HK40027114B - Combination of an erbb-2/erbb-3 bispecific antibody with endocrine therapy for breast cancer - Google Patents

Description

FIELD OF THE INVENTION

The invention relates to the field of antibodies. In particular it relates to the field of therapeutic (human) antibodies for the treatment of diseases involving aberrant cells. More in particular it relates to antibodies that can bind ErbB-2 and ErbB-3 and the treatment of subjects with breast cancer with these antibodies in combination with an endocrine therapy drug.

BACKGROUND

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 them grow. About 2 out of 3 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.

There are various drugs that interfere with this mechanism.

Drugs that block estrogen receptors

These drugs work by stopping estrogen from affecting breast cancer cells. An example of such a drug is Tamoxifen. This drug blocks estrogen receptors in breast cancer cells. This stops estrogen from binding to the cancer cells and telling them to grow and divide. While Tamoxifen acts like an anti-estrogen in breast cells, it acts like estrogen in other tissues, like the uterus and the bones. Because of this, it is called a selective estrogen receptor modulator (SERM). Tamoxifen is among the most well-known SERMs. Other SERMs that have been 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 treatment of hormone receptor positive breast cancer. Many more SERMS have not (yet) been approved but are also functional.

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

In women at high risk of breast cancer, Tamoxifen can be used to help lower the risk of developing breast cancer.

Toremifene (Fareston) is another SERM presently approved to treat metastatic breast cancer. These drugs mostly are taken orally, most often as a pill.

Fulvestrant (Faslodex)

Fulvestrant is a drug that blocks estrogen receptors and also eliminates them temporarily. 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 hormone drugs (like Tamoxifen and often an aromatase inhibitor) have stopped working.

It can be administered by intramuscular injection. Fulvestrant is currently approved for use in post-menopausal women.

Aromatase inhibitors (AIs)

Aromatase inhibitors (AIs) are drugs that stop estrogen production. Before menopause, most estrogen is made by the ovaries. But for women whose ovaries are no longer working, either due to menopause or medical treatments, a small amount of estrogen is still made by an enzyme (called aromatase) in the adipose tissue, breast or skin. It is understood that AIs work by blocking aromatase from making estrogen.

These drugs are used in post-menopausal women, although they can also be used in premenopausal women if combined with ovarian ablation.

There are various AIs. The exemplary AIs include: Letrozole (Femara); Anastrozole (Arimidex) and Exemestane (Aromasin)

These drugs are pills that may be taken daily.

For post-menopausal women whose cancers are hormone receptor-positive, doctors may recommend taking an AI during adjuvant therapy.

Luteinizing hormone-releasing hormone (LHRH) analogs: These drugs are used more often than oophorectomy. It is understood that they stop the signal that the body sends to ovaries to make estrogen, which causes temporary menopause. Common LHRH drugs include Goserelin (Zoladex) and Leuprolide (Lupron). They can be used alone or with other hormone drugs (Tamoxifen, aromatase inhibitors, Fulvestrant) as hormone therapy in pre-menopausal women.

Chemotherapy drugs: Some chemotherapeutic drugs can damage the ovaries of pre-menopausal women so they no longer make estrogen. For some women, ovarian function returns months or years later, but in others, the damage to the ovaries is permanent and leads to menopause.

The drugs mentioned above are collectively referred to as endocrine therapy drugs of breast cancer. Endocrine therapy and the accompanying drugs have recently been reviewed by Lumachi et al (Lumachi, F., et al. "Endocrine therapy of breast cancer." Current medicinal chemistry 18.4 (2011): 513-522). In the context of the present invention endocrine therapy drugs refers to drugs that are used for endocrine therapy of breast cancer.

In the context of an invention described herein, the term endocrine therapy includes therapy drugs that interfere with the action of a hormone, typically the hormone estrogen or progesterone in the cancer cell. This can be directly by the action of the drug in the cancer cell, or indirectly by for instance lowering the amount of estrogen that can reach the cancer cell, including by interfering directly or indirectly with the action of estrogen on the tumor.

Immunotherapy of breast cancer is also available. ErbB-2/ErbB-3 bispecific antibodies are described in WO2015130173 , US2014/056898 . In particular, US2014/056898 discloses MM-111, an ErbB2/ErbB3 bispecific antibody, in combination with tamoxifen, or with fulvestrant; the combinations were used to treat mice implanted with breast cancer-derived BT474-M3 cells. A combination of MM-111 and letrozole was also shown to inhibit spheroid growth of BT474-M3 cells.

SUMMARY OF THE INVENTION

The invention as claimed is provided in the appended set of claims.

The invention provides a combination of an ErbB-2/ErbB-3 bispecific antibody and an endocrine therapy drug for use in the treatment of breast cancer in a subject that has breast cancer or is at risk of having said cancer, wherein the bispecific antibody has an antigen binding site that can bind an extra-cellular part of ErbB-2 and an antigen binding site that can bind an extra-cellular part of ErbB-3, wherein said antibody comprises

• a variable domain with a heavy chain variable region comprising at least the CDR1, CDR2 and CDR3 sequences AYYIN, RIYPGSGYTSYAQKFQG and PPVYYDSAWFAY, respectively, of an ErbB-2 specific heavy chain variable region,
• a variable domain with a heavy chain variable region comprising at least the CDR1, CDR2 and CDR3 sequences GYYMH, WINPNSGGTNYAQKFQG and DHGSRHFWSYWGFDY, respectively, of an ErbB-3 specific heavy chain variable region, and
• a common light chain having sequence and
• wherein the endocrine therapy drug comprises Tamoxifen, Fulvestrant, or Letrozole.

Further embodiments of the invention are provided inter alia in the claims.

The antibody may be MCLA-128.

In one embodiment the cancer is an immunohistochemistry ErbB-2 + cancer or an immunohistochemistry ErbB-2 ++ without ErbB-2 gene amplification cancer.

In one embodiment the breast cancer is ER-positive with low HER2 expression metastatic breast cancer MBC IHC 1+, or IHC 2+ combined with negative FISH.

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 may be, for example, Palbociclib, Ribociclib or Abemaciclib.

In one embodiment the subject that has breast cancer or is at risk of having said cancer which includes subjects at risk of relapse is a subject that had received 1 and preferably 2 endocrine therapy treatments prior to initiation of a treatment with an ErbB-2/ErbB-3 bispecific antibody as described herein. The subject preferably received these prior treatments for treatment of a metastasis. The subject in addition preferably had received a cyclin-dependent kinase inhibitor prior to initiation of a treatment with an ErbB-2/ErbB-3 bispecific antibody as described herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a combination of an ErbB-2/ErbB-3 bispecific antibody and an endocrine therapy drug for use in the treatment of breast cancer, as defined in the appened claims.

The technical disclosure set out below may in some respects go beyond the scope of the invention, which is defined by the appended claims. Elements of the disclosure which do not fall within the scope of the claims are provided for information, namely to place the actual invention claimed in a broader technical context.

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 cancers are routinely tested for the presence of the mentioned hormone receptors and generally accepted classifications and tests are available to the skilled person. Reference is made to Hammond et al (2010: J. of Clinical Oncology Vol 28:pp 2784-2794) which describe suitable tests and provides guidelines therefore. For example, a patient suitable for treatment according to the invention is one in which at least 1% of tumor nuclei, for example in a tumor biopsy, are immunoreactive; positive to estrogen receptor and/or progesterone receptor as determined by immunohistochemistry.

Further, another example of a suitable patient for treatment according to the invention is one with a cancer with documented hormone receptor positive status, estrogen receptor positive [ER+] and/or progesterone receptor positive [PR+]), including ≥ 1% positive stained cells by local standards, based on local analysis on the most recent tumor biopsy.

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

The 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 tests and provide recommendations. A generally accepted stratification of breast cancers on the basis of 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 an ErbB-2+ or an ErbB-2++ without ErbB-2 gene amplification breast cancer, which includes the absence of gene amplification at the level of detection.

Fluorescence in situ hybridization (FISH) may be used to determine the presence or absence of gene amplification. Accordingly, a patient suitable for treatment according to the invention may be one with a cancer which shows no ErbB-2 gene amplification according to FISH analysis, understood by the person of ordinary skill in the art to be FISH negative.

A suitable patient for treatment according to the invention may be one which is ER-positive with low HER2 expression metastatic breast cancer (MBC) (immunohistochemistry (IHC) 1+, or IHC 2+ combined with negative fluorescence in situ hybridization (FISH).

In one embodiment the breast cancer is an ErbB-3 positive breast cancer.

In one embodiment, the bispecific antibody can reduce a ligand-induced receptor function of ErbB-3 on a ErbB-2 and ErbB-3 positive cell.

As used herein, the term "antigen-binding site" refers to a site derived from and preferably as present on an antibody which is capable of binding to antigen. An unmodified antigen-binding site is typically formed by and present in the variable domain of the antibody. The variable domain contains said antigen-binding site. A variable domain that binds an antigen is a variable domain comprising an antigen-binding site that binds the antigen.

An antibody variable domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). The antigen-binding site can be present in the combined VH/VL variable domain, or in only the VH region or only the VL region. When the antigen-binding site is present in only one of the two regions of the variable domain, the counterpart variable region can contribute to the folding and/or stability of the binding variable region, but does not significantly contribute to the binding of the antigen itself.

As used herein, antigen-binding refers to the typical binding capacity of an antibody to its antigen. An antibody comprising an antigen-binding site that binds to ErbB-3, binds to ErbB-3 and, under otherwise identical conditions, not to the homologous 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 that express the receptor(s). An antibody of the invention binds human ErbB-2, and human ErbB-3.

Antigen binding by an antibody is typically mediated through the complementarity regions of the antibody and the specific three-dimensional structure of both the antigen and the variable domain allowing these two structures to bind together with precision (an interaction similar to a lock and key), as opposed to random, non-specific sticking of antibodies. As an antibody typically recognizes an epitope of an antigen, and as such epitope may be present in other compounds as well, antibodies as described in the claims that bind ErbB-2 and ErbB-3 may recognize other proteins as well, if such other compounds contain the same epitope. Hence, the term "binding" does not exclude binding of the antibodies to another protein or protein(s) that contain the same epitope. Such other protein(s) is preferably not a human protein. An ErbB-2 antigen-binding site and an ErbB-3 antigen-binding site as defined in the claims typically do not bind to other proteins on the membrane of cells in a post-natal, preferably adult human. A bispecific antibody as described in the claims is typically capable of binding ErbB-2 or ErbB-3 with a binding affinity of at least 1x10e-6 M, as outlined in more detail below.

The term "interferes with binding" as used herein means that the antibody is directed to an epitope on ErbB-3 and the antibody competes with ligand for binding to ErbB-3. The antibody may diminish ligand binding, displace ligand when this is already bound to ErbB-3 or it may, for instance through steric hindrance, at least partially prevent that ligand from binding to ErbB-3.

The term "antibody" as used herein means a proteinaceous molecule, preferably belonging to the immunoglobulin class of proteins, containing one or more variable domains that bind an epitope on an antigen, where such domains are derived from or share sequence homology with the variable domain of an antibody. Antibodies for therapeutic use are preferably as close to natural antibodies of the subject to be treated as possible (for instance human antibodies for human subjects). Antibody binding can be expressed in terms of specificity and affinity. The specificity determines which antigen or epitope thereof is specifically bound by the binding domain. The affinity is a measure for the strength of binding to a particular antigen or epitope. Specific binding, is defined as binding with affinities (KD) of at least 1x10e-6 M, more preferably 1x10e-7 M, more preferably higher than 1x10e-9 M. Typically, antibodies for therapeutic applications have affinities of up to 1x10e-10 M or higher. Antibodies such as the bispecific antibodies as described in the claims may comprise the constant domains (Fc part) of a natural antibody. An antibody as described in the claims is typically a bispecific full length antibody, preferably of the human IgG subclass. Preferably, an antibody as described in the claims is of the human IgG1 subclass. Such antibodies have good ADCC properties, have favorable half life upon in vivo administration to humans and CH3 engineering technology exists that can provide for modified heavy chains that preferentially form heterodimers over homodimers upon co-expression in clonal cells.

An antibody as described in the claims is preferably a "full length" antibody. The term 'full length' according to the invention is defined as comprising an essentially complete antibody, which however does not necessarily have all functions of an intact antibody. For the avoidance of doubt, a full length antibody contains two heavy and two light chains. Each chain contains constant (C) and variable (V) regions, which can be broken down into domains designated CH1, CH2, CH3, VH, and CL, VL. An antibody binds to antigen via the variable domains contained in the Fab portion, and after binding can interact with molecules and cells of the immune system through the constant domains, mostly through the Fc portion. The terms 'variable domain', 'VH/VL pair', 'VH/VL' are used herein interchangeably. Full length antibodies encompass antibodies wherein mutations may be present that provide desired characteristics. Such mutations should not be deletions of substantial portions of any of the regions. However, antibodies wherein one or several amino acid residues are deleted, without essentially altering the binding characteristics of the resulting antibody are embraced within the term "full length antibody". For instance, an IgG antibody can have 1-20 amino acid residue insertions, deletions or a combination thereof in the constant region. For instance, ADCC activity of an antibody can be improved when the antibody itself has a low ADCC activity, by slightly modifying the constant region of the antibody (Junttila, T. T., 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)

Full length IgG antibodies are preferred because of their favourable half-life and the need to stay as close to fully autologous (human) molecules for reasons of immunogenicity. An antibody as described in the claims is preferably a bispecific IgG antibody, preferably a bispecific full length IgG1 antibody. IgG1 is favoured based on its long circulatory half life in man. In order to prevent any immunogenicity in humans it is preferred that the bispecific IgG antibody as described in the claims is a human IgG1.

The term 'bispecific' (bs) means that one part of the antibody (as defined above) binds to one epitope on an antigen whereas a second part binds to a different epitope. The different epitope is typically present on a different antigen. In an antibody as described in the claims, said first and second antigens are in fact two different proteins. A preferred bispecific antibody as described in the claims is an antibody that comprises parts of two different monoclonal antibodies and consequently binds to two different types of antigen. One arm of the bispecific antibody contains the variable domain of one antibody and the other arm contains the variable domain of another antibody. The heavy chain variable regions of the bispecific antibody as described in the claims are typically different from each other, whereas the light chain variable regions are the same in the bispecific antibodies as described in the claims. A bispecific antibody wherein the different heavy chain variable regions are associated with a common light chain is referred to as a bispecific antibody with a common light chain.

Preferred bispecific antibodies can be obtained by co-expression of two different heavy chains and a common light chain in a single cell. When wildtype CH3 domains are used, 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 engineering can be employed, or in other words, one can use heavy chains with compatible heterodimerization domains, as defined hereunder.

The term 'compatible heterodimerization domains' as used herein refers to protein domains that are engineered such that engineered domain A' will preferentially form heterodimers with engineered domain B' and vice versa, whereas homodimerization between A'-A' and B'-B' is diminished.

The term `common light chain' as described in the claims refers to a light chain having sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC. The terms `common light chain', `common VL', `single light chain', `single VL', with or without the addition of the term `rearranged' are all used herein interchangeably. Preferred as a common light chain is a human light chain that can combine with different heavy chains to form antibodies with functional antigen binding domains ( WO2004/009618 , WO2009/157771 , Merchant et al. 1998 and Nissim et al. 1994). Said common light chain has a germline sequence. Said germline sequence is a light chain variable region that is frequently used in the human repertoire and has good thermodynamic stability, yield and solubility. Said germline light chain is the rearranged germline human kappa 012 light chain IgVκ1-39*01/IGJκ1*01(nomenclature according to the IMGT database worldwide web at imgt.org). The terms rearranged germline human kappa light chain IgVκ1-39*01/IGJκ1*01, IGKV1-39/IGKJ1, huVκ1-39 light chain or in short huVκ1-39 are used interchangeably throughout the application. Said common light chain comprises the sequence

The term `ErbB-2' as used herein refers to the protein that in humans is encoded by the ERBB-2 gene. Alternative names for the gene or protein include CD340; HER-2; HER-2/neu; MLN 19; NEU; NGL; TKR1. The ERBB-2 gene is frequently called HER2 (from human epidermal growth factor receptor 2). Where reference is made herein to ErbB-2, the reference refers to human ErbB-2. An antibody comprising an antigen-binding site that binds ErbB-2, binds human ErbB-2. The ErbB-2 antigen-binding site may, due to sequence and tertiary structure similarity between human and other mammalian orthologs, also bind such an ortholog but not necessarily so. Database accession numbers for the human ErbB-2 protein and the gene encoding it are (NP_001005862.1, NP_004439.2 NC_000017.10 NT_010783.15 NC_018928.2). The accession numbers are primarily given to provide a further method of identification of ErbB-2 as a target, the actual sequence of the ErbB-2 protein bound the antibody may vary, for instance because of a mutation in the encoding gene such as those occurring in some cancers or the like. The ErbB-2 antigen binding site binds ErbB-2 and a variety of variants thereof, such as those expressed by some ErbB-2 positive tumor cells.

The term `ErbB-3' as used herein refers to the protein that in humans is encoded by the ERBB-3 gene. 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. Where reference is made herein to ErbB-3, the reference refers to human ErbB-3. An antibody comprising an antigen-binding site that binds ErbB-3, binds human ErbB-3. The ErbB-3 antigen-binding site, may, due to sequence and tertiary structure similarity between human and other mammalian orthologs, also bind such an ortholog but not necessarily so. Database accession numbers for the human ErbB-3 protein and the gene encoding it are (NP_001005915.1 NP_001973.2, NC_000012.11 NC_018923.2 NT_029419.12 ). The accession numbers are primarily given to provide a further method of identification of ErbB-3 as a target, the actual sequence of the ErbB-3 protein bound by an antibody may vary, for instance because of a mutation in the encoding gene such as those occurring in some cancers or the like. The ErbB-3 antigen binding site binds ErbB-3 and a variety of variants thereof, such as those expressed by some ErbB-2 positive tumor cells.

A bispecific antibody as described in the claims that comprises a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, can reduce or reduces a ligand-induced receptor function of ErbB-3 on an ErbB-2 and ErbB-3 positive cell. In the presence of excess ErbB-2, ErbB-2/ErbB-3 heterodimers may provide a growth signal to the expressing cell in the absence of detectable ligand for the ErbB-3 chain in the heterodimer. This ErbB-3 receptor function is herein referred as a ligand-independent receptor function of ErbB-3. The ErbB-2/ErbB-3 heterodimer also provide a growth signal to the expressing cell in the presence of an ErbB-3 ligand. This ErbB-3 receptor function is herein referred to as a ligand-induced receptor function of ErbB-3.

The term "ErbB-3 ligand" as used herein refers to polypeptides which bind and activate ErbB-3. Examples of ErbB-3 ligands include, but are not limited to neuregulin 1 (NRG) and neuregulin 2, betacellulin, heparin-binding epidermal growth factor, and epiregulin. The term includes biologically active fragments and/or variants of a naturally occurring polypeptide.

The term "ligand-induced receptor function of ErbB-3" preferably refers to ErbB-3 ligand-induced growth of an ErbB-2 and ErbB-3 positive cell. Said cell preferably is an MCF-7 cell (ATCC^® HTB-22^™); an SKBR3 (ATCC^® HTB-30^™) cell; an NCI-87 (ATCC^® CRL-5822^™) cell; a BxPC-3-luc2 cell (Perkin Elmer 125058), a BT-474 cell (ATCC^® HTB-20^™) or a JIMT 1 cell (DSMZ no.: ACC 589).

In a preferred embodiment the ErbB-2 and ErbB-3 positive cell comprises at least 50.000 ErbB-2 receptors on the cell surface. In a preferred embodiment at least 100.000 ErbB-2 receptors. In one preferred embodiment, the ErbB-2 and ErbB-3 positive cell comprises at least 1.000.000 ErbB-2 receptors on the cell surface. In another preferred embodiment the ErbB-2 and ErbB-3 positive cell comprises no more than 1.000.000 ErbB-2 receptors on the cell surface. Currently used therapies such as trastuzumab (Herceptin) and pertuzumab are only prescribed for patients with malignant ErbB-2 positive cells that have more than 1.000.000 ErbB-2 receptors on their cell surface, in order to obtain a clinical response. Patients with ErbB-2 positive tumor cells with more than 1.000.000 ErbB-2 receptors on their cell surface are typically classified as ErbB-2 [+++]. Patients are for instance classified using the HercepTestTM and/or HER2 FISH (pharm Dx^™), marketed both by Dako Denmark A/S, and/or using a HERmark^® assay, marketed by Monogram Biosciences. Trastuzumab and pertuzumab are only prescribed to ErbB-2 [+++] patients because patients with lower ErbB-2 concentrations typically do not exhibit a sufficient clinical response when treated with trastuzumab and pertuzumab. The bispecific antibodies as described in the claims also have an improved binding affinity for cells with a lower ErbB-2 receptor concentration, as compared to trastuzumab. As shown in the Examples, proliferation of such cells with lower ErbB2 expression is effectively counteracted with an antibody as described in the claims. Such lower ErbB-2 receptor concentration is present on malignant cells of patients that are classified as ErbB-2 [++] or ErbB-2 [+]. Also, relapsed ErbB-2 positive tumors often have an ErbB-2 receptor concentration of lower than 1.000.000 receptors per cell. Such ErbB-2 [++] or ErbB-2 [+] patients, as well as patients with a relapsed ErbB-2 positive tumor, are therefore preferably treated with an endocrine therapy drug and a bispecific antibody as referred to in the claims.

Further provided is therefore 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 antibody can reduce ligand-induced growth of an ErbB-2 and ErbB-3 positive cell that has less than 1.000.000 ErbB-2 cell-surface receptors. Also provided is a bispecific antibody as described in the claims and a therapeutically effective amount of an endocrine therapy drug for use in a method for the treatment of a subject having ErbB-2, ErbB-3 or ErbB-2/ErbB-3 positive tumor or at risk of having said tumor, wherein said tumor has less than 1.000.000 ErbB-2 cell-surface receptors per cell, the method comprising administering to the subject said bispecific antibody. Said antibody as described in the claims is typically capable of reducing a ligand-induced receptor function, preferably ligand induced growth, of ErbB-3 on a ErbB-2 and ErbB-3 positive cell. Said antibody as described in the claims comprises a first antigen-binding site that binds domain I of ErbB-2 and a second antigen-binding site that binds domain III of ErbB-3. The affinity of said second antigen-binding site for an ErbB-3 positive cell preferably is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB-2 positive cell, as explained herein below in more detail. The affinity of said second antigen-binding site for an ErbB-3 positive cell is preferably lower than or equal to 2.0 nM, more preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. The affinity of said first antigen-binding site for an ErbB-2 positive cell is preferably lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM preferably lower than or equal to 4.0 nM.

A bispecific antibody as described in the claims preferably comprises an antigen-binding site that binds at least one amino acid of domain I of ErbB-2 selected from the group consisting ofT144, T164, R166, P172, G179, S180 and R181, and surface-exposed amino acid residues that are located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181.

Said antibody as described in the claims preferably comprises an antigen-binding site that binds at least one amino acid of domain III of ErbB-3 selected from the group consisting of R426 and surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB-3 protein.

To establish whether a tumor is positive for ErbB-3 the skilled person can for instance determine the ErbB-3 gene amplification and/or staining in immunohistochemistry. At least 10% tumor cells in a biopsy should be positive. The biopsy can also contain 20%, 30% 40% 50% 60% 70% or more positive cells.

As used herein the ligand-induced receptor function 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%. The reduction is preferably determined by determining a ligand-induced receptor function in the presence of a bispecific antibody as described in the claims, and comparing it with the same function in the absence of the antibody, under otherwise identical conditions. The conditions comprise at least the presence of an ErbB-3 ligand. The amount of ligand present is preferably an amount that induces half of the maximum growth of an ErbB-2 and ErbB-3 positive cell line. The ErbB-2 and ErbB-3 positive cell line for this test is preferably the MCF-7 cell line (ATCC^® HTB-22^™), the SKBR3 cell line (ATCC^® HTB-30^™) cells, the JIMT 1 cell line (DSMZ ACC 589) or the NCI-87 cell line (ATCC^® CRL-5822^™). The test and/or the ligand for determining ErbB-3 ligand-induced receptor function is preferably a test for ErbB-3 ligand induced growth reduction as specified in the examples.

The ErbB-2 protein contains several domains (see for reference figure 1 of Landgraf, R Breast Cancer Res. 2007; 9(1): 202-). The extracellular domains are referred to as domains I-IV. The place of binding to the respective domains of antigen-binding sites of antibodies described herein has been mapped (see examples). Bispecific antibodies with an antigen-binding site (first antigen-binding site) that binds domain I of ErbB-2 (first antigen-binding site) and an antigen-binding site for ErbB-3 (second antigen-binding site) were found to be more effective in reducing a ligand-induced receptor function of ErbB-3 when compared to a bispecific antibody comprising an antigen-binding site (first antigen-binding site) that binds to another extra-cellular domain of ErbB-2. A bispecific antibody comprising an antigen-binding site (first antigen-binding site) that binds ErbB-2, wherein said antigen-binding site binds to domain I of ErbB-2 is used in the claims.. A bispecific antibody with an antigen-binding site (first antigen-binding site) that binds ErbB-2, and that further comprises ADCC was found to be more effective than other ErbB-2 binding antibodies that did not have significant ADCC activity, particularly in vivo. A bispecific antibody as described in the claims which exhibits ADCC is therefore preferred. It was found that antibodies wherein said first antigen-binding site binds to domain IV of ErbB-2 had intrinsic ADCC activity. A domain I binding ErbB-2 binding antibody that has low intrinsic ADCC activity can be engineered to enhance the ADCC activity. Fc regions mediate antibody function by binding to different receptors on immune effector cells such as macrophages, natural killer cells, B-cells and neutrophils. Some of these receptors, such as CD16A (FcyRIIIA) and CD32A (FcyRIIA), activate the cells to build a response against antigens. Other receptors, such as CD32B, inhibit the activation of immune cells. By engineering Fc regions (through introducing amino acid substitutions) that bind to activating receptors with greater selectivity, antibodies can be created that have greater capability to mediate cytotoxic activities desired by an anti-cancer Mab.

One technique for enhancing ADCC of an antibody is afucosylation. (See for instance Junttila, T. T., 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). Further provided is therefore a bispecific antibody as described in the claims, which is afucosylated. Alternatively, or additionally, multiple other strategies can be used to achieve ADCC enhancement, for instance including glycoengineering (Kyowa Hakko/Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis (Xencor and Macrogenics), all of which seek to improve Fc binding to low-affinity activating FcyRIIIa, and/or to reduce binding to the low affinity inhibitory FcyRIIb.

Several in vitro methods exist for determining the efficacy of antibodies or effector cells in eliciting ADCC. Among these are chromium-51 [Cr51] release assays, europium [Eu] release assays, and sulfur-35 [S35] release assays. Usually, a labeled target cell line expressing a certain surface-exposed antigen is incubated with antibody specific for that antigen. After washing, effector cells expressing Fc receptor CD 16 are typically coincubated with the antibody-labeled target cells. Target cell lysis is subsequently typically measured by release of intracellular label, for instance by a scintillation counter or spectrophotometry. A preferred test is detailed in the Examples.

One advantage of the bispecific antibodies as described in the claims is the fact that binding of these antibodies, such as for instance PB4188, to ErbB-2 and ErbB-3 positive cells results in internalization that is to the same extent as compared to trastuzumab. If a combination of trastuzumab and pertuzumab is used, internalization of these antibodies is enhanced. This enhanced internalization, however, results in reduced ADCC. An antibody as described in the claims resulting in internalization that is essentially to the same extent as compared to trastuzumab is, therefore, preferred over a combination of trastuzumab and pertuzumab because with such antibody the ADCC activity is better maintained.

A bispecific antibody as described in the claims, comprising an antigen-binding site that binds ErbB-3, interferes with binding of an ErbB-3 ligand to ErbB-3. Such antibodies are more effective in reducing a ligand-induced receptor function of ErbB-3 on an ErbB-2 and ErbB-3 positive cell line, particularly in the context of a bispecific antibody that also comprises an antigen-binding site that binds ErbB-2.

Preferred embodiments of the current invention as described in the claims provide a bispecific antibody comprising a first antigen-binding site that binds domain I of ErbB-2. In particular, and as shown in the Examples, bispecific antibodies comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein said first antigen-binding site binds domain I of ErbB-2, are well capable of binding ErbB-2 and ErbB-3 positive cells and counteracting their activity (such as the ligand-induced receptor function of ErbB-3 and the ligand-induced growth of an ErbB-2 and ErbB-3 positive cell). Moreover, such bispecific antibodies comprising a first antigen-binding site that binds domain I of ErbB-2 are particularly suitable for use in combination with existing anti-ErbB-2 therapies like trastuzumab and pertuzumab, because trastuzumab and pertuzumab bind different domains of ErbB-2. Trastuzumab binds domain IV of ErbB-2 and pertuzumab binds domain II of ErbB-2. Hence, bispecific antibodies as described in the claims that bind domain I of ErbB-2 do not compete with trastuzumab and pertuzumab for the same epitope.

A bispecific antibody as described in the claims comprises a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein said second antigen-binding site binds domain III of ErbB-3. Such antibody is particularly suitable for combination therapy with currently used anti- ErbB-3 binding molecules that do not bind domain III of ErbB-3, such as MM-121 (Merrimack Pharmaceuticals; also referred to as #Ab6) and RG7116 (Roche) that bind domain I of ErbB-3, because then the different binding molecules do not compete with each other for the same epitope.

A bispecific antibody that comprises a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein said first antigen-binding site binds domain I of ErbB-2 and said second antigen-binding site binds domain III of ErbB-3 is particularly suitable for combination therapy with anti- ErbB-2 binding molecules that do not bind domain I of ErbB-2, such as trastuzumab and pertuzumab, and with anti-ErbB-3 binding molecules that do not bind domain III of ErbB-3, such as MM-121 (#Ab6) and RG7116.

Said bispecific antibody comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein said first antigen-binding site binds domain I of ErbB-2 and said second antigen-binding site binds domain III of ErbB-3 preferably can reduce a ligand-induced receptor function of ErbB-3 on a ErbB-2 and ErbB-3 positive cell. Said antibody can reduce ligand-induced growth of an ErbB-2 and ErbB-3 positive cell.

The bispecific antibody as described in the claims, comprises a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein the affinity (KD) of said second antigen-binding site for an ErbB-3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB-2 positive cell. Contrary to bispecific compounds such as for instance MM 111 from Merrimack Pharmaceuticals, which have a higher affinity for ErbB-2 than for ErbB-3, the bispecific antibodies as described in the claims have an ErbB-3-specific arm with an affinity for ErbB-3 on cells that is higher than the affinity of the ErbB-2-specific arm for ErbB-2 on cells. Such bispecific antibodies are better capable of binding ErbB-3, despite the low cell surface concentration of ErbB-3. This provides the advantage that the functional activity against ErbB-3 is enhanced as compared to prior art compounds, meaning that these bispecific antibodies as described in the claims are better capable of counteracting ErbB-3 activity (such as ligand-induced growth).

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

The affinity (KD) of said second antigen-binding site for an ErbB-3 positive cell is preferably lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, more preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. The affinity of said second antigen-binding site for ErbB-3 on SK BR 3 cells is preferably lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, more preferably lower than or equal to 1.39 nM, preferably lower than or equal to 0.99 nM. Said affinity is preferably within the range of 1.39-0.59 nM. The affinity of said second antigen-binding site for ErbB-3 on BT 474 cells is preferably lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, more preferably lower than or equal to 1.0 nM, more preferably lower than 0.5 nM, more preferably lower than or equal to 0.31 nM, more preferably lower than or equal to 0.23 nM. Said affinity is preferably within the range of 0.31-0.15 nM. The above-mentioned affinities are preferably as measured using steady state cell affinity measurements, wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell-bound radioactivity is measured, as described in the Examples.

The affinity (KD) of said first antigen-binding site for an ErbB-2 positive cell is preferably lower than or equal to 5.0 nM, more preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 3.9 nM. The affinity of said first antigen-binding site for ErbB-2 on SK BR 3 cells is preferably lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 4.0 nM, more preferably lower than or equal to 3.5 nM, more preferably lower than or equal to 3.0 nM, more preferably lower than or equal to 2.3 nM. Said affinity preferably is within the range of 3.0-1.6 nM. The affinity of said first antigen-binding site for ErbB-2 on BT 474 cells is preferably 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. Said affinity is within the range of 4.5-3.3 nM. The above-mentioned affinities are preferably as measured using steady state cell affinity measurements, wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell-bound radioactivity is measured, as described in the Examples.

The affinity (KD) of said bispecific antibody as described in the claims for BT 474 cells is preferably lower than or equal to 5.0 nM, preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 4.0 nM, more preferably lower than or equal to 3.5 nM, more preferably lower than or equal to 3.7 nM, preferably lower than or equal to 3.2 nM. Said affinity preferably is within the range of 3.7-2.7 nM. The affinity of said bispecific antibody as described in the claims for SK BR 3 cells preferably 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 4.0 nM, more preferably lower than or equal to 3.5 nM, more preferably lower than or equal to 3.0 nM, preferably lower than or equal to 2.5 nM, more preferably lower than or equal to 2.0 nM. Said affinity preferably is within the range of 2.4-1.6 nM. Again, the above-mentioned affinities are preferably as measured using steady state cell affinity measurements, wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell-bound radioactivity is measured, as described in the Examples.

The bispecific antibody as described in the claims compres a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein the affinity (KD) of said second antigen-binding site for an ErbB-3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB-2 positive cell, and wherein the antibody can reduce a ligand-induced receptor function of ErbB-3 on a ErbB-2 and ErbB-3 positive cell. Said antibody can preferably reduce ligand-induced growth of an ErbB-2 and ErbB-3 positive cell.

The bispecific antibody as described herein comprises a first antigen-binding site that binds domain I of ErbB-2 and a second antigen-binding site that binds domain III of ErbB-3, wherein the affinity of said second antigen-binding site for an ErbB-3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB-2 positive cell.

Said second antigen-binding site binds domain III of ErbB-3 and has an affinity (KD) for an ErbB-3 positive cell that is preferably lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. Said second antigen-binding site binds domain III of ErbB-3 and has an affinity for ErbB-3 on SK BR 3 cells that is preferably lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, preferably lower than or equal to 1.39 nM, more preferably lower than or equal to 0.99 nM. Said affinity preferably is within the range of 1.39-0.59 nM. Said second antigen-binding site binds domain III of ErbB-3 and has an affinity for ErbB-3 on BT 474 cells that is preferably lower than or equal to 2.0 nM, more preferably lower than or equal to 1.5 nM, more preferably lower than or equal to 1.0 nM, more preferably lower than or equal to 0.5 nM, more preferably lower than or equal to 0.31 nM, more preferably lower than or equal to 0.23 nM. Said affinity preferably is within the range of 0.31-0.15 nM.

Said first antigen-binding site binds domain I of ErbB-2 and has an affinity (KD) for an ErbB-2 positive cell that is preferably lower than or equal to 5.0 nM, more preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 3.9 nM. Said first antigen-binding site binds domain I of ErbB-2 and has an affinity for ErbB-2 on SK BR 3 cells that is preferably lower than or equal to 5.0 nM, more preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 4.0 nM, more preferably lower than or equal to 3.5 nM, more preferably lower than or equal to 3.0 nM, more preferably lower than or equal to 2.5 nM, more preferably lower than or equal to 2.3 nM. Said affinity preferably is within the range of 3.0-1.6 nM. The affinity of said bispecific antibody for SK BR 3 cells is preferably lower than or equal to 5.0 nM, more preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 4.0 nM, more preferably lower than or equal to 3.5 nM, more preferably lower than or equal to 3.0 nM, more preferably lower than or equal to 2.5 nM, more preferably lower than or equal to 2.4 nM, more preferably lower than or equal to 2.0 nM. Said affinity is preferably within the range of 2.4-1.6 nM.

Said first antigen-binding site binds domain I of ErbB-2 and has an affinity (KD) for ErbB-2 on BT 474 cells that is preferably lower than or equal to 5.0 nM, more preferably lower than or equal to 4.5 nM, preferably lower than or equal to 3.9 nM. Said affinity preferably is within the range of 4.5-3.3 nM. The affinity of said bispecific antibody for BT 474 cells is preferably lower than or equal to 5.0 nM, more preferably lower than or equal to 4.5 nM, more preferably lower than or equal to 4.0 nM, more preferably lower than or equal to 3.7 nM, more preferably lower than or equal to 3.2 nM. Said affinity preferably is within the range of 3.7-2.7 nM.

Again, the above-mentioned affinities are preferably as measured using steady state cell affinity measurements, wherein cells are incubated at 4°C using radioactively labeled antibody, where after cell-bound radioactivity is measured, as described in the Examples.

The bispecific antibody as described in the claims preferably does not significantly affect the survival of cardiomyocytes. Cardiotoxicity is a known risk factor in ErbB-2 targeting therapies and the frequency of complications is increased when trastuzumab is used in conjunction with anthracyclines thereby inducing cardiac stress. For instance, the combination of doxycycline (DOX) with trastuzumab induces severe cardiac side effects. Clinical studies have estimated that 5% to 10% of patients who receive trastuzumab in the adjuvant setting of 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, in a retrospective study, it was demonstrated that the risk for developing asymptomatic cardiac dysfunction is actually as high as about 25% when trastuzumab is used in the adjuvant setting with DOX (Wadhwa et al., Breast Cancer Res Treat 2009;117:357-64). As shown in the Examples, the present invention provides antibodies described in the claims that target ErbB-2 and that do not, or to a significantly lesser extent as compared to trastuzumab and pertuzumab, affect the survival of cardiomyocytes. This provides an important advantage since cardiotoxicity is reduced. This is already advantageous for people who do not suffer from an impaired cardiac function, and even more so for people who do suffer from an impaired cardiac function, or who are at risk thereof, such as for instance subjects suffering from congestive heart failure (CHF), left ventricular dysfunction (LVD) and/or a ≥ 10% decreased Left Ventricular Ejection Fraction (LVEF), and/or subjects who have had a myocardial infarction. Bispecific antibodies as described in the claims that do not significantly affect the survival of cardiomyocytes are, therefore, preferred. In vitro, the function of cardiomyocytes is for instance measured by determining the viability of cardiomyocytes, by determining BNP (B-type natriuretic peptide, which is a cardiac biomarker), by determining QT prolongation, and/or by determining mitochondrial membrane potential.

Said antibody that does not significantly affect the survival of cardiomyocytes as described in the claims comprises:

• at least the CDR1, CDR2 and CDR3 sequences, of an ErbB-2 specific heavy chain variable region from MF3958 as depicted in Figure 10A or Figure 10E, and
• at least the CDR1, CDR2 and CDR3 sequences of an ErbB-3 specific heavy chain variable region from MF3178 as depicted in Figure 10B or Figure 10E or Figure 11. In one preferred embodiment, said antibody is PB4188.

Said bispecific antibody as described in the claims, comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, comprises an antigen-binding site that binds at least one amino acid residue of domain I of ErbB-2 selected from the group consisting ofT144, T164, R166, P172, G179, S180 and R181, and surface-exposed amino acid residues that are located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181. The amino acid residue numbering is that of Protein Data Bank (PDB) ID #1S78. As shown in the Examples, antibodies binding this region of domain I of ErbB-2 exhibit particularly good binding characteristics and they are capable of counteracting the activity of ErbB-2 positive cells (such as ligand-induced receptor function of ErbB-3 on a ErbB-2 and ErbB-3 positive cell, and/or ligand-induced growth of such cell). Moreover, such antibodies are particularly suitable for combination therapy with currently known anti-ErbB-2 monoclonal antibodies like trastuzumab (that binds domain IV of ErbB-2) and pertuzumab (that binds domain II of ErbB-2) because they bind different domains of ErbB-2. Hence, these antibodies can be used simultaneously without competition for the same epitope. The term "surface-exposed amino acid residues that are located within about 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181" refers to amino acid residues that are in the primary amino acid sequence located within about the first five amino acid residues adjacent to the recited residues and that are at least in part exposed to the outside of the protein, so that they can be bound by antibodies (see for instance Figure 21B of WO2015/130173 ). Preferably, said amino acid residue located within about 5 amino acid positions from 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, said antibody comprises an antigen-binding site that binds at least 2 or at least 3 amino acid residues of domain I of ErbB-2 selected from the group consisting ofT144, T164, R166, P172, G179, S180 and R181, and surface-exposed amino acid residues that are located within 5 amino acid positions from T144, T164, R166, P172, G179, S180 or R181.

The bispecific antibody as described in the claims preferably comprises an antigen-binding site that binds at least T144, R166 and R181 of domain I of ErbB-2. In another embodiment, the bispecific antibody as described in the claims comprises an antigen-binding site that binds at least T144, R166, P172, G179 and R181 of domain I of ErbB-2. In another embodiment the antibody as described in the claims comprises an antigen-binding site that binds at least T144, T164, R166, P172, G179, S180 and R181 of domain I of ErbB-2.

The bispecific antibody as described in the claims, comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, comprises an antigen-binding site that binds at least one amino acid of domain III of ErbB-3 selected from the group consisting R426 and surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB-3 protein. The amino acid residue numbering is that of Protein Data Bank (PDB) ID #4P59. As shown in the Examples, antibodies binding this region of domain III of ErbB-3 exhibit particularly good binding characteristics and they are capable of counteracting the activity of ErbB-3 positive cells (such as ligand-induced receptor function of ErbB-3 on a ErbB-2 and ErbB-3 positive cell, and/or ligand-induced growth of such cell). The term "surface-exposed amino acid residues that are located within 11.2 Å from R426 in the native ErbB-3 protein" refers to amino acid residues that are in the tertiary structure of the ErbB-3 protein spatially positioned within 11.2 Å from R426 and that are at least in part exposed to the outside of the protein, so that they can be bound by antibodies. Preferably, said amino acid residues that are located within 11.2 Å from R426 in the native ErbB-3 protein are selected from the group consisting of L423, Y424, N425, G427, G452, R453, Y455, E480, R481, L482, D483 and K485 (see for instance Figure 21C and Table 15 of WO2015/130173 ). The bispecific antibody as described in the claims preferably comprises an antigen-binding site that binds at least R426 of domain III of ErbB-3.

A bispecific antibody as described in the claims is preferably afucosylated in order to enhance ADCC activity. A bispecific antibody as described in the claims preferably comprises a reduced amount of fucosylation of the N-linked carbohydrate structure in the Fc region, when compared to the same antibody produced in a normal CHO cell.

A bispecific antibody as described in the claims is preferably used in humans. To this end a bispecific antibody as described in the claims is preferably a human or humanized antibody.

Tolerance of a human to a polypeptide is governed by many different aspects. Immunity, be it T-cell mediated, B-cell mediated or other is one of the variables that are encompassed in tolerance of the human for a polypeptide. The constant region of a bispecific antibody as described in the claims is preferably a human constant region. The constant region may contain one or more, preferably not more than 10, preferably not more than 5 amino-acid differences with the constant region of a naturally occurring human antibody. It is preferred that the constant part is entirely derived from a naturally occurring human antibody. Various antibodies produced herein are derived from a human antibody variable domain library. As such these variable domains are human. The unique CDR regions may be derived from humans, be synthetic or derived from another organism. The variable region is considered a human variable region when it has an amino acid sequence that is identical to an amino acid sequence of the variable region of a naturally occurring human antibody, but for the CDR region. The variable region of an ErbB-2 binding VH, an ErbB-3 binding VH, or a light chain in an antibody as described in the claims may contain one or more, preferably not more than 10, preferably not more than 5 amino-acid differences with the variable region of a naturally occurring human antibody, not counting possible differences in the amino acid sequence of the CDR regions. Such mutations occur also in nature in the context of somatic hypermutation.

Antibodies may be derived from various animal species, at least with regard to the heavy chain variable region. It is common practice to humanize such e.g. murine heavy chain variable regions. There are various ways in which this can be achieved among which there are CDR-grafting into a human heavy chain variable region with a 3D-structure that matches the 3-D structure of the murine heavy chain variable region; deimmunization of the murine heavy chain variable region, preferably done by removing known or suspected T- or B- cell epitopes from the murine heavy chain variable region. The removal is typically by substituting one or more of the amino acids in the epitope for another (typically conservative) amino acid, such that the sequence of the epitope is modified such that it is no longer a T- or B-cell epitope.

Such deimmunized murine heavy chain variable regions are less immunogenic in humans than the original murine heavy chain variable region. Preferably a variable region or domain as described in the claims is further humanized, such as for instance veneered. By using veneering techniques, exterior residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic or substantially non-immunogenic veneered surface. An animal as described herein preferably is a mammal, more preferably a primate, most preferably a human.

A bispecific antibody as described in the claims preferably comprises a constant region of a human antibody. According to differences in their heavy chain constant domains, antibodies are grouped into five classes, or isotypes: IgG, IgA, IgM, IgD, and IgE. These classes or isotypes comprise at least one of said heavy chains that is named with a corresponding Greek letter. An antibody as described in the claims preferably comprises a constant region that is selected from the group of IgG, IgA, IgM, IgD, and IgE constant regions, more preferably said constant region comprises an IgG constant region, more preferably an IgG1 constant region, preferably a mutated IgG1 constant region. Some variation in the constant region of IgG1 occurs in nature, such as for instance the allotypes G1m1, 17 and G1m3, and/or is allowed without changing the immunological properties of the resulting antibody. Typically between about 1-10 amino acid insertions, deletions, substitutions or a combination thereof are allowed in the constant region.

Said antibody comprises at least the CDR1, CDR2 and CDR3 sequences of an MF3958 ErbB-2 specific heavy chain variable region, as depicted in Figure 10A or Figure 10E.

Said antibody comprising a variable domain that binds ErbB-3 comprises at least the CDR1, CDR2 and CDR3 sequences of an MF3178 ErbB-3 specific heavy chain variable region as depicted in Figure 10B or Figure 10E or Figure 11.

The bispecific antibody as described herein, comprises a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein said first antigen-binding site comprises at least the CDR1, CDR2 and CDR3 sequences of MF3958 and wherein said second antigen-binding site comprises at least the CDR1, CDR2 and CDR3 sequence of MF3178.

The VH chain of the variable domain that binds ErbB-2 preferably comprises the amino acid sequence of MF3958; or comprises the amino acid sequence of MF3958 depicted in figure 10A 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 with respect to the VH chain sequence. The VH chain of the variable domain that binds ErbB-3 comprises the amino acid sequence of MF3178; or comprises the amino acid sequence of MF3178 depicted in Figure 10B 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 with respect to the VH chain sequence. The above-mentioned amino acid insertions, deletions and substitutions are not present in the CDR3 region, the CDR1 and CDR2 regions. The above-mentioned amino acid insertions, deletions and substitutions are also preferably not present in the FR4 region.

The ErbB-2/ErbB-3 specific antibody as disclosed herein is a bispecific antibody. The antibody preferably comprises a variable domain with a heavy chain variable region comprising at least the CDR1, CDR2 and CDR3 sequence of an ErbB-2 specific heavy chain variable region of MF3958 as depicted in Figure 10, and a variable domain with a heavy chain variable region comprising at least the CDR1, CDR2 and CDR3 sequence of an ErbB-3 specific heavy chain variable region of MF3178 as depicted in Figure 10.

The ErbB-2/ErbB-3 specific antibody having the aforementioned CDRs of MF3958 and MF3178 preferably comprises an ErbB-2 specific variable domain with a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region of MF3958 as depicted in Figure 10 with 0-10, preferably 0-5, preferably 0, 1 or 2 amino acid substitutions and an ErbB-3 specific variable domain with a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region of MF3178 as depicted in Figure 10 with 0-10, preferably 0-5, preferably 0, 1 or 2 amino acid substitutions. The light chain in these antibodies is

The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use in a method of treating breast cancer in a subject that has breast cancer or is at risk of having breast cancer preferably further comprises determining the expression level of the estrogen receptor, ErbB-2, ErbB-3, or a combination thereof on cells of said cancer.

The subject that has breast cancer or is at risk of having breast cancer is preferably a human. A person is said to be at risk of having breast cancer if that person has had breast cancer in the past but it is in remission thereof such that the cancer cannot be detected with routine check-ups. Such a person can be considered cured but this person has a higher risk of having breast cancer when compared to a normal health y individual of the same age. The breast cancer can be a recurrent cancer at the position of the primary cancer which was in remission or a metastasis of the breast cancer typically at a position different from the position of the primary cancer site.

The invention provides an ErbB-2/ErbB-3 bispecific antibody and an endocrine therapy drug for use in a method of treating breast cancer in a subject that has breast cancer or is at risk of having said cancer, comprising administering to the subject in need thereof a therapeutically effective amount of the bispecific antibody that can bind an extra-cellular part of ErbB-2 and that inhibits ErbB-2/ErbB-3 dimerization on the cancer cell, wherein the cancer preferably is a hormone receptor positive cancer. The antibody is a bispecific antibody that has an antigen binding site that can bind an extra-cellular part of ErbB-2 and an antigen binding site that can bind an extra-cellular part of ErbB-3. Said endocrine therapy drug is preferably combined with the antibody in a therapeutically effective amount for administering to the subject in need thereof. The cancer is preferably an immunohistochemistry ErbB-2 + cancer or an immunohistochemistry ErbB-2 ++ without ErbB-2 gene amplification cancer

Typically, the bispecific antibody and endocrine therapy may be used repeatedly, over a course of treatment, in a method of treatment as described in the claims. For example, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of an endocrine therapy and multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of a bispecific antibody may be used in a method of treating a subject in need of said treatment.

The endocrine therapy for use may be given daily, on several days of the week, weekly, biweekly, every 3 or 4 weeks, or with longer intervals) and the bispecific antibody may be given weekly, biweekly, every 3 or 4 weeks, or with longer intervals. The bispecific antibody and endocrine therapy may be, but are not necessarily administered according to the same regimen of administration. Thus, endocrine therapy and the bispecific antibody may be given the same day or in different days, in different sequences (first endocrine, second bispecific, or vice versa). The endocrine therapy may be administered 1 or more days before or after the bispecific antibody or vice versa.

The dose of the bispecific antibody and/or endocrine therapy may be varied over time. The dose of the endocrine therapy and or the bispecific can be the same along the whole treatment. Alternatively, the dose of the endocrine therapy and/or the bispecific can be higher at the beginning, for example a load higher dose (which could be a unique or several doses) followed by a maintenance dose. Alternatively, the dose of the endocrine therapy and/or the bispecific can be lower at the beginning (which could be a unique or several doses) followed by a maintenance dose. In addition, or alternatively, the initial regimen of the endocrine therapy and/ or the bispecific antibody that started as a weekly regimen can change to a biweekly regimen or other. A clinician may utilize preferred dosages and/or dosage regimes as warranted by the condition of the patient being treated

Treatment with the endocrine therapy may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small amounts until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided in portions during the day if desired. Intermittent therapy (e.g., one week out of three weeks or three out of four weeks) may also be used.

The bispecific antibody for use may be administered at a flat dose of about 750 mg every 3 weeks, but alternatively may be given in a range of dose of from 300mg to 900mg flat dose in a regimen of administration of weekly, biweekly, every 3 weeks, every 4 weeks or with longer intervals.

Where herein ranges are given as between number 1 and number 2, the range includes the number 1 and number 2. For instance a range of between 2-5 includes the numbers 2 and 5.

When herein reference is made to an affinity that is higher than another, the Kd = lower than the other Kd. For the avoidance of doubt a Kd of 10e-9 M is lower than a Kd of 10e-8 M. The affinity of an antibody with a Kd of 10e-9 M for a target is higher than when the Kd is 10e-8 M.

BRIEF DESCRIPTION OF THE DRAWINGS

• Figure 1. Mean and Individual tumor growth changes in human HBCx-34 breast tumor xenograft. Treatments started 36 days post HBCx-34 implantation. Vehicle MCLA-128 and MCLA-128 25 mg/kg were administered on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56 and vehicle Letrozole and Letrozole 2.5 mg/kg were administered daily for 57 days. Initial group size: 9-10 animals.
• Figure 2. T/C% in human HBCx-34 breast tumor xenograft. Treatments started 36 days post HBCx-34 implantation. Vehicle MCLA-128 and MCLA-128 25 mg/kg were administered D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56 and vehicle Letrozole and Letrozole 2.5 mg/kg were administered daily for 57 days. Initial group size: 9-10 animals.
• Figure 3. Mean and Individual relative body weight changes in human HBCx-34 breast tumor xenograft. Treatments started 36 days post HBCx-34 implantation. Vehicle MCLA-128, MCLA-128 25 mg/kg were administered on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56 and vehicle Letrozole and Letrozole 2.5 mg/kg were administered daily for 57 days. Initial group size: 9-10 animals.
• 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 engrafted s.c. with HBCx-34 tumors. Animals supplemented with estrogens were treated with either vehicle of Fulvestrant and animals that did not receive estrogens were treated with vehicle or Letrozole. The supplementation of estrogen induced a faster tumor growth and Letrozole significantly reduced the formation of estrogen-independent tumors. T-tests were performed to compare tumor volumes from estrogen-independent tumors treated with vehicle or Letrozole (*, p<0.05, **, p<0.01, ***, p<0.001).
• Figure 5. Tumor volumes of MCF-7 xenograft mice at day 25. Treatments started MCLA-128 vehicle, MCLA-128 25 mg/kg 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 weekly. Statistical significance for Kruskal Wallis Dunn's or Mann-Whitney U test for comparison with vehicle group: ns indicates non-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.
• Figure 6. Tumor volumes of MCF-7 xenograft mice treated with MCLA-128, Fulvestrant or Tamoxifen as single agent or combination therapies. For each timepoint, a statistical t-test compared Fulvestrant vs Fulvestrant + MCLA-128 and Tamoxifen vs Tamoxifen + MCLA-128. N.s. indicates non-significant, * indicates P<0.05; ** indicates P<0.01.
• Figure 7. Inter-assay correlation between two independent VeraTag assays. Assay scores obtained in assays Round 1 and Round 2 were plotted in an XY graph and compared using x=y linear regression . All assays showed good correlation between the results of the two time points with only the "Gr.7 An.3" sample showing more than 20% coefficient variation (CV%).
• Figure 8. Modulation of levels of total HER2, total HER3 and HER2:HER3 dimers in MCF-7 tumors after 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 thereof. 24 hours after the last dose, tumors were harvested, processed to FFPE and subjected to VeraTag analysis for the indicated assays. Five independent tumors per treatment group were used for the final analysis. A statistical t-test compared all groups independently. ** indicates P<0.01.
• Figure 9. Reverse 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 derived from treatment groups with those derived from the PBS group. ** indicates P<0.01.
• Figure 10. Nucleic acid and amino acid sequences of VH-chains, common light chain and heavy chains of antibodies. Where in this figure a leader sequence is indicated this is not part of the VH chain or antibody, but is typically cleaved of during processing of the protein in the cell that produces the protein.
• Figure 11. Amino acid and nucleotide alignments of the MF3178 variants. CDR regions are indicated.
• Figure 12. Study design for combination therapy: MCLA-128 + endocrine treatment.
• Figure 13. Treatment administration for combination therapy study: MCLA-128 + endocrine treatment - all cycles.

EXAMPLES

As used herein "MFXXXX" wherein X is independently a numeral 0-9, refers to a Fab comprising a variable domain wherein the VH has the amino acid sequence identified by the 4 digits. Unless otherwise indicated the light chain variable region of the variable domain typically has a sequence of the variable region of the light chain of Figure 10C. The light chain is typically the light chain as indicated in figure 10C. "MFXXXX VH" refers to the amino acid sequence of the VH identified by the 4 digits. The MF further comprises a constant region of a light chain and a constant region of a heavy chain that normally interacts with a constant region of a light chain. PG refers to a monospecific antibody comprising identical heavy and light chains. PB refers to a bispecific antibody with two different heavy chains. The VH variable regions of the heavy chains differs and typically also the CH3 region, wherein one of the heavy chains has a KK mutation of its CH3 domain and the other has the complementing DE mutation of its CH3 domain ( WO2013/157954 ).

Example 1

Antitumor efficacy of an HER2/HER3 targeting antibody was determined in the context of a combination treatment together with an aromatase inhibitor. MCLA-128 was used as a preferred example of a bispecific Her2/Her3 targeting antibody. It was used as 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 a breast cancer.

The human tumor xenograft models

Human tumor samples of various histological origins were obtained with informed consent from patients treated at cancer centers and established as transplantable xenografts in immunodeficient mice. The grafted samples are residual material from primary tumors or metastases obtained before or after treatment. These patient-derived xenograft (PDX) models have been established without prior in vitro culture and have been studied for histology, cytogenetics, genetic and other biological markers, and for their response to standard-of-care (SOC) therapies.

The HBCx-34 PDX model was derived from a treatment-naive primary breast infiltrating ductal carcinoma. The HBCx-34 PDX model has a mutated ATM (gene coding for a protein implicated in double strand DNA repair) and wt p53, is ER+/PR+, and is responder to Docetaxel, Capecitabine, Tamoxifen and the combination Adriamycin/ Cyclophosphamide and low responder to Letrozole.

The HBCx-34 tumor model takes about 35 days to obtain the maximum of tumors in the range 60 to 200 mm³ and about 80 days to reach 2000 mm³ from implantation day (with estrogen supplementation). HBCx-34 has got no overt cachectic properties, but not body weight gain is observed in HBCx-34-bearing mice

MCLA-128 is as an ADCC-enhanced IgG1 bispecific antibody that targets the HER2:HER3 dimer. MCLA-128 demonstrates an in vitro potency superior to other anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of heregulin (HRG) thereby overcoming one of the resistance mechanisms of current HER2 therapies.

Tumor-bearing mice received estrogen diluted in drinking water (β-oestradiol, 8.5 mg/l), from the date of tumor implant to the date of inclusion. During the treatment period, mice were not supplemented with estrogen.

Animals and maintenance conditions

Outbred athymic (nu/nu) female mice (<< HSD : Athymic Nude-Foxn1^nu >>) weighing 18-25 grams (Harlan Laboratories, Gannat, France) were allocated to acclimate in the animal facility with access to food and water ad libitum for at least 6 days prior to manipulation (Scheme 4-1).

Scheme 4-1 Animal Characteristics

Mouse (Mus musculus)

Harlan, France

Female

18-25

5 weeks

Test compound and formulations

The MCLA-128 vehicle was ready-to-use and used for dosing from day 0 to day 38 and stored at +4°C. Then, from day 42 to day 56 (end of the study), 0.9% NaCl (CDM Lavoisier batch 6F134) was used as vehicle for dosing and MCLA-128 preparation. The 0.9% NaCl aliquots (CDM Lavoisier batch 6F134) were weekly prepared from day 42 to day 56 and stored at +4°C. The Letrozole vehicle (CDM Lavoisier batch 6F134): 0.9%NaCl was ready-to-use. The 0.9% NaCl aliquots (CDM Lavoisier batch 6F134) were weekly prepared and stored at +4°C.

MCLA-128 was ready-to-use and used for injections from day 0 to day 42. After that, MCLA-128 aliquots were diluted in 0.9% NaCl to obtain the working solutions at 2.5 mg/ml which were prepared before each injection. They were used for dosing from day 42 to day 56 (end of the study). Letrozole tablets (Letrozole Actavis) were dissolved in 0.9%NaCl under magnetic agitation to form the final solution at 0.25 mg/ml. The dosing solution was stable for 7 days and stored at +4°C light protected.

Tumor graft models induction

Tumors of the same passage were transplanted subcutaneously onto 6-24 mice (donor mice, passage (n-1)). When these tumors reached 1000 to 2000 mm³, donor mice were sacrificed by cervical dislocation, tumors were aseptically excised and dissected. After removing necrotic areas, tumors were cut into fragments measuring approximately 20 mm³ and transferred in culture medium before grafting.

95 mice were anaesthetized with 100 mg/kg ketamine hydrochloride (batch 5D92, Exp: 2017/03, Virbac) and 10 mg/kg xylazine (batch KP0AX9X, Exp: 2017/08, Bayer), and then skin was aseptized with a chlorhexidine solution, incised at the level of the interscapular region, and a 20 mm³ tumor fragment was placed in the subcutaneous tissue. Skin was closed with clips. All mice from the same experiment were implanted on the same day.

Treatment phase

40 mice with a subcutaneously growing HBCx-34 tumor between 62.5 and 256 mm³ were allocated, according to their tumor volume to give homogenous mean and median tumor volume in each treatment arm. Treatments were randomly attributed to boxes housing up to 5 mice and were initiated 36 days post implantation of the tumor (42% inclusion rate). The study was terminated following 57 days after the start of treatment.

Tumor measurement and animal observations

Tumor volume was evaluated by measuring tumor diameters, with a calliper, biweekly during the experimental period.

The formula TV (mm3) = [length (mm) × width (mm)2]/2 was used, where the length and the width are the longest and the shortest diameters of the tumor, respectively.

All animals were weighed biweekly during the experimental period. Toxicity of the different treatments was determined as: body weight loss percent (% BWL) = 100 - (mean BWx/mean BW0x 100), where BWx is the mean BW at any day during the treatment and BW0 is the mean BW on the 1st day of treatment.

A total of 4 groups were used as summarized in the scheme below. Each group initially included 10 mice.

Gr.	Nb of included mice	Test Item
1	10	Vehicle MCLA-128	126.9	117.0	16.52
2	10	MCLA-128 25 mg/kg	124.4	117.0	13.31
3	10	Letrozole 2.5 mg/kg	126.9	117.0	16.92
4	10	MCLA-128 25 mg/kg	123.1	126.0	19.72
		Letrozole 2.5 mg/kg

• In group 1, vehicle MCLA-128 and vehicle Letrozole were dosed at 10 ml/kg, i.p. D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56 and p.o. qdx 57 respectively;
• In groups 2 and 4, MCLA-128 was dosed at 25 mg/kg, i.p., D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56;
• In groups 3 and 4, Letrozole was dosed at 2.5 mg/kg, p.o., qd × 57.

All treatment doses were body weight adjusted at each injection.

Dose and dose schedules in the XTS-1521 efficacy study

Gr.	N
		Agent	Dose mg/kg	Route	Volume ml/kg	Schedule	Agent	Dose mg/kg	Route	Volume ml/kg	Schedule
1	10	Vehicle MCLA-128	-	IP	10	D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56	Vehicle Letrozole ∧	-	PO	10	qd×57*
2	10	MCLA-128	25	IP	10	As above	-	-	-	-	-
3	10	-	-	-	-	-	Letrozole	2.5	PO	10	qd×57*
4	10	MCLA-128	25	IP	10	As above	Letrozole	2.5	PO	10	qd×57*

(*) qd × 57: from D0 to D56. (^) Vehicle Letrozole = 0.9% NaCl

Results

Mean percent body weight change during the treatment period are illustrated in Figure 3.

In group 1, MCLA-128 vehicle given i.p. on D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56 and Letrozole vehicle given p.o. qdx57, both administered at 10 ml/kg, were well tolerated with 0.4% of maximum mean body weight loss on day 4 and 3.7% of maximum individual body weight loss on day 49.

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

In group 3, Letrozole dosed p.o. at 2.5 mg/kg, administered at 10 ml/kg, qdx57, was well tolerated with no mean body weight loss and 4.1% of maximum individual body weight loss on day 4.

In group 4, MCLA-128, i.p. D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56 dosed at 25 mg/kg, i.p. and Letrozole dosed at 2.5 mg/kg, p.o. qdx57, both administered at 10 ml/kg, was well tolerated with 0.7% of maximum mean body weight loss on day 4 and 4.2% of maximum individual body weight loss on day 4.

Tumor growth curves (mean tumor volume over time) are illustrated in Figure 1. Percent T/C values for each treatment group are indicated in table 1 and illustrated in Figure 2. Definitions of partial and complete responses are defined in Table 2. Statistical analysis is shown in Table 3 and Table 4. In this study tumors were measured biweekly during the experimental period.

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

In group 3, Letrozole dosed at 2.5 mg/kg, administered at 10 ml/kg, p.o. qdx57, induced a statistically significant tumor growth inhibition (p<0.001 compared with the vehicle group, Mann Whitney test and p<0.05 compared to the group control by Dunn's test) with TGDi >1.1, best T/C% = 18.68% on day 56 (end of the control group) and best TGI%=145.70% at day 35. Moreover, 7/10 partial tumor regression and 1/10 complete tumor regression were observed.

In group 4, the combination MCLA-128, i.p. D0, D3, D7, D10, D14, D17, D21, D24, D28, D31, D35, D38, D42, D45, D49, D52, D56 dosed at 25 mg/kg, i.p. and Letrozole dosed at 2.5 mg/kg, administered at 10 ml/kg, p.o. qdx57, induced a statistically significant tumor growth inhibition (p<0.001 compared with the vehicle group, Mann Whitney test and Dunn's test) with TGDi >1.1, best T/C% = 9.64% on day 56 (end of the control group) and TGI%= 169.03% at day 32. Moreover, 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.

Discussion

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

In the HBCx-34 model, MCLA-128 alone did not induce tumor growth inhibition whereas Letrozole alone induced a statistically significant tumor growth inhibition. Letrozole in combination with MCLA-128 induced a statistically significant synergistic tumor growth inhibition.

Treatment selection for breast cancer patients are guided by three biomarkers: HER2, estrogen (ER) and progesterone (PR) receptors. Patients with HER2 overexpression are eligible for HER2-targeting therapies, such as Trastuzumab and Pertuzumab. ER/PR positive patients will receive antiestrogen therapies or aromatase inhibitors. Antiestrogens (such Tamoxifen and Fulvestrant) modulate ER activity while aromatase inhibitors (AI, such as Letrozole or exemestane) inhibit the conversion of androgens to estrogens, depleting levels of ER stimuli in patients. Antiestrogen and aromatase inhibitors are used in different ER+ breast cancer patient populations, e.g. AI being used as first-line treatment in post-menopausal patients. In example 2 it is demonstrated that adding MCLA-128 to either Tamoxifen or Fulvestrant enhances the activity of the antiestrogen treatments.

Example 2

MCLA-128 was evaluated as monotherapy and in combination with the SERM Tamoxifen and SERD Fulvestrant (Faslodex^®) for efficacy in a nude mouse xenograft model of estrogen responsive MCF-7 human breast carcinoma. The study design also included tumor collection for downstream analysis.

Treatments began on D1 in mice with established subcutaneous MCF-7 tumors. The study endpoint was intended to be a tumor volume endpoint of 1000 mm3 45 days, whichever came first. The study ended on D39, and treatment outcome was based on percent tumor growth inhibition (%TGI), defined as the percent difference between D25 median tumor volumes (MTVs) of treated and control mice. D25 was chosen for analysis as it was the last day before animals exited the study for tumor progression. Treatment response was determined from an analysis of percent tumor growth inhibition (%TGI), defined as the percent difference between final (D25) median tumor volumes (MTVs) of treated and control groups, with differences between groups deemed statistically significant at P ≤ 0.05 using the Mann-Whitney test. Tumor regressions, mean tumor growth, and treatment tolerability also were considered.

Mice

Female athymic nude mice (Crl:NU(NCr)-Foxn1^nu, Charles River) were ten weeks old and had a body weight range of 19.2 to 30.5 grams on D1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated Lab Diet^® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o'cobs^™ Laboratory Animal Bedding in static microisolators on a 12-hour light cycle at 20-22 °C (68-72 °F) and 40-60% humidity.

Tumor Implantation

Three days prior to tumor cell implantation, estrogen pellets (0.36 mg estradiol, 60-day release, Innovative Research of America, Sarasota, FL) were implanted subcutaneously between the scapulae of all test animals using a sterilized trocar.

Xenografts were initiated with cultured MCF-7 human breast carcinoma cells. Tumor cells were grown to mid-log phase in RPMI-1640 medium containing 10% fetal bovine serum, 100 units/mL penicillin G, 100 µg/mL streptomycin sulfate, 2 mM glutamine, 10 mM HEPES, 0.075% sodium bicarbonate and 25 µg/mL gentamicin. On the day of tumor cell implant, the cells were trypsinized, pelleted, 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 tumor growth was monitored as their mean volume approached the desired 100 to 150 mm3 range.

Nineteen days after tumor cell implantation, designated as D1 of the study, the mice were placed into six groups of 15 animals and four groups of six animals. Individual tumor volumes ranged from 75 to 196 mm3 and group mean tumor volumes of 134-137 mm3 on D1. Tumor weight was estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume.

Therapeutic Agents

MCLA-128 was stored at 4oC, was provided pre-formulated at 2.5 mg/mL and ready to dose as 25 mg/kg in a 10 mL/kg dose volume, and was protected from light during storage and handling. Tamoxifen (Sigma-Aldrich, Lot No. WXBB5732V) was received as a powder and was stored at 4 °C protected from light. Each week, a 5 mg/mL dose solution was prepared 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 tradename Faslodex^® (AstraZeneca, Lot No. LV032, LW466, and LX432) was received as 50 mg/mL stock solution that was stored at 4 °C. Each dosing day, the stock was diluted in corn oil to 25 mg/mL, and each mouse received 0.2 mL for a dose of 5 mg/animal.

Treatment

Groups 1 and 7 served as controls for efficacy and sampling, respectively, and received MCLA-128 vehicle intraperitoneally (i.p.) on D1, 4, 8, 11, 15, 18, 22, 25, and 29 and corn oil subcutaneously (s.c.) every other day for fifteen doses (qod × 15). Groups 2 and 8 were administered MCLA-128 at 25 mg/kg, i.p., on the same schedule as MCLA-128 vehicle. Groups 3 and 9 were administered Faslodex^® at 5.0 mg/animal, s.c., once weekly for five weeks (qwk × 5). Group 4 was administered Tamoxifen at 1 mg/animal, s.c., qod × 15. Groups 5 and 10 received MCLA-128 and Faslodex^®, on the regimens described above. Group 6 received MCLA-128 and Tamoxifen on the regimens described above.

MCLA-128 was administered at a 10 mL/kg dose volume, scaled to the individual body weight of each animal. Faslodex^® and Tamoxifen were administered at a fixed volume of 0.2 mL.

Results

Figure 5 is a scatter plot showing the tumor distribution by group. Figure 6 shows the group median tumor growth curves for all groups in the study.

Growth of MCF-7 Tumors in Control Mice (Group 1)

Group 1 mice received MCLA-128 vehicle and corn oil as indicated above and served as the control group for calculation of percent TGI and statistical comparisons. The median tumor volume was 600 mm3 with individual tumor volumes on D25 ranging from 288 to 936 mm3 (Figure 5) in this group. At the time of data analysis, there were two NTRu deaths recorded on D15 and D21, leaving thirteen assessable animals. Additional NTRu deaths were recorded after D25; one on D26 and two on D35. All deaths were due to suspected estrogen-related toxicity.

Response to MCLA-128 (Group 2)

In Group 2, MCLA-128 was administered as indicated above. This treatment resulted in a median tumor volume of 600 mm3, corresponding to a non-significant 0% TGI relative to the control group (P > 0.05). At the time of data analysis, there were six NTRu deaths recorded; two each on D17 and D22, and one each on D19 and D20, leaving nine assessable animals.

Additional NTRu deaths were recorded after D25; one each on D29 and D36. All deaths were due to suspected estrogen-related toxicity.

Response to Faslodex^® (Group 3)

In Group 3, Faslodex^® was administered as indicated above.. This treatment resulted in a median tumor volume of 288 mm3, corresponding to a significant 52% TGI relative to the control group (P < 0.001). There were two NTRu deaths recorded on D20 and D24, leaving thirteen assessable animals. All deaths were due to suspected estrogen-related toxicity.

Response to Tamoxifen (Group 4)

In Group 4, Tamoxifen was administered as indicated above. This treatment resulted in a median tumor volume of 184 mm3, corresponding to a significant 69% TGI relative to the control group (P < 0.001). At the time of data analysis, there were three NTRu deaths recorded on D3, D10, and D22, leaving twelve assessable animals. Two deaths were also recorded on D26 which was after the time of data analysis. All deaths were due to suspected estrogen-related toxicity.

Response to MCLA-128 combined with Faslodex^® or Tamoxifen (Groups 5 and 6)

In Group 5, MCLA-128 was combined with Faslodex^® and administered as indicated above. This treatment resulted in a median tumor volume of 126 mm3 corresponding to a significant 79% TGI. This outcome was significant relative to control and MCLA-128 monotherapy groups (P < 0.001) as well as to Faslodex^® monotherapy (P < 0.05). Three PRs were recorded in this group. One NTRu death was recorded on D17, leaving fourteen assessable animals. This death was due to suspected estrogen-related toxicity.

In Group 6, MCLA-128 was combined with Tamoxifen and administered as indicted. This treatment resulted in a median tumor volume of 108 mm3 corresponding to a significant 82% TGI. This outcome was significant relative to control and MCLA-128 monotherapy (P < 0.001) as well as to Tamoxifen monotherapy (P < 0.05). Four PRs were recorded in this group. At the time of data analysis, there was one NTRu death recorded on D15, leaving fourteen assessable animals. Three additional deaths were also recorded after the time of data analysis; two on D30 and one on D39. These deaths were due to suspected estrogen-related toxicity.

Discussion

This example evaluated the agent MCLA-128 compared to and combined with Faslodex^® and Tamoxifen for efficacy in a nude mouse xenograft model of estrogen responsive MCF-7 human breast carcinoma. Tumors were measured twice per week through D39, and TGI analysis was performed on D25.

On D25, the median tumor volume for the control Group 1 was 600 mm3, with an individual tumor range of 288 to 936 mm3. Groups administered MCLA-128, Faslodex^®, or Tamoxifen resulted in median tumors volumes of 600, 288, and 184 mm3, corresponding to TGIs of 0, 52 and 69% TGI. The outcome of MCLA-128 monotherapy was not significant compared to control (P > 0.05), but the outcomes for Tamoxifen and Faslodex^® were significant compared to control (P < 0.001). MCLA-128 treatment in combination with Tamoxifen or Faslodex^® resulted in median tumor volumes of 126 and 108 mm3, corresponding to 79 and 82% TGI, respectively. Each of these outcomes was significant compared to control and MCLA-128 monotherapy (P < 0.001) as well as to Tamoxifen or Faslodex^® respective therapies (P < 0.05). Three PRs were recorded in the MCLA-128/ Faslodex^® group and four PRs were recorded in the MCLA-128/Tamoxifen group. All treatments were well tolerated. Several deaths across all groups were attributed to estrogen toxicity. In summary, MCLA-128 combination therapy with either Faslodex^® or Tamoxifen offered significant synergistic survival benefit compared to respective monotherapies in the MCF-7 nude mouse xenograft model. All treatments were acceptably tolerated.

MCLA-128 showed no anti-tumor efficacy as a single agent, while Fulvestrant and Tamoxifen both significantly reduced tumor growth. Towards the end of the treatment period at day 25, MCLA-128 combination therapy with either Fulvestrant or Tamoxifen offered significant survival benefit when compared with the respective monotherapies (Figure 5). Interestingly, the beneficial anti-tumor effect of combined treatment continued during the treatment-free observation period (Figure 6). All treatments were acceptably tolerated.

Example 3 Pharmacodynamic analysis VeraTag assay Round 1

All formalin-fixed tumors from example 2 were processed to FFPE blocks. The initial VeraTag assay analysis (round 1) was performed with 3 tumors per group

Prior to the VeraTag analysis, tumors were sectioned and stained with hematoxylin-eosin to determine the percentage of tumor content. Microlaser capture was used to grossly remove any non-tumor content. The initial round of analysis included the following five VeraTag assays: total HER2, total HER3, HER2:HER3 dimers, HER3-PI3K complexes and phosphorylated HER3.

Relative to vehicle, MCLA-128 treatment showed no significant effect in any of the assays. A significant difference between the groups was only observed in the HER2:HER3 assay, where treatment with Fulvestrant significantly upregulated the formation of HER2:HER3 dimers and co-treatment with MCLA-128 reversed the levels of HER2:HER3 dimers to the situation in vehicle-treated tumors.

While not statistically significant, a similar trend was observed in the HER2 and HER3 assays. Since the data included only three tumors per treatment group, more data points were needed in order to confirm whether this trend was an indication of a result similar to that seen for the HER2:HER3 assay. Therefore, the remaining tumors from Table 5 were included in a second round of VeraTag assays for total HER2, total HER3 and HER2:HER3 dimers. Since no significant effect of Fulvestrant or the combination with MCLA-128 was observed in the phospho-HER3 and HER3-PI3K assays, these assays were not included in Round 2.

VeraTag assay Round 2

The tumors analyzed in Round 1 that had scores close to the average value of the specific assays were included in Round 2 to estimate the inter-assay reproducibility and to ensure the data from the two independent experiments could be combined. The results for those tumors analyzed in both rounds correlated well between assays, with a coefficient of variation (CV) below 20%, except for tumor "Gr.7 An.3", which was excluded from the final analysis (Figure 7).

Data from both rounds were combined and the results confirmed that Fulvestrant significantly induced HER2:HER3 dimerization compared to vehicle, and that this was reversed by co-administration of MCLA-128 (Figure 8).

In addition, HER2 expression levels in the Fulvestrant group were significantly higher than in the MCLA-128 single or combined treatment groups. The vehicle group had some variation between the samples and did not show a significant difference with the Fulvestrant group. Finally, HER3 expression were not significantly altered between the different treatment groups, although there was a slight increase in HER3 in the Fulvestrant group relative to all other groups.

RPPA analysis

Tumors from groups 1-6 were collected when they had reached the maximum tolerated volume, or at the end of the experiment at day 39, which was 10 days after the last dose. Per treatment group, six tumors were included in the Reverse phase protein array (RPPA). The selection of tumors was made so that the average tumor size of the 6 specimens was close to that of the whole group. RPPA analysis was performed as follows: tumors were sectioned and placed on immunohistochemistry slides. Stromal and inflammatory content was macrodissected to purify for tumors cells. Protein lysates were prepared and protein content was quantified. Protein samples were then spotted on nitrocellulose slides in quadruplicate at two different concentrations and then incubated with primary antibodies specific for Akt, ERK, HER2 and HER3, in their total or phosphorylated forms.

Reverse phase protein array (RPPA) data were analyzed using one-way ANOVA to detect significant differences between the treatment groups and the vehicle group. Total and phosphorylated Akt levels were increased in the Faslodex group, no significant differences were observed in the other groups. Total ERK levels were increased in the single agent groups only, no differences were measured in the other groups or in the phosphorylated ERK analysis. Total HER2 levels, but not phosphorylated HER2, were significantly increased in the Tamoxifen and Faslodex groups. MCLA-128 co-treatment with hormonal therapy prevented this induction of HER2 expression. HER3 total levels were significantly increased in the Faslodex group only and were reduced with co-treatment of MCLA-128.

Discussion Pharmacodynamic analysis

A finding of the VeraTag assay analysis was the significantly higher levels of HER2 expression in tumors from mice treated with Fulvestrant. Scores on the VeraTag assay correlate with HER2 positivity as determined by immunohistochemistry (IHC): HER2 VeraTag scores below 10.5 are negative in IHC, scores between 10.5 and 17.8 are equivocal and scores above 17.8 are positive (Huang et al., 2010 Am J Clin Pathol. 134(2):303-11). It therefore appears that Fulvestrant treatment can change the HER2 status of a HER2-negative tumor (i.e. scored as 0 or 1+ according to ASCO guidelines, Wolff et al. 2013 J Clin Oncol. 31(31):3997-4013). to a HER2 status that is at least equivocal (typically scored as 2+). Most importantly, the VeraTag analysis showed that Fulvestrant treatment induces the formation of HER2:HER3 dimers, which can be reversed by the addition of MCLA-128 to this treatment regimen.

Biomarker analysis

A finding of this set of experiments - in which we measured levels of a panel of biomarkers using RPPA - was enhanced Akt, HER2 and HER3 expression in hormonal therapy-treated tumors. The fact that this hormonal therapy-induced increase could be reverted by co-administering MCLA-128 suggests that the activation of the Akt signaling pathway is linked to HER2:HER3 dimer activity, which was thus targeted by MCLA-128 in these tumors. This is in line with the data obtained from the VeraTag assay and further substantiates the beneficial effect of co-administering MCLA-128 with hormonal therapy in vivo.

General considerations

The increase in HER2 protein expression after Fulvestrant treatment in an MCF-7 xenograft is in line with mechanisms of resistance against hormonal therapy that have 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 with the RPPA analysis but not the VeraTag analysis. This may be due to different timing for the tumor harvest (at the beginning and end of the treatment period for the VeraTag and RPPA assays, respectively).

The synergy between MCLA-128 and Fulvestrant can be explained by the increased HER2:HER3 dimer formation seen in tumors derived from mice treated with this combination. Also the increased phosphorylation of Akt observed in tumors of mice treated with Fulvestrant alone (RPPA analysis).

Example 4: Phase II study of MCLA-128/endocrine therapy in estrogen receptor positive and low-HER2 expression MBC

While the Example describes the administration of MCLA-128 in combination with endocrine therapy, the Example is not intended to be limiting to the use of the specific therapeutic agents set out, and applies to the disclosed ErbB-2 and ErbB-3 binding bispecific antibodies in combination with an endocrine therapy.

OBJECTIVES: Estrogen receptor [ER]-positive/low HER2 expression MBC): MCLA-128 + endocrine therapy Primary objective:

• Evaluate efficacy of MCLA-128 combined with endocrine therapy in terms of clinical benefit rate (CBR) at 24 weeks based on RECIST 1.1 (per investigator review) in ER-positive and low HER2 expression MBC patients who have previously progressed on the same endocrine therapy

Secondary objectives:

• Evaluate CBR at 24 weeks based on RECIST 1.1 per central review
• Evaluate progression free survival (PFS) (per investigator and central review)
• Evaluate objective response rate (ORR) based on RECIST 1.1 (per investigator and central review)
• Evaluate the time from response (CR or PR) until progression or death due to underlying cancer (DoR) based on RECIST 1.1 (per investigator and central review)
• Evaluate OS
• Evaluate safety and tolerability of MCLA-128 combined with endocrine therapy
• Characterize PK of MCLA-128 combined with endocrine therapy
• Characterize immunogenicity of MCLA-128 combined with endocrine therapy

Exploratory objective:

• Evaluate potential correlations between biomarkers in tumor or blood samples and antitumor activity (including HER2, HER3, HER2:HER3 dimers, heregulin and other potential biomarkers).

STUDY DESIGN

A phase 2, open-label, multicenter international study is performed to evaluate the efficacy of MCLA-128-based combinations in a metastatic breast cancer (MBC) population, ER-positive/low HER2.

Patients are ER-positive with low HER2 expression metastatic breast cancer (MBC) (immunohistochemistry (IHC) 1+, or IHC 2+ combined with negative fluorescence in situ hybridization (FISH)) who have progressed per RECIST v1.1 on the last line of prior endocrine therapy (administered for at least 12 weeks) that included an aromatase inhibitor or fulvestrant.

Patients must have received 1 or 2 prior endocrine therapies in the metastatic setting and have progressed (per RECIST v1.1) on a cyclin-dependent kinase inhibitor (in any line) are eligible.

For enrollment, HER2 and HR status and radiologic documentation of prior progression are based on medical records. Eligibility is confirmed as soon as possible for HER2/HR status by central lab review and for prior disease progression by central imaging review. Patients found to be ineligible retrospectively are not evaluable for the primary objective and may be replaced.

MCLA-128 is administered in combination with the same previous endocrine therapy on which progressive disease is radiologically documented. A total of up to 40 patients evaluable for efficacy is included.

See Figure 12.

STUDY POPULATION Inclusion criteria

Patients must fulfill all of the following requirements to enter the study:

1. 1. Signed informed consent before initiation of any study procedures.
2. 2. Women with histologically or cytologically confirmed breast cancer with evidence of metastatic or locally advanced disease not amenable to any local therapy with curative intent:
   1. a. Documented hormone receptor positive status (estrogen receptor positive [ER+] and/or progesterone receptor positive [PR+]), including ≥ 1% positive stained cells, based on analysis on the most recent tumor biopsy.
   2. b. Documented low-level HER2 expression, defined as IHC HER2 1+, or IHC HER2 2+ combined with negative FISH, based on local analysis on a fresh tumor biopsy or an archival biopsy collected within 12 months before screening (preferably metastatic otherwise primary).
   3. c. One or two lines of prior endocrine therapy (aromatase inhibitor or fulvestrant) for metastatic disease, with radiologically documented disease progression on the last line, after at least 12 weeks of therapy.
   4. d. Progression on a cyclin-dependent kinase inhibitor.
   5. e. No more than one previous chemotherapy regimen for advanced/metastatic disease. Note: Pre/peri-menopausal women can be enrolled if amenable to be treated with the lutenizing hormone-releasing hormone LHRH agonist goserelin. Such patients must have commenced treatment with goserelin or an alternative LHRH agonist at least 4 weeks prior to study entry, and patients who received an alternative LHRH agonist prior to study entry must switch to goserelin for the duration of the trial.
3. 3. Measurable disease as defined by RECIST version 1.1 by radiologic methods on or after the most recent line of therapy. For Cohort 2, imaging must be available for central review.
4. 4. Age ≥ 18 years at signature of informed consent.
5. 5. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
6. 6. Life expectancy of ≥ 12 weeks, as per investigator.
7. 7. Left ventricular ejection fraction (LVEF) ≥ 50% by echocardiogram (ECHO) or multiple gated acquisition scan (MUGA).
8. 8. Adequate organ function:
   1. a. Absolute neutrophil count (ANC) ≥ 1.5 × 109/L
   2. b. Hemoglobin ≥ 9 g/dL
   3. c. Platelets ≥ 100 × 109/L
   4. d. Serum calcium within normal ranges (or corrected with supplements)
   5. 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 normal ranges will be allowed)
   6. f. Serum creatinine ≤ 1.5 × ULN or creatinine clearance ≥ 60 mL/min calculated according to the Cockroft and Gault formula or modification of diet in renal disease (MDRD) formula for patients aged > 65 years (Appendix 19.2)
   7. g. Serum albumin > 3.0 g/dL

INVESTIGATIONAL AND COMPANION THERAPIES

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

Premedication with paracetamol/acetaminophen, antihistamines and corticosteroids (as per standard practices) is mandatory for every MCLA-128 infusion.

Endocrine therapy: Patients receive the same dose and regimen as that administered under the last line of endocrine therapy prior to study entry on which the patient progressed.

TREATMENT REGIMENS

A cycle is 3 weeks (including Cohort 2 which may include q4w fulvestrant dosing). A 6-hour observation period is implemented following infusion start for the initial MCLA-128 administration, and 2 hours for all subsequent administrations.

All patients receive MCLA-128 administration on Day 1 q3w. For endocrine therapy, patients receive the same dose and regimen as that administered under the last line of endocrine therapy prior to study entry and on which the patient progressed.

Fulvestrant is administered on Days 1, 15, 29 and once every 28 days thereafter, or aromatase inhibitor therapy (letrozole, anastrazole and exemestane) is administered daily from Day 1.

Treatment administration (all cycles)

See Figure 13. A 6-hour observation period is implemented following infusion start for the initial MCLA-128 administration, and 2 hours for all subsequent administrations. • Same endocrine therapy under which the patient progressed prior to study entry. Can be administered before, during, or immediately after the MCLA-128 infusion.

Treatment assignment

No specific treatment assignment is required.

Treatment adaptation

• No dose reductions are permitted for MCLA-128 or endocrine therapy agents.
• MCLA-128 infusion will be interrupted in the event of an infusion-related reaction (IRR) and must be stopped definitively for severe IRRs. For mild to moderate events the infusion can be resumed at a 50% infusion rate and infusion duration extended to 4 hours.
• MCLA-128 administration can be delayed for a maximum of 6 weeks between infusions to manage adverse events (AEs), specifically for clinically significant LVEF decreases, signs of congestive heart failure or persistent grade 2 or grade 3-4 diarrhea.
• Hormone therapy drugs are administered according to the summary of product characteristics (SPC) of each drug.

Treatment duration

Study treatment is administered until confirmed progressive disease (as per RECIST 1.1), unacceptable toxicity, withdrawal of consent, patient non-compliance, investigator decision (e.g. clinical deterioration), treatment interruption > 6 consecutive weeks, withdrawal of any study drug. Patients are followed up for safety for at least 35 ± 5 days following the last study drug administration and until recovery/stabilization of related toxicities, and for disease progression and survival status for 12 months.

PROPHYLACTIC AND CONCOMITANT MEDICATION Permitted

• Administration of paracetamol/acetaminophen, antihistamines and corticosteroids is mandatory with every MCLA-128 administration. In the event of an IRR or hypersensitivity, the patient is managed according to local clinical practice, as clinically indicated.
• All medication necessary for the wellbeing of the patient and which is not expected to interfere with evaluation of the study drug, including supportive treatment of symptoms and AEs or standard treatment of concomitant conditions is given at the investigator's discretion.
• Goserelin in pre/peri-menopausal women who started a n (LHRH) agonist >4 weeks prior to study entry.

Prohibited

• Concomitant chronic oral corticosteroids (>10 mg/day prednisone equivalent), TNF-alpha inhibitors, anti-T-cell antibodies (due to risk of immunosuppression).
• Any investigational drugs during the study or 4 weeks prior to the first dose of study treatment.
• Systemic anticancer therapy (other than the last endocrine therapy) during the study or within 3 weeks of the first dose of study treatment.

SAFETY / TOLERABILITY ASSESSMENTS

• AEs (CTCAE version 4.03), SAEs
• Lab parameters: hematology, biochemistry, coagulation, urinalysis, cytokines
• ECG, MUGA/ECHO
• Medical history, vital signs, performance status and physical exam
• Concomitant medications
• Dose modifications (reductions, interruptions, delays), discontinuation due to toxicity

EFFICACY ASSESSMENTS

Tumor assessment is based on CT/MRI with contrast per RECIST 1.1, every 6 weeks after treatment start. Objective responses are confirmed at least 4 weeks after first observation. Central review of imaging by an independent radiologist(s) is performed for all patients (screening and on-study). Bone scans are performed as clinically indicated for patients with bone metastases at baseline or suspected lesions on study.

Tumor markers (CA15-3, CEA, CA27-29) are assessed on Day 1 every cycle.

BIOMARKERS

Candidate exploratory biomarkers are evaluated in tumor tissue (screening, optional after 12 weeks and EOT) and blood (pre-dose on Day 1 every 4 cycles and End of Treatment).

Tumor: HER2, HER3, HER2:HER3 dimerization, downstream signaling proteins (eg PIK3CA), heregulin, phosphorylation of HER2, HER3 and proteins in the MAPK and AKT signaling pathway, expression of inhibitors such as PTEN, mutations in cancer-related genes including HER2 and HER3 signaling, heregulin-gene fusions.

Blood: Fcy receptor polymorphism, plasma circulating tumor DNA mutations, exploratory serum biomarkers (e.g. soluble HER2, heregulin).

PHARMACOKINETICS

Blood samples are collected to measure serum MCLA-128. No PK sampling is performed for fulvestrant or aromatase inhibitors.

PK sampling is performed at the following time points:

• Cycle 1: Day 1, pre-dose, EOI, and at 2, 4, and 22 hours post EOI, then at any time on Day 8;
• Cycle 2, 3, 5: Day 1, pre-dose, EOI
• Every 4 cycles thereafter: pre-dose

IMMUNOGENICITY

Blood samples (5 mL) are collected in all patients to assess serum titers of anti-MCLA-128 antibodies pre-dose on Day 1 pre-dose for Cycles 1, 3, 5, every 4 cycles thereafter, and End of Treatment.

Definitions

All efficacy endpoints will be defined and analyzed based on tumor assessment by RECIST 1.1.

CBR: the proportion of patients with a best overall response of CR, PR or SD ≥ 24 weeks.

ORR: the proportion of patients with best overall response of CR or PR.

PFS: the time from treatment start until radiologic progression or death due to any cause.

PFS ratio: the ratio of PFS with the previous regimen to PFS on study treatment.

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

OS: the time from treatment start until death due to any cause.

Endpoints Primary

CBR per investigator radiologic review at 24 weeks

Key secondary

CBR at 24 weeks per central review, and PFS per investigator and central review

Other secondary

• Safety: Incidence, severity and relationship of AEs, laboratory abnormalities, SAEs, ECG and LVEF measurements and vital signs
• Tolerability: discontinuations due to AEs, dose modifications due to AEs, immunogenicity, and cytokine assessments
• Other efficacy: DoR, PFS ratio, ORR and OS
• Pharmacokinetics: Cmax, C0h, AUC, CL, Vss, tmax and t1/2 for MCLA-128

Analysis populations

Treated population: patients who receive at least one dose of MCLA-128.

Evaluable for efficacy: patients who receive at least 2 complete cycles (6 weeks) of treatment and have undergone baseline assessment and one on-study tumor assessment, or who discontinue early due to disease progression.

Analyses

Patient disposition and demographics are analyzed in the treated population, efficacy will be analyzed in the evaluable for efficacy population, and safety will be analyzed in the treated population.

Quantitative variables will be summarized using descriptive statistics. Continuous variables will be presented as N, mean and/or median, standard deviation, range. Categorical variables will be presented using frequencies and percentage.

Criteria for success primary endpoint: A median PFS of 5 months is assumed as relevant, with the activity threshold for CBR at 24 weeks set to 45%.

CBR and ORR are summarized with accompanying 90% exact binomial CI. For PFS, OS and DoR the survival function is estimated using the Kaplan-Meier product limit method; probability estimates and 90% CI is provided at specified time points; median duration and 90% CI is also be provided. DoR is estimated for responders only. The number and proportion of any patients with a PFS ratio ≥ 1.3 is tabulated for Cohort 2 with 90% exact CI.

AEs are tabulated by the Medical Dictionary for Regulatory Activities (MedDRA^®) preferred term and by organ class according to incidence and severity. Severity of AEs is based on Common Terminology for Adverse Events (CTCAE) 4.03.

PK, immunogenicity and biomarkers are analyzed centrally and reported separately. 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.

GR.7 AN.1	177.2	288
GR.7 AN.2	198.8	365
GR.7 AN.3	150	256
GR.7 AN.4	138.8	196
GR.7 AN.5	306.6	320
GR.7 AN.6	283.7	405
GR.8 AN.1	120.5	144
GR.8 AN.2	98.2	256
GR.8 AN.4	135.5	221
GR.8 AN.5	314	288
GR.8 AN.6	207.7	320
137.9	196
140.1	172
174.4	196
GR.9 AN.5	119.7	144
GR.9 AN.6	117.3	196
GR.10 AN.1	110.1	172
GR.10 AN.2	162.2	196
GR.10 AN.3	190.3	320
GR.10 AN.4	150.9	196
GR.10 AN.6	162.3	196

List of abbreviations in Example 1

bid:

Bidaily

BW:

Body Weight

BWL:

Body Weight Loss

C:

Control mice

CR:

Complete tumor Regression

inj:

Injection

i.v.:

Intravenous

i.p.:

Intraperitoneal

IVC:

Individually Ventilated Cages

kg:

Kilogram

mg:

Milligram

ml:

Milliliter

mut:

Mutated

ns:

Not significant

p.o.:

Per os, by mouth (gavage)

PR:

Partial tumor Regression

PSU:

Polysulfone

qd

Que die, every day

qwk

Once a week

RBW:

Relative Body Weight

RTV:

Relative Tumor Volume

S:

Significant

s.c.:

Subcutaneous

sem:

Standard error of the mean

SoC:

Standard of Care

T:

Treated mice

TGD:

Tumor Growth Delay

TGDI:

Tumor Growth Delay Index

TGI:

Tumor Growth Inhibition

TFS:

Tumor Free Survivor

TS:

Tumor Stabilization

% T/C:

Percent tumor volume change treated over control group

µL:

Microliter

qd:

quotidian

List of abbreviations in Example 2

BW

body weight

CR

complete tumor regression

D

Study Day

i.p.

intraperitoneal(ly)

MTD

maximum tolerated dose

MTV

median tumor volume

NTR

non-treatment-related

PR

partial tumor regression

qod x 15

dosing every other day for fifteen doses

qwk x 5

weekly dosing for five weeks

s.c.

subcutaneously

TGI

tumor growth inhibition

TR

treatment-related death

List of abbreviations in Example 4

AEs

adverse events

ALT

alanine aminotransferase

ANC

absolute neutrophil count

AST

aspartate aminotransferase

AUC

area under the curve

CBR

clinical benefit rate

CHF

congestive heart failure

CR

complete tumor regression

DoR

the time from response (CR or PR) until progression or death due to underlying cancer.

ECHO

echocardiogram

ER

estrogen receptor

FISH

fluorescence in situ hybridization

IHC

immunohistochemistry

IRR

infusion-related reaction

MBC

metastatic breast cancer

MDRD

modification of diet in renal disease

MUGA

multiple gated acquisition scan

LVD

left ventricular dysfunction

LVEF

left ventricular ejection fraction

LHRH

luteinizing hormone-releasing hormone

PFS

progression free survival

PR

progesterone receptor or partial remission depending on context

OCOG

eastern cooperative oncology group

ORR

objective response rate

OS

the time from treatment start until death due to any cause.

SPC

summary of Product Characteristics

ULN

upper limit of normal

Claims (16)

1. An ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use in a method of treating breast cancer in a subject that has breast cancer or is at risk of having said cancer, wherein the bispecific antibody has an antigen binding site that can bind an extra-cellular part of ErbB-2 and an antigen binding site that can bind an extra-cellular part of ErbB-3, wherein the endocrine therapy drug comprises Tamoxifen, Fulvestrant or Letrozole, and wherein said antibody comprises

   a variable domain with a heavy chain variable region comprising at least the CDR1, CDR2 and CDR3 sequences AYYIN, RIYPGSGYTSYAΓaKFΓaG and PPVYYDSAWFAY, respectively, of an ErbB-2 specific heavy chain variable region,

   a variable domain with a heavy chain variable region comprising at least the CDR1, CDR2 and CDR3 sequences GYYMH, WINPNSGGTNYAQKFQG and DHGSRHFWSYWGFDY, respectively, of an ErbB-3 specific heavy chain variable region, and

   a common light chain having sequence
2. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of claim 1, wherein the breast cancer is a hormone receptor positive breast cancer.
3. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of the preceding claims, wherein the breast cancer is an estrogen receptor positive breast cancer.
4. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of the preceding claims, wherein the breast cancer is a progesterone receptor positive breast cancer.
5. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of the preceding claims, wherein the breast cancer is ER-positive with low HER2 expression metastatic breast cancer MBC IHC 1+, or IHC 2+ combined with negative FISH.
6. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of the preceding claims, wherein the bispecific antibody can reduce a ligand-induced receptor function of ErbB-3 on a ErbB-2 and ErbB-3 positive cell.
7. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of the preceding claims, wherein the cancer is an immunohistochemistry ErbB-2 + cancer or an immunohistochemistry ErbB-2 ++ without ErbB-2 gene amplification cancer.
8. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of the preceding claims, further comprising administering to the patient a cyclin dependent kinase 4/6 inhibitor.
9. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use according to any one of the preceding claims, wherein the subject had received one or more endocrine therapy treatments prior to initiation of a treatment with said ErbB-2/ErbB-3 bispecific antibody and said therapeutically effective amount of an endocrine therapy drug.
10. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use according to any one of the preceding claims, wherein the subject had received a cyclin-dependent kinase inhibitor prior to initiation of a treatment with said ErbB-2/ErbB-3 bispecific antibody and said therapeutically effective amount of an endocrine therapy drug.
11. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of the preceding claims, further comprising determining the expression level of an estrogen receptor, ErbB-2, ErbB-3, or a combination thereof on cells of said cancer.
12. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use according to any one of the preceding claims, wherein the subject has a hormone receptor positive status (estrogen receptor positive [ER+] and/or progesterone receptor positive [PR+]), for ≥ 1% positive cells as determined by immunohistochemistry on a tumor biopsy.
13. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of the preceding claims, wherein the endocrine therapy drug comprises Tamoxifen.
14. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of the claims 1-12, wherein the endocrine therapy drug comprises Letrazole.
15. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use of any one of claims 1-12, wherein the endocrine therapy drug comprises Fulvestrant.
16. The ErbB-2/ErbB-3 bispecific antibody and a therapeutically effective amount of an endocrine therapy drug for use according to any one of the preceding claims, wherein the ErbB-2/ErbB-3 bispecific antibody is administered as an intravenous dose of from about 300mg to about 900mg, for example every two to four weeks.