapy and in combination with the SERM tamoxifen and the SERD fulvestrant Efficacy of the combination in a nude mouse xenograft model of estrogen-responsive MCF-7 human breast cancer. The study design also included tumor harvesting for downstream analysis.

[0211] Treatment was initiated at D1 in mice with established subcutaneous MCF-7 tumors. The study endpoint was intended to be 1000 mm 3 The tumor volume endpoint was 10 days or 45 days, whichever came first. The study was terminated at D39, and treatment results were based on the percentage of tumor growth inhibition (%TGI), which was defined as the percentage difference between the median tumor volume (MTV) of treated mice and control mice at D25. D25 was chosen for analysis because it was the last day before animals were withdrawn from the study due to tumor progression.

[0212] Treatment response was determined by analysis of percent tumor growth inhibition (% TGI), defined as the percentage difference between the final (D25) median tumor volume (MTV) of the treatment and control groups, with differences between groups considered statistically significant at P ≤ 0.05 using the Mann-Whitney test. Tumor regression, mean tumor growth, and treatment tolerance were also considered.

[0213] mice

[0214] Female athymic nude mice (Crl: NU(NCr)-Foxn1 nu , Charles River) were ten weeks old and weighed between 19.2 and 30.5 g on D1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and a NIH 31 Modified and Irradiated Laboratory Diet consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. Mice were housed in static microisolators on irradiated Enrich-o'cobs at 20 to 22°C (68 to 72°F) and 40 to 60% humidity on a 12-hour light cycle. TM On laboratory animal bedding.

[0215] Tumor implantation

[0216] Three days prior to tumor cell implantation, an estrogen pellet (0.36 mg estradiol, 60-day release, Innovative Research of America, Sarasota, FL) was implanted subcutaneously between the shoulder blades of all animals using a sterile trocar.

[0217] Xenografts were initiated with cultured MCF-7 human breast cancer cells. Tumor cells were grown to mid-logarithmic phase in RPMI-1640 medium containing 10% fetal bovine serum, 100 units / mL of penicillin G, 100 μg / mL of streptomycin sulfate, 2 mM glutamine, 10 mM HEPES, 0.075% sodium bicarbonate, and 25 μg / mL gentamicin. On the day of tumor cell implantation, cells were trypsinized, precipitated, and resuspended in phosphate buffered saline (PBS) at a concentration of 1 × 10e8 cells / mL. Each test mouse received 1 × 10e7 MCF-7 cells implanted subcutaneously in the right flank and the average volume was close to the desired 100 to 150 mm. 3 Monitor tumor growth from this range.

[0218] Nineteen days after tumor cell implantation, designated as D1 of the study, mice were divided into six groups of 15 animals each and four groups of 6 animals each. On D1, the volume of individual tumors ranged from 75 to 196 mm 3 The mean tumor volume in the group was 134 to 137 mm 3 1mg is equivalent to 1mm 3 Tumor weight was estimated assuming the tumor volume was .

[0219] therapeutic agents

[0220] MCLA-128 was provided pre-formulated at 2.5 mg / mL and was ready for administration as 25 mg / kg in a 10 mL / kg dose volume and stored at 4°C, protected from light during storage and handling. Tamoxifen (Sigma-Aldrich, lot number WXBB5732V) was received as a powder and stored at 4°C, protected from light. A 5 mg / mL dose solution was prepared weekly in corn oil, and each mouse received 0.2 mL for a dose of 1 mg / animal. The dose solution was stored at 4°C. Fulvestrant, trade name (AstraZeneca, lot numbers LV032, LW466, and LX432) was received as a 50 mg / mL stock solution and stored at 4° C. On each dosing day, the stock solution was diluted to 25 mg / mL in corn oil, and each mouse received 0.2 mL for a dose of 5 mg / animal.

[0221] treat

[0222] Groups 1 and 7 served as efficacy and sampling controls, respectively, and received MCLA-128 vehicle intraperitoneally (ip) on D1, 4, 8, 11, 15, 18, 22, 25, and 29, and corn oil subcutaneously (sc) every other day for 15 doses (qod x 15). Groups 2 and 8 were administered MCLA-128 at 25 mg / kg ip on the same schedule as the MCLA-128 vehicle. Groups 3 and 9 were administered sc at 5.0 mg / animal once weekly. The treatment lasted for 5 weeks (qwk×5). Group 4 received tamoxifen at 1 mg / animal, sc, qod×15. In the above regimen, groups 5 and 10 received MCLA-128 and Group 6 received MCLA-128 and tamoxifen in the above regimen.

[0223] MCLA-128 was administered at a dose volume of 10 mL / kg, proportionally adjusted to the individual body weight of each animal. and tamoxifen were administered in a fixed volume of 0.2 mL.

[0224] result

[0225] Figure 5 is a scatter plot showing the distribution of tumors by group. Figure 6 Group median tumor growth curves are shown for all groups in the study.

[0226] Growth of MCF-7 tumors in control mice (Group 1)

[0227] Group 1 mice received MCLA-128 vehicle and corn oil as indicated above and served as the control group for calculation of TGI percentage and statistical comparison. The median tumor volume in this group was 600 mm 3 , where the volume of individual tumors ranged from 288 to 936 mm at D25. 3 ( Figure 5 At the time of data analysis, two NTRu deaths were recorded on D15 and D21, leaving thirteen evaluable animals. Additional NTRu deaths were recorded after D25: one on D26 and two on D35. All deaths were attributed to suspected estrogen-related toxicity.

[0228] Response to MCLA-128 (Group 2)

[0229] In Group 2, MCLA-128 was administered as above. This treatment resulted in a median tumor volume of 600 mm 3, corresponding to a non-significant 0% TGI relative to the control group (P>0.05). At the time of data analysis, six NTRu deaths had been recorded; two each on D17 and D22, and one each on D19 and D20, leaving nine evaluable animals. Additional NTRu deaths were recorded after D25; one each on D29 and D36. All deaths were attributed to suspected estrogen-related toxicity.

[0230] right Response (Group 3)

[0231] In Group 3, the above This treatment resulted in a median tumor volume of 288 mm 3 , corresponding to a significant 52% TGI relative to the control group (P<0.001). Two NTRu deaths were recorded on D20 and D24, leaving thirteen evaluable animals. All deaths were attributed to suspected estrogen-related toxicity.

[0232] Response to tamoxifen (Group 4)

[0233] In Group 4, tamoxifen was administered as above. This treatment resulted in a median tumor volume of 184 mm 3 , corresponding to a significant 69% TGI relative to the control group (P < 0.001). At the time of data analysis, three NTRu deaths were recorded on D3, D10, and D22, leaving twelve evaluable animals. After data analysis, two more deaths were recorded on D26. All deaths were attributed to suspected estrogen-related toxicity.

[0234] To Response to MCLA-128 in combination with tamoxifen (Groups 5 and 6)

[0235] In group 5, MCLA-128 was combined with The treatment resulted in a median tumor volume of 126 mm 3 , corresponding to a significant 79% TGI. The results were significant as single treatments (P < 0.05). Three PRs were recorded in this group. One NTRu death was recorded on D17, leaving fourteen evaluable animals. This death was attributed to suspected estrogen-related toxicity.

[0236] In Group 6, MCLA-128 was combined with tamoxifen and administered as above. This treatment resulted in a median tumor volume of 108 mm 3, corresponding to a significant 82% TGI. This result was significant relative to the control group and MCLA-128 monotherapy (P < 0.001) and tamoxifen monotherapy (P < 0.05). Four PRs were recorded in this group. At the time of data analysis, one NTRu death was recorded on D15, leaving fourteen evaluable animals. After data analysis, three additional deaths were recorded; two on D30 and one on D39. The deaths were attributed to suspected estrogen-related toxicity.

[0237] discuss

[0238] This example evaluates the effect of the agent MCA-128 on Compared with tamoxifen and Efficacy of the combination of tamoxifen and tamoxifen in a nude mouse xenograft model of estrogen-responsive MCF-7 human breast cancer. Tumors were measured twice weekly until D39 and TGI analysis was performed on D25.

[0239] At D25, the median tumor volume of control group 1 was 600 mm 3 , single tumors ranged from 288 to 936 mm 3 . Administration of MCLA-128, or tamoxifen resulted in median tumor volumes of 600, 288, and 184 mm 3 , corresponding to TGI of 0%, 52% and 69% TGI. The results of MCLA-128 monotherapy were not significant compared with the control (P>0.05), but tamoxifen and The results were significant (P < 0.001). MCLA-128 and tamoxifen or Combination therapy resulted in median tumor volumes of 126 and 108 mm 3 , corresponding to 79% and 82% TGI, respectively. Each of these results was significant compared with the corresponding treatment (P < 0.05). Three PRs were recorded in the MCLA-128 / tamoxifen group and four PRs were recorded in the MCLA-128 / tamoxifen group. All treatments were well tolerated. Several deaths in all groups were attributed to estrogen toxicity. In conclusion, MCLA-128 and tamoxifen had a similar effect compared to the corresponding monotherapy in the MCF-7 nude mouse xenograft model. Combination therapy with tamoxifen or tamoxifen provided a significant synergistic survival benefit. All treatments were acceptably tolerated.

[0240] MCLA-128 as a single agent showed no antitumor efficacy, whereas both fulvestrant and tamoxifen significantly reduced tumor growth. At day 25, near the end of the treatment period, the combination of MCLA-128 with fulvestrant or tamoxifen provided a significant survival benefit ( 0.05 % CI 0.04-0.06) compared to the corresponding monotherapy. Figure 5 Interestingly, the beneficial antitumor effects of the combination therapy persisted during the treatment-free observation period ( Figure 6 ). All treatments were acceptably tolerated.

[0241] Example 3

[0242] Pharmacodynamic analysis

[0243] First round of VeraTag assay

[0244] All formalin-fixed tumors from Example 2 were processed into FFPE blocks. Initial VeraTag assay analysis (Round 1) was performed with 3 tumors per group.

[0245] Prior to VeraTag analysis, tumors were sectioned and stained with hematoxylin-eosin to determine the percentage of tumor content. Micro-laser capture was used to substantially remove any non-tumor content. The initial round of analysis included the following five VeraTag assays: total HER2, total HER3, HER2:HER3 dimer, HER3-PI3K complex, and phosphorylated HER3.

[0246] MCLA-128 treatment did not show a significant effect relative to vehicle in any assay. Significant differences between groups were observed only in the HER2:HER3 assay, where treatment with fulvestrant significantly upregulated HER2:HER3 dimer formation, and co-treatment with MCLA-128 reversed the levels of HER2:HER3 dimers to those in vehicle-treated tumors.

[0247] Although not statistically significant, similar trends were observed in the HER2 and HER3 assays. Since the data only included three tumors / treatment groups, more data points are needed to determine whether this trend indicates similar results to those seen in the HER2:HER3 assay. Therefore, the remaining tumors in Table 5 were included in a second round of VeraTag assays for total HER2, total HER3, and HER2:HER3 dimer. Since no significant effects of fulvestrant or in combination with MCLA-128 were observed in the phosphorylated HER3 and HER3-PI3K assays, these assays were not included in round 2.

[0248] Second round of VeraTag assay

[0249] Tumors analyzed in the first round whose scores were close to the mean for a particular assay were included in the second round to assess inter-assay reproducibility and to ensure that data from two independent experiments could be combined. Results for those tumors analyzed in both rounds that correlated well between assays with coefficients of variation (CV) below 20% were excluded from the final analysis ( Figure 7 ).

[0250] The data from both rounds were combined and the results demonstrated that fulvestrant significantly induced HER2:HER3 dimerization compared to vehicle, and that this could be reversed by co-administration of MCLA-128 ( Figure 8 ).

[0251] In addition, HER2 expression levels were significantly higher in the Fulvestrant group compared to either the MCLA-128 alone or the combination treatment groups. The vehicle group showed some variability between samples and did not show significant differences compared to the Fulvestrant group. Finally, although HER3 was slightly increased in the Fulvestrant group relative to all other groups, HER3 expression did not change significantly between the different treatment groups.

[0252] RPPA analysis

[0253] When the tumors from groups 1 to 6 reached the maximum tolerated volume, or at the end of the experiment on day 39 (i.e. 10 days after the last dose), the tumors were collected. Six tumors per treatment group were included in the reverse phase protein array (RPPA). Tumor selection was performed so that the average tumor size of the six samples was close to the average tumor size of the entire group. RPPA analysis was performed as follows: the tumor was sliced and placed on an immunohistochemical slide. Macroscopic dissection of the matrix and inflammatory contents was performed to purify the tumor cells. Protein lysates were prepared and the protein content was quantified. The protein samples were then placed on nitrocellulose slides in quadruplicate at two different concentrations and then incubated with a primary antibody specific for all or phosphorylated forms of Akt, ERK, HER2 and HER3.

[0254] Reverse phase protein array (RPPA) data were analyzed using one-way analysis of variance to detect significant differences between the treated and vehicle groups. Total Akt and phosphorylated Akt levels increased in the Faslodex group, with no significant differences observed in the other groups. Total ERK levels increased only in the single agent group, with no differences measured in the other groups or in the phosphorylated ERK analysis. Total HER2 levels significantly increased in the tamoxifen and Faslodex groups, but phosphorylated HER2 levels did not. Co-treatment of MCLA-128 with hormone therapy prevented this induction of HER2 expression. Total HER3 levels significantly increased only in the Faslodex group, while total HER3 levels decreased in the case of co-treatment with MCLA-128.

[0255] discuss

[0256] Pharmacodynamic analysis

[0257] The finding of the VeraTag assay analysis was that HER2 expression levels were significantly higher in tumors from mice treated with fulvestrant. The score according to the VeraTag assay correlated with HER2 positivity as determined by immunohistochemistry (IHC): a HER2 VeraTag score below 10.5 was negative in IHC, a score of 10.5 to 17.8 was equivocal, and a score above 17.8 was positive (Huang et al., 2010 Am J Clin Pathol. 134(2):303-11). Thus, fulvestrant treatment was shown to change the HER2 status of HER2-negative tumors (i.e., a score of 0 or 1+ according to the ASCO guidelines, Wolff et al., 2013 J Clin Oncol. 31(31):3997-4013) to at least an equivocal (usually a score of 2+) HER2 status. Most importantly, VeraTag analysis showed that fulvestrant treatment induced the formation of HER2:HER3 dimers, which could be reversed by adding MCLA-128 to the treatment regimen.

[0258] Biomarker analysis

[0259] In this set of experiments, in which we measured the levels of a panel of biomarkers using RPPA, we found enhanced expression of Akt, HER2, and HER3 in tumors treated with hormone therapy. The fact that this hormone therapy-induced increase could be reversed by co-administration of MCLA-128 suggests that activation of the Akt signaling pathway is associated with HER2:HER3 dimer activity and is therefore targeted by MCLA-128 in these tumors. This is consistent with data obtained from the VeraTag assay and further confirms the beneficial effects of co-administering MCLA-128 with hormone therapy in vivo.

[0260] General Notes

[0261] Increased HER2 protein expression following fulvestrant treatment in MCF-7 xenografts is consistent with a mechanism of resistance to hormonal therapy that has been observed in patients (Osborne and Schiff 2011 Osborne CK, Schiff R. Annu Rev Med. 62: 233-47). Fulvestrant has also been described to upregulate HER3 expression in MCF-7 cells in vitro and in vivo (Morrison et al. 2013 J Clin Invest. 123(10): 4329-43), which was observed by RPPA analysis but not VeraTag analysis. This may be due to the different timing of tumor harvest (at the beginning and end of the treatment period for VeraTag and RPPA assays, respectively).

[0262] The synergistic effect between MCLA-128 and fulvestrant can be explained by the increased HER2:HER3 dimer formation seen in tumors from mice treated with this combination. Increased phosphorylation of Akt was also observed in tumors from mice treated with fulvestrant alone (RPPA analysis).

[0263] Example 4: MCLA-128 / Endocrine Therapy II in Estrogen Receptor-Positive and Low HER2-Expressing MBC Phase II study

[0264] Although the examples describe administration of MCLA-128 in combination with endocrine therapy, the examples are not intended to limit the use of the specific therapeutic agents described and apply to the disclosed bispecific antibodies that bind ErbB-2 and ErbB-3 in combination with endocrine therapy.

[0265] Target: Estrogen receptor [ER]-positive / low HER2-expressing MBC: MCLA-128 plus endocrine therapy

[0266] Main objectives:

[0267] The efficacy of MCLA-128 in combination with endocrine therapy in patients with ER-positive and low HER2-expressing MBC who had previously progressed on the same endocrine therapy was evaluated based on the clinical benefit rate (CBR) at 24 weeks, according to RECIST 1.1 (according to investigator review).

[0268] Secondary Objectives:

[0269] CBR at 24 weeks based on central review according to RECIST 1.1

[0270] Evaluation of progression-free survival (PFS) (based on investigator and center review)

[0271] Objective response rate (ORR) based on RECIST 1.1 (based on investigator and central review)

[0272] Time from response (CR or PR) to progression or death due to underlying cancer (DoR) based on RECIST 1.1 (based on investigator and central review)

[0273] ·Evaluate OS

[0274] Evaluate the safety and tolerability of MCLA-128 in combination with endocrine therapy

[0275] Characterize the PK of MCLA-128 in combination with endocrine therapy

[0276] Characterize the immunogenicity of MCLA-128 in combination with endocrine therapy

[0277] Exploration objectives:

[0278] • Evaluation of potential correlations between biomarkers in tumor or blood samples and anti-tumor activity (including HER2, HER3, HER2:HER3 dimer, heregulin, and other potential biomarkers).

[0279] Study Design

[0280] A phase 2, open-label, multicenter, international study was conducted to evaluate the efficacy of the MCLA-128-based combination in a population of ER-positive / HER2-low metastatic breast cancer (MBC).

[0281] Patients were ER-positive with low HER2-expressing metastatic breast cancer (MBC) (immunohistochemistry (IHC) 1+ or IHC 2+ combined with negative fluorescence in situ hybridization (FISH)) who had progressed since the last line of prior endocrine therapy containing an aromatase inhibitor or fulvestrant (administered for at least 12 weeks) according to RECIST v1.1.

[0282] Patients were eligible if they had received 1 or 2 prior endocrine therapies in the metastatic setting and had progression (according to RECIST v1.1) on a cyclin-dependent kinase inhibitor (at any step).

[0283] For enrollment, HER2 and HR status, as well as radiographic documentation of prior disease progression, were based on medical records. Eligibility for HER2 / HR status was confirmed as soon as possible by central laboratory review, and for prior disease progression was confirmed as soon as possible by central imaging review. Patients who were retrospectively found to be ineligible were ineligible for the primary endpoint and could be substituted.

[0284] MCLA-128 was administered in combination with the same prior endocrine therapy for which progressive disease was documented radiographically. A total of up to 40 patients evaluable for efficacy were included.

[0285] See also Figure 12 .

[0286] Research group

[0287] Inclusion criteria

[0288] Patients must meet all of the following requirements to enter the study:

[0289] 1. Sign the informed consent form before starting any research procedures.

[0290] 2. Women with histologically or cytologically confirmed breast cancer with evidence of metastatic or locally advanced disease who are not candidates for any local therapy with curative intent:

[0291] a. Documented hormone receptor-positive status (estrogen receptor-positive [ER+] and / or progesterone receptor-positive [PR+]) based on analysis of the most recent tumor biopsy, comprising ≥1% of positively stained cells.

[0292] b. Documented low-level HER2 expression, defined as IHC HER2 1+ or IHC HER2 2+ in combination with negative FISH (preferably metastatic, otherwise primary), based on local analysis of fresh tumor biopsies or archived biopsies collected within 12 months prior to screening.

[0293] c. One or two prior courses of endocrine therapy for metastatic disease (aromatase inhibitor or fulvestrant) with radiographically documented disease progression since the last course of treatment after at least 12 weeks.

[0294] d. Advances in cyclin-dependent kinase inhibitors.

[0295] e. No more than one prior chemotherapy regimen for advanced / metastatic disease.

[0296] Note: Premenopausal / perimenopausal women may be enrolled if they are able to receive treatment with the luteinizing hormone-releasing hormone (LHRH) agonist goserelin. Such patients must have started treatment with goserelin or an alternative LHRH agonist at least 4 weeks prior to study entry, and patients receiving an alternative LHRH agonist prior to study entry must switch to goserelin for the duration of the trial.

[0297] 3. Radiographically measurable disease as defined by RECIST version 1.1 since or after the most recent step of treatment. For Cohort 2, images must be available for central review.

[0298] 4. Aged ≥ 18 years when signing the informed consent form.

[0299] 5. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0300] 6. Life expectancy ≥ 12 weeks according to the investigator.

[0301] 7. Left ventricular ejection fraction (LVEF) ≥ 50% as determined by echocardiogram (ECHO) or multiple gated acquisition scan (MUGA).

[0302] 8. Proper Organ Function:

[0303] a. Absolute neutrophil count (ANC) ≥ 1.5 × 10 9 / L

[0304] b. Hemoglobin ≥9 g / dL

[0305] c. Platelet count ≥100×10 9 / L

[0306] d. Serum calcium within normal range (or corrected by supplementation)

[0307] e. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤ 2.5 × upper limit of normal (ULN) and total bilirubin ≤ 1.5 × ULN (in cases of liver involvement, ALT / AST ≤ 5 × ULN and total bilirubin within the normal range will be allowed)

[0308] f. For patients aged >65 years, serum creatinine ≤1.5 × ULN or creatinine clearance ≥60 mL / min calculated according to the Cockroft and Gault formula or the Modification of Diet in Renal Disease (MDRD) formula (Appendix 19.2)

[0309] g. Serum albumin > 3.0 g / dL

[0310] Investigational treatment and concomitant therapy

[0311] MCLA-128: 750 mg intravenous flat dose over 2 hours, day 1 every 3 weeks (q3w).

[0312] For each MCLA-128 infusion, premedication with paracetamol / acetaminophen, antihistamines, and corticosteroids (according to standard practice) is mandatory.

[0313] Endocrine therapy: Patients received the same dose and regimen before study entry as that administered with the last step of endocrine therapy since which they had progressed.

[0314] Treatment options

[0315] One cycle is 3 weeks (including Cohort 2, which may include q4w fulvestrant dosing). For the initial MCLA-128 administration, a 6-hour observation period will be implemented after the start of the infusion, and for all subsequent administrations, a 2-hour observation period will be implemented.

[0316] All patients received MCLA-128 administration every 3 weeks on day 1. For endocrine therapy, patients received the same dose and schedule before study entry as that administered with the last step of endocrine therapy since which the patient had progressed.

[0317] Fulvestrant was administered on days 1, 15, 29, and every 28 days thereafter, or aromatase inhibitor therapy (letrozole, anastrozole, and exemestane) was administered daily starting on day 1.

[0318] Treatment administration (all cycles)

[0319] See also Figure 13 For the initial MCLA-128 administration, a 6-hour observation period was implemented after the start of the infusion, and for all subsequent administrations, a 2-hour observation period was implemented.

[0320] *In cases where patients have progressed on the same endocrine therapy prior to study entry, the same endocrine therapy may be administered before, during, or immediately after the MCLA-128 infusion.

[0321] Treatment allocation

[0322] No specific treatment assignment was required.

[0323] Treatment adaptation

[0324] • Dose reductions of MCLA-128 or endocrine therapy were not permitted.

[0325] If an infusion-related reaction (IRR) occurs, the MCLA-128 infusion will be interrupted and must be stopped definitively for severe IRRs. For mild to moderate events, the infusion rate may be continued at 50% and the infusion duration extended to 4 hours.

[0326] • MCLA-128 administration may be delayed for up to 6 weeks between infusions to manage adverse events (AEs), particularly for clinically significant decreases in LVEF, signs of congestive heart failure, or persistent Grade 2 or Grade 3 to 4 diarrhea.

[0327] Administer hormone therapy drugs according to the summary of product characteristics (SPC) for each drug.

[0328] Duration of treatment

[0329] Study treatment was administered until confirmed progressive disease (according to RECIST 1.1), unacceptable toxicity, withdrawal of consent, patient non-compliance, investigator decision (e.g., clinical deterioration), treatment interruption for >6 consecutive weeks, or withdrawal of any study drug. Patients were followed for safety for at least 35 ± 5 days after the last dose of study drug until recovery / stabilization of the relevant toxicity, and for 12 months for disease progression and survival.

[0330] Preventive and concomitant medications

[0331] allow

[0332] Administration of paracetamol / acetaminophen, antihistamines, and corticosteroids is mandatory with each administration of MCLA-128. In the event of an IRR or hypersensitivity reaction, manage the patient as clinically indicated according to local clinical practice.

[0333] All medications, including supportive care for symptoms and AEs or standard treatment of accompanying conditions, were administered at the discretion of the investigator as necessary for the patient's well-being and not expected to interfere with the evaluation of the study drug.

[0334] • Goserelin in pre- / perimenopausal women who started an LHRH agonist > 4 weeks prior to study entry.

[0335] prohibit

[0336] Concomitant chronic oral corticosteroids (>10 mg / day prednisone equivalents), TNF-α inhibitors, anti-T-cell antibodies (due to the risk of immunosuppression).

[0337] Any study medication during the study or in the 4 weeks prior to the first dose of study treatment.

[0338] Systemic anticancer therapy (except last endocrine therapy) during the study or within 3 weeks of the first dose of study treatment.

[0339] Safety / tolerability assessment

[0340] AE (CTCAE version 4.03), SAE

[0341] Laboratory parameters: hematology, biochemistry, coagulation, urinalysis, cytokines

[0342] ECG, MUGA / ECHO

[0343] Medical history, vital signs, performance status, and physical examination

[0344] Concomitant medication

[0345] Dose modification (reduction, interruption, delay), discontinuation due to toxicity

[0346] Effectiveness evaluation

[0347] In the present invention, the present invention relates to the treatment of leukemia and leukemia.After treatment starts, every 6 weeks according to RECIST 1.1, carry out tumor evaluation based on the CT / MRI utilizing contrast agent (contrast).After observing for the first time, at least 4 weeks confirm objective response.All patients are all carried out center review (screening and research) to imaging by independent radiologist (radiologist).In research, as clinically indicated, for suffering from the bone metastasis at baseline or the patient with suspected lesion, carry out bone scan.

[0348] Tumor markers (CA15-3, CEA, CA27-29) were assessed on day 1 of each cycle.

[0349] Biomarkers

[0350] Candidate exploratory biomarkers are evaluated in tumor tissue (screening, optionally after 12 weeks, and EOT) and blood (pre-dose and end-of-treatment on Day 1 of every 4 cycles).

[0351] Tumors: HER2; HER3; HER2:HER3 dimerization; downstream signaling proteins (e.g., PIK3CA); heregulin; phosphorylation of HER2, HER3, and proteins in the MAPK and AKT signaling pathways; expression of inhibitors (e.g., PTEN); mutations in cancer-associated genes, including HER2 and HER3 signaling; and heregulin-gene fusions.

[0352] blood: Fcγ receptor polymorphisms, plasma circulating tumor DNA mutations, exploratory serum biomarkers (eg, soluble HER2, heregulin).

[0353] Pharmacokinetics

[0354] Blood samples were collected to measure serum MCLA-128. No PK sampling was performed for fulvestrant or aromatase inhibitors.

[0355] PK sampling was performed at the following time points:

[0356] Cycle 1: Day 1: before dosing, at EOI, and 2, 4, and 22 hours after EOI; then any time on Day 8;

[0357] Cycles 2, 3, and 5: Day 1: Pre-dose, EOI

[0358] Every 4 cycles thereafter: Before administration

[0359] Immunogenicity

[0360] For cycles 1, 3, 5, and every four cycles thereafter, blood samples (5 mL) were collected from all patients before dosing on day 1 and at the end of treatment to assess pre-dose anti-MCLA-128 antibody serum titers.

[0361] definition

[0362] All efficacy endpoints were defined and analyzed based on tumor assessment by RECIST 1.1.

[0363] CBR: proportion of patients with best overall response (CR), PR, or SD for ≥24 weeks.

[0364] ORR: the proportion of patients with a best overall response of CR or PR.

[0365] PFS: the time from the start of treatment until radiographic progression or death due to any cause.

[0366] PFS ratio: the ratio of PFS on the previous regimen to PFS on the study treatment.

[0367] DoR: time from response (CR or PR) until progression or death due to the underlying cancer.

[0368] OS: time from the start of treatment until death due to any cause.

[0369] end

[0370] main

[0371] CBR according to investigator radiological review at week 24

[0372] Key secondary

[0373] CBR at 24 weeks according to central review, and PFS according to investigator and central review

[0374] Other minor

[0375] Safety: incidence, severity, and relationships of AEs, laboratory abnormalities, and SAEs; ECG and LVEF measurements, and vital signs

[0376] Tolerability: discontinuation due to AEs, dose modification due to AEs, immunogenicity, and cytokine assessments

[0377] Other efficacy: DoR, PFS ratio, ORR and OS

[0378] Pharmacokinetics: C of MCLA-128 max 、C 0h , AUC, CL, V ss , t max and t 1 / 2 .

[0379] Analyze groups

[0380] Treated population: Patients who received at least one dose of MCLA-128.

[0381] Efficacy evaluable: Patients who received at least 2 complete cycles (6 weeks) of treatment and underwent a baseline assessment and one on-study tumor assessment, or discontinued early due to disease progression.

[0382] analyze

[0383] Patient disposition and demographics will be analyzed in the treated population, efficacy will be analyzed in the efficacy-evaluable population, and safety will be analyzed in the treated population.

[0384] Quantitative variables will be summarized using descriptive statistics. Continuous variables will be expressed as N, mean and / or median, standard deviation, and range. Categorical variables will be expressed using frequencies and percentages.

[0385] Criteria for successful primary endpoint : A median PFS of 5 months was assumed to be relevant, and the activity threshold for CBR at 24 weeks was set at 45%.

[0386] CBR and ORR are summarized and accompanied by 90% exact binomial confidence intervals.

[0387] For PFS, OS, and DoR, survival functions were estimated using the Kaplan-Meier product limit method; probability estimates and 90% CIs were provided at the specified time points; median duration and 90% CIs were also provided. DoR was estimated only for responders. The number and proportion of patients with a PFS ratio ≥ 1.3 in Cohort 2 were tabulated with 90% exact CIs.

[0388] AE through the Medical Dictionary for Regulatory Activities Terms were preferred and tabulated by organ class based on incidence and severity. The severity of AEs was based on the Common Terminology for Adverse Events (CTCAE) 4.03.

[0389] PK, immunogenicity, and biomarkers were analyzed centrally and reported separately.

[0390] Table 1. Summary of Dunn's Multiple Comparison Test Analysis of Relative Body Weight in the HBCx-34 Tumor Model, Efficacy Study XTS-1521. Group comparisons between treatment and control groups were performed using Dunn's Multiple Comparison Test: ns = not significant, * = P < 0.05, ** = P < 0.01, and *** = P < 0.001.

[0391] Initial group size: 9 to 10 animals.

[0392]

[0393] Table 2. Antitumor activity of 25 mg / kg MCLA-128, 2.5 mg / kg letrozole in HBCx-34 xenografts, efficacy study XTS-1521.

[0394] XenTech T / C = mean tumor volume of treated mice / mean tumor volume of control mice × 100 (calculated at the time of the first ethical sacrifice of the control group); TGD (Tumor Growth Delay) = time required for the median tumor volume to reach the D0 tumor volume × 5; TGDI (Tumor Growth Delay Index) = TGD from treated mice / TGD from control mice; TS (Tumor Stabilization) = number of mice with a constant tumor size during at least 6 consecutive measurements; PR (partial regression) = number of mice with a tumor size lower than the initial tumor size during at least 6 consecutive measurements; CR (complete regression) = number of mice with a tumor size of 0 to 14 mm during at least 3 consecutive measurements. 3 TFS (Tumor Free Survivor) = the number of complete remissions recorded until the end of the group day. Treatment was started 36 days after implantation.

[0395]

[0396] Table 3. Summary of Mann-Whitney analysis of tumor volume in the HBCx-34 tumor model, efficacy study XTS-1521.

[0397] Group comparisons between treatment and control groups were performed using the Mann-Whitney nonparametric test: ns = not significant, * = P < 0.05, ** = P < 0.01, and *** = P < 0.001. Initial group size: 9 to 10 animals.

[0398]

[0399] Table 4. Summary of Dunn's Multiple Comparison Test Analysis of Tumor Volume in the HBCx-34 Tumor Model, Efficacy Study XTS-1521. Group comparisons between treatment and control groups were performed using Dunn's Multiple Comparison Test: ns = not significant, * = P < 0.05, ** = P < 0.01, and *** = P < 0.001. Initial group size: 9 to 10 animals.

[0400]

[0401] Table 5. Weights and volumes of xenograft MCF-7 tumors prepared at Charles River for pharmacodynamic analysis. Tumors highlighted in red were included in the first round of VeraTag analysis.

[0402]

[0403] List of abbreviations in Example 1

[0404] bid: twice a day

[0405] BW: body weight

[0406] BWL: Weight Loss

[0407] C: control mice

[0408] CR: Complete tumor regression

[0409] inj: injection

[0410] iv: intravenous

[0411] ip: intraperitoneal

[0412] IVC: Individually ventilated cage

[0413] kg: kilogram

[0414] mg: milligrams

[0415] ml: milliliters

[0416] mut: mutated

[0417] ns: not significant

[0418] po: per os, through the mouth (tube feeding)

[0419] PR: Partial tumor regression

[0420] PSU: Polysulfone

[0421] qd: daily (Que die), every day

[0422] qwk: once a week

[0423] RBW: relative body weight

[0424] RTV: relative tumor volume

[0425] S: significant

[0426] sc: subcutaneous

[0427] sem: standard error of the mean

[0428] SoC: Standard of Care

[0429] T: treated mice

[0430] TGD: tumor growth delay

[0431] TGDI: tumor growth delay index

[0432] TGI: tumor growth inhibition

[0433] TFS: tumor-free survivor

[0434] TS: tumor stability

[0435] %T / C: Percentage change in tumor volume in the treatment group relative to the control group

[0436] μL: microliter

[0437] qd: daily (quotidian)

[0438] List of abbreviations in Example 2

[0439] BW: body weight

[0440] CR: Complete tumor regression

[0441] D: Research Day

[0442] ip: intraperitoneal

[0443] MTD: Maximum tolerated dose

[0444] MTV: median tumor volume

[0445] NTR: Not Treatment Related

[0446] PR: Partial tumor regression

[0447] qod×15: Administer every other day for 15 doses

[0448] qwk×5: weekly administration for 5 weeks

[0449] sc: subcutaneous

[0450] TGI: tumor growth inhibition

[0451] TR: Treatment-related death

[0452] List of abbreviations in Example 4

[0453] AEs: adverse events

[0454] ALT: alanine aminotransferase

[0455] ANC: absolute neutrophil count

[0456] AST: aspartate aminotransferase

[0457] AUC: Area under the curve

[0458] CBR: clinical benefit rate

[0459] CHF: Congestive heart failure

[0460] CR: Complete tumor regression

[0461] DoR: time from response (CR or PR) until progression or death due to the underlying cancer.

[0462] ECHO: echocardiogram

[0463] ER: estrogen receptor

[0464] FISH: Fluorescence in situ hybridization

[0465] IHC: Immunohistochemistry

[0466] IRR: Infusion-related reaction

[0467] MBC: metastatic breast cancer

[0468] MDRD: Modification of Diet in Renal Disease

[0469] MUGA: Multiple Gated Acquisition Scan

[0470] LVD: Left ventricular dysfunction

[0471] LVEF: left ventricular ejection fraction

[0472] LHRH: Luteinizing hormone-releasing hormone

[0473] PFS: progression-free survival

[0474] PR: progesterone receptor or partial response, depending on the context

[0475] OCOG: Eastern Cooperative Oncology Group

[0476] ORR: objective response rate

[0477] OS: time from the start of treatment until death due to any cause.

[0478] SPC: Summary of Product Characteristics

[0479] ULN: Upper limit of normal

Claims

1. Use of a therapeutically effective amount of an ErbB-2 / ErbB-3 bispecific antibody in combination with a therapeutically effective amount of an endocrine therapy drug in the preparation of a medicament for treating a subject having or at risk of having breast cancer, wherein the bispecific antibody comprises: an ErbB-2-specific heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences of AYYIN, RIYPGSGYTSYAQKFQG, and PPVYYDSAWFAY, respectively; and an ErbB-3-specific heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences of GYYMH, WINPNSGGTNYAQKFQG, and DHGSRHFWSYWGFDY, respectively; and wherein the ErbB-2-specific heavy chain variable region and the ErbB-3-specific heavy chain variable region are paired with a light chain variable region comprising the following sequence: DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; and The endocrine therapy drug is selected from tamoxifen, fulvestrant or letrozole.

2. The method according to claim 1, wherein the endocrine therapy drug is tamoxifen.

3. The method according to claim 1, wherein the endocrine therapy drug is fulvestrant. The method according to claim 1 , wherein the endocrine therapy drug is letrozole. The use according to claim 1 , wherein the breast cancer is hormone receptor-positive breast cancer. The use according to claim 1 , wherein the breast cancer is estrogen receptor-positive breast cancer. The use according to claim 1 , wherein the breast cancer is progesterone receptor-positive breast cancer.

8. The use according to claim 1, wherein the breast cancer is an immunohistochemical ErbB-2+ cancer or an immunohistochemical ErbB-2++ cancer without ErbB-2 gene amplification.

9. The use according to claim 1, wherein the breast cancer is ER-positive metastatic breast cancer (MBC) with low HER2 expression, which is IHC 1+ or IHC 2+ combined with negative FISH.

10. The use according to claim 1, wherein the bispecific antibody is capable of reducing ErbB-3 ligand-induced receptor function on ErbB-2 and ErbB-3 positive cells.

11. Use of a therapeutically effective amount of an ErbB-2 / ErbB-3 bispecific antibody in combination with a therapeutically effective amount of an endocrine therapy drug in the preparation of a medicament for treating a subject with hormone receptor-positive breast cancer, wherein the bispecific antibody inhibits ErbB-2 and ErbB-3 dimerization, wherein the bispecific antibody comprises: an ErbB-2-specific heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences of AYYIN, RIYPGSGYTSYAQKFQG, and PPVYYDSAWFAY, respectively; and an ErbB-3-specific heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences of GYYMH, WINPNSGGTNYAQKFQG, and DHGSRHFWSYWGFDY, respectively; and wherein the ErbB-2-specific heavy chain variable region and the ErbB-3-specific heavy chain variable region are paired with a light chain variable region comprising the following sequence: DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; and The endocrine therapy drug is selected from tamoxifen, fulvestrant or letrozole.

12. The use according to claim 11, wherein the cancer is an immunohistochemical ErbB-2+ cancer or an immunohistochemical ErbB-2++ cancer without ErbB-2 gene amplification.

13. The use according to claim 11, wherein the breast cancer is ER-positive metastatic breast cancer (MBC) with low HER2 expression, IHC 1+ or IHC 2+ combined with negative FISH.

14. The use according to claim 1 or 11, wherein the treatment further comprises a cyclin-dependent kinase 4 / 6 inhibitor.

15. The use according to claim 1 or 11, wherein the subject has been treated with one or more endocrine therapies before starting treatment with the ErbB-2 / ErbB-3 bispecific antibody and the therapeutically effective amount of the endocrine therapy.

16. The use according to claim 1 or 11, wherein the subject has received a cyclin-dependent kinase inhibitor prior to starting treatment with the ErbB-2 / ErbB-3 bispecific antibody and the therapeutically effective amount of the endocrine therapy drug.

17. The use according to claim 1 or 11, further comprising determining the expression level of estrogen receptor, ErbB-2, ErbB-3, or a combination thereof on cells of the cancer.

18. The use according to claim 1 or 11, wherein the breast cancer has a hormone receptor positive status of ≥ 1% positive cells as determined by immunohistochemistry of a tumor biopsy, wherein the hormone receptor positive status refers to estrogen receptor positive [ER+] and / or progesterone receptor positive [PR+].

19. The use according to claim 1 or 11, wherein the treatment comprises administering the ErbB-2 / ErbB-3 bispecific antibody at an intravenous dose of 300 mg to 900 mg.

20. The use according to claim 1 or 11, wherein the treatment comprises administering the ErbB-2 / ErbB-3 bispecific antibody at an intravenous dose of 300 mg to 900 mg every two to four weeks.

21. The use according to claim 1 or 11, wherein the antibody and the endocrine therapeutic drug are for simultaneous or sequential use.

22. The use according to claim 1 or 11, wherein the antibody and the endocrine therapeutic drug are used separately.

23. The use according to claim 1, wherein the breast cancer is hormone receptor positive breast cancer, and wherein the antibody inhibits ErbB-2, ErbB-3 dimerization.

24. The use according to claim 1 or 11, wherein the ErbB-2 specific heavy chain variable region comprises the amino acid sequence: QVQLVQSGAEVKKPGASVKLSCKASGYTFTAYYINWVRQAPGQGLEW IGRIYPGSGYTSYAQKFQGRATLTADESTSTAYMELSSLRSEDTAVYFCARPP VYYDSAWFAYWGQGTLVTVSS.

25. The use according to claim 1 or 11, wherein the ErbB-3 specific heavy chain variable region comprises the amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLE WMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCA RDHGSRHFWSYWGFDYWGQGTLVTVSS.

26. The use according to claim 1 or 11, wherein the ErbB-2-specific heavy chain variable region and the ErbB-3-specific heavy chain variable region are paired with a light chain comprising the following sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

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